. ..

.fi ‘
424.1,-
:2 ‘ 4 3.

Rania... ...
ab... 21‘;
u 0.. '5. ‘4

 

I».
.9!
.o

5....

2.3103}!

 

 

1:330}

 

LIE‘" ‘ "-‘Y
Michi: . State
University

 

 

 

This is to certify that the
dissertation entitled

ACCESSING THE
2-AMINO-5,5-D|SUBST|TUTED-1H-IMIDAZOL-4-ONE
SCAFFOLD FOR NATURAL PRODUCT SYNTHESIS
AND EVALUATION FOR
CHECKPOINT KINASE 2 INHIBITION

presented by

Christopher David Hupp

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Chemistry

 

 

m

 

\- Majorfiofessor’s Signature

6—Ol—oq]

Date

MSU is an Affirmative Action/Equal Opportunity Employer

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5/08 K:/Prolecc&Pres/CIRC/DaleDue.indd

 

 

ACCESSING THE
2-AMINO-5,5-DISUBSTITUTED-1H-IMIDAZOL—4-ONE
SCAFFOLD FOR NATURAL PRODUCT SYNTHESIS
AND EVALUATION FOR CHECKPOINT KINASE 2 INHIBITION
By

Christopher David Hupp

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Chemistry

2009

~"I-Aa
l

""‘v‘\.

t“'* an

a“.
v

a.“

A._ y,
””1 I

I.‘H‘ h
- VI“

(to
(I'-

N-
5 .:\\i

 

(I,

ABSTRACT
ACCESSING THE
2—AMINO-5,5-DlSUBSTITUTED-1H-lMIDAZOL-4-ONE
SCAFFOLD FOR NATURAL PRODUCT SYNTHESIS
AND EVALUATION FOR CHECKPOINT KINASE 2 INHIBITION
By
Christopher David Hupp

This dissertation is primarily focused on the development of a synthetic
method for the preparation of 2-amino-5,5-disubstituted-1H-imidazol-4-ones
through a novel rearrangement and the application toward the first total synthesis
of a marine alkaloid. The first chapter identifies checkpoint kinase 2 (Chk2) as a
viable drug target for adjuvant therapeutics. The second chapter elaborates on
the development of a new rearrangement applicable for the synthesis of potential
Chk2 inhibitors. The third chapter describes the total syntheses of a natural
product and two analogs. Finally, the fourth chapter describes the biological
evaluation of all synthesized compounds.

Chemotherapy and radiation therapy offer effective methods to treat
various forms of cancer. However, these procedures can often lead to negative
side effects resulting in the destruction of healthy cells. Developing adjuvant
drugs that inhibit Chk2, a kinase part of the DNA damage response network, is
believed to help ameliorate these harmful side effects by desensitizing healthy
cells toward treatments such as ionizing radiation.

lndoloazepine, an analog of the natural product debromohymenialdisine,

was found to be a potent inhibitor of Chk2. Additional literature evaluation led to

the identification of a natural product, from the tunicate Dendrodoa grossu/aria,

v
N"
‘1‘
- v' R.
uyA,

l

: U‘e
";\»1p.
5 I y ‘

“A"‘Br
-F A
“ In

C
m “c.
" v»!
T «ah ft
"U‘ I“

5“.

‘ '
Hz? N
V‘ .U

’.
~;PP;F :-
"‘ ,4 II‘

I’

which contained structural similarities to indoloazepine. The indole alkaloid
contained a unique quaternary imidazolone scaffold that required the use of a
novel transformation to attain.

The traditional oxazole rearrangement was discovered by Prof. Steglich in
1975. Unfortunately, this transformation is often plagued by the inability to
remove an N-acyl group resulting from the ring-opened oxazolone product, thus
preventing any further chemical modifications. In order to apply an oxazole
rearrangement to the synthesis of the natural product, a novel rearrangement
was developed. Overall, a one-pot transformation from a thiourea to a quaternary
hydantoin intermediate allowed access to the imidazolone scaffold needed to
complete the first total synthesis of the natural product.

The natural product was synthesized in 12 linear steps with an overall
yield of 11.8%. The synthesis highlighted the use of the newly developed
rearrangement and also allowed for analogs to be synthesized without a major
modification of the synthetic pathway. Finally, biological evaluation of the natural
product, analogs and other heterocycles revealed that all compounds prepared
were inactive against the targets screened. Additionally, all compounds tested for

cell cytotoxicity were found to be non-cytotoxic.

This dissertation is dedicated to my parents, David and Sandra Hupp,
who have always made my education a top priority.

I told you I’d become a doctor someday!

I‘n pl
1

«Jay ‘

‘M u

\
Vt ”it“
a

PM»

(1’
{v
(I)

ACKNOWLEDGMENTS

I would first like to acknowledge my advisor, Prof. Jetze Tepe. It was truly
a daunting experience to come into a large university from a small undergraduate
college. Jetze showed patience (yes, he does Show that quality sometimes)
throughout the first years of me learning the ropes in lab and encouraged me to
not just do chemistry, but understand it. I believe he always had faith in me to
complete my project even though at times the road was very bumpy and the
outcome looked bleak. It is with his faith and my determination that pushed me to
reach my goal. I truly learned a great deal about organic synthesis and medicinal
chemistry but, overall, I believe the most valuable things I learned in his lab were
the lessons on dealing with struggles in lab and how to approach a problem. For
all of this, I thank you Jetze.

I would also like to acknowledge Prof. Robert Maleczka, my second
reader. By far, he is easily one of the best professors I have had for an Organic
Chemistry course. Although his class was the hardest I have ever taken, it is also
the class where I learned the most. I will always remember that class and what it
has done for me on an intellectual level. I also want to acknowledge my other
committee members, Professors William Wulff and Jim McCusker, whom have
been an excellent source of information and guidance throughout my graduate
career.

I want to extend a sincere thanks to other staff at MSU including Bob

Rasico, Melissa Parsons, Bill Flick, Dr. Daniel Holmes and Kermit Johnson. The

Aq:~‘j

» - '

L ‘1‘:
(

 

Ii
..

My
U

‘ I.

I" i)

. "a

”A“
\ VL' i:
"v
la.
.5.“
~ -
U
~11 E
,‘v-
“u

chemistry department is very lucky to have staff such as these people, who were
always willing to lend a hand. MSU is truly blessed with such talented and
thoughtful staff.

The past and present Tepe group members have also shaped the type of
scientist I have become and for that, I am very grateful. I wouldn’t be Where I am
today without the help from all of the group members. It is not every day that co-
workers and great friends coincide. I am blessed to have become friends with
many of the people I worked with including Jason, Sam, Thu and Adam. It has
made the whole process of graduate school much easier and I hope to stay in
touch in the future.

Lastly, but certainly not least, I would like to thank my wife Amber and my
family. I am lucky to have such a wonderful and understanding wife as Amber. I
am truly nobody without her. I want to thank her for being supportive for the late
night study sessions, Saturday classes and every hoop we had to jump through
for graduate school. I want to also thank my parents, Dave and Sandy, for giving
me the means to achieve my goals and always believing in me. I also want to
thank my in-Iaws, Tom and Jille, for all their support and encouragement over the
years. They have treated me like a son and for that, I am grateful. It’s not
everyday that you get two sets of great parents!! I would also like to thank my
sister, Amy, who keeps me grounded by reminding me that I’m still JUST her big
brother. Finally, I would like to extend a big thank you to all of my family who has
been a source of comfort and relaxation throughout the past six years. I truly

could not have done this without all of you.

vi

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................... ix

LIST OF FIGURES ............................................................................................... x

LIST OF SCHEMES ........................................................................................... xii

LIST OF ABBREVIATIONS ............................................................................... xv
CHAPTER I. CHECKPOINT KINASE 2 AS A TARGET FOR ADJUVANT

CANCER THERAPEUTICS ......................................................... 1

LA. Introduction to cancer ......................................................................... 1

LB. Chemo and radiotherapy .................................................................... 3

LC. DNA damage response pathways ...................................................... 6

ID. Developing adjuvant therapeutics for cancer treatment ...................... 7

LE. Chk2 as drug target candidate ............................................................ 9

LP. Validation of Chk2 as drug target ..................................................... 15

LG. Purified drug target and X-ray crystal structure ................................ 17

I.H. Checkpoint kinase 2 inhibitors .......................................................... 19

Li. Chk2 binding pocket properties ........................................................ 23

I.J. Goal of project .................................................................................. 30

I.K. References ....................................................................................... 31

CHAPTER II. DEVELOPMENT OF A SYNTHETIC METHOD FOR THE

PREPARATION OF 2-AMlNO-5,5-DISUBSTITUTED-1H-

IMIDAZOL-4(5H)-ONES ............................................................ 43

"A. Nomenclature and numbering of imidazolones ................................. 43

".8. Reactivity of imidazolones ................................................................ 45

MC. Natural products containing the imidazolone core ............................ 48

MD. Synthetic routes to imidazolones ...................................................... 53

II.D.1. Synthetic routes to imidazolones: Aza-wittig reaction ............... 54
".02. Synthetic routes to imidazolones: Reaction of aziridinone and

thiourea .................................................................................... 54

".03 Synthetic routes to imidazolones: Oxazolone to imidazolone... 55

"DA. Synthetic routes to imidazolones: Oxidation of imidazoles ....... 56

II.D.5. Synthetic routes to imidazolones: Diketone rearrangement ..... 57

II.D.5.a. Synthetic routes to imidazolones: Using diketone

rearrangement to produce imidazolone analogs of natural

product ............................................................................... 61

II.D.6. Synthetic routes to imidazolones: Dehydration reactions ......... 65

"DJ. Synthetic routes to imidazolones: Cyclization with carbodiimide

intermediates ..............................

vii

.............................................. 66

IIF.
II.
II
13.
Hit
III.

CHAPTEI

INA,

IIIE
lllc
III)
I: a,
HIF.
IIIG
IIIH
III I.

CHAPTE

IfA
WE
II”! C

II.D.8. Synthetic routes to imidazolones: Cyclization reactions with

nitriles ....................................................................................... 69
II.D.9. Synthetic routes to imidazolones: Cyclization reactions with N-

cyanoguanidines ...................................................................... 71

NE. Synthesis of quaternary intermediates for the synthesis of the natural
product ............................................................................................. 72

HF. Development of a novel EDCl-mediated oxazole rearrangement ..... 81
l|.F.1. Scope of rearrangement ........................................................... 84
lI.F.2. Proposed reaction mechanism ................................................. 91
".6. General experimental information ..................................................... 95
"H. Experimental procedures and characterization ................................. 96
NJ. References ..................................................................................... 145

CHAPTER III. TOTAL SYNTHESIS OF AN INDOLE ALKALOID FROM

||l.A.
l||.B.
III.C.
|||.D.
III.E.
|I|.F.
||I.G.
III.H.
l|I.|.

THE TUNICATE DENDRODOA GROSSULARIA AND TWO

ANALOGS ............................................................................... 157
Pharmacological value of marine compounds ................................ 157
Background on Dendrodoa grossu/aria .......................................... 158
Retrosynthetic analysis of indole alkaloid III-1 ................................ 162
Synthesis of indole alkaloid III-1 ..................................................... 163
Retrosynthetic analysis of analog 1 and 2 ...................................... 168
Synthesis of analogs 1 and 2 .......................................................... 169
General experimental information ................................................... 172
Experimental procedures and characterization ............................... 173
References ..................................................................................... 1 86

CHAPTER IV. BIOLOGICAL TESTING OF NATURAL PRODUCT,

IV.A.
NB.
NC.
ND.
NE.
IV.F.
IV.G.
IV.H.

ANALOGS AND OTHER HETEROCYCLIC COMPOUNDS 189

Biological target of natural product and hypothesis for activity ....... 189
Kinase screen for natural product and analogs .............................. 190
Other biological testing for natural product and analogs ................. 191
Synthesis of additional heterocycles ............................................... 192
Biological testing for additional heterocycles .................................. 195
General experimental information ................................................... 196
Experimental procedures and characterization ............................... 197
References ..................................................................................... 209

viii

n‘.’

Iazell.

Table I-1
Table ”-1
Table "-2
Table ”—3

Table III-1

Table lV-1
Table IV-2

Table IV-3

LIST OF TABLES

FDA-approved kinase inhibitors ................................................. 5
Oxazole rearrangement with various N-acyl groups ................. 79
Rearrangement containing various R groups ........................... 88
Rearrangement containing various allyl groups ........................ 91

Comparison of spectral data from synthesized natural product

(III-1) and isolated natural product .......................................... 167
Results of kinase screen for III-1, Ill-16 and III-19 .................. 191
Results for cytotoxicity, NF-kB and lL-6 assays ..................... 192

Biological activity results for lV-1, lV-3, IV-6, IV-9 and lV-11.. 195

’ l l
.

D pp |.
Id v '
-

D I F,

.0 ‘u- I

F .
Ind u L

I.
.04 .
Iii-fl
div v
. I
-“'fl.l
~'«
Idv'~
. I
5‘ A.
.- ."
1.4.
i
n
:A
5".
‘-..h
:led
.
~1p;'7
:" .,
d a I
u

D
La , 'A
.I»EI~;I

 

D
-~'Al1
H
:v«.
O
55.
-Pa 4
:‘W‘I

.
'“I M
‘ H
3" v '1
-.
PM

Figure l-1
Figure l-2
Figure I-3
Figure I-4
Figure l-5
Figure l-6
Figure l-7
Figure l-8
Figure I-9
Figure l-1O
Figure H 1
Figure l-12
Figure l-13
Figure l-14
Figure l-15
Figure l-16
Figure I-17
Figure l-18
Figure l-19
Figure "-1

Figure "-2

LIST OF FIGURES

Normal cell division .................................................................... 2
Cancer cell division .................................................................... 3
DNA damage response pathways .............................................. 7
Schematic for drug development ................................................ 9
Components of the damage response network ........................ 13
DNA damage pathway involving Chk2 ..................................... 16

Illustration of Chk2 and crystal structure of kinase domain ...... 18

Squaric acid derivatives and indazoles as Chk2 inhibitors ....... 19
Chk2 inhibitors from compound libraries .................................. 20
Benzimidazole and non-benzimidazole Chk2 inhibitors ........... 21
Natural products and analogs used as Chk2 inhibitors ............ 22
DBH inside the Chk2 active site ............................................... 23

Interactions between southern portion of DBH and Chk2 ......... 24

Indoloazepine modeled inside the Chk2 binding pocket ........... 25
New indole alkaloid and indoloazepine .................................... 26
Comparison of dihedral angles ................................................. 27
Hybridization of carbon connected to indole ring ...................... 28
New indole alkaloid modeled in Chk2 binding pocket ............... 29
Oxazolone chemistry affording various heterocycles ............... 30
Numbering of the 2-amino-imdazol-4-one ring system ............. 44
Representative structures of imidazolones ............................... 44

Figure “-3
Figure "-4
Figure "-5
Figure “-6
Figure "-7
Figure ”-8
Figure III-1
Figure III-2
Figure IV-1

Figure lV-2

Reactions of 2-aminoimidazolone ............................................ 45
Relative rates of deuterium exchange ...................................... 48
pKa’s of differently substituted 2-aminoimidazolones ............... 48
Natural products containing the 2-aminoimidazolone core ....... 50
Natural products containing an imidazolone core ..................... 51

Additional natural products containing 2-aminoimidazolone ..... 52

Alkaloids from Dendrodoa grossu/aria .................................... 158
X-ray crystal structure of indole alkaloid III-1 .......................... 168
The indole natural product III-1 and indoloazepine ................. 189
Kinase screen performed by Millipore .................................... 190

xi

Scheme I-1
Scheme "-1
Scheme ”-2
Scheme "-3
Scheme "-4
Scheme ”-5
Scheme ”-6
Scheme "-7
Scheme "-8
Scheme "-9
Scheme "-10
Scheme "-1 1
Scheme "-12
Scheme "-13
Scheme "-14
Scheme "-15
Scheme "-16
Scheme "-17
Scheme "-18
Scheme "-19

Scheme "-20

LIST OF SCHEMES

DNA damage by UV and IR ...................................................... 11
Alkylation of 2-aminoimidazoI-4-one ......................................... 46
Alkylation at C-5 of an imidazolone .......................................... 47
Aza-wittig reaction to produce imidazolones ............................ 54

Reaction of aziridinones and thiourea to form imidazolones 55

lmidazolones using oxazolones and thioureas ......................... 56
Oxidation of imidazoles to form imidazolones .......................... 57
lmidazolones from 1, 2-diketone and guanidine ....................... 58
Proposed mechanism for diketone rearrangement ................... 59
Alternative mechanism for diketone rearrangement ................. 60
Synthesis of imidazolone II-18 .................................................. 62
Synthesis of imidazolone II-23 .................................................. 64
lmidazolones produced by dehydration .................................... 65
Cyclization from diimide: urea to imidazolone .......................... 67
Cyclization from diimide: thiourea to imidazolone ..................... 67
Cyclization from diimide: azide to imidazolone ......................... 68
Intramolecular cyclization: thiourea to imidazolone .................. 69
Intramolecular cyclization: urea to imidazolone ........................ 69
Imidazolone through cyclization onto nitrile .............................. 70

Imidazolone through cyclization using N-cyanoguanidines ...... 71

Rearrangement developed by Steglich .................................... 73

xii

.n-Mfl
—

ewwl

A
‘FR~I
.-

vdv v

\_

vvv V

A .
Annmfi'
.-

‘
Henna
‘ _

Uri Uh

’ I
.-
.‘ :N‘Q
v 5 Vi
\ "_‘_m:
V. \- I».
I
.
\ ”:3“;
In. , U

I
\"“'!-Al
. I‘
v. ' U
I.
\.‘;Ma
r. '
v 1V
n
\‘n-w‘
v. " Q
‘V
A
\"-~.

c/ D
t —P
",4
(D
J
( I)

(I)
")
I

Scheme "-21
Scheme "-22
Scheme ”-23
Scheme "-24
Scheme "-25
Scheme "-26
Scheme ”-27
Scheme "-28
Scheme ”-29
Scheme "-30
Scheme "-31
Scheme "-32
Scheme "-33
Scheme "-34
Scheme Ill-1
Scheme Ill-2
Scheme Ill-3
Scheme Ill-4
Scheme Ill-5
Scheme Ill-6
Scheme III-7
Scheme III-8

Scheme IV-1

Rearrangement leading to 4-fluoropyridines ............................ 74
Rearrangement affording d-trifluoromethyl a-amino acids ....... 75
Rearrangement leading to Il-26-lI-28 ....................................... 75
Rearrangement to synthesize C-glycosyl o-amino acids .......... 76
Unexpected reaction producing hydantoin II-41 ....................... 8O
Unexpected reaction producing hydantoin Il-42 ....................... 81
Routes from hydantoin to imidazolone ..................................... 82
New rearrangement applied to the synthesis of III-1 ................ 84
General scheme for novel rearrangement ................................ 85
General synthesis of thioureas ll-63-ll-69 ................................ 86
Synthesis of thiourea lI-75 ........................................................ 87
Potential byproduct pathway .................................................... 89
Synthesis of higher substituted allylic thioureas ....................... 90
Proposed mechanism of EDCl-mediated rearrangement ......... 93
Synthesis of derivative Ill-4 .................................................... 161
Retrosynthesis of indole alkaloid Ill-1 ..................................... 163
Synthesis of thiourea III-9 ....................................................... 164
Synthesis of hydantoin III-1O .................................................. 165
Synthesis of alkaloid III-1 ....................................................... 166

Retrosynthesis of analog 1 (Ill-16) and analog 2 (Ill-19) ........ 169

Synthesis of analog 1 (III-16) .................................................. 170
Synthesis of analog 2 (III-19) ................................................. 171
Synthesis of IV-1 and lV-3 ...................................................... 193

xiii

“Ema!
...,

g
V
I!

Scheme IV-2 Synthesis of hydantoin lV-6 .................................................... 193

Scheme IV-3 Synthesis of IV-11 .................................................................. 194

xiv

LIST OF ABBREVIATIONS

AIDS — acquired immune deficiency syndrome
Asn — asparagine

ATM - ataxia telangiectesia mutated

ATP — adenosine triphosphate

ATR - ataxia telangiectesia related

Bn - benzyl

Boc - tert-butyloxycarbonyl

BOP - Benzotriazole-l-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate

Cbz - carbobenzyloxy

Cdc — cell division cycle

CDK — cyclin dependent kinase
Chk# - checkpoint kinase #
DBH - debromohymenialdisine
DCC — dicyclohexylcarbodiimide
DCM — dichloromethane

DIPEA — diisopropylethyl amine
DMAP — dimethylamino pyridine
DMDO — dimethyldioxirane
DMEM - Dulbecco's modified eagle medium
DMF - dimethylformamide

DMSO — dimethyl sulfoxide

XV

DNA — deoxyribonucleic acid

E050 — half maximal effective concentration
EDCI - 1-EthyI-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride
EDTA - ethylenediaminetetraacetic acid

ELISA - enzyme-linked immunosorbent assay

Et — ethyl

FBS — fetal bovine serum

FDA — food and drug administration

Fmoc - 9H-fluoren-9-ylmethoxycarbonyl

Glu - glutamic acid

GSK - glycogen synthase kinase

HMDS — hexamethyl disilazane

HPLC - high performance liquid chromatography
HRMS — high resolution mass spectrometry

IBX - 2-lodoxybenzoic acid

leo - half maximal inhibitory concentration

IL - interleukin

IR — ionizing radiation or infrared

Law. Rgt. - Lawesson's reagent

LCMS - liquid chromatography mass spectrometry
Lys - lysine

Me - methyl

Met - methionine

xvi

141:2 _

l a
:"n -
Ia...

‘I-I
I"‘.;_,

I'll.A A

‘II‘J-I

,4

III:
II

|'\"'A'\'I

‘V~V I

\ Rh
b ‘ -

\- Ugl
I

I.
n J

v ‘ Y“

Q:
u,"
85
'a
3:: IA
0 ‘ L“
V
‘\
~_I
‘\H| \t
TI-
5

MOPS - 3-morpholinopropanesulfonic acid
MS — mass spectrometry

Napth. - naphthalene

NF-KB — nuclear transcription factor — kappa B
NMO — N-methylmorpholine-N-oxide

NMR — nuclear magnetic resonance

NOESY - nuclear overhauser enhancement spectroscopy
OAc — acetate

Ph — phenyl

PIKK - phosphoinositide 3-kinase-like kinase
ppm - parts per million

psi — pounds per square inch

Pyr. — pyridine

RNA - ribonucleic acid

rt — room temperature

SAR — structure activity relationship

Ser - serine

tBu — tert butyl

TEA — triethylamine

TFA - trifluoroacetic acid

TFAA — trifluoroacetic anhydride

THF - tetrahydrofu ran

Thr - threonine

xvii

TLC — thin layer chromatography
TNF — tumor necrosis factor
Ts - tosyl

UV — ultraviolet

xviii

5" Rip It";
' 'VI LI I

”hung,

L:.:l U

-I H: Int
IM ‘
‘tTSEft

HA I
I M,‘ ,
' i A,‘
“dyv .l
' ‘I
PM
“ ‘
i

CHAPTER I

CHECKPOINT KINASE 2 AS A TARGET
FOR ADJUVANT CANCER THERAPEUTICS

l.A Introduction to cancer

Despite the increase in knowledge and therapeutic advances, cancer is
currently one of the highest causes of mortality in the United States.1' 2 Even
though the mortality rate of some forms of cancer (stomach, breast and rectum
cancer) have slowly decreased, other forms of cancer (pancreatic, lung and
bronchus) have seen little improvements over the last several years.2' 3 Cancer
can be defined as an unregulated growth of tissue not governed by the
regulations of normal cell growth.4 The main causes of human cancer include
tobacco/tobacco products (30%), hormones (30%), diet (15%), viruses (10%),
drugs, x-rays and UV light (10%) and occupational carcinogens (5%).4

Normal cell growth and division is systematic and necessary to replace the
aging and dying cells or to repair injuries. Furthermore, normal cell growth and
division can only be conducted in a manner that is cognisant of a neighboring
cell’s growth to efficiently and correctly form tissues and organs.4 The process of
division for a normal cell iS shown below in Figure H. A healthy cell will grow and
divide into two new daughter cells, which can each then grow and divide
themselves, and so on. In the event that a cell is damaged in the process or a
mutation has occurred, the cell can either repair itself or initiate cell suicide
(apoptosis). The apoptotic pathway is essential for eliminating damaged DNA

and preventing mutated cells from passing on the damaged genetic information.4

/v—_:_>@ Form tissues
/'@<:©_——> _, and/or organs

O”@ /.@Repa.r

Normal
Mutation Cell suicide

cell \ © @<:

Damage

Figure l-1. Normal cell division

However, if cells stop cooperating with their neighboring cells, become
autonomous in their growth and are mutated, they can form tumor cells, which
could later become cancer cells. Tumors are generally classified into two
categories: benign and malignant. Benign tumors are localized, do not spread to
other parts of the body and are generally not lethal to their host. However, if a
benign tumor is exerting pressure on a sensitive organ such as the brain, then
complications can arise. Malignant tumors can destroy parts of the body from
which they originate and can invade surrounding tissue. Furthermore, these
tumors can form secondary tumors in new sites and eventually cause destruction
of additional tissue and organs. This large difference in mobility distinguishes
cancer from benign growths.4

Malignant tumor cells, or cancer cells, grow uncontrollably and divide in an
unordered fashion. As shown in Figure I-2 a cancer cells division process
proceeds in a non-systematic way leading to non-structured masses or tumors.
These cells can then spread or metastasize toward other tissues and eventually

cause damage at sites far from the original location.4

 

C§r\@/ @

cell \A @ ' ' "
Uncontrolled and unordered

growth

Figure I-2. Cancer cell division

LB Chemo and radiotherapy

Although uncontrolled cancerous cell growth can lead to solid tumors
(except in cases such as leukemias), cancer can be treated by a variety of
methods including chemotherapeutics and/or radiation therapy.5'18 There are
many different types of chemotherapeutics used to treat cancer such as
alkylating agents, antimetabolites, topoisomerase inhibitors, mitotic inhibitors as
well as other targeted therapies. Alkylating agents are used to directly damage
DNA by alkylating the base pairs and ultimately preventing the cell from
reproducing. Some of the more common types of alkylating agents include the
nitrogen mustards‘o, mitomycin C12 and the powerful platinum drugs cis-platin
and carboplatin.5 Antimetabolites are a class of drugs that interfere with the
growth of DNA and RNA by essentially mimicking the natural metabolites used
for the synthesis of DNA and RNA. Common modes of action for antimetabolites
include inhibition of DNA synthesis enzymes or causing mismatched DNA
synthesis, which could result in cell death. A few common antimetabolites that

are used for chemotherapy include 5-fluorouracil8 and 6-mercaptopurine.15

II

..--A g .
‘~\—g \
..wu V

-.A-'unn

5 j-hvll

I”
J
u)‘
‘
——..

w

’ “'9 i-'
p l
w: 5,"
«1:!- Raf,
"5 U5“;
TT’IQI‘W
"“J~U

Topoisomerase inhibitors are compounds that interact with the enzymes
called topoisomerase I and topoisomerase II, which are responsible for assisting
in the unwinding of DNA for the replication process. Topoisomerase I perfoms a
reversible single strand cleavage of DNA to allow relaxation of the coil and then
reseals the cleaved strand after unwinding. Topoisomerase l inhibitors include,
camptothecin, topotecan and irinotecan.13 The topoisomerase II enzyme acts in a
similar fashion as the topoisomerase I enzyme except that it completes a
reversible double stranded DNA cleavage to aid in the unwinding of DNA for
replication followed by a resealing of the cleaved strands. The most common
topoisomerase II inhibitors include etoposide, teniposide, doxorubicin and
daunorubicin.9' 11' 17' ‘8 Anthracyclines, such as doxorubicin, intercalate between
the base pairs of DNA resulting in a distortion that prevents the DNA replication
process. Funhermore, metabolism of specific anthracyclines, such as
doxorubicin, leads to the formation of free radicals that oxidatively damage DNA
and to reactive intermediates that form covalent adducts with DNA, inhibiting the
overall replication process.18

Mitotic inhibitors interfere with mitosis and prevent the cell from
reproducing. One of the more famous mitotic inhibitors include paclitaxel (Taxol),
a natural product isolated from the pacific yew tree.14 Paclitaxel’s mode of action
is unique from other mitotic inhibitors due to its effect on the polymerization of
tubulin, the building block of microtubules. In general, most mitotic inhibitors
induce the disassembly of microtubules, a component of cells responsible for

aiding in the cell division process. However, paclitaxel was found to induce the

‘6

\I

~_‘

av;

 

polymerization of tubulin, producing dysfunctional microtubules leading to the
death of the cell.14

As researchers have come to understand cancer more thoroughly, new
more specific therapies have developed. Kinases have become an interesting
and challenging target for cancer drug discovery in the past decade. Although
extensive research has been accomplished trying to develop kinase inhibitors,
only a few have been FDA approved in the last ten years (Table M).7 The main
complication that arises from targeted kinase chemotherapy is the formation of
drug resistant tumors.

Table l-1. FDA-approved Kinase inhibitors (adapted from ref. 13)

 

 

 

 

 

 

 

 

 

 

U.S. brand Year Cancer .
name approved type Company Target kinase
Abl, c-Kit,
Gleevec 2001 CML Novartis PDGFRa,
PDGFRb
lressa 2003 NSCLC AstraZeneca EGFR
Tarceva 2004 NSCLC, P Gegesnltgm EGFR
Raf, VEGFRZ,
Nexavar 2005 HC, RCC Bayer, Onyx Vifizfs’
PDFGRb
c-Kit, VEGFR,
Sutent 2006 GIST, RCC Pfizer PDGFR,
FLT3
Abl, c-Kit,
Sprycel 2006 CML BMS PDGFR, Src
Abl, c-Kit,
Tasigna 2007 CML Novartis PDSBrERb’
Ephthrin
Tykerb 2007 BC GSK EGFR, Her-2

 

 

 

 

 

 

CML = chronic myeloid leukemia; NSCLC = non-small-cell lung cancer; P =
pancreatic; HC = hepatocellular carcinoma; RCC = renal cell carcinoma; GIST =
gastrointestinal stromal tumor; BC = breast cancer

h- at
.'-fi1
i S n

r'zn ,‘

't ~U‘l

Spa---.
3" 5'».

.I~h .
.l “7
" VII\

J P

Radiation therapy also offers a highly sophisticated treatment of cancer. The
radiation can be administered externally by a machine, internally by a catheter or
taken orally. The radiation induces unsustainable DNA damage to targeted cells
which results in cell death.6' 16 Overall, one of the impediments of
chemotherapeutic and ionizing radiation therapy is that these treatments are
often limited in efficacy by severe side effects to healthy tissue. Strategies that
address these limitations may have the potential to enhance the efficacy of
current treatments.3
I.C DNA damage response pathways

On average, the induction of DNA damage appears to be the most common
strategy in anticancer treatment and has dramatically increased the survival rate
of patients.19’22 The anticancer effects are even better when these agents are
used in combination with other drugs containing different modes of action.23
Upon treatment of cells with any of the chemotherapeutics described previously
or ionizing radiation, two distinct DNA damage response pathways are induced
(Figure I-3).24 One response includes the activation of the NF-KB pathway, which
is largely responsible for antiapoptotic cell signaling. The second distinct
response is the activation of cell cycle checkpoint kinases, which essentially
mediate the induction of cell cycle arrest and apoptosis.”31 Activation of the NF-
KB signaling pathway induces the expression of a wide range of genes involved
in cell survival responses.”35 Activation of the DNA checkpoint kinases results in
a complex network of Signaling pathways involved in the induction of apoptosis or

cell cycle arrest, which allows for DNA repair and re-initiation of cell cycle

A

h

3

"RI“
fl
, IIOI
4

IF A.
I. 'JII

A...

progression.“ 36"” Unfortunately, the activation of these antiapoptotic signaling

pathways limits the overall efficacy of cancer treatments.3

 

CHEMOTHERAPEUTICS
Ol'
IONIZING RADIATION

I

DNA damage

 

 

 

 

 

 

 

 
  
     
 

  

Cell cycle
checkpoint
kinases

2 I -
ACTIVATION

 

 

 

 

 

Inhibition Cell cycle arrest
of or
apoptosis Apoptosis

(cell survival)

 

 

 

Figure I-3. DNA damage response pathways
Efficacy of treatment is often limited by the narrow therapeutic window in
which drugs are effective due to their inherent toxicity to the patient. Strategies of
combining traditional chemotherapeutics with cell cycle checkpoint inhibitors to
either sensitize cancerous cells or desensitize healthy cells, may widen the
therapeutic window and improve the overall efficacy of the cancer treatment.3
I.D Developing adjuvant therapeutics for cancer treatment
Adjuvant therapeutics offer a strategy to limit the negative side effects of

current cancer treatment practices. It is hypothesized that by targeting a specific

I ‘ ‘
- FR
”IT." Ii "1‘

I. .t-up'v“

leR§~ n‘
val I
.v-J V ‘

E

u ~¢C Bier
dJut U‘ \
V

‘ T’ N .a

:v UL . 'C
“ h

”to.”

find 'I the

checkpoint kinase, healthy cells can be selectively desensitized to radiation
treatment and allow a more effective procedure to manage cancer. Developing a
drug to become an adjuvant therapeutic is a long process as illustrated in Figure
l-4.41 This section (ID) is meant to briefly explain each major step in the process
(Figure I—4) and will be complemented by the application of this process to my
project in the following sections.

As Shown in Figure I-4, the first step in developing therapeutics is to identify
a drug target candidate. After initial studies determine if the candidate is a viable
option, the target is validated. Subsequently, efforts are made to 1) purify the
drug target so that additional information can be gathered and 2) compounds
from a variety of areas are screened to identify potential hits. If a crystal structure
is identified for the target, it can aid in the development of additional compounds
to test and other potential drug candidates through a rational drug design
approach. If a lead compound is designed or found, the process of optimizing the
lead drug can begin through molecular modeling, medicinal chemistry and
combinatorial chemistry techniques. Once optimized, the drug is subjected
through many rounds of preclinical studies and finally clinical studies before

being considered for approval to go on the market.

 

 

Drug target candidate]

I

Validated druflrfil

II ,
Purified I
—> Natural ProductSI—> Screeningl/ drug target

Assays
, Combinatoriall / - l

libraries
X-ray crystal I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Compound structure
’ collections l *
Rational drug design
I I
Drug leads I

 

 

Optimized drug Ieadsl

I

Preclinical studies then
clInIcal trials

 

 

 

 

 

Figure I-4. Schematic for drug development (adapted from ref. 40)
LE Chk2 as drug target candidate

The primary structure of DNA is continuously being altered by
endogenous and exogenous stimuli, which cause abnormalities ranging from
simple base changes to deletions. Despite these continuous DNA damaging
events, nature has recruited reinforcements in the form of checkpoint pathways
that maintain and monitor the integrity of the genome. These pathways were first
observed when it was shown that damage induced by chemotherapeutic agents

resulted in an inhibition of the cell cycle, allowing for cellular repair.“ 43 A Similar

control of cell cycle progression was found in Saccharomyces cerevisiae and
was then coined a ‘checkpoint’.44 It was originally thought that checkpoint
pathways were operational for the sole purpose of regulating cell cycle
transitions. Currently, it is generally accepted that the checkpoints are part of a
cascade of signals that ultimately lead to DNA damage response processes.” 39
These response processes include removal of the DNA damaged sites and
restoration of the DNA duplex. Temporary arrest of the cell cycle allows for repair
and prevention of the duplication of damaged DNA or its transcriptional
response. In the event of heavily damaged or seriously deregulated cells,
induction of apoptosis or terminal cell cycle arrest occurs.” 45' 46 The DNA
damage cell cycle checkpoints will repair, patch, and promote the overall survival
of cells, even cancer cells, resulting in a reduction of the overall anticancer
efficacy.

However, if the induction of checkpoint pathways and DNA repair could be
selectively inhibited in cancerous cells, the efficacy of the treatment may be
enhanced. Alternatively, if DNA repair can be selectively induced in healthy cells,
the therapeutic window of the treatment could be significantly widened, allowing
for a more tolerable and effective anticancer therapy. Consequently, modulating
the DNA checkpoint pathways using small molecules could potentially sensitize
cancer cells and desensitize healthy cells to DNA damage induced by

2

chemotherapeutics or ionizing radiation.4 More specifically, altering the

checkpoint pathways via modulation of two of the key checkpoint kinases, Cth

10

 

“u

'u

g.

and Chk2, has become an increasingly appealing approach to broaden the
therapeutic window of conventional anticancer therapies.47

These checkpoint pathways, rather than being thought of as a molecular
switch, represent a continuous process that is amplified in the presence of DNA
damage, such as from ultraviolet (UV) and ionizing radiation (IR).37 Between UV
and IR, UV radiation is lower in energy and the majority of DNA damage results
from the formation of photoproducts, such as dimers of pyrimidine containing
nucleotides (A, Scheme l-1). These dimmers can inhibit DNA transcription and
replication machinery ultimately leading to cell death.“ 49 Ionizing radiation is
higher in energy and can cause more substantial damage to DNA. IR is absorbed
mostly by surrounding water molecules, which subsequently form highly reactive
radicals that cause severe DNA damage. These radicals can cause nucleotide
damage, crosslinking and strand breaks. Reaction B in Scheme I-1 illustrates an
example of a strand cleavage reaction that could occur due to ionizing
radiation.16

Scheme I-1. DNA damage by UV and IR

O

O
O O
HN I HN I
A A A UV HN NH _,' cell death

0 N O N _’
,2 ' 0%N NAO
/ \ / I

F’O\k——7Base—.fl'+ PC)\:;7Basefi|i> POUasefi FIE—788%

P= phosphate

11

’50 BI

I m
vmnnnpf‘l
W ilv- V ‘

p-Afiflr‘q't

- . .

uni-4 I...
V

'1!!!

I I"
. as 3

a

5: Oil-"I

3.5
l

A v
Uni ans

The DNA damage checkpoint pathways consist of three different main
components: sensors, signal transducers, and effectors (Figure I-5). The sensors
recognize the damaged DNA and initiate subsequent events. Two of the main
sensors are ATM and ATR, which belong to the phosphoinositide 3-kinase-like
kinase (PIKK) family members.” 50

The first DNA damage sensor, ATM (ataxia telangiectesia mutated) exhibits
kinase activity when activated by agents that induce double strand breaks, such
as ionizing radiation.51 ATM will subsequently phosphorylate proteins such as
Chk2 and p5351' 52 (among others) when activated after cells have been exposed
to IR. A deficiency in ATM exhibits phenotypes such as cerebellar degeneration,
immunodeficiency, genome instability, clinical radiosensitivity, and cancer
predisposition.53

The second DNA damage sensor, ATR, has a sequence homology to both
ATM and SpRad3, thus comes the name ATR (ATM and Rad3 related).37' 5‘ ATR
is similar to ATM since it is a kinase that essentially phosphorylates the majority
of the same substrates as ATM. However, ATR is activated in vivo by UV
radiation rather than ionizing radiation.” 55 Unlike ATM, it does not appear that

ATR is activated by double strand breaks.37 Thus, ATR is the main PIKK family

member that initiates a signal transduction pathway after UV radiation damage.55

12

DNA Damage
Sensors

Signal
Transducers

Effectors

W

DNA DAMAGE

/

 

 

 

 

 

 

 

 

ATR I

Chk1 I

CD025
CDK Activation

\

ATM I

II

Chk2 I

ll

 

 

 

 

 

 

 

 

 

 

ll

p53
Pathway

 

 

 

 

Cell cycle arrest
(DNA repair &
replication)

fl

 

 

 

 

Apoptosis
(Cell death)

 

 

Figure I-5. Components of the damage response network

The Signal transducers are components of the DNA damage pathway that

essentially receive the signal from the sensors. The two signal transducers that

will be discussed here in detail are Chk1 and Chk2.56‘58 Chk1 and Chk2 are

structurally unrelated but are essential checkpoint kinases downstream of the

13

It tTillslt
5390'853
320mg:

8386

Chen

' “I90:
tel

.eVed

'Efiajj‘ re

DNA damage sensors and play a critical role in determining the cell’s fate.37' 59‘“
ATM is activated by double strand breaks and the signal is transduced by Chk2
52' 53, while the UV damage signal sensed by ATR is transduced by Chk1.55' 64
Chk1 is primarily responsible for cell arrest in response to DNA damage, allowing
for the initiation of DNA repair65, whereas Chk2 has been implicated with the
phosphorylation and activation of the apoptotic transcription factor p53.4°' 62' 65 It
is important to note the drastic difference in Chk1 and Chk2 null mice. Chk1 (-/-)

mice are not viable and exhibit embryonic lethality” 66

, whereas Chk2 (-/-) mice
still illicit near normal checkpoint responses and are viable.67

The effector components, Cch5 and p53, are involved in the arrest of the
cell cycle and apoptosis, respectively. Three phosphotyrosine phosphatases,
Cch5a-c, are responsible for dephosphorylating cyclin-dependent kinases that
ultimately affect proteins directly involved in cell cycle transitions?7 Essentially,
the checkpoint kinases phosphorylate one or more of the Cdc25 proteins that
results in their inactivation and degradation, thus preventing the Cd025 proteins
to translocate into the nucleus and enable cell cycle progression'58 The tumor
suppressor protein p53 is responsible for arresting the cell cycle and inducing
apoptosis.69

Based on the described pathways it is possible that inhibition of these
checkpoint kinases may be used to enhance the effects of chemotherapeutics by
sensitizing cancer cells or desensitizing healthy cells.6°' 70‘" Inhibition of Chk1 is

believed to sensitize tumor cells by blocking the cell’s ability to initiate DNA

repair, resulting in unsustainable DNA damage.” 65' 73' 74 Inhibition of the G2

14

“It. al-m'

‘ V1 ‘v‘l It»
:55 215 CI

~AAt—r 0‘
up a
. .4.

PM
. .~ A
“WAC. ,
«VCJ
I

DNA checkpoint Chk2 can selectively prevent apoptosis in p53 wild type cells
(healthy cells), thus desensitizing them from cell death during chemotherapeutic
treatment.” 75 Chk2 inhibitors are anticipated not to have an effect on apoptosis
in cells containing mutated p53 (>50% of cancerous cells). We were interested in
focusing our efforts on developing Chk2 inhibitors to desensitize healthy cells
toward ionizing radiation to help widen the therapeutic window of this treatment.
I.F Validation of Chk2 as drug target

Activation of Chk2 is initiated by factors that cause DNA damage, such as
ionizing radiation, chemotherapeutic agents, and telomere initiated senescence.
40' 76' 77 In response to IR induced DNA damage, signals are intercepted by ATM,
which in turn directly phosphorylates Chk2 at Thr68 within the SQ/TQ rich
domain.73' 79 After the initial phosphorylation and dimerization, multiple
intermolecular phosphorylation events occur to complete the activation.” 80 The
phosphorylation of Ser516 was recently found to be of great importance as this
modification is required for full activation of Chk2.4°'81'82 A schematic of the DNA
damage checkpoint pathway involving Chk2 is shown in Figure l-6.

Once activated and all the phosphorylation events of Chk2 have
transpired, Chk2 signals the effector proteins CchSa, Cd025c, and p53, among
others (Figure l-6). The Cch5 family of phosphatases is responsible for the
progression of the cell cycle by dephosphorylating cyclin dependent kinases cdk1
and cdk2 at specific sites.4°' “'86 Chk2 phosphorylates Cdc25a at Ser123 and
consequently renders it inactive‘o' 84' 87, while Chk2 phosphorylates Cdc25c at

Ser216. This results in the sequestering of Cdc25c in the cytoplasm, thus

15

preventing activation of the cdk1/Cyclin B complex, resulting in G2/M arrest.” 88‘

90

 

Apoptosis

I
I

G1-S phase checkpoint

 

 

 

 

 

 

 

 

 

 

 

 

S-phase checkpoint G2-M phase checkpoint

 

 

 

Figure l-6. DNA damage pathway involving Chk2
Another important downstream substrate of Chk2 is tumor suppressor
protein p53. Although the exact relationship between p53 and Chk2 is still
unclear, Chk2 is thought to be upstream of p53 in the reaction pathway that
contains IR induced apoptosis.” 42' 91 It was reported that Chk2 phosphorylates

p53 at Ser20 thereby regulating the amount of activated p53 in response to

16

,‘cv-

vdu'

 

.3.

,.:
..

d4

‘.
no
u

it

 

 

r‘-.

 

double strand breaks.” 62' ”'94 However, this point has been challenged based
on a few observations. First, the p53 derived peptide containing the Chk2
targeted phosphorylation sites is a poor substrate for Chk2 and does not contain
the characteristic sequence for directing a phosphorylation found in other Chk2
substrates. Second, the phosphorylation of p53 induced by DNA damage was
only slightly affected, if at all, by down regulation or knockout experiments of
Chk2.62' 91' 95"” Despite these arguments, most scientific analyses of the Chk2-
p53 link strongly support the role of Chk2 as a p53 kinase.97

The role of Chk2 in DNA damage induced apoptosis is supported by Chk2
deficient mice, which Show an increased resistance to ionizing radiation and
cellular defects in apoptosis.62' 67' 95' 96 Chk2 -/- mice survived significantly longer
than the wild-type mice following whole body irradiation.96 The increased survival
of those mice without Chk2 is attributed to resistance of several cell types to IR
induced apoptosis.95' 96 Furthermore, it is important to remember that Chk2 (-/—)
mice still illicit near normal checkpoint responses and are viable.67 The support
given by the study where the Chk2 null mice survived whole body irradiation and
the study which showed that Chk2 null mice were still viable offer validation for
choosing Chk2 as the drug target.
LG Purified drug target and X-ray crystal structure

As illustrated in Figure l-4, it can be important to obtain information from
the purified drug target to aid in the development Of inhibitors. Fortunately, Oliver

et al. were able to crystallize the kinase domain of Chk2 (Figure I-7) and in effect

17

give an enormous amount of information to the aid in developing inhibitors of

Chk2.99

1 12-175 220-486

1 543
N :26; [ FHA ]——-[ Kinase C

 

 

 

 

 

Figure l-7. Illustration of Chk2 and crystal structure of kinase domain

The top of Figure l-7 displays the linear representation of the whole
checkpoint kinase 2 protein. As illustrated above, Chk2 is a 543 long amino acid
protein that consists of three domains. The first domain is called a serine-

glutamine/threonine-glutamine cluster domain (SCD), which is the substrate for

18

 

lg"!

,m

I. .I.

“F:-

(t)

‘A

”I

 

the upstream protein ATM, as shown in Figure l-6. The second domain is called
the forkhead associated domain (FHA) which has been implicated to help
modulate protein-protein interactions. The last domain, which was crystallized, is
the kinase domain responsible for performing the major requirement of a kinase,
that is, to phosphorylate its substrates.4°' 99 Overall, gaining access to this
information helps in discovering new inhibitors of Chk2 and allows for the fonNard
progress in obtaining an adjuvant drug for radiation therapy.
l.H Checkpoint kinase 2 inhibitors

Until recently, there were only a few reports of potent Chk2 inhibitors that
show good Chk2 selectivity. Within the last decade there has been an increase
into the amount of research being performed to develop checkpoint kinase 2
inhibitors. Some remain highly confidential like XL844, developed by Exelixis,
which is one of the newest checkpoint kinase inhibitors to move into clinical
trials.100 Others have been kept hidden in patents, like the indazole and squaric

acid derivatives shown in Figure l-8, and have not been highly publicizedm'103

O o o R,
HN R4 0
I / R
HN / Fifi I: /R3 N\ \\ 3
\ HN-X X‘<R:7 ”N / / X’ZLN’Ns
R1 R2
Squaric acid derivativesR2 '"daZOIBS

Figure l-8. Squaric acid derivatives and indazoles as Chk2 inhibitors

However, there are more and more reports of novel Chk2 inhibitors
developed through high throughput screening of compound collections and
combinatorial libraries.10‘"110 Pommier et al. described a bis-guanylhydrazone

(NSC 109555) identified after a high throughput screen of over 100,000

19

.,. .— ~, r‘ ‘
'y .. a V

{0’ ‘FF 8
th‘tl)

‘31:..in
U' .

v

U
.A FR;FI
'4 ~4\ .,
'Uu‘v- J

compounds (Figure l-9). The compound was found to be an ATP competitive
inhibitor and displayed an leo of 0.20 IN. When screened against other kinases,
including Chk1, NSC 109555 was found to be fairly selective for Chk2 with IC50

values for a few other kinases at least 6.5 times that for Chk2.106

H H O NH
N N Y 2
YG if (DY ....
I IN 0.. F H 0
‘NH HN’ S
A A “N" ””
HN NH2 H2N NH

NH

NSC 109555 (0.20 pM) HN AZD7762 (~5 nM)
“\g/OH
o
Br
0N
H
VRX0466617 (120 nM)
Figure l-9. Chk2 inhibitors from compound libraries
Wu and Carlessi describe a novel and selective inhibitor of Chk2,
VRX0466617 (Figure 1-9).1°5' ‘07 A series of isothiazole carboxamidine
compounds were found to be potent inhibitors displaying le0 values in the range
of 120 nM to 40 pM. These compounds were also found to be ATP competitive
inhibitors.1°5' ‘07 Zabludoff and co-workers report a similar looking Chk2 inhibitor,
AZD7762 (Figure l-9). The compound was shown to be a potent Chk2 inhibitor
with IC50 values in the range of 5 nM.“°
Researchers at Johnson and Johnson also developed a new class of
benzimidazoles as potent and selective Chk2 inhibitors. High-throughput
screening of purified human Chk2 led to the discovery of a novel series of 2-

arylbenzimidazoles that exhibited potent and selective inhibition of Chk2.104 The

20

 

"Iv

most potent 2-arylbenzimidazole (Figure l-10) gave an leo value of 2 nM and
showed excellent selectivity for Chk2 over 35 other kinases tested. Furthermore,
it was shown that the benzimidazole inhibitors displayed effective radioprotection

of human T-cells against ionizing radiation with ECso values between 3-7.6

pNL104, 109

OH O
O U V\\X/Z —
N I k \ 0 \ \/
H2N >_©_S W/ Y /R
n non-benzimidazole

2-arylbenzimidazole (2 nM) (GpM - 16 nM)

Figure l-10. Benzimidazole and non-benzimidazole Chk2 inhibitors

To help define the scope of the SAR study on the benzimidazole inhibitors, a
series of non-benzimidazole Chk2 inhibitors were developed. The leo values for
the more potent new inhibitors range from 5.8 pM to 16 nM.108 Further studies
are needed to determine if these inhibitors are effective at potentiating the
cytotoxic abilities of current cancer therapeutics.3

Natural products and analogs of natural products have also contributed to the
number of Chk2 inhibitors currently known. Staurosporine (Figure M1) and
analogs, such as UCN-01 (Figure l-11) have shown to be potent checkpoint
inhibitors, however, the are not very selective for checkpoint kinases.1°6' 111' "2
Although UCN-O1 displays a potent inhibition of Chk2 (10 nM)24 and has shown
to potentiate the anticancer activity of a variety of therapeutics such as cis-platin,

113-117

mitomycin and ionizing radiation and augments the cytotoxicity of

temozolomide in human glioblastoma cells118, there are some concerns

associated with UCN-O1 such as its promiscuity as a kinase inhibitor. In addition,

21

,fll

L'Vi‘tul

SM 5,

>85

UCN-01 contains some pharmacokinetic drawbacks including its strong binding

to human plasma protein d1-acid glycoprotein and low bioavailability.“9'121

H
O N

U N N O
. ,, N
; ”OCH,

 

 

- 2 'I’OCH3
NHCH3 NHCH3
Natural Ems:
Staurosporine (80 nM) ”CNN (10 nM)
HZN /N
HN 0
\
/ I
N
H NH
O
W W lndoloazeplne (8 nM)
DBH (183 nM)

Figure I-11. Natural products and analogs used as Chk2 inhibitors
Debromohymenialdisine (DBH) (Figure M1) is a natural product that
displays moderate IC50 values for Chk2 inhibition (3.5 pM (cell culture), 183 nM in
vitro).73' ‘22 However, a low selectivity for Chk2 over other kinases severely limits
the use of debromohymenialdisine as a Chk2 inhibitor.3
A new analog of debromohymenialdisine, indoloazepine (Figure l-11), was
developed in our laboratory and has been shown to be a potent and selective
inhibitor of Chk2.122 It was illustrated that indoloazepine displayed an leo value
of 8 nM for Chk2 kinase inhibition through an in vitro assay and was shown to be

quite selective against a number of purified kinases including Chk1 (30 fold).3'122

22

Fl“ 5n“.
..5', .: :3 {0

Li Ch

f F
ui

v'lv aJ
32:”lnar
. - . ‘Vy

ECI.ESE

h.
W2

It is with the success of indoloazepine as a potent checkpoint kinase 2 inhibitor
that the foundation of my project is built upon.
|.| Chk2 binding pocket properties

With the success of the crystallization of the kinase domain of Chk2 by
Oliver and co-workersQQ, information regarding the binding pocket can be
examined and explored. Oliver et al. were also successful in crystallizing
debromohymenialdisine inside of the binding pocket allowing a scrutinizing eye to
identify possible residues important for binding purposes. It can be seen in Figure

l-12 the important binding interactions between DBH and the Chk2 active site.

