SYNTHESIS AND BIOLOGICAL EVALUATION OF INDOLOAZEPINE AND ITS
ANALOGS AS AN INHIBITOR OF CHK2
By
Thu Ngoc Thi Nguyen

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of
DOCTOR OF PHILOSOPHY

Chemistry
2011

ABSTRACT
SYNTHESIS AND BIOLOGICAL EVALUATION OF INDOLOAZEPINE AND ITS
ANALOGS AS AN INHIBITOR OF CHK2
By
Thu Ngoc Thi Nguyen
This dissertation is focused on two objectives. The first objective comprises the
improvement of the synthesis of (Z)-5-(2-amino-4-oxo-1H-imidazol-5(4H)-ylidene)2,3,4,5-tetrahydroazepino[3,4-b]indol-1(10H)-one, also referred to as indoloazepine.
The second objective is to evaluate indoloazepine as a c ellular inhibitor of checkpoint
kinase 2 and its use as an adjuvant drug for cancer therapy. The first chapter identifies
checkpoint kinase 2 (Chk2) as a viable adjuvant drug target for cancer therapies. The
second chapter discusses the improvement in a previously reported synthesis of
indoloazepine. The third chapter describes the biological evaluation and the cellular
inhibition of Chk2 by indoloazepine. Finally, the fourth chapter describes the use of
indoloazepine as a potential chemosensitizer via the nuclear factor-kappa B (NF-кB)
pathway.
DNA damage induced by ionizing radiation activates the ataxia telangiectasia
mutated pathway, resulting in apoptosis or DNA repair. The serine/threonine checkpoint
kinase, Chk2 is an i mportant transducer of this DNA damage signaling pathway and
mediates the ultimate fate of the cell. Chk2 is an advantageous target for the
development of adjuvant drugs for cancer therapy; because inhibition of Chk2 allows
normal cells to enter cell cycle arrest and carry out DNA repair, whereas many tumors
bypass cell cycle checkpoints. Chk2 inhibitors may thus have a radioprotective affect on
normal cells. Previously, our research group reported the synthesis of hymenialdisine-

derived indoloazepine and its ability to inhibit the kinase activity of Chk2 using purified
kinase.
The previous synthesis of indoloazepine reported by the Tepe research group
yielded indoloazepine in 12%. The synthetic route utilized steps that resulted in unstable
intermediates that were difficult to isolate with the final step requiring purification twice
by column chromatography. The improved synthesis reported here increased the overall
yield to 30% utilizing a thiohydantoin derivative, which also facilitated the isolation of the
indoloazepine. In addition, this route gave rise to new analogs of indoloazepine which
were evaluated as inhibitors of Chk2.
Indoloazepine was evaluated in cell culture and was found to be non-cytotoxic to
cells. Indoloazepine increased survival in normal cells following IR-induced DNA
damage, but not in tumor cells with mutated p53. Additionally, indoloazepine was
directly inhibited Chk2 in cells by inhibition of cis-autophosphorylation of Ser516 of
Chk2. Chk2 also plays a s ignificant role in the inhibition of IR-induced G2 arrest. The
data suggests that inhibition of Chk2 in normal cells protects cells exposed to IR as well
as induce rapid progression into G1 so that cells initiate DNA repair following IR-induced
DNA damage.

This dissertation is dedicated to my mother, Nam Nguyen, and my grandmother, Lieu
Nguyen. Although they did not have the opportunities to advance in their own
educations, they taught me more lessons about life, faith, perseverance and hard work
than I could ever find in a book. Cám ơn nhiều Mẹ va Bà Ngoại.

iv

ACKNOWLEDGEMENTS

I would like to acknowledge my advisor, Prof. Jetze Tepe and to thank him for
being incredibly patient with my many, many, many struggles in my research. My
determination and perseverance along with his patience have carried me through my
research. I did not realize that graduate school would teach me so many things about
life, and have so much influence on my character. I would not have learned these
without my advisor. Thank you, Jetze.
Additionally, I would like to acknowledge Prof. Babak Borhan, my second reader.
After taking his CEM 845 course for a few weeks, I really thought he enjoyed picking on
me and mocking me (this might still be true). However, I like to think that his intentions
were good and that he was pushing me to be a better student. He has always been an
invaluable resource to me. I would like to acknowledge my committee members,
Professors J. Justin McCormick and Kevin Walker for being a wealth information as well
as guidance throughout my graduate career.
Doing biochemistry surrounded by synthetic organic chemists proved to be
challenging. I would like to thank Dr. Sandra O’Reilly, Dr. Elizabeth McNeil and Dr.
Louis King for all the help with my irradiations and flow cytometry experiments and
intellectual discussions. In addition to these people, Teri Lansdell has also been a
tremendous resource for learning different biological techniques, trouble shooting and
helpful discussions.

v

I most genuinely want to thank staff in MSU Chemistry including Nancy Lavrik,
Bob Rasico, Melissa Parsons, Bill Flick, Beth McGaw, and Karen Maki. The chemistry
department is unbelievably lucky to have such a hard-working staff that is always willing
to help. They are an invaluable asset to this department.
I cannot have come as far as I have as a scientist and teacher without the
influence and friendships of the past and present Tepe group members. I have met
remarkable people and made lifelong friends in this lab. It has been an honor and a
privilege to have become such good friends with my lab mates including, Chris, Nicole,
Stacy, Amanda and especially Adam.
I never expected that I would come to graduate school and meet somebody who
would someday become my husband. I want to thank Adam for being there. He has
always been a wonderful source of scientific discussions throughout my graduate
school career and provided infinite support. He has survived all of my empty stomach
mood swings and somehow he still stuck around. Thanks Adam.
I want to thank my mother for coming to America so that her children could have
a better life. She is an amazing woman who worked two jobs in order to raise her 5
children. She’s always been supportive of me achieving my goals. My family and has
incredibly supportive and has always offered a pr ayer when I needed it. I have been
truly blessed to have been surrounded by such good people.

vi

TABLE OF CONTENTS
LIST OF TABLES ........................................................................................................ viii
LIST OF FIGURES......................................................................................................... ix
LIST OF SCHEMES ..................................................................................................... xiii
LIST OF ABBREVIATIONS .......................................................................................... xv
CHAPTER I. CHECKPOINT KINASE 2 AS A TARGET FOR ADJUVANT CANCER
THERAPY ................................................................................................. 1
I.A

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

I.B

Chemotherapy ........................................................................................... 6

I.C

Radiation therapy ....................................................................................... 9

I.D

DNA damage and cell cycle checkpoints ................................................. 13

I.E

Activation of the ATR and ATM pathways................................................ 16

I.F

Checkpoint kinase 2 ................................................................................ 18

I.G

The role of Chk2 and p53 ........................................................................ 20

I.H

Cell cycle response pathways .................................................................. 26

I.I

Chk2 as a drug target candidate .............................................................. 30

I.J

Validation of Chk2 as a drug target ......................................................... 31

I.K

Purified drug target and X-ray crystal structure........................................ 33

I.L

Current Chk2 Inhibitors ............................................................................ 34

I.M

Chk2 binding pocket properties ............................................................... 41

I.N

Goals of project ........................................................................................ 45

I.O

References .............................................................................................. 46

CHAPTER II. IMPROVEMENT OF A SYNTHETIC METHOD FOR THE
PREPARATION OF (Z)-5-(2-AMINO-IMIDAZOL-5(4H)-YLIDENE)-2,3,4,5TETRAHYDROAZEPINO[3,4-b]INDOLE-1(10H)-ONE, A
HYMENIALDISINE ANALOG ................................................................. 70
II.A

Isolation and characterization of hymenialdisine analogs ........................ 70

II.B

Synthesis of hymenialdisine and debromohymenialdisine ....................... 73

vi

II.C

Synthesis of marine sponge alkaloids through a common bicyclic pyrrole
coupling to 2-aminoimidazole .................................................................. 77

II.D

Synthesis of axinohydantoins .................................................................. 84

II.E

Synthesis of hymenialdisine and analogs through thiols ......................... 86

II.F

Synthesis of indole analogs of hymenialdisine......................................... 92

II.G

Synthesis of 2-aminoimidazolone ring through phenyloxazalone ........... 96

II.H

Hymenialdisine analog synthesis via Heck reaction ............................... 97

II.I

Improvement of the synthetic route to indoloazepine II-70 .................... 100

II.J

General experimental information ......................................................... 109

II.K

References ........................................................................................... 124

CHAPTER III. EVALUATION OF INDOLOAZEPINE AS AN INHIBITOR OF
CHECKPOINT KINASE 2 ..................................................................... 129
III.A

Biological target of natural product and hypothesis for activity ............. 129

III.B

In vitro evaluation of Chk2 inhibitors ...................................................... 130

III.C

In vitro evaluation of the effect of indoloazepine in the DNA pathway .. 136

III.D

In vitro evaluation of the cell cycle with indoloazepine .......................... 140

III.E

Cellular evaluation of indoloazepine ..................................................... 147

III.F

Indoloazepine as a radioprotecting agent ............................................. 153

III.G

In vivo evaluation of indoloazepine ....................................................... 163

III.H

Inhibition of Chk2 as a strategy for chemosensitization ........................ 172

III.I

Conclusions .......................................................................................... 174

III.J

General experimental information and procedures ............................... 175

III.K

References ........................................................................................... 184

CHAPTER IV. EVALUATION OF INDOLOAZEPINE AS AN INHIBITOR OF NF-кB 189
IV.A

Introduction to the NF-кB pathway ......................................................... 189

IV.B

Targets for inhibition of NF-кB ............................................................... 193

IV.C

Evaluation of II-70 and analogs as an inhibitor of NF-кB-mediated gene
transcription. .......................................................................................... 199

IV.D

Evaluation of II-70 as an inhibitor of IL-1β induced TNF-α production in
human whole blood ............................................................................... 202

IV.E

Evaluation of II-70 as an inhibitor of NF-кB in vitro ............................... 204

IV.F

General experimental Information ......................................................... 207

III.H

References ........................................................................................... 212
vii

LIST OF TABLES
Table I-1.

FDA-approved kinase inhibitors ................................................. 9

Table I-2.

Some of the possible damage in mammalian cell nucleus
following 1 Gy IR...................................................................... 13

Table II-1.

Nitrogen protecting group variations for indole aldisine ........... 94

Table II-2.

Scope of the Suzuki and Stille coupling for indole aldisine. ..... 95

Table II-3.

Attempted conditions for cyclization....................................... 105

Table III-1.

Kinase profiling data for indoloazepine II-1, II-2 and II-70 ...... 130

Table III-2.

Summary of IC50 values for kinase inhibition by analogs II-70, II84, II-99, III-1 and III-2 ........................................................... 134

Table III-3.

Statistically significant differences in cell cycle distribution of
untreated Chk2 +/+ versus II-70 treated cells, and unt reated
Chk2 +/+ versus Chk2 -/- cells ............................................... 145

Table III-4.

Results of NCI-60 cell line screen with II-70 .......................... 152

Table III-5.

Statistically analysis of vehicle compared to II-70 whole body
irradiated Balb-C mice ........................................................... 171

Table III-6.

Log values, standard error of log values and IC50 values ...... 178

Table III-7.

Mean and standard deviation of IC50 values of Chk1 and Chk2
............................................................................................... 179

Table III-8.

Summary of GI50 values of cells treated with II-70 ................. 180

Table IV-1.

Kinase inhibition by hymenialdisine-derived analogs. ............ 201

viii

LIST OF FIGURES
Figure I-1.

Representation of replication of normal and mutated cells ........ 2

Figure I-2.

Representation of uncontrolled growth of cancer cells .............. 3

Figure I-3.

Causes of cancer ....................................................................... 4

Figure I-4.

DNA damage by IR.. ................................................................ 12

Figure I-5.

Schematic representation of some types of DNA damage in
double-stranded DNA caused by IR ........................................ 12

Figure I-6.

Components of the DNA damage checkpoint pathway............ 15

Figure I-7.

Representation of Chk2 ........................................................... 17

Figure I-8.

Upstream signaling of p53 after IR-induced DNA damage ...... 22

Figure I-9.

Control of the cell division cycle by cyclins and Cdks and their
regulation by cell cycle checkpoints ........................................ 27

Figure I-10.

The G1/S and G2/M checkpoint regulation network following
upstream ATM/ATR activation ................................................. 28

Figure I-11.

Survival rates of Chk2 -/-, Chk2 +/- and Chk2 +/+ mice after 8
Gy whole body ionizing radiation ............................................. 32

Figure I-12.

Illustration of Chk2 and crystal structure of kinase domain ...... 34

Figure I-13.

Squaric acid derivatives and indazoles as Chk2 inhibitors ...... 35

Figure I-14.

Structures of NSC 109555 and PV1019 .................................. 36

Figure I-15.

Structure of carboxamide Chk2 inhibitors. ............................... 37

Figure I-16.

Structure of arylbenzimidazole and non-benzimidazole inhibitors
of Chk2 .................................................................................... 38

Figure I-17.

Structures of aminopyrimidine inhibitors of Chk2. .................... 38

Figure I-18.

Structure of CCT241533. ......................................................... 39

Figure I-19.

Structure of natural product staurosporine and analog UCN-01.
................................................................................................. 40

Figure I-20.

Structure of debromohymenialdisine and its analog
indoloazepine........................................................................... 41

Figure I-21.

Debromohymenialdisine inside the Chk2 active site, top view. 42
ix

Figure I-22.

Debromohymenialdisine inside the Chk2 active site, bottom
view ......................................................................................... 43

Figure I-23.

Indoloazepine modeled in the Chk2 catalytic site .................... 44

Figure II-1.

Structures (Z)-debromohymenialdisine and (Z)-hymenialdisine
................................................................................................. 70

Figure II-2.

Structures
of
the
axinohydantoins
and (
Z)debromohymenialdisine and (Z)-hymenialdisine...................... 71

Figure II-3.

Structure of hymenin and stevensine....................................... 73

Figure II-4.

Azafulvene intermediate to be coupled to 2-aminoimidazole II20............................................................................................. 78

Figure II-5.

Structure of 1-benzoyl-2-methylsulfanyl-1,5-dihydroimidazol-4one........................................................................................... 87

Figure II-6.

Structure of phenyloxazolone .................................................. 96

Figure II-7.

General scaffold for hymenialdisine analogs. ........................ 100

Figure III-1.

Structure of hymenialdisine and hymenialdisine-derived
indoloazepine II-1, II-2 and II-70 ............................................ 129

Figure III-2.

Illustration of radio-active kinase protocol .............................. 131

Figure III-3.

Structures of analogs II-84, II-99, III-1 and III-2 ..................... 132

Figure III-4.

Inhibition of [γ-33P]-ATP phosphorylation by Chk1 or Chk2. .. 133

Figure III-5.

Natural product hymenialdisine in Chk2 catalytic site ............ 135

Figure III-6.

Compounds II-70, III-1 and III-2 modeled in the Chk2 catalytic
site with PyMol ....................................................................... 136

Figure III-7.

Western blot analysis of 184B5 (normal, p53 WT) whole cell
extracts .................................................................................. 138

Figure III-8.

Western blot analysis of MDA-MB-231 (tumor, p53 mut) whole
cell extracts ............................................................................ 139

Figure III-9.

Western blot analysis of 184B5 whole cell untreated (control) or
treated with vehicle or with compounds III-1 and III-1 for 2 hours
prior to IR (10 Gy) .................................................................. 140

Figure III-10.

Cell cycle histograms of HCT116 Chk2 +/+ or Chk2 -/- cells
untreated (control) or pretreated for 2 hours with II-70 (30 µM)
followed by IR (0-5 Gy) .......................................................... 142

Figure III-11.

DNA frequency histograms of HCT116 Chk2 +/+ or Chk2 -/cells in G2............................................................................... 143
x

Figure III-12.

The abbreviated G1/S and G2/M checkpoint regulation network
following upstream ATM/ATR activation ................................ 144

Figure III-13.

DNA frequency histograms of HCT116 Chk2 +/+ or Chk2 -/cells in G1............................................................................... 146

Figure III-14.

Results of trypan blue exclusion test in breast cell lines. ....... 148

Figure III-15.

Results of trypan blue exclusion test in colon cancer cell lines.
............................................................................................... 150

Figure III-16.

Microscopic cell images of HCT116 cell lines following treatment
with II-70 for 24 hours of continuous exposure ...................... 151

Figure III-17.

Clones of 184B5 (normal) cells untreated (top panel) or
pretreated with II-70 for 30 minutes (bottom panel) followed by
IR (0-7.5 Gy) and seeded at the indicated cloning density .... 154

Figure III-18.

Cloning survival of 184B5 (normal) cells untreated or pretreated
with II-70 for 30 minutes followed by IR (0-7.5 Gy) ................ 155

Figure III-19.

Microscopic images (20X) of normal cell colonies (184B5)
treated with II-70 followed by 0 or 10 Gy IR ........................... 156

Figure III-20.

Cloning survival of 184B5 normal cells with various treatment
times. A) Cells post-treated 30 minutes following IR (0-7.5 Gy);
B) Cells pretreated for 2 hours and post-treated for 4 hours after
IR (0-7.5 Gy) .......................................................................... 157

Figure III-21.

Cloning survival of 184B5 and MDA-MB-231 cells. ............... 158

Figure III-22.

Clones of MDA-MB-231 (tumor, p53 mutant) cells untreated (top
panel) or pretreated with II-70 for 30 minutes (bottom panel)
followed by IR (0-7.5 Gy) and s eeded at the indicated cloning
density ................................................................................... 159

Figure III-23.

Microscopic images (20X) of tumor cell colonies (MDA-MB-231)
treated with II-70 followed by 0 or 10 Gy IR ........................... 160

Figure III-24.

Cloning survival of HCT116 cell lines. ................................... 161

Figure III-25.

Comparison of cloning survival of HCT116 treated with II-70
versus HCT116 Chk2 -/- cells following IR ............................ 162

Figure III-26.

LC/MS quantification of biologically available II-70 in mice. ... 166

Figure III-27.

Cytotoxicity of mice treated with varying doses of II-70 by IP. 167

Figure III-28.

Mouse irradiation chamber used for whole body IR. .............. 169

Figure III-29.

Effect of pre-treatment with II-70 (10, 1 or 0.1 mg/kg) for 30
minutes prior to whole body IR with 7.3 Gy on survival of male
Balb-C mice. .......................................................................... 170
xi

Figure III-30.

Trypan blue exclusion assay of OVCAR-3 cells treated with II70........................................................................................... 173

Figure III-31.

Trypan blue exclusion assay of OVCAR-3 cells treated with II-70
in combination with 0.5 µM Topotecan .................................. 174

Figure IV-1.

NF-кB signal cascade ............................................................ 190

Figure IV-2.

IкB Kinase inhibitors .............................................................. 196

Figure IV-3.

IKKβ inhibitors ....................................................................... 196

Figure IV-4.

Structures of GSK-3 Inhibitors ............................................... 198

Figure IV-5.

Structures of II-70, II-84, II-100. ............................................. 199

Figure IV-6.

Structures of IV-1 – IV-4 ........................................................ 200

Figure IV-7.

Dose-response activity of II-70 in inhibition of luciferase
production in HeLa-NF-кB-luc cells ........................................ 202

Figure IV-8.

Dose-response activity of II-70 in inhibition of TNF-α production
in IL-1β stimulated human blood ............................................ 203

Figure IV-9.

Western blot of TNF-α induced p65 after 30 minutes in HeLaNF-кB-luc cells ....................................................................... 204

Figure IV-10.

Western blot of HeLa cells with TNF-α induced p-p65 after 30
minutes ................................................................................. 205

Figure IV-11.

EMSA Assay for NF-кB Activation by TNF-α (10 ng/mL). ...... 206

xii

LIST OF SCHEMES
Scheme I-1.

DNA damage by UV ............................................................... 10

Scheme II-1.

Synthetic route toward II-1 and II-2 of intermediate II-11 ......... 74

Scheme II-2.

Synthetic route toward II-1 and II-2 of intermediate II-14. ........ 75

Scheme II-3.

Synthesis of II-17 toward II-1 and II-2 ...................................... 76

Scheme II-4.

Completed synthesis of II-1 and II-2.. ...................................... 77

Scheme II-5.

Synthesis of azafulvene intermediate II-19a ............................ 78

Scheme II-6.

Synthesis of hymenin and debromohymenin ........................... 79

Scheme II-7.

Synthesis of intermediates II-25 and II-26 ............................... 80

Scheme II-8.

Synthesis of stevensine II-7 ..................................................... 81

Scheme II-9.

Synthesis of II-7 from II-6 ......................................................... 82

Scheme II-10.

Synthesis of II-1 and II-2 from intermediate II-29 and II-28 ...... 83

Scheme II-11.

Synthesis of II-2 using a one-pot, two-step synthesis ............. 84

Scheme II-12.

Synthesis of II-3c, II-4c, II-36 and II-37 from intermediate II-35
................................................................................................. 85

Scheme II-13.

Synthesis of axinohydantoins II-3b and II-4b from II-3c and II-4c
................................................................................................. 86

Scheme II-14.

Synthesis of II-2 via thiohydantoin II-39. ......................................

Scheme II-15.

Improved synthesis of II-11a .................................................... 88

Scheme II-16.

Synthesis of II-1 and II-2 utilizing II-41. .................................... 89

Scheme II-17.

Conversion of II-44 to II-2 under microwave conditions. .......... 90

Scheme II-18.

Synthetic route for II-3a, II-3b and II-4a, II-4b. ......................... 91

Scheme II-19.

Synthesis of endo-pyrrole analogs........................................... 92
xiii

Scheme II-20.

Toward the synthesis of indole analogs of hymenialdisine.. .... 93

Scheme II-21.

Synthesis of mono- and di-triflate protected indole aldisines. .. 94

Scheme II-22.

Synthesis of indoloazepine II-70 .............................................. 97

Scheme II-23.

Synthesis of intermediate II-74 for ring closure via Heck reaction
................................................................................................. 98

Scheme II-24.

Unsuccessful Heck reaction approach to II-70......................... 99

Scheme II-25.

Synthesis of II-82 via hydantoin II-79. .................................... 101

Scheme II-26.

Hydantoin coupling to II-68. ................................................... 102

Scheme II-27.

Attempted HWE synthesis to form II-84. ................................ 102

Scheme II-28.

Alternate synthesis to II-84. ................................................... 102

Scheme II-29.

Synthesis of intermediate II-95. ............................................. 104

Scheme II-30.

Synthesis of II-41 ................................................................... 106

Scheme II-31.

Synthesis of intermediate II-98 .............................................. 107

Scheme II-32.

Synthesis of II-70 and analogs II-84 and II-99. ...................... 108

xiv

LIST OF ABBREVIATIONS
184B5

normal, breast cell line

5-FU

fluorouracil

Abl

tyrosine kinase enzyme

ABI

arylbenzimidazole, Chk2 Inhibitor II

ACN

acetonitrile

ADP

adenosine diphosphate

AP

apyrimidic/apurinic

ARS

acute radiation syndrome

Asn

asparagine

ATM

ataxia telangiectasia mutated

ATP

adenosine triphosphate

ATR

ataxia telangiectasia and Rad3-related

BRCA1

breast cancer type 1 susceptibility protein

BJ-hTERT

foreskin fibroblasts

BM

hematopoietic syndrome

Boc

tert-butyl carbamate
xv

13

C NMR

carbon nuclear magnetic resonance

c-Kit

human proto-oncogene

c-Rel

proto-oncogene protein encoded by Rel

Cdc2

cell division cycle 2

Cdc25A

cell division cycle 25 homolog A

Cdc25C

cell division cycle 25 homolog C

CD4+

cluster of differentiation 4

CD8+

cluster of differentiation 4

Cdk1

cyclin dependent kinase cyclin

Chk1

checkpoint kinase 1

Chk2

checkpoint kinase 2

CK1δ

casein kinase 1

CK2

casein kinase 2

CML

chronic myeloid leukemia

CNS

central nervous system

DBH

Debromohymenialdisine

DCM

dichloromethane

xvi

DMAP

dimethylaminopyridine

DMF

dimethylformamide

DMSO

dimethylsulfoxide

DNA

deoxyribonucleic acid

DNA-PK

DNA dependent protein kinase

DSB

double-strand break

Dex

Dexamethasone

(E)

entgegen, trans double bond

EDCI

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

EGFR

epidermal growth factor receptor

EMSA

Electrophoretic mobility shift assay

EtOAc

ethyl acetate

EtOH

ethanol

eV

electronvolt

FDA

food and drug administration

FHA

forkhead association domain

FLT3

receptor tyrosine kinase

xvii

G1

Gap 1 phase

G2

Gap 2 phase

GI

gastrointestinal syndrome

GIST

gastrointestinal stromal tumor

Glu

gluatamate

GSK-3β

glycogen synthase kinase -3 beta

Gy

Gray, SI unit of absorbed radiation

1

proton nuclear magnetic resonance

H NMR

HOAc

acetic acid

H2AX

gene that codes for histone H2A

HC

hepatocellular carcinoma

HCT116

colon cancer cell line

Her-2

human epidermal growth factor receptor 2

HWE

Horner-Wadsworth-Emmons reaction

IBX

2-Iodoxybenzoic acid

IC50

half maximal inhibitor concentration

IFN-α

interferon-α
xviii

IкB-α

inhibitory kappa B-alpha protein

IKKα

inhibitory kappa B kinase-alpha

IKKβ

inhibitory kappa B kinase-beta

IL-#

interleukin-#

IP

intraperitoneal

IR

Ionizing radiation

KHMDS

Potassium bis(trimethylsilyl)amide

MCF-7

breast cancer cell line (Michigan Cancer Foundation 7)

MDA-MB-231

breast tumor, p53 mutant

MDM2

murine double minute 2 gene

MeCN

acetonitrile

MEF

mouse embryonic fibroblasts

MEK1

mitogen-activated protein kinase kinase/ERK1

MeOH

methanol

Met

methionine

MG-132

proteasome inhibitor

MRE11

meiotic recombination 11 homolog A

xix

mRNA

messenger ribonucleic acid

MSK1

mitogen- and stress-activated protein kinase-1

Myt1

myelin transcription factor

NaOAc

sodium acetate

NBS

N-bromosuccinimide

NBS1

nibrin, Nijmegen breakage syndrome 1

NCI

National Cancer Institute

NES

nuclear export signal

NLS

nuclear localization signal

NF-кB

nuclear factor-kappa B

NSCLC

non-small cell lung cancer

OVCAR

ovarian carcinoma

p21

WAF1

cyclin-dependent kinase inhibitor

p53

protein p53, tumor protein supressor

PARP

poly adenosine diphosphate ribose polymerase

PDGFRa

platelet-derived growth factor receptor, alpha polypeptide

PDGFRb

platelet-derived growth factor receptor, beta polypeptide

xx

Ph

phenyl

PIKK

phosphoinositide 3-kinase related kinases

PKCα

protein kinase C-alpha

PKCβII

protein kinase C-alpha

PPA

polyphosphoric acid

RA

rheumatoid arthritis

Rad50

DNA repair protein

Rb

retinoblastoma protein

RCC

renal cell carcinoma

RelA

v-rel reticuloendotheliosis viral oncogene homolog A

RelB

v-rel reticuloendotheliosis viral oncogene homolog B

RHD

Rel homology domain

RNA

ribonucleic acid

RNAi

ribonucleic acid interfernce

RPA

Replication Protein A

RT-PCR

real-time polymerase chain reaction

S

synthesis phase

xxi

SAR

structural activity relationship

SCD

serine-glutamine/threonine-glutamine cluster domain

SEM

[2-(trimethylsilyl)ethoxy]methyl ether

Ser

serine

siRNA

small interfering RNA

SRB

sulforhodamide B

Src

sarcoma

SQ/TQ

serine-glutamine/threonine-glutamine region

SSB

single-strand break

TADs

transactivation domains

TBAF

tetra butyl ammonium fluoride

TEA

triethylamine

TFA

trifluoroacetic acid

THF

tetrahydrofuran

Thr

threonine

TIPS

triisopropylsilyl ether

TNF-α

tumor necrosis factor-α

xxii

U2OS

human osteosarcoma cell line

VEGFR

vascular endothelial growth factor

Wee1

cell division kinase inhibitor

UV

ultra violet

X-ray

X-radiation

(Z)

zusammen, cis double bond

xxiii

CHAPTER I
CHECKPOINT 2 KINASE AS A TARGET FOR ADJUVANT CANCER THERAPY
I.A.

Introduction to cancer
Currently, one in four deaths in the United States is caused by cancer, making it
1

one of the leading causes of death. Although there has been an increase in knowledge
and advances in cancer therapies, the mortality rate due to cancer has not been greatly
1

affected. However, from 1991 to 2006, US cancer death rates decreased 16% while
rates for other chronic diseases including heart disease (36%), cerebrovascular (31%)
1

and influenza (49%) decreased even more significantly. This is a major public health
problem that plagues not only the United states, but many other parts of the world.

1

Cancer exists in over 100 different forms capable of manifesting in almost every
tissue in the body. Cancer encompasses a group of diseases described by uncontrolled
2

growth and spread of abnormal cells, frequently resulting in death. Each human has
trillions of cells that work together to form networks of tissue and organs. Normal cells
have the ability to divide in a regulated and systematic way. These cells form new
2

daughter cells allowing injured, mutated or dead cells to be replaced (Figure I-1). Cells
that cease to cooperate metabolically and structurally with neighboring cells can
become transformed and eventually become tumor cells which may later result in
cancer cells.

2

1

Figure I-1. Representation of replication of normal and mutated cells. For interpretation
of the references to color in this and all other figures, the reader is referred to the
electronic version of this dissertation.
Benign tumors belong to the first of the two classes of generic tumors. They are
most often enclosed in a protective capsule of tissue

3, 4

and do not spread to other parts

5

of the body. In general, benign tumors are not lethal, unless they exert pressure
against critical organs or tissue, leading to death.

2

Malignant tumors make up the second, more dangerous class of tumors. These
tumors are capable of destroying a part of the body in which they originate, as well as
neighboring tissue. Malignant tumors can also metastasize to other parts of the body,

6

7

initiating the growth of new secondary tumors. These secondary tumors can continue
to destroy additional tissues and organs, distinguishing them from benign tumors, which
do not spread to other parts of the body.

2, 5

As mentioned above, malignant tumor cells (cancer cells), can cause destruction
in the tissue of origin, as well as surrounding and neighboring tissues.

2, 5

Unlike normal

cells, cancer cells divide and grow rapidly, piling up into a mass or tumor as illustrated in
2

Figure I-2.

2-4, 8, 9

These cancer cells can spread and metastasize to distant tissue and

can eventually destroy it.

2, 5

Cancer cells from the primary tumor can travel through the

bloodstream or lymphatic system, spreading to sites near to and far away from the
2, 5

original tumor.

Figure I-2. Representation of uncontrolled growth of cancer cells.
Malignant tumors are comprised of different types including carcinomas,
sarcomas, leukemias and lymphomas. Carcinomas make up 92% of human tumors.
These tumors develop in tissue that line the surfaces of organs such as the lung, liver,
2

skin, and breast. Sarcomas originate from mesenchymal cells, including cells from
bone, cartilage, fat, connective tissues and muscle. Leukemias are malignant tumors
that affect white blood cells. Sarcomas and leukemias combined make up 8% of the
2

total human tumors. Lymphomas develop in the lymphatic system and represent the
smallest percentage of human tumors, developing in the lymphatic system.

2

Because there are so many different types of cancers, there are also several
causes, or factors that contribute to cancer. There are endogenous factors (hormones),
and exogenous factors (occupational and environmental carcinogens) that can
3

2

contribute to the development of human cancer. The major causes include tobacco
smoke and related tobacco products (30%), hormones (30%), diet (15%), viruses
(10%), drugs, x-rays, and ultra violet light (UV) (10%) as well as occupational
carcinogens (5%) (Figure I-3).

2

Occupational

Drugs, Xray UV
Tobacco
Viruses

Diet
Hormones

Figure I-3. Causes of cancer.

2

Several cancers thought to be induced by tobacco smoke exposure include lung
cancer, bladder cancer, pancreatic cancer, liver cancer, cervical cancer, laryngeal
cancer, cancer of the lip, stomach cancer, kidney cancer, and colon cancer.

10

The influence of hormones is believed to be the cause of about 30% of new
cases of cancer.

2

Estrogens without progestogens cause endometrial cancer and

estrogen can also cause breast cancer (progesterone can increase the effect).
4

2, 10

Ovarian cancer can be brought on by the hormone changes involved in ovulation.

10

The

effects of testosterone or its metabolites can be responsible for the onset of prostate
2, 10

cancer.

Dietary factors are a second major contributor to the induction of human cancer.
10-13

2,

Two factors that are believed to contribute to the induction of cancer are excess

caloric content and a diet high in red meats. A diet including more green-leafy and
yellow vegetables protect against various types of cancers, while a diet lacking these
has been correlated with a higher cancer incidence.

10-14

These, along with other dietary

factors are thought to account for 15% of human cancers.

2, 10-13

Other contributing causes of cancer include oncogenic viruses (about 10%) such
as hepatitis B virus leading to liver cancer.

10, 15

In addition, 10% of human cancer can

be due to medical drugs, exposure to radiations such as medical and dental x-rays or
UV light.

10

This also encompasses “secondary tumors induced by specific

chemotherapeutic agents, skin tumors induced by UV light, and tumors induced by
2

specific drugs.”

Lastly, 5% of human cancer can be attributed to by exposure to carcinogens in
the workplace. These include asbestos (lung cancer and mesothelioma), benzene
(acute myelogenous leukemia), and hexavalent chromium (nasal and respiratory
cancer).

9, 16-18

Nearly half (45%) of all human cancer causes can be traced to exposure to
chemical carcinogens and radiation.

10

It is astounding that 60% of all human cancers

are theoretically preventable. Eliminating exposure to tobacco and tobacco products
5

would reduce human cancer by 30%. In addition, having a proper diet with controlled
caloric and fiber intake, including increasing vegetables and limiting intake of animal fat
could help to reduce risks of cancer exposure. Avoiding excess exposure to specific
drugs, x-rays and UV light can also prevent human cancer.
I.B

2, 10, 11, 13, 19, 20

Chemotherapy
Cancer in its many forms of uncontrolled cell growth can lead to solid tumors

(with the exception of a few cases such as leukemia), but can be treated using different
types of therapy. Surgery, when applicable is a viable option to remove solid tumors,
however it does not completely remove the cancer. Chemotherapy and/or radiation
therapy or the combination of them can be used as a treatment method.

21-35

There are

several types of chemotherapeutic agents available including alkylating, topoisomerase
II inhibitors, antimetabolic, biological, hormonal and tubulin-targeting agents.

36

Alkylating agents are polyfunctional compounds that can substitute alkyl groups
for hydrogen ions. Most of these compounds can react with phosphate, amino, hydroxyl,
sulfhydryl, carboxyl, and imidazole groups. Alkylations, which most often occur at the N7 position of guanine, leads to abnormal base pairing and interference with DNA
replication, transcription of RNA as well as the disruption of nucleic acid function.
Alkylating agents can lead to disruptions in the normal cell functions in both cancerous
and normal tissues.

37, 38

The alkylating agents are divided into five classes:

bischloroethylamines (nitrogen mustards), aziridines, nitrosoureas and non-classical
alkylating agents.

36-40

Topoisomerase inhibitors are a class of compounds that interact with
topoisomerase I and topoisomerase II enzymes by assisting in the unwinding of DNA
6

during the replication process. Topoisomerase I can reversibly cleave a single strand of
DNA in order to allow relaxation of the coil, then reseal the cleaved strand after
unwinding.
irinotecan.

28

Topoisomerase

I

inhibitors

include

camptothecin,

topotecan

and

Topoisomerase II has a function similar to topoisomerase I, however it

completes a reversible double stranded DNA cleavage to aid in the unwinding of DNA
for replication, then reseals the cleaved strands. Topoisomerase II inhibitors include
etoposide, doxorubicin and daunorubicin.

25, 32-34

Doxorubicin belongs to a class of

anthracyclines that intercalate between base pairs of DNA causing a distortion that
prevents DNA replication process. The metabolism of this process with compounds like
doxorubicin can also lead to free radical formation that can cause oxidative damage to
DNA. Additionally, the reactive intermediates formed from the radicals can form
covalent adducts with DNA, again repeating the overall replication process.

34

Antimetabolites constitute a large group of anticancer drugs that are able to
interfere with metabolic processes necessary for the proliferation of cancer cells. The
major classes of the antimetabolites include the antifols, purine analogs, and pyrimidine
analogs.

36, 38, 41-43

Nearly all of the antimetabolites in clinical use function as an inhibitor

of the synthesis of purine or pyrimidine nucleotides or directly inhibit enzymes involved
36

in DNA replication.

Newer antimetabolites include gemcitabine and the 5-fluorouracil

(5-FU) prodrug, capecitabine.
Biologic agents or biologic therapy utilizes agents that evoke immune responses,
directly target receptor or signaling pathways, modify the stroma of the tumor, or elicit
tumor regression.

36

This class of therapy includes recombinant cytokines, some of

7

which have immunomodulatory and antitumor activity, including interleukin-2 and
interferon-α (IFN-α). Other cytokines have been evaluated including tumor necrosis
factor-α (TNF-α), interferon-γ, IL-1, IL-4, and IL-12.

36

Hormonal reagents play a significant role in regulating the growth and
development of their target organs. There have been different hormonal reagents used
in treating tumors originating from the breasts, uterus, ovary and prostate. Estrogens,
androgens and progestins function via their respective receptors. Following high-affinity
binding to the receptors, the steroids can then modify the configuration of the receptor
molecules and allow them to bind to a segment of the DNA template (hormone
response element). Once the steroid is bound to the hormone response element, it can
regulate gene transcription and control cellular growth and function.

44

Tubulins are critical in eukaryotic biology during both cell division as well as
nonmitotic phases of the cell cycle.

36

Tubulin-binding drugs play a part in chromosomal

separation during mitosis, making them desirable targets for antitumor drugs. This class
of drugs is made up of a large family of heterogeneous compounds that include the
vinca alkaloids, taxanes, and estramustine phosphate.

45

Kinases have become an attractive and challenging target in cancer drug
discovery as researchers have revealed more information about cancer. Many groups
have researched the development of kinase inhibitors; however few have been
approved by the FDA in the last decade (Table I-1).

8

28

Table I-1. FDA-approved kinase inhibitors
U.S.
Brand
Name

Year
Approved

Cancer
type

Company

Target kinase

Gleevec

2001

CML

Novartis

Abl, c-Kit, PDGFRa, PDGFRb

Iressa

2003

NSCLC

EGFR

Tarceva

2004

NSCLC, P

AstraZeneca
Genetech,
OSIP

Nexavar

2005

HC, RCC

Bayer, Onyx

c-Kit, VEGFR, PDGFR, FLT3

Sutent

2006

GIST, RCC

Pfizer

Abl, c-Kit, PDGFR, Src

Sprycel

2006

CML

BMS

Abl, c-Kit, PDGFR, Src

Tasigna

2007

CML

Novartis

Abl, c-KIT, PDGFRb, Src,
Ephthrin

EGFR

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
I.C

Radiation therapy
Although a large variety of chemotherapeutic agents are available for cancer

treatment, radiation continues to be a method of therapy for nearly half of all cancer
patients. Radiation therapy uses high-energy radiation in order to reduce tumor sizes by
causing instability in the form of DNA damage leading to cell death.

22, 31, 46

There are a

variety of methods in which radiation can be delivered. The first method involves the
delivery of x-rays or γ-rays

46

outside of the body using external-beam radiation therapy.

A patient receiving radiation therapy for breast cancer could be exposed to 45-60 Gray
(Gy) of radiation over a period of weeks.

47

The radiation therapy is administered in

fractionated doses in which the patient receives 1-2 Gy/day for 5 days/week.
9

47

1 Gy of

radiation is approximately equivalent to the radiation exposure of 200,000 dental X-rays
or 140 chest X-rays.

47

Internal radiation therapy can be administered by placing

radioactive material in the body near cancer cells, also referred to as brachytherapy.
Systemic radiation therapy uses substances such as radioactive iodine 131 to treat
thyroid cancers. The radioactivity can travel in the blood stream and accumulate in the
46

thyroid, killing cancer cells in this region.

Ultraviolet radiation is lower in energy and the majority of DNA damage results
from the formation of photoproducts. An example of a photoproduct resulting from UV
damage is a pyrimidine dimer (Scheme I-4). The dimer can inhibit transcription of DNA
and the replication machinery which can ultimately lead to cell death.

48

Scheme I-1. DNA damage by UV.

Ionizing radiation (IR) has been previously reported as a cause of cancer among
other adverse effects. It is a human carcinogen that is among the most extensively
studied, accounting for nearly 3% of all cancers.

49

IR can remove electrons from atoms

resulting in changes in the molecular structures of the cells. The DNA damage incurred
from the ionizing radiation is capable of causing the development of cancer.
damage could also be used as a tool in cancer therapy.
10

49

This DNA

IR is among the various agents that are capable of creating free radicals that
induce DNA damage.

31

DNA damage resulting from •OH are the predominant source of

damage when DNA is irradiated in dilute aqueous media. In an aqueous DNA solution,
only ~0.5% of the energy of IR is actually absorbed by DNA following γ-irradiation. The
remaining ~99.5% of the energy of IR is absorbed by water. The majority of the freeradical damage to DNA is caused by the free-radicals produced via the radiolysis of
water. An almost negligible amount of free-radicals come from the energy absorbed by
DNA.

31

On average, ~100 eV of energy is deposited per ionization event when highenergy electrons are absorbed by water following IR. A water radical cation and an
electron are generated during this process (Figure I-4, Reaction (1)). The water radical
cation can further cause ionizations in the surrounding areas. The areas which contain
further ionizations and sometimes electronic excitations events are referred to as spurs
(Figure I-4, Reaction (2)). The small pockets containing spurs can lead to the formation
of clustered lesions which can be amplified resulting in more than one damaged site
•

caused from just one OH radical.

31

The radicals can cause nucleotide damage,

crosslinking and strand breaks. Figure I-4 shows an example of a strand cleavage
reaction that could occur following IR.

31

11

Figure I-4. DNA damage by IR.
The main types of damage that can be formed in DNA include base damage,
apyrimidic/apurinic (AP) site, single-strand break (SSB), double-strand break (DSB),
tandem lesions and various clustered lesions. These types of damage are summarized
in Figure I-5. With enzymatic treatments, Figure I-5a, b and e could potentially turn into
an SSB, while f and g could lead to a DSB (Figure I-5).

31

Figure I-5. Schematic representation of some types of DNA damage in double-stranded
DNA caused by IR: a) base damage; b) AP site; c) SSB; d) DSB from two close-by
SSBs; e) tandem lesion; f) clustered lesion with two damaged bases at opposite
strands; g) SSB with damaged base on opposite strand; h) clustered lesion with three
damaged bases; i) clustered lesion with a DSB (from two close-by SSBs) and two
31
damaged bases.
12

Although one site of damage can cause other sites of damage, it is important to
note that a one single strand or double strand break does not necessarily constitute
damage that is lethal.

31

Cellular repair enzymes in the DNA damage repair pathway can

often efficiently repair the simple lesions. Lethal DNA damage is commonly the result of
more complex lesions.

31

Table I-2 lists the possible damage as a result of 1 Gy of IR

ranging from a simple, repairable lesion, to more complex lesions.
Table I-2. Some of the possible damage in a mammalian cell nucleus following 1 Gy
50, 51
IR.
Initial phsyical damage

# of events

Ionizations in the cell nucleus

~ 100,000

Ionizations directly in DNA

~ 2,000

Excitations directly in DNA

~ 2000

Selected biochemical damage
SSBs

1,000

8-oxo-A (a typical single-base damage)

700

DSBs

40

DNA-protein cross-links

150

Selected cellular events
Lethal events
Chromosome abberations

I.D

~0.2-0.8
~1

DNA damage and cell cycle checkpoints
DNA damage arises from several sources including UV, IR, background

radiation, environmental mutagens and chemical substances that attack DNA.

52, 53

Cells

have evolved complex mechanisms to protect their genomes from the damage. DNA
damage responses function to correct the damage or minimize the probability of lethal
13

or permanent genetic damage.

53

The cellular responses to DNA damages are made up

of several repair mechanisms and checkpoint responses which can delay the
progression of the cell cycle or modulate DNA replication. These responses are pivotal
in maintaining stability of the genome.

53

DNA checkpoint pathways act as an anticancer barrier to maintain the genetic
integrity from one cell to the next.

54

The pathways were initially observed when

chemotherapeutic agents induced damage to DNA which led to an inhibition of the cell
cycle that subsequently allowed cellular repair.

55, 56

A similar control of cell cycle

progression was observed in Saccharomyces cerevisiae and was then termed a
57, 58

„checkpoint.‟

The original view was that checkpoint pathways functioned only as

regulators of the cell cycle transitions. The current accepted view is that checkpoints are
part of a cascade of signals that are responsible for DNA damage response
59, 60

processes.

The damage responses are capable of removing the damaged DNA

sites in addition to restoring the DNA duplex. Arresting the cell cycle temporarily permits
repair and prevention of further replication of damaged DNA or its transcriptional
response.

57

Cells that have been heavily damaged or seriously deregulated can

undergo apoptosis or terminal cell cycle arrest.

59, 61, 62

The checkpoint pathways consists of three major groups of proteins that work
together (Figure I-6):

63

a) sensor proteins that recognize damaged DNA and signal the

presence of abnormalities, initiating the signal cascade;

63

b) transducer proteins, often

protein kinases that relay and amplify the damage signal obtained from the sensors and
phosphorylating other kinases or downstream targets;
14

63

and c) effector proteins, which

consist of the most downstream targets of the transducer protein kinases, and are
consequently regulated by phosphorylation to prevent cell cycle progression.

63, 64

DNA Damage
DNA Damage
Sensors

ATR

ATM

DNA-PK

Signal
Transducers

Chk1

Chk2

IKK

Effectors

CDC25
CDK activation

p53
pathway

NF-κB
pathway

Cell cycle arrest
(DNA repair &
replication)

Apoptosis
(cell death)

Anti-Apoptosis
(cell survival)

Figure I-6. Components of the DNA damage checkpoint pathway.
In the initial stages of checkpoint activation, DNA damage sensors relay
information to members of a family of phosphoinositide 3-kinase related kinases
(PIKK).

65

Three of the main sensors belong to the PIKK family and include the: ataxia

telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) proteins
66-69

61,

and DNA-PK, all of which play pivotal roles in early signal transmission through cell
64

cycle checkpoints.

DNA-PK activates the NF-кB signaling pathway and will be

described in further detail in Chapter 4. ATM and ATR are large serine-threonine
kinases with a molecular weight of 350 kDa that phosphorylate different substrates in
the DNA damage response signaling networks.
15

After DNA damage, sensor proteins such as ATR/ATM initiate the signal cascade
and the effect is amplified by phosphorylation of the signal transducers checkpoint
kinase 1 (Chk1) and checkpoint kinase 2 (Chk2), respectively. Chk1 and Chk2 are
serine-threonine kinases required for cell cycle arrest in response to DNA damage and
can further phosphorylate downstream targets.

63

The effector components, Cdc25 and

p53, are phosphorylated by Chk1 and Chk2, leading to cell cycle arrest and apoptosis,
respectively.
I.E.

57

Activation of the ATR and ATM pathways
ATR and ATM respond to different forms of DNA damage. ATR is activated by

tracts of single strand breaks in conjunction with its partner protein ATR-interacting
protein (ATRIP).

70-72

ATM is recruited and activated by DSBs in association with the

MRE11:Rad50:NBS1 (MRN) sensor complex.

73, 74

Because ATM and ATR have a

sequence homology, they often have overlapping functions and substrate specificities.

52

ATM has been observed to respond to DNA damage in the early stages and ATR then
responds in the late stages.
ATR is activated most strongly when DNA replication is interrupted, such as
nucleotide depletion or replication-blocking DNA damage lesions often induced by UV
light.

53, 64

Replication-blocking causes DNA polymerases to become uncoupled from the

replicative helicase,

75

which generates SSBs and quickly gets coated with a trimeric

ssDNA-binding protein complex, Replication Protein A (RPA). ATR is then recruited and
activated at the single strand breaks with its partner ATRIP.

53, 72

The activation of ATR

does not require autophosphorylation or posttranslational modification.
16

64

Once ATR is activated, it phosphorylates its downstream effector Chk1 at both
Ser345 and Ser317, in response to UV DNA damage.

76, 77

Phosphorylation at Ser345

is essential for Chk1 biological activity, although the exact modifications that are
required for regulation of the Chk1 catalytic activity are poorly understood.

53, 78

Phosphorylation of Chk1 regulates its subcellular localization and therefore translocates
to centrosomers and inhibits premature activation of Cdk1 (Cdc2)-Cyclin B1 through
78-80

inhibition of Cdc25C.

Chk1 can also be phosphorylated by ATM in response to IR-

induced replication stress, contributing to the degradation of Cdc25A following IR.

81

Chk1 and Chk2 possess overlapping functions of Chk1 and Chk2 despite their lack of
structural similarity.

82-84

The ATM pathway is activated by IR and genotoxins that induce DSBs. They can
also be activated only weakly, if at all, by agents that block DNA replication without
85

inducing damage.

When DNA damage is not present, ATM is believed to exist as

inactive homodimers. When DSBs occur, the inactive ATM homodimers are quickly
induced to autophosphorylate in trans, causing them to dissociate to form partially
active monomers.

67

The first autophosphorylation site identified is Ser1981. This

residue is not necessary for ATM function,
activation of ATM.

67

86

however its modification is linked to the

The monomers of ATM are recruited to DSBs via interactions with

the MRN sensor complex,

87

which fully activates ATM allowing it to act on downstream

substrates. These substrates include the variant histone, H2AX, forming the DNA
damage-associated γ-H2AX histone mark,

88

the MRN complex and Chk2.

17

89

In its active

form, Chk2 can phosphorylate several substrates that control cell cycle progression and
DNA repair as well apoptosis.

90

The difference in functionalities of ATR and ATM was confirmed utilizing their
respective null mice. The ATM null mice were viable, however had a phenotype that
was characterized by infertility and growth retardation.

91, 92

ATR null mice, however,

died early in embroygenesis, demonstrating a phenotype similar to that of mitotic
catastrophe.

93, 94

This illustrates how essential ATR is for life, likely because of its

functions in monitoring DNA replication.
I.F

95

Checkpoint kinase 2
The first example of the „Chk2 family‟ of checkpoint kinases was first discovered

in 1994 as Rad53, which was a kinase involved in multiple checkpoint responses in
budding yeast.
eukaryotes.

97

96

Homologues of Rad53 were later identified in fission yeast and higher

Five laboratories reported identification of the human homologue

and the human gene Chk2 was cloned and named by the Elledge group.
structure of the Chk2 protein is similar in all eukaryotes.

97

98

98-102

The overall

Although all eukaryotes share

a structural homology and share a role as transducers of cell cycle checkpoints, there
are functional differences between the Chk2 homologues.

97

The yeast homologues are

needed for different forms of DNA damage as well as replication blocks,
the mammalian Chk2 responds primarily to DSBs.

96, 103-105

while

98-102

The amino-terminal domain of Chk2 contains a series of serine or threonine
residues followed by glutamine serine-glutamine/threonine-glutamine (SQ/TQ)-rich motif
106

or SCD (Figure I-7a).

These are reported to be the preferred sites for phosphorylation
18

107

by ATM/ATR.

Another key domain of Chk2 is the fork-head associated (FHA)

domain, which seems to bind phospho-threonine residues. It is also thought to be
involved in protein-protein interactions triggered by phosphorylation of proteins
recognized by FHA partners such as Chk2.

97

The kinase domain comprises nearly the

entire carboxy-terminal half of Chk2, and the main functional elements including the
activation loop (Figure I-7a).

98

In response to DSBs, ATM is activated and subsequently phosphorylates Chk2
at Thr68 in the SQ/TQ motif of one Chk2 molecule. This results in recognition by the
phosphopeptide-binding FHA domain of another molecule (Figure I-7b) and leads to
transient homodimerization, intermolecular activation loop autophosphorylation on
Thr383 and Thr387 and cis-autophosphorylation
activation.

108,

109

on Ser516, and full

110-112

a
SCD
(19-69)

N
ATR

b

Th68

FHA
(115-175)

C (543)

KINASE (226-486)
Th383 Th387
(auto-inhibitory loop)

ATM

Ser516

IR
ATM

Inactive
monomer

Th68
phosphorylation

Th68-P induced
dimerization

Activation by
autophosphorylation
Active dimers

Figure I-7. Representation of Chk2. a) Functional domain of Chk2;
activation.

113

Active monomers

b) Model of Chk2

Following activation, Chk2 is believed to dissociate from sites of damage in its
monomeric form to act on multiple substrates resulting in cell cycle arrest, activation of
19

DNA repair, and apoptosis.

53, 89

Substrates of Chk2 in vivo include, but are not limited

to, p53, BRCA1, Cdc25A and Cdc25C.
stabilizes the p53 protein,

114-116

97

Phosphorylation of p53 by Chk2 and/or Chk1

which increases the potential to positively regulate the

factors involved in DNA repair, cell death and cell-cycle control.
BRCA1 is another substrate of Chk2

119

117, 118

and is a key checkpoint controlling

protein that is responsible for regulating homologous recombination repair, transcriptioncoupled repair, the cell cycle, chromatin remodeling and possible, and cell death.

120-122

Chk2 phosphorylates BRCA1 at Ser988, which is required for the release of BRCA1
from its complex with Chk2. This event is important for cell survival following DNA
damage.
I.G

119

The role of Chk2 and p53
Chk2 has been associated with mediating cell cycle arrest in pathways through

p53. p53 is a short-lived transcription factor that has attracted extensive research in its
ability to mediate tumor suppression.

117, 123, 124

Evidence has suggested that p53 is not

responsible for the rate of tumor initiation or mutation. Instead, p53 prevents the
progression of malignant tumor cells.

125-128

It regulates cell growth through activation of

cell-cycle arrest and apoptosis following chemotherapeutic or radiation induced DNA
damage.

129-131

p53 is highly expressed in several normal tissues (lymphoid,

hematopoietic organs, intestinal epithelia, etc.), which are severely damaged during
chemotherapeutic treatment and IR.

132

There is a high level of p53 mutations in

cancerous tissue (> 50% of all cancer contain p53 mutations).

20

133

In nearly half of these

tumors, p53 is inactivated directly resulting from mutations in the p53 gene.

117

In the

majority of the remaining tumors, p53 is inactivated indirectly through viral proteins or
from alterations in genes whose products interact with or transmit information to p53.

117

The p53 network can be activated by the ATM-Chk2 and the ATR-Chk1
pathway,

117

which inhibit degradation of p53, therefore stabilizing high concentrations of

p53. The activated form of p53 can bind to particular DNA sequences and activate
transcription of nearby genes which can ultimately lead to inhibition of cell division or
cell death.
The levels of p53 in cells are dependent on its degradation rate which is
regulated by ubiquitin-mediated proteolysis. The MDM2 protein is an enzyme that is
involved in the attaching ubiquitin to p53, labeling it for degradation. This process is
regulated by a negative feedback loop (Figure I-8).

134

p53 binds to the regulatory region

of MDM2 and stimulates gene transcription to mRNA and subsequent translation into
117

protein.

The MDM2 binds to p53 forming a complex, which allows ubiquitinylation and

subsequent degradation of p53.

117, 126

The degradation of p53 reduces the amount of

p53 and also reduces transcription of MDM2, closing the feedback loop and
consequently allowing p53 levels to increase. Upstream phosphorylation of p53 occurs
in the N-terminal region (Ser15, Thr18, Ser20, Ser37),

116, 135-138

where MDM2 binds.

This affects the interaction with MDM2 in vitro, leading to stabilization of p53.

21

116, 135-138

ATM

Chk2

P

X

P

p53

MDM2

P

X
Ub Ub
Ub
Ub

p53

p53

MDM2

MDM2

Inactived
complex

p53
proteasomal
degradation

Figure I-8. Upstream signaling of p53 after IR-induced DNA damage. Phosphorylation
of p53 and MDM2 disrupt the interaction between the two proteins.
ATM and Chk2 are capable of phosphorylating p53 within the MDM2 region,
which has lead to much controversy over the connection between Chk2 and p53. Chk2
has been placed immediately upstream of p53 through studies involving Chk2 knockout
mice as well as in vivo and in vitro experiments.

89, 114-116, 139-142

Chk2 was found to

phosphorylate p53 at Ser20 thereby regulating p53 levels in response to DSBs.
140, 143

114, 115,

A kinase defective form of Chk2 prevented stabilization and phosphorylation of

Ser20 in co-transfected p53 after DNA damage.

116

U2OS cells having a dominant

negative Chk2 were incapable of stabilizing or phosphorylating endogenous p53 at

22

Ser20 and induced a p53 dependent G1 checkpoint.

116

In further support of the

connection between Chk2 and p53, it was reported that Chk2 was capable of
phosphorylating p53 at Ser20 in vitro and was capable of disrupting pre-complexed p53
and Mdm2.

114, 116

A different study reported that IR-activated immunoprecipitated Chk2

was able to phosphorylate p53.

81

When Chk2 was silenced using engineered zinc finger

transcription factors, decreased p53 transcriptional activation of the MDM2 gene and
reduced p53 Ser20 phosphorylation were observed.

144

Conversely, evidence has also weakened the view that Chk2 regulates p53.
Chk2 that was purified from tumor cell lines prior to and following DNA damage was
unable to phosphorylate various forms of truncated or full-length p53 despite being
markedly activated to phosphorylate the domain of Cdc25C containing Ser216.

145

Other

experiments in vivo have also challenged the relationship of Chk2 and p53. Chk1 and
Chk2 knockout by siRNAs or RNAi of several human tumor cell lines resulted in partial
or no defects in the DNA damage induced phosphorylation and stabilization of p53
Ser20

phosphorylation

or

phosphorylation of Cdc25C.

transcriptional

140-142, 145, 146

activity

despite

preventing

Ser216

Additionally, homozygous deletion of Chk2 in

HCT116 colon carcinoma cells yielded no phenotype with respect to p53, G 1 or G2 cell
cycle arrest, and apoptosis.

113, 146

The region of p53 spanning Ser20 does not share the homology with the
consensus motif, L-X-R-X-X-S/T which is present in other known substrates of Chk2.
Consequently, the peptide covering the p53 Ser20 region is a poor Chk2 substrate,
which weakens the Chk2-p53 link.

147, 148

However, there are two regions of p53, amino
23

acids 117-132 and 272-288, that were proposed as Chk2 docking sites that could
potentially regulate the kinase activity toward p53 allosterically.

143

The purified Chk2

supplemented with these docking-site motifs was able to phosphorylate a Ser20 peptide
147

(P-L-S-Q-E-T-F-S-D-L-D-L-W-K-L-K-K-K)

in a kinase assay.

143

This study showed

that Chk2 could modulate p53 specifically with a protein kinase docking site instead of
by a consensus sequence.
Chk2 knockout mice have been generated independently by two groups which
both support a role for Chk2 in the regulation of p53, however the proposed
mechanisms did not agree. The first reported Chk2 null mouse generated from Hirao
and colleagues

140

demonstrated that thymocytes, splenic T cells, and MEFs lacking

Chk2 were unable to stabilize p53 following γ-IR. When Chk2 was reintroduced, the
defect was corrected. The cells were highly resistant to apoptosis consistent with a
defect in p53 signaling.

140

A follow up study showed that Chk2 knockout mice were

highly resistant to apoptosis in multiple tissue compartments by whole body IR
experiments. These mice also developed chemically-induced skin tumors more
rapidly.

141

At low doses of γ-IR, the G1/S checkpoint of these mice was compromised,

however, the cells arrested normally at doses above 5 Gy.

141

Another group used MEFs

from these same mice, however did not observe a defect in G 1/S arrest at even low Xray doses.

149

Chk2 knockout mice generated by another research group was reported

yielding different results.

142

The second generation of mice had many tissues that were

resistant to apoptosis induced by γ-IR, which was in agreement with the original Chk2
null mice, strongly supporting the role of Chk2 in DNA damage induced apoptosis.
24

142

These authors observed a deficient G1/S arrest consistent with impaired p53
signaling.

113

Unlike results reported by Hirao et al, only a 30-50% reduction in p53

stabilization and human p53 introduced into Chk2 -/- MEFs was phosphorylated
normally on Ser20 after IR.

142

These results indicated that Chk2 in murine tissue made

only a partial contribution to p53 stabilization and that other factors contributed to the
stabilization. Only a partial p53 stabilization defect was observed upon analysis of five
p53 targets genes by quantitative RT-PCR in thymocytes and MEF‟s. There was no
upregulation of mRNA observed in the Chk2 -/- cells after IR, possibly providing a
mechanistic explanation for the G1/S arrest and apoptotic defects in the cells.

142

The relationship between Chk2 and p53 is likely to be stimuli- as well as cell
type-specific as cell death in primary neuronal cultures induced by etoposide or
camptothecin was shown to be ATM-dependent but Chk2 independent.

113, 150

It is

evident that Chk2 plays a pivotal role in γ-IR induced apoptosis, however the exact
mechanism

remains

unclear.

Chk2

can

regulate

p53

transcriptional

activity

independently of Ser20 phosphorylation and stabilization in murine tissues after IR.

113

It

is important to consider that many types of cellular stresses that activate p53 do not
activate Chk2 and can subsequently signal to p53 normally even in the absence of
Chk2.

81

Chk2 also has molecular targets other than p53, in the event that p53 or Chk2

is not present, the other protein remains a participant in other checkpoint pathways.
Chk1 has been observed to undergo activation compensating for the absence of
Chk2.

142

These features could support why Chk2 deletion or mutation does not affect

p53 inactivation in human cancer cells.

81

25

Although the DNA damage pathway in

response to DSBs is not a linear pathway, cancer cells defective in Chk2 are under
pressure to inactivate p53, which is consistent with the observed concomitant mutations
of p53 and Chk2 in some tumors.
I.H

139, 151-153

Cell cycle response pathways
Checkpoints function by arresting or delaying the progression of the cell cycle in

response to DNA damage.

93

The DNA damage response can operate in several ways:

it regulates cell cycle arrest, activates DNA repair mechanisms, induces cell cycle death
by apoptosis, regulates telomere length and activates pivotal transcriptional
programs.

64, 154

Checkpoint genes have also been demonstrated to be necessary for

the survival of organisms, causing embryonic lethality in mice when particular genes are
knocked out.

76, 155

This provides strong evidence that checkpoint genes are critical for

regulation of DNA damage, and more importantly, essential for functions in cellular
survival.