 

Figure l-12. DBH inside the Chk2 active site

DBH is shown in the center of Figure l-12 and the yellow dashed lines
represent hydrogen bonding interactions with the side chains and main chain of

Chk2. It is observed that the main interactions that make DBH a fairly potent

23

inhibitor include the imidazolone moiety, which shows interactions with threonine,
arginine and glutamic acid residues. Furthermore, there are additional hydrogen
bonding interactions that are present in the southern portion of DBH. Figure l-13
illustrates these interactions and it can be seen that the amide functional group of
the seven—membered ring plays a large role in bonding to a glutamic acid and

methionine residue along the main chain in Chk2.

   
 

MET 304

Figure l-13. Interactions between southern portion of DBH and Chk2

Interpreting this information and applying it to the DBH analog,
indoloazepine, it can be imagined that indoloazepine may have similar hydrogen
bonding interactions within Chk2. In fact, according to Figure l—14, indoloazepine
could potentially bind in a similar fashion as DBH. The hydrogen bonding

interactions between the imidazolone ring of indoloazepine and the residues

24

-..,“ N

l
:4 av ‘-

surrounding it could interact to help hold indoloazepine in the active site.
Furthermore, Figure ”-14 also shows that indoloazepine could have the same
interactions between the amide of the seven membered ring and the same

residues that were proposed to hold DBH inside the active site.

 

Figure l-14. lndoloazepine modeled inside the Chk2 binding pocket

Although the exact nature of the binding of indoloazepine and the active
site of Chk2 is not known, it can be hypothesized based on the known binding
properties of a very closely related structure (DBH). Furthermore, we can use this
information to rationalize why it is thought that a new indole alkaloid, the focal
point of my research, could also be a potential Chk2 inhibitor. As shown
previously in Figure l-4, the point at which drug leads have been identified can

lead to a rational design approach, which feeds back into the natural product tank

25

.- fif‘fli‘ [‘
w a. "JV '

“map: F;
a, ...‘UIJ a

T1

.
"en Men

A A ‘-
I. a I)- x.

to uncover new natural products of interest as well as a new library of
compounds.

The success of indoloazepine as a Chk2 inhibitor has led to a reevaluation
of another natural product, shown in Figure l-15. This indole alkaloid possesses
some structural features that are similar to that of indoloazepine. As a result, it

warranted further examination to determine its potential inhibition of Chk2.

 

   

Indole alkaloid

 

 

lndoloazepine

 

Figure M5 New indole alkaloid and indoloazepine

At first glance the structural similarities between the indole alkaloid and
indoloazepine may not be obvious, besides the fact that they both contain an
imidazolone ring and indole moiety. However, after some calculations using
Spartan Pro®, it can be seen that there are some more important correlations. As
illustrated in Figure I-16, the dihedral angle between the plane containing the
imidazolone ring and the plane containing the indole ring for indoloazepine is
calculated to be 50°. Furthermore, according to the crystal structure of the
indoloazepine analog where the indole nitrogen is methylated, the dihedral angle
is 56°. When examining the new indole alkaloid, it was determined that the
dihedral angle between the imidazolone ring and indole is also very similar with

an angle of 54°. It is important to remember that spatial arrangements inside the

26

binding pocket are very important when considering how the molecule fits into
the active site. More importantly, the imidazolone moiety seemed to be very
important to the binding of DBH and indoloazepine considering the number of

hydrogen bonding interactions that were present.

 

Dihedral angle = 54°

Dihedral angle = 50°

Figure I-16. Comparison of dihedral angles

Further analysis of both compounds using Spartan Pro® revealed that the
carbon connected to the indole ring (at position 3) for both compounds were
surprisingly similar even though the two compounds contained different
hybridizations at that carbon. It is shown in Figure l-17 that the hybridization of
indoloazepine at carbon 3 of the indole ring, although technically sp2 hybridized,
actually exhibits more of a sp3 hybridization, possibly due to the partial single
bond character that the bond has as a result of the donating nature of the indole
nitrogen’s lone pair of electrons. The angle at the indicated carbon of
indoloazepine was calculated to be 111°, while the angle at the carbon on

position 3 of the indole from the new indole alkaloid was calculated to be 110°.

27

 

3
Angle = 110.. Hybridization = sp2
Angle = 111°

Hybridization = sp

Figure l-17. Hybridization of carbon connected to indole ring

Placing the new indole alkaloid inside the binding pocket of Chk2 (Figure
l-18) reveals that there is potential for the imidazolone ring of the alkaloid to
hydrogen bond to the same residues responsible for binding DBH and possibly
indoloazepine. Although the indole alkaloid lacks the amide functional group that
seems to play a role in the binding of DBH and indoloazepine, it does contain a
ketone, which was thought to potentially interact with a lysine residue to form a
Schiff base and result in a covalent bonding interaction, possibly allowing for a
more potent inhibition.

It is understood that these potential interactions and reason for possible
potency are purely speculative. There is no definite way to know exactly which
compounds are active and which are not. Our hypothesis is that this natural
product has the potential and structural features that may allow it to be a
checkpoint kinase 2 inhibitor. Thus, our hypothesis is the impetus for my project,

which is focused on the new indole alkaloid and its potential biological activity.

28

    

LYS 249

Figure I-18. New indole alkaloid modeled in Chk2 binding pocket

29

l.J Goal of project

The main goal of my project includes the synthesis of the natural product.
Furthermore, it was thought that the synthesis of the natural product could be
obtained through an oxazolone intermediate, a scaffold that our laboratory has
explored quite extensively (Figure l-19).123‘135 It was thought that new
methodology could be developed extending our chemistry to grant access to
quaternary imidazolones, the core of the natural product. Additional goals were to
synthesize analogs of the natural product, as well as other heterocyclic
compounds that could be tested for Chk2 activity. The following chapters
elaborate on the development of a new synthetic method that gives access to the
quaternary imidazolone scaffold, the first total synthesis of the indole alkaloid and
two analogs and the biological evaluation of the indole alkaloid, analogs and

other heterocyclic compounds synthesized for testing.

 

 

 

 

 

 

 

 

'34
HZNYH R4 R5 . R2
. _ Ix
\ CO H
NWRS / R1 N 2
0
Re 0R4 R1 0 T5
RI R2 \]|/ O R4/N /N
5 \ +—— N R _____ ... Y O
o N C02” R2 3 HN72:
H 3
R2
Oxazolone t
5\N/S;R2 F/ R4
)eN COZH
R1 R2

Figure I-19. Oxazolone chemistry affording various heterocycles

30

|.K

10.

11.

References

Jemal, A.; Thun Michael, J.; Ries Lynn, A. G.; Howe Holly, L.; Weir
Hannah, K.; Center Melissa, M.; Ward, E.; Wu, X.-C.; Eheman, C.;
Anderson, R.; Ajani Umed, A.; Kohler, B.; Edwards Brenda, K., Annual
report to the nation on the status of cancer, 1975-2005, featuring trends in
lung cancer, tobacco use, and tobacco control. J Natl Cancer Inst 2008,
100, 1672-94.

Greenlee, R. T.; Murray, T.; Bolden, S.; Wingo, P. A., Cancer statistics,
2000. CA CancerJ Clin 2000, 50, 7-33.

Sharma, V.; HUpp, C. D.; Tepe, J. J., Enhancement of chemotherapeutic
efficacy by small molecule inhibition of NF-kB and checkpoint kinases.
Curr. Med. Chem. 2007, 14, 1061-1074.

Warshawsky, D.; Landolph, J. R., Molecular Carcinogenesis and The
Molecular Biology of Human Cancer. CRC Taylor & Francis: Boca Raton,
FL, 2006.

Aletras, V.; Hadjiliadis, D.; Hadjiliadis, N., On the Mechanism of Action of
the Antitumor Drug cis-Platin (cis-DDP) and its Second Generation
Derivatives. Met Based Drugs 1995, 2, 153-85.

Bernier, J.; Hall, E. J.; Giaccia, A., Timeline: Radiation oncology: a century
of achievements. Nat. Rev. Cancer 2004, 4, 737-747.

Bikker, J. A.; Brooijmans, N.; Wissner, A.; Mansour, T. S., Kinase Domain
Mutations in Cancer: Implications for Small Molecule Drug Design
Strategies. J. Med. Chem. 2009, 52, 1493-1509.

Ghoshal, K.; Jacob, S. T., An alternative molecular mechanism of action of
5-fluorouracil, a potent anticancer drug. Biochem. Pharmacol. 1997, 53,
1569—1575.

Hande, K. R., Etoposide: four decades of development of a topoisomerase
II inhibitor. Eur. J. Cancer 1998, 34, 1514-1521.

Katsoulas, A.; Rachid, Z.; Brahimi, F.; McNamee, J.; Jean-Claude, B. J.,
Cytokinetics and mechanism of action of AKO4: a novel nitrogen mustard
targeted to bcr-abl. Leuk. Res. 2005, 29, 565-572.

Krohn, K.; Ed., Topics in Current Chemistry; Anthracycline Chemistry and

Biology II; Mode of Action, Clinical Aspects and New Drugs. Springer-
Verlag: 2008; Vol. 283, p 222 pp.

31

..
yw

5.).

l\"
—L

R”

K‘u'i
and
red

R”
i Uh
Or:

his

Tie
WC
ail
201

to;

 

 

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Kumar, G. S.; Lipman, R.; Cummings, J.; Tomasz, M., Mitomycin C-DNA
adducts generated by DT-diaphorase. Revised mechanism of the enzymic
reductive activation of mitomycin C. Biochemistry 1997, 36, 14128-14136.

Rothenberg, M. L., Topoisomerase | inhibitors: review and update. Ann.
Oncol. 1997, 8, 837-55.

Rowinsky, E. K.; Donehower, R. C., Paclitaxel (taxol). N Engl J Med 1995,
332, 1004-14.

Sahasranaman, 8.; Howard, D.; Roy, 8., Clinical pharmacology and
pharmacogenetics of thiopurines. Eur. J. Clin. Pharmacol. 2008, 64, 753-
767.

Sonntag, C. v., Free-Radical—lnduced DNA Damage and Its Repair.
Springer-Verlag: 2006; p 523.

Thorburn, A.; Frankel, A. E., Apoptosis and anthracycline cardiotoxicity.
Mol. Cancer Ther. 2006, 5, 197-199.

Trevino, A. V.; Woynarowska, B. A.; Herman, T. S.; Priebe, W.;
Woynarowski, J. M., Enhanced topoisomerase ll targeting by annamycin
and related 4-demethoxy anthracycline analogues. Mol. Cancer Ther.
2004, 3, 1403-1410.

Foye, W. 0., Cancer Chemotherapeutic Agents. ACS: Washington, 1995.

Pourquier, P.; Pommier, Y., Topoisomerase l-mediated DNA damage.
Adv. Cancer Res. 2001, 80, 189-216.

Sordet, O.; Khan, Q. A.; Kohn, K. W.; Pommier, Y., Apoptosis induced by
topoisomerase inhibitors. Curr Med Chem Anticancer Agents 2003, 3,
271-90.

Rajski, S. R.; Williams, R. M., DNA cross-linking agents as antitumor
drugs. Chem. Rev. 1998, 98, 2723-2795.

Hurley, L. H., DNA and its associated processes as targets for cancer
therapy. Nat. Rev. Cancer 2002, 2, 188-200.

Yu, 0.; La Rose, J.; Zhang, H.; Takemura, H.; Kohn, K. W.; Pommier, Y.,
Ucn-01 inhibits p53 up-regulation and abrogates g-radiation-induced G2-M
checkpoint independently of p53 by targeting both of the checkpoint
kinases, chk2 and chk1. Cancer Research 2002, 62, 5743—5748.

32

25.

26.

27.

28.

29.

30.

31.

32.

33.

35.

36.

37.

38.

Janssens, S.; Tschopp, J., Signals from within: the DNA-damage-induced
NF-kappaB response. Cell Death Differ 2006, 13, 773-84.

Kastan, M. B.; Bartek, J., Cell-cycle checkpoints and cancer. Nature 2004,
432, 316-23.

Webster, G. A.; Perkins, N. D., Transcriptional cross talk between NF-
kappaB and p53. Mol. Cell Biol. 1999, 19, 3485-95.

Ryan, K. M.; Ernst, M. K.; Rice, N. R.; Vousden, K. H., Role of NF-kappaB
in p53-mediated programmed cell death. Nature 2000, 404, 892-7.

Meek, D. W., The p53 response to DNA damage. DNA Repair (Amst)
2004, 3, 1049-56.

Sherr, C. J., Cancer cell cycles. Science 1996, 274, 1672-7.

Makin, G.; Hickman, J. A., Apoptosis and cancer chemotherapy. Cell
Tissue Res 2000, 301, 143-52.

Karin, M.; Lin, A., NF—kappaB at the crossroads of life and death. Nat.
Immunol. 2002, 3, 221-7.

Liu, 0.; Guntuku, S.; Cui, X.-S.; Matsuoka, S.; Cortez, D.; Tamai, K.; Luo,
G.; Carattini-Rivera, S.; DeMayo, F.; Bradley, A.; Donehower, L. A.;
Elledge, S. J., Chk1 is an essential kinase that is regulated by Atr and
required for the G2/M DNA damage checkpoint. Genes Dev. 2000, 14,
1448-1459.

Beg, A. A.; Baltimore, D., An essential role for NF-kappaB in preventing
TNF-alpha-induced cell death. Science 1996, 274, 782-4.

Boland, M. P., DNA damage signalling and NF-kappaB: implications for
survival and death in mammalian cells. Biochem. Soc. Trans. 2001, 29,
674-8.

Pommier, Y.; Weinstein, J. N.; Aladjem, M. l.; Kohn, K. W., Chk2
molecular interaction map and rationale for Chk2 inhibitors. Clin Cancer
Res 2006, 12, 2657-61.

Sancar, A.; Lindsey-Boltz, L. A.; Unsal-Kacmaz, K.; Linn, 8., Molecular
Mechanisms of Mammalian DNA Repair and the DNA Damage
Checkpoints. Annu. Rev. Biochem. 2004, 73, 39-85.

Zhou, B. B.; Elledge, S. J., The DNA damage response: putting
checkpoints in perspective. Nature 2000, 408, 433-9.

33

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

Elledge, S. J., Cell cycle checkpoints: preventing an identity crisis.
Science 1996, 274, 1664-72.

Ahn, J.; Urist, M.; Prives, C., The Chk2 protein kinase. DNA Repair 2004,
3, 1039-1047.

Liu, R.; Hsieh, C.-Y.; Lam, K. S., New approaches in identifying drugs to
inactivate oncogene products. Semin. Cancer Biol. 2004, 14, 13-21.

Zhou, B. B. S.; Bartek, J., Targeting the Checkpoint Kinases:
Chemosensitization versus Chemoprotection. Nature Rev. 2004, 4, 1-10.

Tobey, R. A., Different drugs arrest cells at a number of distinct stages in
G2. Nature 1975, 254, 245-7.

Weinert, T. A.; Hartwell, L. H., The RAD9 gene controls the cell cycle
response to DNA damage in Saccharomyces cerevisiae. Science 1988,
241, 317-22.

Zhou, B.-B. S.; Elledge, S. J., The DNA damage response: putting
checkpoints in perspective. Nature 2000, 408, 433-439.

Khanna, K. K.; Jackson, S. P., DNA double-strand breaks: signaling,
repair and the cancer connection. Nat. Genet. 2001, 27, 247-254.

Bartek, J.; Lukas, J., Chk1 and Chk2 kinases in checkpoint control and
cancer. Cancer Cell 2003, 3, 421-9.

Batista, L. F. 2.; Kaina, B.; Meneghini, R.; Menck, C. F. M., How DNA
lesions are turned into powerful killing structures: Insights from UV-
induced apoptosis. Mutat. Res., Rev. Mutat. Res. 2009, 681, 197-208.

Cadet, J.; Sage, E.; Douki, T., Ultraviolet radiation-mediated damage to
cellular DNA. Mutat. Res., Fundam. Mol. Mech. Mutagen. 2005, 571, 3-17.

Durocher, D.; Jackson, S. P., DNA-PK, ATM and ATR as sensors of DNA
damage: variations on a theme? Curr. Opin. Cell Biol. 2001, 13, 225-231.

Banin, S.; Moyal, L.; Shieh, S. Y.; Taya, Y.; Anderson, C. W.; Chessa, L.;
Smorodinsky, N. l.; Prives, C.; Reiss, Y.; Shiloh, Y.; Ziv, Y., Enhanced
phosphorylation of p53 by ATM in response to DNA damage. Science
1998, 281, 1674-1677.

Canman, C. E.; Lim, D.-S.; Cimprich, K. A.; Taya, Y.; Tamai, K.;
Sakaguchi, K.; Appella, E.; Kastan, M. B.; Siliciano, J. D., Activation of the

34

 

J

17"

(J)

‘1’.’

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

ATM kinase by ionizing radiation and phosphorylation of p53. Science
1998, 281 , 1677-1679.

Shiloh, Y., ATM and related protein kinases: safeguarding genome
integrity. Nat. Rev. Cancer 2003, 3, 155-168.

Cimprich, K. A.; Shin, T. B.; Keith, C. T.; Schreiber, S. L., cDNA cloning
and gene mapping of a candidate human cell cycle checkpoint protein.
Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 2850-5.

Abraham, R. T., Cell cycle checkpoint signaling through the ATM and ATR
kinases. 2001; Vol. 15, p 2177-2196.

Melo, J.; Toczyski, D., A unified view of the DNA-damage checkpoint.
Curr. Opin. Cell Biol. 2002, 14, 237-245.

Rhind, N.; Russell, P., Chk1 and Cds1: linchpins of the DNA damage and
replication checkpoint pathways. J. Cell Sci. 2000, 113, 3889-3896.

McGowan, C. H., Checking in on cds1 (chk2): a checkpoint kinase and
tumor suppressor. BioEssays 2002, 24, 502-511.

Zhou, B. B.; Bartek, J., Targeting the checkpoint kinases:
Chemosensitization versus Chemoprotection. Nat Rev Cancer 2004, 4,
216-25.

Zhou, B. B.; Anderson, H. J.; Roberge, M., Targeting DNA checkpoint
kinases in cancer therapy. Cancer Biol Ther 2003, 2, 816-22.

Blagden, S.; de Bono, J., Drugging cell cycle kinases in cancer therapy.
Curr Drug Targets 2005, 6, 325-35.

Hirao, A.; Kong, Y.-Y.; Matsuoka, S.; Wakeham, A.; Ruland, J.; Yoshida,
H.; Liu, D.; Elledge, S. J.; Mak, T. W., DNA damage-induced activation of
p53 by the checkpoint kinase Chk2. Science 2000, 287, 1824-1827.

Matsuoka, S.; Rotman, G.; Ogawa, A.; Shiloh, Y.; Tamai, K.; Elledge, S.
J., Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro.
Proc. Natl. Acad. Sci. USA 2000, 97, 10389-10394.

Zhao, H.; Piwnica-Worms, H., ATR-mediated checkpoint pathways
regulate phosphorylation and activation of human Chk1. Mol. Cell. Biol.
2001, 21, 4129-4139.

Zhou, B. B.; Sausville, E. A., Drug discovery targeting Chk1 and Chk2
kinases. Prog Cell Cycle Res 2003, 5, 413-21.

35

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

Takai, H.; Tominaga, K.; Motoyama, N.; Minamishima, Y. A.; Nagahama,
H.; Tsukiyama, T.; Ikeda, K.; Nakayama, K.; Nakanishi, M.; Nakayama, K.-
l., Aberrant cell cycle checkpoint function and early embryonic death in
Chk1-l- mice. Genes Dev. 2000, 14, 1439-1447.

Jack, M. T.; Woo, R. A.; Hirao, A.; Cheung, A.; Mak, T. W.; Lee, P. W. K.,
Chk2 is dispensable for p53-mediated G1 arrest but is required for a latent
p53-mediated apoptotic response. Proc. Natl. Acad. Sci. USA 2002, 99,
9825-9829.

Bartek, J.; Lukas, J., Mammalian G1- and S-phase checkpoints in
response to DNA damage. Curr. Opin. Cell Biol. 2001, 13, 738-747.

Pietrancosta, N.; Garino, C.; Laras, Y.; Quelever, G.; Pierre, P.; Clavarino,
G.; Kraus, J.-L., Are p53 inhibitors potentially useful therapeutics? Drug
Dev. Res. 2005, 65, 43-49.

Suganuma, M.; Kawabe, T.; Hori, H.; Funabiki, T.; Okamoto, T.,
Sensitization of cancer cells to DNA damage-induced cell death by
specific cell cycle G2 checkpoint abrogation. Cancer Res 1999, 59, 5887-
91.

Tenzer, A.; Pruschy, M., Potentiation of DNA-damage-induced cytotoxicity
by G2 checkpoint abrogators. Curr Med Chem Anti-Canc Agents 2003, 3,
35—46.

Sausville, E. A., Cyclin-dependent kinase modulators studied at the NCI:
pre-clinical and clinical studies. Curr Med Chem Anti-Cane Agents 2003,
3, 47-56.

Curman, D.; Cinel, B.; Williams, D. E.; Rundle, N.; Block, W. D.; Goodarzi,
A. A.; Hutchins, J. R.; Clarke, P. R.; Zhou, B.-B.; Lees-Miller, S. P.;
Andersen, R. J.; Roberge, M., Inhibition of the G2 DNA damage
checkpoint and of protein kinases Chk1 and Chk2 by the marine sponge
alkaloid debromohymenialdisine. J. Biol. Chem. 2001, 276, 17914-17919.

Prudhomme, M., Novel checkpoint 1 inhibitors. Rec. Pat. Anti. Cancer
Drug Discov. 2006, 1, 55-68.

Lukas, C.; Bartkova, J.; Latella, L.; Falck, J.; Mailand, N.; Schroeder, T.;
Sehested, M.; Lukas, J.; Bartek, J., DNA damage-activated kinase Chk2 is
independent of proliferation or differentiation yet correlates with tissue
biology. Cancer Res. 2001, 61, 4990-4993.

36

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

d'Adda di Fagagna, F.; Reaper, P. M.; CIay-Farrace, L.; Fiegler, H.; Carr,
F.; von Zglinicki, T.; Saretzki, G.; Carter, N. R; Jackson, S. P., A DNA
damage checkpoint response in telomere-initiated senescence. Nature
2003, 426, 194-198.

Oh, H.; Wang, S. C.; Prahash, A.; Sano, M.; Moravec, C. S.; Taffet, G. E.;
Michael, L. H.; Youker, K. A.; Entman, M. L.; Schneider, M. D., Telomere
attrition and Chk2 activation in human heart failure. Proc. Natl. Acad. Sci.
USA 2003, 100, 5378-5383.

Ahn, J.-Y.; Schwarz, J. K.; Piwnica-Worms, H.; Canman, C. E., Threonine
68 phosphorylation by ataxia telangiectasia mutated is required for
efficient activation of Chk2 in response to ionizing radiation. Cancer Res.
2000, 60, 5934-5936.

Melchionna, R.; Chen, X.-B.; Blasina, A.; McGowan, C. H., Threonine 68
is required for radiation-induced phosphorylation and activation of Cds1.
Nat. Cell Biol. 2000, 2, 762-765.

Lee, C.-H.; Chung, J. H., The ths1 (Chk2)-FHA domain is essential for a
chain of phosphorylation events on ths1 that is induced by ionizing
radiation. J. Biol. Chem. 2001, 276, 30537-30541.

Schwarz, J. K.; Lovly, C. M.; Piwnica-Worms, H., Regulation of the Chk2
protein kinase by oligomerization-mediated cis- and trans-phosphorylation.
Mol. Cancer Res. 2003, 1, 598-609.

Wu, X.; Chen, J., Autophosphorylation of Checkpoint Kinase 2 at Serine
516 Is Required for Radiation-induced Apoptosis. J. Biol. Chem. 2003,
278, 36163-36168.

Matsuoka, S.; Huang, M.; Elledge, S. J., Linkage of ATM to cell cycle
regulation by the Chk2 protein kinase. Science 1998, 282, 1893-1896.

Falck, J.; Mailand, N.; Syljuasen, R. G.; Bartek, J.; Lukas, J., The ATM-
Chk2-Cdc25a checkpoint pathway guards against radioresistant DNA
synthesis. Nature 2001, 410, 842-847.

Brown, A. L.; Lee, C.-H.; Schwarz, J. K.; Mitiku, N.; Piwnica-Worms, H.;
Chung, J. H., A human Cds1-related kinase that functions downstream of
ATM protein in the cellular response to DNA damage. Proc. Natl. Acad.
Sci. USA 1999, 96, 3745-3750.

Blasina, A.; Van de Weyer, |.; Laus, M. C.; Luyten, W. H. M. L.; Parker, A.

E.; McGowan, C. H., A human homolog of the checkpoint kinase Cds1
directly inhibits Cd025 phosphatase. Curr. Biol. 1999, 9, 1-10.

37

ii

Sowv
LzKi
phase
raca‘

247i

Zfiou
M Shl
asoh
ataxi;

fl

lu34
Pen;
Worr
had:
1501

Data
local
6nd“

Ahn,
Intt
204E

Che
Pho
res;
137'

Shh
how-
053
300

Cra
Elie
Ehj

file
ma!

 

87.

88.

89.

90.

91.

92.

93.

94.

95.

 

Sorensen, C. S.; Syljuasen, R. G.; Falck, J.; Schroeder, T.; Ronnstrand,
L.; Khanna, K. K.; Zhou, B.-B.; Bartek, J.; Lukas, J., Chk1 regulates the S
phase checkpoint by coupling the physiological turnover and ionizing
radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell 2003, 3,
247-258.

Zhou, B.-B. S.; Chaturvedi, P.; Spring, K.; Scott, S. P.; Johanson, R. A.;
Mishra, R.; Mattern, M. R.; Winkler, J. D.; Khanna, K. K., Caffeine
abolishes the mammalian G2/M DNA damage checkpoint by inhibiting
ataxia-telangiectasia-mutated kinase activity. J. Biol. Chem. 2000, 275,
1 0342-1 0348.

Peng, C.-Y.; Graves, P. R.; Thoma, R. 8.; Wu, 2.; Shaw, A. S.; Piwnica-
Worms, H., Mitotic and G2 checkpoint control: regulation of 14-3-3 protein
binding by phosphorylation of CchSC on serine-216. Science 1997, 277,
1501-1505.

Dalal, S. N.; Schweitzer, C. M.; Gan, J.; DeCaprio, J. A., Cytoplasmic
localization of human cdc25C during interphase requires an intact 14-3-3
binding site. Mol. Cell. Biol. 1999, 19, 4465-4479.

Ahn, J.; Urist, M.; Prives, C., Questioning the Role of Checkpoint Kinase 2
in the p53 DNA Damage Response. J. Biol. Chem. 2003, 278, 20480-
20489.

Chehab, N. H.; Malikzay, A.; Stavridi, E. S.; Halazonetis, T. D.,
Phosphorylation of Ser—20 mediates stabilization of human p53 in
response to DNA damage. Proc. Natl. Acad. Sci. U. S. A. 1999, 96,
1 3777-13782.

Shieh, S.-Y.; Ahn, J.; Tamai, K.; Taya, Y.; Prives, C., The human
homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate
p53 at multiple DNA damage-inducible sites. Genes Dev. 2000, 14, 289-
300.

Craig, A.; Scott, M.; Burch, L.; Smith, G.; Ball, K.; Hupp, T., Allosteric
effects mediate CHK2 phosphorylation of the p53 transactivation domain.
EMBO Rep. 2003, 4, 787-792.

Hirao, A.; Cheung, A.; Duncan, G.; Girard, P.-M.; Elia, A. J.; Wakeham, A.;
Okada, H.; Sarkissian, T.; Wong, J. A.; Sakai, T.; De Stanchina, E.;
Bristow, R. G.; Suda, T.; Lowe, S. W.; Jeggo, P. A.; Elledge, S. J.; Mak, T.
W., Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia
telangiectasia mutated (ATM)-dependent and an ATM-independent
manner. Mol. Cell. Biol. 2002, 22, 6521-6532.

38

 

35'

”Di,

-..L
C)
U?

lake
Andi
sa‘V‘r
tad}:

1 l

Bari
can

SLI

mmCUZID 0’

mmrnn

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

Takai, H.; Naka, K.; Okada, Y.; Watanabe, M.; Harada, M; Saito, S. L;
Anderson, C. W.; Appella, E.; Nakanishi, M.; Suzuki, H.; Nagashima, K.;
Sawa, H.; Ikeda, K.; Motoyama, N., Chk2-deficient mice exhibit
radioresistance and defective p53-mediated transcription. EMBO J. 2002,
21 , 5195-5205.

Bartek, J.; Lukas, J., Chk1 and Chk2 kinases in checkpoint control and
cancer. Cancer Cell 2003, 3, 421-429.

Jallepalli, P. V.; Lengauer, C.; Vogelstein, B.; Bunz, F., The Chk2 Tumor
Suppressor Is Not Required for p53 Responses in Human Cancer Cells. J.
Biol. Chem. 2003, 278, 20475-20479.

Oliver, A. W.; Paul, A.; Boxall, K. J.; Barrie, S. E.; Aherne, G. W.; Garrett,
M. D.; Mittnacht, S.; Pearl, L. H., Trans-activation of the DNA-damage
signalling protein kinase Chk2 by T-loop exchange. EMBO J. 2006, 25,
3179-3190.

Matthews, D. J.; Yakes, F. M.; Chen, J.; Tadano, M.; Bornheim, L.; Clary,
D. 0.; Tai, A.; Wagner, J. M.; Miller, N.; Kim, Y. D.; Robertson, 8.; Murray,
L.; Karnitz, L. M., Pharmacological abrogation of S-phase checkpoint
enhances the anti-tumor activity of gemcitabine in vivo. Cell Cycle 2007, 6,
104-110.

Preparation of 3-aminoindazoles as CHK1/CHK2 kinase inhibitors. 2006-
1020060051791020060051 79, 20060206., 2007.

Janetka, J. W.; Ashwell, S., Checkpoint kinase inhibitors: a review of the
patent literature. Expert Opin. Ther. Pat. 2009, 19, 165-197.

Mederski, W.; Gericke, R.; Klein, M.; Beier, N.; Lang, F. Preparation of 3-
oxoindazolyl-substituted squaric acid derivatives as CHK1, CHK2 and/or
SGK kinase inhibitors. 2006-EP76502007022858, 20060802., 2007.

Arienti, K. L.; Brunmark, A.; Axe, F. U.; McClure, K.; Lee, A.; Blevitt, J.;
Neff, D. K.; Huang, L.; Crawford, 8.; Pandit, C. R.; Karlsson, L.;
Breitenbucher, J. G., Checkpoint Kinase Inhibitors: SAR and
Radioprotective Properties of a Series of 2-Arylbenzimidazoles. J. Med.
Chem. 2005, 48, 1873- 1885.

Carlessi, L.; Buscemi, G.; Larson, G.; Hong, Z.; Wu, J. 2.; Delia, D.,
Biochemical and cellular characterization of VRX0466617, a novel and
selective inhibitor for the checkpoint kinase Chk2. Mol. Cancer Ther. 2007,
6, 935-944.

39

106.

107.

108.

109.

110.

111.

112.

113.

114.

Jobson, A. G.; Cardellina, J. H., II; Scudiero, D.; Kondapaka, S.; Zhang,
H.; Kim, H.; Shoemaker, R.; Pommier, Y., Identification of a bis-
guanylhydrazone [4,4'-diacetyldiphenylurea-bis(guanylhydrazone); NSC
109555] as a novel chemotype for inhibition of Chk2 kinase. Mol.
Pharmacol. 2007, 72, 876-884.

Larson, G.; Yan, S.; Chen, H.; Rong, F.; Hong, Z.; Wu, J. 2., Identification
of novel, selective and potent Chk2 inhibitors. Bioorg. Med. Chem. Lett.
2007, 17, 172-175.

McClure, K. J.; Huang, L.; Arienti, K. L.; Axe, F. U.; Brunmark, A.; Blevitt,
J.; Guy Breitenbucher, J., Novel non-benzimidazole chk2 kinase inhibitors.
Bioorg. Med. Chem. Lett. 2006, 16, 1924-1928.

Neff, D. K.; Lee-Dutra, A.; Blevitt, J. M.; Axe, F. U.; Hack, M. D.; Buma, J.
C.; Rynberg, R.; Brunmark, A.; Karlsson, L.; Breitenbucher, J. G., 2-Aryl
benzimidazoles featuring alkyl-linked pendant alcohols and amines as
inhibitors of checkpoint kinase Chk2. Bioorg. Med. Chem. Lett. 2007, 17,
6467-6471.

Zabludoff, S. D.; Deng, C.; Grondine, M. R.; Sheehy, A. M.; Ashwell, S.;
Caleb, B. L.; Green, S.; Haye, H. R.; Horn, C. L.; Janetka, J. W.; Liu, D.;
Mouchet, E.; Ready, 8.; Rosenthal, J. L.; Queva, C.; Schwartz, G. K.;
Taylor, K. J.; Tse, A. N.; Walker, G. E.; White, A. M., AZD7762, a novel
checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates
DNA-targeted therapies. Mol. Cancer Ther. 2008, 7, 2955-2966.

Kohn, E. A.; Yoo, C. J.; Eastman, A., The Protein Kinase C Inhibitor
Goe6976 Is a Potent Inhibitor of DNA Damage-induced S and G2 Cell
Cycle Checkpoints. Cancer Res. 2003, 63, 31-35.

Yu, 0.; La Rose, J.; Zhang, H.; Takemura, H.; Kohn, K. W.; Pommier, Y.,
Ucn-01 inhibits p53 up-regulation and abrogates g-radiation-induced G2-M
checkpoint independently of p53 by targeting both of the checkpoint
kinases, chk2 and chk1. Cancer Res. 2002, 62, 5743-5748.

Lara, P. M, Jr.; Mack, P. C.; Synold, T.; Frankel, P.; Longmate, J.;
Gumerlock, P. H.; Doroshow, J. H.; Gandara, D. R., The Cyclin-
Dependent Kinase Inhibitor UCN-01 Plus Cisplatin in Advanced Solid
Tumors: A California Cancer Consortium Phase I Pharmacokinetic and
Molecular Correlative Trial. Clin. Cancer Res. 2005, 11 , 4444—4450.

Akinaga, S.; Nomura, K.; Gomi, K.; Okabe, M., Enhancement of antitumor

activity of mitomycin C in vitro and in vivo by UCN-01, a selective inhibitor
of protein kinase C. Cancer Chemo. Pharmacol. 1993, 32, 183-9.

40

115.

116.

117.

118.

119.

120.

121.

122.

123.

124.

Pollack, I. F.; Kawecki, S.; Lazo, J. S., Blocking of glioma proliferation in
vitro and in vivo and potentiating the effects of BCNU and Cisplatin: UCN-
01, a selective protein kinase C inhibitor. J. Neurosurg. 1996, 84, 1024-
1032.

Wang, 0.; Fan, 8.; Eastman, A.; Worland, P. J.; Sausville, E. A.;
O'Connor, P. M., UCN-01: A potent abrogator of G2 checkpoint function in
cancer cells with disrupted p53. J. Natl. Cancer Inst. 1996, 88, 956-965.

Hu, B.; Zhou, X. Y.; Wang, X.; Zeng, 2. C.; lliakis, G.; Wang, Y., The
radioresistance to killing of A1-5 cells derives from activation of the Chk1
pathway. J. Biol. Chem. 2001, 276, 17693-8.

Hirose, Y.; Berger, M. S.; Pieper, R. O., Abrogation of the Chk1-mediated
G2 checkpoint pathway potentiates temozolomide-induced toxicity in a
p53-independent manner in human glioblastoma cells. Cancer Res. 2001,
61 , 5843-5849.

Fuse, E.; Tanii, H.; Kurata, N.; Kobayashi, H.; Shimada, Y.; Tamura, T.;
Sasaki, Y.; Tanigawara, Y.; Lush, R. D.; Headlee, D.; Figg, W. D.; Arbuck,
S. G.; Senderowicz, A. M.; Sausville, E. A.; Akinaga, S.; Kuwabara, T.;
Kobayashi, S., Unpredicted clinical pharmacology of UCN-01 caused by
specific binding to human a1-acid glycoprotein. Cancer Res. 1998, 58,
3248-3253.

Sausville, E. A.; Arbuck, S. G.; Messmann, R.; Headlee, D.; Bauer, K. S.;
Lush, R. M.; Murgo, A.; Figg, W. D.; Lahusen, T.; Jaken, S.; Jing, X.-x.;
Roberge, M.; Fuse, E.; Kuwabara, T.; Senderowicz, A. M., Phase I trial of
72-hour continuous infusion UCN-01 in patients with refractory neoplasms.
J. Clin. Oncol. 2001, 19, 2319-2333.

Fuse, E.; Kuwabara, T.; Sparreboom, A.; Sausville, E. A.; Figg, W. D.,
Review of UCN-01 development: a lesson in the importance of clinical
pharmacology. J Clin Pharmacol 2005, 45, 394-403.

Sharma, V.; Tepe, J. J., Potent inhibition of checkpoint kinase activity by a
hymenialdisine-derived indoloazepine. Bioorg. Med. Chem. Lett. 2004, 14,
4319-4321.

Mosey, R. A.; Tepe, J. J., New synthetic route to access (+-)-
salinosporamide A via an oxazolone-mediated ene-type reaction.
Tetrahedron Lett. 2009, 50, 295-297.

Mosey, R. A.; Fisk, J. S.; Friebe, T. L.; Tepe, J. J., Synthesis of tert-Alkyl

Amino Hydroxy Carboxylic Esters via an Intermolecular Ene-Type
Reaction of Oxazolones and Enol Ethers. Org. Lett. 2008, 10, 825-828.

41

125.

126.

127.

128.

129.

130.

131.

132.

133.

134.

135.

Fisk, J. S.; Mosey, R. A.; Tepe, J. J., The diverse chemistry of oxazol-5-
(4H)-ones. Chem. Soc. Rev. 2007, 36, 1432-1440.

Fisk, J. S.; Tepe, J. J., Intermolecular Ene Reactions Utilizing Oxazolones
and Enol Ethers. J. Am. Chem. Soc. 2007, 129, 3058-3059.

Sharma, V.; Peddibhotla, S.; Tepe, J. J., Sensitization of Cancer Cells to
DNA Damaging Agents by Imidazolines. J. Am. Chem. Soc. 2006, 128,
9137-9143.

Sharma, V.; Tepe, J. J., Diastereochemical diversity of imidazoline
scaffolds via substrate controlled TMSCl-mediated cycloaddition of
azlactones. Org. Lett. 2005, 7, 5091-5094.

Keni, M.; Tepe, J. J., One-Pot Friedel-Crafts/Robinson-Gabriel Synthesis
of Oxazoles Using Oxazolone Templates. J. Org. Chem. 2005, 70, 4211-
4213.

Sharma, V.; Lansdell, T. A.; Peddibhotla, S.; Tepe, J. J., Sensitization of
Tumor Cells toward Chemotherapy: Enhancing the Efficacy of
Camptothecin with Imidazolines. Chem. Biol. 2004, 11 , 1689-1699.

Peddibhotla, S.; Tepe, J. J., Stereoselective Synthesis of Highly
Substituted Delta 1-Pyrrolines: exo—Selective 1,3-Dipolar Cycloaddition
Reactions with Azlactones. J. Am. Chem. Soc. 2004, 126, 12776-12777.

Sharma, V.; Tepe, J. J., Potent inhibition of checkpoint kinase activity by a
hymenialdisine-derived indoloazepine. Bioorg. Med. Chem. Lett. 2004, 14,
4319-4321.

Sharma, V.; Lansdell, T. A.; Jin, G.; Tepe, J. J., Inhibition of Cytokine
Production by Hymenialdisine Derivatives. J. Med. Chem. 2004, 47, 3700-
3703.

Peddibhotla, S.; Tepe, J. J., Multicomponent synthesis of highly
substituted imidazolines via a silicon mediated 1,3-dipolar cycloaddition.
Synthesis 2003, 1433-1440.

Peddibhotla, S.; Jayakumar, S.; Tepe, J. J., Highly Diastereoselective
Multicomponent Synthesis of Unsymmetrical Imidazolines. Org. Lett.
2002, 4, 3533-3535.

42

l
1 ‘Wfi I
5;" Saul;

Tel. '19 I
‘5’7'; for
Ci‘E'Eht
"31W
Li88 Va

lidazol

CHAPTER II

DEVELOPMENT OF A SYNTHETIC METHOD FOR THE PREPARATION
OF 2-AMINO-5, 5-DISUBSTITUTED-1H-lMlDAZOL-4(5H)-ONES

II.A Nomenclature and numbering of imidazolones

The general structure for a 2-amino-imidazolone is shown in Figure “-1. It
can be observed that there are actually three tautomeric forms for a 2-amino-
imidazolone where the double bond is switched between the three nitrogens
making up the guanidine portion of the heterocycle. The preferred tautomeric
form for the imidazolone was shown by Matsumoto and co-workers1 and in a
different set of experiments by Kenyon and co-workers2 to be tautomer "-2.
Traditional nomenclature for these types of molecules encountered in literature
has varied,3 with names such as 2-amino-imidazoI-4-ones (2-amino-
imidazolone), glycocyamidines and 2-iminohydantoins being some of the more
recent common terms. The numbering of the ring system varies depending on
the tautomer specified as shown in Figure "-1.3 When the double bond is
exocyclic as in the case of "-1 or when the double bond is endocyclic and in
conjugation with the carbonyl (II-2), the numbering of the ring begins with the
nitrogen furthest from the carbonyl and is given position 1. Numbering the ring
clockwise from that point will result in the second nitrogen contained in the ring
being in position 3, the carbonyl carbon in position 4 and the methylene carbon in

position 5.3

43

HI
\

l -
\v-I- fps
v, '
I ‘aZfi.

 

HN

"-1 "-2 "-3

Figure "-1. Numbering of the 2-amino-imdazoI-4-one ring system

However, when the double bond is endocyclic and not in conjugation with
the carbonyl (II-3), the numbering of the ring system starts with the opposite
nitrogen in the ring. A counter-clockwise numbering scheme follows which puts
the second nitrogen in position 3, the methylene in position 4 and the carbonyl
carbon in position 5.3

When an amino group is not present in the 2 position, the structure is still
considered an imidazolone, however, it simply contains a different substitution at
the 2 position. For the purposes of consistency, the term imidazolone will be
used for the nomenclature of the general ring structure. When substitution of the
imidazolone ring is present, the numbering scheme will follow that for structures
"-1 and "-2, which is the most commonly accepted numbering format.3 In
addition, the name of the substituent will follow the number at which it is found.
For example, structure "-4 in Figure "-2, would have the name 2-
aminoimidazolone where structure "-5 (also in Figure "-2) would have the name
2, 5, 5-trimethylimidazolone.

HN
"-4 “-5

Figure "-2. Representative structures of imidazolones

44

vet:
"J's
a

later

a
fiDldi
01

".3 Reactivity of imidazolones

The 2—aminoimidazolone ring system can undergo a number of reactions
in a variety of locations such as at the carbonyl carbon, alpha to the carbonyl and
at the guanidine nitrogens (Figure Il-3).3'7

H2N N
Wig: o HZN HN/N

NH Oxidation
JL OH Reduction
H N N
2 H/\[()]/

or
H drol sis , Alkylation
H2 N VCOZH < y y HZNJ/f

/
H2N N
or RX 7 11:):0

H
O
YN O Condensation Acylation
HN

HZNHNKHORJLH :fSI/fo

R
R

 

 

 

 

 

 

Figure "-3. Reactions of 2-aminoimidazolone
Alkylation of imidazolones using alkyl halides such as methyl iodide
occurs at N-3 primarily, although if N-1 and N-3 are already alkylated, N-2 can be
alkylated.2 It was shown by Kenyon and co-workers that the nitrogen in position 3
is more reactive and the alkylated product can undergo a reversible
rearrangement to put the double bond back into conjugation with the carbonyl,

affording N-2 bearing the alkyl group (Scheme "-1).2

45

Scheme "-1. Alkylation of 2-aminoimidazol-4-one

 

Mel, EtOH _ H2

HZN /N N N/
Hl/fo 90% If f0
'lMeOH

J1 J. \ U n
be *— 0 1r, \

Further support for N-3 being the most basic nitrogen, and hence most
nucleophilic, comes from a study by Reddick and co-workers where they
identified via 15N-NMR that the nitrogen in position 3 is primarily protonated at a
low pH.8 Acylation reactions occur in a similar fashion?" 5 lmidazolones can also
undergo a condensation reaction with aldehydes producing a 5-arylidene
imidazolone (Figure ll-3).3' 4' 9'12 The success of the reaction is dependent on the
nucleophilic character of the imidazolone and the electrophilic character of the
aldehyde. Furthermore, the reaction often produces a mixture of E/Z isomers,
some of which can isomerizes to one other depending on the product and
conditions.3' 4

Alkylation is also possible at carbon 5 of an 2-aminoimidazolone as shown
by Danishefsky and co-workers (Scheme ”-2).13 The alkylation was completed
using a imidazolone core similar to "-1 (Figure “-1) and a benzyl bromide giving

the quaternary imidazolone in 91% yield.13

46

Scheme "-2. Alkylation at C-5 of an imidazolone

OMe OMe

/N LiHMDS, THF, 91% /N O 03”
o

 

Hydrolysis of the 2-aminoimidazolone ring can lead to a variety of
products depending on the reaction conditions (Figure II-3).3' 4' 6 Cleavage of the
amide bond of the heterocyclic ring can lead to products that resemble a glycine
derivative with a guanidine moiety instead of a primary amine. It has also been
observed that under certain conditions, the imidazolone ring can hydrolyze at a
different position and form urea and glycine.3 Lastly, hydrolysis can also
transform the 2-aminoimidazolone into the hydantoin analog under basic
conditions.4' 6

Oxidation of the methylene group at position 5 of the heterocyclic ring is
possible and the resulting dicarbonyl compound is formed (Figure ”-3). However,
these reactions are often run under alkali conditions and the resulting dicarbonyl
heterocycle undergoes further chemical modifications and is known to hydrolyze
giving the oxalate and derivatives of guanidine.3 Reduction of the carbonyl at
position 5 can occur using hydrogenation under a platinum catalyst. The typical
reaction conditions include 50 mol% of a platinum catalyst and a hydrogen
pressure of 15-35 psi. The result of this reaction is the formation of cyclic
guanidine structures (Figure "-3).7

The acidity of imidazolones have been studied by a few different groups."

3' ‘4 Chandrasekhar and co-workers compared the relative rates of a base

47

catalyzed deuterium exchange for an imidazolone, oxazolone and pyrrolone. It
was found that the rate of deuterium exchange was greatest for the imidazolone
and worst for the pyrrolone (Figure ”-4),14 leading to conclude that based on

these results the imidazolone is more acidic than the other analogs tested.

N N

lmidazolone Oxazolone Pyrrolone

Figure "-4. Relative rates of deuterium exchange
The pKa’s of multiple 2-aminoimidazolone hydrochloride compounds have
been identified initially by Matsumoto and co-workers and then to a greater
extent by Kenyon and co-workers.1' 2 It was found that the pKa’s of the
protonated imidazolones range from 4.48-9.01 (in H20) depending on the
substitution on the heterocyclic ring. There is a clear distinction between the
pKa’s of imidazolones that contain the double bond in conjugation with the

carbonyl group and those that do not, such as between "-8 and "-9 (Figure ”-5).2

 

 

 

 

 

 

 

 

- r 1 F '1 r 1
HCI- HCI- I HCI- I HCI- /
HZN\I4\N)=OW /NYN/ /N\l./’N /N\j/N O
|
“N Mfo HN\)=O fo
ll-6 _ "-7 a _ II-8 _ L "-9 _
9K3 = 4-30 pKa = 7.57 pKa = 4.48 pKa = 9-01

Figure "-5. pKa’s of differently substituted 2-aminoimidazolones
ll.C Natural products containing the imidazolone core
There are many accounts of natural products that contain the imidazolone
core in a variety of different forms.”28 One of the most common imidazolones is

an end product of nitrogen metabolism in the human body, a compound called

48

creatinine (Figure ”-6).8 Hymenialdisine is a natural product that contains the 2-
aminoimidazolone core with an alkenic substitution at position 5 of the
heterocyclic core (Figure ”-6). The compound comes from the sponges AxineI/a
verrucosa and Acanthella aurantiaca and was isolated in 198218 and first
synthesized in 1995.29 De-bromohymenialdisine, an analog of hymenialdisine
without the bromine on the pyrrole ring, also contains the imidazolone core.

Dispacamide 1 is a novel bromo pyrrole alkaloid that was isolated from a
Caribbean sponge in 199616 and was synthesized in 1997.30
Debromodispacamides B and D were isolated from the marine sponge Age/as
mauritiana and synthesized in 200827 , while the related compounds
polyandrocarpamines A and B were isolated from the ascidian Polyandrocarpa
sp. and synthesized in 2002.19 All of the dispacamide and polyandrocarpamine
compounds exhibit the same type of imidazolone core that hymenialdisine
possesses, the 2-aminoimidazoI-4-one core with an alkenic substitution pattern
at carbon 5 (Figure "-6).