52

Cell cycle division in mammals is controlled by numerous cyclin-dependent
kinase (Cdk) complexes (Figure I-9). Cdk4/6-Cyclin D complexes are active in the early
phase of the G1 phase of the cell cycle. Entry into and progression of cells through the
S-phase are regulated by Cdk-2-Cyclin E and Cdk2-Cyclin A, respectively. Whether or
not cells proceed into mitosis is governed by Cdk1 (Cdc2)-Cyclin B. The activities of the
Cdk‟s can be inhibited by several cyclin-dependent kinase inhibitors, such as p21

WAF1

.

In addition to the existing cell cycle phases G1, S, G2 and mitosis, there are also
transitional points: G1/s, G2/M as well as intra-S phase and the mitotic checkpoint,
which are all tightly controlled checkpoints.

61, 156

26

A key function of Chk1 and Chk2

following activation by ATR and ATM centers around the inactivation of members of the
Cdc25 family by phosphorylation. This results in inhibition of cell cycle progression after
52

DNA damage in the G1/S phase or the G2/M phase.
G2/M DNA damage checkpoint

Mitotic checkpoint

Cdk4/6-Cyclin D

M
Cdk1 (Cdc2) -Cyclin B

G2

G1

Cdk1 (Cdc2) -Cyclin A

Cdk2-Cyclin E
S

Cdk2-Cyclin A

G1/S DNA damage checkpoint

Intra S phase DNA damage checkpoint

Figure I-9. Control of the cell division cycle by cyclins and Cdks and their regulation by
cell cycle checkpoints.

Progression of the G1/S checkpoint is dependent upon the Chk1/Chk2-Cdc25ACdk2 pathway (Figure I-10, pathway A). When active, Cdc25A is unphosphorylated and
functions as a phosphatase for the Cdk2-Cyclin E complex. The Cdk2-Cyclin E complex
remains active and allows the cell cycle to progress from G 1 to S.

52

In response to DNA

damage, Chk1 and Chk2 can phosphorylate Cdc25A resulting in its degradation. The
degradation of Cdc25A causes the Cdk2-Cyclin E complex to stay in its inactive
hyperphosphorylated form and halts the G1/S progression.

27

52

ATM

ATM-P

ATR-P

inactive

inactive

Chk2-P

DNA damage
sensors

ATR

Chk1-P

DNA damage
sensors

Chk1

Chk2
active

active

Inactive/degraded

inactive

Cdc25A-P

Cdc25C-P
p53

Cdc25A
active

active

p53Mdm2
degraded

PP P P

P

Cdk2
Cyclin E

Cdk2
Cyclin E

inactive

p53-P

inactive

active

p21WAF1
P

Cdk4
Cyclin D

Cdk1 (Cdc2)
Cyclin B

active

Rb-P

inactive
Wee
Myt1

Rb

active

inactive

G1

P PPP

Cdk1 (Cdc2)
Cyclin B

active

S

Cdc25C

C

G2

A

M

B

Figure I-10. The G1/S and G2/M checkpoint regulation network following upstream
52
ATM/ATR activation.
The G2/M checkpoint relies on the Chk1/Chk2-Cdc25C-Cdk1 pathway (Figure I10, pathway B). Activation of Cdk1-Cyclin B1 can subsequently activate Cdc25C,
creating a positive feedback loop allowing progression into mitosis.

157, 158

The Cdk1-

Cyclin B1 complex can also inactivate Wee1, which would result in two positive
feedback loops.

159

Mitosis that is triggered by Cdk1 is the most attractive target of the

G2 regulation. Cdk1 is phosphorylated at two sites by protein kinases Wee1 and Myt1
then subsequently dephosphorylated by Cdc25C phosphatase.

52

Increased amounts of

phosphorylation of Cdk1 by Wee1/Myt1, or inhibition of dephosphorylation by Cdc25C
28

52

activated by Chk1 results in G2/M DNA damage checkpoint arrest.

The Cdk1-Cyclin

B1 complex when inactive blocks entry into mitosis. Consequently, Chk1 and Chk2 can
inhibit the cell cycle by controlling the Cdk1-Cyclin B1 complex via Cdc25C following
DNA damage (Figure I-10, pathway B).

52

The Chk1/Chk2 pathway involving p53 regulates the G1/S checkpoint (Figure I10, pathway B, C). p53 can be phosphorylated at many sites by various protein kinases
following DNA damage

160, 161

and is catalyzed by ATM, ATR, DNA-PK, and Chk1/Chk2.

The phosphorylated form of p53 reduces the ability of MDM2 to bind to p53, which
increases accumulation of p53 as well as its phosphorylation after DNA damage as
mentioned above.

135, 162

The activation of p53 upregulates several target genes

involved in the DNA damage response, one of which his p21

WAF1

WAF1

. Build up of p21

downregulates Cdk2-Cyclin E, resulting in G1 arrest (Figure I-10, pathway C).

163

Inhibition of the G1/S phase is a result of preventing phosphorylation of Rb by inhibiting
Cdk2.

52

WAF1

Activated p53 can also play a role in the G2/M checkpoint by inducing p21

,

which blocks the formation of Cdk1-Cyclin B1 and therefore inhibiting its activity (Figure
I-10, pathway B).

164-166

Checkpoint control following DNA damage can be influenced by p53 which is
phosphorylated directly by ATM or ATR,
Chk1 and Chk2.

114, 140

69, 162, 167, 168

or indirectly by ATM and ATR via

The Chk2 gene can have cancer-related mutations which

prevents p53 from being phosphorylated.

139, 169

Cancer cells are dependent on the S

and G2 checkpoints in order to repair DNA damage. This is in part due to the defective
G1 checkpoint mechanisms. The S-phase checkpoint does not arrest in response to
29

DNA damage, instead it slows the progression of the cell cycle, then arrests at G 2.
Consequently, the G2 checkpoint is the key regulator of cancer cells and hence has
been an attractive target for cancer therapy. Arresting cancer cells at G 2 prevents them
from repairing DNA damage and forces them into mitotic catastrophe and apoptosis.
I.I

170

Chk2 as a drug target candidate
Radiation

and

genotoxic

chemotherapies

remain

primary

methods

of

conventional cancer treatment and are likely to remain so. These therapies are
imperfect often incurring severe side effects. Consequently, there has been interest
focused on understanding how normal and tumor cells respond to DNA damage and
determining whether the responses to DNA damage could be used for therapeutic
purposes.

53

Based on the described DNA damage pathways above, it is possible that

inhibiting the checkpoint kinases could be used to augment the effects of
chemotherapeutics by sensitizing cancer cells, or desensitizing normal cells.

171-174

It is

believed that inhibiting Chk1 sensitizes tumor cells by blocking the ability of cells to
initiate DNA repair, resulting in DNA damage beyond repair.

171 175-177

Inhibition of Chk2

could selectively inhibit apoptosis in p53 wild type or normal cells, therefore
desensitizing them from cell death during chemotherapeutic treatment.

173, 178

Inhibition

of Chk2 is not anticipated to have effects on apoptosis in cells containing mutated p53
(> 50% of cancer cells).

133

Efforts have been focused on developing Chk2 inhibitors in

order to desensitize normal cells toward IR and thus widen the therapeutic window of
treatment.
I.J

Validation of Chk2 as a drug target

30

Chk2 has become an attractive target for cancer therapy given its pivotal role in
the regulation of the cell cycle, apoptosis and DNA repair following DNA damage.

179, 180

Ionizing radiation and DNA damaging chemotherapeutics continue to be the
conventional form of cancer therapy often resulting in severe side effects. Inhibiting the
DNA damage checkpoints could potentially augment the current chemo- or radiation
therapies by sensitizing cancer cells to the cytotoxic effects of the therapy. Conversely,
checkpoint inhibition could potentially allow an enhancement in survival of normal cells
by desensitizing normal cells from IR or DNA damaging agents.

2

Chk2 inhibition is involved in eliciting radio- or chemoprotection of normal tissue
via the inhibition of p53-dependent apoptosis.

179, 180

Most tumor cells bypass normal

cell cycle responses following DNA damage if p53 is mutated.

181

In normal cells, p53 is

activated by Chk2 following IR, resulting in cell cycle arrest and apoptosis.

182-184

Thus,

inhibition of Chk2 in normal cells may prevent p53-mediated apoptosis, DNA repair and
potential cell survival. This hypothesis was supported by a study in which Chk2 -/transgenic mice showed a reduction in p53-dependent apoptotic responses.

142

Increased survival of the Chk2 -/- mice was observed following exposure to whole body
IR as compared to Chk2 +/+ mice (Figure I-11).
thymocytes

185

142

Inhibition of Chk2 in mouse

and isolated CD4+ and CD8+ human lymphocytes

186

also showed a

decrease in apoptosis in response to IR. These and other elegant studies

66, 185-189

therefore indicate that inhibition of Chk2 in normal cells, may increase survival following
IR by selectively reducing p53-mediated cell death.

31

Survival of Mice following IR
Chk2 -/Chk2 +/-

survival (%)

100

Chk2 +/+

75
50
25
0

0

10

20

30

Time after IR (days)

Figure I-11. Survival rates of Chk2 -/-, Chk2 +/- and Chk2 +/+ mice after 8 Gy whole
142
body ionizing radiation.
In addition to the radioprotective affects in normal cells, Chk2 inhibitors have
been shown to enhance the effects of cytotoxic drugs
with Chk2 siRNA

145

190-192

Transfection of MCF-7 cells

was found to enhance the effect of paclitaxel.

192

Chk2 inhibition

augmented the levels of mitotic catastrophe when used together with doxorubicin
cisplatin

194

193

or

by releasing mitochondrial pro-apoptotic proteins. Additionally, small

molecule Chk2 inhibitors have shown potentiation of DNA-damaging agents topotecan
and camptothecin in OVCAR-4 and OVCAR-5 human tumor cells.

195

Recent studies also demonstrate that inhibition of Chk2 without an additional
genotoxic agent may also be advantageous for therapeutic development in tumors
possessing increased levels of activated Chk2.

180, 195, 196

Tumor cells in which Chk2 is

constitutively activated have plausibly adapted a Chk2 dependence in order to survive.

32

A recent study by Pommier et al demonstrated the antiproliferative activity of a Chk2
inhibitor (discussed further in Chapter I.L) and Chk2 siRNA in cancer cell lines that were
over-expressed in Chk2.

195

The use of Chk2 inhibitors for potentiating DNA-damage agents continues to be
exciting albeit somewhat controversial
demonstrate these effects.

185, 188

179, 180, 197

as not all inhibitors of Chk2

There are only a few small molecule inhibitors of Chk2

that have been reported in the literature, thus interest in discovering a potent and
selective inhibitor remains high. Of these reported inhibitors, high-throughput screening
has proven to be very useful resulting in the identification of several ATP-competitive
Chk2 inhibitors including 2-arylbenzimidazole-5-carboxamides (ABI),
carboxamidines

185, 188

186, 187

isothiazole

and bis-guanylhydrazones (discussed further in Chapter I.L).

66,

195

I.K

Purified drug target and X-ray crystal structure
It can be important and beneficial to have information about the purified drug

target as a tool in developing inhibitors. Oliver et al. were able to crystallize the kinase
domain of Chk2 (Figure I-12) offering an enormous amount of information toward the
development of Chk2 inhibitors.

112

Several Chk2 inhibitors have since been co-

crystallized within the Chk2 binding domain.

111, 196, 198-200

The top of Figure I-12 illustrates the linear representation of the whole Chk2
protein. Chk2 is a 543 long amino acid protein that consists of three domains which
were described in Chapter I.F. Briefly, The first domain is known as the SQ/TQ cluster
domain (SCD), which is the substrate of the upstream protein ATM. The second domain
33

is the FHA which has been implicated in the modulation of protein-protein interactions.
The last domain, which was crystallized, is the kinase domain which is responsible for
performing the phosphorylation of substrates.

112, 113

The information the Chk2 crystal

structure has provided has been invaluable toward the discovery of new Chk2 inhibitors
and progress toward an adjuvant drug for chemo- and radiation therapy.
1

112-175

N

19-69

SCD

220-486

FHA

KINASE

543

C

Figure I-12. Illustration of Chk2 and crystal structure of kinase domain.
I.L

Current Chk2 Inhibitors
Although there has been an increase in the synthesis and discovery of Chk2

inhibitors within the last decade, few potent and selective Chk2 inhibits were reported
until recently. Some of these inhibitors remain highly confidential such as XL844,
developed by Excelixis, which is currently in clinical trials.

34

201-203

While the structure of

XL844 is not publicly known, other inhibitors have been hidden in patents such as the
indazole and squaric acid derivatives shown in Figure I-13, and have not been highly
publicized.

204-206

Figure I-13. Squaric acid derivatives and indazoles as Chk2 inhibitors.
An increasing number of novel Chk2 inhibitors have been reported through high
throughput screening of compound collections and combinatorial libraries.
198, 199, 207, 208

66, 185-188, 195,

A bis-guanylhydrazone (NSC 109555) was identified after a high

throughput screen of over 100,000 compounds (Figure I-14) and was co-crystallized in
the Chk2 binding pocket.

199

The compound was found to be an ATP competitive

inhibitor of Chk2 with an IC50 of 240 nM. NSC 109555 was relatively selective for Chk2
showing at least 6.5 times selectivity over other kinases. When evaluated in cellular
studies, NSC 109555 was unable to inhibit topotecan-induced autophosphorylation of
66

Chk2 at Ser516 as well as abrogation of the G2/M block induced by camptothecin.

The lack of cellular inhibition was possibly due to pharmacokinetics or DNA binding
interference of the polyamine.

66

A further SAR study of the bis-guanylhydrazones

produced a derivative of NSC 109555, which demonstrated similar potent inhibition of
Chk2 in vitro. PV1019 was at least 75 times more selective for Chk2 when evaluated
against a screen of 50 kinases.

195

PV1019 inhibited Chk2 autophosphorylation in
35

addition to its downstream substrates. Chk2 inhibitor PV1019 protected normal mouse
thymocytes against IR-induced apoptosis and showed synergistic antiproliferative
activity with chemotherapeutics in tumor cell lines. Inhibition of Chk2 with PV1019 alone
in cancer cells with high Chk2 expression resulted in antiproliferative activity.

Figure I-14. Structures of NSC 109555 and PV1019.
A series of isothiazole carboxamidine compounds were described by Wu and
Carlessi as novel and selective and potent inhibitors of Chk2 (IC 50 = 120 nM to 40
µM).

185

Of these, VRX0466617 (Figure I-15) was found to be the most potent and

selective, exhibiting radioprotection in BJ-hTERT cells and mouse thymocytes.
VRX0466617 did not modify the cytotoxicity of anticancer drugs, nor did it have a
significant effect on G2/M arrest. However, VRX0466617 did show cellular inhibition of
Chk2 at Ser 19 and Ser 33-25 in addition to other downstream substrates.

185

Zabludoff

et al. introduced a thiophene carboxamide, AZD7762, which is currently in phase I
clinical trials in combination with gemcitabine and IR in pancreatic tumor models.

207, 209

AZD7762 has been shown in multiple assays to inhibit DNA damage-induced cell cycle
checkpoints. This Chk2 inhibitor has been evaluated extensively and has demonstrated
its ability to increase the response to multiple DNA-damaging agents in several tumor
cell lines. Although AZD7762 is an equally potent inhibitor of Chk1 and Chk2, the

36

cellular studies performed point to mechanisms targeted at Chk1 in vivo and not
Chk2.

207, 209

AZD7762 inhibited Chk1 autophosphorylation (Ser296), stabilized Cdc25A

and increased ATR/ATM-mediated Chk1 phosphorylation (Ser345).

209

AZD7762

showed radio- and chemosensitizing properties comparable to those observed by Chk1
inhibitors PD-321852 and PF-00477736 (100-fold more selective for Chk1 than
210-212

Chk2).

Figure I-15. Structure of carboxamide Chk2 inhibitors.
Researchers at Johnson and Johnson developed a new class of benzimidazoles
as inhibitors of Chk2. A high-throughput screening of purified human Chk2 led to a
novel series of 2-arylbenzimidazoles which demonstrated potent and selective inhibition
of Chk2.

186

The most potent inhibitor is illustrated in Figure I-16, resulting in an IC50

value of 2 nM and showing excellent selectivity for Chk2 over 35 kinases evaluated. The
2-arylbenzimidazole inhibitor also showed effective radioprotection of human T-cells
against IR with EC50 values between 3-7.6 µM. Additionally, isolated CD4+ and CD8+
human lymphocytes also showed a decrease in apoptosis.

186, 187

An SAR study was performed with a series of non-benzimidazole Chk2 inhibitors
(Figure I-16). The IC50 values for the new inhibitors ranged from 5.8 µM-16 nM.
37

187

Studies with these inhibitors evaluating the potentiation of cytotoxic abilities with current
cancer therapeutics have not yet been performed.

57, 187

Figure I-16. Structure of arylbenzimidazole and non-benzimidazole inhibitors of Chk2.
Hilton et al. identified a class of 3,5-diaryl-2-aminopyridine inhibitors through
high throughput screening of a kinase-focused compound library. Over 60 compounds
were synthesized and characterized in addition to displaying the interaction of the
compounds using co-crystallization with Chk2. Many of these compounds were reported
to be Chk2 inhibitors. Of the many nanomolar inhibitors, three compounds (Figure I-17)
showed

selectivity

toward

Chk2

autophosphorylation of Chk2 at Ser516.

and

demonstrated

cellular

inhibition

of

198

Figure I-17. Structures of aminopyrimidine inhibitors of Chk2.
Structure-based design was used to optimize a new class of 2-(quinazoline-2yl)phenols in order to generate potent and selective inhibitors of Chk2.
38

196

The SAR

study gave rise to nearly 50 compounds which were screened for Chk2/Chk1 potency
and selectivity. The most potent compound (IC50 = 3 nM), CCT241533 (Figure I-18),
had a 63-fold selectivity of Chk2 over Chk1 and demonstrated cellular inhibition of
autophosphorylation of Ser516.

196

Additionally, CCT241533 enhanced the cytotoxicity

of PARP inhibitors in p53-deficient cell lines and inhibited etoposide-induced biomarkers
of Chk2 activity in human tumor cell lines.

213

CCT241533 showed a dose dependent

decrease in apoptosis of mouse thymocytes when treated before IR.

196

Caldwell et al

were also able to identify minor differences in the Chk1 and Chk2 binding pockets,
however were unable to determine if these changes were significant.

196

Figure I-18. Structure of CCT241533.
Natural products and analogs of natural products have contributed to the
number of current Chk2 inhibitors. Staurosporine and its analog UCN-01 (Figure I-19)
were shown to be potent inhibitors of Chk2, however, did not demonstrate selectivity for
66, 214, 215

the checkpoint kinase.
214

nM)

UCN-01 showed potent inhibition of Chk2 (IC50 = 10

and an ability to potentiate the anticancer activity of a variety of therapeutics

including cis-platin, mitomycin and IR.

216-219

39

Additionally, UCN-01 has been found to

augment the cytotoxicity of chemotherapeutic agents in human glioblastoma cells.

220

The use of UCN-01 has been questionable due to its promiscuity as a kinase inhibitor.
The structure of UCN-01 also contains pharmacokinetic disadvantages such as its
strong binding to human plasma protein and its low bioavailability.

221-223

Figure I-19. Structure of natural product staurosporine and analog UCN-01.
Debromohymenialdisine (Figure I-20) is a natural product that inhibited Chk1 and
Chk2 (IC50 = 3 and 3.5 µM, in vitro).

176, 224

Debromohymenialdisine also exhibited

moderate cytotoxicity (25 µM) and inhibition of the G 2 checkpoint at 8 µM in vivo.

176

Despite the moderate inhibition, its lack of selectivity for Chk2 over other kinases limits
the use of debromohymenialdisine as a Chk2 inhibitor.

40

57

Figure I-20. Structure of debromohymenialdisine and its analog indoloazepine.
A debromohymenialdisine-derived analog, indoloazepine, was developed in our
laboratory and has been shown to be a potent and selective inhibitor of Chk2.

224

An in

vitro assay identified an IC50 = 8 nM for Chk2 inhibition by indoloazepine, which was
also ~30 fold more selective over Chk1 and other purified kinases.

57, 224

Indoloazepine

is a potent and selective inhibitor in the kinase assay, however, we wanted to further
evaluate the drug to determine if it also showed cellular inhibition of Chk2, which is the
foundation of my project.
I.M

Chk2 binding pocket properties
Crystallization of the kinase domain of Chk2 by Oliver et al.

112

allowed

exploration and examination within the binding pocket. Debromohymenialdisine (DBH)
was successfully crystallized within the binding pocket making it possible to identify
possible residues important for binding purposes. Important hydrogen bonding
interactions can be seen between DBH and the Chk2 (Figure I-21) using PyMol to
visualize the active site.

225

41

THR 367

ASN 352

GLU 351

GLU 308
Figure I-21. Debromohymenialdisine inside the Chk2 active site, top view.
DBH is shown in the center of Figure I-21 in blue, and the purple dashed lines
represent hydrogen bonding interactions made with the side chains and main chain of
Chk2. The potent inhibition of DBH can be attributed to the hydrogen binding
interactions to the glycocyamidine portion, which include threonine, asparagine and
glutamic acid residues. Additional hydrogen bonding interactions were observed in the
southern portion of DBH as shown in Figure I-22. The interactions are made between
the amide functional group of the seven-membered ring to glutamine and methionine
residues, contributing further to the potency of DBH.

42

GLU 302

MET 304

Figure I-22. Debromohymenialdisine inside the Chk2 active site, bottom view.
Information obtained from DBH and Chk2 was applied to the DBH analog,
indoloazepine, which could have similar binding interactions within Chk2. Figure I-23
shows the potential binding interactions could be very similar to those of DBH.

225

The

hydrogen bonding interactions between the glycocyamidine ring of indoloazepine and
the residues surrounding it could interact to help hold indoloazepine in the active site.
The seven-membered ring of indoloazepine could also form hydrogen binding
interactions between the amide and those residues that held DBH in the active site.
The exact binding nature of indoloazepine in the active site of Chk2 remains
unknown, however it can be hypothesized based on the binding properties of a closely
related structure (DBH).
43

ASN 352
THR 367
GLU 351

GLU 308

GLU 302

MET 304

Figure I-23. Indoloazepine modeled in the Chk2 catalytic site.

44

I. N

Goals of project
The first goal of this project was directed toward optimizing a scale up synthesis

of indoloazepine, which would allow for further biological evaluation. The synthetic route
previously synthesized in our lab produced indoloazepine in 12% total yield.

224, 226

In

the interest of obtaining a more potent/selective inhibitor, analogs were synthesized to
assist in understanding the importance of the free amine moiety of the glycocyamidine
ring in the ATP binding pocket.
Chk2 was inhibited by indoloazepine in an in vitro kinase assay, however Chk2
had not been confirmed as the target of indoloazepine in cell culture. The second goal
of the project was to elucidate cellular mechanisms and radioprotection abilities.

45

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Parsels, L. A.; Morgan, M. A.; Tanska, D. M.; Parsels, J. D.; Palmer, B. D.;
Booth, R. J.; Denny, W. A.; Canman, C. E.; Kraker, A. J.; Lawrence, T. S.;
Maybaum, J., Gemcitabine sensitization by checkpoint kinase 1 inhibition
correlates with inhibition of a Rad51 DNA damage response in pancreatic cancer
cells. Mol. Cancer Ther. 2009, 8 (1), 45-54.

212.

Blasina, A.; Hallin, J.; Chen, E. H.; Arango, M. E.; Kraynov, E.; Register, J.;
Grant, S.; Ninkovic, S.; Chen, P.; Nichols, T.; O'Connor, P.; Anderes, K.,
Breaching the DNA damage checkpoint via PF-00477736, a novel smallmolecule inhibitor of checkpoint kinase 1. Mol. Cancer Ther. 2008, 7 (8), 23942404.

213.

Anderson, V. E.; Walton, M. I.; Eve, P. D.; Boxall, K. J.; Antoni, L.; Caldwell, J. J.;
Aherne, W.; Pearl, L. H.; Oliver, A. W.; Collins, I.; Garrett, M. D., CCT241533 Is a
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Potent and Selective Inhibitor of CHK2 that Potentiates the Cytotoxicity of PARP
Inhibitors. Cancer Res. 2011, 71 (2), 463-472.
214.

Yu, Q.; La Rose, J.; Zhang, H.; Takemura, H.; Kohn, K. W.; Pommier, Y., UCN01 inhibits p53 up-regulation and abrogates gamma-radiation-induced G(2)-M
checkpoint independently of p53 by targeting both of the checkpoint kinases,
Chk2 and Chk1. Cancer Res. 2002, 62 (20), 5743-5748.

215.

Kohn, E. A.; Yoo, C. J.; Eastman, A., The protein kinase c inhibitor Go6976 is a
potent inhibitor of DNA damage-induced S and G(2) cell cycle checkpoints.
Cancer Res. 2003, 63 (1), 31-35.

216.

Akinaga, S.; Nomura, K.; Gomi, K.; Okabe, M., Enhancement of antitumoractivity of mitomycin-C in vitro and in vivo by UCN-01, a selective inhibitor of
protein-kinase-C. Cancer Chemother. Pharmacol. 1993, 32 (3), 183-189.

217.

Wang, Q. Z.; Fan, S. J.; Eastman, A.; Worland, P. J.; Sausville, E. A.; Oconnor,
P. M., UCN-01, a potent abrogator of G(2) checkpoint function in cancer cells
with disrupted p53. J. Nat. Cancer Inst. 1996, 88 (14), 956-965.

218.

Hu, B. C.; Zhou, X. Y.; Wang, X.; Zeng, Z. C.; Iliakis, G.; Wang, Y., The
radioresistance to killing of A1-5 cells derives from activation of the Chk1
pathway. J. Biol. Chem. 2001, 276 (21), 17693-17698.

219.

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 (6), 1024-1032.

220.

Hirose, Y.; Berger, M. S.; Pieper, R. O., Abrogation of the Chk1-mediated G(2)
checkpoint pathway potentiates temozolomide-induced toxicity in a p53independent manner in human glioblastoma cells. Cancer Res. 2001, 61 (15),
5843-5849.

221.

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 (4), 394-403.

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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.; Kuwahara, A.; Kobasashi, S.,
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Unpredicted clinical pharmacology of UCN-01 caused by specific binding to
human alpha(1)-acid glycoprotein. Cancer Res. 1998, 58 (15), 3248-3253.
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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
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hymenialdisine-derived indoloazepine. Bioorg. Med. Chem. Lett. 2004, 14 (16),
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DeLano, W. L. The PyMol Molecular Graphics System., 2009.

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by Hymenialdisine Derivatives. J. Med. Chem. 2004, 47 (14), 3700-3703.

69

CHAPTER II
IMPROVEMENT OF A SYNTHETIC METHOD FOR THE PREPARATION OF (Z)-5-(2AMINO-4-OXO-1H-IMIDAZOL-5(4H)-YLIDENE-2,3,4,5-TETRAHYDROAZEPINO[3,4b]-1(10H)-ONE, A HYMENIALDISINE ANALOG
II.A

Isolation and characterization of hymenialdisine and analogs
Debromohymenialdisine (structure II-1), shown in Figure II-1, was first isolated
1

from the Great Barrier Reef sponge Phakellia sp. in 1980. Natural products from this
sponge have been known to possess antimicrobial activities.
originally elucidated with

1

2, 3

The structure was

H NMR, UV and chemical degradation.

1

Hymenialdisine

(structure II-2) was later extracted from the Red Sea sponge Acanthella sp. and the
Mediterranean sponge Axinella sp. in 1982 as well as the from Hymeniacidon sp. in
1983, and characterized by X-ray crystallography.

4-6

Several pyrrolic metabolites in

addition to II-1 and II-2 were isolated and confirmed in X-ray studies from
7

Pseudaxinyssa sp. in 1985. II-1 was reported to be slightly cytotoxic against murine
p388 lymphocytic cells,

5

and had no antimicrobial activity. II-2 was found to be

moderately cytotoxic in PV4 cells (32.5% at 100 µg/mL).

8

Figure II-1. Structures (Z)-debromohymenialdisine and (Z)-hymenialdisine.

70

II-1 and II-2 are structurally similar compounds, differing only by the presence of
bromine at the C2 position. Both compounds contain a pyrroloazepine ring that is
connected to a glycocyamidine ring through a double bond between C10 and C11.
Endocyclic torsion angles for the azepine group suggests a distorted boat
4

conformation. Although the pyrrole ring is planar, the adjacent amide bond at C6 is not
4

planar due to a slight rotation in the amide carbonyl and nitrogen, forming a 43.80º
dihedral angle between the pyrroloazepine plane and the glycocyamidine ring.

6

Figure II-2. Structures of the axinohydantoins and (Z)-debromohymenialdisine and (Z)hymenialdisine.
Axinohydantoins II-3 and II-4, illustrated in Figure II-2 are structurally related to II2 consisting of the same fused pyrroloazepine ring. Instead of the glycocyamidine ring,
71

the pyrroloazepine ring is bonded to a hydantoin ring via a (Z) or (E) double bond. (E)axinohydantoin II-3a was isolated in 1990 from the sponge Axinella sp. and the
9

10

structure was determined by X-ray crystallography. (Z)-axinohydantoin II-4a
bromine derivatives II-4b and II-4c
respectively.

10, 11

11

and the

were isolated and characterized in 1997 and 1998,

Like the axinohydantoins, II-2 and II-1 have also been isolated as the

(E) isomers II-5a and II-5b,
thermodynamic stability.

13, 14

12

although the (Z) isomer is predominant due to its

Isolation of crystalline samples of the free bases of II-2
1

13

and II-1 produced two sets of H and

C NMR signals, which were determined to be

the (10E)- and (10Z)-isomers of both II-2 and II-1. Higher concentrations of the (10E)and (10Z)- isomers resulted in a single set of signals, while dilution of the sample
caused the signals to revert back to two sets. The NH protons in the compounds were
found to act as a base in the tautomerization. As was expected, addition of any protic
solvent to the NMR sample also resulted in a single set of signals matching that of the
(Z)-isomer.

12

Hymenin II-6 (Figure II-3) was isolated from an Okinawan sponge Hymeniacidon
sp. in 1984 and characterized by NMR.

15

Unlike II-2, hymenin contains two bromines on

the pyrrole ring and contains a single bond to a 2-aminoimidazole ring instead of a
glycocyamidine ring. Stevensine II-7 (Figure II-3) isolated from Pseudaxinyssa sp. in
1985,

7, 16

is another natural product similar to hymenin, but contains an additional

unsaturation site between C9 and C10.

7, 16

72

Figure II-3. Structure of hymenin and stevensine.
II.B

SYNTHESIS OF HYMENIALDISINE AND DEBROMOHYMENIALDISINE
II-2 and II-1 were first synthesized by Annoura and Tatsuoka in 1995.

17

This

synthetic route sought an efficient way to make the 2-amino-4-oxo-2-imidazoline-5-(Z)disubstituted -ylidine ring system. The synthesis began with treatment of pyrrole-2carboxylic acid II-8 with SOCl2 and catalytic DMF in toluene at 60ºF, followed by
condensation with β-alanine methylester, as seen in Scheme II-1. Bromination of 2pyrrole derivative II-9 was accomplished using NBS, resulting in II-10. Formation of the
aldisine ring II-11 was synthesized with a Friedel-Craft acylation with PPA following
hydrolysis of the methyl ester II-10.

73

Scheme II-1. Synthetic route toward II-1 and II-2 of intermediate II-11.
The rings II-11a and II-11a’ were formed in 65% yield as a mixture of isomers.
This may have resulted from a 1,2-migration of the bromine atom, which was likely
caused by the strong acidic environment. The bromine isomers II-11a and II-11a’ were
not easily isolated until after the pyrrole and amide nitrogen were protected with SEM
group to yield II-12a, II-12a’ (Scheme II-2). Aldisine II-11b was also SEM protected to
give II-12b. The Horner-Wadsworth-Emmons (HWE) reaction proceeded with ethyl
diethylphosphonoacetate giving a mixture of α,β and β,γ-unsaturated esters II-13. The
ester II-13 was subsequently deprotonated with KHMDS to generate ester anions,
which then reacted with 2-benzenesulfonyl-3-phenyloxaziridine
unsaturated esters II-14 as a single regioisomer.

74

18

to give the α-β-

Scheme II-2. Synthetic route toward II-1 and II-2 of intermediate II-14.
Reacting II-14 with guanidine and AlMe3 as a Lewis acid resulted in the
hydantoin derivative II-17 as seen in Scheme II-3. Experimentation with intermediate II16 (not isolated) under various conditions did not result in cyclization to form the
glycocyamidine ring.

75

Scheme II-3. Synthesis of II-17 toward II-1 and II-2.
In order to avoid formation of the hydantoin, compound II-14 was mesylated
under basic conditions to yield II-15 quantitatively (Scheme II-4). Treatment of II-15 with
guanidine cyclized to form the glycocyamidine ring followed by isomerization of the
double bond into conjugated system to give II-18a and II-18b in 42% and 52% yield,
respectively. The NMR data of the products were compared with those of the natural
products and the Z-stereochemistry was confirmed. The SEM groups were deprotected
followed by purification over silica gel to give II-1 70% and II-2 75%.

17

Tatsuoka demonstrated an efficient route to the synthesis of II-2 and II-1.

Annoura and
17

As we will

see in the next few sections, other groups have found different, perhaps more versatile
76

methods for the synthesis of the above mentioned natural products as well as their
unnatural analogs.

Scheme II-4. Completed synthesis of II-1 and II-2.
II.C SYNTHESIS OF MARINE SPONGE ALKALOIDS THROUGH A COMMON
BICYCLIC PYRROLE COUPLING TO 2-AMINOIMIDAZOLE
Horne, D.A. et al discovered a methodology to synthesize hymenialdisine,
debromohymenialdisine, hymenin and stevensine from a common bicyclic pyrrole[2,3c]azepine-8-one ring system.

13, 14, 19

The key step in the synthesis of these compounds

was coupling the azafulvene unit II-19 to 2-aminoimidazole II-20 (Figure II-4) under
acidic conditions to form the carbon-carbon bond between the two rings of the natural
products.

13

77

Figure II-4. Azafulvene intermediate to be coupled to 2-aminoimidazole

One obstacle to the synthesis of hymenin was to prevent or suppress the
dimerization of the monomer with the azafulvene unit, resulting in the formation of II-22
as seen in Scheme II-5. Several acidic conditions did not result in the coupling of II-20
with II-19a.

Scheme II-5. Synthesis of azafulvene intermediate II-19a.
However, when bromine was introduced to the pyrrole II-19b, it functioned in
blocking the nucleophilic site and prevented the dimerization and formation of II-22
(Scheme II-6). After forming the fused bicyclic pyrrole, coupling II-20 with II-19b went
smoothly in methanesulfonic acid (23ºC, 7 d) to give racemic hymenin II-6. Further
hydrogenation of II-6 over Pd/C and NaOAc in methanol gave (±)-2,3-debromohymenin
II-23 in quantitative yield.

13

Wan et. al utilized the same synthetic strategy to synthesize

II-2 as well as indolo- analogs of II-2, which will be discussed later.

78

20

Scheme II-6. Synthesis of hymenin and debromohymenin.
Intermediate II-19a proved to be useful toward the synthesis of stevensine II-7
and its analogs. Recalling that II-7 is saturated at C9-C10, it is possible to visualize the
formation of an alkene through halogen elimination. An addition of bromine to
pyrroloazepine II-19a was performed in methanol to give II-24 (Scheme II-7). When the
elimination was performed in methanesulfonic acid, II-25 was obtained in 46% yield
along with the bromo olefin II-26 (20%).

79

Scheme II-7. Synthesis of intermediates II-25 and II-26.
In order to synthesize II-7 from II-25, elimination of HBr was necessary. Acid
catalysis was used to facilitate the syn elimination, which otherwise proved to be
challenging under basic conditions (Scheme II-8). Heating II-25 in methanesulfonic acid
in a sealed tube gave two analogs of II-7: II-27 and II-28. The formation of these two
products eliminated HBr initially to give II-7. HBr then acted as a reducing agent causing
protodebromination of the β-pyrrole position to produce olefin II-27, which, when
combined with the molecular bromine present, afforded II-28. Horne et al found that
eliminating the HBr and molecular bromine by leaving the flask unsealed, prevented II-7
13

from going on to II-27 and II-28.

80

Scheme II-8. Synthesis of stevensine II-7.
Alternatively, scheme II-9 illustrates how II-7 can be obtained in two steps from II6. Although a direct oxidation of II-6 to II-7 was not possible, treatment with bromine in
trifluoroacetic acid gave the expected 4’-bromohymenin II-29 in 95% yield. When the
bromination is performed in methanesulfonic acid, the reaction gave II-29 as a product
as well as 5-bromostevensine II-30 and starting material.

13, 21

II-7 could not be directly

obtained from hymenin, however, debromination of II-29 in methanesulfonic acid gave
II-7 in 20% yield.

13

81

Scheme II-9. Synthesis of II-7 from II-6.
In addition to the chemistry of protodebromination and transbromination,
hydrolysis was also used in synthesizing II-1 and II-2 from intermediate II-29. As shown
in Scheme II-10, II-29 was heated in HBr forming II-2 directly in 40% yield along with
2,3-debromostevensine II-31 (21% yield). Under the same conditions, II-1 was not
formed due to the competitive nature of the hydrolysis and protodebromination. There
was not a direct way to obtain II-1 from precursor II-29, however, milder conditions
using a mixture of 1:1 H2O/CH3CO2H gave the hydrolysis product II-32. A selective
protodebromination in methanesulfonic acid in a sealed tube produced II-1 and II-2 in
33% and 27% yield, respectively. II-1 could also be formed in 65% yield as the major
product by hydrolyzing II-28 in aqueous acetic acid (Scheme II-10).

82

13

Scheme II-10. Synthesis of II-1 and II-2 from intermediate II-29 and II-28.
In an effort to streamline the syntheses, Sosa et al

14

designed a one-pot, two-

step procedure to synthesize II-6 from II-33 in Scheme II-11. The first reported
syntheses of (Z)-3-bromohymenialdisine was obtained, while further hydrogenation
gives debromohymenialdisine.

14

In a one-pot reaction, acid-catalyzed deprotection of

acetal II-33 and cyclization of the resultant aldehyde occurred to give intermediate II19b without isolation. Intermediate II-19b was then coupled in the same pot to II-20 to
give II-6. The overall reaction was performed on a multi-gram scale giving II-6 in 60%
overall yield. Oxidation of II-6 cleanly gave (Z)-3-bromohymenialdisine II-34 followed by
hydrogenation to give II-2.

14

83

Scheme II-11. Synthesis of II-2 using a one-pot, two-step synthesis.
II.D

SYNTHESIS OF AXINOHYDANTOINS
The first reported synthesis of axinohydantoins was reported by Sosa et al in

2002.

19

The authors had envisioned synthesizing these compounds using hymenin

analog II-19b. However, accessing a hydantoin nucleus from an imidazolone precursor
was problematic and an intramolecular cyclization was instead utilized to remedy
problems associated with the use of II-19b as seen in Scheme II-12.

19

This key step

formed the tricyclic pyrroloazepinone framework present in the axinohydantoins. Thus,
II-35 was treated with trifluoroacetic acid to form II-36. Cyclizations of α-debromopyrrole
analogs were observed to have a higher yield than cyclization of α-bromopyrrole
analogs, which underwent hydrolysis to form a spirolactam (not shown).

19

Utilizing

protodebromination conditions developed for the other marine alkaloid syntheses
II-37, II-3c and II-4c were synthesized from II-36.

84

19

13, 14

,

Scheme II-12. Synthesis of II-3c, II-4c, II-36 and II-37 from intermediate II-35.
Hydrogenation of II-3c and II-4c afforded debromoaxinohydantoins as seen in
Scheme II-13. Hydrogenation of II-4c gave II-4b in 80% yield, however, the reduction of
the (E)-isomer II-3c gave a 1:1.3 mixture of expected (E)-debromoaxinohydantoin II-3b,
and the unexpected mono-reduction product, (E)-isoaxinohydantoin II-38. Compound II3b was found to isomerize slowly at room temperature in DMSO-d6 to the (Z)-isomer.
19

Similar observations were seen with II-38.

85

Scheme II-13. Synthesis of axinohydantoins II-3b and II-4b from II-3c and II-4c.

II.E

SYNTHESIS OF HYMENIALDISINE AND ANALOGS THROUGH THIOLS
In an effort to improve the synthesis of hymenialdisine, a few groups considered

alternative heterocyclic coupling partners, such as thiohydantoin II-39 as seen in
Scheme II-14.

22-24

(Scheme II-11),

13

Portevin et al repeated the synthesis as performed by Horne

however, the cyclization to form II-19b followed by coupling of II-19b

to 2-aminoimidazole II-20 occurred slowly, requiring 4 and 5 days, respectively.

13, 22

Although II-2 was obtained, there was room for improvement in the synthesis. The
improved synthesis began with the same aldisine precursor II-11b, which was coupled
with thiohydantoin II-39 using BF3•OEt2 (Scheme II-14). Using TiCl4 as a coupling
reagent only gave the product in trace amounts, whereas the use of BF 3•OEt2 under
unoptimized reaction conditions gave 31% of II-40. Reacting II-40 with aqueous

86

ammonium hydroxide in the presence of tert-butylhydroperoxide gave optimal results
affording II-2 in 74% yield.

22

Scheme II-14. Synthesis of II-2 via thiohydantoin II-39.

Alternatively, Papeo et al synthesized II-1 as well as II-2 using II-41, an Smethylated, N-benzoylated thiohydantoin, as seen in Figure II-5.

23

The syntheses of II-1

and II-2 involved a condensation between II-11b and II-41, which was synthesized
readily from N-benzoylthiohydantoin on a gram scale.

Figure II-5. Structure of 1-benzoyl-2-methylsulfanyl-1,5-dihydroimidazol-4-one.
Before the condensation, Papeo et al improved the overall yield of the Friedel17

Craft cyclization II-11a

to 53% as illustrated in Scheme II-15. Carboxylic acid II-10 was

reacted with phosgene to give the acyl chloride, and in the same one-pot process, AlCl3
and 4 Å molecular sieves were added as an acid scavenger.

87

23

Using this method, II-11a

was prepared without the problematic 1,2-bromine migration that was observed in
17

Annoura’s synthesis.

Scheme II-15. Improved synthesis of II-11a.
Scheme II-16 illustrates the condensation of II-41 with II-11 to give II-42.
Intermediate II-42 was discovered to be sensitive to nucleophilic ring-opening as well as
chromatography and the synthesis proceeded without isolation of II-42. Ammonia in
dioxane was used to displace the methylthio group, giving compound II-43. However
these conditions were not successful in displacing the benzoyl-protecting group, even
after 2 days. Evaporation of dioxane and exposure to 7 N NH3 in methanol removed the
benzoyl protecting group and gave II-1 and II-2.

88

Scheme II-16. Synthesis of II-1 and II-2 utilizing II-41.
In the case of II-43b, endo-isomer II-44 was also formed in 10%, however, this
25

was easily converted to the more thermodynamically stable exo-(Z)-isomer
diluted aqueous ammonia using microwave conditions (Scheme II-17).

89

23

with

Scheme II-17. Conversion of II-44 to II-2 under microwave conditions.

The same intermediate II-2a and II-42b were also used in the first total synthesis
of (Z)-axinohydantoin II-4a (seven steps, 8% yield overall) following demethylation
under acidic conditions and cleavage of the N-benzoyl protecting group using
NH2NH2•H2O (Scheme II-18). The stereoselective synthesis also afforded (Z)debromoaxinohydantoin as well as the (E) isomers II-3a, II-3b) as the minor product as
seen in Scheme II-15.

26

90

Scheme II-18. Synthetic route for II-3a, II-3b and II-4a, II-4b.

Scheme II-19 shows that 2-substituted endo-analogs of hymenialdisine can be
synthesized using analogs of II-46 as was demonstrated by Papeo.

23, 24

The synthesis

of this series of analogs began with either pyrrole derivatives or alkylated carboxylic
acid derivatives of pyrrole (synthesis not shown).

24

Compound II-46 was obtained

following procedures similar to the previous syntheses of hymenialdisine.

13, 14, 17, 22, 23

The expected exo-isomer was not obtained following reaction with ammonia, instead
the endo-isomer was observed. Converting the isomers from endo to exo under
microwave conditions (Scheme II-17) did not afford the exo-isomer. It was reported that
having substituents in the 2-position on the pyrrole ring likely require higher energy in
order to isomerize than unsubstituted pyrroles.
91

24

Scheme II-19. Synthesis of endo-pyrrole analogs.

II.F

SYNTHESIS OF INDOLE ANALOGS OF HYMENIALDISINE
2-substituted hymenialdisine analogs are accessible as well as fused indole-

pyrrole analogs of hymenialdisine.

27

The synthesis of the indole-aldisine intermediate

began with the coupling of indole-2-carboxyllic acid with β-alanine ethyl ester in the
presence of DMAP and EDCI to afford II-50a as seen in Scheme II-20

27

N-methylation

of II-50a, followed by hydrolysis under mild conditions yielded II-50c which subsequently
was cyclized to II-51 using PPA and P2O5.

27, 28

92

Scheme II-20. Toward the synthesis of indole analogs of hymenialdisine.

The amide nitrogen was protected with various groups as seen in Table II-1.
Following protection, indoloaldisine II-52 was reacted under basic conditions with
trifluoromethanesulfonic anhydride (Scheme II-21). Utilizing II-52a and II-52e, three
compounds were isolated: the expected triflate II-53 (21% yield from II-53a and 53%
yield from II-53e), the monotriflate II-54 with unprotected amide (30% yield from II-53a
and 20% from II-53e), and ditriflate II-55 (13% yield from II-53a and 23% yield from II53e) (Scheme II-21).

27

93

Table II-1. Nitrogen protecting group variations for indole aldisine.

Scheme II-21. Synthesis of mono- and di-triflate protected indole aldisines.

II-53d was the most reactive of the series with the added benefit of the ability to
deprotect the amide nitrogen easily.

27

Thus, the indole-hymenialdisine analogs were

prepared from II-53d using either Stille conditions or Suzuki conditions (Table II-2).
94

Table II-2. Scope of the Suzuki and Stille coupling for indole aldisine.

95

II.G

SYNTHESIS
OF
PHENYLOXAZALONE

2-AMINOIMIDAZALONE

RING

THROUGH

Sharma et al synthesized an indolo- analog of hymenialdisine, which was obtained
via a condensation with phenyloxazolone II-66 and indoloaldisine II-67 or II-51, following
a displacement with S-benzylthiourea (Figure II-6).

29, 30

Figure II-6. Structure of phenyloxazolone.
The syntheses of II-70a and II-70b were achieved initially by amidation of indole2-carboxylic acid with β-alanine ethyl ester in the presence of DMAP and EDCI
27

(Scheme II-22).

Compound II-68 was obtained via hydrolysis of II-67 and II-50c.

Friedel-Craft cyclization was completed using P2O5 and methanesulfonic acid instead of
PPA.

29, 30

Compound II-68 and II-51 underwent a TiCl4 mediated aldol condensation

with II-66 to obtain the key intermediate II-69. Intermediate II-69 was treated with Sbenzylthiourea under basic conditions to give II-70a and II-70b in modest yields
(Scheme II-22).

29, 30

96

Scheme II-22: Synthesis of indoloazepine II-70.

II.H

HYMENIALDISINE ANALOG SYNTHESIS VIA HECK REACTION
Another route of synthesis of the compound II-70a was proposed combining the

Wittig reaction as well as the Heck reaction to close the 7-membered azepine ring.

31-33

Synthesis of the glycocyamidine ring was modeled after Annoura and Tatsuoka’s
synthesis.

17

Shown in Scheme II-23, indole-2-carboxylic acid II-49, was reacted with

iodine to give I-substituted II-71.

31-33

Compound II-71 was subsequently coupled with

(E)-5-amino-2-pentoate hydrochloride II-72 in the presence of EDCI and DMAP in
97

33

dichloromethane to produce II-73.
to give II-74.

Boc protection of the free nitrogen atoms followed

31, 33

Scheme II-23. Synthesis of intermediate II-74 for ring closure via Heck reaction.

With II-74 in hand, it was possible to perform the catalytic Heck cyclization to
form the indole-azepine ring under optimized conditions giving II-75 (Scheme II-24).

31, 33

Dihydroxylation of II-75 followed by dehydration afforded allyl alcohol II-77, which
subsequently was converted to an allylic chloride II-78.

31

Additionally, several attempts

to obtain II-70a from II-78 through reaction with guanidine in DMF were unsuccessful.

98

31

Scheme II-24. Unsuccessful Heck reaction approach to II-70.
The natural and unnatural analogs of hymenialdisine described here have a
similar fused bicyclic pyrroloazepine ring system bonded to another 5-membered ring.
With the exception of the indole analogs, these have in common with the marine sponge
alkaloids a C11N5 core. Figure II-7 illustrates the scaffolds and positions that were
modified to create analogs.

20

Using this scaffold, Wan et al were able to synthesize over

50 analogs for biological evaluation.

99

Figure II-7. General scaffold for hymenialdisine analogs.

II.I

IMPROVEMENT OF THE SYNTHETIC ROUTE TO INDOLOAZEPINE II-70.
As more information about the biological activity of II-70 became available,

greater quantities of the compound was needed to perform in vitro studies. The original
synthesis of II-70 as performed in the Tepe group afforded the desired final product in a
28% yield (Scheme II-22) as the free amine. When the same synthesis was repeated as
originally proposed, only 8% yield was obtained in the final step. Although this method
provided material temporarily for biological testing, there was much room for
improvement in the synthetic route.
Various attempts were made in the last step to replace the thiourea with a
reagent that would introduce the guanidine portion of the imidazolone in higher yields.
However, this method proved to be unfruitful with the phenylazlactone remaining intact.
With the phenylazlactone remaining seemingly untouched, it was thought that perhaps
the phenylazlactone was the troublesome portion of the molecule. Replacing the
100

phenylazlactone with a hydantoin had potential for providing higher yields of II-70. A
previously reported total synthesis of a marine alkaloid from the Tunicate Dendrodoa in
the Tepe group demonstrated that the imidazolone could be accessed via the hydantoin
(Scheme II-25).

34

This route could allow access to the imidazolone with the free amine

using ammonium hydroxide instead of dimethyl amine. Use of dimethyl amine however,
would facilitate access to an additional analog of II-70.

Scheme II-25. Synthesis of II-82 via hydantoin II-79.

With the possibility of transforming a hydantoin into the glycocyamidine ring,
efforts were put into coupling a hydantoin with aldisine II-68. Coupling II-68 to hydantoin
with TiCl4 did not yield the desired compound II-84 (Scheme II-26). Instead, hydantoin

101

was recovered along with starting materials. Protecting the amines of hydantoin did not
improve the coupling efforts to II-68.

Scheme II-26. Hydantoin coupling to II-68.

Due to lack of success in a direct coupling of hydantoin to II-68, a HornerWadsworth Emmons (HWE) approach was instead pursued. Phosphonate II-85 was
synthesized as previously reported by Cativiela et al (Scheme II-27).

35

The HWE

reaction was performed under various previously reported conditions, however did not
yield the desired product II-84.

35, 36

Scheme II-27. Attempted HWE synthesis to form II-84.

102

With several unsuccessful attempts at synthesizing II-84, another route was
proposed as seen in Scheme II-28. The proposed route begins with iodination at C3 of
indole-2-carboxylic acid to give II-87. II-87 was then coupled with II-88 and the resulting
27, 31-33

alcohol II-89 was protected with TIPSCl to yield II-90.

Scheme II-28. Alternate synthesis to II-84.
In Scheme II-29, the nitrogens of aldehyde II-90 were Boc protected before the
TIPS group was removed. II-92 was oxidized to the aldehyde with IBX before coupling
to II-85 utilizing conditions for a similar reaction previously published.

37

Following

coupling, Boc protection of remaining nitrogens and a Heck reaction would give the
desired compound.

103

Scheme II-29. Synthesis of intermediate II-95.
Cyclization of II-95 (R=H and R=Boc) was attempted under various conditions
involving halogen-metal exchange (Table II-3). It was envisioned that a lithium ion would
exchange with the iodine and perform an intramolecular conjugate addition in order to
close the ring (entry 1). However, this did not occur. Attempts at forming a Grignard
intermediate (entry 2) to perform the ring closure also failed. Formation of a cuprate in
situ (entry 3) was unsuccessful in producing the hydantoin analog II-84. Entry 4 shows
an attempted Heck reaction using conditions optimized for a similar substrate.

32, 33

This

reaction gave decomposition products upon filtration. The salts contained starting
104

material. When Pd(OAc)2 was used in molar quantities as well as in excess, there was
still no cyclized product observed. It was speculated that there was not enough base
and triphenylphosphine to get to Palladium(0). These amounts were increased and
there was still no observance of the cyclized product.
Table II-3. Attempted conditions for cyclization.

105

The Heck reaction was also attempted using Pd(PPh3)4. The desired product
was not observed. Different methods of work up were also performed, however, the
product was not isolated. Sterics very likely played a major role in the reaction not
producing the II-84. There were several bulky Boc groups that although could assist in
orienting the chain to cyclize, could also provide enough bulkiness to inhibit the ring
closure.
After several various failed attempts at closing the ring on II-95, another method
was attempted. An improved synthesis was found utilizing Papeo’s thiohyandantoin II41. II-41 was easily synthesized on a multigram scale from hippuric acid and ammonium
thiocyanate (Scheme II-30).

Scheme II-30. Synthesis of II-41.
With II-41 in hand, a TiCl4 coupling was possible with indoloaldisine II-68 which
was synthesized as previously reported (Scheme II-22).

28, 29

The resultant product (II-

97) of the coupling was prone to decomposition during purification over silica, so was
debenzoylated without further purification. II-98b was isolated cleanly using silica
chromatography.

106

Scheme II-31. Synthesis of intermediate II-98.

Intermediate

II-98b was a

stable

compound

that proved synthetically

advantageous, providing easy access to three compounds. Upon reaction with
ammonia in a sealed tube, II-70 resulted as a precipitate which was filtered and washed
with 10% HCl before being filtered again (Scheme II-32). The resultant bright yellow
solid was collected as HCl salt form of II-70 in 67% yield, a vast improvement from the
previously reported 28% yield and nearly 10-fold greater yield when repeated in the lab.
A dimethylamino analog II-99 was synthesized substituting dimethylamine for ammonia.
This reaction mixture formed a similar precipitate, which was filtered, washed with 10%
HCl, then filtered again. To form the hydantoin analog II-84, intermediate II-98a or II-98b
was refluxed with NaOH in a 1:1 H2O/THF mixture overnight. Unlike II-70 or II-99, this
compound required column chromatography over silica in order to isolate II-100 from
impurities.

107

Scheme II-32. Synthesis of II-70 and analogs II-99 and II-84.

The improved synthetic route employed in Scheme II-32 provided more facile
access to II-70 in which was precipitated out of the reaction. Column chromatography
was not required to isolate the compound from impurities. Multiple grams of II-70 was
obtained using this synthesis which was then taken on to be examined as a checkpoint
kinase 2 (Chk2) inhibitor in vitro and in vivo (discussed in Chapter 3).

108

II.J

General experimental information of procedures and characterization
Reactions were carried out in flame-dried glassware under nitrogen atmosphere.

All reactions were magnetically stirred and monitored by TLC with 0.25 µm pre-coated
silicaa gel plates using either UV light or iodine to visualize compounds. Column
chromatography was carried out on Silica Gel 60 (2330-400 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
1

spectrometer. H NMR and

13

C NMR spectra were recorded on a Varian Unity Plus-500

spectrometer or a Varian Inova-300, as noted in the experimental for each compound.
Chemical shifts are reported relative to the residue peaks for the solvent (CDCl 3: 7.24
1

ppm for H and 77.0 ppm for
1

13

1

C); (DMSO-d6: 2.49 ppm for H and 39.5 ppm for

(Acetone-d6: 2.09 ppm for H and 30.60 for

13

13

C);

C) . The following abbreviations are used

1

to denote the multiplicities in H NMR: s = singlet, d = doublet, dd = doublet of doublets,
t = triplet, m = multiplet, q = quartet. 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 compound, whose elemental
1

analyses were above the ACS tolerated 0.4% deviation, were confirmed by H NMR
and

13

C 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 was dispensed from a delivery system which passes solvents through a column

109

packed with dry neutral alumina. Anhydrous tetrahydrofuran was distilled over sodium
and benzophenone.
1-benzoyl-2-(methylthio)-1H-imidazol-4(5H)-one (II-41). Compound II-97 (10.0
g, 45.4 mmol) was dissolved in dichloromethane (500 mL) in a flame-dried RBF. To this
mixture was added iodomethane (8.5 mL, 136.2 mmol), dimethylaminopyridine (553 mg,
4.54 mmol), and N,N-diisopropylethylamine (40 mL, 227 mmol) and the mixture was
stirred at room temperature for 3 hours. 1% HCl was added to the reaction mixture and
stirred for 10 minutes. The organics were then extracted and dried with anhydrous
Na2SO4 and concentrated. The product was crystallized from ethyl acetate to yield
white crystals (10.4 g, >99%). The spectral data matches that previously reported in the
literature.

23

1

m.p. 156-158. H NMR (500 MHz, DMSO-d6) δ 2.53 (s, 3H), 4.48 (s, 2H),

7.40-7.72 (m, 5H).

13

C NMR (128.2 MHz, DMSO-d6) δ 16.1, 55.0, 127.7, 128.7, 132.0,

133.6, 166.8, 182.5, 186.1. MS: m/z calcd for C11H11N2O2S [M+H], 235.0541; found
[M+H] 235.0551.
Ethyl 3-(1H-indole-2-carboxamido)propanoate (II-50a). This reaction was
performed according to the known procedure.

29

To a mixture of 2-indolecarboxylic acid

(2.41 g, 14.9 mmol) and 4-(dimethylamino)pyridine (3.24 g, 26.4 mmol) in 75 mL of
CH2Cl2

was

added

N-(3-dimethylaminopropyl)-N-ethylcarbodiimide

hydrochloride

(EDCI·HCl) (3.46 g, 18 mmol) and β-alanine ethyl ester hydrochloride (2.91 g, 18 mmol)
at 0°C. The mixture was then stirred at 0°C for 4 hours and allowed to warm to room
temperature for 20 hours. The solution was washed with water (25 mL) and 10% HCl
(25 mL). The organics were dried over anhydrous Na2SO4, filtered and concentrated to
110

dryness in vacuo to give a white solid (3.52g, 93%) which was recrystallized in
methanol. The spectral data matches that previously reported in the literature.

28

m.p.

1

158-160°. H NMR (500 MHz, CDCl3) δ 1.17 (t, J = 7.5 Hz, 3H), 2.58 (t, J = 6.5 Hz, 1H),
3.67 (q, J = 6.0 Hz, 2H), 4.08 (q, J = 7 Hz, 2H), 6.81 (s, 1H), 7.01 (t, J = 7.5 Hz, 1H),
7.09 (s, 1H), 7.14 (t, J = 7 Hz, 1H), 7.35 (d, J = 8.5 Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H),
9.86 (s, 1H).

13

C NMR (74.47 MHz, CDCl3) δ 13.9, 33.9, 35.0, 60.6, 102.3, 111.9,

120.3, 121.7, 124.2, 127.5, 130.6, 136.4, 161.7, 172.3. IR (NaCl): 3377, 3352, 1715,
1624, 1550, 1320, 1200, 750 cm

-1.

3-(1H-indole-2-carboxamido)propanoic
performed according to the known procedure.

28

acid

(II-67).

This

reaction

was

A mixture of II-50a (1.38 g, 5.3 mmol)

and LiOH·H2O (0.70 g, 10.6 mmol) was dissolved in ethanol (100 mL) and stirred at
room temperature for 20 hours. The ethanol was evaporated to dryness in vacuo. The
residue was dissolved in water (15 mL), and the solution was acidified to pH = 3 with
concentrated HCl to form a white solid. The mixture was allowed to stand at 0°C for 30
min, and the precipitate was isolated by filtration. The resulting solid was washed with
water (20 mL) and recrystallized with a minimal amount of methanol to give 1.201 g,
97%. The spectral data matches that previously reported in the literature.
1

28

m.p. 232°C.

6

H NMR (300 MHz, d -DMSO) δ 2.55 (t, 2H, J = 7.2 Hz), 3.47 (q, J = 6.9 Hz, 2H), 7.01,

(t, J = 7.2 Hz, 1H), 7.04 (s, 1H), 7.13 (t, J = 7.2 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.57
(d, J = 8.1 Hz, 1H), 8.52(s, 1H), 11.55 (s, 1H);

13

6

C NMR (74.47 MHz, d -DMSO) δ 34.6,

35.9, 103.2, 112.9, 120.3, 122.1, 123.9, 127.8, 132.4, 137.1, 161.8, 173.4. IR (NaCl):
-1

3420, 3268, 1750, 1702, 1640, 1545, 1415, 1340, 1249, 740 cm .
111

3,4-dihydroazepino[3,4-b]indole-1,5(2H,10H)-dione (II-68). This reaction was
performed according to the known procedure.

28

II-67 (6 g, 26.6 mmol) was added to a

clear solution of P2O5 (4.1 g, 32 mmol) in MeSO3H (300 g) at 60°C. The reaction
mixture was heated to 110°C and stirred for 1.5 hours, then cooled to room
temperature. The mixture was poured into ice water and stirred for 30 min. The purplecolored solid was then filtered. The solid was set aside. The filtrate was extracted with
ethyl acetate (3 x 150 mL), dried with anhydrous Na2SO4 and concentrated in vacuo.
The solid from filtration was dissolved in acetone, filtered again and the filtrate was
combined with the organics of the extraction and concentrated. The light brown solid
was purified further by column chromatography (20% acetone in CH 2Cl2) on silica gel to
yield the desired product (4.6 g, 81%). The spectral data matches that previously
reported in the literature.

28

1

6

m.p. 257-260°C. H NMR (300 MHz, d -DMSO) δ 2.80-2.85

(m, 2H), 3.40-3.46 (m, 2H), 7.22-7.38 (m, 2H), 7.51 (d, J = 9.0 Hz, 1H), 8.28 (d, J = 9,0
Hz, 1H), 8.72 (m, 1H), 12.41 (s, 1H).

13

6

C NMR (74.47 MHz, d -DMSO) δ 36.5, 44.0,

112.6, 113.7, 122.6, 122.7, 124.7, 126.0, 134.5, 135.6, 162.3, 195.0. IR (KBr-pellet):
-1

3160, 1660, 1620, 1530, 1430, 1400 cm .
(Z)-4-(1-oxo-1,2,3,4-tetrahydroazepino[3,4-b]indol-5(10H)-ylidene)-2phenyloxazol-5(4H)-one (II-69). This reaction was performed according to the known
procedure.