The last members of natural products that contain this type of heterocyclic
core are the Ieucettamines B and C and Ieucettamidine (Figure ”-6).
Leucettamine B and Ieucettamidine were isolated from the sponge Leucetta
microraphis in 1993,17 while Ieucettamine C was isolated from Leucetta avocado
in 2003.31 To date, only Ieucettamine B has been synthesized with the first

synthesis reported in 1994.32

49

an

N:
L t
:1 HCH‘ ‘
vvuvULUK
My“
Jv 5

In...
3d
pk v

dflfias;

HZNYffO \
N
/ Br / \
Creatinine N NH
H O
Hymenialdisine
O \ O
/ O n 1 .—
N \
N Br
yNH H \ / HZN N o
HzN . . )
Dispacamide 1 Br

Leucettamine B 0

Figure "-6. Natural products containing the 2-aminoimidazolone core

Natural products that contain the imidazolone core without the 2-amino
substitution have also been reported.15' 23' 25 Rhopaladins A-D (Figure "-7)
contain a unique imidazolone core consisting of an alkenic substitution at carbon
5 and an acyl group at carbon 2. These natural products come from the
Okinawan tunicate Rhopalaea sp. and were isolated in 1998.25 The first synthesis
of rhopaladin D was completed in 200033, while the total synthesis of rhopaladins
A-C were completed shortly after in 2002.34

Kottamides A-D (Figure "-7) are structurally unusual due to the quaternary
substitution pattern on carbon 2. These natural products are optically active,
were isolated from the New Zealand ascidian Pycnoclavella kottae in 2002 and
have not been synthesized to date.15 The last example of a natural product that
has the imidazolone core but lacking the 2-amino functionality is a novel
bis(indole) alkaloid that was isolated from the sponge Dragmacidon sp. in 1996.

The structure of this compound (Figure "-7) is very unique with a quaternary

50

center at carbon 5 and has also not been synthesized to date. Due to the light
absorbing tendencies of the compound in solution, chiroptical data was hard to

establish.23

HO

Kottamide A

 

Novel bis(lndole) alkaloid

Figure "-7. Natural products containing an imidazolone core

The next group of natural products contains the imidazol—4-one
heterocyclic core and also the 2-amino functionality. However, these compounds
do not contain the alkenic substitution pattern at carbon 5 as those in Figure "-8.
Instead, most of these natural products contain a quaternary center in the
imidazolone cycle. Compound 2096A (Figure "-8) and an analog 20963, a
diastereomer of 2096A, were isolated in 2000 from extracts of Streptomyces
sp.28 It is found that these two compounds can interconvert due to the acidity of
the proton at the quaternary center of the bicyclic heterocycle and as a result, the
stereochemistry is not defined. Additionally, the compounds were found to

decompose readily in aqueous solutions making the synthesis of these

51

“0qu

Iva-
NIP

J

3an let's;

Fl

compounds a challenge.28 To date, there are no reported syntheses of 2096A or
B.

Oxysceptrin (Figure "-8) and related alkaloids have been isolated from the
sponge Age/as conifera in 1991 .35' 36 Oxysceptrin contains an unique cyclobutane
core flanked by imidazole, pyrrole and imidazolone appendages. The synthesis
of oxysceptrin as well as other alkaloids from the same family was completed by

O’Malley and co-workers in 2007.37

2096A and B

   

Indole alkaloid
(:l:)-CalcaridineA (III-1) kN O
H2N
Mauritlamine
Figure "-8. Additional natural products containing 2-aminoimidazolone
Another natural product related to oxysceptrin is mauritiamine, a
compound with a quaternary imidazolone containing imidazole and pyrrole

appendages at position 5 (Figure ”-8). The natural product comes from the

marine sponge Age/as mauritiana and was isolated in 1996. Furthermore, the

52

natural product contains a quaternary center but is optically inactive and was
isolated at a racemate.26 The first racemic synthesis of mauritiamine was
completed by Olofson and co-workers in 1997 utilizing an oxidative dimerization
of two imidazole containing intermediates.38

Calcaridine A (Figure ”-8) was isolated from a Fijian sponge Leucetta sp.
in 2003.20 The natural product contains a uniquely substituted 2-aminoimidazol-4-
one core with a quaternary center at carbon 5. The natural product is optically
active although the absolute stereochemistry is not yet defined.20 The first
racemic total synthesis of calcaridine A was completed by Koswatta and co-
workers in 2008 using a novel rearrangement to afford the quaternary
stereocenter.39

The last natural product that contains the 2-aminoimidazol-4-one core and
also a quaternary center at carbon 5 is the indole alkaloid (Ill-1) shown in Figure
”-8. It was isolated from the tunicate Dendrodoa grossularia in 1998 and the
stereochemistry at the quaternary carbon is also not defined.22 Further detail on
this molecule is given in chapter Ill. The synthesis of this natural product is the
goal of my project and thus a synthetic route to form the uniquely substituted
quaternary imidazolone is needed.
".0 Synthetic routes to imidazolones

lmidazolones with and without a quaternary center at position 5 have been
synthesized in a variety of ways.”‘73 The following section illustrates the various
routes that have been used previously to obtain imidazolones as well as the

routes that were attempted to create quaternary imidazolones for synthesizing

53

the indole alkaloid, the focal point of my project. Finally, the new synthetic
method developed for gaining access to the quaternary imidazolone scaffold
needed for the total synthesis of the indole alkaloid will be discussed.
II.D.1 Synthetic routes to imidazolones: Aza-wittig reaction

The first route that will be discussed is the synthesis of imidazolones via
an intramolecular aza-wittig reaction (Scheme ”-3). Takeuchi and co-workers
developed an efficient aza-wittig reaction which reacts an azide and
triphenylphosphine to obtain an intermediate that attacks the carbonyl of the
imide to produce imidazolones in good yields.69 They were able to use alkyl, aryl
and heteroaryl groups at R1; alkyl and aryl groups in the R2 position; and
hydrogen and aryl groups in the R3 position. The reaction proceeds at room
temperature in two hours and produces yields ranging from 70-99%.69

Scheme "-3. Aza-wittig reaction to produce imidazolones

*- 1

R
O 0 PPh )3: O cPPh3 0 hi 2
1 l benzene, 1 I />‘R1
R2 R3 rt R2 R3 R3 N

 

 

70-99%

".02 Synthetic routes to imidazolones: Reaction of aziridinone and
thiourea

Talaty and co-workers use a reaction between an aziridinone and a
thiourea to produce imidazolones in yields up to 55% (Scheme ”—4).70 The
aziridinones were substituted at the R1 and R2 position with either a tert-butyl or
adamantly substituent and reacted with a thiourea to attack the carbonyl of the
aziridinone. After the ring opening of the aziridinone it is proposed that the

reaction eventually produces a guanidine thioester intermediate.7O After a final

54

attack of the guanidine on the thioester, the imidazolone product is proposed to
be formed.70

Scheme “-4. Reaction of aziridinones and thiourea to form imidazolones

-I

 

 

 

 

 

o f o 69NH2 0
/ N~ THF reflux r ,N
R2 R1 . R1/ No R1 Nl H‘ngz
O 0 O

sz/lL

RBKKIN L R2 —§NH N i:
N ‘ N ; R/

R? NH2 R’1 NH 1 Y

 

 

II.D.3 Synthetic routes to imidazolones: Oxazolone to imidazolone
Oxazolones have been used to produce imidazolones using a thiourea as
well (A, Scheme “-5).” 64' 65 Tepe and co-workers found that by treating
oxazolones with a thiouronium salt under basic conditions, they were able to
isolate 28% of the corresponding imidazolone.“ 65 Flygare and co-workers
developed a solid-phase synthesis of imidazolones using the same type of
chemistry giving yields ranging from 46-91% yields (B, Scheme "-5).49 The
oxazolone consisted of aromatic R1 substituents while the thiourea contained

alkyl and benzyl R2 substituents.49

55

Scheme "-5. lmidazolones using oxazolones and thioureas

 

 

N O
s’Bn H2N~</
A HN \
A HCI-HN NH2 ;

LiH, EtOH, reflux, \ NH

28% N
H o

8/

¥
7

DMF, EtONa, 100°C

0 O A R2 N O
B QR “” "“0 ~~</
N \ R2 OJ HN \
R1 46-91% R1
II.D.4 Synthetic routes to imidazolones: Oxidation of imidazoles
An oxidative rearrangement of imidazoles is also known to produce

imidazolones.39' 61'

67' 68' 73' 74 Lovely and co-workers used this type of
rearrangement recently in the racemic total synthesis of calcaridine A as the key
step in forming the quaternary imidazolone core. The reaction (A, Scheme ”-6)
utilized the appropriate benzyl groups in the R2 and R3 position and the yield was
around 93%, containing a mixture of diastereomers.39 The nucleobase guanine
has also been reported to undergo a similar rearrangement under oxidative
conditions?“ 67' 68' 73 Ye and co-workers report that DMDO can oxidize guanine
and produce a spiro imidazolone intermediate. Subsequent hydrolysis of the

spiro imidazolone affords a final quaternary imidazolone in 71% yield (B, Scheme

II-b).73

56

Scheme "-6. Oxidation of imidazoles to form imidazolones

\
\
\
\
O \

 

A N R2 go Ph N02 R3 R2
2
H2~~<’.1 = $0
R o
/ 3 MeOH, 40 C H2N N\

93%

o o
N N
HN DMDO HN
I \ /U\/Eo\
HZNN ,9 -——> x ,,>

 

 

H H2N N H
B
0 NH2 00
HN/ngo H20 HN/lggk'N
HNékfi 215:0 HNéLfi {3H

71%

II.D.5 Synthetic routes to imidazolones: Diketone rearrangement

Chalcones, a,B-unsaturated ketones, o,B—epoxy ketones and 1,2-
diketones have been reported to undergo a rearrangement with guanidine and
substituted guanidines to form imidazolones.4°'43' 62' 7‘ Darvas and co-workers
reported that a, B-unsaturated ketones (R1 and R2 = aryl or heteroaryl) rearrange
with guanidine to form imidazolones in yields ranging from 29-87% (A, Scheme
"-7).71 Dhar et al. reported that a similar reaction occurs with a, B-epoxy ketones
(R1 and R2 = aryl) under basic conditions with yields between 40-83% (B,
Scheme “-7).43 The rearrangement of 1,2-diketones with guanidines to form
imidazolones has been studied quite thoroughly.4°42' 62' 63' 75' 76 Nishimura
reported the formation of imidazolones from the reaction of diketones with

guanidine and 1, 1-disubstituted guanidines with yields ranging from 70-92% (C,

57

Scheme "-7).62 Like the previous examples, R1 and R2 were mostly aryl.62

However, when R1 = methyl the rearrangement failed to produce the imidazolone
and made a deep colored solution, which was thought to have gone through a
62. 75

completely different reaction.

Scheme "-7. lmidazolones from 1, 2-diketone and guanidine

 

 

 

O G 'd' H o HZNYN
/ uanI me, 2 2 _ / O
A R1/U\/\R2 o r HN
KOH, EtOH, 80 c
2n,29-87% R1 R2
0 H2N
M Guanidine ‘ YN O
3 R1 0 R2 NaH, THF, reflux ”N7:
4-12 h, 40-83% R, R
2
o H2N
MR2 Guanidine _ YN O
C R1 ROH,A,2-4h, ”N72;
0 70-92% R1

The proposed mechanism of the reaction between o,B-unsaturated
ketones, a,B-epoxy ketones and 1,2-diketones with guanidine is related to each
other based on the intermediates proposed to be formed throughout the
mechanism.“ 62' 71 It is proposed that when the starting material is an 0,8-
unsaturated ketone, the mechanism starts with the formation of an o,j3-epoxy
ketone using hydrogen peroxide. Next, a base mediated rearrangement occurs
from the epoxy ketone to afford a 1,2-diketone.77'81 Subsequently, the guanidine
then reacts with a diketone to produce intermediates that rearrange to the final

imidazolone (Scheme "-8).“ 62' 71

58

Scheme "-8. Proposed mechanism for diketone rearrangement

I- -I

 

 

 

 

 

 

o - o 1 o
/ H202 0 Base
0 O O O O 0 D
Guanidine
HZNYN )N\HZ NHZ
HN O N \N o N \N o
\ CO or O \ O

 

 

When the starting material is an (LB-epoxy ketone, the mechanism is
truncated and begins with the rearrangement of the epoxy ketone to a 1,2-
diketone and continues on as shown in Scheme “-8. Furthermore, when starting
with the 1,2-diketone, the rearrangement occurs after the guanidine forms the
heterocyclic intermediate with the diketone. In summary, whether the starting
material is an a,j3-unsaturated ketone, (LB-epoxy ketone or 1,2-diketone, the
reaction mechanism consists of similar intermediates and simply begins at
different points in the mechanism shown in Scheme ”-8.

Alternatively, it is proposed by Nishimura that when an alkyl group is used
in the rearrangement (R1 = methyl; see C, Scheme "-7) a different reaction
occurs (Scheme "-9).” 75 It is thought that instead of rearranging to the
imidazolone, the methyl group is deprotonated under the basic conditions leading
to intermediate "-10 (Scheme "-9). After addition of water to form "-12, it is

surmised that the imidazole is transformed into a diimidazole compound (ll-13)

59

similar to the Voges-Proskauer pigment, which is red in color.75 Although the
exact mechanism of this transformation is not fully understood, it offers an
explanation to the red color observed when Nishimura attempted the
rearrangement with a methyl substituent.

Scheme "-9. Alternative mechanism for diketone rearrangement

'- '7
R2 HO R2 Ho 9 R2
CH3 Aqueous H042 H

/ e / ——. N

N /N Base N /N N /
Y Y Y "-10

R1 R1 R1

R R
2 2 R2 R2
NWN H_ 7—1—
‘— N Ne
:>\/NH N\<N <— N\ NH Y
R1 "-13 E R1 "-11
(Red color)R

Voges-Proskauer pigment when "-12
R2 = Ph; R1 = N(CH3)(Benzyl)

 

 

 

I
O
69

Although the 1, 2-diketone rearrangement had not been successful with
substitutents other than aryl, benzyl or heteroaryl, I proposed that this
rearrangement would, at the very least, provide a relatively quick access to
analogs of the natural product (Ill-1). Optimistically speaking, I hypothesized that
there was a possibility that the rearrangement could even afford the correct
quaternary imidazolone scaffold needed for the natural product. As a result of the
natural product containing an indole group at the quaternary center it was
decided to first independently synthesize an analog with an indole and benzyl

substitution. Having the benzyl substituent already determined to be compatible

60

with the rearrangement, the success of the reaction to the corresponding
imidazolone would indicate that the indole moiety is also compatible.

l|.D.5.a Synthetic routes to imidazolones: Using diketone
rearrangement to produce imidazolone analogs of natural product

Scheme ”-10 outlines the synthetic route used to create the 1,2-diketone
with an indole and benzyl substitution pattern. An aldol reaction between 3-acetyl
indole and benzaldehyde under basic conditions yielded the o, B-unsaturated
ketone "-14 in a 65% yield.82' 83 Oxidation of “-14 to the corresponding 0, B-
epoxy ketone "-15 was completed using hydrogen peroxide in a 65% yield. The
a, B-epoxy ketone "-15 was transformed into the a, B-hydroxy ketone "-16 using
hydrogen and a palladium catalyst in a 89% yield, which was oxidized to the 1, 2-
diketone (II-17) using IBX in a 88% yield. The rearrangement of "-17 with
dimethyl guanidine hydrogen sulfate under basic conditions afforded the
corresponding quaternary imidazolone (II-18) in a moderate yield (53%). It is
important to note that the rearrangement with dimethyl guanidine was also
attempted with the a, B-unsaturated ketone "-14 as well as the a, B-epoxy ketone
"-15. However, in both cases, the reaction never produced the corresponding
imidazolone in any appreciable amount. It was found that starting with the
diketone efficiently reduced the amount of side products and conveniently
produced the desired quaternary imidazolone in a sufficient yield to where the
product would precipitate out of solution, making isolation and purification much

easier.

61

Scheme "-10. Synthesis of imidazolone "-18

o O/O

\ Benzaldehyde, KOH ‘

N Dioxane, reflux, 65%? O
H

 

II-14

IZ/

H202

NaOH, MeOH,
reflux, 65%

Pd/C, EtOH,
89%

   

IBX

 

NaH, THF, reflux,
53%

   

“-18

The success of this reaction was encouraging because it meant that the
indole was compatible with the rearrangement (the indole had not been tested in
any of the previous refs for the rearrangement). It was then decided that because
the indole was compatible, the benzyl group should be replaced with another
group that may allow for the formation of the 2-oxopropyl moiety needed for the
natural product. However, all attempts to synthesize 1,2-diketones that provided
a substituent that would give access to the 2—oxopropyl moiety needed for the

natural product (Ill-1) were fruitless. The two groups attempted in the

62

rearrangement to access the 2-oxopropyl moiety were an allyl group and a
ethylene glycol protected analog of the 2-oxopropyl moiety. Degradation of the
starting materials and/or formation of multiple by-products were observed
indicating that the diketone rearrangement may not be suitable for the synthesis
of the natural product.

It was discovered in the process of finding successful diketones for the
rearrangement that an isobutyl substituent is compatible to a small degree. The
synthesis of the appropriate diketone is shown in Scheme ”-11 and begins with a
Wittig reaction between N-tosyl-indole-3-carboxaldehyde and
isoamyltriphenylphosphine bromide to provide the cis alkene "-19 in an 89%
yield. The geometry of the alkene was confirmed by NOESY experiments
(Figure ll-15). Dihydroxylation of alkene "-19 using osmium tetroxide and NMO
led to diol "-20 in a 99% yield. An IBX oxidation was used to oxidize both
alcohols to the corresponding diketone "-21 in a 57% yield. The next step was to
deprotect the nitrogen of the indole group using potassium carbonate and
methanol to yield diketone "-22 in a 53% yield. Finally, the rearrangement of
diketone "-22 and dimethyl guanidine hydrogen sulfate under basic conditions
afforded the corresponding quaternary imidazolone "-23 in a low yield (19%,
Scheme ll-11). The reaction was not expected to give a high yield, if any, of the
corresponding imidazolone since alkyl groups had not been previously shown to
work with the rearrangement. To our delight, enough of the imidazolone was

produced to have a sample to test for Chk2 inhibition, if needed. Overall, the

63

diketone rearrangement allowed for the synthesis of a few quaternary
imidazolones that are analogs of the natural product (Ill-1).

Scheme "-11. Synthesis of imidazolone II-23

 

 

 

 

/k/\® Bre
aPPh3_
\
NaHMDS THF " 19
N ,48°Cmn, Ts
Ts 98%
0504, NMO
AcetoneNVater,
99%
ll
0 O H O OH
\ 4 IBX \
N “-21 EtOAc, 57% N ".20
TS TS
K2003
MeOH,
rt, 53W0 e
@NHHSO4 N
2 / N
0 Y
o \N/U\NH2 HN O
l
\ a l
N NaH, THF/ reflux, N
19
H ° H "-23
"-22

Since the diketone rearrangement was unsuccessful in the formation of an
imidazolone that could be applied to the total synthesis of the natural product,
other methods to prepare an imidazolone were evaluated. One of the most
common synthetic routes to producing imidazolones (non-quaternary and

quaternary) is through some type of ring closure.45'48' 51'55' 58'60' 65' 72 In the case of

64

quaternary imidazolones, the quaternary stereocenter is formed prior to a final
ring closure that forms the heterocyclic ring.
II.D.6 Synthetic routes to imidazolones: Dehydration reactions

The first type of ring closure used to produce imidazolones that will be
discussed is a cyclization through a dehydration reaction. Gillman et al. and Liu
et al. used the dehydration of an amide to produce various quaternary
imidazolones (Scheme ”42).” 72 Gillman synthesized amino amides and then
coupled the amine moiety to a carboxylic acid to form a diamide intermediate.
Under basic conditions, he was able to dehydrate one of the amides and produce
a quaternary imidazolone in yields ranging from 2-42°/o (Scheme ll-12).53 R1, R2
and R4 remained constant throughout the study (R1 and R2 were phenyl groups
and R4 was a hydrogen), while R3 was varied between aryl groups, heterocyclic
groups and a few alkyl groups.53

Scheme "-12. lmidazolones produced by dehydration

 

0 o
82 ,R4 1. NaOH, EtOH, rt, 2 h, R,
R, N 242% _ N284
HN H or 7 R1 N=<
>50 2.uw, 10-15 min, 77-98% R
R3 3

Liu used microwave technology to speed up the dehydration process and
produce imidazolones in 10-15 min,72 where Gillman’s reactions took 2 h.53 Liu
produced a diamide intermediate in a different fashion as Gillman and used the
energy of microwaves to effect the dehydration and produce quaternary
imidazolones (Scheme Il-12). R1 and R2 for the majority of the examples Liu

illustrated were connected through a cyclic alkane producing spiro-imidazolones

65

as the final product in yields ranging from 77-98%.72 There were a few cases
were R1 and R2 were alkyl and not tethered together and those yields were
between 85-95%. Additionally, R3 and R4 were varied between alkyl, phenyl and
benzyl and afforded imidazolones in yields between 77-98%. The yields reported
were determined by HPLC from LC-MS results of the reaction mixture. The
isolated yields of the imidazolones greatly decreased and were reported to be
between 36-78%.72

II.D.7 Synthetic routes to imidazolones: Cyclization with carbodiimide
intermediates

The main type of reactions used to produce imidazolones are cyclizations
where a carbodiimide intermediate is attacked internally or externally by a
nucleophile, which causes a subsequent formation of the imidazolone product.“
46' 54' 55' 6° The main differences in most of the examples that use this type of ring
closure is in the nucleophile used or if similar nucleophiles are used, such as
amines to produce a guanidine moiety, then the difference is in the formation of
that guanidine moiety.

Lee et al. and Lange et al. have used the dehydration of ureas with
triphenylphosphine/Bromine and Burgess’ reagent, respectively, to produce in
situ a carbodiimide intermediate, which is attacked by an external nucleophile.55'
6° Lee used alkyl and benzyl substituents for R2; phenyl and heteroaryl grignards
for the nucleophile (R3); and benzyl and phenyl substituents for R4 (Scheme II-
13). Yields ranged from 56-99% and reactions took about 2 h to complete. There
were no reports on producing any quaternary imidazolones.55 On the other hand,

Lange used a solid phase technique where R1 was connected to a solid support,

66

which helped in the isolation of the imidazolones (Scheme ll-13).60 Benzyl and
alkyl substituents were used for R2, while R3 tended to be various amines giving
products that were a variety of 2-aminoimidazolones. R4 was strictly confined to
aryl groups and the yields ranged from 47-93°/o.60

Scheme "-13. Cyclization from diimide: urea to imidazolone

- 2 0
,R4 1.dehydrating ,R4
R2 '1 agent _ O R2 1 -R1OH R2 N’R4
RI/OVN O 2. Nucleo. (R3) R1/ Wj/kN R3 solvent, rt, NZ<
O H O H R3

 

 

 

 

Drewry and co-workers used a thiourea instead of a urea to form the
guanidine moiety. A desulfurization of the thiourea using Mukaiyama’s reagent
led to a carbodiimide intermediate which was attacked by various amines to
produce 2-aminoimidazolones.46 Like Lange, Drewry used a solid support
connected at R1 allowing an easier isolation of the resulting imidazolones.
Furthermore, R2 was typically a benzyl group; R3 was a range of primary and
secondary amines; and R4 was primarily aryl groups (Scheme ll-14). The
reactions took longer than solution phase reactions with times around the 24 h
mark and yields ranged from 34-94%.46

Scheme “-14. Cyclization from diimide: thiourea to imidazolone

 

 

 

' ' 0
,R4 1.desulfurinzing ,R4
R2 HN agent R7- )NL -R1OH R2 N’R4
O k = /O ——» “1%
R1’ N S 2. amine (R3) RI ” R3 solvent, rt,
0 H 0 34-94% R3

Aza-Wittig reactions have also been used to produce a carbodiimide

intermediate, which ultimately gets attacked by a nucleophile to produce the

67

guanidine moiety (Scheme ll-15).45' 54 Villalgordo used the aza-Wittig reaction
between an azide and an isocyanate to form a highly reactive carbodiimide
intermediate. Subsequently, alkyl and benzyl amines, as well as, secondary
amines were used to attack the carbodiimide, form the guanidine and cyclize on
an ethyl ester to produce the 2-aminoimidazolone products. R2 was typically an
alkyl or aryl substituent, while R4 was always aryl substituents. The yields for this
reaction were between 72-89°/o and reaction times varied between 6-15 h.54 Ding
used a similar technique to produce the guanidine and yields for the 2-
aminoimidazolones ranged from 40-86%. Similar substituents to Villalgordo were
used, i.e. primary and secondary amines, Aryl R4 substituents and alkyl and
hydrogen at R245

Scheme "-15. Cyclization from diimide: azide to imidazolone

 

 

 

 

R2 1. PPh3, DCM F R2 N’th O
BOW/\N rt' 8 h = EtO i 'EtOH RZTQLN’R“
3 2. isocyanate (R4) \ll/kfi R3 solvent, rt, N=<
0 DCM, rt, 2 h 0 R3
3. amine (R3) ‘ ‘

Alternatively, Batey and co-workers used an opposite approach to those
described above. Batey utilized the reactivity of a thiourea to form a carbodiimide
intermediate in situ using HgCl2, which was subsequently attacked internally by
an amide, closing up to form an imidazolone (Scheme ”46).” 52 Multiple amino
acid amides were used as starting materials allowing R2 to correspond to the
appropriate amino acid. Peptides could be used in this reaction, allowing R1 to be
a long peptidic chain, as well as linked to a solid phase support ameliorating the

isolation process. R3 was typically aryl groups and yields ranged from 68-99%.47

68

Scheme "-16. Intramolecular cyclization: thiourea to imidazolone

 

 

 

_ 2 O
O O ,N.
H H HgCl 20’ R3 R2¥LWR1
R1\ )KKN N. ___2_, R1\ )KrN : _
1:1 \fl/ R3 n solvent, rt, N
R2 3 R2 18h [NH
_ _. R3
68-99%

Frutos and co—workers used a dehydration of a urea to mediate the
cyclization to an imidazolone product (Scheme ll-17).48 The urea was proposed
to be transformed into a carbodiimide intermediate, which was spontaneously
attacked intramolecularly by the secondary amide present in the molecule. As a
result, the dehydration mediated by triphenylphosphine and carbon tetrachloride
afforded the substituted imidazolone in a 81% yield.48 The quaternary substitution
pattern at position 5 consisted of R1 and R2 being a benzyl and methyl group,
while R3 was an aryl group.48

Scheme "-17. Intramolecular cyclization: urea to imidazolone

 

0 0
R2 R R
R1 N, 3 PPh;,, col4 22(1LN'R3
H = R
TEA, rt, 12 h, 1 —
HMro 81% N—(NH
HNVCOZEt EtOzC—/

II.D.8 Synthetic routes to imidazolones: Cyclization reactions with nitriles
A second type of ring closure to form imidazolones involves the
intramolecular cyclization of an amine or amide on a nitrile (Scheme ll-18).
Nagasawa and Kwon describe a facile synthesis of imidazolones using a ring
closure between an amine and nitrile using primarily heat although sometimes a
little acid was used (A, Scheme “48).” 59 The amine moiety was protected with

a carbamate group (Cbz) and the R2 and R3 substituents were varied between

69

hydrogen, alkyl, benzyl and phenyl. Quaternary imidazolones have been
successfully prepared and yields for all imidazolones produced using this method
ranged from 20-50%.58 The reactive intermediate for the reaction is not clear due
to IR bands observed at 2140 cm'1 and 2260 cm", depending on the pH.58 A
band at 2140 cm'1 hints at a carbodiimide intermediate, while the band at 2260
cm‘1 suggests more of a nitrile intermediate. The tautomeric relationship between
the acylcyanamide and carbodiimide makes it hard to specify which intermediate
is responsible for the cyclization. Regardless, the overall product in the reaction
is variously substituted 2-aminoimidazolones.

Lempert and co-workers report a similar cyclization where an amide
attacks a nitrile and forms an imidazolone product (B, Scheme ll-18).66 Lempert
successfully produces a variety of imidazolones, where R = alkyl, benzyl and
differently substituted aryl groups, in yields ranging from 90-98%. It is also
possible to remove the tert-butyl protecting group using aqueous HCI to provide
t.66

the free 2-aminoimidazolone as the final produc

Scheme "-18. lmidazolone through cyclization onto nitrile

0
R2 R3 H

3
A CBz\N)Kn/N\\ 1.solvent, A, 20-50%_ R Xk NH
' 2

I)

 

 

 

H o \N 2. H2/Pd HN
NH
0
5‘ 0 J4 P“ J<
B //N>(1LN TEA , PhXLN
N/ ph Ph H solvent, A, 90-98% N
R NH

70

ll.D.9 Syn
ryanogua

siren-me
seen-me
m fiat, m
ital lmic
fi'aazolo
were 789-2

Schem

 

 

II.D.9 Synthetic routes to imidazolones: Cyclization reactions with N-
cyanoguanidines

The last type of cyclization reaction to produce imidazolones involves a
rarely seen cyclization between an N-cyanoguanidine and a carboxylic acid
(Scheme lI-19).51 Garratt proposes a reaction mechanism where under acidic
conditions, a N-cyanoguanidine is attacked by a carboxylic acid to form the
seven-membered ring intermediate (ll-24). Subsequent rearrangement of the
seven-membered ring to the five-membered ring (imidazolone lI-25), followed by
elimination of the amide protecting group on N-3 results in the formation of the
final imidazolone product (Scheme ll-19).51 There were only a couple
imidazolones made through this route where R = ethyl and benzyl and the yields
were 78% and 79%, respectively.

Scheme "-19. lmidazolone through cyclization using N-cyanoguanidines

G

 

 

N o OTf
filing o HE’S:
N I TFA N/ff)
Bn‘N’LkN R Bnt A f0
l H N) N
l H R
”-24
OTf
N BgN/ C)>\‘~NH
Bn’ YN n \\ (:1 2
o
HN HN$O
R R ”-25

78%
To sum up thus far, many different types of reactions have been used to

create imidazolones. Many have used unique routes to obtain quaternary (5, 5-

71

disubstitution on the imidazolone ring) and non-quaternary imidazolones.
However, the entirety of the cyclization reactions that produced quaternary 2-
aminoimidazolones contained the quaternary stereocenter before the cyclization.
With this in mind, it was thought that the best route to obtain the desired
quaternary imidazolone for the natural product (Ill-1) would be to first set the
quaternary center and then cyclize to form the imidazolone.

II.E Synthesis of quaternary intermediates for the synthesis of the
natural product

Steglich and co-workers first described a rearrangement in 1975 where an
allyl ester of an N-acyl amino acid was converted into a quaternary oxazolone
using a variety of dehydrating reagents (Scheme ll-20).84' 85 The proposed
mechanism is shown below in Scheme "-20 and starts with a cyclodehydration of
an allyl ester to afford an oxazole intermediate, which upon a Claisen-type
reaction produces an oxazolone.“ 85 The oxazole intermediate had not been
isolated in the original reports, but is presumed based on the reaction of alkyl
esters of N-acyl amino acids.” 87 Two acyl groups were used for the
rearrangement, with R = phenyl and isopropyl, while R1 varied between aryl and
alkyl substituents. The substitution on the allyl group (R2 and R3) varied between
hydrogen, alkyl and aryl groups with yields for the quaternary oxazolone ranging
from 26-820/o.84’ 85 Like the Claisen rearrangement, it was observed that the
oxazole rearrangement produced only one diastereomer in cases where two
stereocenters were able to be formed.88 Steglich discusses an additional hetero-
Cope rearrangement that occurred (when R2 or R3 at H) after the formation of the

quaternary oxazolone to afford oxazolin-5-one products in yields ranging from

72

 

12-100%. Depending on the substitution of R2 and R3, the hetero-cope

rearrangement sometimes needed additional heating to complete.“ 85

Scheme "-20. Rearrangement developed by Steglich

r— I-

H 0 R2 R 0 n
RTNW/KOMRs Reagent F\/8~O’E
0 R1

WR3

_ _

 

 

oxazole intermediate

 

[Claisen
R3

R 0 o

R \ /
2 O O A hetero-Cope my
I R=phenyl,alkyl R‘, R2R3

R N R oxazolone
12-100% 26-82%

Steglich’s oxazole rearrangement is a useful tool to create a quaternary
stereocenter and has been used to produce a variety of different compounds.”97
Haufe et al. used the oxazole rearrangement to lead to 4-fluoropyridines
(Scheme ll-21).97 Fluoro allyl esters of N-acyl amino acids were cyclodehydrated
using triphenylphosphine and carbon tetrachloride to produce the corresponding
quaternary oxazolones in yields ranging from 98-100%. Subsequently, Haufe
exposed the oxazolones to decahydronaphthalene and air for 20 h at 160°C

which transformed the oxazolones to the corresponding 4-fluoropyridines in

yields between 21-55% (Scheme ll-21).97

73

Sct

 

Bu.
oxazole l
"alltl‘tls e

Vifime

“a l», ,
~eir|l€e

“ta
.‘Vrme

 

Scheme "-21. Rearrangement leading to 4-fluoropyridines
R

0 Ph F c
/U\ )YO Ph3P,CCI4,TEA_ WNT o
R Q CH3CN,rt,16h,
0 R1

98-100% Ph

 

 

Ph F 0
R1 "r -co2 o
F R R1 R

decahydronaphthalene
air, 160°C, 20 h

l
Ph F F
/ ——’ —"
\
R1 R Ph N R Ph N/ R

21 -55%

 

Burger et al. described a unique method for the formation of the allyl
oxazole intermediate by coupling the fluorinated oxazole starting material with
various alcohols (Scheme ll-22).89'91 Subsequently, the oxazole rearrangement
produced a variety of a-trifiuoromethyl q-amino acids (Scheme ll-22) in yields
between 40-69%.90 Interestingly, Burger also identified that the allyl oxazole
intermediate could be spectroscopically characterized when R = (CH3)3Si-
CH=CH-CH2OH. This is the first example of the allylic oxazole intermediate being
observable spectroscopically.90 Additionally, Burger showed that benzyl alcohols
and hydroxymethylheterocycles, such as 2-hydroxymethylthiophene, also are
compatible in the oxazole rearrangement.”91 Furthermore, Burger along with
Krantz, illustrated that propargyl alcohols could be used in place of allylic

alcohols to produce q-allenic a-amino acids.” 90' 92- 93

74

Scheme "-22. Rearrangement affording a-trifluoromethyl a-amino acids

 

 

 

CF3 R1 CF3/\)\
NiF HO/VRR NaH or KOH “Ito/J
ph/ko\ Ph/ko\
oxazole
F3C Ph
CO H O
NHCOCSHS N R,
R R1
F3C R \
40-69%

The oxazole rearrangement has been utilized in the synthesis of a-benzyl
v-lactam derivatives, a-benzyl 6-Iactam derivatives and d-benzylproline

derivatives (Scheme "-23, "-26, "-27, "-28, respectively).96 Holladay and co-

workers used phosgene to carry out the oxazole rearrangement to afford a

quaternary oxazolone that is quite unstable. By treating the oxazolone formed in

situ with ammonium hydroxide, a primary amide was formed. Subsequent

treatment of the amide with a combination of reagents led to each of the desired

heterocycles (Scheme ll-23).96

Scheme “-23. Rearrangement leading to lI-26-ll-28

 

 

Ph Ph /
0 o
F CAN/KNOW 1'C'ZCO'TEA : JL NH2 —> lI-26-il-28
3 H 2. NH4OH, 70% F30 fl '
0 0
Ph
NH
RHN
o NH2
"-26 "-27 “-23

 

 

 

75

Colombo et al. applied the oxazole rearrangement to the synthesis of C-
glycosyl a-amino acids, and q-D-C-mannosyl-(R)-a|anine.94' 95 It was found that
the cyclodehydration/rearrangement reaction afforded a 2.6:1 mixture of two
diastereomeric oxazolones (Scheme ll-24). It was proposed that the major
isomer is produced through a boat-like transition state during the oxazole
rearrangement possibly due to steric interactions that would accompany a chair-
Iike transition state for this specific case.95

Scheme "-24. Rearrangement to synthesize C-glycosyl a-amino acids

*0 )VO
0 o 0
/ Ph3P, ccu, TEA _

0

)fi/ rt, 12 h, 86%
N
\
/
o /N 04
>’ Ph
Ph

The majority of the examples of the oxazole rearrangement only produced

  

 

 

quaternary oxazolones with one stereocenter.“ 85' 91' 96' 97 However, it was
reported that a mixture of diastereomers were observed in the examples that had

the potential to produce them, as in the case of Burger and Krantz with the

89, 90, 92, 93 94, 95 In

allenic oxazolones and Colombo with the glycosyl oxazolones.
the case of the allenic oxazolones, it was described that having a substitution on
the methylene of the propargyl alcohol could influence the diastereomeric ratio
depending on the size of the substituent.88 Moreover, in the glycosyl oxazolone

cases, the stereochemistry of the sugar moiety helps dictate the stereochemical

outcome of the rearrangement.“ 95

76

We proposed that this oxazole rearrangement is a unique and efficient
way to create a quaternary center and would be excellent to utilize the
rearrangement in the synthesis of the natural product (III-1). Furthermore, it
seemed to augment and highlight the theme of our group’s work dealing with the
development of oxazolone chemistry?8 The oxazole rearrangement was
attempted with various N-acyl groups as shown in Table ”-1. The dehydrating
reagents varied depending on the N-acyl group used.

Trifluoroacetic anhydride worked the best when R = phenyl (Table ”-1)
affording the oxazolone (ll-36) in a 76% yield. POC|3 was the dehydrating
reagent used when R = methyl and dimethylamino to give the quaternary
oxazolones "-37 and "-38 in a 69% and 86% yield, respectively. However, when
the N-acyl derivative was used, a different isomer was observed as the product
(Table ”-1). The product isolated was actually the lactone (lsomer B, Table "-1)
in which a hetero-Cope rearrangement occurred after the initial Claisen
rearrangement, which had been seen before in Steglich’s initial study.85

The physical evidence that supports this structural determination comes
from the absorptions seen in the IR for oxazolone "-37 compared to those
reported for the oxazolone product (lsomer A) and lactone (lsomer B). For "-37,
the signals observed in the carbonyl region are at 1779 cm'1 and 1627 cm'1,
which match very closely the reported lR signals for the lactone product (1780-
1770 cm" and 1645-1610 cm'1)84' 85. The IR signals associated with the
oxazolone product tend to be in the range of 1850-1810 cm'1 and 1660-1650 cm‘

1.84' 85 It is also noteworthy to mention that when the dimethylamino urea (II-31)

77

was used in the rearrangement, the resulting oxazolone was so labile that
isolation could only occur after treatment with methanol. This resulted in the
isolation of the quaternary ring-opened methyl ester (Structure C, Table ”-1).

Electron withdrawing acyl groups attached to the nitrogen, such as the
trifiuoromethyl acetyl group and 2-ethoxy-2-oxoacetyl group (compounds "-32
and "-33, respectively) require a different dehydrating reagent altogether. TFAA,
POCI3, PCI3, PC|5 and PPhg/CCI4 have all been evaluated and failed to effect the
rearrangement. It was found that phosgene was the only reagent that could
produce the corresponding quaternary products (ll-39 and "-40, Table ”-1) in
yields of 75 and 55%, respectively. However, due to the lability of the resulting
oxazolones, they would readily ring-open at the carbonyl carbon even on silica
gel. As a result, treatment of the quaternary oxazolones immediately with a
nucleophile was necessary to be able to isolate the corresponding ring-opened
products (Structure C, Table "-1).

A carbonate and pivaloyl group were also attempted in the rearrangement
(entries "-34 and II-35) but failed to produce the quaternary oxazolone using any
of the previously described dehydrating reagents. It was observed that when a
carbonate was used as the acyl group, the reaction gave back starting material
after heating for 24 h with each dehydrating reagent. Conversely, when the
pivaloyl group was used as the acyl group, the starting material seemed to
decompose in almost every reaction condition, presumably due to the acidic

nature of the reagents.

78

Table "-1. Oxazole rearrangement with various N-acyl groups

 

 

 

 

 

 

 

 

 

 

 

 

 

o ' R
TFAA, POCI3, Y0 R
| O
H 0 55-86% R. N‘
R'= N-tosyl indole A B R'
ll-29-II-35
|so| ted Dehydrat. 0
Compound R Strui'ture Reagent Yield Product R—( O
HN OMe
"-29 A TFAA 76% "-36 R-
c
"-30 )1, B POC|3 69% 11.37
"-31 \N)" c POCI3 86% "-38
11.32 F3C5‘t c cocr2 75% "-39
EtO
11.33 c cocr2 55% "-40
o
"-34 C|3CAO/1LL NR NA NA NA
"-35 NA NA NA NA

As stated before, the majority of the techniques used to create quaternary
imidazolones involved the synthesis of the stereocenter prior to heterocycle

formation. This technique was thought to be employed in the synthesis of the

natural product. Therefore, since the quaternary center has now been achieved

through the oxazole rearrangement, the next step was to form the imidazolone
heterocycle.

The quaternary oxazolone products (ll-36 and ll-39) were subjected to
different reaction conditions to open the quaternary oxazolone and remove the

resulting acyl group on the nitrogen to move ahead in the synthesis of the natural

79

 

product. However, basic reaction conditions usually only opened the oxazolone
and/or removed the tosyl protecting group, while acidic reaction conditions
resulted in degradation of the starting material and/or formation of multiple by-
products. Surprisingly, attempts to manipulate the urea quaternary product (II-38)
provided some interesting results. When treated with reagents intended to
activate the urea carbonyl and subsequently substitute with an aminegg, an
unexpected result was observed (Scheme ll-25). Instead of forming the intended
guanidine, the final product corresponded to the quaternary hydantoin "-41 which
was isolated in good yields (71 %).

Scheme "-25. Unexpected reaction producing hydantoin II-41

 

Bn\ 0

|
N
/ e

O \ NH
I OM BOP DBU /N—<\N OMe HN
0 ' 96* 0
BnNHz, CH3CN, \
\ reflux, 16 h \ N
N N
+3

 

. “-38 +3 "-41
Ts

 

 

71%

 

An even more unusual reaction was observed when the amine was left out
of the reaction and water was added instead. it was believed that the unexpected
reaction might have proceeded through an isocyanate intermediate and the water
was intended to hydrolyze the intermediate and upon basic workup, afford an
amine (Scheme ll-26). However, a quaternary hydantoin with an incorporation of

DBU was observed ih a 55% yield (Scheme ll-26).

80

Scheme "-26. Unexpected reaction producing hydantoin Il-42

 

 

 

l
/ N O OMe

o

”N o BOP, DBU V O:<N

A:
\ CH3CN/H20, N

N reflux, 16h H \

' "'38 ll-42

Ts N\
Ts

55%

 

 

 

It has been reported in multiple accounts that use the oxazole
rearrangement to make quaternary products that the largest downfall in the
reaction is the inability to remove the acyl group (of the ring-opened oxazolone)
after the rearrangement.92'93"100 This main disadvantage was the driving force to
create a new rearrangement in which the product could be more amenable
toward synthesizing the natural product. Furthermore, the formation of the
unexpected quaternary hydantoins sparked an interesting idea to create a
method where an unprotected quaternary hydantoin is formed after an oxazole
rearrangement. This would allow access to the imidazolone scaffold through
known chemistry.“ 50' 56' 57' ‘01
ll.F Development of a novel EDCI-mediated oxazole rearrangement

The restraint of the traditional oxazole rearrangement was the stimulus for
developing a novel reaction that incorporates aspects of the oxazole
rearrangement to afford a quaternary product capable of leading to the 5,5-

(disubstituted)-imidazol-4-one scaffold of the natural product, which is the main

goal of my project. It was imagined that forming a 5,5-(aryl, allyl) hydantoin after

81

an oxazole rearrangement85

would be novel and ideal for the synthesis of the
indole alkaloid (Ill-1) for three reasons. First, the hydantoin intermediate would
allow access to the imidazolone scaffold using known chemistry either directly to
the imidazolone50 or through a thiohydantoin” 44' 56' 57' 101 (Scheme ll-27).
Gadwood and co—workers described a method in which a hydantoin was
converted into the imidazolone using Meerwein’s reagent to alkylate the carbonyl
in the 2 position followed by replacement of the newly formed alkoxy group with

an amine (Path 1, Scheme ll-27).50

Scheme "-27. Routes from hydantoin to imidazolone

RZNYN
O
tBuOOH HN .

 

 

NHR
2 2 R"
H
(Di/N Law R t YN 3 NHR2, reflux RZN N
o ——9—. 2 we 0
HN , HN HN
.. R R' R
R R" R"
4
1.Meerwein's Rgt. \ R2N N
1. I, /
2.NHR2 Me Base: Y O
2. NHR2 HN R,
R"
RZNYN
HN7Z:O
R.

R"

Alternatively, a thiohydantoin can be transformed into an imidazolone
using a variety of methods.” 44' 56' 57' 101 Ireland used an oxidation of a
thiohydantoin followed by treatment of an amine to afford an imidazolone19 (Path
2, Scheme ll-27), while Kiec—Kononowicz and co-workers simply refluxed a

thiohydantoin in the presence of the desired amine to afford imidazolones (Path

82

3, Scheme ll-27).57 However, the most widely used method for transforming a
thiohydantoin into an imidazolone is to alkylate the thiocarbonyl first and then
replace it with the desired amine as illustrated by Overmanm‘, Khodair56 and
lverson44 (Path 4, Scheme ll-27).

The second advantage of forming a 5,5-(aryl, allyl) hydantoin after an
oxazole rearrangement would be that it could act as a synthetic handle with
which the carbonyl in the 2-position of the hydantoin could be replaced with
potentially any amine, giving access to a number of analogs of the natural
product, if desired. Finally, the allyl group was thought to give access to the keto
functionality found in the indole alkaloid. To achieve the formation of a quaternary
hydantoin it was thought to combine certain aspects of the oxazole
rearrangement and the cyclization reaction Batey"'7 used to create imidazolones.
In the end, a new reaction could be developed giving access to uniquely
substituted quaternary hydantoins.

We chose to implement a thiourea moiety in place of the N-acyl group
from the original oxazole rearrangement and use a desulfurizing agent to achieve
a quaternary oxazolone product that could be manipulated into the corresponding
quaternary hydantoin we desired. Overall, a novel rearrangement (Scheme ll-28)
that gives access to a unique marine alkaloid scaffold was developed.102 The
one-pot reaction converts a thiourea into a 5,5-(aryl, allyl) oxazolone through a
new twist on the oxazole rearrangement, which upon further treatment with

sodium methoxide yields a 5,5-(aryl, allyl) hydantoin. As a result, this hydantoin is

83

able to offer a concise synthetic route to molecules such as the natural product,
indole alkaloid Ill-1.

Scheme "-28. New rearrangement applied to the synthesis of Ill-1

 

H
H 0 0 N
S N
Y \l/lLO/Wl/ 1.EDCI, TEA, DCM> HN O
. 2. NaOMe (excess) .
R,NH R R

 

Natural Product (Ill-1)

ll.F.1 Scope of rearrangement

It is hypothesized that the new rearrangement initiates after the thiourea is
activated and converted into a carbodiimide, which undergoes a 5-exo—dig
cyclization to form an oxazole intermediate (based on data discussed in the
mechanism section). There are a variety of reagents that have been reported to
transform a thiourea into a carbodiimide intermediate and subsequently allow for
attack by an external or internal nucleophile.1°3'107 We screened the typical
reagents chosen for this type of chemistry including HgCl2,1°4' 108' "’9
Mukaiyama’s reagent,1°7' “0 and EDCI105' "1 and found that the success of the
reaction was highly dependent on the reagent choice. Mercuric chloride did
provide a reasonable yield of the quaternary hydantoin (49%), while
Mukaiyama’s reagent failed to produce any desired hydantoin. EDCI was the
most effective at yielding the quaternary hydantoin in good yields (70%).
Furthermore, the hazard of mercury waste removal and the ease of workup with

EDCI solidified the reason to use EDCI as the reactant for the rest of the study. A

more cost efficient carbodiimide, DCC, was also attempted in the rearrangement

84

but failed to provide any reasonable yields (<5%). Polymer supported EDCI was
also used although longer reaction times were necessary. A brief solvent screen
revealed that dichloromethane provided the best results (70%), while solvents
such as acetonitrile, benzene, tetrahydrofuran, and dichloroethane all yielded
102

poorer results (50%, 30%, 47%, and 12%, respectively).

Scheme "-29. General scheme for novel rearrangement

5 Kg: R' 80% 0 H
, NaOMe
Y O EDCI ”YO (excess) Y O
0 NH R HN
TEA R R'

The first structural aspect we investigated in the rearrangement was the
different groups compatible at the R position (Scheme ll-29). The different
thiourea starting materials were synthesized through standard amino acid

chemistry (Scheme ll-30). The Boc protected amino acids (ll-43-lI-48) (R =

u 114

methyl,112 benzyl,113 pheny, l "5

pOMe-pheny, | “6

pF-pheny ,

and napthyl,117

respectively) were esterified with the appropriate allylic alcohol using
dicyclohexylcarbodiimide (DCC). Deprotection of the Boc group with a mixture of
TFA and DCM (1:1) led to the TFA salt of the amino allylic esters (ll-56-Il-62).
Upon treatment of the amine salts with ethyl isothiocyanatoformate under basic

conditions the desired thioureas (ll-63-Il-69) were produced.102

85

Scheme "-30. General synthesis of thioureas ll-63-Il-69

 

 

 

R = Me; R' = allyl, "-63

R = Bn; R' = allyl, "-64

R = Ph; R' = allyl, "-65

R = pOMe-Ph; R' = allyl, “-66
R = pF-Ph; R' = allyl, "-67

R = Napth.; R' = allyl, "-68

n O H O '
Boc’ OH R'OH. DCC V Boc’N\(u\O’R
R DMAP, DCM, rt R
R = Me, "-43 R = Me; R' = allyl, "-49
R = Bn, "-44 R = Bn; R' = allyl, "-50
R = Ph, "-45 R = Ph; R' = allyl, "-51
R = pOMe-Ph. "-46 R = pOMe-Ph; R' = allyl, "-52
R = pF-Ph, "-47 R = pF-Ph; R' = allyl, "-53
R = Napth.. "-48 R = Napth.; R' = allyl, "-54
R = Ph; R' = 1,1-disub. allyl, ll-55
TFA/DCM
(1 :1 ), rt
0 O
as ii H R- JL 'TFA O
\n, T \l/‘LO’ ‘L EtO NCS HzN 0’ R'
o s R TEA, DCM, rt R

R = Me; R' = allyl, "-56

R = Bn; R' = allyl, "-57

R = Ph; R' = allyl, "-58

R = pOMe-Ph; R' = allyl, "-59
R = pF-Ph; R' = allyl, "-60

R = Napth.; R' = allyl, "-61
R = Ph; R' = 1,1-disub. allyl, "-62

R = Ph; R' = 1,1-disub. allyl, "-69
The synthesis of thiourea (ll-75, when R = N-tosylindole) followed a

different synthetic pathway (Scheme ll-31). 2-(1H-indol-3-yl)-2-oxoacetyl

chloridem' “9

(ll-70) was treated with allyl alcohol in CH3CN to yield keto-ester
(ll-71) in a 95% yield. Protection of the indolic nitrogen with p-toluene sulfonyl
chloride under basic conditions afforded keto-ester (ll-72). The transformation of
the ketone functional group in (ll-72) to oxime (ll-73) was completed by refluxing
the starting ketone with hydroxyl amine and pyridine in dioxane. The resulting
oxime was a mixture of isomers and both were reduced to amine (ll-74) using
zinc and acetic acid and subsequently reacted with TFA to produce the TFA salt.