28

A 1 mM solution of TiCl4 in CH2Cl2 (19 mL, 19 mmol) was added to THF

(150 mL) at -10°C. II-68 (1.07 g, 5 mmol) and 2-phenylazlactone (1.46 g, 9 mmol) were
subsequently added and allowed to stir at 0°C for 1 hour. Pyridine (3.2 mL, 40 mmol)
was then added over a 30 min period. The reaction mixture was stirred at 0°C for an
112

additional 2 hours and then the reaction was allowed to stir overnight at room
temperature. NH4Cl (80 mL saturated solution in water) was added, and the mixture
was stirred for 10 min and subsequently extracted with ethyl acetate (3 x 80 mL). The
organic extracts were combined, dried with anhydrous Na2SO4 and concentrated in
vacuo. The residue was purified using column chromatography on silica gel (7%
MeOH/43% Hexanes/50% EtOAc) to give 1.02 g, 57% yield as a reddish-yellow solid.
The spectral data matches that previously reported in the literature.
1

28, 29

m.p. 247-

6

250°C. H NMR (300 MHz, d -DMSO) δ 3.36-3.47 (m, 4H), 7.14 (t, J = 8.1 Hz, 1H),
7.30 (t, J = 9.0 Hz, 1H), 7.49-7.58 (m, 4H), 7.81-7.87 (m, 3H), 8.45-8.56 (m, 1H), 12.11
(s, 1H);

13

6

C NMR (74.47 MHz, d -DMSO) δ 37.7, 38.6, 112.9, 115.3, 121.0, 125.1,

125.3, 126.5, 127.4, 129.0, 133.2, 134.0, 136.9, 146.3, 158.4, 165.0, 166.1. IR (KBr-1

pellet): 3350, 3190, 1750, 1650, 1640, 1450 cm .
(Z)-5-(2-amino-4-oxo-1H-imidazol-5(4H)-ylidene)-2,3,4,5-tetrahydroazepino
[3,4-b]indol-1(10H)-one (II-70). Compound II-98b was dissolved in THF (10 mL) and
stirred with ammonium hydroxide (30 mL) at 90°C in a sealed tube for 24 hours. The
reaction mixture was allowed to cool to room temperature at which point the precipitate
was filtered from the reaction mixture. The solid was collected and washed in 10% HCl
and filtered a second time. The bright yellow solid was filtered and dried in vacuo (617
mg, 69.2%). Further purification of II-70, if necessary can also be performed with silica
column chromatography with a gradient elution system of 15% MeOH: 85% CH2Cl2
followed by 4% TEA:20%MeOH: 76% CH2Cl2. Washing the resulting solid with 10% HCl
yields II-70 after filtration and drying in vacuo. m.p.: decomposes over 260°C.
113

1

H NMR.

(500 MHz, DMSO-d6) δ 3.32-3.39 (br, 4H), 7.19 (t, 1H, J = 7.9 Hz), 7.31 (t, 1H, J = 7.9
Hz), 7.52-7.57 (m, 2H), 8.36 (br, 2H), 9.05 (br, 1H) 10.31 (br, 1H), 12.45 (s, 1H);

13

C

NMR (128.2 MHz, DMSO-d6) δ 36.6, 39.2, 112.7, 113.6, 121.9, 122.4, 122.9, 124.6,
125.0, 128.9, 132.9, 132.9, 137.0, 154.6, 165.5. IR: (KBr-pellet) 3209, 1701, 1618,
1529, 1469, 1250. HRMS (ESI): m/z calcd for C15H12N5O2 [M+H], 296.1069; found,
296.1144.
(Z)-5-(1-oxo-1,2,3,4-tetrahydroazepino[3,4-b]indol-5(10H)ylidene)imidazolidine-2,4-dione (II-84). Compound 10 (500 mg, 1.2 mmol) was
dissolved in THF (50 mL) and added to a mixture of sodium hydroxide (233 mg, 5.8
mmol) in water, which was then stirred for 24 hours. The reaction mixture was cooled
and neutralized with sat. NH4Cl (50 mL). The organics were extracted with ethyl acetate
(2 x 50 mL). The organics were then dried with Na2SO4 and concentrated. The product
was isolated as a brown solid over silica with MeOH:CH2Cl2 (10:90). (Rf = 0.3) (240 mg,
1

60.7%). m.p. decomposes over 320°C. H NMR. (500 MHz, DMSO-d6) δ 3.50-3.53 (m,
4H), 7.24 (t, J = 8 Hz, 1H), 7.45 (t, J = 8.2, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.99 (s, 1H),
8.09 (d, J = 8 Hz, 1H), 10.54 (s, 1H), 12.47 (s, 1H);

13

C NMR (128.2 MHz, DMSO-d6) δ

23.1, 38.6, 112.9, 114.2, 117.0, 120.5, 120.9, 122.26, 122.37, 122.4, 126.2, 130.5,
139.4, 154.0, 160.8. IR: (KBr-pellet) 3372, 3059, 1674, 1454, 1342, 1201. HRMS (ESI):
m/z calcd. for C15H13N4O3, 297.0988; found 297.0979.
Diethyl (2,5-dioxoimidazolidin-4-yl)phosphonate (II-85). This reaction was
performed according to the known procedure.

35

To a 100 mL 3-neck round bottom flask

was added acetic acid (18 mL) and hydantoin (4.01 g, 40 mmol). The reaction mixture
114

was heated to 100°C and bromine (2.2 mL, 43 mmol) was added drop-wise with an
addition funnel and the mixture was allowed to stir for 45 minutes at 100°C. The mixture
was then cooled to 30°C in an ice/water bath at which point triethyl phosphite (9.6 mL,
55.5 mmol) was added slowly to the reaction mixture while maintaining an internal
temperature between 40-45°C. The reaction mixture was stirred an additional 90
minutes and the solvent was removed under high vacuum. The resultant residue was
dissolved in ether (10 mL) and placed in the freezer to precipitate the product which was
then filtered and washed with cold ether to yield a white solid (6.0 g, 63.7% yield). The
35 1

spectral data matches that previously reported in the literature.

6

H NMR (500 MHz, d -

DMSO) δ 1.23 (t, J = 3.6 Hz, 6H), 4.08 (m, 4H), 4.75 (d, J = 14.6 Hz, 1H), 8.42 (s, 1H),
10.92 (s, 1H).

13

6

C NMR (148.9 MHz, d -DMSO) δ 16.2, 55.6, 56.8, 62.9, 63.1, 157.5,

169.5.
3-iodo-1H-indole-2-carboxylic acid (II-87). Indole-2-carboxylic acid (1.05 g, 6.2
mmol) was dissolved in acetone (50 mL). In a separate flask, N-iodosuccinimide (1.4 g,
6.2 mmol) was dissolved in acetone (10 mL) and this mixture was added drop wise to
the indole-2-carboxylic acid/acetone mixture. The reaction mixture was stirred for 1.5
hours at which time the solvent was removed in vacuo. The solid was filtered and
washed with water to remove succinimide to give a brownish-orange solid (1.5 g, 85%).
1

6

H NMR (500 MHz, d -acetone) δ 3.34 (s, 1H), 7.12 (t, J = 6 Hz, 1H), 7.30 (t, J = 6 Hz,

1H), 7.416 (t, J = 8 Hz, 2H), 12.16 (s, 1H), 13.2 (s, 1H);

13

6

C NMR (148.9 MHz, d -

acetone) δ 64.97, 112.9, 120.9, 125.5, 127.9, 130.7, 136.8 161.8. IR (NaCl): 3400,

115

-1

3270, 3125, 3050, 2950, 1700, 1510, 1470, 1422, 1400, 1220 cm . HRMS (ESI): m/z
calcd for C9H5INO2 [M-H], 285.9370; found [M-H], 285.9362.
N-(3-hydroxypropyl)-3-iodo-1H-indole-2-carboxamide (II-89). Compound II-87
(9.98 g, 34.8 mmol) and 4-(dimethylamino)pyridine (6.49 g, 53 mmol) were dissolved in
CH2Cl2 and stirred at 0°C for 5 minutes. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide
hydrochloride (EDCI·HCl) (7.6 g, 39.6 mmol) and 3-aminopropan-1-ol (3 mL, 39 mmol)
were then added and stirred at 0°C for 4 hours, allowed to warm to room temperature
and stirred overnight. The reaction mixture was washed with water (150 mL) and
extracted. The mixture was washed with 10% HCl (150 mL) and stirred for 10-15
1

minutes. Organics were extracted and dried with anhydrous Na2SO4. H NMR (500
6

MHz, d -DMSO) δ 1.72 (quint, J = 6.5 Hz, 2H), 3.37 (q, J = 6.9 Hz, 2H), 3.54 (t, J = 6.2
Hz, 2H), 4.53 (bs, 1H), 7.14 (t, J = 7.7 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 7.35 (d, J = 7.9
Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.94 (s, 1H), 11.97 (s, 1H).

13

6

C NMR (148.9 MHz, d -

DMSO) δ 32.1, 36.5, 58.6, 112.5, 120.7, 121.6, 124.4, 130.2, 132.1, 135.9, 160.5. IR
-1

(NaCl): 3300, 3952, 2873, 1620, 1550, 1420, 1330, 1282, 825 cm . HRMS (ESI): m/z
calcd for C12H14IN2O2 [M+H], 345.0100; found [M+H], 345.0107.
3-iodo-N-(3-((triisopropylsilyl)oxy)propyl)-1H-indole-2-carboxamide

(II-90).

In a flame dried RBF, II-89 (1.53 g, 4.4 mmol) and triisopropylsilyl chloride (1.2 g, 5.2
mmol) were dissolved in DMF (10 mL) and stirred for 10 minutes at room temperature.
Imidazole (0.856 g, 11 mmol) was then added to the mixture and stirred at room
temperature for an additional 16 hours. 1% HCl was then added to the mixture which
was extracted with ethyl acetate (3 x 10 mL). The organics were dried over Na2SO4 and
116

solvent removed in vacuo. The resulting residue was purified using silica column
chromatography with 15% EtOAc/85% hexanes to give a clear residue (1.8 g, 82%
1

6

yield). H NMR (500 Mhz, d -DMSO) δ 1.01 (d, J = 8.0 Hz, 21H), 1.80 (quint, J = 6.5 Hz,
2H), 3.40 (dd, J = 6.9, 12.7 Hz, 2H), 3.78 (t, J = 6.3 Hz, 2H), 7.13 (ddd, J = 0.96, 7.0,
8.0 Hz, 1H), 7.25 (ddd, J = 1.2, 7.0, 8.2 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.41 (d, J =
8.2 Hz, 1H), 7.91 (t, J = 5.4 Hz, 1H), 11.96 (s, 1H).

13

6

C NMR (148.9 MHz, d -DMSO) δ

11.4, 17.9, 32.3, 36.3, 59.6, 60.8, 112.5, 120.7, 124.5, 130.1, 132.1, 160.6. IR (KBr):
-1

3400, 3250, 2950, 2875, 1630, 1560, 1470, 1422, 1400, 1240 cm . MS: m/z calcd for
C21H33N2O2ISi [M+H], 500.4; found [M+H] 457.1 [-CH3CHCH3]. HRMS (ESI): m/z calcd
for C21H34IN2O2SI [M+H], 501.1434; found, 501.1415.
tert-butyl

2-((tert-butoxycarbonyl)(3-

((triisopropylsilyl)oxy)propyl)

carbamoyl) -3-iodo-1H-indole-1-carboxylate (II-91). In a flame dried RBF, II-90 (1.8
g, 4 mmol), and di-tert-butyl-dicarbonate (2.2 g, 10 mmol) were dissolved in acetonitrile
(125 mL) and stirred for 10 minutes. 4-dimethylaminopyridine (1.7 g, 12 mmol) was then
added to the mixture which was stirred at room temperature for an additional 18 hours.
Acetonitrile was then removed in vacuo and the reaction mixture was purified using
silica chromatography in 10% EtOAc/90% hexanes to yield a light yellow foam. (2.7 g,
1

96% yield). H NMR (500 Mhz, CDCl3) δ 1.06 (d, J = 11.0, 21H), 1.11 (s, 9H), 1.58 (s,
9H), 2.02-2.08 (m, 2H), 3.83 (quint, J = 6.5 Hz, 2H), 4.07-4.15 (m, 2H), 7.24-7.58 (m,
4H), 8.09 (d, J = 8.3 Hz, 1H).

13

C NMR: (148.9 MHz, CDCl3) δ 11.98, 18.0, 27.6, 27.9,

31.1, 31.8, 42.5, 61.7, 64.99 83.5, 85.2, 109.7, 115.5, 121.6, 123.7, 126.1, 134.6,
-1

151.5,151.7, 164.1. IR (KBr): 2975, 2850, 1730 1680, 1460, 1430 cm . MS: m/z calcd
117

C31H49N2O6ISi [M+H], 570.72, found, 657.1 [-CH3CHCH3]. HRMS (ESI): m/z calcd for
C31H50IN2O6Si [M+H], 701.2483; found, 701.2484.
tert-butyl

2-((tert-butoxycarbonyl)(3-hydroxypropyl)carbamoyl)-3-iodo-1H-

indole-1-carboxylate (II-92). In a flame-dried RBF, compound II-91 (4.4 g, 6.3 mmol),
and tetrabutylammonium fluoride (12.6 mL, 12.6 mmol) was dissolved in anhydrous
THF (100 mL). The reaction mixture was stirred at room temperature for 4 hours. Sat.
NH4Cl (100 mL) was then added and stirred for 10 minutes at which point the organics
were extracted with EtOAc (3 x 75 mL). The organics were dried with Na2SO4 and
solvent removed in vacuo. The alcohol was purified using silica chromatography with
1

10% acetone/90% dichloromethane to give a light yellow foam. (2.7 g, 79.4% yield). H
NMR (300 Mhz, CDCl3) δ 1.06 (s, 9H), 1.60 (s, 9H), 1.96-2.02 (m, 2H), 2.68 (s, 1H),
3.78 (s, 2H), 3.89-3.98 (m, 1H), 4.13-4.21 (m, 1H), 7.38-7.40 (m, 3H), 8.05 (d, J = 9.15
Hz, 1H).

13

C NMR (74.47 MHz, CDCl3) δ 27.4, 28.0, 31.0, 40.9, 59.3, 65.1, 83.9, 85.4,

115.4, 121.7, 123.9, 126.2, 130.8, 134.3, 136.4, 152.3, 164.4. IR (KBr): 3443, 2950,
-1

2920, 1730, 1675, 1470, 1370, 1280, 1100 cm . MS: m/z calcd for C22H29N2O6I [M+H],
544.11, found, 544.0. HRMS (ESI): m/z calcd for C22H30IN2O6 [M+H], 545.1149; found,
545.1155.
tert-butyl

2-((tert-butoxycarbonyl)(3-oxopropyl)carbamoyl)-3-iodo-1H-

indole-1-carboxylate (II-93). In a flame-dried RBF, compound II-92 (2.25 g, 4.13 mmol)
was dissolved in EtOAc (150 mL). IBX (2.31, 8.26 mmol) was added to the reaction
mixture which was then refluxed for 20 hours. The mixture was filtered over celite and
the filtrate concentrated in vacuo. The product was isolated using silica column
118

chromatography with 30% EtOAc/70% Hexanes to yield a clear residue (1.7 g, 75%
yield).

1

H NMR (500 Mhz, CDCl3) δ 1.05 (s, 9H), 1.56 (s, 9H), 2.94 (t, J = 7.6 Hz, 2H),

4.09 (dt, J = 7.29, 14.3, 2H), 4.33 (dt, J = 6.99, 13.6 Hz, 2H, ), 7.30 (dd, J = 6.41, 15.55
Hz, 3H), 8.03 (d, J = 8.79 Hz, 1H), 9.84 (s, 1H).

13

C NMR (148.9 MHz, CDCl3) δ 27.2,

27.7, 38.2, 42.2, 65.0, 83.6, 85.2, 115.2, 121.4, 123.7, 126.1, 130.5, 134.2, 136.0,
-1

148.0, 151.1, 163.8, 200.0. IR (KBr): 2975, 2900 1725, 1730, 1675, 1450,1375 cm .
MS: m/z calcd for C22H27N2O6I [M+H]; 542.11, found, 542.0.
(Z)-tert-butyl

2-((tert-butoxycarbonyl)(3-(2,5-dioxoimidazolidin-4-

ylidene)propyl)carbamoyl)-3-iodo-1H-indole-1-carboxylate (II-94). In a flame dried
flask, compound II-85 (739 mg, 3.13 mmol) and lithium bromide (326 mg, 3.76 mmol)
were dissolved in anhydrous acetonitrile (75 mL) and stirred for 5 minutes.
Triethylamine (0.43 mL, 3.76 mmol) was added to the mixture and stirred for 10 minutes
followed by the addition of II-93 (1.7 g, 3.13 mmol). The reaction mixture was stirred 24
hours at room temperature. 1N HCl (50 mL) was added to the reaction mixture and
stirred for 10 minutes. Organics were extracted with CH2Cl2 (3 x 100 mL), dried over
Na2SO4 and concentrated to dryness in vacuo. The product was isolated from impuries
with silica chromatography with 20% acetone/dichloromethane to yield a light yellow
1

4

foam. (1.6g, 82% yield (2:1 Z:E)). H NMR (500 Mhz, d -Acetone) (mixture of isomers,
not separated) δ 1.15 (s, 9H), 1.18 (s, 9H), 1.61 (s, 9H), 1.62 (s, 9H), 2.11 (dt, J = 1.46,
8.39, 8.28 Hz, 2H), 3.17-3.21 (m, 2H), 3.89-3.98 (m, 2H), 4.16-4.21(m, 2H), 5.70 (t, J =
8.05 Hz, 1H), 5.80 (t, J = 7.83 Hz, 1H), 7.49-7.39 (m, 5H), 8.14 (d, J = 8.33 Hz, 1H),
9.03 (s, 1H), 9.18 (s, 1H) , 9.80 (s, 1H).

13

119

4

C NMR (148.9 MHz, d -Acetone) δ 25.7,

26.4, 27.7, 28.0, 43.8, 44.4, 64.8, 84.2, 86.2, 108.7, 114.3, 122.3, 124.8, 131.4, 131.9,
-1

135.3, 149.0, 152.4, 154.9, 164.4. IR (KBr): 3250, 2975, 1730, 1450, 1350 cm . MS:
m/z calcd C25H29N4O7I [M+H] 624.42; found [M+H ], 524.0 [-Boc]. HRMS (ESI):m/z
calcd for C25H30IN4O7 [M+H], 625.1159; found, 625.1166.
(Z)-di-tert-butyl

4-(3-(N,1-bis(tert-butoxycarbonyl)-3-iodo-1H-indole-2-

carboxamido)propylidene)-2,5-dioxoimidazolidine-1,3-dicarboxylate

(II-95).

Compound II-94 (750 mg, 1.2 mmol) and di-tert-butyl-dicarbonate (654 mg, 3 mmol)
were dissolved in anhydrous THF (35 mL) followed by the addition of 4dimethylaminopyridine (29.4 mg, 0.24 mmol). Triethylamine (140 μl, 1.2 mmol) was
added to the mixture which was then stirred at room temperature for 3 hours at which
time an additional portion of DMAP (0.98 g, 0.008 mmol) was added and the mixture
stirred for an additional 20 hours. The mixture was then concentrated to dryness in
vacuo and dissolved in CH2Cl2, and subsequently washed with 1 N HCl, saturated
Na2CO3 and brine. The organics were dried with anhydrous Na2SO4 and concentrated
in

vacuo.

The

product

was

purified

with

silica

EtOAc/Hexanes to yield a yellow foam (4.4 g, 98% yield).

chromatography
1

with

25%

H NMR (500 Mhz, CDCl3) δ

1.08 (s, 9H), 1.55 (s, 18H), 1.56 (s, 9H), 3.19-3.39 (m, 1H), 3.32-3.42 (m, 1H), 3.92-4.01
(m, 1H), 4.18-4.27 (m, 1H), 7.16 (t, J = 7.93 Hz, 1H), 7.28 (t, J = 7.01 Hz, 1H), 7.34 (t, J
= 7.1 Hz, 2H), 8.03 (d, J = 8.4 Hz, 1H).

13

C NMR (148.9 MHz, CDCl3) δ 26.1, 27.5, 27.7,

27.9, 43.4, 65.0, 83.5, 85.2,85.5, 56.4, 115.4, 121.7, 123.7,124.6, 127.8, 145.3,145.7,
151.4, 157.3, 163.9. IR (KBr): 2980, 2950, 1720, 1690, 1490, 1450, 1270, 1160, 1050

120

-1

cm .

MS: m/z calcd for C35H45N4O11I [M+H] 824.66, found [M+H] 523.3 [-3 Boc].

HRMS (ESI): m/z calcd for C35H46N4O11I [M-H], 823.2057; found, 823.2035.
1-benzoyl-2-thioxoimidazolidin-4-one (II-97). This reaction was performed
23

according to the known procedure . Hippuric acid (8.0g, 44.6 mmol) and ammonium
thiocyanate (8.5 g, 111.5 mmol) were thoroughly ground together in a mortar and
suspended in a mixture of glacial acetic acid (4 mL) and acetic anhydride (36 mL). The
reaction mixture was heated for one hour at 60ºC and for an additional hour at 100ºC.
The red solution was cooled, diluted with cold water (200 mL) and stirred overnight for
18 hours. The reaction mixture was concentrated in vacuo. The crude residue was then
crystallized from EtOH affording the product (8.07 g, 82%) as an orange solid. The
spectral data matches that previously reported in the literature.

23

1

m.p. 156-158. H

NMR. (500 MHz, DMSO-d6) δ 4.60 (s, 2H), 7.4-7.7 (m, 5H), 12.50 (bs, 1H).

13

C NMR

(128.2 MHz, DMSO-d6) δ 52.9, 127.9, 128.5, 131.7, 134.4, 169.0, 170.7, 182.6. MS:
+

+

m/s calcd for C10H9N2O2S [M ] 221.0385; found [M ], 221.0383.
(Z)-5-(2-(methylthio)-4-oxo-1H-imidazol-5(4H)-ylidene)-2,3,4,5tetrahydroazepino[3,4-b]indol-1(10H)-one (II-98b). A 1 M solution of TiCl4 in CH2Cl2
(1.9 mL, 1.9 mmol) was added to THF (25 mL) at -10°C. II-68 (200 mg, 0.5 mmol) and
II-41 (219 mg, 0.93 mmol) were subsequently added and allowed to stir at 0°C for 30
minutes. Pyridine (0.3mL, 3.7 mmol) was then added drop wise over 30 minutes. The
reaction mixture was stirred at 0°C for an additional 2 hours and allowed to stir
overnight at room temperature. Sat. NH4Cl (25 mL) was added, and the mixture was
stirred for 10 min followed by subsequent extractions with ethyl acetate (3 x 25 mL). The
121

organic extracts were then washed with water (25 mL), followed by brine (25 mL). The
organic extracts were then dried with anhydrous NaSO4 and concentrated. The
resultant residue (200 mg, 0.47 mmol) was dissolved in THF (10 mL) and stirred with
sodium hydroxide (94 mg, 2.35 mmol) in water (10 mL) at room temperature for 20
hours. The mixture was neutralized with 1% HCl and extracted with ethyl acetate (50
mL). Organics were dried and concentrated. Product was isolated from impurities on
silica with 5% methanol/dichloromethane to yield a reddish-yellow solid (161 mg, 53%
overall yield). m.p. decomposes over 190°C.

1

H NMR (500 MHz, DMSO-d6) 2.33 (s,

3H), 3.34-3.40 (m, 4H), 7.06 (t, J = 7.2 Hz, 1H), 7.21 (t, J = 7.0 Hz, 1H), 7.41 (d, J = 8.2
Hz, 1H), 8.33 (t, J = 5.1 Hz, 1H), 11.59 (s, 1H), 12.19 (s, 1H).

13

C NMR (128.2 MHz,

DMSO-d6) δ 11.9, 35.5, 38.9, 111.9, 115.3, 119.2, 123.8, 125.2, 125.9, 131.7, 134.8,
135.2, 135.9, 157.4, 164.7, 169.7. IR: (KBr-pellet) 3220, 1700, 1650, 1540, 1480.
HRMS (ESI): m/z calcd for C16H15N4O2S [M+H], 327.0916; found, 327.0914.
(Z)-5-(2-(dimethylamino)-4-oxo-1H-imidazol-5(4H)-ylidene)-2,3,4,5tetrahydroazepino[3,4-b]indol-1(10H)-one (2) (II-99). Compound II-98b (514 mg, 1.6
mmol) was added to a solution of dimethylamine (12 mL (2M in THF), 24 mmol). The
mixture was heated to 90°C in a sealed tube for 24 hours. The reaction mixture was
allowed to cool to room temperature and the resulting precipitate was filtered and
washed with 10% HCl. The bright yellow solid was dried in vacuo. (212mg, 41.4%). m.p.
1

decomposes over 250°C. H NMR. (500 MHz, DMSO-d6) δ 2.90 (s, 3H), 3.15 (s, 3H),
3.19 (s, 4H), 7.14 (t, 1H, J = 7.5 Hz), 7.29 (t, 1H, J = 7.6 Hz), 7.54 (d, 1H, J = 8.2 Hz),
8.43 (s, 1H), 10.35 (s, 1H), 12.49 (br, 1H);

13

122

C NMR (128.2 MHz, DMSO-d6) δ 25.5,

35.9, 38.4, 67.0, 112.4, 112.9, 120.5, 122.1, 122.5, 124.1, 129.2, 132.4, 136.3, 153.1,
163.3, 164.7. IR: (KBr) 3385, 3224, 2940, 1676, 1603, 1441, 1287. HRMS (ESI): m/z
calcd for C17H18N5O2 [M+H], 324.1460; found, 324.1463.

123

REFERENCES

124

II.K

References

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Sharma, G. M.; Buyer, J. S.; Pomerantz, M. W., Characterization of a Yellow
Compound Isolated from the Marine Sponge Phakellia-Flabellata. J. Chem. Soc.
Chem. Comm. 1980, (10), 435-436.

2.

Sharma, G.; Magdoff-Fairchild, B., Natural products of marine sponges. 7. The
constitution of weakly basic guanidine compounds, dibromophakellin and
monobromophakellin. J. Org. Chem. 1977, 42 (25), 4118-24.

3.

Sharma, G. M.; Schem, G. F.; Pastore, A. T., Natural products of marine
sponges. VIII: Partial structure of a novel compound isolated from Phakellia
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4.

Mattia, C. A.; Mazzarella, L.; Puliti, R., 4-(2-Amino-4-Oxo-2-Imidazolin-5Ylidene)-2-Bromo-4,5,6,7-Tetrahydropyrrolo-[2,3-C]Azepin-8-One
Methanol
Solvate - a New Bromo Compound from the Sponge Acanthella-Aurantiaca. Acta
Crystallographica Section B-Structural Science 1982, 38 (Sep), 2513-2515.

5.

Cimino, G.; De Rosa, S.; 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 (7), 767-8.

6.

Kitagawa, I.; Kobayashi, M.; Kitanaka, K.; Kido, M.; Kyogoku, Y., Marine natural
products. XII. On the chemical constituents of the Okinawan marine sponge
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7.

Denanteuil, G.; Ahond, A.; Guilhem, J.; Poupat, C.; Dau, E. T. H.; Potier, P.;
Pusset, M.; Pusset, J.; Laboute, P., Marine-Invertebrates from Neo-Caledonian
Lagoons .5. Isolation and Identification of Metabolites from a New Species of
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8.

Endo, M.; Nakagawa, M.; Hamamoto, Y.; Ishihama, M., Pharmacologically Active
Substances from Southern Pacific Marine-Invertebrates. Pure Appl. Chem. 1986,
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9.

Pettit, G. R.; Herald, C. L.; Leet, J. E.; Gupta, R.; Schaufelberger, D. E.; Bates,
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10.

Patil, A. D.; Freyer, A. J.; Killmer, L.; Hofmann, G.; Randall, K., Z-axinohydantoin
and debromo-z-axinohydantoin from the sponge Stylotella aurantium: Inhibitors
of protein kinase C. Natural Product Letters 1997, 9 (3), 201-207.

11.

Inaba, K.; Sato, H.; Tsuda, M.; Kobayashi, J., Spongiacidins A-D, new
bromopyrrole alkaloids from Hymeniacidon sponge. J. Nat. Prod. 1998, 61 (5),
693-695.

12.

Williams, D. H.; Faulkner, D. J., Isomers and tautomers of hymenialdisine and
debromohymenialdisine. Nat. Prod. Lett. FIELD Full Journal Title:Natural Product
Letters 1996, 9 (1), 57-64.

13.

Xu, Y.-z.; Yakushijin, K.; Horne, D. A., Synthesis of C11N5 Marine Sponge
Alkaloids:
(+-)-Hymenin,
Stevensine,
Hymenialdisine,
and
Debromohymenialdisine. J. Org. Chem. 1997, 62 (3), 456-464.

14.

Sosa, A. C. B.; Yakushijin, K.; Horne, D. A., A Practical Synthesis of (Z)Debromohymenialdisine. J. Org. Chem. 2000, 65 (2), 610-611.

15.

Kobayashi, J.; Ohizumi, Y.; Nakamura, H.; Hirata, Y.; Wakamatsu, K.; Miyazawa,
T., Hymenin, a novel a-adrenoceptor blocking agent from the Okinawan marine
sponge Hymeniacidon sp. Experientia 1986, 42 (9), 1064-5.

16.

Kobayashi, J.; Nakamura, H.; Ohizumi, Y., Alpha-Adrenoceptor Blocking Action
of Hymenin, a Novel Marine Alkaloid. Experientia 1988, 44 (1), 86-87.

17.

Annoura, H.; Tatsuoka, T., Total syntheses of hymenialdisine and
debromohymenialdisine: stereospecific construction of the 2-amino-4-oxo-2imidazolin-5(Z)-disubstituted ylidene ring system. Tetrahedron Lett 1995, 36 (3),
413-16.

18.

Davis, F. A.; Vishwakarma, L. C.; Billmers, J. M.; Finn, J., Synthesis of AlphaHydroxy Carbonyl-Compounds (Acyloins) - Direct Oxidation of Enolates Using 2Sulfonyloxaziridines. J. Org. Chem. 1984, 49 (17), 3241-3243.

19.

Sosa, A. C. B.; Yakushijin, K.; Horne, D. A., Synthesis of Axinohydantoins. J.
Org. Chem. 2002, 67 (13), 4498-4500.

126

20.

Wan, Y.; Hur, W.; Cho, C. Y.; Liu, Y.; Adrian, F. J.; Lozach, O.; Bach, S.; Mayer,
T.; Fabbro, D.; Meijer, L.; Gray, N. S., Synthesis and target identification of
hymenialdisine analogs. Chem. Biol. 2004, 11 (2), 247-59.

21.

Xu, Y. Z.; Yakushijin, K.; Horne, D. A., Transbromination of brominated pyrrole
and imidazole derivatives: Synthesis of the C11N5 marine alkaloid stevensine.
Tetrahedron Lett. 1996, 37 (45), 8121-8124.

22.

Portevin, B.; Golsteyn, R. M.; Pierre, A.; De Nanteuil, G., An expeditious
multigram
preparation
of
the
marine
protein
kinase
inhibitor
debromohymenialdisine. Tetrahedron Lett. 2003, 44 (52), 9263-9265.

23.

Papeo, G.; Posteri, H.; Borghi, D.; Varasi, M., A new glycociamidine ring
precursor: Syntheses of (Z)-hymenialdisine, (Z)-2-debromohymenialdisine, and
(+/-)-endo-2-debromohymenialdisine. Org. Lett. 2005, 7 (25), 5641-5644.

24.

He, Q. F.; Chen, W.; Qin, Y., Synthesis of 2-substituted endo-hymenialdisine
derivatives. Tetrahedron Lett. 2007, 48 (11), 1899-1901.

25.

Eder, C.; Proksch, P.; Wray, V.; Steube, K.; Bringmann, G.; van Soest, R. W. M.;
Sudarsono; Ferdinandus, E.; Pattisina, L. A.; Wiryowidagdo, S.; Moka, W., New
alkaloids from the Indopacific sponge Stylissa carteri. J. Nat. Prod. 1999, 62 (1),
184-187.

26.

Tutino, F.; Posteri, H.; Borghi, D.; Quartieri, F.; Mongelli, N.; Papeo, G.,
Stereoselective synthesis of (Z)-axino- and (Z)-debromoaxinohydantoin.
Tetrahedron 2009, 65, 2372-2376.

27.

Chacun-Lefevre, L.; Joseph, B.; Merour, J.-Y., Synthesis and reactivity of
azepino[3,4-b]indol-5-yl trifluoromethanesulfonate. Tetrahedron 2000, 56 (26),
4491-4499.

28.

Sharma, V.; Lansdell, T. A.; Jin, G.; Tepe, J. J., Inhibition of Cytokine Production
by Hymenialdisine Derivatives. J. Med. Chem. 2004, 47 (14), 3700-3703.

29.

Sharma, V.; Tepe, J. J., Potent inhibition of checkpoint kinase activity by a
hymenialdisine-derived indoloazepine. Bioorg. Med. Chem. Lett. 2004, 14 (16),
4319-4321.

127

30.

Sharma, V.; Lansdell, T. A.; Peddibhotla, S.; Tepe, J. J., Sensitization of Tumor
Cells toward Chemotherapy: Enhancing the Efficacy of Camptothecin with
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31.

Montagne, C.; Laurent, N.; Joseph, B.; Merour, J. Y., Preparation and reactivity
of 5-substituted azepino[3,4-b]indoles. J. Heterocycl. Chem. 2005, 42 (7), 14331441.

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Chacun-Lefevre, L.; Beneteau, V.; Joseph, B.; Merour, J.-Y., Ring closure
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Chacun-Lefevre, L.; Joseph, B.; Merour, J.-Y., Intramolecular Heck coupling of
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Hupp, C. D.; Tepe, J. J., Total synthesis of a marine alkaloid from the tunicate
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35.

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reagents for the mild and efficient preparation of C-5 unsaturated hydantoin
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Shabica, A. C.; Howe, E. E.; Ziegler, J. B.; Tishler, M., Improved synthesis of 3indolealdehyde. J. Am. Chem. Soc. 1946, 68, 1156-7.

128

CHAPTER III
EVALUATION OF INDOLOAZEPINE AS AN INHIBITOR OF CHECKPOINT KINASE 2
III.A

Biological target of natural product and hypothesis for activity
As discussed in detail in Chapter 1, Chk2 has been the biological target from the

start of this project. The function of Chk2 has been proposed to act as a barrier to
tumorigenesis by preventing or delaying progression through the cell cycle following the
1, 2

activation of the DNA damage response in cancer lesions.

Activation of ATM-Chk2

by IR leads to phosphorylation of several downstream substrates involved in many
cellular processes including cell cycle arrest, apoptosis and DNA repair.

3-9

The use of Chk2 inhibitors for potentiating DNA-damage agents continues to be
exciting but controversial

10-12

as not all inhibitors of Chk2 demonstrate a synergistic

cytotoxic effect with chemotherapeutic drugs.

13

There are a few small molecule

inhibitors of Chk2 that have been reported in the literature, and interest in discovering a
potent and selective inhibitor remains high.

Figure III-1. Structure of debromohymenialdisine, hymenialdisine and hymenialdisinederived indoloazepine II-70.

129

Natural product debromohymenialdisine has some structural characteristics
similar to indoloazepine II-70 (Figure III-1). It has been hypothesized that the majority of
the hydrogen bonding contacts believed to be important for the natural product are
present

in

indoloazepine

(See

Figure

I-20

–

I-22).

Cellular

inhibition

of

debromohymenialdisine and hymenialdisine (Figure III-1) was reported by Roberge and
14

co-workers showing inhibition of the G2 checkpoint.

Debromohymenialdisine inhibited

Chk1 and Chk2 in vitro with an IC50 value of 3 and 3.5 µM, respectively.
III.B

14

In vitro evaluation of Chk2 inhibitors
In order to determine the selectivity of II-70 for Chk2, in vitro kinase-profiling

experiments were performed (Table III-1). The kinase listed in table III-1 were selected
from a large panel of kinases known to be affected by the hymenialdisines at low
micromolar concentrations.

15, 16, 17

Table III-1. Kinase profiling data for indoloazepine debromohymenialdisine,
hymenialdisine and II-70. Values are reported at IC50 values. Experiments were
performed by Millipore KinaseProfiler Service using a radioactive assay method.
Kinase
debromohymenialdisine hymenialdisine
II-70
Chk1 (nM)

725

1950

237

Chk2 (nM)

183

42

8

GSK-3β (nM)
CK1δ(h) (nM)
CK2 (h) (nM)
IKKα(h) (nM)

NA
NA
NA
NA

10
15, 16
35
15, 16
7000
NA

86
1,352
> 10,000
> 10,000

IKKβ(h) (nM)

NA

NA

> 10,000

6
15, 16
700
15, 16
1200

89
2,539
3,381

MEK1(h) (nM)
PKCα(h) (nM)
PKCβII(h) (nM)

824

18

18

NA
NA

130

The compounds were screened for multiple kinases so that insight into the
selectivity against other kinases could be evaluated. The kinases screened are listed in
Table III-1. An in vitro kinase assay was performed against the listed kinases with
debromohymenialdisine, hymenialdisine and II-70 as seen in Table III-1 (Millipore,
Billerica, MA).

17

A general illustration of the protocol used for the kinase screen is

shown in Figure III-2. Briefly, a known substrate for the kinase is incubated with a
33

compound, such as II-70, along with radio-labeled [γ- P]-ATP and the kinase. The
kinase phosphorylates the substrate with

33

P-ATP (or not, if inhibited) and the amount

of radio-activity bound to the negatively charged filter mat is directly related to the
incorporation of

33

P.

++

P

P

33P

ATP

Substrate

33P

++

Substrate
Kinase

+ Inhibitor
Figure III-2. Illustration of radio-active kinase protocol.
II-70 exhibited potent inhibition of Chk1 and Chk2 with IC50 values of 237 nM and
8 nM, respectively.

17

II-70 showed a greater potency toward Chk2 compared to

hymenialdisine. However, hymenialdisine was more selective for Chk2 than Chk1 (~45fold selectivity) when compared to II-70 (~30-fold selectivity). II-70 also demonstrated

131

inhibition and at least 10-fold selectivity over GSK-3β (IC50 = 86 nM), MEK1 (IC50 = 89
nM).

Figure III-3. Structures of analogs II-84, II-99, III-1 and III-2.

17, 19

The Tepe lab reported the original synthesis of II-70,

and the improved

synthesis was discussed in Chapter 2. With the newly acquired II-70 collected as the
HCl salt, it was necessary to re-evaluate the inhibitor to ensure it was still able to inhibit
Chk1 and Chk2. Two other hymenialdisine derived analogs III-1, and III-2 (Figure III-3)
were synthesized by two members of the Tepe research group (Micah Luderer,
Rahman Saleem, respectively). The analogs were synthesized in order to learn more
about the structural requirements for Chk2 binding activity. At this time, analogs II-84, II99, III-1 and III-2 (Figure III-3) were also evaluated for their ability to Chk1 and Chk2.

132

A

B

100

II-70 and Chk2
100

% Relative CPM

% Relative CPM

II-70 and Chk1

50

C
hk
C 2+
hk
2 H3
+ PO
Ve
hi 4
c
10 le
M
1
0.  M
1
0.  M
01
0.
00  M
1
M

0

C
hk
C 1+
hk
1 H3
+ PO
Ve
hi 4
c
10 le
M
1

0. M
1
0.  M
01
0.  M
00
1
M

0

50

33

Figure III-4. Inhibition of [γ- P]-ATP phosphorylation by Chk1 or Chk2. H3PO4 was
33
used as a negative or untreated control. A) Inhibition of [γ- P]-ATP phosphorylation of
33
Chk1 by indoloazepine II-70. B) Inhibition of [γ- P]-ATP phosphorylation of Chk2 by
indoloazepine II-70.
The radio-labeled filter mat assay that was described above was used to
evaluate II-70, II-84 II-99 and III-1 and III-2. The results of the inhibition of Chk1 and
Chk2 are illustrated in Figure III-4. The graphs clearly indicate a dose dependent
inhibition of Chk1 (Figure III-4A) and Chk2 (Figure III-4B) by II-70, and that II-70 was a
more selective inhibitor of Chk2 than for Chk1. The values for IC50 were calculated
based on the phosphoric acid control and the vehicle control and found to be 220.4 nM
and 13.5 nM for Chk1 and Chk2, respectively (Table III-2). Neither analogs II-84 or II-99
indicated any inhibition of Chk1 or Chk2 at relevant concentrations (IC 50 >10 µM).
However, compounds III-1 and III-2 were also found to be potent inhibitors of Chk2,
albeit with somewhat less selectivity toward the related Chk1. The results of the Chk1
and Chk2 inhibition by these compounds are summarized in Table 1.
133

TABLE III-2. Summary of mean IC50 values for kinase inhibition by analogs II-70, II-84,
II-99, III-1 and III-2.
II-70
220.4
13.5

Kinase
Chk1 (nM)
Chk2 (nM)

II-84
> 10,000
> 10,000

II-99
> 10,000
> 10,000

III-1
581.4
65.9

III-2
626.8
53.9

Replacement of the exocyclic amine with an oxygen (compound II-84) abrogated
the ability of the indoloazepine analog to inhibit Chk1 or Chk2. The lack of Chk2
inhibition by compound II-99 suggests that the methyl groups add steric bulk to the
exocyclic nitrogen of the glycocyamidine, which could prevent a proper fit in the active
pocket of Chk2 and eliminate the hydrogen bonding interactions with ASN352, as seen
in Figure III-5.

20

The lack of activity of compounds III-84 and III-99 indicates that the free

amine group is necessary for binding and could also act as a hydrogen bond donor,
consistent with the binding of hymenialdisine to Chk2. Compounds II-70, III-1 and III-2
all possess the free amine on the glycocyamidine ring allowing for the hydrogen
bonding that is not present in II-84 and II-99 (Figure III-6). The decrease in potency of
compound III-1 relative to II-70 (Figure III-6a) may potentially be explained by the
location of the double bond of the glycocyamidine ring, which is not conjugated with the
carbonyl in the ring, but instead is conjugated to the indoloaldisine portion, thus
competing for hydrogen bonding with GLU308. The indole hydrogen could form an
intramolecular hydrogen bond with the proximal nitrogen, potentially reducing hydrogen
bond opportunities to the Chk2 catalytic site (Figure III-6b). The intramolecular hydrogen

134

A

B
THR 367

ASN 352

GLU 351

GLU 302
GLU 308

MET 304

Figure III-5. Natural product hymenialdisine in Chk2 catalytic site. A) Hymenialdisine
pictured with surface interactions. B) Hymenialdisine pictured with surface interactions
as well as with hydrogen bonds to residues. Green regions represent hydrophobic
20
areas, blue indicates electropositive areas and red areas are electronegative.
bonding could also contribute to the change in the way III-1 fits in the active site when
the glycocyamidine ring is aligned with glycocyamidine ring of hymenialdisine (Figure III6b). As anticipated, phenyl pyrrole III-2, was capable of inhibiting Chk2 effectively, but
unfortunately displayed a reduced activity and selectivity compared to indoloazepine II70. The hydrophobic phenyl ring may have steric interactions with an electronegative
area instead of fitting into the hydrophobic region of the catalytic site.
A radio-labeled filter mat experiments demonstrate that II-70, III-1 and III-2 are
inhibitors of Chk1 and Chk2. The structural similarities of the glycocyamidine ring of
these analogs compared to the natural product hymenialdisine can explain the ability of
these analogs to inhibit Chk1 and Chk2. As noted above, analogs II-84 and II-99 are
poor inhibitors of both of these kinases due to their lack of hydrogen bond donors and
steric methyl groups.

135

A

B

C

Figure III-6. Compounds II-70, III-1 and III-2 modeled in the Chk2 catalytic site with
21
PyMol. A) II-70 pictured with surface interactions. B) III-1 pictured with surface
interactions. C) III-2 pictured with surface interactions. Green regions represent
hydrophobic areas, blue indicates electropositive areas and red areas are
20
electronegative.
III.C

In vitro evaluation of the effect of indoloazepine in the DNA pathway
In order to determine support for cellular inhibition of Chk2 by II-70, Chk2 levels

(total protein levels) were evaluated, in addition to Chk2 Th68 and Chk2
autophosphorylation at Ser516. A normal breast cell line, 184B5 was untreated or
pretreated for 2 hours prior to IR with vehicle (DMSO), the positive control Chk2 inhibitor
ABI (Chapter 1, Figure I-16A, 30 µM ), the negative control Chk2-inactive indoloazepine
136

II-99 (30 µM), and the Chk2 inhibitor II-70 (30 µM). The cells were irradiated (10 Gy)
and treated with fresh media and vehicle or 30 µM ABI, II-99 or II-70 for 4 hours at
which time whole cell extracts were collected. Whole cell extracts were then evaluated
by Western blot for cellular inhibition of Chk2. Total Chk2 levels were probed with antiChk2 antibody. No changes in the level of Chk2 were observed upon exposure to low
(0.1 µM) and high (30 µM) concentrations of II-70 (Figure III-7A). ABI (30 µM) also did
not have an effect on the total levels of Chk2. The IR-induced activation of Chk2 was
confirmed by observing phosphorylation of Chk2 at Thr68, which occurred when cells
were exposed to IR (Figure III-7A, Lanes 3, 5-8, 10, and 12).

22

As anticipated, inhibition

of Chk2 by II-70 or ABI did not affect phosphorylation of Thr68 by ATM (Figure III-7A,
Lanes 5 and 12), indicating that the ATM function was not affected by compound II-70.
However, there was a dose-dependent effect of II-70 on IR-induced Chk2
autophosphorylation at Ser516 shown in 184B5 cells (Figure III-7A and B, lanes 5-8).
As expected, Chk2 autophosphorylation at Ser516 was also seen with ABI (Figure III23

7A and B, lane 12), a known Chk2 inhibitor . Figure III-7B shows the relative levels of
phosphorylated forms of Chk2 pSer516 after normalization for total β-actin levels as
determined by densitometry. This further illustrates the dose dependent inhibition of
autophosphorylation by II-70. ABI also showed a comparable level of Chk2 inhibition at
30 µM compared to that seen with II-70. There was no inhibition of Chk2
phosphorylation of Thr68 or Ser516 with the inactive Chk2 agent II-99 (IC50 > 10 µM),
which was used in the assay as a negative control (Figure III-7A, Lane 10).

137

A

184B5

1 2 3 4 5 6 7 8 9 10 11 12
ABI (µM)
2 (µM)
1 (µM)
Vehicle (DMSO)
IR (10 Gy)

B

-

+
-

+

- - - - 30 30 10
- - - + +

1

1
+

- - - 30
- 30 30 0.1 - - - - - + - + -

30
-

+

Chk2
Actin
Chk2 pThr68
Actin
Chk2 pSer516
Actin

0.8

Relative
0.7
intensity

Chk2 pSer516

0.6

0

1 2 3 4 5 6 7
9 101112
1 2 3 4 5 6 7 88 9 10 11 12

Figure III-7. Western blot analysis of 184B5 (normal, p53 WT) whole cell extracts. A)
Cells were untreated (control) or treated with vehicle or with compounds ABI, II-99, and
II-70 for 2 hours prior to IR (10 Gy). Cells were incubated for 4 hours after IR when
extracts were collected. The western blot was evaluated with Chk2 antibodies. B)
Quantification of relative densities of bands for pSer516. These blots and data are
representative of 3 independent experiments.
To further investigate the effect of II-70 on Chk2 inhibition, we evaluated II-70 in
a breast cancer cell line, MDA-MB-231 with a mutated p53. DNA damage was induced
with IR in MDA-MB-231 cells and inhibition of Chk2 phosphorylation was evaluated
(Figure III-8A). As in the normal breast cell line, levels of total Chk2 in MDA-MB-231
remain unchanged when treated with IR, ABI, II-99 or II-70. Chk2 Thr68 was
phosphorylated following treatment with IR and remained unchanged when pretreated
with ABI, II-99 or II-70 (Figure III-8A, Lanes 3, 5-8, 10, 12). Chk2 inhibition with II-70
inhibited autophosphorylation at Ser516 in a dose dependent manner following IR
(Figure III-8A, Lanes 5-8). Figure III-8B shows the relative levels of phosphorylated
138

forms of Chk2 pSer516 after normalization for total β-actin levels as determined by
densitometry.

This

further

illustrates

the

dose

dependent

inhibition

of

autophosphorylation by II-70. Levels of Chk2 inhibition were again compared to ABI,
which also inhibited cis-autophosphorylation of Chk2 at Ser516 (Figure III-8A, Lane 12).
As anticipated, inactive analog II-99 did not inhibit Chk2 autophosphorylation (Figure III8A, Lane 10). This data illustrates that the cellular inhibition of Chk2 by II-70 is
independent of the p53 status of the cell lines and that Chk2 is inhibited in both the
normal breast line, 184B5 as well as the breast tumor cell line, MDA-MB-231.

A

MDA-MB-231

1 2 3 4 5 6 7 8 9 10 11 12
ABI (µM)
2 (µM)
1 (µM)
Vehicle (DMSO)
IR (10 Gy)

B

-

+
-

+

- - - - 30 30 10
- - - + +

1
+

- - - 30
- 30 30 0.1 - - - - - + - + -

30
-

+

1

Chk2
Actin
Chk2 pThr68
Actin
Chk2 pSer516
Actin

1.1
1.0
0.9

Chk2 pSer516

0.8

1

9

7

5

3

1

3

2

2

1

1

1

1

1

9

7

1

1 2 3 4 5 6 7 8 9 10 11 12
5

0

0.7

3

Relative
intensity

Figure III-8. Western blot analysis of MDA-MB-231 (tumor, p53 mut) whole cell extracts.
Cells untreated (control) or treated with vehicle or with compounds ABI, II-99, and II-70
for 2 hours prior to IR (10 Gy). Cells were incubated for 4 hours after IR when extracts
were collected. The western blot was evaluated with Chk2 antibodies. B) Quantification
of relative densities of bands for pSer516. These blots and data are representative of 3
independent experiments.

139

Two other hymenialdisine derived analogs III-1 (IC50 =68.8 nM) and III-2 (IC50 =
43.9 nM), were evaluated as cellular inhibitors of autophosphorylation of Chk2 at
Ser516 (Figure III-9). Both analogs III-1 and III-2 demonstrated a dose dependent effect
on inhibition of Chk2 Ser516, supporting cellular inhibition of Chk2 by III-1 and III-2.
1 2 3 4 5 6 7 8 9 10 11

IR (10 Gy) -

-

- 30 - - - - 30 10 1
- 30
- 30 10 1 - - + - - - - - - - - -

III-2 (µM) III-1 (µM) Vehicle (DMSO) -

-

-

- + + + + + + +

Chk2 pSer516
Actin

Figure III-9. Western blot analysis of 184B5 whole cell untreated (control) or treated
with vehicle or with compounds III-1 and III-1 for 2 hours prior to IR (10 Gy). Cells were
incubated for 4 hours after IR when extracts were collected. The western blot was
evaluated with Chk2 antibodies. These blots are representative of 3 independent
experiments.
Thus, this data indicates that the in vitro activity of inhibitor II-70, III-1 and III-2,
translates in cell culture. In addition, the more potent and selective Chk2 inhibitor II-70
abrogates Chk2 activity in both the normal breast cell line, 184B5 as well as the breast
tumor cell line MDA-MB-231.
III.D

In vitro evaluation of the cell cycle with indoloazepine
Checkpoint signaling pathways are activated following DNA double strand breaks

and result in the arrest of cells at the G1/S, S or G2/M transitions, which allows cells time
24

to repair the damage or execute apoptosis.

In response to IR, the activated ATM-

Chk2 pathway phosphorylates their downstream substrates resulting in a G 2 arrest. To
determine whether inhibition of Chk2 by II-70 would modulate the G2 checkpoint, we
monitored the cell cycle using single parameter flow cytometry. We utiliazed two
140

HCT116 cell lines which were either a Chk2 +/+ or Chk2 -/- genotype. This allowed us
to further verify that Chk2 is the target of II-70. We hypothesized that inhibiting Chk2
with II-70 would yield similar cell cycle effects as the Chk2 -/- cells following IR-induced
DNA damage.
HCT116 Chk2 +/+ cells were pretreated with nothing, vehicle or II-70 (30 µM) for
2 hours, and then the cells were irradiated with various doses of IR. HCT116 Chk2 -/remained untreated or were irradiated at the same doses of IR as HCT116 Chk2 +/+
cells (Figure III-10, Figure III-11). The cells were allowed to incubate for 0-22 hours at
which point cells were harvested and fixed. Consistent with the early and temporary
activation of the ATM-Chk2 pathway,

5, 6, 25

untreated HCT116 Chk2 +/+ cells resulted

in G2 arrest 22 hours after IR, relative to the control cells (Figure III-10B; Figure III-11E).
HCT116 Chk2 +/+ cells treated with Chk2 inhibitor II-70, displayed a significant (**P <
0.0071) decrease at the G2 checkpoint (Figure III-10, Figure III-11) compared to the
untreated control. A similar abrogation of the G2 checkpoint was demonstrated in the
Chk2 -/- cell line (Figure III-11E, **P < 0.0012), again, compared to the Chk2 +/+
untreated cells. Table III-4 summarizes the values of significance between the untreated
Chk2 +/+ and II-70 treated cells as well as the untreated Chk2 +/+ and Chk2 -/- cells.
The inhibition of the IR-induced G2 checkpoint in the Chk2 -/- cell line clearly indicates
that II-70 inhibits Chk2. Abrogation of the IR-induced G2 arrest with Chk2 inhibitor II-70,
allows normal cells to progress to the G1 checkpoint where DNA replication is initiated
and carried out.

141

0 Gy

1 Gy

2.5 Gy

0 Gy

0 Hour
1 Gy 2.5 Gy

5 Gy

0 Gy

4 Hour
1 Gy 2.5 Gy

B

5 Gy

5 Gy

2.5 Gy

5 Gy

2 Hour
1 Gy 2.5 Gy

5 Gy

HCT116 Chk2 +/+
II-70 Untreated

0 Gy

HCT116
Chk2 -/-

8 Hour

HCT116
Chk2 -/-

HCT116 Chk2 +/+
II-70 Untreated

E

1 Gy

HCT116
Chk2 -/-

HCT116
Chk2 -/-

D

HCT116
Chk2 -/-

HCT116 Chk2 +/+
II-70 Untreated

C

0 Gy

HCT116 Chk2 +/+
II-70 Untreated

HCT116 Chk2 +/+
II-70 Untreated

A

22 Hour

Figure III-10. Cell cycle histograms of HCT116 Chk2 +/+ or Chk2 -/- cells untreated
(control) or pretreated for 2 hours with II-70 (30 µM) followed by IR (0-5 Gy). Cells were
fixed at indicated times after IR. A) 0 hr; B) 2 hr. C) 4 hr; D) 8 hr; E) 22 hr. These
histograms are representative of 3 independent experiments.
142

A

B

50

% cells in G 2

% cells in G 2

50

25

0

0

C

1
2.5
IR dose (Gy)

% cells in G 2

% cells in G 2
% cells in G 2

0

1
2.5
IR dose (Gy)

5

50

***

0

E
50

***

D

p < 0.0001

0

25

0

5

50

25

p < 0.0001

1
2.5
IR dose (Gy)

*

p < 0.0071

p < 0.0025
25

0

5

**

0

1
2.5
IR dose (Gy)

5

p < 0.0012

**

Without II-70

25

With 30 M II-70
Chk2 -/-

0

0

1

2.5

5

IR dose (Gy)

Figure III-11. DNA frequency histograms of HCT116 Chk2 +/+ or Chk2 -/- cells in G2.
Cells were either untreated (control) or pretreated for 2 hours with II-70 (30 µM) followed
by IR (0-5 Gy). A) Cells at time 0. B) Cells 2 hours after IR. C) Cells 4 hours after IR. D)
Cells 8 hours after IR. E) Cells 22 hours after IR. Data for G2 were expressed as mean
± SEM from 3 independent experiments.

143

After IR-induced DNA damage, the ATM-ATR pathway is activated and can
trigger a checkpoint at both G1 and G2 (Figure III-12). When Chk2 is activated normally,
it phosphorylates p53 which can activate p21, resulting in arrest at both G 1 and G2.
When Chk2 is inhibited, G2 checkpoint arrest can only occur if it is going through the
Chk1 pathway (Figure III-12). Because Chk1 tends to be activated later, inhibition of
Chk2 also inhibits the G2 arrest and leads to a buildup of cells at G1.
DNA Damage

ATM

ATR

Chk2Th68

p53

Ser20

Chk1-P

Cdc25A

Ser123

Cdc25C
Ser216

p21
Cdk2/CycE

G1

Cdk1/CycB
S

G2

M

Figure III-12. The abbreviated G1/S and G2/M checkpoint regulation network following
upstream ATM/ATR activation.
In response to IR-induced cellular damage, the G1 arrest has been established in
response to a signal transmitted via ATM and p53.

26, 27

The increase in G1 arrest has

been observed in wild-type, but not mutant p53. Transgenic p53 knockout mice have
demonstrated that G1 arrest in response to radiation requires p53.
144

27

Because Chk2 has

been implicated in the G1 cell cycle checkpoint and predicted to act as a downstream
substrate of ATM, the role of Chk2 was evaluated in this capacity.
Table III-3. Statistically significant differences in cell cycle distribution of untreated Chk2
+/+ versus II-70 treated cells, and untreated Chk2 +/+ versus Chk2 -/- cells.
G2
Time (hr)
0
2
4
8
22

II-70

Chk2 -/-

*P < 0.0071

***P < 0.0001
***P < 0.0001
**P < 0.0025
**P < 0.0012

G1
Time (hr)
0
2
4
8
22

II-70

**P < 0.0053

Chk2 -/***P < 0.0005
***P < 0.0001
***P < 0.0001
***P < 0.0001
***P < 0.0001

In response to treatment with IR, untreated HCT116 Chk2 +/+ cells demonstrated
a G1 arrest comparable to those cells treated with II-70 up to 8 hours following 1 Gy IR
(Figure III-13A-C). At 8 hours after IR, the cells treated with II-70 and IR started to
demonstrate a slightly higher percentage of cells in G 1 compared to the untreated
control. At 22 hours, there is a significant (Table III-4) increase in the amount of cells in
G1 when treated with II-70 and 1 Gy IR. However, inhibition of Chk2 by compound II-70
did not have a statistically significant effect on IR-induced change in the G1 arrest when
compared to the no-IR treated controls (Figure III-13E). The difference in G1 is less
noticeable and statistically insignificant following 2.5 and 5 Gy IR. It is unclear if the

145

increase in G1 population is due to the combination of IR and inhibition of Chk2 with II70. Chk2 +/+ cells that were treated with II-70 alone for 22 hours demonstrated a G1

A

B
% cells in G 1

50

0

0

1

2.5

100

1

2.5

IR dose (Gy)

% cells in G 1

E

100

2.5

5

*** p < 0.0001

50

0

5

1

IR dose (Gy)

100

*** p < 0.0001

0

0

D

50

0

*** p < 0.0001

50

0

5

IR dose (Gy)

C
% cells in G 1

100

*** p < 0.0005

% cells in G 1

% cells in G 1

100

0

1

2.5

5

IR dose (Gy)

p < 0.0001

***

** p < 0.0053
Without II-70
With 30 M II-70

50

Chk2 -/0

0

1
2.5
IR dose (Gy)

5

Figure III-13. DNA frequency histograms of HCT116 Chk2 +/+ or Chk2 -/- cells in G1.
Cells were either untreated (control) or pretreated for 2 hours with 1 (30 µM) followed by
IR (0-5 Gy). A) Cells at time 0. B) Cells 2 hours after IR. C) Cells 4 hours after IR. D)
Cells 8 hours after IR. E) Cells 22 hours after IR. Data for G1 were expressed as mean
± SEM from 3 independent experiments.
146

arrest without the induction of DNA damage. The Chk2 -/- cells had an increased
percentage of cells present in G1 compared to the Chk2 +/+ cells at all times following
no IR and with IR.
The cell cycle experiments indicate that inhibition of Chk2 allows the abrogation
of the G2 checkpoint, which could result in the prevention of replication of cancer cells.
This effect was also confirmed with the Chk2 -/- cells. Additionally, these experiments
also indicated that inhibition Chk2 does not have a significant effect in the arrest of G1
following IR.
III.E

Cellular evaluation of indoloazepine
With the knowledge that II-70 inhibited Chk2 at nanomolar levels in vitro, it was

necessary to determine if those results translated into cell culture. Before cellular
inhibition of Chk2 was examined, the affect of II-70 on cell proliferation and cytotoxicity
was determined in normal and tumor cell lines using a trypan blue exclusion assay of
cell viability. Cells were treated with vehicle (final DMSO concentration, < 0.1%) or II-70
(various concentrations) continuously for up to 60 hours. At each time point, cells were
trypsanized and counted with the diazo dye, trypan blue, which passes through the
membranes of dead cells. Figure III-7 shows the results of this assay in breast cell lines.
It is clear that II-70 is not toxic to these cell lines in the concentrations that were tested.

147

A

B

184B5 (normal, p53 wt)

MCF-7 (tumor, p53 wt)

C

M
1

0.

M
1

M
3

M

M

10

e
cl

30

d
te

Ve

nt
re
a
U

MDA-MB-231 (tumor, p53 mut)

% Cell Growth

hi

M
1

0.

M
1

M
3

10

30

hi
c

te

Ve

U

nt
re
a

M

0

M

50

le

50

d

100

0

% Cell Growth

100

100

0 hour
12 hours
24 hours

50

48 hours
60 hours
M
1

0.