Finally, treatment of the amine salt with ethyl isothiocyanatoformate led to the

desired thiourea (ll-75).102

86

Scheme "-31. Synthesis of thiourea ll-75

 

 

 

N CH3CN, 95% N “41
H 11.70 H
TsCl,DlPEA
DMAP, DCM, 86%
H0vN\ o O
\ o < NHZOH-HZOmyr. \ O
N "73 Dioxane/H2O, 93% N "-72
‘rs ' ‘Ts
1.Zn,AcOH
2. TFA, 85%
EtO
H N o ‘—
2 EtO NOS 0 8%NH 0
\ O TEA, DCM, 89% \ o
N, "-74 N "-75
Ts Ts

The results of the rearrangement with the various R groups are illustrated
in Table “-2. It is shown that an alkyl group such as a methyl provided no
product, while a benzyl only performed slightly better by producing the
quaternary hydantoin (ll-76) in low yields (19%). When R = aryl (ll-65-lI-68), the
rearrangement occurred in good yields (Table "-2) except for when R = napthyl,
which could be attributed to the steric bulk of the napthyl group. The
rearrangement was also successful using a heterocycle in the R position with the

N-tosyl indole thiourea (ll-75) providing the corresponding hydantoin (ll-81) in

87

good yields. This particular example is important for the synthesis of indole
alkaloid (Ill-1).102

Table "-2. Rearrangement containing various R groups

 

 

 

H H O o H
VOTNTNfl/Ko/m 1.EDCI,TEA,DCM,O°C : Ifi
O S R 2. NaOMe, MeOH,rt R
ll-63-Il-68, "-75 \
ll-76-lI-81
entry R Yield (%) Product
"-63 Me 0 NA
"-64 Bn 19 "-76
"-65 Ph 7o "-77
ll-66 p-OMe-Ph 67 "-78
ll-67 p-F-Ph 57 "-79
"-68 Napth 31 "-80
"-75 N-tosyl-lndole 7o "-81

It was observed that when R = methyl and benzyl, there appeared to be
the formation of byproducts. It is surmised that a potential reason for the
formation of byproducts and low (if any) yield of product could be an elimination
of the activated thiourea affording an intermediate that could potentially go
through side reactions (Scheme Il-32). Likewise, when the rearrangement was
first attempted with R = indole (no nitrogen protection), the rearrangement failed
to give the desired quaternary hydantoin and produced multiple byproducts. As
shown in Scheme ”-32, it is postulated that a similar elimination reaction could
occur when R = indole giving rise to possible side reactions and byproducts.
Protection of the indole nitrogen with an electron-withdrawing group (tosyl)

appeared to hinder the side reactions and allow the desired rearrangement to

88

occur. Overall, it is believed that the reaction works well under conditions where
a potential elimination reaction cannot occur and when the aromatic oxazole

intermediate (discussed in the mechanism section) is stabilized by aromatic R

 

 

groups.
Scheme "-32. Potential byproduct pathway
H
s N EDC|\ H o 0
Y O/\|l EDCI 693‘ N o
A 0 NH R ———» UT” 3 /\|| _. o
TEA 0 NH H I |
OEt TB R ‘3. R
When R = methyl, benzyl _ B _
o H COZEt O n 3323 o
(\O N\n/NH EDCI (\O VY O/\l
B I S TEA Q (3‘9 I l
\ j EDCI \ /
NH '3’ N
._ H .—

 

 

We then examined the different allyl groups compatible with the
rearrangement. The synthesis of the thiourea with a 1, 1-disubstituted allylic
moiety (ll-69) followed the synthetic route that is outlined in Scheme "-30.
However, when trisubstituted allylic thioureas were synthesized using the
corresponding trisubstituted allylic alcohols, a different route was adopted. It was
found that the higher substituted allylic esters would decompose to the
corresponding carboxylic acid when treated with TFA.120 As a result, instead of
using the Boc protecting group, the Fmoc group was used for the synthesis of

those thioureas (Scheme ll-33). Using Fmoc phenyl glycine121

as the starting
material, the allylic esters (II-83, ll-84) were produced under the same conditions

as before using DCC and DMAP. Deprotection of the Fmoc group from the amine

89

was completed using piperidine to afford the free amines (ll-85, ll-86). Upon
treatment of the amines with ethyl isothiocyanatoformate, the corresponding

thioureas (II-87, II-88) were formed in moderate yields.102
Scheme "-33. Synthesis of higher substituted allylic thioureas

H O 0

N H R
Fmoc OH ROH, DCC t Fmoc O
DCM, DMAP, rt

"-82

 

, "-83

m

Piperidine,
CH3CN
0°C to rt

 

R=m,l|87 Rszl-85
R: M , "-88 R: M , "-86

The rearrangement was successful with all of the differently substituted
allylic esters that were synthesized (Table "-3). When the 1, 1-disubstituted allyl
group was used (entry ll-69) the rearrangement gave about the same yield of
corresponding quaternary hydantoin as the allyl group (entry II-65). Entry "-87,
which had a 1, 1, 2~trisubstitution pattern on the allylic group was also as
successful as the other entries. However, unlike the two previous entries, the

product from the rearrangement (ll-90) contained two stereocenters allowing for

90

db.

il'

.‘v

we.

u'J

1.,

J v"-

-

I4

the formation of diastereomers. It was observed by 1H NMR and 13c NMR that
two unseparatable diastereomers were formed from the reaction in about a 121.3
ratio. The last entry in Table "-3 includes an allyl moiety containing a 1, 2, 2-
trisubstitution pattern. It is hypothesized that the lower yield resulting from this
rearrangement could be attributed to the steric bulk of this particular trisubstituted
allyl group. An electron withdrawing group (ethyl carbamate) on the thiourea was
only used since it was previously found that electron withdrawing groups
accelerate the reaction of a thiourea and desulfurizing reagent and increase the
102, 122

reactivity of the corresponding carbodiimide toward a nucleophile.

Table "-3. Rearrangement containing various allyl groups

0 H H O CY“
\/ \n/Nm/NEERle 1.EDCl,TEA,DCM,0°C HN o
o s T

 

 

 

2. NaOMe, MeOH, rt R1
"-65. ll-69, "-87, "-88 "-77, "-89
. o H
entry R1 Yield (%) Product H.,N
o
HN
"-65 m 7o "-75 O .
"-69 W 66 "-89 "-90
H
CY”
"-87 m 66 "-90 o
HN
\
"-88 q 47 "-91
"-91

"£2 Proposed reaction mechanism
The proposed mechanism is illustrated below in Scheme ”-34. The first

step is thought to be the transformation of the starting thiourea to a carbodiimide

91

rzerlledlate (ll-92
artermentally, 'lt'
Sereral reports sr
sch as EDCl, Hg
'lermediale.‘6' 1
aiacled by an e
rill various 5le
:ferenl heleroc
rlcleoph'lle on
Batey and co-“
tlaloccured aft
31 all intern;
resullur'lzallon
arlllolm‘ldazol
'easonab‘ll C(
image” of a
PEHCllVe, lhu
mobselllable
leactlv'lty‘ lh E
the GarbOdlir

34)?”

intermediate (II-92). Although this intermediate has not been isolated or observed
experimentally, it is presumed to be formed based on previous studies.122
Several reports suggest that thioureas undergo a desulfurization with reagents
such as EDCI, HgCl2, Mukaiyama’s reagent, and others to afford a carbodiimide

122' ‘23 Subsequently, this transient carbodiimide could be

intermediate.46'
attacked by an external nucleophile, such as an amine, to produce guanidines
with various substitutions.1°3'1°6' 1084‘" 122' 123 Furthermore, cyclizations to form
different heterocycles have also been shown to occur through an attack of a
nucleophile on a carbodiimide formed from the desulfurization of a thiourea.
Batey and co—workers produced various iminohydantoins through a cyclization
that occured after a carbodiimide intermediate, formed using HgCl2, was attacked
by an internal amide.47 Additionally, Drewry and co-workers utilized a
desulfurization of a thiourea with Mukaiyama’s reagent to form 2-
aminoimidazolinones.46 These examples serve as supportive evidence to
reasonably conclude that the initial step in the rearrangement involves the
formation of a carbodiimide intermediate. The carbodiimide is believed to be very
reactive, thus giving reason to a short lifespan and consequently being
unobservable spectroscopically. As a consequence of the carbodiimide’s high
reactivity, the carbonyl of the ester is believed to be nucleophilic enough to attack

the carbodiimide, in a similar fashion as the amide in the report by Batey,47 to

cause a cyclization reaction affording an oxazole intermediate (ll-93) (Scheme l|-

34)_102

92

Scheme “-34. Proposed mechanism of EDCI-mediated rearrangement

H H O O>_ O
EtO N N EDCI N==N
\ll/ \ll/ \l/lLO/Y —’ EtO j/lLo/Y
o s R R

carbodiimide intermediate

 

 

 

(II-92)
EtO PEtoH _
O
0 _ \n/N “‘Ifi/W
NaOMe R\'/

(excess)
oxazolone ("_94) oxazole intermediate (II-93)

 

ll

0
H H
N N OEt H
EtO\n/ \ll/ OMe iffo 0 N
O O R HN HN 0
"-95 R

"-96

 

After oxazole (ll-93) undergoes a Claisen rearrangement, oxazolone

intermediate (ll-94) is formed (Scheme Il-34). This intermediate was isolated and
characterized with 1HNMR, 13CNMR and IR, which showed the characteristic
absorption bands for the oxazolone (see experimental section for spectral
information). It is proposed that from the oxazolone intermediate (ll-94), the
mechanism of the novel rearrangement continues with the nucleophilic opening
of the oxazolone by sodium methoxide to yield urea (ll-95). As a consequence of
having excess sodium methoxide in the reaction mixture, urea (ll-95) is

deprotonated and cyclizes to form hydantoin (ll-96), which is further modified by

93

sodium methoxide to yield the final quaternary hydantoin upon removal of the
carbamate group (Scheme ll-34).

The step in which the quaternary stereocenter was formed illustrates an
oxazole rearrangement first described by Steglich and co-workers.84' 85 Steglich
discusses an additional hetero-Cope rearrangement that occurs with the
oxazolone products to form oxazolin-5-ones, depending on the substitution on
the allyl ester.“ 85 However, it is noteworthy to mention that this additional
hetero-Cope rearrangement was not observed in our rearrangement.

Overall, a novel rearrangement was developed that allows access to the
scaffold needed for the synthesis of the natural product. The success of the N-
tosyl indole and the 1, 1-disubstituted allylic moiety in the rearrangement ensures
that the quaternary hydantoin formed from the pairing of those two structural
features will lead to an intermediate capable of transforming into the natural
product (Ill-1). The next chapter will discuss, in depth, the marine organism from
which the natural product comes from as well as the first total synthesis of the

natural product and two analogs.

94

ll.G General experimental information

Reactions were carried out in flame-dried glassware under nitrogen
atmosphere. All reactions were magnetically stirred and monitored by TLC with
0.25 pm pre-coated silica gel plates using either UV light or iodine to visualize the
compounds. Column chromatography was carried out on Silica Gel 60 (230-400
mesh) supplied by EM Science. Yields refer to chromatographically and
spectroscopically pure compounds unless otherwise noted. Infrared spectra
were recorded on a Nicolet IR/42 spectrometer. 1H and 13C NMR spectra were
recorded on a Varian Unity Plus-500 spectrometer or a Varian lnova-300, as
noted in the experimental for each compound. Chemical shifts are reported
relative to the residue peaks of the solvent (CDCI3: 7.24 ppm for 1H and 77.0
ppm for 13C) (Acetone-d5: 2.04 ppm for 1H and 29.8 ppm for 13C) (DMSO-d5:
2.49 ppm for 1H and 39.5 ppm for 13C). The following abbreviations are used to
denote the multiplicities: s = singlet, d = doublet, dd = doublet of doublets, t =
triplet, and m = multiplet. HRMS were obtained with a Micromass Q-ToF Ultima
API LC-MS/MS mass spectrometer. Elemental analysis data were obtained on a
Perkin Elmer 2400 Series II CHNS/O analyzer. Purity of compounds, whose
elemental analyses were above the ACS tolerated 0.4% deviation, were
confirmed by 1H NMR and 13C NMR. Melting points were obtained using an
Electrothermal® capillary melting point apparatus and are uncorrected.
Reagents and solvents were purchased from commercial suppliers and used

without further purification. Anhydrous methylene chloride and toluene were

95

dispensed from a delivery system which passes the solvents through a column
packed with dry neutral alumina.
ll.H Experimental procedures and characterization

(ll-14).124 To a flame dried 25 mL round bottom flask was added dry
dioxane (10.0 mL) and KOH (0.352 g, 6.29 mmol). Then acetyl indole (1.00 g,
6.29 mmol) and benzaldehyde (1.27 mL, 12.6 mmol) were added and the mixture
refluxed for 24 h under nitrogen. The precipitate was filtered off and recrystallized
from EtOH to give yellow solid crystals. Yield (1.00 g, 65.0%). 1H NMR (500
MHz), d-Acetone: 6 7.20-7.30 (m, 2H), 7.38-7.45 (m, 3H), 7.50-7.55 (m, 1H),
7.75-7.80 (m, 4H), 8.50 (m, 1H), 8.57 (s, 1H), 11.00 (bs, 1H); 13C NMR (125
MHz), d-Acetone: 8 112.6, 119.4, 122.7, 123.2, 124.1, 125.3, 127.2, 129.0,
129.6, 130.4, 133.9, 136.6, 138.0, 140.6, 184.5. ms: calculated for C17H13NO
(M+) = 247.1 and found (M+) = 247.1.

(ll-15). To a 50 mL round bottom flask was added "-14 (0.700 g, 2.83
mmol) and MeOH (20.0 mL). Then NaOH (1.95 mL of a 10% solution) was added
to the reaction followed by H2O2 (1.95 mL, 17.0 mmol). The reaction refluxed for
about 5 h under nitrogen. The reaction mixture was reduced to about 5.00 mL
and then H2O (20.0 mL) was added and the solid product precipitated out of
solution and was filtered off. The product was recrystallized from EtOH. Yield
(0.483 g, 65.0%). 1H NMR (500 MHz), DMSO: 8 4.20 (d, J = 1.8 Hz, 1H), 4.49 (d,
J = 1.9 Hz, 1H), 7.24 (m, 2H), 7.40 (m, 5H), 7.51 (m, 1H), 8.22 (dd, J = 1.2, 6.6
Hz. 1H), 8.65 (s, 1H), 12.20 (s, 1H); 13c NMR (125MHz), DMSO: 8 57.9, 59.9,

112-2, 115.2, 121.2, 122.1, 123.3, 125.3, 126.2, 128.4, 128.5, 135.1, 136.2,

96

136.4, 187.3. lR (NaCl) 3190 cm'1, 1625 cm”, 1612 cm". M.S: calculated for
C17H13NO2 (M+) = 263.2 and found (M+) = 263.4. Melting Point = 232-234°C.

(II-16). To a 100 mL round bottom flask was added EtOH (30.0 mL) and ll-
15 (0.250 g, 0.950 mmol). Then 10% Pd/C (1 scoop) was added and the round
bottom was fitted with a hydrogen balloon and the mixture stirred at room
temperature for 30 min. The mixture was filtered over celite and the filtrate
concentrated. The crude material was purified by column chromatography (silica
gel, 50% EtOAc; 50°/o hexane) affording the product as an oil. Yield (0.225 g,
89.0%). 1H NMR (500 MHz), acetone: 8 2.95 (dd, J = 7.9, 13.9 Hz, 1H), 3.22 (dd,
J = 4.2, 13.9 Hz, 1H), 4.24 (d, J = 6.6 Hz, 1H), 5.06 (s, 1H), 7.14-7.28 (m, 7H),
7.53 (m, 1H), 8.33 (m, 1H), 8.38 (s, 1H), 11.10 (s, 1H); 13c NMR (125 MHz),
Acetone: 8 43.9, 75.5, 112.8, 114.5, 122.7, 122.9, 124.1, 126.9, 127.0, 128.7,
130.4, 134.5, 137.6, 139.2, 196.6. IR (NaCl) 3389 cm", 3261 cm", 1633 cm".
HRMS: [M + H]+ = 266.1181, calculated for C17H16NO2, 266.1181.

(II-17). To a 50 mL round bottom flask was added "-16 (0.169 g, 0.640
mmol) and EtOAc (20.0 mL). Then IBX (0.357 g, 1.28 mmol, synthesized
according to Frigerio‘zs) was added and the mixture refluxed overnight under
nitrogen. The yellow mixture was filtered over celite and the filtrate concentrated.
The crude material was purified by column chromatography (silica gel, 30%
EtOAc; 70% hexane) affording the product as an oil. Yield (0.147 g, 88.0%). 1H
NMR (500 MHz), CDCI3: 8 4.30 (s, 2H), 7.24-7.40 (m, 8H), 8.28 (d, J = 3.2 Hz,
1H), 8.43 (dd, J = 1.1, 8.6 Hz, 1H), 8.78 (s, 1H); 13c NMR (125 MHz), cocr3: 8

44.1 , 111.5, 112.7, 122.4, 123.4, 124.3, 126.1, 127.0, 128.6, 129.8, 133.2, 135.8,

97

136.4, 183.7, 199.6. lR (NaCl) 3273 cm'1, 1718 cm", 1602 cm". HRMS: [M + H]+
= 264.1027, calculated for C17H14NO2, 264.1025.

(ll-18). To a flame dried 25 mL round bottom flask was added "-17
(0.0740 g, 0.280 mmol) and dry THF (5.00 mL). Then dimethylguanidine
hydrogen sulfate (0.114 g, 0.420 mmol) was added followed by NaH (0.0390 mg,
0.980 mmol). The reaction stirred at room temperature for 30 min and then at
reflux for 2-3 h and again at room temperature overnight all under nitrogen. A
precipitate was filtered off and washed with water and extracted with EtOAc (10 x
20.0 mL). The organics were combined and dried using anhydrous sodium
sulfate and concentrated to give the product as a solid. Yield (0.0490 g, 53.0%).
1H NMR (500 MHz), DMSO: 8 2.85 (s, 6H), 3.28 (d, J = 13.2 Hz, 1H), 3.46 (d, J =
12.9 Hz, 1H), 6.94 (t, J = 7.4 Hz, 1H), 7.07 (t, J = 7.3 Hz, 1H), 7.15-7.25 (m, 5H),
7.37 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 7.9 Hz, 1H), 7.46 (s, 1H); 13C NMR (125
MHz), DMSO: 8 37.8, 41.9, 68.8, 111.7, 115.4, 118.8, 119.9, 121.3, 123.4, 125.3,
126.5, 127.6, 130.6, 136.0, 136.8, 169.7, 187.6. IR (KBr) 3279 cm", 1681 cm",
1615 cm'1. M.S: calculated for C20H20N4O (M+) = 332.4 and found (M+) = 332.2.
Melting Point = 298-300°C decomposed.

(II-19). To a flame dried 25 mL round bottom flask was added
isoamyltriphenylphosphonium bromide (0.690 g, 1.67 mmol) and anhydrous THF
(6.00 mL). The mixture was cooled to -78°C and then NaHMDS (1.25 mL, 1.25
mmol) was added slowly and the resulting mixture stirred at -78°C for 1 h under
nitrogen. Then a solution of N-tosyl-1H-indole-3-carboxaldehyde126 (0.250 g,

0-836 mmol) in anhydrous THF (6.00 mL) was added and the mixture stirred at -

98

Tinor 3 h ar
Hazel (5.00 it
"moved and '
rainwater (l )
arrjdrous 50
05mm chr0l
:‘iliact as ar
=55 Hz. 6H
3H) 5.86
8.1 Hz. 2H),
7H). 7.58 (s
liZSlile)
123.3. 124
”“1 (weak
("-2
”1100,30
and 030:
Wemlglit
Strifile SO
‘v‘u’ere W8
sodium 1

99 001/0).

78°C for 3 h and then warmed to room temperature overnight all under nitrogen.
Water (5.00 mL) was then added and stirred for 30 min. The solvent was
removed and the residue was extracted with DCM (2 x 20.0 mL) and washed
with water (1 x 20.0 mL) and brine (1 x 20.0 mL). The organics were dried using
anhydrous sodium sulfate and concentrated. The crude residue was purified by
column chromatography (silica gel, 50°/o ether; 50°/o hexane) affording the
product as an oil. Yield (0.264 g, 89.0%). 1H NMR (500 MHz), CDC13: 8 1.00 (d, J
= 6.5 Hz, 6H), 1.80 (sep, J = 6.7 Hz, 1H), 2.26 (dt, J = 1.8 Hz, 6.8 Hz, 2H), 2.31
(s, 3H), 5.86-5.92 (dt, J = 7.0 Hz, 11.5 Hz, 1H), 6.46-6.49 (m, 1H), 7.20 (d, J =
8.1 Hz, 2H), 7.27 (t, J = 7.4 Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 7.6 Hz,
1H), 7.58 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 8.06 (d, J = 8.3 Hz, 1H); 13c NMR
(125MHz), CDCl;,: 8 21.3, 22.4, 28.6, 38.6, 113.5, 117.9, 119.3, 119.4, 123.1,
123.3, 124.7, 126.6, 129.7, 130.8, 133.6, 134.5, 135.0, 144.7. IR: (NaCl) 1610
cm'1 (weak). HRMS: [M + HT = 354.1536, calculated for C21H24NO2S, 354.1528.
(ll-20). To a 50 mL round bottom flask was added "-19 (0.264 g, 0.748
mmol), acetone (18.0 mL) and water (2.00 mL). Then NMO (0.131 g, 1.12 mmol)
and 0504 (0.760 mL, 0.0748 mmol) were added and the mixture stirred at RT
overnight under nitrogen. The mixture was quenched with a saturated potassium
sulfite solution (10.0 mL) and extracted with EtOAc ( 2 x 60.0 mL). The organics
were washed with a brine solution (1 x 60.0 mL) and dried using anhydrous
sodium sulfate and concentrated to give the product as a solid. Yield (0.288 g,
99.0%). 1H NMR (500MHz), acetone: 8 0.75 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 6.6

Hz. 3H), 1.24 (m, 1H), 1.32 (m, 1H), 1.80 (m, 1H), 2.32 (s, 3H), 3.68 (s, 1H), 3.98

99

(s, 1H), 4.32 (s, 1H), 4.84 (d, J = 4.4 Hz, 1H), 7.21 (t, J = 8.1 Hz, 1H), 7.28-7.35
(m, 3H), 7.64 (s, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.97 (d, J
= 8.5 Hz, 1H); 13C NMR (125MHz), acetone: 8 21.3, 21.8, 24.2, 25.1, 41.9, 72.2,
72.8, 114.3, 121.9, 123.8, 124.9, 125.2, 125.4, 127.6, 130.7, 131.0, 136.13,
136.17, 146.1. IR: (NaCl) 3425 (br) cm". HRMS: [M + H]+ = 370.1482, calculated
for C21H24N038, 370.1477 (ll-20 minus H2O). Melting Point = 136-138°C.

(ll-21). To a 50 mL round bottom flask was added "-20 (0.288 g, 0.744
mmol), EtOAc (20.0 mL) and IBX (0.833 g, 2.98 mmol). The mixture was refluxed
for 18 h under nitrogen and then the IBX byproduct was filtered off. The filtrate
was concentrated and the crude residue was purified by column chromatography
(silica gel, 30% EtOAc; 70% hexane) affording the product as an oil. Yield (0.163
g, 57.0%). 1H NMR (500 MHz), CDCI3: 8 0.99 (d, J = 6.7 Hz , 6H), 2.23 (m, 1H),
2.33 (s, 3H), 2.80 (d, J = 6.5 Hz, 2H), 7.24-7.26 (m, 2H), 7.34-7.37 (m, 2H), 7.83-
7.85 (m, 2H), 7.93-7.95 (m, 1H), 8.32-8.34 (m, 1H), 8.74 (s, 1H); 13C NMR (125
MHz), 00013: 8 21.5, 22.6, 24.3, 45.9, 113.1, 115.2, 122.8, 125.0, 125.9, 127.2,
127.9, 130.2, 134.3, 134.4, 136.4, 146.0, 184.9, 201.1. IR: (KBr) 3175 cm", 1712
cm‘1, 1651 cm‘1. HRMS: [M + H]+ = 384.1273, calculated for C21H22NO4S,
384.1270.

(ll-22). To a 25 mL round bottom flask was added "-21 (0.0540 g, 0.141
mmol) and MeOH (10.0 mL). Then K2CO3 (0.0490 g, 0.352 mmol) was added
and the mixture stirred at RT for 1.5 h under nitrogen. The MeOH was taken off
and the residue was put into solution with EtOAc (30.0 mL) and washed with

1%HCl (1 x 10.0 mL), water (1 x 10.0 mL) and brine (1 x 10.0 mL). The organics

100

were dried using anhydrous sodium sulfate and concentrated. The crude residue
was purified by column chromatography (silica gel, 30% EtOAc; 70% Hexane)
affording the product as a yellow solid. Yield (0.0170 g, 53.0%). 1H NMR (500
MHz), CDCl3: 8 0.97 (d, J = 6.6 Hz, 6H), 2.24 (m, 1H), 2.83 (d, J = 7.0 Hz, 2H),
7.29-7.42 (m, 3H), 8.31 (d, J = 3.2 Hz, 1H), 8.42 (m, 1H), 9.15 (bs, 1H); 13C NMR
(125 MHz), CDC13: 8 22.6, 24.4, 46.3, 111.5, 112.5, 122.4, 123.3, 124.3, 126.1,
136.0, 136.1, 185.0, 203.1. IR (NaCl) 3200 cm'1, 1712 cm", 1608 cm", 1584 cm'
1. M.S: calculated for C14H15NO2 (M+) = 229.1 and found (M+) = 229.1. Melting
Point = 130-132’C.

(II-23). To a flame dried 10 mL round bottom flask was added "-22
(0.0750 g, 0.328 mmol) and dry THF (5.00 mL). Then dimethylguanidine
hydrogen sulfate (0.134 g, 0.491 mmol) was added followed by NaH (0.0460 g,
1.15 mmol). The reaction stirred at room temperature for 30 min and then
refluxed for 2-3 h and again at room temperature overnight all under nitrogen. A
precipitate was filtered off and washed with water and extracted with EtOAc (10 x
20.0 mL). The filtrate was concentrated and the residue was extracted with
EtOAc (3 x 20.0 mL) and washed with water (1 x 10.0 mL) and brine (1 x 10.0
mL). The organics were combined and dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 90% DCM; 10% MeOH) affording the product as a solid. Yield (0.0190 g,
19.0%). 1H NMR (500 MHz), CDC|32 8 0.87 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.6
Hz, 3H), 1.65 (m, 1H), 2.01 (dd, J = 5.6 Hz, 11.1 Hz, 1H), 2.07 (dd, J = 5.6 Hz, 11

1Hz, 1H), 3.00-3.08 (bs, 3H), 3.09-3.14 (bs, 3H), 6.91 (t, J = 7.1 Hz, 1H), 7.03 (t,

101

J = 7.1 Hz, 1H), 7.24 (d, J = 2.7 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 7.8
Hz, 1H), 8.26 (s, 1H), 10.9 (s, 1H); 13c NMR (125 MHz), c003: 8 23.7, 24.0,
24.5, 36.1, 38.3, 44.5, 67.7, 111.3, 115.7, 118.3, 119.8, 120.8, 122.5, 124.8,
136.6, 169.7, 188.5. IR: (KBr) 3304 cm", 1681 cm", 1608 cm'1. HRMS: [M + H]+
= 299.1879, calculated for C17H23N4O, 299.1872. Melting Point = 274-276°C
decomposes.

(ll-29). To a flame dried 50 mL round bottom flask was added Ill-8 (0.104
g, 0.261 mmol) and anhydrous DCM (20.0 mL). Then TEA (0.0700 mL, 0.522
mmol) was added and the reaction was cooled to 0°C. Then benzoyl chloride
(0.0400 mL, 0.392 mmol) was added slowly and the mixture warmed up to RT
while stirring overnight under nitrogen. The mixture was washed with saturated
NaHC03 (1 x 10.0 mL) and brine (1 x 10.0 mL) and the organics were dried using
anhydrous sodium sulfate and concentrated. The crude material was purified
using column chromatography (silica gel, 7:3 hexanes/ethyl acetate) to yield a
solid (0.111 g, 85.0%). 1H NMR (500 MHz), 00013: 8 1.57 (s, 3H), 2.30 (s, 3H),
4.59 (q, J = 13.1 Hz, 2H), 4.85 (d, J = 7.1 Hz, 2H), 6.08 (d, J = 7.4 Hz, 1H), 6.96
(d, J = 7.3 Hz, 1H), 7.18-7.25 (m, 3H), 7.30-7.5 (m, 4H), 7.64-7.77 (m, 6H), 7.95
(d, J = 7.7 Hz, 1H) ; 13C NMR (125 MHz), CDCI3: 8 19.3, 21.5, 49.4, 69.2, 113.7,
113.9, 117.7, 119.9, 123.6, 124.9, 125.2, 126.9, 127.1, 128.5, 128.6, 129.9,
131.9, 133.4, 134.9, 135.1, 138.9, 145.2, 166.8, 170.1. IR: (NaCI) 3374 cm'1 ,
1748 cm", 1657 cm". HRMS: [M + H]"’ = 503.1642, calculated for C23H27N205S,

503.1641. Melting Point = 118-120°C.

102

 

(ll-30). To a flame dried 50 mL round bottom flask was added Ill-8 (0.250
g, 0.628 mmol) and anhydrous DCM (20.0 mL). Then TEA (0.200 mL, 1.57
mmol) was added and the reaction was cooled to 0°C. Then acetyl chloride
(0.0900 mL, 1.26 mmol) was added slowly and the mixture warmed up to RT
while stirring overnight under nitrogen. The mixture was washed with saturated
NaHCOa (1 x 10.0 mL) and brine (1 x 10.0 mL) and the organics were dried using
anhydrous sodium sulfate and concentrated. The crude residue was purified by
column chromatography (silica gel, 80% DCM; 20% EtOAc) affording the product
as a solid. Yield (0.264 g, 95.0%). 1H NMR (500 MHz), CDCI3: 8 1.58 (s, 3H),
2.02 (s, 3H), 2.34 (s, 3H), 4.55 (d, J = 12.9, 1H), 4.59 (d, J = 13.0 Hz, 1H), 4.85
(s, 2H), 5.91 (d, J = 7.6 Hz, 1H), 6.40 (d, J = 6.6 Hz, 1H), 7.20-7.36 (m, 4H), 7.61
(s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.96 (d, J = 8.3 Hz,
1H); 13C NMR (125 MHz), CDCI3: 8 19.2, 21.5, 22.9, 48.9, 69.0, 113.6, 113.7,
117.6, 119.9, 123.5, 124.8, 125.2, 126.8, 128.4, 129.9, 134.9, 135.0, 138.9,
145.1, 169.4, 170.1. lR: (NaCI) 3291 cm", 1748 cm", 1651 cm". HRMS: [M + H]"
= 441.1486, calculated for C23H25N2058, 441.1484. Melting Point = 118-120°C.

(ll-31). To a flame dried 25 mL round bottom flask was added Ill-8 (1.48 g,
3.73 mmol) and anhydrous DCM (50.0 mL). Then TEA (3.29 mL, 23.8 mmol) was
added and the reaction was cooled to 0°C. Then dimethylcarbamoyl chloride
(1.50 mL, 16.4 mmol) was added slowly and the mixture warmed to RT overnight
under nitrogen. The mixture was washed with saturated NaHC03 (1 x 10.0 mL)
and brine (1 x 10.0 mL) and the organics were dried using anhydrous sodium

sulfate and concentrated. The crude residue was purified by column

103

chromatography (silica gel, 70% DCM; 30% EtOAc) affording the product as an
oil. Yield (1.38 g, 79.0%). 1H NMR (500 MHz), CDC|3I 8 1.55 (s, 3H), 2.29 (s, 3H),
2.86 (s, 6H), 4.48 (d, J = 13.1 Hz, 1H), 4.57 (d, J = 13.0 Hz, 1H), 4.78 (d, J = 10.5
Hz, 2H), 5.30 (d, J = 7.5 Hz, 1H), 5.77 (d, J = 7.5 Hz, 1H), 7.15 (m, 4H), 7.55 (s,
1H), 7.65 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 8.1 Hz, 2H), 7.91 (d, J = 8.5 Hz, 1H);
13C NMR (125 MHz), CDCI3: 8 19.1, 21.3, 36.0, 50.4, 68.6, 113.3, 113.4, 118.5,
120.0, 123.3, 124.4, 124.9, 126.7, 128.7, 129.7, 134.8, 134.9, 139.0, 144.9,
157.1, 171.1. IR: (NaCI) 3425 cm", 3334 cm", 1748 cm", 1645 cm". HRMS: [M
+ H]+ = 470.1750, calculated for C24H28N3058, 470.1750.

(II-32). To a 250 mL flame dried round bottom flask was added Ill-8 (2.00
g, 5.03 mmol) and anhydrous DCM (0.100 L). Then anhydrous TEA (1.60 mL,
11.6 mmol) was added and the mixture was cooled to 0°C. Trifluoroacetic
anhydride (1.06 mL, 7.54 mmol) was then added dropwise and the reaction
mixture was warmed to room temperature while stirring under nitrogen overnight.
Saturated NaHC03 was added to quench the excess TFAA. The organics were
separated from the aqueous solution and then washed with brine (1 x 50.0 mL).
The organics were dried using anhydrous sodium sulfate and concentrated. The
crude residue was purified by column chromatography (silica gel, 80% hexanes;
20% EtOAc) affording the product as a solid. Yield (2.00 g, 80.0%). 1H NMR (500
MHz), CDCl3: 8 1.55 (s, 3H), 2.32 (s, 3H), 4.58 (d, J = 12.8 Hz, 1H), 4.64 (d, J =
12.9 Hz, 1H), 4.85 (s, 2H), 5.92 (d, J = 7.2 Hz, 1H), 7.22 ( d, J = 8.7 Hz, 2H),
7.27 (t, J = 8.1 Hz, 1H), 7.36 (t, J = 8.3 Hz, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.63 (d,

J = 7.2 Hz, 1H), 7.74 (s, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.98 (d, J = 8.5 Hz, 1H);

104

13C NMR (125 MHz), CDC13: 8 19.0, 21.3, 49.2, 69.6, 112.0, 113.6, 114.0, 114.3,
115.6, 116.5, 118.8, 119.4, 123.7, 125.3, 125.5, 126.7, 127.8, 129.8, 134.5,
134.8, 138.4, 145.3, 156.0, 156.3, 156.6, 156.9, 168.5. lR: (NaCI) 3346 cm”,
1742 cm", 1718 cm". HRMS: [M + H]° = 495.1206, calculated for
C23H22N205SF3, 495.1202. Melting Point = 119-121°C.

(ll-33). To a flame dried 50 mL round bottom flask was added Ill-8 (0.250
g, 0.628 mmol) and anhydrous DCM (20.0 mL). Then TEA (0.170 mL, 1.26
mmol) was added and the reaction was cooled to 0°C. Then ethyl
(chlorocarbonyl)formate (0.100 mL, 0.942 mmol) was added slowly and the
mixture stirred at 0°C for 1.5 h under nitrogen. Water (5.00 mL) was added and
the organics were separated, washed with brine (1 x 20.0 mL), dried using
anhydrous sodium sulfate and concentrated. The crude residue was purified by
column chromatography (silica gel, 70% hexane; 30% EtOAc) affording the
product as an oil. Yield (0.172 g, 55.0%). 1H NMR (500 MHz), CDC13: 8 1.32 (t, J
= 7.3 Hz, 3H), 1.54 (s, 3H), 2.29 (s, 3H), 4.29 (q, J = 7.5 Hz, 2H), 4.55 (m, 2H),
4.82 (m, 2H), 5.87 (d, J = 8.1 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 7.21-7.31 (m,
3H), 7.59 (m, 1H), 7.64 (s, 1H), 7.74 (m, 2H), 7.93 (m, 2H); 130 NMR (125 MHz),
CDCI3: 8 13.8, 19.1, 21.4, 49.2, 63.3, 69.2, 113.6, 113.9, 116.3, 119.7, 123.5,
125.24, 125.26, 126.8, 128.0, 129.9, 134.8, 134.9, 138.6, 145.1, 155.8, 159.6,
168.8. IR: (NaCI) 3360 cm“, 1750 cm", 1615 cm". HRMS: [M + H]+ = 499.1544,
calculated for C25H27N2078, 499.1539.

(II-34). To a 100 mL flame dried round bottom flask was added Ill-8 (0.311

g. 0.781 mmol) and anhydrous DCM (50.0 mL). Then DIPEA (0.270 mL, 1.56

105

mmol) was added and the mixture was cooled to 0°C. Then Troc-Cl (0.160 mL,
1.17 mmol) was then added dropwise and the reaction mixture was warmed to
room temperature while stirring under nitrogen overnight. The solvent was
removed and the residue was resolvated in ether (50.0 mL) and washed with 1%
HCI (30.0 mL). The organics were separated and washed with brine (1 x 30.0
mL). The organics were dried using anhydrous sodium sulfate and concentrated.
The crude residue was purified by column chromatography (silica gel, 80%
hexanes; 20% EtOAc) affording the product as a solid. Yield (0.264 g, 59.0%). 1H
NMR (500 MHz), CDCI3: 8 1.57 (s, 3H), 2.34 (s, 3H), 4.57 (d, J = 12.7 Hz, 1H),
4.61 (d, J = 13.0 Hz, 1H), 4.72 (d, J = 12.2 Hz, 1H), 4.76 ( d, J = 11.9 Hz, 1H),
4.85 (s, 2H), 5.70 (d, J = 7.8 Hz, 1H), 6.11 (d, J = 7.5 Hz, 1H), 7.20-7.28 (m, 3H),
7.34 (t, J = 8.3 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.66 (s, 1H), 7.76 (d, J = 8.3 Hz,
2H), 7.98 (d, J = 8.3 Hz, 1H); 13C NMR (125 MHz), CDCI3: 8 19.1, 21.4, 50.9,
69.2, 74.6, 95.1, 113.65, 113.69, 113.8, 117.1, 119.8, 123.5, 124.9, 125.2, 126.7,
126.81, 126.88, 128.1, 129.84, 129.89, 129.94, 134.8, 135.0, 138.7, 145.1,
153.6, 169.5. lR: (NaCI) 3370 cm", 1742 cm". HRMS: [M + H]+ = 573.0430,
calculated for C24H24N2058Cl3, 573.0421. Melting Point = 84-87°C.

(ll-35). To a flame dried 50 mL round bottom flask was added Ill-8 (0.250
g, 0.628 mmol) and anhydrous DCM (20.0 mL). Then TEA (0.200 mL, 1.57
mmol) was added and the reaction was cooled to 0°C. Then pivaloyl chloride
(0.150 mL, 1.26 mmol) was added slowly and the mixture stirred at 0°C for 1.5 h
under nitrogen. The mixture was washed with saturated NaHC03 (1 x 10.0 mL)

and brine (1 x 10.0 mL) and the organics were dried using anhydrous sodium

106

sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 70% hexane; 30% EtOAc) affording the product as a
solid. Yield (0.244 g, 80.0%). 1H NMR (500MHz), CD013: 8 1.20 (s, 9H), 1.57 (s,
3H), 2.32 (s, 3H), 4.53 (d, J = 13.0 Hz, 1H), 4.62 (d, J = 13.0 Hz, 1H), 4.84 (m,
2H), 5.87 (d, J = 7.1 Hz, 1H), 6.55 (d, J = 7.2 Hz, 1H), 7.20-7.34 (m, 4H), 7.6-
7.62 (m, 2H), 7.75 (d, J = 8.5 Hz, 2H), 7.96 (d, J = 8.5 Hz, 1H); 13C NMR
(125MHz), CDC13: 8 19.1, 21.4, 26.9, 27.2, 38.5, 49.0, 68.9, 113.6, 117.8, 119.7,
123.4, 124.6, 125.1, 126.7, 128.4, 129.8, 134.8, 135.0, 138.8, 145.0, 170.0,
177.8. IR: (NaCI) 3352 cm" , 1748 cm", 1663 cm". HRMS: [M + H]° = 483.1961,
calculated for C26H31N2058, 483.1954. Melting Point = 114-116°C.

(ll-36). To a 100 mL round bottom flask was added "-29 (0.492 g, 0.980
mmol), dichloroethane (60.0 mL), and trifluoroacetic anhydride (1.37 mL, 9.80
mmol). The yellowish solution stirred at room temperature under nitrogen for 24 h
and then was brought to reflux for 6 h and then stirred overnight at room
temperature. The organics were washed with sodium bicarbonate (2 x 20.0 mL)
and brine (2 x 10.0 mL), combined, dried using anhydrous sodium sulfate, and
concentrated. The crude material was purified using column chromatography
(silica gel, 8:2 hexanes/ethyl acetate) to yield a yellowish oil (0.360 g, 76.0%). 1H
NMR (500MHz), CDCI3: 8 1.74 (s, 3H), 2.31 (s, 3H), 2.93 (dd, J = 5.8, 13.4 Hz,
2H), 4.80 (d, J = 17.8 Hz, 2H), 7.20-7.32 (m, 5H), 7.49-7.59 (m, 3H), 7.72 (s, 1H),
7.75 (d, J = 8.3 Hz, 2H), 7.94 (d, J = 9 Hz, 1H), 8.04-8.06 (m, 2H) ; 13C NMR
(125MHz), CDCI3: 8 21.5, 24.3, 46.1, 72.8, 113.6, 116.6, 119.3, 122.0, 123.3,

123.5, 124.9, 125.7, 126.9, 127.8, 128.1, 128.8, 129.9, 132.9, 135.1, 135.4,

107

139.2, 145.1
935.1542, 02

(ll-37
g. 0.227 ml
[.794 mmol
solvent was
:3 slasher
:8 organic
3336 resid

EtOAc

 

139.2, 145.1, 160.7, 177.6. lR: (NaCI) 1821 cm", 1657 cm". HRMS: [M + H]° =
485.1542, calculated for C28H25N2O4S, 485.1535.

(II-37). To a flame dried 25 mL round bottom flask was added "-30 (0.100
g, 0.227 mmol) and anhydrous benzene (12.0 mL). Then POC13 (0.0600 mL,
0.704 mmol) was added and the mixture refluxed overnight under nitrogen. The
solvent was removed and the residue was put into solution with EtOAc (20.0 mL)
and washed with saturated NaHCO3 (1 x 10.0 mL) and brine (1 x 10.0 mL) and
the organics were dried using anhydrous sodium sulfate and concentrated. The
crude residue was purified by column chromatography (silica gel, 80% Hexane;
20% EtOAc) affording the product as a solid. Yield (0.0690 mg, 72.0%). 1H NMR
(300MHz), CDCI3: 8 1.72 (s, 3H), 1.79 (s, 3H), 2.39 (s, 3H), 2.73 (dd, J = 14.1 Hz,
22.5 Hz, 2H), 4.82 (s, 1H), 4.97 (s, 1H), 7.28-7.46 (m, 4H), 7.91 (d, J = 8.7 Hz,
2H), 8.06-8.09 (m, 1H), 8.37-8.40 (m, 1H), 8.92 (s, 1H); 13C NMR (125MHz),
CDCI3: 8 21.5, 24.0, 24.7, 46.8, 106.6, 111.1, 113.3, 117.4, 122.5, 124.4, 125.7,
127.1, 127.7, 130.0, 131.5, 134.6, 134.7, 138.5, 145.6, 151.5, 164.7. IR: (NaCI)
1779 cm", 1627 cm". HRMS: [M + H]+ = 423.1379, calculated for C23H23N2O4S,
423.1379. Melting Point = 121-123°C.

(ll-38). To a flame dried 50 mL round bottom flask was added "-31 (0.150
g, 0.320 mmol) and anhydrous benzene (20.0 mL). Then POCI3 (0.0900 mL,
0.991 mmol) was added and the mixture was refluxed overnight under nitrogen.
The solvent was then taken off and the residue was put into solution with EtOAc
(20.0 mL) and washed with saturated NaHC03 (1 x 10.0 mL) and brine (1 x 10.0

mL) and the organics were dried using anhydrous sodium sulfate and

108

 

{133080173161
159011 (5.01
Cree gas E
335 purifiel
afardng tr

,5. 3H), 3.7

m»

“blame

concentrated. The residue was then resuspended in benzene (20.0 mL) and
MeOH (5.00 mL) and TMSCHN2 (0.480 mL, 0.960 mmol) was added slowly.
Once gas evolution ceased, the solvents were taken off and the crude residue
was purified by column chromatography (silica gel, 90% DCM; 10% EtOAc)
affording the product as an oil. Yield (0.134 g, 86.0%). 1H NMR (500 MHz),
CDC13: 8 1.63 (s, 3H), 2.27 (s, 3H), 2.84 (s, 6H), 3.10 (d, J = 13.5 Hz, 1H), 3.61
(s, 3H), 3.76 (d, J = 13.0 Hz, 1H), 4.71 (s, 1H), 4.90 (s, 1H), 5.96 (s, 1H), 7.12-
7.22 (m, 4H), 7.49 (d, J = 9.1 Hz, 1H), 7.70-7.72 (m, 3H), 7.85 (d, J = 9.5 Hz,
1H); 130 NMR (125 MHz), coca: 8 21.4, 23.4, 36.0, 41.3, 52.9, 61.3, 113.6,
115.8, 120.1, 122.1, 123.1, 124.2, 125.1, 126.8, 128.4, 129.7, 135.1, 140.7,
144.6, 155.8, 173.4. lR: (NaCI) 3443 cm", 1736 cm", 1663 cm". HRMS: [M + H]°
= 484.1915, calculated for C25H30N3058, 484.1906.

(ll-39). To a flame dried 25 mL round bottom flask was added "-32 (0.100
g, 0.202 mmol) and anhydrous acetonitrile (10.0 mL). Then anhydrous TEA
(0.140 mL, 1.03 mmol) was added and the mixture was cooled down to 0°C.
Then phosgene (0.240 mL, 0.465 mmol) was added and the reaction turned
yellow and stirred at 0°C for 2 h. The reaction was quenched by being poured
into an ice water mix (10.0 mL). EtOAc was added (3 x 20.0 mL) to extract the
organics, which were dried and concentrated. The crude residue was resolvated
into a basic methanolic solution and stirred for 20 min. The reaction was
neutralized with a 1% HCI solution and the solvent was removed. The crude
residue was purified was purified by column chromatography (silica gel, 90%

hexanes; 10% EtOAc) affording the product as a solid. Yield (0.0530 mg, 55.0%).

109

'11 111R (5001
3.3 is. 3H). 1
arrow
Hm1H)‘
EHJBQJ
1299. 134.8.
WOMM
C;:H;.-.Nzoss
m4m
"0,3860 mg,
7611,0120
iC.Then pl
reiow and t
in"0 an ice \
03816913)
(Rainy
Rwflnd
residue Wa
reranes; 41
:H NMR (51
RU83H
”213, J =

3J=95

1H NMR (500MHz), CDC13: 8 1.71 (s, 3H), 2.30 (s, 3H), 3.20 (d, J = 13.4 Hz, 1H),
3.68 (s, 3H), 3.78 (d, J = 13.6 Hz, 1H), 4.81 (s, 1H), 4.99 (s, 1H), 7.14-7.27 (m,
4H), 7.32 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.79 (s, 1H), 7.87 (d, J =
9.3 Hz, 1H), 7.91 (s, 1H); 13C NMR (125MHz), CDCI3: 8 21.4, 22.8, 40.2, 53.7,
61.3, 113.9, 114.1, 116.3, 117.3, 118.9, 119.2, 123.6, 124.9, 126.0, 126.8, 127.4,
129.9, 134.8, 135.1, 138.6, 145.1, 154.8, 155.1, 172.0. IR: (NaCI) 3389 cm",
1730 (with shoulder) cm". HRMS: [M + H]+ = 509.1358, calculated for
C24H24N2058F3, 509.1358. Melting Point = 166-168°C.

(ll-40). To a flame dried 25 mL round bottom flask was added "-33
(0.0860 mg, 0.173 mmol) and anhydrous acetonitrile (10.0 mL). Then anhydrous
TEA (0.120 mL, 0.882 mmol) was added and the mixture was cooled down to
0°C. Then phosgene (0.210 mL, 0.397 mmol) was added and the reaction turned
yellow and stirred at 0°C for 2 h. The reaction was quenched by being poured
into an ice water mix (10.0 mL). EtOAc was added (3 x 20.0 mL) to extract the
organics, which were dried and concentrated. The crude residue was resolvated
into a basic methanolic solution and stirred for 20 min. The reaction was
neutralized with a 1% HCI solution and the solvent was removed. The crude
residue was purified was purified by column chromatography (silica gel, 60%
hexanes; 40% EtOAc) affording the product as a solid. Yield (0.0470 mg, 55.0%).
1H NMR (500MHz), CDCI3: 8 1.68 (s, 3H), 2.30 (s, 3H), 3.18 (d, J = 13.4 Hz, 1H),
3.67 (s, 3H), 3.79 (d, J = 11.9 Hz, 1H), 3.80 (s, 3H), 4.78 (s, 1H), 4.93 (s, 1H),
7.12 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 9.5 Hz, 2H), 7.39 (d, J = 8.1 Hz, 1H), 7.72

(d. J = 9.5 Hz, 2H), 7.77 (s, 1H), 7.84 (d, J = 8.3 Hz, 1H), 8.61 (s, 1H); 13c NMR

110

12531112)"
034.124
.5 171
4&15459

0L4
133409'
0.118 mm‘
brsrmn.
were it
removed 2
ramr(10
sodium 8
ea'o'rlatol
ail. Yield
3H12190
1H)117E
3H), 7.95
412.63.
1285,12
3258 cm
CzeHzeN:

(ll
{005005

(125MHz), CDCI3: 8 21.5, 23.0, 40.2, 53.4, 53.5, 61.3, 113.7, 116.8, 119.6, 119.7,
123.4, 124.7, 125.6, 126.8, 127.7, 129.9, 134.9, 135.0, 139.2, 145.0, 154.3.
160.5, 171.9. lR: (NaCI) 3388 cm", 1742 cm", 1712 cm". HRMS: [M + H]° =
499.1545, calculated for C25H27N2078, 499.1539. Melting Point = 180-182°C.