M
1

M
3

M

10

30

M

e
hi
cl

Ve

U

nt
re
a

te

d

0

Figure III-14. Results of trypan blue exclusion test in breast cell lines. A) 184B5 cells,
normal, breast, p53 wt; B) MCF-7 cells, tumor, breast, p53 wt; C) MDA-MB-231 cells,
tumor, breast, p53 mutant.
In the normal breast cell line 184B5 (Figure III-14A), a cytostatic effect (or no
growth compared to untreated 12 hour control) was observed at the highest
concentration tested (30 µM), after 12 hours of continuous exposure. The cells grew
very slowly following 48 hours exposure, and II-70 became slightly cytotoxic following
60 hours of exposure. At 10 µM of II-70, a similar cytostatic effect was observed, but to
a lesser degree, and did not seem to be cytotoxic after 60 hours. Concentrations less

148

than 10 µM did not significantly hinder cell growth in the normal cell line. MCF-7 tumor
breast cells (p53 wt) displayed a cytostatic effect following continuous treatment with II70 (30 µM and 10 µM), and as with the normal cells, growth was not significantly
inhibited at concentrations below 10 µM (Figure III-14B). The MDA-MB-231 cells (tumor,
breast, p53 mutant) also experienced cytostatic growth with continuous exposure to II70 and slight cytotoxicity after 60 hours (Figure III-14C). From these data, it was
concluded that experiments can be performed in breast cell lines with continuous
exposure to II-70 for 48 hours without the cells being affected by cytotoxicity.
There was an interest in performing experiments in the HCT116 colon cell lines
due to the availability of Chk2 +/+, Chk2 -/-, p53 +/+, p53 +/- and p53 -/- phenotypes.
HCT116 colon cell lines were also evaluated with the trypan blue exclusion assay
(Figure III-15). If II-70 inhibits Chk2 in cell culture, these cell lines would theoretically
mimic the results seen in the Chk2 -/- cell line. All of these colon cell lines displayed
cytotoxicity after 24 hours when exposed to a high concentration (100 µM) of II-70.
Growth inhibition was observed at 30 µM concentrations after 12 hours. No significant
growth inhibition was observed at concentrations below 10 µM. The physiological
effects of II-70 (100 µM, 30 µM) on the cell lines following 24 hours of exposure is
illustrated in Figure III-16. An enlargement was observed in the cytosol in the cells
treated with II-70, as compared to untreated cells. In the HCT116 parent cells (p53 wt,
Chk2 p53), a cytostatic effect was observed at 24 and 48 hours and cytotoxic effect
after 60 hours of continuous exposure (Figure 15A). A cytostatic effect was also
observed with 10 µM treatment in the HCT116-40-16 (p53 wt) for up to 60 hours (Figure
III-15B).

149

A

HCT116 (p53 wt, Chk2 wt)

B

HCT116-40-16 (p53 +/+)

50

0

at
U

nt
re

at
nt
re
U

e
Ve d
hi
c
10 le
0
M
30
M
10
M
3
M
1
M

50

e
Ve d
hi
c
10 le
0
M
30
M
10
M
3
M
1
M

100

0

% Cell Growth

100

C

D

HCT116-386 (p53 +/-)

HCT116-379.2 (p53 -/-)

50

0

at
re
U
nt

at
re
U
nt

E

e
Ve d
hi
c
10 le
0
M
30
M
10
M
3
M
1
M

50

e
Ve d
hi
c
10 le
0
M
30
M
10
M
3
M
1
M

100

0

% Cell Growth

100

% Cell Growth

HCT116-67 (CHk2 -/-)

0 hour

100

12 hours
24 hours

50

48 hours
60 hours

U

nt

re

at

e
Ve d
hi
c
10 le
0
M
30
M
10
M
3
M
1
M

0

Figure III-15. Results of trypan blue exclusion test in colon cancer cell lines. A)
HCT116-parent cells (p53 wt, Chk2 wt); B) HCT116-40-16 cells (p53 +/+); C) HCT116386 cells (p53 +/-); D) HCT116-379.2 cells (p53 -/-); HCT116-67 cells (Chk2 -/-).

150

The cell lines in which p53 (Figure III-15C, HCT116-386 (p53 +/-); Figure III-15D,
HCT116-379.2 (p53 -/-)) or Chk2 (Figure III-15D, HCT116-67 (Chk2 -/-) were modified
and treated with 10 µM of II-70 did not grow during the first 24 hours, and demonstrated
slowed growth following that time. Therefore, additional experiments on the effect of
cells with II-70 will be limited to exposures that are not cytotoxic to cells.
HCT116

HCT116-40-16

HCT116-386

HCT116-379.2

HCT116-67

0 µM
II-70

30 µM
II-70

100 µM
II-70

Figure III-16. Microscopic cell images of HCT116 cell lines following treatment with II-70
for 24 hours of continuous exposure.
Indoloazepine II-70 was sent to the National Cancer Institute and screened in the
NCI-60 Developmental Therapeutics Program Human Tumor Cell line screen.

28

Cytotoxicity of particular tumor cell lines were evaluated using a sulforhodamide B
(SRB) colorimetric assay. SRB is an aminoxanthene dye with two sulfonic groups that
can bind to basic amino-acid residues under mild acid conditions, and dissociate under
basic conditions.

29

Human tumor cells are incubated with drug (II-70, 10 µM) for 48

hours at which point cells are fixed to the plate with trichloroacetic acid and stained with
SRB. Cells are washed, removing the excess dye. Protein-bound dyes is dissolved and
151

Table III-4. Results of NCI-60 cell line screen with II-70.
Panel/Cell Line

Growth
Percent

Panel/Cell Line

Leukemia
CCRF-CEM
83.50
HL-60 (TB)
82.28
K-562
92.42
MOLT-4
85.50
RPMI-8226
93.48
SR
51.05
Non-Small Lung Cancer
A549/ATCC
98.73
EKVX
15.24
HOP-62
118.10
HOP-92
119.60
NCI-H226
89.39
NCI-H23
103.46
NCI-H322M
87.93
NCI-H460
97.28
NCI-H522
44.82
Colon Cancer
COLO-205
89.02
HCC-2998
92.05
HCT-116
54.43
HCT-15
85.80
HT29
65.75
KM12
91.48
SW-620
106.59
CNS Cancer
SF-268
102.62
SF-295
106.10
SF-539
81.69
SNB-19
116.40
SNB-75
79.23
U251
102.46

Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer
786-0
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate Cancer
PC-3
DU-145
Breast Cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468

152

Growth
Percent
72.71
88.29
72.12
87.62
52.87
121.55
98.22
118.75
91.46
80.66
118.4
89.16
107.96
93.84
118.14
51.55
69.6
70.27
57.46
83.5
104.43
59.85
93.35
100.4
101.88
104.39
115.29
77.73
88.73
114.9
78.2

Optical density is determined with a microplate reader.
is directly proportional to the cell mass.

29

29

The amount of dye in the cells

The results of the NCI-60 cell line screen are

listed in Table III-3. Compound II-70 did not demonstrate any significant inhibition of
growth in the majority of the cell lines screened. The handful of cell lines which were
inhibited by II-70 did exhibit growth inhibition. The growth percentage was reported as
44.52% in a non-small lung cancer cell line, NCI-H522, the cell line most affected by II70.
III.F

Indoloazepine as a radioprotecting agent
Chk2 inhibition has been indicated to elicit radio- or chemoprotection of normal

tissue via inhibition of p53-dependent apoptosis.

11, 12

Therefore, we wanted to perform

a clonogenic survival assay to determine if pretreatment of normal breast cells (184B5
cells, p53 wt) with Chk2 inhibitor II-70 (30 µM) was capable of protecting only the
normal cells from the cytotoxic effects of various doses of IR (0-7.5Gy). Conversely, we
hypothesized that the inhibition of Chk2 would have an insignificant effect on cell
survival in p53 mutant cancer cells (MDA-MB-231 cells), compared to the vehicle
control.
In order to evaluate this hypothesis, 184B5 and MDA-MB-231 cells were
pretreated with nothing, vehicle (DSMO), or 1 (30 µM) for 30 minutes. The cells were
irradiated in suspension (0-7.5 Gy) and seeded at cloning density. Cells were allowed to
incubate for 7 days at which point fresh media was given to the clones. After 10-14
days, clones were fixed and stained with crystal violet. Surviving clones were scored (ie,
colonies containing > 50 cells) and viability of the cells was determined (Figure III-17).

153

0 gy
50 cells
50 cells

1 gy
70 cells
70 cells

2.5 gy
400 cells
375 cells

5 gy
1000 cells
960 cells

7.5 gy
9000 cells
5000 cells

No drug

30 µM
II-70

Figure III-17. Clones of184B5 (normal) cells untreated (top panel) or pretreated with II70 for 30 minutes (bottom panel) followed by IR (0-7.5 Gy) and seeded at the indicated
cloning density.
The results of the clonogenic assay revealed that the control treatment (IR and
vehicle only) resulted in a decrease in the number of viable cells with increasing doses
of IR (Figure 18a) as anticipated. However, treatment with Chk2 inhibitor II-70 protected
cells from IR-induced cell death in the normal cell line (p53 wt), as compared to the
vehicle control treatments with nearly 75% survival at doses up to 5 Gy IR, or compared
to the untreated control. The radio-protective effect was also evaluated at
concentrations below 30 µM (Figure III-18B). Decreased cell death was observed 10
and 3 µM II-70, with less radioprotection at the lower dose. A radioprotective effect was
not demonstrated with 1 µM at IR doses of 1-5 Gy. However, a very slight effect was
observed at 7.5 Gy.

154

Surviving fraction

A

B
100

100

10

10

1
0

2

4

6

8

1
0

Dose IR (Gy)

Untreated
Vehicle (DMSO)
II-70 (30 M)
II-70 (10 M)
II-70 (3 M)
II-70 (1 M)
2

4
6
Dose IR (Gy)

8

Figure III-18. Cloning survival of 184B5 (normal) cells untreated or pretreated with II-70
for 30 minutes followed by IR (0-7.5 Gy). A) Cells treated with 30 µM II-70 followed by
IR (0-7.5 Gy); B) Cells treated with 10, 3, or 1 µM II-70 followed by IR (0-7.5 Gy).
A closer look at the colonies illustrates the physiological differences in the normal
cells treated with II-70 and those that remained untreated prior to IR (Figure III-19). The
morphology of the normal cells that were treated with II-70 had an enlarged cytoplasm,
making the cell appear larger than those that were exposed to IR alone. This was also
observed above in the HCT116 cells that were not exposed to IR (Figure III-19).
However, those cells were continuously treated with II-70 for a period of 24 hours. The
normal cells (184B5) were treated for only 30 minutes before IR, and still demonstrated
a slight, but noticeable change in morphology. The exact reason why II-70 causes this
expansion in cell cytoplasm is unclear.

155

0 Gy

10 Gy

0 µM II-70

30 µM II-70

Figure III-19. Microscopic images (20X) of normal cell colonies (184B5) treated with II70 followed by 0 or 10 Gy IR.
With just a 30 minute pretreatment of II-70, a decrease in cell death was
observed. There was a question of whether modifying the order of treatment and
modifying the length of time would change the radioprotective effect. Figure III-20A
shows the cloning survival of normal breast cells treated with II-70 (30 µM) 30 minutes
after IR. Although there was radioprotective effect observed with this treatment, the
percentage of survival was less with a 30 minute post-treatment (69.2% at 2.5 Gy,
24.5% at 5 Gy) than with a 30 minute pretreatment (72.8% at 2.5 Gy, 43.0% at 5 Gy) as
seen in Figure III-20A. II-70 is slightly less effective when treated after IR.
Normal cells were then pretreated with vehicle or II-70 (30 µM) for 2 hours
followed by IR and post-treated for 4 hours (Figure III-20B). These cells were exposed
to the Chk2 inhibitor II-70 for a total of 6 hours. It was anticipated that increased
exposure time to II-70 would strengthen the radioprotection. However, the decrease in
cell death (67.9% at 2.5 Gy, 43.1% at 5 Gy) was very similar to that of cells pretreated
156

for only 30 minute indicating that increased exposure time before and after IR did not

surviving fraction

further radioprotect normal cells as compared to the 30 minute pretreatment.

100

100

10

10

1

0

2

Untreated

4
6
Dose IR (Gy)

8

1

0

Vehicle (DMSO)

II-70 (30 M) 2 hr pre-, 4 hr post-treat

2

4
6
Dose IR (Gy)

8

II-70 (30 M) 0.5 hr pre-treat
II-70 (30 M) 0.5 hr post-treat

Figure III-20. Cloning survival of 184B5 normal cells with various treatment times. A)
Cells post-treated 30 minutes following IR (0-7.5 Gy); B) Cells pretreated for 2 hours
and post-treated for 4 hours after IR (0-7.5 Gy).
As a negative control, analog II-99 was evaluated to make sure that a compound
lacking in vitro Chk2 inhibition activity could not enhance survival following radiation.
The negative control, inactive Chk2 inhibitor analog II-99 did not exhibit any
radioprotective effects in the IR challenged 184B5 cells (Figure III-21A). This data
strongly indicates that the enhanced survival seen in this cell line is due to the
abrogation of Chk2 activity by Chk2 inhibitor II-70.

157

Surviving fraction

A

B
100

100

10

10

1
0

Untreated
Vehicle (DMSO)
II-99 (30 M)
2

4
6
Dose IR (Gy)

8

1
0

Untreated
Vehicle (DMSO)
II-70 (30 M)
2

4
6
Dose IR (Gy)

8

Figure III-21. Cloning survival of 184B5 and MDA-MB-231 cells. A) 184B5 cells
pretreated with II-99 (30 µM) for 30 minutes, followed by various doses of IR (0-7.5 Gy);
B) MDA-MB-231 cells pretreated with II-70 (30 µM) for 30 minutes, followed by various
doses of IR (0-7.5 Gy).

Importantly, pretreatment of the p53 mutant MDA-MB-231 cells for 30 minutes

with nothing, vehicle (DMSO) or II-70 (30 µM) followed by the same doses of IR (0-7.5
Gy) did not show a decrease in cell death in the presence of Chk2 inhibitor II-70 (Figure
III-22). Figure III-21B clearly indicates that there was no detectable induction of cell
survival as the untreated, vehicle and treated cells showed statistically identical survival
curves. These results in the two cell lines implicate the p53 pathway in the survival
mechanism of Chk2 inhibitor II-70, in cells treated with IR.

158

0 gy
200 cells

1 gy
400 cells

2.5 gy
1,000 cells

5 gy
4,000 cells

7.5 gy
12,000 cells

No drug

30 µM
II-70

Figure III-22. Clones of MDA-MB-231 (tumor, p53 mutant) cells untreated (top panel) or
pretreated with II-70 for 30 minutes (bottom panel) followed by IR (0-7.5 Gy) and
seeded at the indicated cloning density.
The morphological differences in the tumor cell line, MDA-MB-231 are more
obvious in the cells receiving both 30 minute pretreatment of II-70 and IR (Figure III-23).
These cells are grossly enlarged as is illustrated in the lower right panel. There are
approximately 5-6 cells in this window and each cell’s nucleus and cytoplasm are larger
than cells that did not receive the pretreatment of II-70. The cell morphology was
affected here, however, survival remained unaffected.

159

0 Gy

10 Gy

0 µM II-70

30 µM II-70

Figure III-23. Microscopic images (20X) of tumor cell colonies (MDA-MB-231) treated
with II-70 followed by 0 or 10 Gy IR.
The HCT116 tumor colon cell lines were also evaluated using the clonogenic
survival assay (Figure III-24). The HCT116 parent cell line demonstrated a very slight
decrease in cell death when pretreated with II-70 for 30 minutes (Figure III-24A). The
radioprotection in the tumor cell line was less than the protection observed in normal
cells (Figure III-18A). The p53 wild type (Figure III-24B), p53 +/- (Figure III-24C), and
p53 -/- (Figure 24D) had no detectable radioprotection when treated with the Chk2
inhibitor II-70. Interestingly, the two untreated p53 mutant cell lines (Figure III-24C,D)
had survival curves that were slightly higher than those seen for the p53 wild type cell
lines (Figure III-24,B). These results may further support the p53 pathway in the survival
mechanism in cells treated with IR.

160

A

surviving fraction

HCT116 (Chk2 +/+, p53 +/+)

2

4
6
Dose IR (Gy)

1
0

8
D

HCT116-386 (p53 +/-)
100

2

4
6
Dose IR (Gy)

8

HCT116-379.2 (p53 -/-)
100

10

1
0

Untreated
Vehicle (DMSO)
II-70 (30 M)

10

10

1
0

HCT116-40-16 (p53 +/+)
100

100

C

surviving fraction

B

Untreated
Vehicle (DMSO)
II-70 (30 M)

10

2

4
6
Dose IR (Gy)

8

1
0

2

4
6
Dose IR (Gy)

8

Figure III-24. Cloning survival of HCT116 cell lines. A) HCT116 (Chk2 +/+, p53 +/+)
pretreated for 30 minutes with II-70 (30 µM) followed by various doses of IR (0-7.5 Gy);
B) HCT116-40-16 (p53 +/+) pretreated for 30 minutes with II-70 (30 µM) followed by
various doses of IR (0-7.5 Gy); C) HCT116-386 (p53 +/-) pretreated for 30 minutes with
II-70 (30 µM) followed by various doses of IR (0-7.5 Gy); D) HCT116-379.2 (p53 -/-)
pretreated for 30 minutes with II-70 (30 µM) followed by various doses of IR (0-7.5 Gy).
To further understand the role of Chk2 inhibition in cell survival, the cloning
survival assay was performed in the Chk2 -/- cell line and compared to the HCT116
parent cell line (Figure III-25). There was a slight increase in survival in HCT116 cells
treated with II-70 (44.7% at 2.5 Gy, 8.0% at 5 Gy) as compared to the untreated
HCT116 cells (30.9% at 2.5 Gy, 6.1% at 5 Gy). It was anticipated that the Chk2 -/- cell
line would show a greater protective effect or one similar to that observed in the normal
cell line treated with II-70 (Figure III-18A). However, the Chk2 -/- demonstrated a very
161

little radioprotection when compared to Chk2 +/+ cells treated with II-70 (Figure III-24A).
It is not clear why removing Chk2 completely from the cell line did not protect the cells
from IR. The tumor status of the colon cells versus the normal cell status of the breast
cells may contribute to these results. Additionally, another explanation could be due to
the redundant functions of the checkpoint present in normal cells that could mask any
checkpoint defects. Increases in cell apoptosis immediately following IR have been
observed between the Chk2 +/+ cell line versus the Chk2 -/-.

30, 31

Cloning survival has

been increased in Chk2 siRNA studies as compared to Chk2 wild type cell lines
following IR-induced DNA damage,

30

however these differences have not yet been

demonstrated in the HCT116 Chk2 -/- cell line. Additionally, this protective effect in
cloning survival has also been observed in Chk2 -/- mouse embryonic fibroblasts
(MEFs).

31

However, the cloning assay performed here with the Chk2 +/+ and Chk2 -/-

cell lines have not indicated differences in long term survival as observed in the
31

surviving fraction

MEFs.

100

Untreated (Chk2 +/+)
II-70 (30 M) (Chk2 +/+)
Chk2 -/-

10

1

0

2

4
6
Dose IR (Gy)

8

Figure III-25. Comparison of cloning survival of HCT116 treated with II-70 versus
HCT116 Chk2 -/- cells following IR.

162

The clonogenic survival assays in the normal and tumor cell lines demonstrated
that inhibition of Chk2 in p53 mutant cells did not have any cellular effect on cell
survival, whereas Chk2 inhibition by II-70 increased cell survival following IR-induced
DNA damage in the normal breast p53 wt cell line. The graphs in Figure III-18A clearly
indicate the difference in IR protection by II-70 in the normal p53 wt cells over the p53
mutant cells (Figure III-21b).
III.G

In vivo evaluation of indoloazepine
With cloning survival demonstrating the radioprotective effect of II-70, and

western blot data supporting cellular inhibition of Chk2, we wanted to evaluate the
protective effect of II-70 in a mouse model. We were interested in mimicking the results
of the Chk2 -/- mice exposed to whole body IR.

32

The hypothesis was that treatment of

Balb C mice with Chk2 inhibitor II-70 30 minutes prior to whole body IR would allow
mice to survive longer than those mice not receiving the inhibitor. Mouse handling and
studies were performed by Dr. Sandra O’Reilly of Osteopathic Medicine and Research
with my assistance.
In order to evaluate the mice exposed to IR and II-70, it was important to monitor
the mice for acute radiation syndrome (ARS). There are three syndromes associated
with ARS: the central nervous system (neurovascular) syndrome (CNS), the
gastrointestinal syndrome (GI) and the hematopoietic syndrome (BM).

33

These

syndromes can occur after whole body or partial body IR. The effects may appear within
minutes of IR exposure, or may present symptoms years after exposure, depending on
the type, dose and dose-rate of the irradiation.

33

Cells that are proliferating are sensitive

to the effects of whole body IR. These effects are mainly associated with changes in the
163

germinal epithelium, gastrointestinal epithelium and the bone marrow progenitor cells.
The gastrointestinal epithelium and the bone marrow progenitor cells are pivotal for the
maintenance of life.

33

Damage to either of these cell types can have an adverse effect

on survival due to the drastic disruption of normal physiological mechanisms.

33

Although the bone marrow progenitor cells are more sensitive to the damaging effects
of whole body IR compared to the gastrointestinal epithelium, the effects on the GI
system are expressed more rapidly compared to the effects on the hematopoietic
system due to the rapid transit time of the cells in the GI tract.

33

In mice receiving whole

body IR the lethal (death) effects are seen within 10 days post-irradiation for the
gastrointestinal system whereas changes in the hematopoietic system are seen
between days 11 to 30 post-irradiation.

33

Before subjecting mice to whole body IR, it was necessary to first determine if II70 was observed in the blood stream. Mice were initially given II-70 dissolved in 30%
propylene glycol/water by oral gavage. Blood from the mice was collected at various
time points. The serum from the mice was then analyzed by Liquid Chromatography
Mass Spectrometry (LC/MS). It was concluded that II-70 was not orally available in
mice.
Mice were then given an intraperitoneal (IP) injection of varying doses (100, 10, 1
mg/kg) of II-70 dissolved in a 33% propylene glycol/water solution. Males and females
were treated with II-70 (100 mg/kg, Figure III-26A,B) or vehicle (data not shown, II-70
was not observed in vehicle mice) to determine if there was a difference in absorption
between the two sexes. Figure III-26A illustrates that there is not a statistical
significance in the amount of II-70 that is absorbed in the blood. There was
164

approximately 8 mg/kg, or ~800 µM of II-70 remaining in the blood 30 minutes after the
injection, and was still detected 12 hours after injection (Figure III-26A,B). Because the
differences in absorption in the blood were not significant, there was no preference to
which group (male or female) would undergo IR treatment. Mice that were injected IP
with 10 mg/kg had ~ 80 µM of II-70, (Figure III-26C) present 30 minutes after injection.
Mice injected with 1 mg/kg had ~8 µM of II-70 remaining in the blood after 30 minutes
(Figure III-26D). Indoloazepine II-70 was not present at detectable levels past 4 and 2
hours when injected with 10 mg/kg and 1 mg/kg doses, respectively. However, the
concentrations of Chk2 inhibitor II-70 present 30 minutes after the injection time well
above the Chk2 in vitro IC50 (10 nM).

165

12

1.2

0.

0.

Time (hr)

D
~ 80 M II-70

0.12

Time (hr)

12

6

4

2

5
0.

0.

12

0.00
6

0.0
4

0.04

2

0.4

1

0.08

5

0.8

~ 8 M II-70

1

[II-70] (mg/kg)

Time (hr)

12

0
6

4

4

4

5

8

5
1
2
4
6
12
0.
5
1
2
4
6
12

8

0

C

~ 800 M II-70

Female

2

[II-70] (mg/kg)

B

Male

12

1

A

Time (hr)

Figure III-26. LC/MS quantification of biologically available II-70 in mice. n = 2 for each
time point. A) Male and female mice injected IP with 100 mg/kg II-70; B) Females
injected IP with 100 mg/kg II-70; C) Female mice injected with 10 mg/kg II-70; D)
Female mice injected with 1 mg/kg II-70.
Following the bioavailability experiment, a toxicity study with the mice was
performed next to evaluate the long term affects of mice treated with II-70 (Figure III27). Mice were treated with various concentrations of II-70 and were monitored over 30
days. At the highest concentration (100 mg/kg), two mice died within the first two days
of the study, this may have been in part due to an injection to a vital organ.
Nevertheless, the majority of the mice survived out to 30 days, in addition to the mice
treated with 50, 10 and 1 mg/kg. There were no cytotoxic effects observed the mice

166

treated with II-70 as compared to those treated with vehicle only (30% propylene
glycol/water).

Survival of Mice treated with II-70 (IP)

survival (%)

100

Vehicle (propylene glycol)
100 mg/kg (n = 5)
50 mg/kg (n = 5)
10 mg/kg (n = 5)
1 mg/kg (n = 5)

75
50
25
0

0

10
20
30
Time after treatment (days)

Figure III-27. Cytotoxicity of mice treated with varying doses of II-70 by IP.
The bioavailability data and cytotoxicity studies encouraged us to move forward
with mouse whole body irradiations in order to investigate the protective effect of II-70.
The radioprotective effect of II-70 was determined not by using death as an end point,
but instead evaluation of how well II-70 alleviated symptoms associated with the GI and
BM syndromes. The animals were monitored for physical changes that would indicate
changes in the GI and BM syndromes including: ruffled fur coat, loss of body weight,
labored breathing, lethargy, lack of curiosity, dehydration, evidence of diarrhea,
hunched posture, closed eyes, or discharge around eyes, change in color of tails, and
ears or any obvious hematomas. Mice showing several or all severe symptoms were
sacrificed and removed from the study. The spleens and intestines of mice dosed with
II-70 and those receiving IR only were compared following necropsies of the mice.

167

Initially, mice aged 6-8 weeks, were pretreated with II-70, then given avertin
(2,2,2-tribromoethanol) an anesthetic drug, before being transferred in an irradiation
chamber, to the veterinary oncology clinic at MSU. Irradiations were performed by Dr.
Elizabeth McNeil, of the College of Veterinary Medicine. The mice were then irradiated
using a 6 MV linear accelerator between a 1.5 cm sheet of bolus material. Mice were
returned to a pathogen free room before they awoke from the anesthetic. In some
experiments, the avertin did not successfully anesthetize the mice. In others, the mice
were already deceased upon returning from IR, or died shortly after IR. It was later
discovered that avertin can degrade in the presence of heat, light or changes in pH,
leading to degradation products that can be nephrotoxic and hepatotoxic.

34

In order to

avoid these unnecessary and sometimes lethal side effects, a new chamber was
designed that was made with polystyrene which could be autoclaved. This new
chamber did not require the use of anesthesia (Figure III-28). The lid of the chamber
was capable of rotating and also contained a sliding wedge in which the mice could be
put in and taken out of each of the sections of the chamber.

168

Figure III-28. Mouse irradiation chamber used for whole body IR.
Although Motoyama et al used a sub lethal irradiation dose of 8 Gy,

32

this dose

was too toxic to see any improvement in survival of the mice when we performed the
experiment with the same dosage. The mice suffered from the effects of damage to the
BM and GI systems before any radioprotection could be observed (~10 days). The
intestines of mice treated with II-70 were yellow in appearance instead of the normal
pale green/whitish color. Some of the pretreated mice intestines were observed to be
empty, indicating a depletion of the epithelial layer and damage to tissue that was not
replenished by the stem cells.
A slightly lower dose of 7.3 Gy allowed the IR-only mice to survive for more than
15 days, past the initial stages of GI syndrome effects (Figure III-29). Mice that were
pretreated for 30 minutes with 10 mg/kg II-70 were observed to starting at 8 days, likely
from the beginning stages of GI syndrome. At day 13, ~50% of the II-70-IR mice and
about 80% of the vehicle-IR mice were still alive, indicating that II-70 was having a
negative effect on the survival of the mice, contrary to the radioprotective effect seen in
169

the cloning assay with normal cells. Statistical analysis was performed on the vehicle
mice as compared to the II-70 mice and there was no significant difference in the two
curves (Table

A

B
Percent survival

Percent survival

100
75
50
25
0

0

5

10
15
20
Days
Vehicle Data (n = 25)
10 mg/kg II-70 (n = 10)

100
75
50
25
0

0

5

10
15
20
Days
Vehicle Data (n = 25)
1 mg/kg II-70 (n = 8)
0.1mg/kg II-70 (n = 8)

Figure III-29. Effect of pre-treatment with II-70 (10, 1 or 0.1mg/kg) for 30 minutes prior
to whole body IR with 7.3 Gy on survival of male Balb-C mice.
Mice that received lower concentrations of 1 mg/kg or 0.1 mg/kg saw an increase
in survival between days 13-17 (Figure III-29) showing some protection or delay in the
effects of the GI syndrome. These slight increases in mouse survival between days 1317 were not statistically significantly different from the vehicle treated mice (Table III-5).
By day 20, it is likely that both the GI and BM syndromes have affected the II-70 treated
and vehicle treated mice. Necropsies of sacrificed mice treated with II-70 revealed the
same yellowed intestines that were observed in the mice irradiated with 8.3 Gy IR.

170

Table III-5. Statistically analysis of vehicle compared to II-70 whole body irradiated
Balb-C mice.
Comparison of survival curves

Vehicle vs 10
mg/kg II-70

Vehicle vs 1.0
mg/kg II-70

Vehicle vs 0.1
mg/kg II-70

2.872
1
0.0901
ns
No

1.475
1
0.2245
ns
No

1.466
1
0.226
ns
No

1.565
1
0.211
ns
No

2.722
1
0.099
ns
No

3.198
1
0.0737
ns
No

Log-rank (Mantel-Cox) Test
Chi square
df
P value
P value summary
Significant?
Gehan-Breslow-Wilcoxon Test
Chi square
df
P value
P value summary
Significant?

These results were puzzling, to say the least. It was anticipated that inhibiting
Chk2 with II-70 would increase the survival of the mice following IR. However, in some
cases (10 mg/kg, Figure III-29A), treatment with II-70 sensitized the mice to IR. II-70 is
also an inhibitor of Chk1 (IC50 = 220 nM), as well as NF-кB (IC50 = 10 µM, Chapter IV).
Inhibiting either of these proteins in vitro can result in sensitization of cells to DNA
damage, leading to cell death. With a treatment of 10 mg/kg II-70, there is
approximately 80 µM, which is well above the IC50 values. It is possible that at this dose
in the mice, II-70 is inhibiting Chk1 and/or NF-кB and therefore sensitizing mice to cell
death following IR-induced DNA damage.
The lower doses (1 mg/kg, 0.1 mg/kg, Figure III-29B) did not completely increase
survival of mice over the 20 day period. The protective effect was observed between
days 13-17 (Figure III-29B), however, it is unclear why all of the mice in this study died
171

at the same time as the vehicle mice. It is possible that the mice treated with II-70
started to suffer from side effects of whole body irradiation.
Although these results are promising, it is important to note that the whole body
irradiation experiments with mice and II-70 were not optimized, and that many more
experiments need to be performed. These include optimal pretreatment time for dosage,
concentration administered, multiple administrations of drug, IR dose rate, or multiple
low IR doses over a period of time, etc.
III.H

Inhibition of Chk2 as a strategy for chemosensitization
The radiosensitization results of the mouse irradiation (Figure III-30A) prompted

an investigation of using Chk2 inhibition as a means for sensitizing cells to DNA
damage. Many human tumors have increased levels of activated Chk2.

35

It is

conceivable that the human tumor cells with increased levels of Chk2 have become
dependent on Chk2 in order to survive.

35

Consequently, inhibition of Chk2 in these cells

would result in cell death. Jobson et al demonstrated a sensitization to a
chemotherapeutic (topotecan) in colon and ovarian cancer cell lines having increased
levels of Chk2 when treated with PV1019 (Chapter I, Figure I-13).
also able to inhibit growth in the Chk2-overexpressing cells.

35

PV1019 alone was

35

Chk2 inhibitor II-70 was evaluated in one of the ovarian cancer cell lines with
increased levels of Chk2 to determine if it was cytotoxic to these cells alone, or if it had
a synergistic effect with the cytotoxic agent topotecan. The trypan blue exclusion assay
was first performed in the ovarian cancer cell line, OVCAR-3, with II-70 (Figure III-30). It
is evident from the cell counts that after 24 hours of continuous exposure, the cells

172

treated with 30 µM of II-70 begin to display cytotoxic effects (Figure III-30). Cytotoxicity
at 10 µM is also observed following 72 hours of exposure.

% Cell Growth

100

0 hour
24 hours
48 hours
72 hours

50

M
1
0.