(ll-41). To a flame dried 25 mL round bottom flask was added "-38
(0.0440 9, 0.0910 mmol) and anhydrous CH3CN (10.0 mL). Then BOP (0.0520 g,
0.118 mmol) and DBU (0.200 mL, 1.37 mmol) were added and the mixture stirred
for 5 min. Then benzyl amine (0.0100 mL, 0.137 mmol) was added and the
mixture refluxed overnight under a nitrogen atmosphere. The solvent was
removed and the residue was resolvated with EtOAc (20.0 mL) and washed with
water (10.0 mL) and brine (10.0 mL). The organics were dried using anhydrous
sodium sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 4% MeOH; 96% DCM) affording the product as an
oil. Yield (0.0330 g, 71.0%). 1H NMR (500MHz), CDC13: 8 1.41 (s, 3H), 2.31 (s,
3H), 2.90 (m, 2H), 4.61 (d, J = 14.5 Hz, 1H), 4.67 (d, J = 14.5 Hz, 1H), 4.70 (s,
1H), 4.76 (s, 1H), 6.75 (s, 1H), 7.16-7.38 (m, 9H), 7.63 (s, 1H), 7.71-7.78 (m,
3H), 7.95 (d, J = 9.0 Hz, 1H); 13c NMR (125MHz), coch: 8 21.5, 23.7, 42.5,
44.2, 63.8, 113.7, 117.3, 120.2, 121.5, 123.5, 123.9, 125.1, 126.8, 127.4, 127.8,
128.5, 128.6, 129.9, 134.9, 135.5, 135.6, 138.3, 145.2, 156.6, 173.1. IR: (NaCI)
3258 cm", 1774 cm“, 1715 cm". HRMS: [M + Hr = 514.1807, calculated for
C29H28N304S, 514.1801.

(ll-42). To a flame dried 25 mL round bottom flask was added "-38

(0.0500 g, 0.104 mmol) and anhydrous CH3CN (10.0 mL). Then BOP (0.0590 g,

111

3.135 mmo'
stredforS
'efluxed 0vr
re'esidue
mL) and b
s.iiate an
verrematogl
01. Yield (C
arr 6H), 1.
3J:13;
1H), 4.92
hiz, 2H), 7
kai5.
1140‘ 11
1359. 13:
Cm“. 162
577.2485
(Il-
9-529 m
mW) ar
brOught c
was am)“

The Whit

0.135 mmol) and DBU (0.0230 mL, 0.156 mmol) were added and the mixture
stirred for 5 min. Then water (0.400 mL, 22.2 mmol) was added and the mixture
refluxed overnight under a nitrogen atmosphere. The solvent was removed and
the residue was resolvated with EtOAc (20.0 mL) and washed with water (10.0
mL) and brine (10.0 mL). The organics were dried using anhydrous sodium
sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 4% MeOH; 96% DCM) affording the product as an
oil. Yield (0.0330 g, 55.0%). 1H NMR (500MHz), CDCI3: 8 1.58 (s, 3H), 1.59-1.73
(m, 6H), 1.84 (m, 2H), 2.35 (s, 3H), 2.50 (m, 2H), 2.93 (d, J = 13.7 Hz, 1H), 2.99
(d, J = 13.7 Hz, 1H), 3.28 (m, 2H), 3.41 (m, 2H), 3.53 (t, J = 7.1 Hz, 2H), 4.82 (s,
1H), 4.92 (s, 1H), 6.96 (s, 1H), 7.22-7.36 (m, 4H), 7.67 (s, 1H), 7.78 (d, J = 8.5
Hz, 2H), 7.87 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 8.3 Hz, 1H); 13C NMR (125MHz),
CDC13: 8 21.8, 23.6, 24.3, 26.9, 28.8, 30.1, 37.0, 37.4, 44.4, 45.8, 49.7, 64.0,
114.0, 117.4, 120.5, 121.8, 123.8, 124.2, 125.3, 127.1, 127.7, 130.2, 135.1,
135.9, 138.9, 145.5, 157.0, 173.6, 176.1. lR: (NaCI) 3243 cm", 1779 cm", 1718
cm", 1621 cm". HRMS: [M + H]+ = 577.2490, calculated for C31H37N4058,
577.2485.

(II-49). To a flame dried 100 mL round bottom flask was added "-43 (1.00
g, 5.29 mmol) and anhydrous DCM (50.0 mL). Then allyl alcohol (0.720 mL, 10.6
mmol) and DMAP (0.0650 g, 0.529 mmol) were added and the mixture was
brought down to 0°C. DCC (1.63 g, 7.94 mmol) was then added and the mixture
was allowed to warm to room temperature overnight while stirring under nitrogen.

The white precipitate that formed was filtered off and the DCM filtrate was

112

washed with brine (1 x 20.0 mL), dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 20% EtOAc; 80% Hexane) affording the product as an oil. Yield (1.09 g,
90.0%). 1H NMR (500 MHz), CDCI3: 8 1.33 (d, J = 7.1 Hz, 3H), 1.37 (s, 9H), 4.26
(bs, 1H), 4.56 (m, 2H), 5.08 (bs, 1H), 5.17-5.27 (m, 2H), 5.81-5.87 (m, 1H); 13c
NMR (125 MHz), CDCI3: 8 18.4, 28.2, 49.1, 65.6, 79.6, 118.4, 131.6, 155.0,
172.9. IR: (NaCI) 3380 cm", 1750 cm“, 1710 cm". HRMS: [M + H1" = 230.1401,
calculated for C11H20NO4, 230.1392. Anal. Calcd. For C11H19NO4: C, 57.62; H,
8.35; N, 6.11. Found: C, 58.89; H, 8.22; N, 5.90.

(ll-50). To a flame dried 100 mL round bottom flask was added "-44 (1.38
g, 5.23 mmol) and anhydrous DCM (50.0 mL). Then allyl alcohol (0.710 mL, 10.5
mmol) and DMAP (0.0640 g, 0.523 mmol) were added and the mixture was
brought down to 0°C. DCC (1.62 g, 7.85 mmol) was then added and the mixture
was allowed to warm to room temperature overnight while stirring under nitrogen.
The white precipitate that formed was filtered off and the DCM filtrate was
washed with brine (1 x 20.0 mL), dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 20% EtOAc; 80% Hexane) affording the product as a whitish solid. Yield
(1.34 g, 84.0%). 1H NMR (500MHz), CDCI3: 8 1.39 (s, 9H), 3.02-3.14 (m, 2H),
4.58 (d, J = 6.6 Hz, 2H), 4.96 (bs, 1H), 5.20-5.30 (m, 2H), 5.80-5.88 (m, 1H), 7.12
(m, 2H), 7.20-7.29 (m, 3H); 13c NMR (125MHz), cocra: 8 28.2, 38.3, 54.4, 65.8,
79.8, 118.8, 126.9, 128.4, 129.3, 131.5, 135.9, 155.0, 171.5. IR: (NaCI) 3380 cm“

1 , 1750 cm", 1710 cm". HRMS: [M + H]* = 306.1703, calculated for C17H24NO4,

113

 

 

336.1705. 7
53.29: H, 7.
(ll-5‘
5.97 mm
3301) and
33.ng do
was allowe
"re white
trashed w
:ercentrat
Get, 20%
11.49 g, 8
5.12-5.17
7.26-7.36
127, 123:
710 cm"
Anal. Calc
“1.4.78, 1).
(Il-
170992 9,
mL, 7-06
was brou

”Entire W

306.1705. Anal. Calcd. For C17H23NO4: C, 66.86; H, 7.59; N, 4.59. Found: C,
68.29; H, 7.41; N, 4.62. Melting Point = 62-64°C.

(ll-51). To a flame dried 100 mL round bottom flask was added "-45 (1.50
g, 5.97 mmol) and anhydrous DCM (50.0 mL). Then allyl alcohol (0.820 mL, 12.0
mmol) and DMAP (0.0730 g, 0.597 mmol) were added and the mixture was
brought down to 0°C. DCC (1.85 g, 8.96 mmol) was then added and the mixture
was allowed to warm to room temperature overnight while stirring under nitrogen.
The white precipitate that formed was filtered off and the DCM filtrate was
washed with brine (1 x 20.0 mL), dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 20% EtOAc; 80% Hexane) affording the product as a whitish solid. Yield
(1.49 g, 86.0%). 1H NMR (500MHz), CDCI3: 8 1.41 (s, 9H), 4.58-4.60 (m, 2H),
5.12-5.17 (m, 2H), 5.32 (d, J = 7.3 Hz, 1H), 5.56 (bs, 1H), 5.75-5.83 (m, 1H),
7.26-7.36 (m, 5H); 13c NMR (125MHz), CDC13: 8 28.2, 57.6, 65.9, 80.0, 118.3,
127, 128.3, 128.7, 131.3, 136.8, 154.7, 170.7. lR: (NaCI) 3390 cm“ , 1750 cm",
1710 cm". HRMS: [M + H]°° = 292.1559, calculated for C16H22NO4, 292.1549.
Anal. Calcd. For C16H21NO4: C, 65.96; H, 7.27; N, 4.81. Found: C, 66.78; H, 7.14;
N, 4.78. Melting Point = 40-42°C.

(II-52). To a flame dried 100 mL round bottom flask was added "-46
(0.992 g, 3.53 mmol) and anhydrous DCM (50.0 mL). Then allyl alcohol (0.480
mL, 7.06 mmol) and DMAP (0.0430 g, 0.353 mmol) were added and the mixture
was brought down to 0°C. DCC (1.09 g, 5.30 mmol) was then added and the

mixture was allowed to warm to room temperature overnight while stirring under

114

733383. The
res washed
:errerltratec
331.30% Et
$07g.94l
.1: 5.3 Hz,
8.7112, 2H
567, 65.4.
180‘) 339
RTCRHEJ
FOUHd; C,
(“-1
{3.625 g,
11‘4.65
was bfoL
“Emits l
”13880
was alga.
when.
'36], 20
37.0% 1
1013 21:4

nitrogen. The white precipitate that formed was filtered off and the DCM filtrate
was washed with brine (1 x 20.0 mL), dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 30% EtOAc; 70% hexane) affording the product as a whitish solid. Yield
(1.07 g, 94.0%). 1H NMR (500MHz), CDC13: 8 1.32 (s, 9H), 3.63 (s, 3H), 4.48 (d,
J = 5.3 Hz, 2H), 5.14 (m, 2H), 5.19 (d, J = 7.5 Hz, 1H), 5.69 (m, 2H), 6.74 (d, J =
8.7 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H); 13c NMR (125MHz), CDC13: 8 27.9, 54.7,
56.7, 65.4, 79.4, 113.8, 117.8, 128.0, 128.5, 131.1, 154.5, 159.2, 170.6. IR:
(NaCI) 3395 cm", 1743 cm", 1711 cm". HRMS: [M + H]'° = 322.1653, calculated
for C17H24N05, 322.1654. Anal. Calcd. For C17H23N05: C, 63.54; H, 7.21; N, 4.36.
Found: C, 63.69; H, 7.14; N, 4.61. Melting Point = 60-62'C.

(ll-53). To a flame dried 100 mL round bottom flask was added "-47
(0.625 g, 2.32 mmol) and anhydrous DCM (50.0 mL). Then allyl alcohol (0.320
mL, 4.65 mmol) and DMAP (0.0280 g, 0.232 mmol) were added and the mixture
was brought down to 0°C. DCC (0.717 g, 3.48 mmol) was then added and the
mixture was allowed to warm to room temperature overnight while stirring under
nitrogen. The white precipitate that formed was filtered off and the DCM filtrate
was washed with brine (1 x 20.0 mL), dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 20% EtOAc; 80% hexane) affording the product as an oil. Yield (0.626 g,
87.0%). 1H NMR (500MHz), CDCI3: 8 1.40 (s, 9H), 4.59 (d, J = 5.5 Hz, 2H), 5.15
(m, 2H), 5.29 (d, J = 6.9 Hz, 1H), 5.57 (s, 1H), 5.79 (m, 1H), 6.09-7.02 (m, 2H),

7.31-7.33 (m, 2H); 13c NMR (125MHz), coca: 8 28.2, 56.9, 66.1, 80.2, 115.7 (d,

115

J = 11.7 Hz), 118.6,,128.83 (d, J = 8.3 Hz), 131.1, 132.8, 154.7, 162.6 (d, J =
247.5 Hz), 170.5. IR: (NaCI) 3383 cm'1 , 1749 cm", 1711 cm". HRMS: [M + H]* =
310.1463, calculated for C15H21FNO4, 310.1455. Anal. Calcd. For C15H20FNO4:
C, 62.12; H, 6.52; N, 4.53. Found: C, 61.93; H, 6.29; N, 4.49.

(ll-54). To a flame dried 100 mL round bottom flask was added "-48 (1.30
g, 4.32 mmol) and anhydrous DCM (50.0 mL). Then allyl alcohol (0.590 mL, 8.64
mmol) and DMAP (0.0530 g, 0.432 mmol) were added and the mixture was
brought down to 0°C. DCC (1.33 g, 6.48 mmol) was then added and the mixture
was allowed to warm to room temperature overnight while stirring under nitrogen.
The white precipitate that formed was filtered off and the DCM filtrate was
washed with brine (1 x 20.0 mL), dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 20% EtOAc; 80% hexane) affording the product as an oil. Yield (1.13 g,
77.0%). 1H NMR (500MHz), CDC13: 8 1.42 (s, 9H), 4.61-4.64 (m, 2H), 5.08—5.15
(m, 2H), 5.49 (d, J = 7.5 Hz, 1H), 5.77 (m, 1H), 6.09 (d, J = 7.8 Hz, 1H), 7.40-
7.58 (m, 4H), 7.82 (m, 1H), 7.86 (d, J = 8.7 Hz, 1H), 8.17 (d, J = 8.4 Hz, 1H); 13C
NMR (125MHz), CDC|32 8 28.2, 54.7, 66.1, 80.2, 118.4, 123.3, 125.2, 125.4,
126.0, 126.9, 128.8, 129.3, 131.0, 131.3, 132.8, 134.0, 155.0, 171.5. IR: (NaCI)
3389 cm'1 , 1749 cm", 1711 cm". HRMS: [M + Hr = 342.1706, calculated for
C20H24NO4, 342.1705. Anal. Calcd. For C20H23NO4: C, 70.36; H, 6.79; N, 4.10.
Found: C, 67.48; H, 6.43; N, 3.85.

(ll-55). To a flame dried 100 mL round bottom flask was added "-45 (1.50

g, 5.97 mmol) and anhydrous DCM (50.0 mL). Then 2-methyI-2-propen-1-ol (1 .00

116

mL, 12.0 mmol) and DMAP (0.0730 g, 0.597 mmol) were added and the mixture
was brought down to 0°C. DCC (1.85 g, 8.96 mmol) was then added and the
mixture was allowed to warm to room temperature overnight while stirring under
nitrogen. The white precipitate that formed was filtered off and the DCM filtrate
was washed with brine (1 x 20.0 mL), dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 20% EtOAc; 80% hexane) affording the product as a whitish solid. Yield
(1.39 g, 76.0%). %). 1H NMR (500MHz), CDCI3: 8 1.41 (s, 9H), 1.58 (s, 3H),
4.46-4.58 (m, 2H), 4.78 (s, 1H), 4.81 (s, 1H), 5.33 (d, J = 7.0 Hz, 1H), 5.56 (s,
1H), 7.27-7.37 (m, 5H); 13c NMR (125MHz), cocrgz 8 19.1, 28.2, 57.7, 68.6,
80.0, 113.2, 127.1, 128.3, 128.8, 136.9, 139.1, 154.7, 170.8. lR: (NaCI) 3389 cm‘
1 , 1743 cm", 1711 cm". HRMS: [M + H]" = 306.1705, calculated for C17H24NO4,
306.1705. Anal. Calcd. For C17H23NO4: C, 66.86; H, 7.59; N, 4.59. Found: C,
66.95; H, 7.66; N, 4.64. Melting Point = 38-40°C.

(II-56). To a 100 mL round bottom flask was added "-49 (1.32 g, 4.32
mmol), DCM (2.50 mL) and TFA (2.50 mL). The resulting solution stirred for 30
min. The excess TFA and DCM were removed and CHC|3 (3 x 15.0 mL) was
added and subsequently taken off to remove any residual TFA. The crude
material (oil) was taken on without further purification. Yield (1.00 g, 88.0%). 1H
NMR (500MHz), CDCI3: 8 1.56 (d, J = 7.3 Hz, 3H), 4.10 (q, J = 7.1 Hz, 1H), 4.63
(m, 2H), 5.25-5.32 (m, 2H), 5.8-5.9 (m, 1H), 8.24 (s, 3H); 13C NMR (125MHz),

CDCI3: 6 15.6, 49.2, 67.1, 115.8 (0, J = 290.9 Hz), 119.7, 130.5, 161.8 (q, J =

117

359112111
#390872

(ll-5'
moi), DC
Ti'r, The I
33:93 am
areeipitatt
lit-'educt E

3.34-3.1E

:m“_ (.4
Calcd.

1.31, r

36.9 Hz), 169.9. IR: (KBr) 3100 (br) cm°1, 1748cm", 1675 cm". HRMS: [M + Hr
= 130.0872, calculated for C6H11NO2, 130.0868.

(ll-57). To a 100 mL round bottom flask was added “-50 (1.32 g, 4.32
mmol), DCM (2.50 mL) and TFA (2.50 mL). The resulting solution stirred for 30
min. The excess TFA and DCM were removed and CHCI3 (3 x 15.0 mL) was
added and subsequently taken off to remove any residual TFA. The product was
precipitated out of the crude residue using ether/petroleum ether to afford the
product as a white solid. Yield (1.27 g, 92.0%). 1H NMR (500MHz), DMSO: 8
3.04-3.19 (m, 2H), 4.32 (t, J = 6.1 Hz, 1H), 4.58 (m, 2H), 5.18-5.26 (m, 2H), 5.73-
5.81 (m, 1H), 7.20-7.36 (m, 5H), 8.64 (bs, 3H); 13c NMR (125MHz), DMSO: 8
36.1, 53.2, 65.8, 117.1 (q, J = 299.2 Hz), 118.6, 127.2, 128.5, 129.3, 131.4,
134.6, 158.4 (q, J = 31.3 Hz), 168.7. IR: (KBr) 3100 (br) cm'1, 1745 cm", 1660
cm". HRMS: [M + H]’° = 206.1188, calculated for C12H15NO2, 206.1181. Anal.
Calcd. For C14H16F3NO4: C, 52.67; H, 5.05; N, 4.39. Found: C, 52.69; H, 4.91; N,
4.31. Melting Point = 88-90°C.

(ll-58). To a 100 mL round bottom flask was added "-51 (1.44 g, 4.95
mmol), DCM (2.50 mL) and TFA (2.50 mL). The resulting solution stirred for 30
min. The excess TFA and DCM were removed and CHCl3 (3 x 15.0 mL) was
added and subsequently taken off to remove any residual TFA. The product was
precipitated out of the crude residue using ether/petroleum ether to afford the
product as a white solid. Yield (1.48 g, 98.0%).1H NMR (500MHz), DMSO: 8 4.66
(m, 2H), 5.12-5.16 (m, 2H), 5.33 (s, 1H), 5.80 (m, 1H), 7.44-7.50 (m, 5H), 9.07

(bs, 3H); 13c NMR (125MHz), DMSO: 8 55.4, 66.0, 117.1 (q, J = 299.7 Hz),

118

((3.1, 128.1
31511310fr
0.1131102,
91.30205
(ll-SS
mmol). DC
31:3. The t
aaeed anr
precipitate
era-duet a
3.75 (8,?
1": 8.9
DMSO:
131.5, ‘
1650 c
Anal. 1

4.64; I

118.1, 128.1, 129.0, 129.5, 131.5, 132.6, 158.3 (q, J = 31.3 Hz), 168.1. IR: (KBr)
3150 (br) cm", 1745 cm", 1675 cm°1. HRMS: [M + H]° = 192.1027, calculated for
C11H14NO2, 192.1025. Anal. Calcd. For C13H14F3NO4: C, 51.15; H, 4.62; N, 4.59.
Found: C, 50.95; H, 4.51; N, 4.45. Melting Point = 96-98°C.

(II-59). To a 100 mL round bottom flask was added "-52 (1.02 g, 3.17
mmol), DCM (2.00 mL) and TFA (2.00 mL). The resulting solution stirred for 30
min. The excess TFA and DCM were removed and CHC13 (3 x 15.0 mL) was
added and subsequently taken off to remove any residual TFA. The product was
precipitated out of the crude residue using ether/petroleum ether to afford the
product as a white solid. Yield (0.953 g, 90.0%). 1H NMR (500MHz), DMSO: 8
3.76 (s, 3H), 4.66 (m, 2H), 5.14-5.18 (m, 2H), 5.26 (s, 1H), 5.82 (m, 1H), 7.01 (d,
J = 8.9 Hz, 2H), 7.41 (d, J = 8.8 Hz, 2H), 8.91 (s, 3H); 13C NMR (125MHz),
DMSO: 8 54.8, 55.2, 65.9, 114.3, 117.2 (d, J = 300.3 Hz), 118.2, 124.4, 129.6,
131.5, 158.1 (q, J = 30.6 Hz), 160.0, 168.4. IR: (KBr) 3088 (br) cm", 1749 cm",
1680 cm'1. HRMS: [M + H]° = 222.1140, calculated for C14H15N03, 222.1130.
Anal. Calcd. For C14H16F3N05: C, 50.15; H, 4.81; N, 4.18. Found: C, 50.29; H,
4.64; N, 4.19. Melting Point = 88-90°C.

(ll-60). To a 100 mL round bottom flask was added "-53 (0.600 g, 1.94
mmol), DCM (2.00 mL) and TFA (2.00 mL). The resulting solution stirred for 30
min. The excess TFA and DCM were removed and CHC13 (3 x 15.0 mL) was
added and subsequently taken off to remove any residual TFA. The product was
precipitated out of the crude residue using ether/petroleum ether to afford the

product as a white solid. Yield (0.424 g, 68.0%). 1H NMR (500MHz), DMSO: 8

119

4.67 (m, 2H), 5.13-5.19 (m, 2H), 5.40 (s, 1H), 5.82 (m, 1H), 7.33 (m, 2H), 7.56
(m, 2H), 8.98 (s, 3H); 13C NMR (125MHz), DMSO: 8 54.6, 66.1, 115.9 (d, J =
22.1 Hz), 117.2 (q, J = 300.1 Hz), 118.2, 128.9 (d, J = 3.2 Hz), 130.6 (d, J = 8.8
Hz), 131.4, 158.0 (q, J = 30.9 Hz), 162.6 (d, J = 246.2 Hz), 168.0. IR: (KBr) 3100
(br) em", 1749 cm", 1680 cm". HRMS: [M + H]+ = 210.0933, calculated for
C11H13FNO2, 210.0930. Anal. Calcd. For C13H13F4NO4: C, 48.30; H, 4.05; N,
4.33. Found: C, 48.33; H, 3.77; N, 4.23. Melting Point = 68-70°C.

(ll-61). To a 100 mL round bottom flask was added "-54 (1.11 g, 3.26
mmol), DCM (2.00 mL) and TFA (2.00 mL). The resulting solution stirred for 30
min. The excess TFA and DCM were removed and CHC|3 (3 x 15.0 mL) was
added and subsequently taken off to remove any residual TFA. The product was
precipitated out of the crude residue using ether/petroleum ether to afford the
product as a white solid. Yield (0.962 g, 83.0%). 1H NMR (500MHz), DMSO: 8
4.65 (m, 2H), 5.02-5.09 (m, 2H), 5.75 (m, 1H), 6.19 (s, 1H), 7.58-7.72 (m, 4H),
8.04 (t, J = 9.0 Hz, 2H), 8.31 (d, J = 8.6 Hz, 1H), 9.15 (s, 1H); 13C NMR
(125MHz), DMSO: 8 51.3, 66.1, 117.2 (q, J = 300.3 Hz), 118.0, 123.3, 125.3,
125.9, 126.5, 127.2, 128.8, 129.1, 130.2, 130.5, 131.4, 133.5, 158.2 (q, J = 31.1
Hz), 168.5. IR: (KBr) 3090 (br) cm", 1730 cm", 1667 cm". HRMS: [M + Hr =
242.1192, calculated for C15H15NO2, 242.1181. Anal. Calcd. For C17H16F3NO4: C,
57.47; H, 4.54; N, 3.94. Found: C, 57.33; H, 4.47; N, 3.87. Melting Point = 112-
114°C.

(ll-62). To a 100 mL round bottom flask was added "-55 (1.39 g, 4.56

mmol), DCM (3.00 mL) and TFA (3.00 mL). The resulting solution stirred for 30

120

min. The excess TFA and DCM were removed and CHCI3 (3 x 15.0 mL) was
added and subsequently taken off to remove any residual TFA. The product was
precipitated out of the crude residue using ether to afford the product as a white
solid. Yield (1.32 g, 91.0%). 1H NMR (500MHz), DMSO: 8 1.53 (s, 3H), 4.58 (dd,
J = 13.4, 33.9 Hz, 2H), 4.76 (s, 1H), 4.82 (s, 1H), 5.35 (s, 1H), 7.42-7.53 (m, 5H),
9.00 (s, 3H); 130 NMR (125MHz), DMSO: 8 18.7, 55.3, 68.4, 112.9, 117.2 (q, J =
300.1 Hz), 128.0, 129.0, 129.5, 132.6, 139.0, 158.0 (q, J = 31.1 Hz), 168.1. IR:
(KBr) 3150 (br) cm'1, 1743 cm“, 1680 cm". HRMS: [M + H]+ = 206.1182,
calculated for C12H16NO2, 206.1181. Anal. Calcd. For C14H16F3NO4: C, 52.67; H,
5.05; N, 4.39. Found: C, 52.63; H, 4.99; N, 4.34. Melting Point = 142-144°C.
(II-63). To a flame dried 100 mL round bottom flask was added "-56 (1 .00
g, 4.11 mmol) and anhydrous DCM (50.0 mL). Then the solution was brought
down to 0°C and anhydrous TEA (0.630 mL, 4.52 mmol) was added followed by
dropwise addition of ethoxycarbonyl isothiocyanate (0.560 mL, 4.93 mmol). The
solution was allowed to warm to room temperature overnight while stirring under
a nitrogen atmosphere. The solvent was removed and ether (30.0 mL) was
added to the crude residue and was washed with sat. sodium bicarbonate. The
organics were dried using anhydrous sodium sulfate and concentrated. The
crude residue was purified by column chromatography (silica gel, 30% EtOAc;
70% hexane) affording the product as a whitish solid. Yield (1.04 g, 97.0%). 1H
NMR (500MHz), CDC13: 8 1.28 (t, J = 7.1 Hz, 3H), 1.54 (d, J = 7.2 Hz, 3H), 4.21
(q, J = 7.1 Hz, 2H), 4.65 (m, 2H), 5.00 (p, J = 7.2 Hz, 1H), 5.22-5.26 (m, 1H),

5.30-5.35 (m, 1H), 5.89 (m, 1H), 8.15 (s, 1H), 10.14 (d, J = 5.9 Hz, 1H); 13c NMR

121

droowis

solutior

(125MHz), CDCI3: 8 14.0, 17.5, 53.6, 62.7, 66.0, 118.7, 131.3, 152.5, 171.4,
178.8. IR: (KBr) 3295 cm", 3240 cm", 1750 cm", 1725 cm". HRMS: [M + Hr
=261.0920, calculated for C10H17N2O4S, 261.0909. Anal. Calcd. For
C10H15N2O4S: C, 46.14; H, 6.20; N, 10.76. Found: C, 46.88; H, 6.06; N, 10.80.
Melting Point = 43-45°C.

(ll-64). To a flame dried 100 mL round bottom flask was added "-57 (1 .27
g, 3.98 mmol) and anhydrous DCM (50.0 mL). Then the solution was brought
down to 0°C and anhydrous TEA (0.610 mL, 4.38 mmol) was added followed by
dropwise addition of ethoxycarbonyl isothiocyanate (0.540 mL, 4.78 mmol). The
solution was allowed to warm to room temperature overnight while stirring under
a nitrogen atmosphere. The solvent was removed and ether (30.0 mL) was
added to the crude residue and was washed with sat. sodium bicarbonate. The
organics were dried using anhydrous sodium sulfate and concentrated. The
product was recrystallized from the crude residue with EtOAc/hexanes affording
the product as a whitish solid. Yield (1.08 g, 81.0%). 1H NMR (500MHz), CDC13: 8
1.27 (t, J = 7.1 Hz, 3H), 3.20-3.34 (m, 2H), 4.19 (q, J = 7.1 Hz, 2H), 4.60 (m, 2H),
5.24 (m, 3H), 5.82 (m, 1H), 7.15-7.30 (m, 5H), 8.10 (s, 1H), 10.08 (d, J = 5.5 Hz,
1H); 13C NMR (125MHz), CDC13: 8 14.0, 37.2, 59.2, 62.8, 66.1, 118.9, 127.1,
128.5, 129.2, 131.2, 135.3, 152.3, 170.0, 179.1. IR: (KBr) 3290 cm'1 , 3225 cm",
1730 cm'1(with shoulder). HRMS: [M + H]’° =337.1227, calculated for
C16H21N2O4S, 337.1222. Anal. Calcd. For C16H20N2O4S: C, 57.12; H, 5.99; N,

8.33. Found: C, 55.80; H, 5.80; N, 8.30. Melting Point = 79-81°C.

122

0
g. 4.85
33310
:mpws
solution
amrog
abedt
eqaoc
emde r
79%lm

r1119 (1

(ll-65). To a flame dried 100 mL round bottom flask was added "-58 (1.48
g, 4.85 mmol) and anhydrous DCM (50.0 mL). Then the solution was brought
down to 0°C and anhydrous TEA (0.740 mL, 5.33 mmol) was added followed by
dropwise addition of ethoxycarbonyl isothiocyanate (0.660 mL, 5.82 mmol). The
solution was allowed to warm to room temperature overnight while stirring under
a nitrogen atmosphere. The solvent was removed and ether (30.0 mL) was
added to the crude residue and was washed with sat. sodium bicarbonate. The
organics were dried using anhydrous sodium sulfate and concentrated. The
crude residue was purified by column chromatography (silica gel, 30% EtOAc;
70% hexane) affording the product as a whitish solid. Yield (1.39 g, 89.0%). 1H
NMR (500MHz), CDC13: 8 1.29 (t, J = 7.1 Hz, 3H), 4.23 (m, 2H), 4.64 (m, 2H),
5.14-5.22 (m, 2H), 5.81 (m, 1H), 5.98 (d, J = 6.9 Hz, 1H), 7.31-7.45 (m, 5H), 8.06
(s, 1H), 10.59 (d, J = 6.4 Hz, 1H); 13c NMR (125MHz), CDCI3: 8 14.0, 61.8, 62.8,
66.2, 118.5, 127.5, 128.7, 128.9, 131.1, 135.0, 152.5, 169.3, 178.9. IR: (KBr)
3290 cm" , 3225 cm", 1750 cm", 1720 cm". HRMS: [M + H]+ =323.1070,
calculated for C15H19N2O4S, 323.1066. Anal. Calcd. For C15H13N2O4S: C, 55.88;
H, 5.63; N, 8.69. Found: C, 55.46; H, 5.51; N, 8.68. Melting Point = 44-46’C.

(II-66). To a flame dried 100 mL round bottom flask was added "-59
(0.900 g, 2.69 mmol) and anhydrous DCM (50.0 mL). Then the solution was
brought down to 0°C and anhydrous TEA (0.410 mL, 2.96 mmol) was added
followed by dropwise addition of ethoxycarbonyl isothiocyanate (0.360 mL, 3.22
mmol). The solution was allowed to warm to room temperature overnight while

stirring under a nitrogen atmosphere. The solvent was removed and ether (30.0

123

mL) was added to the crude residue and was washed with sat. sodium
bicarbonate. The organics were dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 30% EtOAc; 70% hexane) affording the product as an oil. Yield (0.917 g,
97.0%). 1H NMR (500MHz), CDCl3: 8 1.31 (t, J = 7.2 Hz, 3H), 3.80 (s, 3H), 4.22-
4.26 (m, 2H), 4.60-4.72 (m, 2H), 5.19-5.25 (m, 2H), 5.85 (m, 1H), 5.93 (d, J = 6.8
Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 8.20 (s, 1H), 10.55 (d,
J = 6.5 Hz, 1H); 13C NMR (125MHz), CDCI3: 8 14.0, 55.2, 61.2, 62.8, 66.2, 114.3,
118.5, 127.1, 128.8, 131.2, 152.5, 159.8, 169.5, 178.7. IR: (KBr) 3289 cm'1 ,
3226 cm", 1751 cm", 1724 cm". HRMS: [M + Hr = 353.1180, calculated for
C16H21N2O5S, 353.1171. Anal. Calcd. For C15H20N205S: C, 54.53; H, 5.72; N,
7.95. Found: C, 52.68; H, 5.45; N, 7.55.

(II-67). To a flame dried 50 mL round bottom flask was added "-60 (0.394
g, 1.22 mmol) and anhydrous DCM (20.0 mL). Then the solution was brought
down to 0°C and anhydrous TEA (0.190 mL, 1.34 mmol) was added followed by
dropwise addition of ethoxycarbonyl isothiocyanate (0.160 mL, 1.46 mmol). The
solution was allowed to warm to room temperature overnight while stirring under
a nitrogen atmosphere. The solvent was removed and ether (30.0 mL) was
added to the crude residue and was washed with sat. sodium bicarbonate. The
organics were dried using anhydrous sodium sulfate and concentrated. The
crude residue was purified by column chromatography (silica gel, 30% EtOAc;
70% hexane) affording the product as an oil. Yield (0.383 g, 92.0%). 1H NMR

(500MHz), cocr: 8 1.29 (t, J = 7.1 Hz, 3H), 4.20-4.26 (m, 2H), 4.59-4.69 (m,

124

2H), 5.1
7.3-87.4
CDClgz 1
Hz}. 13‘

IR: (KB

2H), 5.15-5.25 (m, 2H), 5.81 (m, 1H), 5.96 (d, J = 6.8 Hz, 1H), 7.02-7.07 (m, 2H),
7.38-7.42 (m, 2H), 8.10 (s, 1H), 10.61 (d, J = 6.3 Hz, 1H); 13C NMR (125MHz),
CDCI3: 8 14.1, 61.0, 62.9, 66.4, 115.9 (d, J = 22.1 Hz), 118.8, 129.3 (d, J = 8.8
Hz), 131.07 (d, J = 2.3 Hz), 131.1, 152.6, 162.8 (d, J = 248.1 Hz), 169.2, 178.9.
IR: (KBr) 3282 cm'1 , 3232 cm", 1751 cm", 1724 cm". HRMS: [M + H]+ =
341.0974, calculated for C15H13FN2O4S, 341.0971. Anal. Calcd. For
C15H17FN2O4S: C, 52.93; H, 5.03; N, 8.23. Found: C, 51.76; H, 4.89; N, 8.07.
(II-68). To a flame dried 100 mL round bottom flask was added “-61
(0.902 g, 2.54 mmol) and anhydrous DCM (50.0 mL). Then the solution was
brought down to 0°C and anhydrous TEA (0.390 mL, 2.79 mmol) was added
followed by dropwise addition of ethoxycarbonyl isothiocyanate (0.340 mL, 3.05
mmol). The solution was allowed to warm to room temperature overnight while
stirring under a nitrogen atmosphere. The solvent was removed and ether (30.0
mL) was added to the crude residue and was washed with sat. sodium
bicarbonate. The organics were dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 30% EtOAc; 70% hexane) affording the product as an oil. Yield (0.897 g,
95.0%). 1H NMR (500MHz), CDCI3: 8 1.24 (t, J = 7.1 Hz, 3H), 4.12-4.22 (m, 2H),
4.58-4.72 (m, 2H), 5.10-5.18 (m, 2H), 5.78 (m, 1H), 6.80 (d, J = 7.3 Hz, 1H),
7.42-7.60 (m, 4H), 7.85 (t, J = 8.6 Hz, 2H), 8.20 (m, 2H), 10.57 (d, J = 7.1 Hz,
1H); 13c NMR (125MHz), coca: 8 14.0, 58.9, 62.8, 66.3, 118.5, 123.2, 125.2,
126.0, 126.1, 127.0, 128.8, 129.7, 131.0, 131.1, 131.2, 134.0, 152.5, 169.7,

179.2. IR: (KBr) 3289 cm" , 3226 cm", 1749 cm“, 1718 cm". HRMS: [M + Hr =

125

373.1222.
C. 61.27; I
(ll-I
3 3.13 rr
torn 10C
dropwise
solution v
a nitroge
added 10
organics
product .
the prod
(51.28 (1
5.8 Hz,
: 5-5 H;
127.5, .
3232 Cl
CliHati

8.33, F,

373.1222, calculated for C19H21N204S, 373.1222. Anal. Calcd. For C19H20N204S:
C, 61.27; H, 5.41; N, 7.52. Found: C, 58.71; H, 4.85; N, 7.15.

(II-69). To a flame dried 100 mL round bottom flask was added "-62 (1 .00
g, 3.13 mmol) and anhydrous DCM (50.0 mL). Then the solution was brought
down to 0°C and anhydrous TEA (0.480 mL, 3.45 mmol) was added followed by
dropwise addition of ethoxycarbonyl isothiocyanate (0.420 mL, 3.76 mmol). The
solution was allowed to warm to room temperature overnight while stirring under
a nitrogen atmosphere. The solvent was removed and ether (30.0 mL) was
added to the crude residue and was washed with sat. sodium bicarbonate. The
organics were dried using anhydrous sodium sulfate and concentrated. The
product was recrystallized from the crude residue with EtOAc/hexanes affording
the product as a whitish solid. Yield (0.960 g, 91 .0%). 1H NMR (500MHz), CDCI3:
5 1.28 (t, J = 7.2 Hz, 3H), 1.59 (s, 3H), 4.23 (m, 2H), 4.55 (m, 2H), 4.83 (d, J =
5.8 Hz, 2H), 5.98 (d, J = 6.9 Hz, 1H), 7.31-7.44 (m, 5H), 8.08 (s, 1H), 10.62 (d, J
= 6.6 Hz, 1H); 13C NMR (125MHz), CDCI3: 5 14.1, 19.1, 61.9, 62.9, 68.9, 113.5,
127.5, 128.8, 128.9, 135.1, 139.0, 152.5, 169.4, 178.9. IR: (KBr) 3289 cm‘1 ,
3232 cm“, 1749 cm‘1, 1724 cm". HRMS: [M + H]+ = 337.1230, calculated for
C16H21N204S, 337.1222. Anal. Calcd. For C16H20N204S: C, 57.12; H, 5.99; N,
8.33. Found: C, 55.96; H, 5.84; N, 8.03. Melting Point = 62-64’C.

(ll-71). To a flame dried 50 mL round bottom flask was added "-70118'119
(1.55 g, 7.49 mmol) and anhydrous CH3CN. Then allyl alcohol (2.56 mL, 37.5
mmol) was added and the mixture stirred at room temperature under nitrogen

overnight. The solvent was removed and the residue was put into solution with

126

EtOAc (0.200 L) and washed with sat. sodium bicarbonate (1 x 0.100 L). The
organics were dried using anhydrous sodium sulfate and concentrated to give
pure product as a solid. Yield (1.70 g, 99.0%). 1H NMR (500MHz), acetone: 5
4.85 (m, 2H), 5.27-5.31 (m, 1H), 5.42-5.48 (m, 1H), 6.07 (m, 1H), 7.27-7.32 (m,
2H), 7.56-7.60 (m, 1H), 8.32 (m, 1H), 8.46 (s, 1H), 11.35 (s, 1H); 13C NMR
(125MHz), Acetone: 5 66.5, 113.2, 114.2, 119.0, 122.5, 123.6, 124.7, 126.8,
132.8, 137.7, 138.2, 164.0, 179.5. IR: (KBr) 3200om'1 , 1743om", 1620 cm".
HRMS: [M + H]+ =230.0819, calculated for C13H12NO3, 230.0817. Anal. Calcd.
For C13H11N03: C, 68.11; H, 4.84; N, 6.11. Found: C, 66.14; H, 4.80; N, 5.93.
Melting Point = 159-161°C.

(ll-72). To a flame dried 250 mL round bottom flask was added "-71 (1.70
g, 7.42 mmol) and anhydrous DCM (0.100 L). Then TsCl (2.82 g, 14.8 mmol),
DMAP (2.26 g, 18.6 mmol), and DIPEA (3.20 mL, 18.6 mmol) were added and
the mixture stirred at room temperature overnight under a nitrogen atmosphere.
The resulting brown solution was washed with 5% HCI (1 x 30.0 mL) and brine (1
x 30.0 mL) and the organics were dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 30% EtOAc; 70% Hexanes) and recrystallized with EtOAc/hexanes to afford
the product as a solid. Yield (2.46 g, 86.0%). 1H NMR (500MHz), CDCI3: 8 2.35
(s, 3H), 4.86 (m, 2H), 5.35 (m, 1H), 5.46 (m, 1H), 6.03 (m, 1H), 7.27 (d, J = 8.0
Hz, 2H), 7.37 (m, 2H), 7.85 (d, J = 7.4 Hz, 2H), 7.94 (m, 1H), 8.34 (m, 1H), 8.82
(s, 1H); 13C NMR (125MHz), CDCI3: 6 21.6, 66.9, 113.1, 116.9, 120.0, 122.9,

125.2, 126.1, 127.3, 127.6, 130.3, 130.7, 134.2, 134.4, 136.8, 146.2, 161.2,

127

178.3. IR: (KBr) 1730cm", 1674 cm". HRMS: [M + H]+ = 384.0914, calculated for
C20H18N058, 383.0906. Anal. Calcd. For ConnNOsS: C, 62.65; H, 4.47; N, 3.65.
Found: C, 62.47; H, 4.48; N, 3.64. Melting Point = 104-106°C.

(II-73). To a 250 mL round bottom flask was added "-72 (2.30 g, 6.01
mmol) and dioxane (0.100 L). Then hydroxylamine hydrochloride (1.24 g, 18.0
mmol) and pyridine (1.55 mL, 19.2 mmol) were added and the mixture refluxed
under nitrogen overnight (enough water to dissolve the hydroxylamine salt was
added). The solvent was taken off and the residue was put into solution with
EtOAc (0.100 L). The organics were washed with 1% HCI (1 x 30.0 mL) and
brine (1 x 30.0 mL), then dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 30% EtOAc; 70% Hexanes) affording the product as an oil (mixture of
isomers). Yield (2.22 g, 93.0%). Isomer A: 1H NMR (500MHz), CDCI3: 6 2.32 (s,
3H), 4.83 (d, J = 5.9 Hz, 2H), 5.28 (d, J = 10.5 Hz, 1H), 5.38 (d, J = 17.2 Hz, 1H),
5.96 (m, 1H), 7.21 (d, J = 8.5 Hz, 2H), 7.25 (t, J = 8.0 Hz, 1H), 7.35 (t, J = 8.0 Hz,
1H), 7.52 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.3 Hz, 1H),
8.25 (s, 1H), 10.59 (s, 1H); 13C NMR (125MHz), CDCI3: 8 21.4, 66.8, 109.5,
113.2, 119.6, 122.4, 123.3, 124.8, 126.9, 128.0, 129.9, 130.1, 130.9, 133.9,
134.7, 143.1, 145.3, 162.7. Isomer B: 1H NMR (500MHz), CDCI3: 5 2.25 (s, 3H),
4.91 (d, J = 5.9 Hz, 2H), 5.33 (d, J = 10.3 Hz, 1H), 5.45 (d, J = 17.2 Hz, 1H), 6.02
(m, 1H), 7.14-7.21 (m, 3H), 7.32 (t, J = 7.5 Hz, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.76
(s, 1H), 7.94 (d, J = 8.5 Hz, 1H), 8.00 (d, J = 8.1 Hz, 1H), 8.99 (s, 1H); 13C NMR

(125MHz), CDCI3: 5 21.3, 66.5, 113.2, 113.6, 119.7, 122.9, 124.1, 125.6, 126.8,

128

126.9, 127.3, 129.9, 130.7, 134.4, 135.0, 145.4, 146.5, 162.4. IR: (NaCI) 3276
cm", 17300m'1. HRMS: [M + H]+ = 399.1017, calculated for C20H19N2058,
399.1015. Anal. Calcd. For C20H18N205SI C, 60.29; H, 4.55; N, 7.03. Found: C,
58.91; H, 4.30; N, 6.82.

(ll-74). To a 250 mL round bottom flask was added water (55.0 mL) and
AcOH (55.0 mL). Then "-73 (2.12 g, 5.33 mmol) was dissolved in THF (25.0 mL)
and was added to the aqueous acid. The mixture was brought down to 0°C and
zinc (3.46 g, 53.3 mmol) was then slowly added in small portions over 20 min.
The suspension stirred at 0°C for 2 h. The solid was filtered off and the filtrate
was reduced and then brought to a pH of 8 using concentrated ammonium
hydroxide. The amine was extracted into ethyl acetate (4 x 50.0 mL), dried using
anhydrous sodium sulfate and concentrated. The crude residue was then put into
solution with DCM (5.00 mL) and TFA (3.00 mL) was added. The mixture stirred
for 10 min and then the solvent and excess TFA was removed. CHCI3 (3 x 15.0
mL) was added and subsequently taken off to remove any residual TFA. The
product was precipitated out of the crude residue using ether/petroleum ether to
afford the product as a white solid. Yield (2.25 g, 85.0%). 1H NMR (500MHz),
DMSO: 6 2.31 (s, 3H), 4.66 (m, 2H), 5.06 (m, 1H), 5.09 (m, 1H), 5.71 (s, 1H),
5.75 (m, 1H), 7.33 (t, J = 8.1 Hz, 1H), 7.40 (m, 3H), 7.75 (d, J = 8.1 Hz, 1H), 7.85
(d, J = 7.5 Hz, 2H), 7.94 (d, J = 7.3 Hz, 1H), 8.09 (s, 1H), 9.12 (s, 3H); 130 NMR
(125MHz), DMSO: 5 20.9, 47.8, 66.2, 113.1, 114.1, 117.2 (q, J = 299.2 Hz),
118.1, 120.3, 123.6, 125.5, 126.7, 126.8, 127.8, 130.3, 131.3, 133.7, 133.8.

145.9, 158.3 (q. J = 31.3 Hz), 167.5. IR: (KBr) 3100 (br)cm'1, 1736 cm", 1574

129

cm". HRMS: [M + H]+ = 385.1235, calculated for C20H21N204S, 385.1222. Anal.
Calcd. For C22H21F3N2068: C, 53.01; H, 4.25; N, 5.62. Found: C, 52.95; H, 4.05;
N, 5.50. Melting Point = 170-172°C.

(ll-75). To a flame dried 100 mL round bottom flask was added "-74 (1.30
g, 2.61 mmol) and anhydrous DCM (50.0 mL). Then the solution was brought
down to 0°C and anhydrous TEA (0.400 mL, 2.87 mmol) was added followed by
dropwise addition of ethoxycarbonyl isothiocyanate (0.350 mL, 3.13 mmol). The
solution was allowed to warm to room temperature overnight while stirring under
a nitrogen atmosphere. The solvent was removed and ether (30.0 mL) was
added to the crude residue and was washed with sat. sodium bicarbonate. The
organics were dried using anhydrous sodium sulfate and concentrated. The
product was recrystallized from the crude residue with EtOAc/Hexanes affording
a whitish solid. Yield (1.19 g, 89.0%). 1H NMR (500MHz), CDCI3: 5 1.26 (t, J =
7.2 Hz, 3H), 2.31 (s, 3H), 4.20 (m, 2H), 4.64 (m, 2H), 5.13-5.22 (m, 2H), 5.78 (m,
1H), 6.26 (d, J = 7.9 Hz, 1H), 7.19-7.25 (m, 3H), 7.30 (t, J = 8.3 Hz, 1H), 7.63 (d,
J = 7.5 Hz, 1H), 7.72 (s, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 7.3 Hz, 1H),
8.04 (s, 1H), 10.59 (d, J = 6.9 Hz, 1H); 13c NMR (125MHz), CDCI3: 8 14.1, 21.5,
54.7, 62.9, 66.6, 113.7, 115.9, 119.0, 119.9, 123.6, 125.1, 126.2, 126.9, 128.2,
129.9, 131.0, 134.8, 135.0, 145.1, 152.4, 168.7, 179.0. IR: (KBr) 3282 cm", 3232
cm", 1749 cm", 1724 cm". HRMS: [M + H]* = 516.1273, calculated for
C24H25N30582, 516.1263. Anal. Calcd. For C24H25N30682: C, 55.91; H, 4.89; N,

8.15. Found: C, 55.91; H, 4.79; N, 8.09. Melting Point = 117-119°C.

130

g. 0.45

1.34 W

hen re
the so
938%
and til.
then tr
{3 x 3
sodium
chrom;
Whitish
= 7.4,

3.06 (c

5H). 9.

(II-76). To a flame dried 50 mL round bottom flask was added "-64 (0.150
g, 0.446 mmol) and anhydrous DCM (15.0 mL). Then anhydrous TEA (0.200 mL,
1.34 mmol) was added and the mixture was cooled to 0°C. EDCI (0.189 g, 0.982
mmol) was then added and the mixture stirred at 0°C under nitrogen for 1 h and
then refluxed for 15 h. After the first step was completed, (as indicated by TLC),
the solution was cooled to 0°C and a solution of NaH (0.0890 g, 2.33 mmol) in
MeOH (5.00 mL) was added dropwise. The cloudy mixture stirred at 0°C for 1 h
and then at room temperature for 2 h. The mixture was acidified with 5% HCl and
then the solvents were removed. The aqueous mixture was extracted with EtOAc
(3 x 30.0 mL) and then the organics were combined, dried using anhydrous
sodium sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 20% EtOAc; 80% DCM) affording the product as a
whitish solid. Yield (0.0200 g, 19.0%). 1H NMR (500MHz), Acetone: 5 2.51 (dd, J
= 7.4, 13.8 Hz, 1H), 2.61 (dd, J = 7.3, 13.9 Hz, 1H), 2.90 (d, J = 13.7 Hz, 1H),
3.06 (d, J = 13.7 Hz, 1H), 5.15 (m, 2H), 5.79 (m, 1H), 7.07 (s, 1H), 7.20-7.28 (m,
5H), 9.28 (s, 1H); 13C NMR (125MHz), Acetone: 5 42.1, 42.9, 67.9, 120.2, 127.6,
128.8, 131.1, 132.3, 138.0, 158.5, 178.7. IR: (KBr) 3220 cm'1 (broad), 1761cm'1,
1711 cm". HRMS: [M + H]+ = 231.1141, calculated for C13H15N202, 231.1134.
Anal. Calcd. For C13H14N202: C, 67.81; H, 6.13; N, 12.17. Found: C, 66.59; H,
6.14; N, 12.09. Melting Point = 203-205‘C.

(ll-77). To a flame dried 50 mL round bottom flask was added "-65 (0.150
g, 0.466 mmol) and anhydrous DCM (15.0 mL). Then anhydrous TEA (0.200 mL,

1.40 mmol) was added and the mixture was cooled to 0°C. EDCI (0.197 g, 1.02

131

mmol) was then added and the mixture stirred at 0°C under nitrogen for 1 h and
then refluxed for 15 h. After the first step was completed, (as indicated by TLC),
the solution was cooled to 0°C and a solution of NaH (0.0930 g, 2.33 mmol) in
MeOH (5.00 mL) was added dropwise. The cloudy mixture stirred at 0°C for 1 h
and then at room temperature for 2 h. The mixture was acidified with 5% HCI and
then the solvents were removed. The aqueous mixture was extracted with EtOAc
(3 x 30 mL) and then the organics were combined, dried using anhydrous sodium
sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 20% EtOAc; 80% DCM) affording the product as a
whitish solid. Yield (0.0700 g, 70.0%). 1H NMR (500MHz), acetone: 5 2.78 (dd, J
= 7.4, 14.0 Hz, 1H), 2.95 (dd, J = 7.3, 13.9 Hz, 1H), 5.13 (d, J = 10.1 Hz, 1H),
5.21 (d, J = 17.1 Hz, 1H), 5.73 (m, 1H), 7.33 (t, J = 7.4 Hz, 1H), 7.40 (t, J = 8.2
Hz, 2H), 7.64 (d, J = 7.9 Hz, 2H), 7.68 (s, 1H), 9.65 (s, 1H); 13C NMR (125MHz),
acetone: 5 43.5, 68.6, 120.8, 126.3, 128.7, 129.3, 132.0, 139.7, 157.0, 175.9. IR:
(KBr) 3245 cm'1 (broad), 1774cm'1, 1724 cm". HRMS: [M + H]+ = 217.0979,
calculated for C12H13N202, 217.0977. Anal. Calcd. For C12H12N202: C, 66.65; H,
5.59; N, 12.96. Found: C, 66.43; H, 5.49; N, 12.90. Melting Point = 172-174°C.
(ll-78). To a flame dried 50 mL round bottom flask was added "-66 (0.155
g, 0.440 mmol) and anhydrous DCM (15.0 mL). Then anhydrous TEA (0.180 mL,
1.32 mmol) was added and the mixture was cooled to 0°C. EDCI (0.186 g, 0.968
mmol) was then added and the mixture stirred at 0°C under nitrogen for 1 h and
then refluxed for 15 h. After the first step was completed, (as indicated by TLC),

the solution was cooled to 0°C and a solution of NaH (0.0880 g, 2.20 mmol) in

132

MeOH (5.00 mL) was added dropwise. The cloudy mixture stirred at 0°C for 1 h
and then at room temperature for 2 h. The mixture was acidified with 5% HCI and
then the solvents were removed. The aqueous mixture was extracted with EtOAc
(3 x 30.0 mL) and then the organics were combined, dried using anhydrous
sodium sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 30% EtOAc; 70% DCM) affording the product as a
whitish solid. Yield (0.0720 mg, 67.0%). 1H NMR (500MHz), acetone: 5 2.74 (dd,
J = 7.3, 14.1 Hz, 1H), 2.92 (dd, J = 7.9, 14.1 Hz, 1H), 3.78 (s, 3H), 5.12 (m, 1H),
5.20 (m, 1H), 5.72 (m, 1H), 6.94 (d, J = 9.0 Hz, 2H), 7.52 (d, J = 9.0 Hz, 2H),
7.64 (s, 1H), 9.63 (s, 1H); 13C NMR (125MHz), Acetone: 5 43.4, 55.5, 68.2,
114.6, 120.6, 127.6, 131.7, 132.2, 156.9, 160.3, 176.1. IR: (KBr) 3251 cm'1
(broad), 1774cm“, 1724cm". HRMS: [M + H]* = 247.1088, calculated for
C13H15N203, 247.1083. Anal. Calcd. For C13H14N203: C, 63.40; H, 5.73; N, 11.38.
Found: C, 62.74; H, 5.53; N, 11.35. Melting Point = 166-168°C.

(ll-79). To a flame dried 50 mL round bottom flask was added "-67 (0.151
g, 0.444 mmol) and anhydrous DCM (15.0 mL). Then anhydrous TEA (0.180 mL,
1.33 mmol) was added and the mixture was cooled to 0°C. EDCI (0.188 g, 0.977
mmol) was then added and the mixture stirred at 0°C under nitrogen for 1 h and
then refluxed for 15 h. After the first step was completed, (as indicated by TLC),
the solution was cooled to 0°C and a solution of NaH (0.0880 g, 2.20 mmol) in
MeOH (5.00 mL) was added dropwise. The cloudy mixture stirred at 0°C for 1 h
and then at room temperature for 2 h. The mixture was acidified with 5% HCI and

then the solvents were removed. The aqueous mixture was extracted with EtOAc

133

(3 x 30.0 mL) and then the organics were combined, dried using anhydrous
sodium sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 30% EtOAc; 70% DCM) affording the product as a
whitish solid. Yield (0.0600 g, 57.0%). 1H NMR (500MHz), acetone: 5 2.76 (dd, J
= 7.3, 14.0 Hz, 1H), 2.95 (dd, J = 7.2, 14.1 Hz, 1H), 5.14 (dd, J = 2.1, 10.1 Hz,
1H), 5.21 (dd, J = 2.1, 10.2 Hz, 1H), 5.72 (m, 1H), 7.16 (m, 2H), 7.67 (m, 2H),
7.75 (s, 1H), 9.74 (s, 1H); 13C NMR (125MHz), Acetone: 5 43.6, 68.2, 115.9 (d, J
= 21.7 Hz), 121.0, 128.6 (d, J = 8.2 Hz), 131.8, 135.9 (d, J = 3.1 Hz), 156.8,
183.2 (d, J = 245.4 Hz), 175.8. IR: (KBr) 3220 cm" (broad), 1780cm", 1730cm".
HRMS: [M + H]+ = 235.0876, calculated for C12H12FN202, 235.0883. Anal. Calcd.
For C12H11FN202: C, 61.53; H, 4.73; N, 11.96. Found: C, 61.48; H, 4.61; N,
11.81. Melting Point = 178-180’C.

(ll—80). To a flame dried 50 mL round bottom flask was added "-68 (0.156
g, 0.419 mmol) and anhydrous DCM (15.0 mL). Then anhydrous TEA (0.170 mL,
1.26 mmol) was added and the mixture was cooled to 0°C. EDCI (0.177 g, 0.922
mmol) was then added and the mixture stirred at 0°C under nitrogen for 1 h and
then refluxed for 15 h. After the first step was completed, (as indicated by TLC),
the solution was cooled to 0 °C and a solution of NaH (0.0840 g, 2.09 mmol) in
MeOH (5.00 mL) was added dropwise. The cloudy mixture stirred at 0°C for 1 h
and then at room temperature for 2 h. The mixture was acidified with 5% HCI and
then the solvents were removed. The aqueous mixture was extracted with EtOAc
(3 x 30.0 mL) and then the organics were combined, dried using anhydrous

sodium sulfate and concentrated. The crude residue was purified by column

134

chromatography (silica gel, 30% EtOAc; 70% DCM) affording the product as an
oil. Yield (0.0340 g, 31.0%). 1H NMR (500MHz), CDCI3: 5 3.07 (dd, J = 7.5, 14.2
Hz, 1H), 3.26 (dd, J = 6.9, 14.1 Hz, 1H), 5.12 (d, J = 11.1 Hz, 1H), 5.21 (d, J =
18.0 Hz, 1H), 5.71 (m, 1H), 7.33 (t, J = 7.7 Hz, 1H), 7.42-7.53 (m, 3H), 7.67 (d, J
= 7.3 Hz, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 7.6
Hz, 1H), 9.68 (s, 1H); 13C NMR (125MHz), CDCI3: 5 42.1, 69.7, 121.4, 124.4,
124.8, 125.6, 126.5, 129.6, 130.05, 130.08, 130.14, 132.3, 134.8, 157.6, 175.3.
IR: (KBr) 3245 cm" (broad), 1774cm'1, 1724cm". HRMS: [M + H]* = 287.1134,
calculated for C16H15N202, 267.1134.

(ll-81). To a flame dried 50 mL round bottom flask was added "-75 (0.150
g, 0.291 mmol) and anhydrous DCM (15.0 mL). Then anhydrous TEA (0.120 mL,
0.874 mmol) was added and the mixture was cooled to 0°C. EDCI (0.123 g,
0.640 mmol) was then added and the mixture stirred at 0°C under nitrogen for 1 h
and then refluxed for 15 h. After the first step was completed, (as indicated by
TLC), the solution was cooled to 0°C and a solution of NaH (0.0580 g, 1.46
mmol) in MeOH (5.00 mL) was added dropwise. The cloudy mixture stirred at
0°C for 1 h and then at room temperature for 2 h. The mixture was acidified with
5% HCI and then the solvents were removed. The aqueous mixture was
extracted with EtOAc (3 x 30.0 mL) and then the organics were combined, dried
using anhydrous sodium sulfate and concentrated. The crude residue was
purified by column chromatography (silica gel, 20% EtOAc; 80% DCM) affording
the product as a whitish solid. Yield (0.0830 g, 69.0%). 1H NMR (500 MHz),

acetone: 5 2.33 (s, 3H), 2.99 (dd, J = 7.2, 14.1 Hz, 1H), 3.05 (dd, J = 7.3, 14.2

135

Hz, 1H), 5.15 (d, J = 10.1 Hz, 1H), 5.22 (d, J = 17.5 Hz, 1H), 5.75 (m, 1H), 7.27
(t, J = 8.0 Hz, 1H), 7.36 (m, 3H), 7.65 (s, 1H), 7.83 (s, 1H), 7.88 (d, J = 8.3Hz.
3H), 8.01 (d, J = 8.5 Hz, 1H), 9.83 (s, 1H); 13C NMR (125 MHz), Acetone: 5 21.3,
41.5, 65.7, 114.4, 121.1, 121.8, 122.4, 124.2, 125.4, 125.7, 127.8, 128.7, 130.9,
131.4, 135.6, 136.3, 146.5, 156.7, 175.0. IR: (KBr) 3232 cm“, 17800m'1, 1730
cm". HRMS: [M + H]+ = 410.1178, calculated for C21H20N3O4S, 410.1175. Anal.
Calcd. For C21H19N304S: C, 61.60; H, 4.68; N, 10.26. Found: C, 61.59; H, 4.60;
N, 9.80. Melting Point = 226-228°C.

(II-83). To a flame dried 100 mL round bottom flask was added "-82 (1.21
g, 3.24 mmol) and anhydrous DCM (50.0 mL). Then cyclopent-1-enylmetnanol127
(0.477 g, 4.87 mmol) and DMAP (0.0400 g, 0.324 mmol) were added and the
mixture was brought down to 0°C. DCC (1 .00 g, 4.87 mmol) was then added and
the mixture was allowed to warm to room temperature overnight while stirring
under nitrogen. The white precipitate that formed was filtered off and the DCM
filtrate was washed with brine (1 x 20.0 mL), dried using anhydrous sodium
sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 20% EtOAc; 80% hexane) affording the product as a
whitish solid. Yield (0.997 g, 68.0%). 1H NMR (500MHz), CDCl3: 5 1.82 (p, J =
7.5 Hz, 2H), 2.13 (s, 2H), 2.27 (s, 2H), 4.20 (t, J = 6.6 Hz, 1H), 4.38 (m, 2H), 4.70
(m, 2H), 5.40 (d, J = 7.4 Hz, 1H), 5.52 (s, 1H), 5.89 (d, J = 6.9 Hz, 1H), 7.28-7.40
(m, 9H), 7.57 (d, J = 7.3 Hz, 2H), 7.74 (d, J = 7.5 Hz, 2H); 13c NMR (125MHz),
CDCI3: 5 23.1, 32.3, 32.5, 47.1, 58.0, 64.4, 67.1, 119.9, 125.0, 127.0, 127.1.

127.6, 128.5, 128.8, 129.1, 136.6, 138.1, 141.2, 143.7, 143.8, 155.3, 170.6. IR:

136

(NaCI) 3350 cm'1 , 1725 cm'1 (broad). HRMS: [M + H]+ = 454.2020, calculated for
C29H28NO4, 454.2018. Anal. Calcd. For C29H27NO4: C, 76.80; H, 6.00; N, 3.09.
Found: C, 76.97; H, 5.84; N, 3.12. Melting Point = 96-98°C.

(ll-84). To a flame dried 100 mL round bottom flask was added "-82 (1.25
g, 3.35 mmol) and anhydrous DCM (50.0 mL). Then 3-methyl-2-buten-1-ol (0.690
mL, 6.70 mmol) and DMAP (0.0410 g, 0.335 mmol) were added and the mixture
was brought down to 0°C. DCC (1.04 g, 5.03 mmol) was then added and the
mixture was allowed to warm to room temperature overnight while stirring under
nitrogen. The white precipitate that formed was filtered off and the DCM filtrate
was washed with brine (1 x 20.0 mL), dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 20% EtOAc; 80% hexane) affording the product as a whitish solid. Yield
(1.15 g, 77.0%). 1H NMR (500MHz), CDCI3: 5 1.62 (s, 3H), 1.71 (s, 3H), 4.21 (t, J
= 7.1 Hz, 1H), 4.35-4.44 (m, 2H), 4.54-4.72 (m, 2H), 5.27 (m, 1H), 5.40 (d, J =
7.6 Hz, 1H), 5.91 (d, J = 7.3 Hz, 1H), 7.27-7.41 (m, 9H), 7.58 (d, J = 7.3 Hz, 2H),
7.75 (d, J = 7.6 Hz, 2H); 13C NMR (125MHz), CDCI3: 5 17.9, 25.6, 47.1, 57.9,
62.7, 67.0, 117.7, 119.9, 125.0, 127.0, 127.1, 127.6, 128.4, 128.8, 136.7, 140.0,
141.2, 143.7, 143.8, 155.3, 170.7. IR: (NaCI) 3351 cm", 1724 cm’1 (broad).
HRMS: [M + H]+ = 442.2019, calculated for C23H28NO4, 442.2018. Anal. Calcd.
For C23H27NO4: C, 76.17; H, 6.16; N, 3.17. Found: C, 75.87; H, 6.10; N, 3.19.
Melting Point = 108-110°C.

(II-85). To a 100 mL round bottom flask was added “-83 (0.947 g, 2.09

mmol) and DCM (8.00 mL). The solution was brought down to 0°C and then

137

3385033
330gen
empvec
wthsat
333g 8
pudfied
90% DC
lst G
222-21

13C N13"

”3000
pipertd
Whoge
rem0v
wth S;
Umng
Dufifie
90$; [

Chara

piperidine (2.00 mL, 20.9 mmol) was added and the mixture stirred under
nitrogen for 1 h and then 1 h at room temperature. The solvent was then
removed and the crude residue was taken up in EtOAc (40.0 mL) and washed
with sat. NH4CI (1 x 10.0 mL) and brine (1 x 10.0 mL). The organics were dried
using anhydrous sodium sulfate and concentrated. The crude residue was
purified by column chromatography (silica gel, 30% EtOAc; 70% hexane, then
90% DCM 10% MeOH) affording the product as an oil. Yield (0.434 g, 90.0%).1H
NMR (500MHz), CDCI3: 5 1.77-1.83 (m, 2H), 1.91 (s, 2H), 2.10-2.15 (m, 2H),
2.22-2.28 (m, 2H), 4.59 (s, 1H), 4.63 (m, 2H), 5.49 (m, 1H), 7.24-7.37 (m, 5H);
13c NMR (125MHz), c0013: 5 23.4, 32.5, 32.9, 59.0, 84.1, 127.0, 128.1, 128.9,
138.8, 140.6, 174.0. IR: (KBr) 3385 (br)cm'1, 3321 cm", 1733 cm". HRMS: [M +
H]+ = 232.1341, calculated for C14H18N02, 232.1338. Anal. Calcd. For
Cr4H17N02: C, 72.70; H, 7.41; N, 6.06. Found: C, 72.05; H, 7.30; N, 6.09.

(ll-86). To a 100 mL round bottom flask was added “-84 (1.15 g, 2.61
mmol) and DCM (10.0 mL). The solution was brought down to 0°C and then
piperidine (2.60 mL, 26.1 mmol) was added and the mixture stirred under
nitrogen for 1 h and then 1 h at room temperature. The solvent was then
removed and the crude residue was taken up in EtOAc (50.0 mL) and washed
with sat. NH4C| (1 x 20.0 mL) and brine (1 x 20.0 mL). The organics were dried
using anhydrous sodium sulfate and concentrated. The crude residue was
purified by column chromatography (silica gel, 30% EtOAc; 70% hexane, then
90% DCM 10% MeOH) affording the product as an oil. Yield (0.488 g, 85.0%).

Characterization is for the TFA salt. 1H NMR (500MHz), DMSO: 5 1.58 (s, 3H),

138

1.66 (s, 3H), 4.60 (dd, J = 7.3, 12.2 Hz, 1H), 4.67 (dd, J = 7.0, 11.7 Hz, 1H), 5.22
(m, 1H), 5.26 (s, 1H), 7.45 (m, 5H), 8.89 (s, 3H); 13C NMR (125MHz), DMSO: 5
17.7, 25.2, 55.3, 62.6, 117.2 (q, J = 300.5 Hz), 117.5, 128.1, 128.9, 129.5, 132.5,
139.8, 157.9 (q, J = 30.9 Hz), 168.3. IR: (KBr) 3164 (br) cm", 1736 cm", 1675
cm". HRMS: [M + H]+ = 220.1341, calculated for C13H13N02, 220.1338. Anal.
Calcd. For C15H18F3NO4: C, 54.05; H, 5.44; N, 4.20. Found: C, 53.92; H, 5.26; N,
4.18. Melting Point = 118-120°C.

(II-87). To a flame dried 100 mL round bottom flask was added "-85
(0.406 g, 1.76 mmol) and anhydrous DCM (50.0 mL). Then the solution was
brought down to 0°C and ethoxycarbonyl isothiocyanate (0.240 mL, 2.11 mmol)
was added dropwise. The solution was allowed to warm to room temperature
overnight while stirring under a nitrogen atmosphere. The solvent was removed
and ether (30.0 mL) was added to the crude residue and was washed with sat.
sodium bicarbonate. The organics were dried using anhydrous sodium sulfate
and concentrated. The crude residue was purified by column chromatography
(silica gel, 20% EtOAc; 80% hexane) and the product was recrystallized with
EtOAc/hexanes affording the product as a white solid. Yield (0.518 g, 81.0%). 1H
NMR (500MHz), CDCI3: 5 1.29 (t, J = 7.1 Hz, 3H), 1.81 (m, 2H), 2.13 (m, 2H),
2.26 (m, 2H), 4.23 (m, 2H), 4.69 (s, 2H), 5.53 (s, 1H), 5.97 (d, J = 6.9 Hz, 1H),
7.30-7.42 (m, 5H), 8.08 (s, 1H), 10.62 (d, J = 6.5 Hz, 1H); 13C NMR (125MHz),
CDCI3: 5 14.1, 23.1, 32.3, 32.5, 61.8, 62.8, 64.5, 127.5, 128.7, 128.9, 129.1.
135.2, 138.1, 152.5, 169.4, 178.8. IR: (KBr) 3289 cm'1, 3226 cm", 1743cm‘1,

1724 cm". HRMS: [M + H]+ = 383.1387, calculated for C13H23N204S, 383.1379.

139

Anal. Calc
5.78: N, 7
(Il-
10428 g.
Drought c
.185 add
31813190
and ethe
sodium
and con
lislllca gr
9. 99.00..

is. 3H),

910.44

mlnot)

Anal. Calcd. For C18H22N204S: C, 59.65; H, 6.12; N, 7.73. Found: C, 59.36; H,
5.78; N, 7.69. Melting Point = 58-60°C.

(II-88). To a flame dried 100 mL round bottom flask was added "-86
(0.428 g, 1.95 mmol) and anhydrous DCM (50.0 mL). Then the solution was
brought down to 0°C and ethoxycarbonyl isothiocyanate (0.260 mL, 2.35 mmol)
was added dropwise. The solution was allowed to warm to room temperature
overnight while stirring under a nitrogen atmosphere. The solvent was removed
and ether (30.0 mL) was added to the crude residue and was washed with sat.
sodium bicarbonate. The organics were dried using anhydrous sodium sulfate
and concentrated. The crude residue was purified by column chromatography
(silica gel, 20% EtOAc; 80% hexane) affording the product as an oil. Yield (0.678
g, 99.0%).1H NMR (500MHz), CDCI3: 5 1.29 (t, J = 7.1 Hz, 3H), 1.60 (s, 3H), 1.69
(s, 3H), 4.23 (m, 2H), 4.55 (dd, J = 7.3, 12.4 Hz, 1H), 4.68 (dd, J = 7.1, 12.2 Hz,
1H), 5.26 (m, 1H), 5.95 (d, J = 6.8 Hz, 1H), 7.30-7.44 (m, 5H), 7.96 (s, 1H), 10.59
(d, J = 6.6 Hz, 1H); 13C NMR (125MHz), CDCI3: 5 14.1, 18.0, 25.6, 61.9, 62.88,
62.89, 117.7, 127.5, 128.6, 128.9, 135.3, 140.0, 152.5, 169.6, 178.8. IR: (KBr)
3282 cm" , 3232 cm", 1724 cm'1(with shoulder). HRMS: [M + H]* = 351.1380,
calculated for C17H23N204S, 351.1379. Anal. Calcd. For C17H22N204S: C, 58.27;
H, 6.33; N, 7.99. Found: C, 54.58; H, 5.86; N, 7.46.

(II-89). To a flame dried 50 mL round bottom flask was added "-69 (0.150
g, 0.446 mmol) and anhydrous DCM (15.0 mL). Then anhydrous TEA (0.190 mL,
1.34 mmol) was added and the mixture was cooled to 0°C. EDCI (0.188 g, 0.981

mmol) was then added and the mixture stirred at 0°C under nitrogen for 1 h and

140

Immref
fiesoh

MeOHl

then refluxed for 15 h. After the first step was completed, (as indicated by TLC),
the solution was cooled to 0°C and a solution of NaH (0.0890 g, 2.23 mmol) in
MeOH (5.00 mL) was added dropwise. The cloudy mixture stirred at 0°C for 1 h
and then at room temperature for 2 h. The mixture was acidified with 5% HCI and
then the solvents were removed. The aqueous mixture was extracted with EtOAc
(3 x 30.0 mL) and then the organics were combined, dried using anhydrous
sodium sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 10% acetone; 90% DCM) affording the product as a
whitish solid. Yield (0.0680 g, 66.0%). 1H NMR (500MHz), acetone: 5 1.70 (s,
3H), 2.70 (d, J = 13.2 Hz, 1H), 2.99 (d, J = 13.7 Hz, 1H), 4.85 (m, 1H), 4.91 (m,
1H), 7.30-7.42 (m, 3H), 7.66 (m, 2H), 7.69 (s, 1H), 9.64 (s, 1H); 13C NMR
(125MHz), acetone: 5 24.1, 46.8, 68.7, 116.7, 126.3, 128.7, 129.2, 140.4, 140.7,
158.8, 178.1. IR: (KBr) 3243 cm", 1772 cm'1, 1724 cm". HRMS: [M + Hr =
231.1139, calculated for C13H15N202, 231.1134. Anal. Calcd. For C13H14N202: C,
67.81; H, 6.13; N, 12.17. Found: C, 66.99; H, 5.86; N, 11.81. Melting Point = 169-
171°C.

(lI-90). To a flame dried 50 mL round bottom flask was added "-87 (0.150
g, 0.414 mmol) and anhydrous DCM (15.0 mL). Then anhydrous TEA (0.170 mL,
1.24 mmol) was added and the mixture was cooled to 0°C. EDCI (0.175 g, 0.911
mmol) was then added and the mixture stirred at 0°C under nitrogen for 1 h and
then refluxed for 15 h. After the first step was completed, (as indicated by TLC),
the solution was cooled to 0°C and a solution of NaH (0.0830 g, 2.07 mmol) in

MeOH (5.00 mL) was added dropwise. The cloudy mixture stirred at 0°C for 1 h

141

arc-then.
tre'ltlles
3X3OC
333m 8
mromato
.nsepara
"01.3) 1

3.45 (t, J

(II
9. 0.429

129 mlr
m”‘01) VI.
thenrefi
the sol”
IlleOH (

and ther

and then at room temperature for 2 h. The mixture was acidified with 5% HCI and
then the solvents were removed. The aqueous mixture was extracted with EtOAc
(3 x 30.0 mL) and then the organics were combined, dried using anhydrous
sodium sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 10% acetone; 90% DCM) affording the two
unseparatable diastereomers (1.321 ratio) as a whitish solid. Yield (0.0700 g,
66.0%). 1H NMR (500MHz), acetone: 5 1.28-1.96 (m, 8H), 2.26-2.38 (m, 4H),
3.45 (t, J = 7.4 Hz, 1H), 3.56 (t, J = 7.6 Hz, 1H), 4.03 (s, 1H), 4.73 (s, 1H), 4.94
(s, 1H), 5.03 (s, 1H), 7.32-7.43 (m, 6H), 7.55 (s, 1H), 7.64-7.71 (m, 4H), 7.87 (s,
1H), 9.66 (s, 1H); 13C NMR (125MHz), acetone: 5 24.9, 25.2, 35.9, 36.6, 49.9,
50.0, 71.2, 72.3, 108.4, 109.2, 126.7, 127.0, 128.63, 128.69, 129.21, 129.22,
139.7, 139.8, 150.7, 151.7, 157.2, 157.4, 175.7, 176.3. IR: (KBr) 3289 cm“, 1774
cm", 1718 cm'1. HRMS: [M + H]+ = 257.1295, calculated for C15H17N202,
257.1290. Anal. Calcd. For C15H16N202: C, 70.29; H, 6.29; N, 10.93. Found: C,
68.27; H, 6.37; N, 10.48. Melting Point = 234-236°C.

(II-91). To a flame dried 50 mL round bottom flask was added "-88 (0.150
g, 0.429 mmol) and anhydrous DCM (15.0 mL). Then anhydrous TEA (0.180 mL,
1.29 mmol) was added and the mixture was cooled to 0°C. EDCI (0.181 g, 0.944
mmol) was then added and the mixture stirred at 0°C under nitrogen for 1 h and
then refluxed for 15 h. After the first step was completed, (as indicated by TLC),
the solution was cooled to 0°C and a solution of NaH (0.0860 g, 2.15 mmol) in
MeOH (5.00 mL) was added dropwise. The cloudy mixture stirred at 0°C for 1 h

and then at room temperature for 2 h. The mixture was acidified with 5% HCI and

142

renthe
13x 30
$3031
mmmd

wodsh

HRMS
ForC.

91125
0874
0.640
and u
by Tl
Ol'gar
Conce
98L 1
OH,‘}

3H).

then the solvents were removed. The aqueous mixture was extracted with EtOAc
(3 x 30.0 mL) and then the organics were combined, dried using anhydrous
sodium sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 10% acetone; 90% DCM) affording the product as a
whitish solid. Yield (0.0490 g, 47.0%). 1H NMR (500MHz), acetone: 5 1.09 (s,
3H), 1.14 (s, 3H), 4.99 (dd, J = 1.0, 17.3 Hz, 1H), 5.07 (dd, J = 1.2, 11.0 Hz, 1H),
6.02 (dd, J = 10.7, 17.3 Hz, 1H), 7.35 (m, 3H), 7.75 (m, 2H), 7.95 (s, 1H), 9.70 (s,
1H); 13C NMR (125MHz), acetone: 5 22.1, 22.8, 44.6, 72.7, 115.3, 128.1, 128.3,
128.6, 136.8, 142.9, 156.6, 175.2. IR: (KBr) 3243 cm“, 1772 cm", 1718 cm'1.
HRMS: [M + H]+ = 245.1292, calculated for C14H17N202, 245.1290. Anal. Calcd.
For C14H16N202: C, 68.83; H, 6.60; N, 11.47. Found: C, 68.67; H, 6.39; N, 11.26.
Melting Point = 229-231’C.

(II-94). To a flame dried 50 mL round bottom flask was added "-75 (0.150
g, 0.291 mmol) and anhydrous DCM (15.0 mL). Then anhydrous TEA (0.120 mL,
0.874 mmol) was added and the mixture was cooled to 0°C. EDCI (0.123 g,
0.640 mmol) was then added and the mixture stirred at 0°C under nitrogen for 1 h
and then refluxed for 15 h. After the rearrangement was completed (as indicated
by TLC), the reaction was quenched by addition of ice water (10.0 mL). The
organic layer was separated, dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 10% Ethyl acetate; 90% DCM; ARr = 0.69) affording the product as a thick
oil. 1H NMR (500MHz), CDCI3: 5 1.38 (t, J = 7.2 Hz, 3H), 1.52 (s, 3H), 2.36 (s,

3H), 3.00 (d, J = 13.9 Hz, 1H), 3.04 (d, J = 13.8 Hz, 1H), 4.42 (dq, J = 2.8, 7.1

143

Hz. 2t

ti‘fR

Hz, 2H), 4.85 (s, 1H), 4.98 (m, 1H), 8.48 (s, 1H), 7.24-7.38 (m, 4H), 7.64 (s, 1H),
7.79 (d, J = 8.5 Hz, 2H), 7.91 (d, J = 8.9 Hz, 1H), 8.00 (d, J = 8.3 Hz, 1H); 130
NMR (125MHz), once: 8 13.9, 21.5, 23.8, 44.4, 83.2, 84.5, 113.7, 118.0, 119.3,
121.6, 123.7, 124.1, 125.3, 127.0, 127.1, 130.0, 134.7, 135.7, 138.2, 145.4,
147.5, 151.4, 189.4. IR: (KBr) 3340 cm", 1812 cm", 1770 cm“, 1728 cm".

HRMS: [M + H]+ = 496.1548, calculated for C25H26N3068, 496.1542.

144

g)

11.

10.

11.

References

Matsumoto, K.; Rapoport, H., Preparation and properties of some acyl-
guanidines. J. Org. Chem. 1968, 33, 552-8.

Kenyon, G. L.; Rowley, G. L., Tautomeric preferences among
glycocyamidines. J. Am. Chem. Soc. 1971, 93, 5552-60.

Lempert, C., The chemistry of the glycocyamidines. Chem. Rev. 1959, 59,
667-736.

Mukerjee, A. K.; Joseph, K.; Homami, S. S.; Tikdari, A. M., Triethylamine,
ethanol-mediated disciplined reactions of S-benzylisothiouronium chloride
with unsaturated 2-oxazolin-5-ones: synthesis of (Z)-2-amino-4-
arylmethylene-2-imidazolin-5-ones, 5-benzoylamino-2-benzylthio-6-oxo-
4,4-spirocyclohexyl-1,4,5,6-tetrahydropyrimidine, and their structures.
Heterocycles 1991, 32, 1317-25.

Prager, R. H.; Tsopelas, C., Approaches to the synthesis of 5-
benzylidene-2-imidazolin-4-ones. Aust. J. Chem. 1990, 43, 367-74.

Tikdari, A. M.; Tripathy, P. K.; Mukerjee, A. K., Reactions of some 1,3-
diamino nucleophiles with azlactones. J. Chem. Soc., Perkin Trans. 1
1988, 1659-62.

Wegner, M. M.; Rapoport, H., Catalytic hydrogenation of some
acylguanidines. J. Org. Chem. 1978, 43, 3840-4.

Reddick, R. E.; Kenyon, G. L., Syntheses and NMR studies of specifically
labeled [2-15N]phosphocreatine, [2-15N]creatinine, and related 15N-
labeled compounds. J. Am. Chem. Soc. 1987, 109, 4380-7.

Guella, G.; Mancini, I.; Zibrowius, H.; Pietra, F., Aplysinopsin-type
alkaloids from Dendrophyllia sp., a scleractinian coral of the family
Dendrophylliidae of the Philippines. Facile photochemical (Z/E)
photoisomerization and thermal reversal. Helv. Chim. Acta 1989, 72,
1444-50.

Hu, B.; Malamas, M.; Ellingboe, J.; Largis, E.; Han, S.; Mulvey, R.; Tillett,
J., New oxadiazolidinedione derivatives as potent and selective human
beta 3 agonists. Bioorg. Med. Chem. Lett. 2001, 11, 981-984.

Pardasani, R. T.; Pardasani, P.; Jain, A.; Kohli, 8., Synthesis and
semiempirical calculations of thiazolidinone and imidazolidinone
derivatives of alpha -diones. Phos., Sulf. Sil. Relat. Elem. 2004, 179,
1569-1575.

145

19.

20.

21

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

Ryabukhin, S. V.; Plaskon, A. S.; Volochnyuk, D. M.; Pipko, S. E.;
Tolmachev, A. A., Chlorotrimethylsilane mediated synthesis of 5-(2-

hydroxybenzoyl)pyrimidines from 3-formylchromones. Heterocycles 2008,
75, 583-597.

Li, C.; Danishefsky, S. J., Studies directed toward the synthesis of the
guanidine alkaloid, spiroleucettadine: some observations at the level of
structure. Tetrahedron Lett. 2005, 47, 385-387.

Chandrasekhar, S.; Karri, P., Aromaticity in azlactone anions and its
significance for the Erlenmeyer synthesis. Tetrahedron Lett. 2006, 47,
5763-5766.

Appleton, D. R.; Page, M. J.; Lambert, G.; Berridge, M. V.; Copp, B. R.,
Kottamides A-D: novel bioactive imidazolone-containing alkaloids from the
New Zealand ascidian Pycnoclavella kottae. J. Org. Chem. 2002, 67,
5402-5404.

Cafieri, F.; Gattorusso, E.; Mangoni, A.; TaglialateIa-Scafati, 0.,
Dispacamides, anti-histamine alkaloids from Caribbean Agelas sponges.
Tetrahedron Lett. 1996, 37, 3587-3590.

Chan, G. W.; Mong, S.; Hemling, M. E.; Freyer, A. J.; Offen, P. H.;
DeBrosse, C. W.; Sarau, H. M.; Westley, J. W., New leukotriene B4
receptor antagonist: Ieucettamine A and related imidazole alkaloids from
the marine sponge Leucetta microraphis. J. Nat. Prod. 1993, 56, 116-21.

Cimino, G.; De Rosa, 8.; De Stefano, S.; Mazzarella, L.; Puliti, R.;
Sodano, G., Isolation and x-ray crystal structure of a novel bromo
compound from two marine sponges. Tetrahedron Lett. 1982, 23, 767-8.

Davis, R. A.; Aalbersberg, W.; Meo, S.; Moreira da Rocha, R.; Ireland, C.
M., The isolation and synthesis of polyandrocarpamines A and B. Two
new 2-aminoimidazolone compounds from the Fijian ascidian,
Polyandrocarpa sp. Tetrahedron 2002, 58, 3263-3269.

Edrada, R. A.; Stessman, C. C.; Crews, P., Uniquely modified imidazole
alkaloids from a calcareous Leucetta sponge. J. Nat. Prod. 2003, 66, 939-
942.

Keenan, M.; Jones, K.; Hibbert, F., Highly efficient and flexible total
synthesis of coelenterazine. Chem. Commun. (Cambridge) 1997, 323-
324.

146

25.

27.

28.

29.

30.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

Loukaci, A.; Guyot, M.; Chiaroni, A.; Riche, C., A new indole alkaloid from
the marine tunicate Dendrodoa grossularia. J. Nat. Prod. 1998, 61 , 519-
522.

Mancini, l.; Guella, G.; Debitus, C.; Dubet, D.; Pietra, F., lmidazolone and
imidazolidinone artifacts of a pivotal imidazolthione, zyzzin, from the
poecilosclerid sponge Zyzzya massalis from the Coral Sea. The first
thermochromic systems of marine origin. Helv. Chim. Acta 1994, 77,
1886-94.

Olofson, A.; Yakushijin, K.; Horne, D. A., Unusually Large 13C NMR
Chemical Shift Differences between Neutral and Protonated
Glycocyamidines. New Insights on Previously Reported Chemical Shift
Assignments and Chemical Properties. J. Org. Chem. 1998, 63, 5787-
5790.

Sato, H.; Tsuda, M.; Watanabe, K.; Kobayashi, J. i., Rhopaladins A-D,
new indole alkaloids, from marine tunicate Rhopalaea sp. Tetrahedron
1998, 54, 8687-8690.

Tsukamoto, S.; Kato, H.; Hirota, H.; Fusetani, N., Mauritiamine, a New
Antifouling Oroidin Dimer from the Marine Sponge Agelas mauritiana. J.
Nat. Prod. 1996, 59, 501-3.

Vergne, C.; Appenzeller, J.; Ratinaud, C.; Martin, M.-T.; Debitus, C.;
Zaparucha, A.; Al-Mourabit, A., Debromodispacamides B and D: Isolation
from the Marine Sponge Agelas mauritiana and Stereoselective Synthesis
Using a Biomimetic Proline Route. Org. Lett. 2008, 10, 493—496.

Wang, H.; Lim, K. L.; Yeo, S. L.; Xu, X.; Sim, M. M.; Ting, A. B; Wang, Y.;
Yee, 8.; Tan, Y. H.; Pallen, C. J., Isolation of a Novel Protein Tyrosine
Phosphatase Inhibitor, 2-MethyI-Fervenulone, and Its Precursors from
Streptomyces. J. Nat. Prod. 2000, 63, 1641-1646.

Annoura, H.; Tatsuoka, T., Total syntheses of hymenialdisine and
debromohymenialdisine: stereospecific construction of the 2-amino-4-oxo-
2-imidazolin-5(Z)-disubstituted ylidene ring system. Tetrahedron Lett.
1995, 36, 413-16.

Lindel, T.; Hoffmann, H., Synthesis of dispacamide from the marine
sponge Agelas dispar. Tetrahedron Lett. 1997, 38, 8935-8938.

Crews, P.; Clark, D. P.; Tenney, K., Variation in the alkaloids among lndo-
Pacific Leucetta sponges. J. Nat. Prod. 2003, 66, 177-182.

147

32.

R
30.

39.

40.

41.

42.

43.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

Molina, P.; Almendros, P.; Fresneda, P. M., An iminophosphorane-
mediated efficient synthesis of the alkaloid Ieucettamine B of marine
origin. Tetrahedron Lett. 1994, 35, 2235-6.

Fresneda, P. M.; Molina, P.; Sanz, M. A., The first synthesis of the
bis(indole) marine alkaloid rhopaladin D. Synlett 2000, 1190-1192.

Janosik, T.; Johnson, A.-L.; Bergman, J., Synthesis of the marine alkaloids
Rhopaladins A, B, C and D. Tetrahedron 2002, 58, 2813-2819.

Keifer, P. A.; Schwartz, R. E.; Koker, M. E. S.; Hughes, R. G., Jr.;
Rittschof, D.; Rinehart, K. L., Bioactive bromopyrrole metabolites from the
Caribbean sponge Agelas conifera. J. Org. Chem. 1991, 56, 2965-75.

Kobayashi, J.; Tsuda, M.; Ohizumi, Y., A potent actomyosin ATPase
activator from the Okinawan marine sponge Agelas cf. nemoechinata.
Experientia 1991, 47, 301-4.

O'Malley, D. R; Li, K.; Maue, M.; Zografos, A. L.; Baran, P. 8., Total
Synthesis of Dimeric Pyrrole-lmidazole Alkaloids: Sceptrin, Ageliferin,
Nagelamide E, Oxysceptrin, Nakamuric Acid, and the Axinellamine
Carbon Skeleton. J. Am. Chem. Soc. 2007, 129, 4762-4775.

Olofson, A.; Yakushijin, K.; Horne, D. A., Synthesis of Mauritiamine. J.
Org. Chem. 1997, 62, 7918-7919.

Koswatta, P. B.; Sivappa, R.; Dias, H. V. R.; Lovely, C. J., Total Synthesis
of (+-)—Calcaridine A and (+-)-epi-Calcaridine A. Org. Lett. 2008, 10, 5055-
5058.

Anzai, K., alpha -Diketone rearrangement. Reaction of 1,2-
cyclohexanedione with arginine and with guanidine. Bull. Chem. Soc. Jap.
1969, 42, 3314-17.

Atwood, J. L.; Barbour, L. J.; Heaven, M. W.; Raston, C. L., Synthesis of
2-imino-5-phenylimidazolidin-4-one and the structure of its trifluoroacetate
salt. J. Chem. Crystallogr. 2003, 33, 175-179.

Bengelsdorf, I. S., A reaction of guanidine with glyoxals in aqueous
solution. The preparation of glycocyamidines. J. Am. Chem. Soc. 1953,
75, 3138-40.

Bhat, B. A.; Dhar, K. L.; Puri, S. C.; Spiteller, M., A novel one-pot
rearrangement reaction of 2,3-epoxydiaryl ketones: synthesis of (+-)-5,5-
disubstituted imidazolones and 5,5-disubstituted hydantoins. Synlett 2006,
2723-2726.

148

48.

49.

50.

51.

52.

53.

54.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

Chu, Y.; Lynch, V.; Iverson, B. L., Synthesis and DNA binding studies of
bis-intercalators with a novel spiro-cyclic linker. Tetrahedron 2006, 62,
5536-5548.

Ding, M.-W.; Tu, H.-Y.; Liu, Z.—J., Application of carbodiimide in
heterocyclic synthesis: new facile synthesis of 2-aminoimidazolinone
derivatives. Synth. Commun. 1997, 27, 3657-3662.

Drewry, D. H.; Ghiron, C., Solid-phase synthesis of 2-
aminoimidazolinones. Tetrahedron Lett. 2000, 41, 6989-6992.

Evindar, G.; Batey, R. A., Peptide Heterocycle Conjugates: A Diverted
Edman Degradation Protocol for the Synthesis of N-Terminal 2-
lminohydantoins. Org. Lett. 2003, 5, 1201-1204.

Frutos, R. P.; Johnson, M., Regiocontrolled synthesis of highly-
functionalized fused imidazoles: a novel synthesis of second generation
LFA-1 inhibitors. Tetrahedron Lett. 2003, 44, 6509-6511.

Fu, M.; Fernandez, M.; Smith, M. L.; Flygare, J. A., Solid-Phase Synthesis
of 2-Aminoimidazolones. Org. Lett. 1999, 1, 1351-1353.

Gadwood, R. C.; Kamdar, B. V.; Dubray, L. A. C.; Wolfe, M. L.; Smith, M.
P.; Watt, W.; Mizsak, S. A.; Groppi, V. E., Synthesis and biological activity
of spirocyclic benzopyran imidazolone potassium channel openers. J.
Med. Chem. 1993, 36, 1480-7.

Garratt, P. J.; Thorn, S. N.; Wrigglesworth, R., Preparation of
imidazolones from N-cyano-N'-methylcarboxyguanidines. An unusual
carbon-nitrogen bond formation in the hydrogenolysis of a benzyl ester in
an attempted synthesis of an inhibitor of carboxypeptidase A. Tetrahedron
1993, 49, 6885-98.

Gavrilyuk, J. I.; Evindar, G.; Batey, R. A., Peptide-Heterocycle Hybrid
Molecules: Solid-Phase Synthesis of a 400-Member Library of N-Terminal
2-lminohydantoin Peptides. J. Comb. Chem. 2006, 8, 237-246.

Gillman, K. W.; Higgins, M. A.; Poindexter, G. S.; Browning, M.; Clarke, W.
J.; Flowers, 8.; Grace, J. E.; Hogan, J. B.; McGovern, R. T.; lben, L. G.;
Mattson, G. K.; Ortiz, A.; Rassnick, 8.; Russell, J. W.; AntaI-Zimanyi, |.,
Synthesis and evaluation of 5,5-diphenylimidazolones as potent human
neuropeptide Y5 receptor antagonists. Bioorg. Med. Chem. 2006, 14,
5517-5526.

Heras, M.; Ventura, M.; Linden, A.; Villalgordo, J. M., Reaction of a-
iminomethylene amino esters with mono- and bidentate nucleophiles: a

149

55.

56.

57.

58.

59.

60.

62.

63.

64.

65.

86.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

straightforward route to 2-amino-1H-5-imidazolones. Tetrahedron 2001,
57, 4371—4388.

Kang, J. H.; Moon, J. T.; Kim, J.; Joo, D. J.; Lee, J. Y., Synthesis of 2-
arylsubstituted imidazolone derivatives. Bull. Korean Chem. Soc. 2007,
28, 913-914.

Khodair, A. l.; Bertrand, P., A new approach to the synthesis of substituted
4-imidazolidinones as potential antiviral and antitumor agents.
Tetrahedron 1998, 54, 4859-4872.

Kiec-Kononowicz, K.; Zejc, A., Syntheses of some 2-amino-5,5-
diphenylimidazolin-4-one derivatives. Pol. J. Chem. 1980, 54, 2217-24.

Kwon, C. H.; Iqbal, M. T.; Wurpel, J. N. D., Synthesis and anticonvulsant
activity of 2-iminohydantoins. J. Med. Chem. 1991, 34, 1845-9.

Kwon, C. H.; Nagasawa, H. T., Facile synthesis of substituted 2-
iminohydantoins. Synth. Commun. 1987, 17, 1677-82.

Lange, U. E. W., Solid-phase synthesis of 2,3,5-trisubstituted 4H-
imidazolones. Tetrahedron Lett. 2002, 43, 6857-6860.

Mueller, H.; Carell, T., A carbocyclic analog of the oxidatively generated
DNA lesion spiroiminodihydantoin. Eur. J. Org. Chem. 2007, 1438-1445.

Nishimura, T.; Kitajima, K., Reaction of guanidines with alpha -diketones.
Syntheses of 4,5-disubstituted-Z-aminoimidazoles and 2,6-
unsymmetrically substituted imidazo[4,5-d]imidazoles. J. Org. Chem.
1979, 44, 818-24.

Nishimura, T.; Nakano, K.; Shibamoto, S.; Kitajima, K., Novel synthesis of
2-(disubstituted amino)-5(4)-phenylimidazoles. J. Heterocycl. Chem. 1975,
12, 471-6.

Sharma, V.; Lansdell, T. A.; Jin, G.; Tepe, J. J., Inhibition of Cytokine
Production by Hymenialdisine Derivatives. J. Med. Chem. 2004, 47, 3700-
3703.

Sharma, V.; Tepe, J. J., Potent inhibition of checkpoint kinase activity by a
hymenialdisine-derived indoloazepine. Bioorg. Med. Chem. Lett. 2004, 14,
4319-4321.

Simig, G.; Lempert, K.; Tamas, J.; Czira, G., Hydantoins, thiohydantoins,

and glycocyamidines. 41. Reaction of N-cyano amines with 1-(tert-butyl)-
3,3-diphenylaziridinone. General method for the synthesis of 1-alkyl-, 1-

150

69.

71.

72.

73.

74.

75.

76.

77.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

aralkyI-, and 1-aryl-5,5-diphenyl hydantoins and -g|ycocyamidines.
Tetrahedron 1975, 31, 1195-200.

Slade, P. G.; Priestley, N. D.; Sugden, K. D., Spiroiminodihydantoin as an
Oxo—Atom Transfer Product of 8-Oxo-2'-deoxyguanosine Oxidation by
Chromium(V). Org. Lett. 2007, 9, 4411-4414.

Stover, J. S.; Ciobanu, M.; Cliffel, D. E.; Rizzo, C. J., Chemical and
Electrochemical Oxidation of C8-Arylamine Adducts of 2'-
Deoxyguanosine. J. Am. Chem. Soc. 2007, 129, 2074-2081.

Takeuchi, H.; Hagiwara, S.; Eguchi, S., A new efficient synthesis of
imidazolinones and quinazolinone by intramolecular aza-Wittig reaction.
Tetrahedron 1989, 45, 6375-86.

Talaty, E. R.; Gomez, J. A.; Dillon, J. A.; Palomino, E.; Agho, M. 0.; Batt,
B. C.; Gleason, K. J.; Park, S.; Hernandez, J. R., Reaction of aziridinones
with thiourea: a novel synthesis of specifically substituted glycocyamidines
and hydantoins. Synth. Commun. 1987, 17, 1063-70.

Varga, L.; Nagy, T.; Kovesdi, |.; Benet-Buchholz, J.; Dorman, G.; Urge, L.;
Darvas, F., Solution-phase parallel synthesis of 4,6-diaryI-pyrimidine-2-
ylamines and 2-amino-5,5-disubstituted-3,5-dihydro-imidazol-4-ones via a
rearrangement. Tetrahedron 2003, 59, 655-662.

Ye, P.; Sargent, K.; Stewart, E.; Liu, J.-F.; Yohannes, D.; Yu, L., Novel
and Expeditious Microwave-Assisted Three-Component Reactions for the
Synthesis of Spiroimidazolin-4-ones. J. Org. Chem. 2006, 71, 3137-3140.
Ye, W.; Sangaiah, R.; Degen, D. E.; Gold, A.; Jayaraj, K.; Koshlap, K. M.;
Boysen, G.; Williams, J.; Tomer, K. B.; Ball, L. M., A 2-Iminohydantoin
from the Oxidation of Guanine. Chem. Res. Toxicol. 2006, 19, 506-510.

Lovely, C. J.; Du, H.; He, Y.; Dias, H. V. R., Oxidative rearrangement of
imidazoles with dimethyldioxirane. Org. Lett. 2004, 6, 735-738.

Nishimura, T.; Toku, H.; Ueno, T.; Shibamoto, 8.; Imai, T., Mechanism of
Voges-Proskauer reaction. Chem. Lett. 1972, 649-52.

Toi, K.; Bynum, F.; Norris, E.; ltano, H. A., J. Biol. Chem. 1967, 242, 1036.

Bach, R. D.; Domagala, J. M., The effect of Lewis acid and solvent on
concerted 1,2-acyl migration. J. Org. Chem. 1984, 49, 4181-8.

151

.78.

80.

82.

83.

85.

86.

87.

88.

89.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

Chang, C.-L.; Kumar, M. P.; Liu, R.-S., A Highly Efficient Ruthenium-
Catalyzed Rearrangement of alpha ,beta -Epoxyketones to 1,2-Diketones.
J. Org. Chem. 2004, 69, 2793-2796.

House, H. O.; Ryerson, G. D., The rearrangement of alpha ,beta -epoxy
ketones. VIII. Effect of substituents on the rate of rearrangement. J. Am.
Chem. Soc. 1961, 83, 979-83.

House, H. 0.; Wasson, R. L., Rearrangement of alpha ,beta -
epoxyketones. V. Rearrangements resulting in ring contraction. J. Am.
Chem. Soc. 1957, 79, 1488-92.

Rao, T. B.; Rao, J. M., Silica gel catalyzed rearrangement of alpha -epoxy
ketones to 1,2-diketones. Synth. Commun. 1993, 23, 1527-33.

Bajaj, K.; Srivastava, V. K.; Lata, S.; Chandra, R.; Kumar, A., Synthesis of
some new benzothiazepinyl- and benzoxazepinyl indoles as antipsychotic
agents. Indian J. Chem, Sect. B: Org. Chem. Incl. Med. Chem. 2003,
423, 1723-1728.

Martinez, R.; Ramon, D. J.; Yus, M., Easy alpha -a|kylation of ketones with
alcohols through a hydrogen autotransfer process catalyzed by
RuCI2(DMSO)4. Tetrahedron 2006, 62, 8988-9001.

Engel, N.; Kubel, B.; Steglich, W., C-C Linkage of Carboxylic Acids with
Allyl Alcohols Using 2-Phenylglycine as Vehicle. Angew. Chem. Int. Ed.
1977, 16, 394-396.

Kubel, B.; Hofle, G.; Steglich, W., Hetero Cope Rearrangements in the
Cyclization of Allyl and Propargyl Esters of N-Acyl Amino Acids to
Oxazolin-5-ones. Angew. Chem. Int. Ed. 1975, 14, 58-59.

Firestone, R. A.; Harris, E. E.; Reuter, W., Synthesis of pyridoxine by
Diels-Alder reactions with 4-methyl-5-alkoxyoxazoles. Tetrahedron 1967,
23, 943-55.

Maeda, |.; Takehara, M.; Togo, K.; Asai, S.; Yoshida, R., Synthetic
intermediate of pyridoxine. l. A novel synthesis of 5-alkoxy-2-carboxy-4-
methyloxazole. Bull. Chem. Soc. Jap. 1969, 42, 1435-7.

Fischer, J.; Kilpert, G; Klein, U.; Steglich, W., Stereochemistry of [3.3]-
sigmatropic rearrangements in the oxazole series. Tetrahedron 1986, 42,
2063-74.

Burger, K.; Geith, K.; Gaa, K., A Simple Access to 2-Substituted 3,3,3-
Trifluoroalanine Derivatives. Angew. Chem. Int. Ed. 1988, 27, 848-849.

152

90. Burger, K.; Hennig, L.; Tsouker, P.; Spengler, J.; Albericio, F.; Koksch, B.,
Domino reactions with fluorinated five-membered heterocycles. alpha -
Trifluoromethyl alpha -amino acids with unsaturated side-chains. Amino
Acids 2006, 31, 427-433.

91. Burger, K.; Hennig, L.; Tsouker, P.; Spengler, J.; Albericio, F.; Koksch, 8.,
Synthesis of alpha -trifluoromethyl alpha -amino acids with aromatic,
heteroaromatic and ferrocenyl subunits in the side chain. Amino Acids
2006, 31 , 55-62.

92. Castelhano, A. L.; Home, 8.; Taylor, G. J.; Billedeau, R.; Krantz, A.,
Synthesis of alpha-amino acids with beta, gamma-unsaturated side
chains. Tetrahedron 1988, 44, 5451-5466.

93. Castelhano, A. L.; Pliura, D. H.; Taylor, G. J.; Hsieh, K. C.; Krantz, A.,
Allenic suicide substrates. New inhibitors of vitamin B6 linked
decarboxylases. J. Am. Chem. Soc. 1984, 106, 2734-5.

94. Colombo, L.; Casiraghi, G.; Pittalis, A., Stereoselective Synthesis of C-
Glycosyl alpha-Amino Acids. J. Org. Chem. 1991, 56, 3897-3900.

95. Colombo, L.; di Giacomo, M.; Ciceri, P., Practical stereoselective
synthesis of alpha -D-C-mannosyl-(R)-alanine. Tetrahedron 2002, 58,
9381-9386.

96. Holladay, M. W.; Nadzan, A. M., Synthesis of alpha-Benzyl gamma-
Lactam, alpha-Benzyl delta-Lactam, and alphaBenzylproline Derivatives
as Conformationally Restricted Analogues of Phenylalaninamide. J. Org.
Chem. 1991, 56, 3900-3905.

97. Wittmann, U.; Tranel, F.; Froehlich, R.; Haufe, 6., Novel synthesis of 4-
fluoropyridines based on 2-fluoroallylic alcohols by succeeding Ireland-

Claisen and aza-Cope rearrangements as key steps. Synthesis 2006,
2085-2096.

98. Fisk, J. S.; Mosey, R. A.; Tepe, J. J., The diverse chemistry of oxazol-5-
(4H)-ones. Chem. Soc. Rev. 2007, 36, 1432-1440.

99. Wan, Z.-K.; Wacharasindhu, S.; Binnun, E.; Mansour, T., An efficient
direct amination of cyclic amides and cyclic ureas. Org. Lett. 2006, 8,
2425-2428.

100. Bartlett, P. A.; Tanzella, D. J.; Barstow, J. F., Ester-Enolate Claisen
Rearrangement of Lactic Acid Derivatives. J. Org. Chem. 1982, 47.

153

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

Overman, L. E.; Rogers, B. N.; Tellew, J. E.; Trenkle, W. C.,
Stereocontrolled Synthesis of the Tetracyclic Core of the Bisguanidine
Alkaloids Palau'amine and Styloguanidine. J. Am. Chem. Soc. 1997, 119,
7159-7160.

Hupp, C. D.; Tepe, J. J., 1-EthyI-3-(3-dimethylaminopropyl)carbodiimide
Hydrochloride-Mediated Oxazole Rearrangement: Gaining Access to a
Unique Marine Alkaloid Scaffold. J. Org. Chem. 2009, 74, 3406-3413.

Kent, D. R.; Cody, W. L.; Doherty, A. M., Two new reagents for the

guanylation of primary, secondary and aryl amines. Tetrahedron Lett.
1996, 37, 8711-8714.

Kim, K. S.; Qian, L., Improved Method for the Preparation of Guanidines.
Tetrahedron Left. 1993, 34, 7677-80.

Linton, B. R.; Carr, A. J.; Orner, B. P.; Hamilton, A. D., A Versatile One-
Pot Synthesis of 1,3-Substituted Guanidines from Carbamoyl
Isothiocyanates. J. Org. Chem. 2000, 65, 1566-1568.

Ramadas, K.; Srinivasan, N., An expedient synthesis of substituted
guanidines. Tetrahedron Lett. 1995, 36, 2841-4.

Shibanuma, T.; Shiono, M.; Mukaiyama, T., A convenient method for the
preparation of carbodiimides using 2-chloropyridinium salt. Chem. Lett.
1977, 575-6.

Levallet, C.; Lerpiniere, J.; Ko, S. Y., The HgCI2-promoted guanylation
reaction: the scope and limitations. Tetrahedron 1997, 53, 5291-5304.

Yu, Y.; Ostresh, J. M; Houghten, R. A., Solid-Phase Synthesis of 1,5-
Disubstituted 2-Aryliminoimidazolidines. J. Org. Chem. 2002, 67, 3138-
3141.

Yong, Y. F.; Kowalski, J. A.; Lipton, M. A., Facile and Efficient Guanylation
of Amines Using Thioureas and Mukaiyama's Reagent. J. Org. Chem.
1997, 62, 1540-1542.

Manimala, J. C.; Anslyn, E. V., A highly efficient method for the synthesis
of guanidinium derivatives. Tetrahedron Lett. 2002, 43, 565-567.

Alvarado, C.; Diaz, E.; Guzman, A., Total synthesis of ulongamide A, a

cyclic depsipeptide isolated from marine cyanobacteria Lyngbya sp.
Tetrahedron Lett. 2007, 48, 603-607.

154

118.

119.

120.

1.22.

123.

I24.

113.

114.

115.

116.

117.

118.

119.

120.

121.

122.

123.

124.

Kojima, S.; Chabayashi, T.; Umeda, Y.; lwamoto, A.; Tanabe, K.; Ohkata,
K., Stereoselective synthesis of a novel chiral piperazine. Heterocycles
2008, 75, 1493-1501.

Shimizu, M.; Kume, K.; Fujisawa, T., Switchover of the diastereoselectivity
induced by a simple selection of titanium(lV) halides in the addition
reaction of silyl ketene acetals to a chiral imine. Tetrahedron Left. 1995,
36, 5227-30.

Oertqvist, P.; Peterson, S. D.; Aakerblom, E.; Gossas, T.; Sabnis, Y. A.;
Fransson, R.; Lindeberg, G.; Danielson, U. H.; Karlen, A.; Sandstroem, A.,
Phenylglycine as a novel P2 scaffold in hepatitis C virus NS3 protease
inhibitors. Bioorg. Med. Chem. 2007, 15, 1448-1474.

Yan, S.; Larson, 6.; Wu, J. 2.; Appleby, T.; Ding, Y.; Hamatake, R.; Hong,
2.; Yao, N., Novel thiazolones as HCV NSSB polymerase allosteric
inhibitors: Further designs, SAR, and X-ray complex structure. Bioorg.
Med. Chem. Left. 2007, 17, 63-67.

Barberis, C.; Voyer, N.; Roby, J.; Chenard, S.; Tremblay, M.; Labrie, P.,
Rapid access to N-Boc phenylglycine derivatives via benzylic lithiation
reactions. Tetrahedron 2001, 57, 2965—2972.

Garg, N. K.; Sarpong, R.; Stoltz, B. M., The First Total Synthesis of
Dragmacidin D. J. Am. Chem. Soc. 2002, 124, 13179-13184.

Kharasch, M. 8.; Kane, S. S.; Brown, H. 0., Presence of indole in
\"practical\" a-methylnaphthalene. J. Am. Chem. Soc. 1940, 62, 2242-3.

Nishizawa, M.; Yamamoto, H.; Seo, K.; Imagawa, H.; Sugihara, T., TMS
Triflate-Catalyzed Cleavage of Prenyl (3-Methylbut-2-enyl) Esters. Org.
Lett. 2002, 4, 1947-1949.

Geistlinger, T. R.; Guy, R. K., Novel Selective Inhibitors of the Interaction
of Individual Nuclear Hormone Receptors with a Mutually Shared Steroid
Receptor Coactivator 2. J. Am. Chem. Soc. 2003, 125, 6852-6853.

Katritzky, A. R.; Rogovoy, B. V., Recent developments in guanylating
agents. ARKIVOC (Gainesville, FL, United States) 2005, 49-87.

Martin, N. I.; Woodward, J. J.; Marletta, M. A., NG-Hydroxyguanidines
from Primary Amines. Org. Left. 2006, 8, 4035-4038.

Rani, P.; Srivastava, V. K.; Kumar, A., Synthesis and anti-inflammatory
activity of heterocyclic indole derivatives. Eur. J. Med. Chem. 2004, 39,
449-452.

155

125. Frigerio, M.; Santagostino, M.; Sputore, S., A user-friendly entry to 2-
iodoxybenzoic acid (IBX). J. Org. Chem. 1999, 64, 4537-4538.

126. Yang, L.; Deng, G.; Wang, D.-X.; Huang, Z.-T.; Zhu, J.-P.; Wang, M.-X.,
Highly Efficient and Stereoselective N-Vinylation of Oxiranecarboxamides
and Unprecedented 8-endo-Epoxy-arene Cyclization: Expedient and
Biomimetic Synthesis of Some Clausena Alkaloids. Org. Lett. 2007, 9,
1387-1390.

127. Kim, D. D.; Lee, S. J.; Beak, P., Asymmetric Lithiation-Substitution

Sequences of Substituted Allylamines. J. Org. Chem. 2005, 70, 5376-
5386.

156

CHAPTER III

TOTAL SYNTHESIS OF AN INDOLE ALKALOID FROM THE TUNICATE
DENDRODOA GROSSULARIA AND TWO ANALOGS

III.A Pharmacological value of marine compounds

The biodiversity found in the marine environment offers a wealth of
organisms each containing unique metabolites and secondary metabolites with
chemically diverse structures.“ 2 Efforts to explore this rich and diverse set of
compounds have led to the opening of a “marine pipeline,” which allows for the
flow of these compounds into the pharmaceutical arena. The pharmaceutical
industry has benefited from the study of these organisms and the introduction of
the marine natural products they contain in many areas of research. Compounds
coming from marine organisms have shown to possess biological activity for
diseases such as cancer, arthritis, AIDS and have potential as anti-viral, anti-
microbial, anti-parasitic, analgesic and anti-inflammatory agentsf"5

The extraordinary biological properties that compounds from natural
products possess justify the isolation and harvesting of the sources for these
compounds. Unfortunately, these secondary metablites found in the marine
organisms are usually present in trace amounts making acquisition of an
appropriate amount of these compounds for testing very difficult. In order to
determine the biological potential of a particular compound, there are two options
commonly used: 1) massive recollection of the organism and reisolation of the
molecule or 2) development of a concise synthetic route to the molecule.6

Factors concerning the availablility of the marine organism and ease of isolation

157

will determine which method will access the desired compound the easiest and
fastest.
III.B Background on Dendrodoa grossularia

Invertebrate marine animals such as tunicates, which include sea squirts
and sea cucumbers, have been a prosperous source of structurally unique and
biologically interesting secondary metabolites. In particular, these marine
organisms are known to contain indole alkaloids, nitrogen containing compounds
that also posses an indole structural moiety.7 Furthermore, the novel frameworks
that these indole alkaloids possess often attracts the attention of chemists
interested in developing synthetic routes to the scaffolds. The tunicate
Dendrodoa grossularia (baked bean sea squirt), a red marine organism that
grows along the coasts of Brittany and in the Baltic and North Seas, contains

some heterocyclic alkaloids with unique scaffolds (Figure III-1).8'13

 

\ ‘N/ 0
N /
N y \ N
O N \ k
S’N o
\ \
\
N N
H H H
Dendrodoine .
3-IndonI-4H-imidazoI-4-one “bomon
\N/
N2\NH
__‘_ O
/
O \ N R
N
H
R = Indole (Grossularine 1) Indole Alkaloid
R = pOH-phenyl (Grossularine 2) III-1

Figure III-1. Alkaloids from Dendrodoa grossularia

158

Dendrodoine was isolated by Heitz and coworkers in 198010 and first
synthesized in 198414 and is a unique molecule that exhibited the first example of
a divalent sulfur-nitrogen bond found in a natural product, coming from its 1, 2, 4-
thiadiazole heterocyclic nucleus.12 Results from a brief biological study on the
compound revealed moderate cytotoxicity for L1210 leukemia cells with an ID50
of 10 pg/mL.13 An additional report suggests that dendrodoine inhibits the
synthesis of DNA in leukemia cells through preventing the incoporation of
thymidine into DNA.15

3-lndonI-4H-imidazoI-4-one was isolated and then synthesized in 1986 by
Guyot and coworkers.9 The compound was found to be light sensitive in solution
which resulted in the rearrangment of the molecule to yield additional
byproducts.9 Furthermore, a cytotoxicity assay revealed that the compound was
void of activity.13 Alboinon isolated in 1997 by Steffan and coworkers contains a
rare 1, 3, 5-oxadiazin-2-one scaffold and represents the first natural product
bearing this moiety.8 The synthesis of alboinon was attempted by the same
group using a protecting group on the indolic nitrogen, however, all attempts to
remove the protecting group resulted in the attack on the oxadiazinone ring.
Fortunately, it was found that oxidation of 3-lndonI-4H-imidazol-4-one with m-
chloroperbenzoic acid produced alboinon in a good yield.8 This result offers
insight into a potential biosynthetic route to alboinon suggesting that perhaps it is
synthesized in the tunicate by oxidation of 3-IndolyI-4H-imidazoI-4-one.

Grossularines 1 and 2 were isolated in 1989 and were originally given an

alternative structure. X-ray studies and the availability of larger amounts of the

159

compounds allowed the definite determination of the physical structure.“ 13' 16
Furthermore, these compounds represent the first natural products to contain an
o-carboline skeleton.13 The first total synthesis of both grossularine 1 and 2 was
achieved in 1995 using a thermal electrocyclic reaction to obtain the heterocyclic
core of both compounds.17 Grossularines 1 and 2 were found to be cytotoxic
toward L1210 leukemia cells displaying ID50 values of 6 and 4 pg/mL,
respectively. A closer investigation revealed that these compounds caused
accumulation of these cells in the G1 phase at concentrations of 10 ug/mL
(grossularine 1) and 1.5 pg/mL (grossularine 2).13 The mode of action for
grossularine 2’s cytotoxicity was examined and it was found that grossularine 2
intercalates into the DNA and also inhibits the incorporation of thymidine into
DNA. Grossularine 1 was found to have a somewhat more ambiguous mode of
action by giving results reminiscent of drugs that show intercalation as well as
alkylation.15 Furthermore, they were found even more cytotoxic toward the solid
tumor cell lines WiDr (colon) and MCF7 (breast) showing activity in the 10 ng/mL
range.13

The latest indole alkaloid (III-1) to come from Dendrodoa grossularia was
isolated in 1998 by Guyot and coworkers and contains a unique quaternary
imidazolone core.11 The structure was determined by 1D and 2D NMR
techniques as well as by information given by the X-ray structure of a derivative
of the indole alkaloid (Ill-4), obtained by treating the natural product with acetic
anhydride and pyridine (Scheme III-1). After an initial acetylation of the indolic

nitrogen and nitrogen contained within the imidazolone ring of III-1 affords III-2, it

160

is proposed that an aldol reaction occurs to give III-3. The derivative III-4 is finally
formed after a Bayliss-Hillman reaction occurs between III-3 and acetic anhydride
(Scheme Ill-1).11

Scheme III-1. Synthesis of derivative III-4

 

   

O O
/U\O/U\
pyridine
Indole Alkaloid Derivative III-4
Acetylation Bayliss-Hillman

reaction

 

   

III-2 III-3

The crystal structure of III-4 was ineffective at identifying the absolute
stereochemistry at the only stereogenic center in the natural product due to the
racemic nature of the crystal. It is suggested that the optical rotation observed for
the indole alkaloid III-1 and derivative III-4 ([010 = -15 and -12, respectively) is
due to the presence of an excess of one enantiomer. Furthermore, indole
alkaloid III-1 could be considered to be derived from 3-Indolyl-4H—imidazoI-4-one
another alkaloid from the same tunicate.11 As a result of the known biological

properties of other alkaloids from Dendrodoa grossularia and the physical

161

similarities the new indole alkaloid has with the Chk2 inhibitor, indoloazepine, a
total synthesis for the indole alkaloid was developed.
III.C Retrosynthetic analysis of indole alkaloid III-1

As a continuation of our laboratory’s focus on the development of new
heterocyclic methodologies for the syntheses of pharmacologically significant
scaffolds,18 the racemic total synthesis of indole alkaloid Ill-1 was completed
along with two additional analogs. The key step in forming the quaternary
stereocenter in the alkaloid and analogs utilizes a novel oxazole rearrangement19
(discussed in Chapter II) producing an oxazolone intermediate, ultimately leading
to a quaternary hydantoin.

Scheme III-2 illustrates our retrosynthetic strategy for the total synthesis of
indole alkaloid III-1. It was envisioned that the imidazolone moiety of the natural
product could be accessed from the hydantoin intermediate III-10.2“ 2‘ Through a
newly developed EDCI-mediated oxazole rearrangement, hydantoin III-10 was

thought to be formed from thiourea III-919' 22 In turn, thiourea III-9 could be

obtained from keto allyl ester III-5 after a few functional group manipulations.

162

Scheme III-2. Retrosynthesis of indole alkaloid III-1

 

   

Oxazole
Rearrangement

o H 0
DY .- OYNH
- /
\
OEt /N
n III-5 Ts

III-9

 

 

III.D Synthesis of indole alklaloid III-1

Keto allyl ester III-5 (Scheme III-3) was synthesized in excellent yields
(94%) through an esterification of 2-(1H-indol-3-yI)-2-oxoacetyl chloride, which is
the product of a known reaction between indole and oxalyl chloride,23' 24 with 2-
methyl-Z-propen-1-ol at room temperature for 16 h. Subsequent protection of the
indolic nitrogen with p-toluene sulfonyl chloride under basic conditions for 16 h
gave keto ester III-6 in a 77% yield, which when treated with hydroxylamine and
pyridine in dioxane, produced oxime III-7 in a 96% yield as a mixture of E and Z
isomers. Each isomer was isolated and both were carried on in the synthesis by
reducing the oximes using zinc powder and aqueous acetic acid at 0°C for 2 h to
amine III-8 in a 84% yield. Thiourea III-9 was formed in a 87% yield after treating
amine III-8 with commercially available ethoxy carbonyl isothiocyanate for 16 h at

room temperature (Scheme III-3).22

163

Scheme III-3. Synthesis of thiourea III-9

O O

 

 

 

 

O O
HO
Cl /\H/ o/\H/
\ EtOAc, rt, 18 h, \
N 94% N III-5
H H
TsCl, DMAP,
DIPEA, DCM
rt, 16 h, 77%
OH
2 o O O
N \
O/T A NHZOH-HCI (VT
\ Dioxane/HZO, \
N III-7 Pyr, reflux N III-6
. 16 h, 96% Ts
Ts
Zn, AcOH
0 °C, 2 h,
84%
H N o s H 0
2 0 N
CY A 0 Y O/\"/
\ EtO NCS = NH /
O
N Ill-8 DCM. rt, 16 h, 87 /o OEt /N
Ts T5

Ill-9

Subsequent treatment of thiourea III-9 with EDCI followed by sodium
methoxide yielded hydantoin III-10 in a 71% yield, resulting from the EDCI-
mediated oxazole rearrangement (Scheme III-4). The abbreviated proposed
mechanism for the formation of hydantoin III-10 is also highlighted in Scheme III-
4. After the thiourea moiety was converted into a carbodiimide intermediate using
EDCI, a 5-exo-dig cyclization occured between the carbonyl oxygen of the ester

and the carbodiimide to form a transient oxazole intermediate.22

164

Scheme III-4. Synthesis of hydantoin III-10

 

 

 

 

 

H
H 0 OT”
SYN O/\H/ 1.EDCI, TEA, DCM, HN O
0°C to reflux,9h
0 NH s
Y / 2. NaOMe, MeOH, \
OEt /N rt,4h,71%
Ts N
III-9 TS III-10
S
R’ 1’0 G
\
N. ,
TS TS
—- oxazolone

oxazole intermediate—

Through a Claisen-type rearrangement, the oxazole was transformed into
a quaternary oxazolone intermediate. Upon completion of the rearrangement, as
indicated by TLC, a solution of sodium methoxide in methanol was added to
complete the transformation from the oxazolone to hydantoin III-1t).22 Treatment

of hydantoin III-10 with Lawesson’s reagent25

for 24 h under refluxing conditions
produced thiohydantoin III-11 (Scheme Ill-5) in a 82% yield, which was
selectively methylated at the thiocarbonyl using methyl iodide, DMAP, and
diisopropylethylamine at room temperature for 2 h to furnish imidazolone Ill-12 in
a 83% yield. After heating imidazolone III-12 with a THF solution of

dimethylamine in a sealed tube at 75 °C for 14 h, imidazolone III-13 was

produced in a 94% yield.22

165

Scheme III-5. Synthesis of alkaloid III-1

 

H H
08/“ Sm“
O L . O
HN awesson S HN
Rgt
\ toluene, reflux, \
24 h, 82%
"l‘ III-10 "l' III-11
Ts Ts
Mel, DMAP,
DIPEA, DCM
f1, 2 h, 83%

 

   

\
NH
/ in THF
sealed tube
N 75 °C, 14 h, 94%
\ .
Ts II|13
KOEt, EtOH
reflux, 24 h,
86%

 

1. 0804, NMO,
rt, 4 h

2. NalO4, 0 °C,
2 h, 62%

 

   

N
H III-14 ""1

 

 

 

The final two steps for the total synthesis of III-1 began with the
deprotection of the indole nitrogen of III-13 using potassium ethoxide and ethanol
under refluxing conditions to produce imidazolone III-14 in an 86% yield. Finally,
oxidation of the terminal alkene of III-14 was achieved through a two-step/one-
pot modified Johnson-Lemieux26 reaction to give indole alkaloid Ill-1 in a 62%
yield (Scheme Ill-5). A tabulated spectral comparison of the synthesized alkaloid

(III-1) and the isolated natural product is given below in Table III-1. Fortunately, a

166

suitable crystal of III-1 was able to be formed and allowed 3 X-ray crystal
structure of III-1 to be determined (Figure III-2), which also confirms the structure

of the natural produc

t22

Table III-1. Comparison of spectral data from synthesized natural product
(III-1) and isolated natural product

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Spectrum Indole Alkaloid (III-1) Isolated Natural Product A5
2.18 (s, 3H) 2.18 (s, 3H) 0.00

3.05 (s, 3H) 3.04 (s, 3H) 0.01

3.23 (s, 3H) 3.22 (s, 3H) 0.01

3.48 (dd, J =18, 37 Hz, 2H) 3.45 (dd, J =16, 8 Hz, 2H) 0.01
1H NMR 8.97 (t, J = 7 Hz, 1H) 6.9 (t, J = 8 Hz, 1H) 0.07
(ppm in 7.09 (t, J = 7 Hz, 1H) 7.1 (t, J = 8 Hz, 1H) 0.01
00300) 7.28 (s, 1H) 7.28 (s, 1H) 0.00
7.33 (d, J = 8 Hz, 1H) 7.33 (d, J = 8 Hz, 1H) 0.00

7.45 (d, J = 8 Hz, 1H) 7.48 (d, J = 8 Hz, 1H) 0.01
8.24 (s, 1H, DMSO-d5) 8.2 (s, 1H, DMSO-d5) 0.04
11.01 (s, 1H, DMSO-d6) 11.01 (s, 1H, DMSO-d6) 0.00

31.0 31 0.0

38.9 37 0.1

38.8 38.8 0.0

49.0 48 1.0

87.0 87 0.0

112.8 112.6 0.0

114.5 114.8 0.1

13.31: :33: 133-3 9.3
CD300) 122.7 122.7 0.0
124.1 124.1 0.0

125.8 125.8 0.0

138.8 138.6 0.0

170.8 170.8 0.0

191.7 191.7 0.0

207.3 207.3 0.0

IR (cm") 3234, 1720, 1703, 1811 3200, 1710, 1840 NA

HRMS

[Mm]. 299.1508 299.1483 NA

 

167

 

 

Figure III-2. X-ray crystal structure of indole alkaloid III-1
III.E Retrosynthetic analysis of analogs 1 and 2
The versatility of the synthetic pathway developed for the natural product
was illustrated by the synthesis of 2 analogs that required no major modification
of the original synthetic route. Hypothetically, a number of analogs could be

developed with the synthetic route as a result of the diversity allowed in the

168

rearrangement and the variety of amines that could be used to create the final
imidazolone.

Our retrosynthetic strategy for the total synthesis of analog 1 and analog 2
is illustrated in Scheme III-6. It was envisioned that the imidazolone moiety of the
analog 2 (Ill-19) could be accessed from the hydantoin intermediate Ill-10
utilizing classical chemical modifications.” 21 Hydantoin III-1O could be formed
from thiourea III-9 using the protocol developed for the EDCI-mediated
rearrangement. Analog 1 (Ill-16) was also envisioned to be formed from thiourea
III-9 by slightly modifying the rearrangement to include a removal of the tosyl
protecting group.

Scheme III-6. Retrosynthesis of analog 1 (III-16) and analog 2 (III-19)

N
H Ill-19

 

III.F Synthesis of analogs 1 and 2
The first analog, hydantoin Ill-16 shown in Scheme III-7, was synthesized
using the same thiourea (III-9) used to produce the natural product. Instead of

carefully monitoring the second step of the rearrangement to avoid removal of

169

the tosyl protecting group, a mixture of potassium ethoxide and ethanol was used
and refluxed overnight to yield hydantoin III-15 in a 83% yield. Using the same
procedure as the natural product synthesis, the terminal olefin on hydantoin III-15
was oxidized to the ketone in a 61% yield affording hydantoin III-16 (analog 1).19

Scheme III-7. Synthesis of analog 1 (III-16)

 

H O
SYN O/T 1.EDCI, TEA, DCM,
O NH / 0 °C to reflux, 9 h :
2. KOEt, EtOH,
OEt N reflux, 83%
/
Ts
Ill-9

 

1. 0804, NMO, rt
2. NaIO4, 0°c, 81%

 

 

The second analog shown in Scheme III-8, imidazolone III-19, took
advantage of the synthetic handle that S-methylimidazolone III-12 provided due
to its reactivity and tendency to be substituted with a nucleophile. In this instance,
the S-methyl group was replaced using ammonium hydroxide to give imidazolone
III-17 in a 72% yield. The deprotection method previously described with the
natural product synthesis was also used to afford imidazolone III-18 in a 47%
yield. Finally, the synthesis of analog 2 (Ill-19) was completed after the terminal
alkene of III-18 was oxidized under previously determined conditions.
Unfortunately, due to the difficult nature of isolating the product, only 19% of
imidazolone Ill-19 was recovered although the overall conversion was higher.19

In general, the free amino imidazolones at every step were very hard to isolate

170

due to their tendency to stay in the aqueous phase during extractions, even after
using n-butanol as an extracting solvent.

Scheme III-8. Synthesis of analog 2 (III-19)

 

 

NH4OH, THF, _
sealed tube,
90°C,72%
KOEt, EtOH
reflux, 47%
1. OsO4, NMO, rt

 

‘ 2. NalO4, 0°c, 19%

   

171

III.G General experimental information

Reactions were carried out in flame-dried glassware under nitrogen
atmosphere. All reactions were magnetically stirred and monitored by TLC with
0.25 pm pre-coated silica gel plates using either UV light or iodine to visualize the
compounds. Column chromatography was carried out on Silica Gel 60 (230-400
mesh) supplied by EM Science. Yields refer to chromatographically and
spectroscopically pure compounds unless otherwise noted. Infrared spectra
were recorded on a Nicolet IR/42 spectrometer. 1H and 13C NMR spectra were
recorded on a Varian Unity Plus-500 spectrometer or a Varian lnova-300, as
noted in the experimental for each compound. Chemical shifts are reported
relative to the residue peaks of the solvent (CDCI3: 7.24 ppm for 1H and 77.0
ppm for 13C) (Acetone-d5: 2.04 ppm for 1H and 29.8 ppm for 13C) (DMSO-d5:
2.49 ppm for 1H and 39.5 ppm for 13C). The following abbreviations are used to
denote the multiplicities: s = singlet, d = doublet, dd = doublet of doublets, t =
triplet, and m = multiplet. Low resolution mass spectra were recorded on a
Hewlet—Packard 5890 Series II gas chromatograph connected to a TRIO-1 EI
mass spectrometer. HRMS were obtained with a Micromass Q-ToF Ultima API
LC-MS/MS mass spectrometer. Elemental analysis data were obtained on a
Perkin Elmer 2400 Series II CHNS/O analyzer. Purity of compounds, whose
elemental analyses were above the ACS tolerated 0.4% deviation, were
confirmed by 1H NMR and 13C NMR. Melting points were obtained using an
Electrothermal® capillary melting point apparatus and are uncorrected.

Reagents and solvents were purchased from commercial suppliers and used

172

without further purification. Anhydrous methylene chloride and toluene were
dispensed from a delivery system which passes the solvents through a column
packed with dry neutral alumina.
III.H Experimental procedures and characterization

(III-5). To a flame dried 100 mL round bottom flask was added EtOAc
(50.0 mL) and 2-(1H-indol-3-yI)-2-oxoacetyl chloride23' 2“ (2.00 g, 9.88 mmol).
The yellow mixture was put under an N2 atmosphere. Then 2-methyI-2-propene-
1-ol (4.00 mL, 48.3 mmol) was added. Within minutes the yellow cloudy mixture
became an orange clear solution. The solution stirred overnight at room
temperature under N2. The EtOAc was evaporated by rotary evaporation and the
solid was re-dissolved in EtOAc and washed with brine (2 x 30.0 mL). The
aqueous layer was extracted with EtOAc (2 x 30.0 mL) and the organics were
combined, dried using anhydrous sodium sulfate, and concentrated to give a the
product as a dark reddish solid. Yield: (2.21 g, 94.0%). 1H NMR (300MHz),
Acetone: 5 1.82 (s, 3H), 4.78, (s, 2H), 4.99 (s, 1H,), 5.10 (s, 1H), 7.26-7.33 (m,
2H), 7.55-7.59 (m, 1H), 8.30-8.33 (m, 1H), 8.43 (s, 1H), 11.33 (s, 1H); 13C NMR
(75MHz), Acetone: 5 19.6, 69.0, 113.2, 113.7, 114.2, 122.5, 123.7, 124.8, 126.9,
137.8, 138.1, 140.7, 164.1, 179.6. IR: (NaCI) 1610 cm", 1740 cm'1, 3190 (broad)
cm". M.S: calculated for C14H13N03 [M+] = 243 and found [M+] = 243.0; Anal.
Calcd. For C14H13N03: C, 69.12; H, 5.39; N, 5.76. Found: C, 67.78; H, 5.32; N,
5.26. Melting Point = 122-124°C.

(Ill-6). To a 500 mL flame dried round bottom flask was added III-5 (10.7

g, 44.0 mmol) and anhydrous DCM (0.250 L). Then TsCl (16.7 g, 88.1 mmol),

173

re

W

(I!

In

DMAP (13.4 g, 0.110 mol) and DIPEA (19.2 mL, 0.110 mol) were added and the
mixture stirred at room temperature under nitrogen overnight. The solvent was
removed and diethyl ether (0.200 L) was added to the residue and was washed
with 1% HCI (2 x 80.0 mL) and brine (1 x 80.0 mL). The organics dried with
anhydrous sodium sulfate and concentrated. The crude residue was purified by
column chromatography (silica gel, 30% EtOAc; 70% Hexane) affording the
product as a whitish solid. Yield (13.6 g, 77.0%). 1H NMR (500MHz), CDCI3: 5
1.84 (s, 3H), 2.34, (s, 3H), 4.78 (s, 2H), 5.03 (s, 1H), 5.11 (s, 1H), 7.27 (d, J = 8.0
Hz, 2H), 7.36 (m, 2H), 7.84 (d, J = 8.3 Hz, 2H), 7.94 (d, J = 7.3 Hz, 1H), 8.33 (d,
J = 7.33 Hz, 1H), 8.80 (s, 1H); 13C NMR (125MHz), CDCI3: 5 19.5, 21.6, 69.4,
113.1, 114.5, 117.0, 122.9, 125.2, 126.1, 127.2, 127.6, 130.3, 134.2, 134.5,
136.7, 138.6, 146.1, 161.3, 178.4. IR: (NaCI) 1670 cm", 1732 cm". HRMS: [M +
H]+ = 398.1075, calculated for 021H20N058, 398.1062. Anal. Calcd. For
C21H19N058: C, 63.46; H, 4.82; N, 3.52. Found: C, 63.46; H, 5.00; N, 3.39.
Melting Point = 86-88°C.

(III-7). To a 250 mL round bottom flask was added III-6 (2.10 g, 5.29
mmol) and dioxane (0.100 L). Then hydroxylamine hydrochloride (1.09 g, 15.9
mmol) was added along with a little water (5.00 mL). Then pyridine (1.36 mL,
16.9 mmol) was added and the mixture refluxed overnight under nitrogen. The
solvents were removed and diethyl ether (0.100 L) was added to the residue and
was washed with brine (1 x 50.0 mL). The organics were dried with anhydrous
sodium sulfate and concentrated. The crude residue was purified by column

chromatography (silica gel, 30% EtOAc; 70% Hexane) affording the product as a

174

thick clear oil. Yield (2.10 g, 96.0%). Isomer A: 1H NMR (500MHz), CDCI3: 5 1.83
(s, 3H), 2.32 (s, 3H), 4.82 (s, 2H), 5.02 (s, 1H), 5.15 (s, 1H), 7.14 (d, J = 8.5 Hz,
2H), 7.17 (t, J = 8.0 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.73 (d, J = 8.5 Hz, 2H),
7.77 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 9.14 (s, 1H); 13C
NMR (125MHz), CDCI3: 5 19.4, 21.3, 69.1, 113.1, 113.7, 114.5, 123.0, 124.1,
125.6, 126.8, 126.9, 127.2, 129.9, 134.4, 135.0, 138.7, 145.4, 146.5, 162.5.
Isomer B: 1H NMR (500MHz), CDCI3: 5 1.78 (s, 3H), 2.33 (s, 3H), 4.75 (s, 2H),
4.97 (s, 1H), 5.04 (s, 1H), 7.18 (d, J = 8.5 Hz, 2H), 7.25 (t, J = 8.0 Hz, 1H), 7.35
(t, J = 8.0 Hz, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.5 Hz, 2H), 8.02 (d, J =
8.0 Hz, 1H), 8.28 (s, 1H), 9.78 (s, 1H); 130 NMR (125MHz), c0013: 8 19.3, 21.3,
69.4, 109.6, 113.1, 114.2, 122.4, 123.2, 124.7, 126.8, 128.0, 129.8, 130.0, 133.9,
134.6, 138.8, 143.0, 145.3, 162.8. IR: (NaCI) 3460 cm", 1736 cm'1. HRMS: [M +
H]+ = 413.1167, calculated for C21H21N2058, 413.1171. Anal. Calcd. For
021H20N2058: C, 61.15; H, 4.89; N, 6.79. Found: C, 60.17; H, 4.87; N, 6.56.
(III-8). To a 1 L round bottom flask was added water (0.165 L) and AcOH
(0.165 L). Then III-7 (13.6 g, 33.0 mmol) was dissolved in THF (0.100 L) and was
added to the aqueous acid. The mixture was brought down to 0°C and zinc (21.5
g, 0.330 mol) was then slowly added in small portions over 20 min. The
suspension stirred at 0°C for 2 h. The solid was filtered off and the filtrate was
reduced and then brought to a pH of 8 using concentrated ammonium hydroxide.
The amine was extracted into ethyl acetate (4 x 0.150 L), dried using anhydrous
sodium sulfate and concentrated. The crude residue was purified by column

chromatography (silica gel, 30% EtOAc; 70% DCM) affording the product as a

175

thick oil. Yield (11.1 g, 84.0%). 1H NMR (500MHz), CDCI3: 5 1.51 (s, 3H), 1.93 (s,
2H), 2.30 (s, 3H), 4.51 (dd, J = 14.0, 19.2 Hz, 2H), 4.77 (m, 2H), 4.85 (s, 1H),
7.16-7.32 (m, 4H), 7.58 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 2H),
7.94 (d, J = 8.0 Hz, 1H); 13c NMR (125MHz), once: 8 19.1, 21.4, 51.8, 88.5,
113.3, 113.6, 120.1, 121.5, 123.2, 123.8, 124.9, 126.7, 128.7, 129.8, 135.1,
135.2, 139.2, 144.9, 172.9. IR: (NaCI) 3389 cm“, 3350 cm‘, 1740 cm". HRMS:
[M + H]+ = 399.1369, calculated for C21H23N204S, 399.1379. Anal. Calcd. For
C21H22N204S: C, 63.30; H, 5.56; N, 7.03. Found: C, 63.18; H, 5.70; N, 6.87.
(III-9). To a flame dried 500 mL round bottom flask was added III-8 (10.4
g, 26.1 mmol) and anhydrous DCM (0.250 L). The solution was brought down to
0°C and ethoxycarbonyl isothiocyanate (3.54 mL, 31.4 mmol) was added
dropwise. The solution was warmed to room temperature and stirred overnight
under nitrogen. The solvents were removed and the product was recrystallized
from EtOAc/Hexanes to give a white powder. Yield (12.1 g, 87.0%). 1H NMR
(500MHz), CDCI3: 5 1.27 (t, J = 7.1 Hz, 3H), 1.52 (s, 3H), 2.31 (s, 3H), 4.21 (m,
2H), 4.55 (s, 2H), 4.81 (d, J = 8.0 Hz, 2H), 6.26 (d, J = 7.1 Hz, 1H), 7.18-7.33 (m,
4H), 7.62 (d, J = 7.9 Hz, 1H), 7.72-7.76 (m, 3H), 7.92 (d, J = 8.3 Hz, 1H), 8.02 (s,
1H), 10.60 (d, J = 7.1 Hz, 1H); 13C NMR (125MHz), CDCI3: 5 14.1, 19.1, 21.5,
54.8, 62.9, 69.3, 113.7, 113.8, 116.0, 119.9, 123.6, 125.1, 126.1, 126.9, 128.2,
129.9, 134.9, 135.1, 138.8, 145.1, 152.4, 168.8, 179.0. IR: (NaCI) 3250 cm",
3227 cm", 1740 cm", 1724 cm", 1527 cm". HRMS: [M + H]: = 530.1575,
calculated for C25H28N30582, 530.1420. Anal. Calcd. For C25H27N30682: C, 56.69;

H, 5.14; N, 7.93. Found: C, 56.22; H, 5.13; N, 7.80. Melting Point = 138-140°C.

176

(III-10). To a flame dried 50 mL round bottom flask was added III-9 (0.265
g, 0.501 mmol), anhydrous DCM (20.0 mL), and anhydrous TEA (0.210 mL, 1.50
mmol). The solution was cooled to 0°C and then EDCI (0.212 g, 1.10 mmol) was
added and the mixture stirred at 0°C for 1 h and then refluxed until
disappearance of the starting material, as indicated on TLC. A solution of NaH
(0.100 g, 2.51 mmol) in MeOH (10.0 mL) was then added to the mixture and
stirred at room temperature for 2 h. The reaction was washed with 1% HCI (1 x
10.0 mL) and brine (1 x 10.0 mL) and the organics were dried using anhydrous
sodium sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 20% EtOAc; 80% DCM) affording the product as a
white solid. Yield (0.151 g, 71.0%). 1H NMR (500MHz), Acetone: 5 1.69 (s, 3H),
2.32 (s, 3H), 2.92 (d, J = 14.7 Hz, 1H), 3.10 (d, J = 14.7 Hz, 1H), 4.85 (s, 1H),
4.92 (s, 1H), 7.27 (m, 1H), 7.36 (m, 3H), 7.66 (s, 1H), 7.83 (s, 1H), 7.87 (d, J =
8.3 Hz, 2H), 7.94 (d, J = 7.8 Hz, 1H), 8.01 (d, J = 8.3 Hz, 1H), 9.81 (s, 1H);13C
NMR (125MHz), Acetone: 5 21.3, 24.2, 44.6, 66.0, 114.4, 117.1, 122.6, 122.8,
124.2, 125.2, 125.7, 127.8, 128.8, 130.9, 135.7, 136.4, 140.2, 146.5, 156.6,
175.2. IR: (NaCI) 3390 cm“, 3283 cm'1, 1778 cm", 1728 cm". HRMS: [M + H]+ =
424.1301, calculated for C22H22N304S, 424.1331. Anal. Calcd. For C22H21N304S:
C, 62.40; H, 5.00; N, 9.92. Found: C, 62.08; H, 5.15; N, 9.67. Melting Point =
236-238°C.

(III-11). To a 100 mL flame dried round bottom flask was added III-10
(0.595 g, 1.41 mmol) and anhydrous toluene (50.0 mL). Then Lawesson’s

reagent (0.341 g, 0.844 mmol) was added and the mixture was refluxed for 24 h.

177

 

The toll
mL). T
annyd
colon
as a
2.32

4.96

21 .z
128
176
44C

60.

The toluene was then taken off and the crude residue was put into EtOAc (50.0
mL). The organic solution was then washed with brine (1 x 20.0 mL), dried using
anhydrous sodium sulfate and concentrated. The crude residue was purified by
column chromatography (silica gel, 4% EtOAc; 96% DCM) affording the product
as a solid. Yield (0.506 g, 82.0%). 1H NMR (500MHz), Acetone: 5 1.77 (s, 3H),
2.32 (s, 3H), 3.01 (d, J = 13.6 Hz, 1H), 3.21 (d, J = 13.5 Hz, 1H), 4.90 (s, 1H),
4.96 (s, 1H), 7.26-7.40 (m, 4H), 7.76 (d, J = 8.0 Hz, 1H), 7.88-7.92 (m, 3H), 8.03
(d, J = 8.4 Hz, 1H), 9.46 (s, 1H), 10.80 (s, 1H); 13C NMR (125MHz), CDCI3: 5
21.4, 24.1, 43.8, 68.8, 114.4, 117.4, 120.8, 121.8, 124.4, 125.4, 125.9, 127.8,
128.3, 130.9, 135.5, 136.1, 139.6, 146.6, 175.5, 183.0. IR: (NaCI) 3190 cm",
1783 cm", 1895 cm". HRMS: [M + H]* = 440.1167, calculated for C22H22N3O382,
440.1103. Anal. Calcd. For C22H21N30382: C, 60.11; H, 4.82; N, 9.56. Found: C,
60.07; H, 4.75; N, 9.58. Melting Point = 224-226°C.

(III-12). To a flame dried 25 mL round bottom flask was added III-11
(0.105 g, 0.239 mmol), anhydrous DCM (10.0 mL), DMAP (0.00300 9, 0.0239
mmol) and DIPEA (0.210 mL, 1.20 mmol). Then Mel (0.0450 mL, 0.717 mmol)
was added and the solution stirred at room temperature under nitrogen for 2 h.
The DCM was taken off and diethyl ether (30.0 mL) was added to the residue.
The organic solution was washed with 1% HCI (1 x 10.0 mL) and brine (1 x 10.0
mL), dried using anhydrous sodium sulfate and concentrated. The crude residue
was purified by column chromatography (silica gel, 5% EtOAc; 95% DCM)
affording the product as an oil. Yield (0.0900 g, 83.0%). 1H NMR (500MHz),

Acetone: 5 1.76 (s, 3H), 2.31 (S, 3H), 2.60 (8, 3H), 2.77 (d, J = 13.2 Hz, 1H), 2.89

178

(d, J = 13.2 Hz, 1H), 4.66 (s, 1H), 4.72 (s, 1H), 7.22-7.35 (m, 4H), 7.70 (s, 1H),
7.83 (d, J = 8.3 Hz, 2H), 7.98 (d, J = 8.5 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 10.27
(s, 1H); 13c NMR (125MHz), CDCI3: 8 12.3, 21.3, 24.8, 48.2, 75.1, 114.2, 115.4,
122.3, 123.5, 123.8, 124.0, 125.4, 127.6, 129.7, 130.8, 135.8, 136.2, 141.3,
148.3, 180.8, 183.0. IR: (NaCI) 3239 cm’1 (broad), 1743 cm", 1701 cm". HRMS:
[M + H]+ = 454.1276, calculated for C23H24N30382, 454.1259. Anal. Calcd. For
023H23N30382: C, 60.90; H, 5.11; N, 9.26. Found: C, 59.91; H, 5.19; N, 8.90.
(III-13). To a flame dried 50 mL sealed tube was added III-12 (0.0900 g,
0.199 mmol) and dimethylamine (10.0 mL of a 2.0M solution in THF). The tube
was sealed and heated to an external sand bath temperature of 75°-90° for 14 h.
Once cooled, the tube was opened and nitrogen was purged into the tube to
expel any methane thiol that was formed. The whitish product was filtered off and
the filtrate was concentrated and the residue was triturated with diethyl ether to
obtain additional product, which was then filtered off. Yield (0.0810 g, 91 .0%). 1H
NMR (500MHz), DMSO: 5 1.65 (s, 3H), 2.31 (s, 3H), 2.79 (dd, J = 13.1, 26.0 Hz,
2H), 3.00 (s, 3H), 3.08 (s, 3H), 4.76 (s, 1H), 4.8 (s, 1H), 7.22 (t, J = 8.1 Hz, 1H),
7.32 (t, J = 8.2 Hz, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.58 (d, J = 8.0 Hz, 1H), 7.75 (s,
1H), 7.87-7.92 (m, 3H), 8.33 (s, 1H); 13C NMR (125MHz), DMSO: 5 20.9, 23.9,
36.0, 38.0, 43.2, 67.1, 113.0, 115.3, 121.5, 122.8, 123.0, 123.5, 124.6, 126.7,
127.9, 130.2, 134.1, 134.5, 139.7, 145.5, 169.8, 186.2. lR: (NaCI) 3210 cm'1
(broad), 1715 cm“, 1814 cm". HRMS: [M + H]+ = 451.1818, calculated for
C24H27N403S, 451.1804. Anal. Calcd. For C24H25N4038: C, 63.98; H, 5.82; N,

12.44. Found: C, 62.82; H, 5.96; N, 11.76. Melting Point = 142-144°C.

179

(III-14). To a 50 mL round bottom flask was added III-13 (0.200 g, 0.444
mmol) and EtOH (20.0 mL). Then KOEt (0.186 g, 2.22 mmol) was added and the
solution refluxed overnight. The solvent was taken off and EtOAc (30.0 mL) was
added to the residue. Then 1% HCI (30.0 mL) was added to acidity the solution.
The EtOAc layer was discarded and the acidic aqueous layer was extracted
again with EtOAc (1 x 10.0 mL) and that organic layer was also discarded. The
acidic aqueous layer was neutralized with solid NaHC03 and then extracted with
EtOAc (2 x 50.0 mL) and n-BuOH ( 2 x 50.0 mL). The organics were then dried
using anhydrous sodium sulfate and concentrated. The crude solid was washed
with acetone (10.0 mL) to remove a colored impurity to leave a white solid as
product. Yield (0.113 g, 86.0%). 1H NMR (500MHz), CD3OD: 5 1.80 (s, 3H), 2.96
(d, J = 13.2 Hz, 1H), 3.03 (s, 3H), 3.07 (d, J = 13.2 Hz, 1H), 3.20 (s, 3H), 4.85 (s,
1H, hidden by MeOH), 4.90 (s, 1H), 6.96 (t, J = 8.0 Hz, 1H), 7.08 (t, J = 8.0 Hz,
1H), 7.30 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H); 13C NMR
(125MHz), CD30D: 5 24.6, 36.9, 38.7, 44.4, 69.7, 112.5, 115.2, 116.2, 120.1,
122.6, 124.2, 126.2, 138.5, 141.4, 170.1, 192.1. IR: (NaCI) 3321 cm"1 (broad),
3284 cm'1 (broad), 1884 cm", 1611 cm‘l. HRMS: [M + H]+ = 297.1723, calculated
for C17H21N4O, 297.1715. Melting Point = 267-269°C.

(III-1). To a 25 mL round bottom flask was added III-14 (0.0500 g, 0.169
mmol), DMF (3.00 mL), THF (4.00 mL) and water (1 .00 mL). Then NMO (0.0290
g, 0.253 mmol) and OsO4 (0.171 mL of a 0.0980 M solution in toluene, 0.0169
mmol). The solution stirred at room temperature for 4 h and then was cooled to

0°C before a solution of NalO4 (0.108 g, 0.507 mmol) in water (2.00 mL) was

180

added a
mL)lNa
lnkflne
aqueot
waec
BuOH
gwe a
the n
(500k
37411
(d,J
36£L
170,
(s, 3
H2,
1th
111
018
299
642

248

9.1

added and stirred for 2 h at 0°C. The solvents were removed and EtOAc (10.0
mL) was added along with a sat. solution of K2803 (10.0 mL). This biphasic
mixture stirred for 10 min and then the organic layer was separated and the
aqueous layer was extracted with EtOAc (10.0 mL) again. The EtOAc layers
were combined, and discarded. The aqueous layer was then extracted with n-
BuOH (3 x 20.0 mL), dried using anhydrous sodium sulfate and concentrated to
give a whitish powder. The crude product was recrystallized from EtOH to give
the natural product as a white powder. Yield (0.0310 g, 62.0%). 1H NMR
(500MHz), CDgOD: 5 2.16 (s, 3H), 3.05 (s, 3H), 3.23 (s, 3H), 3.46 (dd, J = 16.6,
37.4 Hz, 2H), 6.97 (t, J = 7.0 Hz, 1H), 7.09 (t, J = 7.1 Hz, 1H), 7.26 (s, 1H), 7.33
(d, J = 8.2 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H); 13C NMR (125MHz), CD300: 5 31.0,
36.9, 38.8, 49.0, 67.0, 112.6, 114.5, 120.2, 120.3, 122.7, 124.1, 125.8, 138.6,
170.8, 191.7, 207.3. 1H NMR (500MHz), DMSO: 5 2.08 (s, 3H), 2.99 (s, 3H), 3.11
(s, 3H), 3.24 (dd, J = 15.8, 38.3 Hz, 2H), 6.93 (t, J = 8.0 Hz, 1H), 7.05 (t, J = 8.0
Hz, 1H), 7.27 (s, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 8.24 (s,
1H), 11.01 (s, 1H); 13C NMR (125MHz), DMSO: 5 30.9, 36.0, 37.9, 48.7, 65.2,
111.5, 114.3, 118.5, 119.6, 121.0, 123.0, 124.5, 136.6, 170.1, 187.6, 205.1. IR:
(NaCI) 3234 cm1 (broad), 1720 cm", 1703 cm", 1611 cm". HRMS: [M + H]+ =
299.1508, calculated for C16H19N402, 299.1508. Anal. Calcd. For C15H13N402: C,
64.41; H, 6.08; N, 18.78. Found: C, 64.33; H, 6.22; N, 18.42. Melting Point = 246-
248°C.

(III-15). To a flame dried 250 mL round bottom flask was added Ill-9 (1 .00

g, 1.89 mmol), anhydrous DCM (75.0 mL), and anhydrous TEA (0.780 mL, 5.67

181

 

mn

adt

dis

ITII’

W E

on

Oil

8C

mmol). The solution was cooled to 0°C and then EDCI (0.798 g, 4.16 mmol) was
added and the mixture stirred at 0°C for 1 h and then refluxed until
disappearance of the starting material, as indicated on TLC. A solution of NaH
(0.750 g, 18.9 mmol) in MeOH (70.0 mL) was then added to the mixture and
refluxed for 8 h. Since the de-tosylation was sluggish with NaOMe the solvent
was removed and ethanol (0.120 L) was added followed by KOEt (0.793 g, 9.45
mmol) and the mixture refluxed overnight. The solvent was removed and residue
was acidified using 1% HCI and extracted using EtOAc (3 x 50.0 mL). The
organics were dried using anhydrous sodium sulfate and concentrated. The
crude residue was purified by column chromatography (silica gel, 20% Acetone;
80% DCM) affording the product as a white solid. Yield (0.426 g, 83.0%). 1H
NMR (500MHz), DMSO: 5 1.75 (s, 3H), 2.72 (d, J = 13.4 Hz, 1H), 3.01 (d, J =
13.5 Hz, 1H), 4.82 (s, 1H), 4.91 (s, 1H), 6.99 (t, J = 6.8 Hz, 1H), 7.09 (t, J = 7.1
Hz, 1H), 7.37 (m, 2H), 7.57 (d, J = 8.1 Hz, 1H), 8.38 (s, 1H), 10.70 (s, 1H), 11.13
(s, 1H); 13C NMR (125MHz), Acetone-d6: 5 23.9, 44.1, 65.9, 111.9, 114.7, 115.9,
119.5, 120.4, 121.9, 123.1, 125.2, 137.6, 140.3, 156.6, 175.8. IR: (NaCI) 3300
cm“, 1790 cm“, 1720 cm". HRMS: [M + H]+ = 270.1255, calculated for
C15H16N302, 270.1243. Anal. Calcd. For C15H15N302: C, 66.90; H, 5.61; N, 15.60.
Found: C, 66.38; H, 5.69; N, 15.11. Melting Point = 238-240°C.

(III-16). To a 25 mL round bottom flask was added III-15 (0.172 g, 0.639
mmol), THF (8.00 mL) and water (1.00 mL). Then NMO (0.112 g, 0.959 mmol)
and 0804 (0.650 mL of a 0.0980 M solution in THF, 0.0639 mmol) were added.

The solution stirred at room temperature for 2 h and then was cooled to 0°C

182

before a solution of Na|O4 (0.410 g, 1.92 mmol) in water (3.00 mL) was added
and stirred at room temperature overnight. The solvents were removed and
EtOAc (10.0 mL) was added along with a sat. solution of K2803 (10.0 mL). This
biphasic mixture stirred for 10 min and then the organic layer was separated and
the aqueous layer was extracted with n-BuOH (3 x 10.0 mL). The EtOAc layer
and nBuOH layers were combined, dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by column chromatography (silica
gel, 10% MeOH; 90% DCM) affording the product as an off white solid. Yield
(0.107 g, 61.0%). Product was recrystallized over 1 week with EtOAc. 1H NMR
(500MHz), CDgOD: 5 2.15 (s, 3H), 3.49 (d, J = 7.9 Hz, 1H), 3.59 (d, J = 7.9 Hz,
1H), 7.02 (t, J = 8.0 Hz, 1H), 7.11 (t, J = 8.2 Hz, 1H), 7.27 (s, 1H), 7.35 (d, J = 8.1
Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H); 13C NMR (125MHz), CD3OD: 5 30.5, 49.2, 64.1,
112.7, 113.9, 120.5, 120.6, 122.9, 123.9, 125.6, 138.8, 160.0, 179.0. IR: (KBr)
3364 (br) cm'1, 1774 cm", 1711 cm“. HRMS: [M + H]+ = 272.1055, calculated for
C14H14N303, 272.1035. Anal. Calcd. For C14H13N303: C, 61.99; H, 4.83; N, 15.49.
Found: C, 61.10; H, 4.82; N, 15.38. Melting Point = 234-236°C.

(III-17). To a sealed tube was added III-12 (0.850 g, 1.88 mmol) in THF
(5.00 mL) and NH4OH (15.0 mL). The mixture was heated at 90°C until the
disappearance of the starting material, as indicated by TLC. The precipitate that
formed was filtered, acidified with 5% HCI and extracted with nBuOH (3 x 20.0
mL), dried using anhydrous sodium sulfate and concentrated. The crude residue
was purified by column chromatography (silica gel, 10% MeOH; 90% DCM)

affording the product as an off white solid. Yield (0.575 g, 72.0%). 1H NMR

183

 

(500MHz), DMSO: 5 1.62 (s, 3H), 2.30 (s, 3H), 2.72 (d, J = 13.5 Hz, 1H), 2.81 (d,
J = 13.5 Hz, 1H), 4.72 (s, 1H), 4.77 (s, 1H), 7.22 (t, J = 8.3 Hz, 1H), 7.32 (t, J =
8.3 Hz, 1H), 7.37 (d, J = 9.5 Hz, 2H), 7.64 (d, J = 8.1 Hz, 1H), 7.69 (s, 1H), 7.85
(d, J = 9.5 Hz, 2H), 7.90 (d, J = 8.5 Hz, 1H), 8.25 (s, 1H); 13C NMR (125MHz),
DMSO: 5 20.9, 23.7, 43.1, 66.0, 113.0, 115.2, 121.5, 123.0, 123.3, 124.7, 126.7,
128.0, 130.2, 134.0, 134.5, 139.9, 145.5, 170.9, 187.5. IR: (KBr) 3470 cm", 3351
cm“, 3307 cm", 3088 cm'1, 1707 cm‘1, 1857 cm‘1. HRMS: [M + H]+ = 423.1494,
calculated for C22H23N4038, 423.1491. Anal. Calcd. For C22H22N4038: C, 62.54;
H, 5.25; N, 13.25. Found: C, 61.21; H, 5.17; N, 13.05. Melting Point = 273-275’C.

(III-18). To a 100 mL round bottom flask was added III-17 (0.500 g, 1.18
mmol) and EtOH (60.0 mL). Then KOEt (0.991 g, 11.8 mmol) was added and the
mixture refluxed for 24 h. The EtOH was taken off and the pH of an aqueous
mixture of the crude residue was adjusted to 8. The aqueous mixture was then
extracted with nBuOH (3 x 40.0 mL). The organics were combined, dried using
anhydrous sodium sulfate and concentrated. The crude residue was purified by
column chromatography (silica gel, 20% IVleOH; 90% DCM) affording the product
as an off white solid. The product was recrystallized from EtOH. Yield (0.150 g,
47.0%). 1H NMR (500MHz), DMSO: 5 1.67 (s, 3H), 2.75 (d, J = 13.4 Hz, 1H),
2.81 (d, J = 13.4 Hz, 1H), 4.75 (s, 1H), 4.78 (s, 1H), 6.92 (t, J = 7.1 Hz, 1H), 7.04
(t, J = 7.0 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.48 (d, J =
8.0 Hz, 1H), 8.03 (s, 1H), 10.96 (s, 1H); 13C NMR (125MHz), DMSO: 5 23.9,
43.4, 66.3, 111.4, 114.7, 115.3, 118.3, 119.7, 120.9, 122.6, 124.8, 136.5, 140.7,

170.8, 189.1. IR: (KBr) 3470 cm'l, 3390 (br) cm'1, 3200 (br) cm’1, 1892 cm",

184

1650 cm'i. HRMS: [M + H]* = 269.1405, calculated for C15H17N4O, 269.1402.
Anal. Calcd. For C15H15N4O: C, 67.15; H, 6.01; N, 20.88. Found: C, 63.98; H,
5.89; N, 19.84. Melting Point = 264-266°C.

(III-19). To a 25 mL round bottom flask was added III-18 (0.0990 g, 0.366
mmol), DMF (7.00 mL), THF (1.00 mL) and water (1.00 mL). Then NMO (0.0640
g, 0.549 mmol) and OsO4 (0.370 mL of a 0.0980 M solution in THF, 0.0366
mmol) were added. The solution stirred at room temperature for 3 h and then
was cooled to 0°C before a solution of NaIO4 (0.235 g, 1.10 mmol) in water (2.00
mL) was added and stirred at room temperature overnight. The solvents were
removed and EtOAc (10.0 mL) was added along with a sat. solution of K2803
(10.0 mL). This biphasic mixture stirred for 10 min and then the organic layer was
separated and the aqueous layer was extracted with n-BuOH (6 x 10.0 mL). The
EtOAc layer and nBuOH layers were combined, dried using anhydrous sodium
sulfate and concentrated. The crude residue was purified by column
chromatography (silica gel, 20% MeOH; 90% DCM) affording the product as an
off white solid. The product was recrystallized from EtOH. Yield (0.0190 g,
19.0%). 1H NMR (500MHz), CD3OD: 5 2.18 (s, 3H), 3.19 (d, J = 7.3 Hz, 1H), 3.64
(d, J = 7.1 Hz, 1H), 6.97 (t, J = 8.0 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 7.20 (s, 1H),
7.33 (d, J = 8.2 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H); 13C NMR (125MHz), CD3OD: 5
30.9, 66.5, 112.5, 114.1, 120.28, 120.29, 122.7, 124.0, 125.9, 138.5, 171.7,
192.5, 207.7. IR: (KBr) 3470 cm", 3420 cm'l, 3270 (br) cm", 3178 (br) cm",
1720 cm“, 1890 cm‘1, 1850 cm". HRMS: [M + H]: = 271.1191, calculated for

C14H15N402, 271.1195. Melting Point = 283-2850C.

185

 

10.

11.

12.

References

Baker, D. D.; Alvi, K. A., Small-molecule natural products: new structures,
new activities. Curr. Opin. Biotechnol. 2004, 15, 576-583.

Hill, R. A., Marine natural products. Annu. Rep. Prog. Chem, Sect. B: Org.
Chem. 2005, 101, 124-136.

Jha, R. K.; Xu, Z.-r., Biomedical compounds from marine organisms. Mar.
Drugs 2004, 2, 123-146.

Molinski, T. F.; Dalisay, D. S.; Lievens, S. L.; Saludes, J. P., Drug
development from marine natural products. Nat. Rev. Drug Discovery
2009, 8, 69-85.

Simmons, T. L.; Andrianasolo, E.; McPhail, K.; Flatt, P.; Gerwick, W. H.,
Marine natural products as anticancer drugs. Mol. Cancer Ther. 2005, 4,
333-342.

Barsby, T., Drug discovery and sea hares: bigger is better. Trends
Biotechnol. 2006, 24, 1-3.

Gul, W.; Hamann, M. T., Indole alkaloid marine natural products: An
established source of cancer drug leads with considerable promise for the
control of parasitic, neurological and other diseases. Life Sci. 2005, 78,
442-453.

Bergmann, T.; Schories, D.; Steffan, B., Alboinon, an oxadiazinone
alkaloid from the ascidian Dendrodoa grossularia. Tetrahedron 1997, 53,
2055-2060.

Guyot, M.; Meyer, M., An 3-indonI-4H-imidazol-4-one from the tunicate
Dendrodoa grossularia. Tetrahedron Left. 1986, 27, 2621-2.

Heitz, S.; Durgeat, M.; Guyot, M.; Brassy, C.; Bachet, B., New indolic
derivative of 1,2,4-thiadiazole, isolated from a tunicate (Dendrodoa
grossular). Tetrahedron Left. 1980, 21 , 1457-8.

Loukaci, A.; Guyot, M.; Chiaroni, A.; Riche, C., A new indole alkaloid from
the marine tunicate Dendrodoa grossularia. J. Nat. Prod. 1998, 61, 519-
522.

Moquin, C.; Guyot, M., Grossularine, a novel indole derivative from the

marine tunicate, Dendrodoa grossularia. Tetrahedron Left. 1984, 25,
5047-8.

186

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

Moquin-Pattey, C.; Guyot, M., Grossularine-1 and grossularine-2,
cytotoxic a-carbolines from the tunicate Dendrodoa grossularia.
Tetrahedron 1989, 45, 3445-50.

Hogan, I. T.; Sainsbury, M., The synthesis of dendrodoine, 5-[3-(N,N-
dimethylamino-1,2,4-thiadiazolyI]-3-indolylmethanone, a metabolite of the
marine tunicate Dendroda grossular. Tetrahedron FIELD Full Journal
Tif/e:Tetrahedron 1984, 40, 681-2.

Helbecque, N.; Moquin, C.; Bernier, J. L.; Morel, E.; Guyot, M.; Henichart,
J. P., Grossularine-1 and grossularine-2, a-carbolines from Dendrodoa
grossularia, as possible intercalative agents. Cancer Biochem. Biophys.
1987, 9, 271-9.

Loukaci, A.; Guyot, M., Revised assignments of the 13C NMR spectra of
grossularine-1 and -2 using 2D heteronuclear 1H-1BC correlations. Magn.
Reson. Chem. 1996, 34, 143-5.

Choshi, T.; Yamada, S.; Sugino, E.; Kuwada, T.; Hibino, 8., Total
synthesis of grossularines-1 and -2. J. Org. Chem. 1995, 60, 5899-904.

Fisk, J. S.; Mosey, R. A.; Tepe, J. J., The diverse chemistry of oxazol-5-
(4H)-ones. Chem. Soc. Rev. 2007, 36, 1432-1440.

Hupp, C. D.; Tepe, J. J., 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
Hydrochloride-Mediated Oxazole Rearrangement: Gaining Access to a
Unique Marine Alkaloid Scaffold. J. Org. Chem. 2009, 74, 3406-3413.

Gadwood, R. C.; Kamdar, B. V.; Dubray, L. A. C.; Wolfe, M. L.; Smith, M.
P.; Watt, W.; Mizsak, S. A.; Groppi, V. E., Synthesis and biological activity
of spirocyclic benzopyran imidazolone potassium channel openers. J.
Med. Chem. 1993, 36, 1480-7.

Kiec-Kononowicz, K.; Zejc, A., Syntheses of some 2-amino—5,5-
diphenylimidazolin-4-one derivatives. Pol. J. Chem. 1980, 54, 2217-24.

Hupp, C. D.; Tepe, J. J., Total Synthesis of a Marine Alkaloid from the
Tunicate Dendrodoa grossularia. Org. Left. 2008, 10, 3737-3739.

Garg, N. K.; Sarpong, R.; Stoltz, B. M., The First Total Synthesis of
Dragmacidin D. J. Am. Chem. Soc. 2002, 124, 13179-13184.

Kharasch, M. 8.; Kane, S. S.; Brown, H. C., Presence of indole in
\"practical\" a-methylnaphthalene. J. Am. Chem. Soc. 1940, 62, 2242-3.

Scheibye, S.; Pedersen, B. S.; Lawesson, S. 0., Studies on
organophosphorus compounds. XXI. The dimer of p-

187

 

26.

methoxyphenylthionophosphine sulfide as thiation reagent. A new route to
thiocarboxamides. Bull. Soc. Chim. Belg. 1978, 87, 229-38.

Pappo, R.; Allen, D. 8., Jr.; Lemieux, R. U.; Johnson, W. S., Osmium
tetroxide-catalyzed periodate oxidation of olefinic bonds. J. Org. Chem.
1956, 21 , 478-9.

188

 

CHAPTER IV

BIOLOGICAL TESTING OF NATURAL PRODUCT, ANALOGS
AND OTHER HETEROCYCLIC COMPOUNDS

IV.A Biological target of natural product and hypothesis for activity

The biological target from the start of the project was checkpoint kinase 2
(Chk2), as discussed in chapter I. This kinase is responsible for helping to
maintain the integrity of the genome by interpreting certain cellular distress
signals and activating other proteins downstream to initiate a response. These
responses can include cell cycle abrogation and allow for cellular repair or it can
include programmed cell death, or apoptosis.1

It was hypothesized that the natural product (III-1) had some structural
characteristics similar to that of a known Chk2 inhibitor, indoloazepine (Figure IV-
1). Furthermore, the majority of the hydrogen bonding contacts thought to be
important for indoloazepine’s activity was present in the natural product. As a
result, the curiosity of elucidating potential biological activity of this uniquely
substituted imidazolone (III-1) prompted an endeavor to determine if there was
any similarity in biological properties to indoloazepine, specifically, as a

checkpoint kinase 2 inhibitor.

   

lndoloazepine

Figure IV-1. The indole natural product III-1 and indoloazepine

189

IV.B Kinase screen for natural product and analogs

The natural product (III-1) and the two analogs synthesized were sent to a
kinase screening company (Millipore). The compounds were screened for more
than just Chk2 inhibition so that in the event of activity, insight into the degree of
selectivity against other kinases could be evaluated. The kinases screened
included Chk1, Chk2, GSK3B, IKKd and IKKB. A general illustration of how the
kinase screen works is shown in Figure IV-2. Essentially, a known substrate for
the kinase is incubated with a compound, such as III-1, along with radio-labelled
ATP. The amount of radio-activity in the end is directly related with the
incorporation of 33P, which can be correlated to how well the compound is at
inhibiting the kinase. For example, if the compound is a good inhibitor of the

kinase, there will be very little radio-activity in the end.

Filter Scintillation

counfing

 

Substrate Substrate

 

Figure IV-2. Kinase screen performed by Millipore
The results from the kinase screen are shown below in Table IV-1. The
numerical value in each of the cells of the table is the percent activity of that
kinase after being incubated with the corresponding compound. The lower the
percent activity, the better of an inhibitor the compound is. The natural product

and analogs were tested at 50 (M to identify it there was any activity of kinase

190

inhibition. As shown in Table lV-1, all three compounds were shown to be
inactive for all five kinases tested. Taking into account the experimental error, all
compounds essentially resulted in 100% activity for all kinases. Although these
results are disappointing, it doesn’t unequivocally prove that the compounds are
inactive.

Table IV-1. Results of kinase screen for Ill-1, III-16 and Ill-19

 

   

 

 

 

 

 

 

 

 

 

 

 

OT“
HN O
Kinase
\ 0
fl III-16
Chk1 1 12 103 107
Chk2 102 106 101
GSK3fi 100 94 99
IKKa 103 103 105
IKKB 96 93 102
Biological . . . . . .
result Inactive Inactive Inactive

 

IV.C Other biological testing for natural product and analogs

The natural product and two analogs were also tested in our laboratory in
other assays to determine any biological activity. Compounds III-1, III-16 and III-
19 were tested in a cell proliferation assay to determine if they were cytotoxic to
MCF-7 cells, a breast cancer cell line. Furthermore, they were tested in a
Iuciferase and whole blood assay for inhibition of NF-KB and IL-6 production,
respectively, which are other biological targets of our laboratory. The two other
quaternary imidazolones synthesized via the diketone rearrangement,

compounds "-18 and "-23, were also tested for NF-KB inhibition. Table IV-2

191

shows the results of all compounds for these assays. It can be seen that all
compounds tested are inactive for the inhibition of NF-KB, inactive for the
inhibition of IL-6 production and non-cyctotoxic for the MCF-7 cancer cell line.

Table lV-2. Results for cytotoxicity, NF-KB and IL-6 assays

 

 

 

 

 

 

 

 

 

 

Compound Target
NF-KB lL-6 MCF-7 cells
III-1 inactive inactive non-cytotoxic
III-16 -- inactive non-cflotoxic
III-19 -- inactive non-cytotoxic
ll-18 inactive -- --
lI-23 inactive -- --

 

 

 

IV.D Synthesis of additional heterocycles

Throughout the process of developing a synthetic method to gain access
to the imidazolone scaffold found in the natural product, other heterocycles were
synthesized and were subsequently tested for biological activity. The majority of
the heterocycles formed contained the indole and hydantoin moiety in various
constitutions. Additionally, an indoloazepine analog was also synthesized. The
following section describes the synthesis of these heterocycles.

Heterocycles such as indoles substituted in the 3-position by a hydantoin
moiety were synthesized through a reaction between an aldehyde, ammonium
carbonate and potassium cyanide (Scheme IV-1).2 Hydantoin IV-13 was
synthesized in a 36% yield through a reaction between indole-3-carboxaldehyde
and the reagents described above, while a N-tosyl analog was synthesized in a
18% yield through a similar reaction. After the indolic nitrogen of indole-3-
carboxaldehyde was protected with a tosyl group, the reaction using ammonium

carbonate and potassium cyanide was used to produce the final hydantoin (IV-3).

192

Scheme IV-1. Synthesis of lV-1 and IV-3

O

O
H HN/Q

\ (NH4)ZCO3, KCN _ N”
H 0/ EtOH '
N 2 i 0

R l‘l
IV-2: R = Ts R
IV-1: R = H, 38%
IV-3: R = Ts, 18%

 

A unique compound was synthesized using a hydantoin phosphonate
reagent developed by Meanwell4 (Scheme lV-2). Protection of the indolic
nitrogen of lV-4 with p-toluene sulfonyl chloride gave keto ester lV-5 in a 90%
yield. A subsequent Horner—Wadsworth-Emmons reaction produced hydantoin
lV-6 in about an 8:1 mixture of E and Z isomers in a 91% yield (Scheme IV-2).
Geometric isomers were identified using NOESY experiments (Figure IV-9).

Scheme IV-2. Synthesis of hydantoin IV-6

 

O O O O
OEt TsCI, DMAP _ OEt
\ DIPEA DCM? \
H 'V'4 90% N IV-5
Ts
O Phosphonate,
HN o NaOEt, EtOH, rt
Phosphonate= 04 PI, 91%,~8:1(E22)
HEtd OEt H
o N o
HN \ O
OEt
\
N IV-6
Ts

Since indoloazepine was found to be a potent checkpoint kinase 2

inhibitor,5 it was thought that synthesizing an analog could help identify the

193

 

structural features necessary for activity. It was decided to synthesize an analog
that did not contain the seven membered ring between the indole and
imidazolone moieties. To synthesize this analog, lndole-2-carboxylic acid was
coupled to methylamine using EDCI to afford IV-7 in a 81% yield. Following a
Vilsmeier-Haacka' 7 reaction to produce aldehyde IV-8, a condensation between
thiohydantoin and IV-8 produced thiohydantoin IV-9 in a 86% yield.
Subsequently, after an S-methylation, the final imidazolone, IV-11, was produced
in a 26% yield by replacing the S-methyl moiety with an NH2 group using
ammonium hydroxide.

Scheme IV-3. Synthesis of IV-11

 

 

 

. O
N EDCI, DMAP, _
H DCM, 0°c to rt, N ”N
81% IV-7
(COC|)2. DMF, DCM
H 0°C to rt, 75%
N
S O H
:h 88’” O H
\ O ‘ piperidine, EtOH, N HN—
N 86% H
H HN— IV-8
N-9
Mel, NaOH,
MeOH, 96%

 

NH4OH

THF, sealed 7
tube, 26%

 

   

194

 

IV.E Biological testing for additional heterocycles

The biological results for compounds IV-1, IV-3, IV-6, IV-9 and lV-11 are

illustrated in Table lV-3. Unfortunately, all the compounds tested for kinase

inhibition were found to be inactive. Additionally, none of the compounds

exhibited any propensity for inhibition or abrogation of lL-6 production. Further

testing is needed to determine if any of the compounds synthesized have any

biological value.

Table IV-3. Biological activity results for IV-1, IV-3, IV-6, IV-9 and lV-11

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cmp d Tagget
' Chk1 Chk2 GSK3B IKKa IKKB NF-KB IL-6
IV-1 inactive inactive inactive inactive inactive inactive inactive
IV-3 -- -- -- -- -- inactive inactive
IV-6 -- -- -- -- -- inactive --
IV-9 -- -- -- -- -- inactive --
IV-1 1 inactive inactive -- -- -- -- --

 

195

 

 

IV.F General experimental information

Reactions were carried out in flame-dried glassware under nitrogen
atmosphere. All reactions were magnetically stirred and monitored by TLC with
0.25 pm pre-coated silica gel plates using either UV light or iodine to visualize the
compounds. Column chromatography was carried out on Silica Gel 60 (230-400
mesh) supplied by EM Science. Yields refer to chromatographically and
spectroscopically pure compounds unless otherwise noted. Infrared spectra
were recorded on a Nicolet IR/42 spectrometer. 1H and 13C NMR spectra were
recorded on a Varian Unity Plus-500 spectrometer or a Varian lnova-300, as
noted in the experimental for each compound. Chemical shifts are reported
relative to the residue peaks of the solvent (CDCI3: 7.24 ppm for 1H and 77.0
ppm for 13C) (Acetone-d5: 2.04 ppm for 1H and 29.8 ppm for ‘30) (DMSO-de:
2.49 ppm for 1H and 39.5 ppm for 13C). The following abbreviations are used to
denote the multiplicities: s = singlet, d = doublet, dd = doublet of doublets, t =
triplet, and m = multiplet. HRMS were obtained with a Micromass Q-ToF Ultima
API LC-MS/MS mass spectrometer. Elemental analysis data were obtained on a
Perkin Elmer 2400 Series II CHNS/O analyzer. Purity of compounds, whose
elemental analyses were above the ACS tolerated 0.4% deviation, were
confirmed by 1H NMR and 13C NMR. Melting points were obtained using an
Electrothermal® capillary melting point apparatus and are uncorrected.
Reagents and solvents were purchased from commercial suppliers and used

without further purification. Anhydrous methylene chloride and toluene were

196

 

dispensed from a delivery system which passes the solvents through a column
packed with dry neutral alumina.
IV.G Experimental procedures and characterization
Cell Proliferation Assay

The cell proliferation study was performed by Thu Nguyen and all detailed
experimental information is found within her notebook. The general procedure is
as follows. Adherent cells were seeded with 500 uL of approximately 2.5 x 105
cells/mL in a 24-well plate. When cells were near 80% confluent, cells were
treated with the appropriate drug in various doses. Cells were incubated for 12,
24 and 48 hours. At each time point, cells were released from the plate with 100
pL of 0.25% trypsin/EDTA and diluted to 500 pL, which was then counted via a
cell counter (Beckman Coulter Z1 Coulter Particle Counter).
Luciferase Assay

The Iuciferase assay was performed by Thu Nguyen, Teri Lansdell and/or
Behnaz Shafii. All detailed experimental information is located within their
respective notebooks. The general experimental procedure is as follows. HeLa
NF-KB cell line was cultured in DMEM, complemented with 10% Fetal Bovine
Serum, 1 mM sodium pyruvate, 0.1 mg/mL Hygromycin, 2mM-L-glutamine, 1mM
sodium pyruvate, 100 units/mL penicillin, 4.5 g/L D-glucose, and no phenol red.
The day before the Iuciferase assay, the medium was switched to DMEM with
2% FBS in addition to 2mM-L-glutamine, 1mM sodium pyruvate, and 100
units/mL penicillin. The day of the experiment, the medium utilized was serum

free DMEM with 2mM-L-glutamine, 1mM sodium pyruvate, and 100 units/mL

197

ex
prr
110

CH

001
010
Ser
biOI
tEst
hog

200

penicillin. Cells were propagated at 37°C with 5% C02, and ambient oxygen.
Cells were seeded in a 96-well plate between 35,000-50,000 cells per well with
DMEM 2% FBS. The outside rows and columns of the plate were not used. Cell
were allowed to adhere and grow overnight at 37°C with 5% CO2, and ambient
oxygen. 0n the day of the experiment, the media was removed and replaced
with serum free DMEM. Cells were pretreated with 2 (in 1 [JL DMSO/well) for 30
minutes before activation with 25 ng/mL TNF-o and incubated an additional 8
hours. Steady glo reagent (Promega) was added to each well and data was
collected using a Iuminometer.
Whole Blood IL-6 Assay

The whole blood assay was performed by Teri Lansdell and all detailed
experimental information is located in her notebooks. The general experimental
procedure is as follows. After obtaining the appropriate approval for de-identified
human cell lines, human whole blood was obtained through the Jasper Research
Clinic, Kalamazoo, MI, from a single healthy, fasted volunteer and was collected
in glass citrated tubes by venipuncture. Only samples with a white blood cell
count falling within the normal range (4800-10,800 per liter) were used. The
blood was diluted 1:10 in RPMI-1640 media supplemented with 10% fetal bovine
serum, 100 U/mL penicillin, and 100 pg/mL streptomycin. Aliquots of diluted
blood (1 mL) were preincubated with vehicle (0.1% DMSO, final concentration) or
test agent (final concentrations were 20, 10, 5, 2.5, 1.25 and 0.625 pM) for two
hours at 37 °C, 5 % C02. lL-1B (Roche) was added to a final concentration of

200 U/mL and the samples were further incubated for 18 hours at 37 °C, 5 %

198

 

C02. At the end of the incubation period, the blood samples were centrifuged at
3000 X g, 4 °C, for 10 minutes. The plasma was removed, snap frozen and
stored at -80 C. IL-6 levels were determined by ELISA (R & D Systems).

Kinase Profiler (Millipore)

The kinase testing was performed by Millipore. All detailed experimental
information can be found on their website.8 The general experimental procedure
for each kinase is described below.

CHK1 (h)

CHK1 (h) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 (M
KKKVSRSGLYRSPSMPENLNRPR, 10 mM MgAcetate and [v-33P-ATP]
(specific activity approx. 500 cpm/pmol, concentration as required). The reaction
is initiated by the addition of the MgATP mix. After incubation for 40 minutes at
room temperature, the reaction is stopped by the addition of 3% phosphoric acid
solution. 10 pL of the reaction is then spotted onto a P30 filtermat and washed
three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to
drying and scintillation counting.

CHK2 (h)

CHK2 (h) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 pM
KKKVSRSGLYRSPSMPENLNRPR, 10 mM MgAcetate and [v-33P-ATP]
(specific activity approx. 500 cpm/pmol, concentration as required). The reaction
is initiated by the addition of the MgATP mix. After incubation for 40 minutes at
room temperature, the reaction is stopped by the addition of 3% phosphoric acid

solution. 10 pL of the reaction is then spotted onto a P30 filtermat and washed

199

three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to
drying and scintillation counting.
GSK3B (h)

GSKBB (h) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 20 uM
YRRAAVPPSPSLSRHSSPHQS( p) EDEEE (phospho G82 peptide), 10 mM
MgAcetate and [v-33P-ATP] (specific activity approx. 500 cpm/pmol,
concentration as required). The reaction is initiated by the addition of the MgATP
mix. After incubation for 40 minutes at room temperature, the reaction is stopped
by the addition of 3% phosphoric acid solution. 10 pL of the reaction is then
spotted onto a P30 filtermat and washed three times for 5 minutes in 50 mM
phosphoric acid and once in methanol prior to drying and scintillation counting.
IKKo (h)

IKKa (h) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 pM
peptide, 10 mM MgAcetate and [v-33PATP] (specific activity approx. 500
cpm/pmol, concentration as required). The reaction is initiated by the addition of
the MgATP mix. After incubation for 40 minutes at room temperature, the
reaction is stopped by the addition of 3% phosphoric acid solution. 10 pL of the
reaction is then spotted onto a P30 filtermat and washed three times for 5
minutes in 75 mM phosphoric acid and once in methanol prior to drying and
scintillation counting.

IKKB (h)
IKKB (h) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 100 pM

peptide, 10 mM MgAcetate and [v-33P-ATP] (specific activity approx. 500

200

cpm/pmol, concentration as required). The reaction is initiated by the addition of
the MgATP mix. After incubation for 40 minutes at room temperature, the
reaction is stopped by the addition of 3% phosphoric acid solution. 10 pL of the
reaction is then spotted onto a P30 filtermat and washed three times for 5
minutes in 75 mM phosphoric acid and once in methanol prior to drying and
scintillation counting.
Experimentals for compounds IV-1-IV-11

(IV-1). To a flame dried 250 mL round bottom flask was added indole-3-
carboxaldehyde (1.00 g, 6.90 mmol) and ethanol (70.0 mL). Then ammonium
carbonate (2.00 g, 20.7 mmol) and water (70.0 mL) were added and the mixture
was heated in an oil bath to 50°C. Once everything was in solution, KCN (0.583
g, 8.97 mmol in 10.0 mL H2O) was added over 25 min. The light orange solution
stirred at 60°C overnight and became a darker orange color. The ethanol was
taken off by rotary evaporation and the solid that precipitated out was filtered off.
The filtrate was neutralized with 1% HCI (aq.) and extracted with ethyl acetate (3
x 50.0 mL). The organics were dried using anhydrous sodium sulfate and
concentrated to give a yellowish oil. The crude material was purified by column
chromatography (silica gel, 4:1 ethyl acetate/hexanes) affording the product as
an off white powder. Yield (0.537 g, 36.0%). 1H NMR (500 MHz), Acetone: 5 5.47
(s, 1H), 7.03-7.06 (m, 1H), 7.12-7.16 (m, 1H), 7.32 (bs, 1H), 7.42-7.44 (m, 2H),
7.57-7.59 (m, 1H), 9.80 (bs, 1H), 10.30 (bs, 1H); 13c NMR (125 MHz), Acetone: 8
57.1, 110.8, 112.6, 119.7, 120.2, 122.7, 125.4, 126.5, 137.9, 157.9, 174.8; IR

(NaCI) 1710 cm", 1770 cm'1, 3300 cm". M.S: calculated for C11H9N302 [M+] =

201

 

215 and found [M+] = 214.9. Anal. Calcd. For C11H9N302: C, 61.40; H, 4.20; N,
19.50. Found: C, 60.90; H, 3.60; N, 18.90. Melting Point = 220-222°C.

(IV-2).9 To a flame dried 500 mL round bottom flask was added indole-3-
carboxaldehyde (5.00 g, 34.5 mmol), dry DCM (0.300 L), TsCl (13.1 g, 69.0
mmol), DMAP (10.5 g, 86.2 mmol), and DIPEA (15.0 mL, 86.2 mmol). The
solution stirred at room temperature overnight under nitrogen. The reaction was
quenched and washed with 1% HCI (aq.) and the organics were dried using
anhydrous sodium sulfate and concentrated. A silica plug was done to isolate
product (silica gel, 100% ethyl acetate). Yield (10.3 g, 99.0%). 1H NMR (500
MHz), CDCI3: 5 2.35 (s, 3H), 7.26-7.41 (m, 4H), 7.83 (d, J = 8.9 Hz, 2H), 7.93 (d,
J = 8.1 Hz, 1H), 8.21 (s, 1H), 8.23 (d, J = 8.9 Hz, 1H), 10.10 (s, 1H); 13C NMR
(125 MHz), CDCI3: 5 21.6, 113.2, 122.3, 122.5, 124.9, 126.2, 127.1, 130.2, 134.2,
135.1, 136.1, 148.1, 185.2; IR (NaCI) 1710 cm“, 2750 cm", 2850 cm".

(IV-3). To a flame dried 250 mL round bottom flask was added IV-2 (3.30
g, 11.0 mmol) and ethanol (75.0 mL). Then ammonium carbonate (3.18 g, 33.1
mmol) and water (75.0 mL) were added and the mixture was heated in an oil
bath to 50°C. Once everything was in solution, KCN (1.08 g, 16.6 mmol in 10.0
mL H20) was added over 25 min. The light orange solution stirred at 60°C
overnight and became a darker orange color. The ethanol was taken off by rotary
evaporation and the solid that precipitated out was filtered off. The filtrate was
neutralized with 1% HCI (aq.) and extracted with ethyl acetate (3 x 50.0 mL). The
organics were dried using anhydrous sodium sulfate and concentrated. The

crude material was purified by column chromatography (silica gel, 3:2 ethyl

202

acetate/hexanes) affording the product as an off white powder. Yield (0.717 g,
18.0%). 1H NMR (500 MHz), Acetone: 5 2.34 (s, 3H), 5.53 (s, 1H), 7.27 (t, J = 7.3
Hz, 1H), 7.35-7.40 (m, 4H), 7.64 (d, J = 7.8 Hz, 1H), 7.84 (s, 1H), 7.89 (d, J = 8.3
Hz, 2H), 8.01 (d, J = 8.3 Hz, 1H), 9.78 (s, 1H); 13C NMR (125 MHz), Acetone: 5
21.4, 56.3, 114.4, 118.5, 121.3, 124.2, 125.9, 126.0, 127.9, 129.3, 131.0, 135.8,
138.2, 148.8, 157.5, 173.3; IR (NaCI) 3249 cm", 1772 cm", 1724 cm". M.S:
calculated for C18H15N304S [M+] = 369 and found [M+] = 369.0. Anal. Calcd. For
C13H15N304S: C, 58.53; H, 4.09; N, 11.38. Found: C, 57.68; H, 4.16; N, 11.25.
Melting Point = 226-228 °C.

(IV-5). To a 500 mL flame dried round bottom flask was added IV-4 (4.47
g, 20.6 mmol), dry dichloromethane (0.225 L), TsCI (7.83 g, 41.2 mmol), DMAP
(6.28 g, 51.5 mmol), and DIPEA (9.00 mL, 51.5 mmol), The reaction stirred under
nitrogen overnight. The reaction was washed with sat. NaHC03 ( 2 x 50.0 mL)
and brine (2 x 50.0 mL) and the organic layer was dried using anhydrous sodium
sulfate and concentrated. The crude material was purified by column
chromatography (silica gel, DCM) affording the product as a solid. Yield (6.80 g,
90.0%). 1H NMR (500 MHz), CDCI3: 5 1.43 (t, J = 7.2 Hz, 3H), 2.34 (s, 3H), 4.43
(q, J = 7.0 Hz, 2H), 7.26-7.41 (m, 4H), 7.85 (d. J = 8.5 Hz, 2H), 7.94 (m, 1H),
8.34 (m, 1H), 8.83 (s, 1H); 13C NMR (125 MHz), CDCI3: 5 14.0, 21.6, 62.5, 113.1,
116.9, 122.9, 125.2, 126.1, 127.2, 127.6, 130.3, 134.2, 134.4, 136.7, 146.1,
181.8, 178.7. IR (NaCI) 1725 cm", 1870 cm". HRMS: [M + H]+ = 372.0907,

calculated for C19H18NO5S, 372.0906. Melting Point = 106-108°C.

203

 

(IV-6). To a flame dried 50 mL round bottom flask was added anhydrous
EtOH (20.0 mL) and Na° (0.0740 g, 3.23 mmol). Once all the sodium metal
reacted and the formation of bubbles ceased, diethyl 2,5-dioxoimidazolidin-4-
ylphosphonate4 (0.763 g, 3.23 mmol, borrowed from Thu Nguyen) was added
and stirred at room temperature for 30 min. Then III-5 (1.00 g, 2.70 mmol) was
added to the mixture and stirred for 18 h at room temperature under a nitrogen
atmosphere. The solvent was then removed and the crude residue was
neutralized with 1% HCI and extracted with EtOAc (2 x 40.0 mL). The organics
were washed with brine, dried using anhydrous sodium sulfate and concentrated.
The crude material was purified by column chromatography (silica gel, 96%
DCM; 4% MeOH) affording the product as an oil (mixture of diastereomers (8:1;
E:Z)). Yield (1.11 g, 91.0%). 1H NMR (500 MHz), CDCI3: (Isomer B) 5 1.28 (t, J =
7.2 Hz, 3 H), 2.28 (s, 3H), 4.35 (q, J = 7.1 Hz, 2H), 7.16-7.22 (m, 3H), 7.29 (t, J =
7.3 Hz, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.78 (d, J = 8.5 Hz, 2H), 7.87 (s, 1H), 7.89
(d, J = 8.5 Hz, 1H), 8.32 (bs, 1H), 9.01 (bs, 1H); 13C NMR (125 MHz), CDCI3: 5
13.7, 21.5, 62.4, 111.7, 113.0, 113.5, 120.4, 123.9, 125.5, 126.7, 127.0, 127.6,
127.9, 130.0, 134.5, 134.7, 145.5, 153.9, 161.6, 165.7. (Isomer A) 1H NMR (500
MHz), CDCI3: 5 1.07 (t, J = 7.0 Hz, 3H), 2.28 (s, 3H), 4.15 (q, J = 7.0 Hz, 2H),
7.10-7.22 (m, 5H), 7.58 (s, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.83 (d, J = 8.2 Hz, 1H),
9.70 (bs, 1H); 130 NMR (125 MHz), CDC13: 8 13.8, 21.4, 81.8, 103.1, 112.4,
113.3, 120.0, 123.0, 124.3, 126.9, 127.3, 129.7, 130.5, 134.3, 135.0, 137.6,
144.7, 152.9, 181.9, 187.2. IR (NaCI) 3281 cm", 1779 cm", 1738 cm", 1657 cm'

1. HRMS: [M + H]+ = 454.1078, calculated for C22H20N3053, 454.1073.

204

(IV-7).10 To a flame dried 100 mL round bottom flask was added
methylamine hydrochloride (1.00 g, 14.9 mmol) and dry DCM (55.0 mL). The
mixture was cooled to 0°C and then DMAP (3.79 g, 31.1 mmol), indole-2-
carboxylic acid (2.00 g, 12.4 mmol) and EDCI (2.86 g, 14.9 mmol) were added.
The mixture stirred at 0°C under nitrogen for 4 h and then at room temperature
overnight. The clear brown solution was washed with sat. sodium bicarbonate
solution (1 x 60.0 mL), 1% HCI (1 x 60.0 mL) and brine (1 x 60.0 mL). The
organics were combined, dried using anhydrous sodium sulfate and
concentrated. A small amount of DCM was then added and the product
precipitated out of solution to give an off white solid. Yield (1.57 g, 73.0%).‘H
NMR (500 MHz), Acetone: 5 2.94 (dd, J = 1.3, 4.7 Hz, 3H), 7.05 (t, J = 7.5 Hz,
2H), 7.20 (dd, J = 7.1, 8.3 Hz, 1H), 7.56-7.61 (m, 2H), 7.81 (s, 1H), 11.01 (s, 1H);
13C NMR (125 MHz), Acetone: 5 26.2, 102.5, 113.0, 120.7, 122.3, 124.3, 128.7,
132.8, 137.6, 162.8. M.S: calculated for C10H10N20 (M+) = 174.2 and found (M+)
= 174.3.

(IV-8). To a flame dried 50 mL round bottom flask was added anhydrous
DMF (3.50 mL) and dry DCM (25.0 mL). The solution was cooled to 0°C and then
oxalyl chloride (0.280 mL, 3.16 mmol) was added and the solution became a
thick white mixture. IV-7 (0.500 g, 2.87 mmol) was then added and the mixture
turned from yellow to red. The solution stirred at room temperature for 8 h under
nitrogen. The precipitate that formed was filtered off and washed with water (20-
30.0 mL) and extracted with EtOAc (5 x 30.0 mL). The organics were combined

and dried using anhydrous sodium sulfate and concentrated to give the product

205

as a solid. Yield (0.398 g, 69.0%). 1H NMR (500 MHz), DMSO: 5 2.91 (d, J = 4.7
Hz, 3H), 7.28 (t, J = 7.1 Hz, 1H), 7.34 (t, J = 7.1 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H),
8.19 (d, J = 8.8 Hz, 1H), 9.82 (s, 1H), 10.30 (s, 1H), 12.77 (s, 1H); 130 NMR (125
MHz), DMSO: 5 26.1, 113.0, 113.9, 120.5, 122.9, 124.7, 126.5, 134.9, 138.0,
159.9, 186.5. IR (NaCI) 3182 cm", 1651 cm“, 1590 cm". M.S: calculated for
C11H10N202 (M+) = 202.2 and found (M+) = 202.4. Anal. Calcd. For C11H10N202:
C, 65.34; H, 4.98; N, 13.85. Found: C, 64.08; H, 4.80; N, 13.58. Melting Point =
257-259 °C.

(IV-9). To a 25 mL round bottom flask was added IV-8 (0.306 g, 1.51
mmol) and EtOH (8.00 mL). Then thiohydantoin (0.193 g, 1.66 mmol) and
piperidine (0.900 mL, 9.10 mmol) were added and the mixture was stirred under
nitrogen at room temperature overnight. The mixture was filtered off and the
precipitate was stirred in acetone for 5 min and then filtered. The precipitate was
collected to give the product as a solid. Yield (0.390 g, 86.0%). 1H NMR (500
MHz), DMSO: 5 2.83 (d, J = 4.6 Hz, 3H), 6.95 (s, 1H), 7.17 (t, J = 7.4 Hz, 1H),
7.29 (t, J = 7.5 Hz, 1H), 7.47 (d, J = 8.3 Hz, 1H), 7.58 (d, J = 8.2 Hz, 1H), 8.14
(bs, 1H), 11.71 (bs, 1H), 11.98 (bs, 1H); 13C NMR (125 MHz), DMSO: 5 26.2,
105.5, 109.5, 112.5, 120.6, 120.8, 124.1, 126.1, 128.8, 131.1, 135.5, 161.6,
185.4, 177.6. IR (KBr) 3370 cm", 3194 cm", 1730 cm", 1883 cm", 1815 cm".
M.S: calculated for C14H12N402S (M+) = 300.3 and found (M+) = 300.3. Anal.
Calcd. For C14H12N402S: C, 55.99; H, 4.03; N, 18.65. Found: C, 54.91; H, 4.16;

N, 17.95. Melting Point = 380 °C decomposes.

206

(IV-10). To a 25 mL round bottom flask was added lV-9 (0.250 g, 0.833
mmol), MeOH (5.00 mL) and a solution of NaOH (0.0370 g, 0.917 mmol in 0.300
mL H20). Then Mel (0.0600 mL, 0.917 mmol) was added and the reaction stirred
under nitrogen over night at room temperature. The product precipitated out of
solution after stirring overnight and the orange solid was filtered off. The crude
product was recrystallized in MeOH to give pure product as a solid. Yield (0.252
g, 96.0%). 1H NMR (500 MHz), DMSO: 5 2.66 (s, 3H), 2.84 (d, J = 4.7 Hz, 3H),
7.14 (t, J = 8.1 Hz, 1H), 7.26 (t, J = 8.1 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.55 (s,
1H), 8.42 (d, J = 5.7 Hz, 1H), 9.11 (d, J = 8.0 Hz, 1H), 11.66 (s, 1H), 12.06 (s,
1H); 13C NMR (125 MHz), DMSO: 5 12.2, 26.1, 112.0, 112.5, 116.7, 120.6,
124.2, 125.1, 125.5, 135.2, 135.6, 135.9, 160.0, 161.8, 170.7. IR (KBr) 3267 cm'
I, 1708 cm", 1827 cm", 1802 cm". HRMS: [M + H]+ = 315.0918, calculated for
C15H15N402S, 315.0916. Anal. Calcd. For C15H14N402S: C, 57.31; H, 4.49; N,
17.82. Found: C, 57.10; H, 4.29; N, 17.58. Melting Point = 268-270°C
decomposes.

(IV-11). To a sealed tube was added IV-10 (0.420 g, 1.34 mmol) and THF
(4.00 mL). Then ammonium hydroxide (4.00 mL) was added and the mixture was
sealed and heated to 90°C for 16 h. The solution was cooled and the solvent was
removed. Methanol (15.0 mL) was added and the product precipitated out. Yield
(0.100 g, 26.0%). 1H NMR (500 MHz), DMSO: 5 2.79-2.95 (m, 3H), 6.90 (bs, 1H),
7.22 (t, J = 8.0 Hz, 1H), 7.43 (t, J = 8.2 Hz, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.74-
7.80 (m, 1H), 7.90-8.18 (m, 2H), 8.32-8.72 (bs, 1H), 10.42 (bs, 1H), 12.25 (bs,

1H); 13c NMR (125 MHz), DMSO: 8 27.5, 101.2, 101.5, 112.6, 120.13, 120.16,

207

121.3, 122.5, 123.8, 126.5, 129.6, 129.7, 132.70, 132.76, 139.4, 153.8, 161.9,
182.3, 188.2, 169.0. IR (KBr) 3488 cm'l, 3304 cm", 3218 cm", 1839 cm", 1808
cm", 1578 ml. HRMS: [M + H]‘“ = 284.1150, calculated for C14H14N502,
284.1147. Anal. Calcd. For C14H13N502: C, 59.36; H, 4.63; N, 24.72. Found: C,

57.30; H, 4.08; N, 24.33. Melting Point = 382-386°C decomposes.

208

IV.H References

1.

10.

Sharma, V.; Hupp. C. D.; Tepe, J. J., Enhancement of chemotherapeutic
efficacy by small molecule inhibition of NF-kB and checkpoint kinases.
Curr. Med. Chem. 2007, 14, 1061-1074.

Arnusch, C. J.; Pieters, R. J., Solid phase synthesis of vancomycin
mimics. Eur. J. Org. Chem. 2003, 3131-3138.

Mancini, |.; Guella, G.; Debitus, C.; Dubet, D.; Pietra, F., lmidazolone and
imidazolidinone artifacts of a pivotal imidazolthione, zyzzin, from the
poecilosclerid sponge Zyzzya massalis from the Coral Sea. The first
thermochromic systems of marine origin. Helvefica Chimica Acta 1994,
77, 1886-94.

Meanwell, N. A.; Roth, H. R.; Smith, E. C. R.; Wedding, D. L.; Wright, J. J.
K., Diethyl 2,4-dioxoimidazolidine-5-phosphonates: Homer-Wadsworth-
Emmons reagents for the mild and efficient preparation of C-5 unsaturated
hydantoin derivatives. J. Org. Chem. 1991, 56, 6897-904.

Sharma, V.; Tepe, J. J., Potent inhibition of checkpoint kinase activity by a
hymenialdisine-derived indoloazepine. Bioorg. Med. Chem. Lett. 2004, 14,
4319-4321.

Chacun-Lefevre, L.; Beneteau, V.; Joseph, B.; Merour, J.-Y., Ring closure
metathesis of indole 2-carboxylic acid allylamide derivatives. Tetrahedron
2002,58, 10181-10188.

Vilsmeier, A.; Haack, A., Action of phosphorus halides on
alkylformanilides. A new method for the preparation of secondary and
tertiary p-aIkylaminonobenzaldehydes. Ber. Dtsch. Chem. Ges. 1927,
608, 119-22.

http://www.millipore.com/druqdiscovery/dd3/KinaseProfiler.

Yang, L.; Deng, G.; Wang, D.-X.; I-Iuang, Z.-T.; Zhu, J.-P.; Wang, M.-X.,
Highly Efficient and Stereoselective N-Vinylation of Oxiranecarboxamides
and Unprecedented 8-endo-Epoxy-arene Cyclization: Expedient and
Biomimetic Synthesis of Some Clausena Alkaloids. Org. Lett. 2007, 9,
1387-1390.

Dutcher, J. D.; Kjaer, A., The C11H8NZOS degradation product of
gliotoxin. J. Am. Chem. Soc. 1951, 73, 4139-41.

209

 

67

ml
M“
B"
Uiii
I
Hi
8”
u
”it
lill
I
“3

03062 78

129

 

lllllllllllllllllllll