M
1

M
3

M
10

M
30

SO
M

D

U
nt

re
a

te

d

0

Figure III-30. Trypan blue exclusion assay of OVCAR-3 cells treated with II-70.
OVCAR-3 cells treated with II-70 only. B) OVCAR-3 cells treated in combination with 0.5
µM Topotecan.
Topotecan alone demonstrates a cytotoxic effect at 48 and 72 hours of
continuous exposure (Figure III-31). Compound II-70 (30 µM) seems to be slightly more
toxic alone than topotecan (0.5 µM). However, when topotecan is combined with II-70
for 48 hours, II-70 increases the cell cytotoxicity compared to topotecan alone (Figure
III-31). The combination of topotecan and II-70 in OVCAR-3 cells also demonstrates cell
death at doses as low as 0.1 µM. These data provide evidence as to the capabilities of
Chk2 inhibitor II-70 for sensitization of human tumor cells with increased levels of
activated Chk2. This increased cell cytotoxicity was only evaluated in one ovarian
cancer cell line with increased expression of Chk2 and would of course need to be
173

repeated in other cell lines with similar Chk2 properties in order to support the use of II70 as a sensitization drug for human tumor cells.

0.5 M Topotecan and II-70

100

0 hour
24 hours
48 hours
72 hours

50

M

0.

1

M
1

M
3

te
ca
n
30
M
10
M

M

po

To

30

SO
M

D

0.

5

M

U

nt

re
a

te

d

0

Figure III-31. Trypan blue exclusion assay of OVCAR-3 cells treated with II-70 in
combination with 0.5 µM Topotecan.
III.I

Conclusions
The inhibition of Chk2 by II-70 has been demonstrated at nanomolar levels in a

radioactive

filter-based

kinase

assay.

Additionally,

II-70

inhibited

cis-

autophosphorylation at Ser516 of Chk2 in vitro demonstrating direct cellular inhibition of
Chk2. Chk2 inhibitor II-70 also demonstrated inhibition of G2 arrest following treatment
with IR and HCT116 Chk2 +/+ cells, which was confirmed with Chk2 -/- cells. Inhibition
of Chk2 did not have an effect on IR-induced arrest of G1. Modifications of the
glycocyamidine moiety in compounds II-99 and II-100 indicated the potential importance
174

of the free amine for inhibition of Chk2, which corresponds to the previously reported
binding motive of the structurally related natural product debromohymenialdisine.
Indoloazepine II-70 was found to increase survival in normal cells exposed to IR,
however tumor cells having mutated p53 had no noticeable enhancements in survival.
II-70 was also able to sensitize human ovarian cancer cells having overexpression of
Chk2 to cell death induced by topotecan.
Chk2 inhibitor II-70 has demonstrated its potential as an adjuvant drug for cancer
therapy. However, several questions have yet to be probed concerning the mechanism
of inhibition of II-70. II-70 also inhibits other kinases at nanomolar concentrations which
have possibly affected its ability to protect the mice from the toxic effects of IR. It will
become important to determine the preferred mode of inhibition and possibly use that
information to create more effective inhibitors for radio-protection.
III.J

General experimental information and procedures
All cell lines were cultured at 37°C with 5% bone dry CO 2 atmosphere, 97%

humidity and ambient O2 in a Napco water-jacketed incubator. Adherent cells were
routinely passaged by 0.25% trypsin-EDTA (Invitrogen, Fredrick, MD) using sterile
technique and materials in a Nuaire biological safety cabinet Class II type A2. For all
lysate and whole cell experiments, the inhibitors were dissolved in DMSO and the final
DMSO concentration of the experiments was maintained at ≤ 0.1% in the reaction buffer
or culture medium. Cells were observed using a 1200CMI series spectrum inverted
microscope. Cells were counted with a hemacytometer or using the Beckman Coulter
Z1 Coulter® Particle Counter. Centrifugation was performed using the Beckman Coulter
Allegra X-15R Centrifuge or Spectrafuge 16M microcentrifuge. Buffers, solutions and
175

media were made with water purified with the Millipore Quantum® Ex filtration system.
Buffers requiring pH adjustments were monitored with a Mettler Toledo SevenEasy pH
e

meter. Protein assays were read using the Molecular Devices SpectraMax M5 plate
reader. Western blots were run on hand-poured SDS PAGE gels using the BioRad mini
protean pouring and electrophoresis system with the BioRad PowerPac HC. Gels were
transferred to polyvinylidene fluoride membrane (BioRad) with the BioRad Criterion
blotter wet transfer system. Blots and gels were quantified using the BioRad Molecular
Imager FX. Luminescence in the luciferase assays were monitored and quantified using
the Turner Biosystems Veritas Microplate Luminometer. Cells in the cloning survival
assay and western blots were irradiated with a

60

Co source providing 1.93 Gy/min. Cells

used in the cell cycle analysis were irradiated using a 6 MV linear accelerator (Clinac
2100 C, Varian Inc) at a dose rate of 300 cGy/minute. The quantity of incorporated [γ33

P]-ATP on the P30 filter mat was detected using a Wallac 1450 Microbeta Plus Liquid

Scintillation counter. Cell cycle analysis was performed using a Vantage TurboSort SE
cytometer (Becton Dickinson, San Jose, CA).
Cell Culture Methods. The 184B5 and MDA-MB-231 cell lines were cultured in
Dulbecco’s Modified Essential Medium (DMEM) supplemented with 5% heat inactivated
bovine calf serum with supplemented with 100 U/ml penicillin, 100 µg/mL streptomycin,
1 mM sodium pyruvate, 0.2 mM L-glutamine, and 100 mM MEM non-essential amino
acids. HCT116 cells were cultured in McCoy’s 5A with 5% bovine calf serum and
supplemented with 100 U/ml penicillin, 100 µg/mL streptomycin. The HCT116 cell lines
were a generous gift from Bert Vogelstein (Howard Hughes Medical Institute, Chevy
Chase, MD). OVCAR-3 cells were cultured in RPMI-8226 media supplemented with 5%
176

heat inactivated bovine calf serum with 100 U/ml penicillin, 100 µg/mL streptomycin, 1
mM sodium pyruvate, and 0.2 mM L-glutamine. For all lysate and whole cell
experiments, the inhibitors were dissolved in DMSO and the final DMSO concentration
of the experiments was maintained at ≤ 0.1% in the reaction buffer or culture medium.
Cells were cultured at 37° in a 5% CO2 incubator. Cells in the cloning survival assay
and western blots were irradiated with a

60

Co source providing 1.93 Gy/min. Cells used

in the cell cycle analysis were irradiated using a 6 MV linear accelerator (Clinac 2100 C,
33

Varian Inc) at a dose rate of 300 cGy/minute. The quantity of incorporated [γ- P]-ATP
on the P30 filter mat was detected using a Wallac 1450 Microbeta Plus Liquid
Scintillation counter. Cell cycle analysis was performed using a FACS-Calibur-Vantage
cytometer (Becton Dickinson, San Jose, CA).
In vitro kinase assay. In a final reaction volume of 25 µL, Chk1 (h) or Chk2 (h)
(Millipore) (5-10 mU) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 100 µM
CHKtide (KKKVSRSGLYRSPSMPENLNRPR) (Millipore), [γ-33P-ATP] (Perkin Elmer)
(approximately 1 µCi diluted in 25 mM MgAcetate and 0.25 mM ATP) and inhibitor. The
reaction was initiated by the addition of the MgATP mix. After incubation for 10 minutes
at 30°C, the reaction was stopped by addition of 5 µL of a 3% phosphoric acid solution.
10 µL of the reaction was spotted onto a p30 filtermat and washed three times for 5
minutes in 75 mM phosphoric acid and once in methanol for 2 minutes before the
addition of scintillation cocktail. The filtermat was read in a scintillation counter.

177

Table III-6. Log values, standard error of log values and IC50 values.
Compound

Chk1 Log
a
IC50

Chk1 Std.
Error Log
b
IC50

Chk1 Std.
c
IC50 (µM)

Chk2 Log
d
IC50

Chk2 Std.
Error Log
e
IC50

Chk2 Std.
f
IC50 (µM)

II-70

-0.6594

0.1412

0.2204

-1.893

0.06599

0.0135

II-84

NA

NA

> 1,000

NA

NA

> 1,000

II-99

NA

NA

> 1,000

NA

NA

> 1,000

III-1

-0.2540

0.08176

0.5814

-1.194

0.08455

0.06589

III-2

-0.2024

0.09818

0.6268

-1.347

0.08789

0.05393

a

33

Log EC50 values for inhibition of phosphorylation of purified active Chk1 by [γ- P]b
ATP. Standard error of log EC50 values inhibition of phosphorylation of purified active
33
c
Chk1 by [γ- P]-ATP. EC50 values calculated from the log EC50 values for of
33
d
phosphorylation of purified active Chk1 by [γ- P]-ATP. Log EC50 values for inhibition
33
e
of phosphorylation of purified active Chk1 by [γ- P]-ATP. Standard error of log EC50
33
f
values inhibition of phosphorylation of purified active Chk1 by [γ- P]-ATP. EC50 values
calculated from the log EC50 values for of phosphorylation of purified active Chk1 by [γ33
P]-ATP. NA: Not available.

178

Table III-7. Mean and standard deviation of IC50 values of Chk1 and Chk2.

Compound

II-70

II-84

II-99

III-1

III-2

Chk1
Experiment #
1
2
3
Mean
Std Dev
1
2
3
Mean
Std Dev
1
2
3
Mean
Std Dev
1
2
3
Mean
Std Dev
1
2
3
Mean
Std Dev

Chk1 EC50
(nM)
354.9
83.8
222.6
220.43
135.56
> 10,000
> 10,000
> 10,000
>10,000
> 10,000
> 10,000
> 10,000
>10,000
358.1
629.7
756.4
581.40
203.50
819.5
434.4
626.5
626.80
192.55

Chk2
Experiment #
1
2
3
Mean
Std Dev
1
2
3
Mean
Std Dev
1
2
3
Mean
Std Dev
1
2
3
Mean
Std Dev
1
2
3
Mean
Std Dev

Chk2 EC50
(nM)
21.3
7.7
11.6
13.53
7.00
> 10,000
> 10,000
> 10,000
>10,000
> 10,000
> 10,000
> 10,000
>10,000
100.5
27.07
70.1
65.89
36.90
79.2
15.7
66.9
53.93
33.68

Kinase Profile Assay. The Kinase profile was performed by the Millipore Kinase
ProfilingServices (Millipore, Billerica, MA). This technology is a radioisotope-based P30
filter-binding assay. 1 was dissolved in pure DMSO to make a 10 mM stock. 1 was then
diluted in pure DMSO to make serial dilutions based on the IC50 ranges. The purified
enzyme (e.g., Chk2) was diluted with 20 mM MOPS/NaOH pH7.0, 1 mM EDTA, 0.01%
Triton-X-100, 5% glycerol, 0.1% 2-mercaptoethanol, 1 mg/ml BSA and incubated with 8
179

mM MOPS pH 7.0, 0.2 mM EDTA, substrate [e.g., Chktide (100 µM in H20)
33

(KKKVSRSGLYRSPSMPENLNRPR)] and 10 mM MgAcetate and [γ- P-ATP] (specific
activity approx. 500 cpm/pmol, concentration as required). The reaction was initiated by
the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the
reaction was stopped by the addition of 3% phosphoric acid solution. 10 µL of the
reaction was 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.
5

Trypan blue exclusion assay: Cells were seeded at 2.5 x 10 cells/ml in a 24well plate the day before treatment. Cells were then treated with vehicle or II-70 in
varying concentrations. At the appropriate time points, media was removed and cells
were washed with PBS. Cells were then removed from the wells with 0.25%
Trypsin/EDTA (50 µl) and diluted with complete media (200 µl). 10 µl of this mixture
was then mixed with 10 µl of Trypan blue. Cells were then counted with a
hemacytometer.

Table III-8. Summary of GI50 values of cells treated with II-70.
Cell Line
184B5
MCF-7
MDA-MB-231

GI50 (µM)

Std Error
LogGI50

> 30
~ 30
> 30

Cell Line
HCT116
HCT116-40-16
HCT116.386
HCT116-379.2
HCT116-67

180

GI50 (µM)
> 30
20.63
22.84
28.58
8.19

Std Error
LogGI50
0.09771
0.08592
0.07148
0.07945
0.1262

Western Blot analysis whole cell extracts of cells treated with Chk2
inhibitor.

184B5 (or MDA-MB-231) cells in T25 flasks were 80% confluent when

incubated with II-70 (in DMSO) for 2 hours, then activated in situ with 10 Gy of IR. Cells
were given fresh media and inhibitor and incubated for 4 hours.

Media was then

removed from the plates and cells were washed two times with ice-cold PBS. Whole
cell lysis buffer (20 mM Tris HCl (pH =7.5), 150 mM NaCl, 1mM Na2EDTA, 1 mM
EGTA, 1% Triton X 100, 20μM DTT, 2 μM NaVO4, 2 μM NaF, 2 μM PMSF, 2 μM βglycerophosphate, 0.02 μg/ml Aprotinin, 0.02 μg/ml Leupeptin) was then added to the
plates which were then rocked for 10 minutes at 4°C. Cells were then scraped and
transferred to a tube followed by brief sonication. Cells were centrifuged at 500 x g for 3
min. 50 μg of protein (quantified with Bradford assay) was diluted with 6X SDS-PAGE
buffer. Equal amounts of denatured protein were electrophoresed on SDSpolyacrylamide gels and transferred to PVDF membranes using wet transfer.
Membranes were blocked with 4% blocking grade non-fat dairy milk (NFDM) for 1 hour
at room temperature and washed with Tris-buffered saline-Tween 20 (TBS-T)
containing 10X Tris Buffer and 0.1% Tween 20 for 10 minutes. Blots were then probed
with primary AB 1:1000 in 5% BSA TBS-T (overnight, 4ºC) and were washed with TBST (3 x 5 minutes). They were then incubated with horse radish peroxidase conjugated
to secondary antibody. Actin antibody (1:10,000 dilution) (Amersham) was incubated in
4% NFDM in TBS-T, washed and incubated with horse radish peroxidase conjugated to
secondary

antibody

chemiluminescence

mouse.
(ECL)

Complexes

technique

(Amersham) on XAR film.
181

were

according

to

detected

by

manufacturer’s

enhanced
directions

Cloning Survival Assay: 184B5 cells were cultured and when 80% confluent,
cells were treated with II-80 for 30 minutes and irradiated in suspension with varying
doses (0-7.5 Gy) of irradiation (Cobalt60) on ice. Cells were diluted and were plated at
cloning density in triplicate, and were allowed to incubate at 37°C with 5% CO 2 and
ambient oxygen. Medium was replaced at 7 days and clones were incubated another 37 days at which point clones were fixed with 0.9% saline and stained with 3% crystal
violet in 70% ethanol.
Flow Cytometry: The distribution of HCT116 cells at different stages of the cell
5

cycle was estimated using flow cytometric DNA analyses. 5 x 10 cells were incubated
overnight in McCoy 5A medium containing 5% BCS, then treated with or without
compound for 2 hours in 6-well plates. Tissue culture plates were placed on a 1.5 cm
sheet of tissue equivalent bolus material, to provide for dose build up on the radiation
treatment couch. The linac gantry was positioned at 180 degrees to irradiate the plates
using a single portal from below. Dose was calculated to the bottom of the tissue culture
plates. Cells were irradiated with 1.0, 2.5, or 5.0 cGy at a dose rate of 3.0 Gy/min. Cell
were allowed to incubate for varying lengths of time (0, 2, 4, 8, 22 hours) postirradiation. Cells were then harvested and washed with phosphate-buffered saline (PBS;
pH 7.4) containing 5% BCS, fixed with 70% ethanol/30% water at 4°C. The cells were
washed with 5%BCS/PBS twice and the pellet was resuspended in a solution containing
RNAse (20µg/ml) and propidium iodide (Invitrogen) and incubated for 15 minutes in the
dark at 37°C. For each sample, at least 10,000 events were analyzed using a Vantage
TurboSort SE cytometer (Becton Dickinson, San Jose, CA) with 488 nM excitation,
fluorescence at 630/22 bandpass filter. The percentage of cells in each cell cycle phase
182

was calculated using CellQuest Ver3.3, and the WinList6 and ModFITLT software
packages (Becton Dickinson).

183

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184

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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.
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15.

Wan, Y.; Hur, W.; Cho, C. Y.; Liu, Y.; Adrian, F. J.; Lozach, O.; Bach, S.; Mayer,
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hymenialdisine analogs. Chem. Biol. 2004, 11 (2), 247-59.

16.

Meijer, L.; Thunnissen, A. M. W. H.; White, A. W.; Garnier, M.; Nikolic, M.; Tsai,
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Mandelkow, E. M.; Kim, S. H.; Pettit, G. R., Inhibition of cyclin-dependent
kinases, GSK-3 beta and CK1 by hymenialdisine, a marine sponge constituent.
Chem. Biol. 2000, 7 (1), 51-63.

17.

Sharma, V.; Tepe, J. J., Potent inhibition of checkpoint kinase activity by a
hymenialdisine-derived indoloazepine. Bioorg. Med. Chem. Lett. 2004, 14 (16),
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18.

Tasdemir, D.; Mallon, R.; Greenstein, M.; Feldberg, L. R.; Kim, S. C.; Collins, K.;
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Sharma, V.; Lansdell, T. A.; Jin, G.; Tepe, J. J., Inhibition of Cytokine Production
by Hymenialdisine Derivatives. J. Med. Chem. 2004, 47 (14), 3700-3703.
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Ahn, J. Y.; Schwarz, J. K.; Piwnica-Worms, H.; Canman, C. E., Threonine 68
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23.

Arienti, K. L.; Brunmark, A.; Axe, F. U.; McClure, K.; Lee, A.; Blevitt, J.; Neff, D.
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24.

Yang, J.; Yu, Y. N.; Hamrick, H. E.; Duerksen-Hughes, P. J., ATM, ATR and
DNA-PK: initiators of the cellular genotoxic stress responses. Carcinogenesis
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188

CHAPTER IV
EVALUATION OF INDOLOAZEPINE AS AN INHIBITOR OF NF-кB
IV.A

Introduction to the NF-кB pathway
Nuclear transcription factor kappa B (NF-кB) is a mammalian nuclear

transcription factor involved in many cellular processes including inflammation, immune
responses, cell proliferation and cell death.

1-6

NF-кB is believed to be a cell death

suppressor and plays an important role in regulating the inducible activation of anti7

apoptotic signaling pathways. NF-кB is made up of five distinct subunits found in
mammalian cells including NF-кB1 (p105/50), NF-кB2 (p100/p52), RelA (p65), RelB and
8

c-Rel. The subunits can form a variety of homo- or heterodimers that are used to
control the specificity and selectivity of specific DNA control elements.

9, 10

form, NF-кB is sequestered in the cytoplasm by the inhibitory protein IкB-α.

In its inactive
11

These IкB-

α-NF-кB complexes constantly shuttle between the cytoplasm and the nucleus in cells
that have not been activated. The back and forth transport between the nucleus and
cytoplasm is regulated by a nuclear export signal (NES) located near the amino
terminus of IкB-α and also by an unmasked nuclear localization signal (NLS) of the p50
proteins that is likely masked.

12

IкB-β does not have the NES and can provide a more

effective mask of the NLS due to the exclusive presence of the IкB-β-NF-кB complexes
in the cytoplasm.

13

189

Figure IV-1. NF-кB signal cascade.
The IKK complex regulates IкB phosphorylation and is activated by extracellular
stimuli including pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) and IL-1β (Figure IV-1).

14

Signal-induced ubiquitinylation and proteasomal
190

degradation of IкBα by the 26S proteasome allows the irreversible release of NF-кB
7, 14-18

which translocates into the nucleus following degradation of IкB.

After NF-кB is

released, it translocates into the nucleus where the subunits are able to bind with
specific DNA control elements, initiating the transcription of several immune and proinflammatory signaling genes.

14, 19

In most cells, NF-кB bypasses apoptosis by inducing

survival genes. However, there are special conditions in which NF-кB can mediate
antiapoptotic responses including responses induced by cellular
mechanism.

DNA repair

20, 21

Transactivation of NF-кB has recently received attention.

22-24

The Rel proteins

share a Rel homology domain (RHD), which controls DNA-binding activity. p65, RelB
and c-Rel have COOH-terminal transactivation domains (TADs).

11, 25

Only TAD in the

p65 can regulate the transcriptional activity of the p65/p50 NF-кB complex. Studies
have suggested that phosphorylation of the p65 subunit plays a key role in the
transcriptional activation after stimulus-induced nuclear translocation.
reported in RHD,

26, 28

while Ser529/Ser536 are in TAD.

31-33

26-30

Ser276 was

Protein kinase A and

mitogen- and stress-activated protein kinase-1 (MSK1) have been identified as proteins
that phosphorylate Ser276.
phosphorylation for Ser536.
of p65 on Ser536.

32

26,

34

IKK has been reported as the source of

TNF-α has been shown to induce phosphorylation by IKK

32

NF-кB regulates genes directly involved in the pathogenesis of inflammatory
diseases including rheumatoid arthritis (RA),
helicobacter pylori-associated gastritis,

40

35, 36

inflammatory bowel disease,
41

atheroscelerosis,
191

multiple sclerosis

42

37-39

and

43

asthma.

NF-кB plays a significant role in cytokine responses making it an attractive

target for therapy of these inflammatory diseases.

44-46

Additionally, constitutively active NF-кB has been observed in cancer cell lines,
as well as in human tumor tissues stemming from patients with multiple myeloma,
acute myelogenous leukemia,
leukemia,

50

prostate,

51

48

52

breast

acute lymphocyte leukemia,

49

47

chronic myelogenous

53

and pancreatic cancers.

NF-кB mediated chemoresistance has been shown via activation by many
clinically

used

chemotherapeutic

agents.

54-61

Effectiveness

of

treatment

with

chemotherapeutic agents often is reduced due to a NF-кB mediated double stimulus,
which causes chemoresistance leading to uncontrolled cell growth.

62, 63

Inhibition of

NF-кB with a mutated form of IкB-α, a natural inhibitor, was shown to control inducible
63

chemoresistance.

Overexpression of IкB, in combination with camptothecin had an

increased cytotoxicity when compared to camptothecin alone.

64

Inhibition of NF-кB has

become an attractive strategy in combination chemotherapy that has been proven to
improve efficacy of anticancer treatment.

63, 65-69

Inhibition of NF-кB through the IKK complex regulates IкB phosphorylation and is
activated by extracellular stimuli including pro-inflammatory cytokines such as tumor
necrosis factor alpha (TNF-α) and IL-1β (Figure IV-1).

14

Signal-induced ubiquitinylation

and proteasomal degradation of IкBα by the 26S proteasome allows the irreversible
release of NF-кB which translocates into the nucleus following degradation of IкB.

7, 14-18

After NF-кB is released, it translocates into the nucleus where the subunits are able to
192

bind with specific DNA control elements, initiating the transcription of several immune
and pro-inflammatory signaling genes.

14, 19

In most cells, NF-кB bypasses apoptosis by

inducing survival genes. However, there are special conditions in which NF-кB can
mediate antiapoptotic responses including responses induced by cellular DNA repair
mechanism.

20, 21

Inhibition of NF-кB by genetic or chemical inhibitors induces the apoptotic
response of various tumor cells and/or inhibits the anti-apoptotic response following
treatment with ionizing radiations or other chemotherapeutic agents. This, in turn
reverses any NF-кB-linked radioresistance or chemoresistance in many models.

67, 70-74

These and other studies have made developing inhibitors of NF-кB activity an attractive
target for chemotherapy.
IV.B Targets for inhibition of NF-кB
As mentioned above, in order for NF-кB to become activated, a phosphorylation
event must occur on IкB-α followed by subsequent ubiquitinylation and degradation.
Inhibition of IкB-α would eventually lead to the inhibition of the transcriptional activity of
NF-кB.

75, 76

Genes responsible for the anti-apoptotic response as well as the immune

and inflammatory response would also be inhibited as a result of inhibition of IкB-α.

6, 76

The group of kinases involved in phosphorylation of IкB are also known as the IKK
complex.

46

There are several subunits that make up the IKK complex including the kinase
subunits IKKα and IKKβ, and the regulatory subunit IKKγ (NEMO).

77

The classical

pathway of the activation of NF-кB involves phosphorylation of the p50/p65 heterodimer
193

by the IKKβ subunit.

75

Many lymphoid or myeloid tumors including multiple myeloma,

Hodgkin diseases, and various solid tumors, including breast cancers and glioblastomas
have constitutively activated NF-кB.

70, 71, 76, 78, 79

The constitutive activity is often linked

to genetic rearrangements which can lead to abnormal gene expression of NF-кB and
consequently mutations of the IкB-α inhibitor or to increased IKK activity.

71, 80-82

Animal

models demonstrated that developments of cancers due to chronic inflammations was
dependent on IKK activity and activation of NF-кB.

83, 84

The combination of this with

inducing the anti-apoptotic gene expression provide potentially useful anticancer
therapy by inhibiting IKKβ mediated NF-кB activation.

80

The lack of selective kinase

inhibitors of IKK have made this potential therapeutic strategy limited, despite the many
examples in literature.

80

Evodiamine is an indole alkaloid that is a major constituent of the Chinese herb
Evodiae fructus having significant antitumor activity (Figure IV-2). Although it is effective
as a single agent, it more importantly has a more additive cytotoxic effect when used in
85-87

combination with chemotherapeutics.

Evodiamine was found to inhibit NF-кB

activation in the induced as well as constitutive form in a variety of cell lines.

87

Mechanistic studies have demonstrated that Evodiamine functions by inhibiting IкB-α
phosphorylation via inhibiting TNF-α induced IKK activation. Evodiamine decreased the
expression of anti-apoptotic genes as well as enhanced the cytotoxicity of TNF-α and
chemotherapeutics including taxol and cis-platin.

87

Parthenolide, a sesquiterpene lactone was initially isolated from Tanacetum
parthenium and is a potent inhibitor of the covalent binding of the IKK complex (Figure
194

IV-2).

88-90

An increase in apoptosis was observed when parthenolide was used in

combination with roscovitine, a Cdk2 and Cdk5 kinase inhibitor.
observed when used in combination with paclitaxel.

91

This affect was not

92

MLN120B is a highly selective inhibitor of IKKβ (IC50 = 60 nM) over IKKα (IC50 =
>100,000 nM) (Figure IV-2).

93

This compound was able to decrease paw swelling in a

dose-dependent manner with an ED50 = 7 mg/kg and 12 mg/kg, respectively when
dosed orally twice daily in either prophylactic or therapeutic adjuvant induced arthritis
models in rats.

94, 95

LPS-induced TNF-α production was also inhibited in rats by

MLN120B with a similar EC50. A significant protection against bone and cartilage
destruction was observed in the diseased controls when monitored by histology and
micro-CT (computerized tomography) imaging. When compared to the diseased
controls, NF-кB regulated gene expression in the joints was significantly decreased. A
dose-dependent decrease in clinical symptoms compared to control animals in a
collagen antibody-induced BALBc model of arthritis was observed.

94, 96

Thienopyridine (Figure IV-2), another potent inhibitor of IKKβ (IC50 = 26 nM) was
evaluated in a rat collagen induced arthritis model.

94, 97

Rats were dosed orally at 10

mg/kg and demonstrated a 44% decrease in paw weight when compared to control
animals.

195

Figure IV-2. IкB Kinase inhibitors.
Several other classes of IKKβ inhibitors have also been identified including
benzamides and thiophenes (Figure IV-3).

94

An increasing number of IKKβ inhibitors

continue to be discovered with various scaffolds using SAR studies.

94

Figure IV-3. IKKβ inhibitors.

In addition to inhibiting IKK, glycogen synthase-3 (GSK-3) has also been targeted
for inhibition. GSK-3 exists in two homologous isoforms that are encoded by different
98

genes, GSK-3α and GSK-3β.

Research has suggested that GSK-3 could be positively

regulating NF-кB. Inhibition of GSK-3 has also significantly decreased proinflammatory
196

responses.

99

Hoeflich et al.

100

demonstrated the necessary role of GSK-3β, and not

GSK-3α, in the NF-кB-mediated survival response after TNF-α stimulation.

100-102

It was

also discovered that GSK-3 played a role in suppressing apoptosis induced by TNFα.

100

Inhibition of GSK-3β with a known inhibitor, lithium chloride caused a decrease in
NF-кB-dependent gene transcription. The same treatment, however, did not interfere
with the degradation of IкB-α or activity of IKK. The translocation of the NF-кB
heterodimer into the nucleus and its binding to DNA were also unaffected.

103

Another

study knocked down GSK-3β resulting in a suppression of TNF-α-induced IKK
activation, IкB-α phosphorylation, and IкB-α ubiquitinylation.

104

GSK-3β was again

shown to inhibit NF-кB-dependent transcription and DNA binding activity by inhibiting
the phosphorylation and degradation of IкB-α following TNF-α induction.

105

As evidence

indicates that GSK-3β plays a part in NF-кB activation, the site of phosphorylation on
the p65 subunit has not been determined. Several protein kinases have demonstrated
phosphorylation of p65 at Ser276,

27

Ser 311,

106

31

Ser529,

and Ser 536.

32

The total

number of phosphorylation sites of p65 has yet to be determined. The Ser468 of p65
was proposed as a site of phosphorylation for GSK-3β and is believed to negatively
25

control NF-кB.

When GSK-3β was inhibited with increasing amounts of LiCl,

107

decreased levels of phosphorylation of Ser468 was observed in a dose-dependent
manner.

25

Following inhibition of GSK-3β, high levels of β-catenin have been observed,
resulting in inhibition NF-кB activity at the level of DNA binding, though the mechanism
197

is unclear.

108, 109

When β-catenin is phosphorylated by GSK-3, it is subsequently

targeted for ubiquitin-mediated proteasomal degradation, which limits the accumulation
of free β-catenin in the nucleus.

110, 111

MEFs and non-transformed rat intestinal

epithelial cells did not exhibit increased levels of β-catenin when decreased levels of
GSK-3 were observed when compared to control cells.

15

These cell lines also

demonstrated the importance of GSK-3β for efficient localization of p65 to the promoter
region of NF-кB-regulated genes during cytokine stimulation.

15, 112

Lithium selectively inhibits the activity of purified GSK-3α and GSK-3β as well as
their functions in intact cells.

113, 114

Its selectively has been used to evaluate the role of

GSK-3 in pharmacological cellular processes.
inhibitor of GSK-3α and GSK-3β.

116-118

115

Valproic acid is a dose-dependent

ATP-competitive inhibitors of GSK-3 have been

identified during screenings of other protein kinase inhibitors including: paullones,
hymenialdisine and staurosporine derivatives. Nanomolar IC50 values were observed
with these inhibitors.

119, 120

Two maleimides, SB216763 and SB415286 (Figure IV-4)

were identified as selective, ATP-competitive inhibitors of both GSK-3α and GSK-3β.

Figure IV-4. Structures of GSK-3 Inhibitors.

198

121

IV.C

Evaluation of II-70 and analogs as an inhibitor of NF-кB-mediated gene
transcription.
Previous biological evaluation of II-70 demonstrated inhibition effects against IL-2
122

and TNF-α production.

This was an indication that it could also be an inhibitor of NF-

кB. Compounds II-70, II-84, and II-99 were evaluated for their ability to inhibit NF-кBmediated gene transcription (Figure IV-5).

Figure IV-5. Structures of II-70, II-84, II-100.
The marine alkaloid IV-8 from the tunicate Dendrodoa grossularia was
synthesized in addition to a few analogues (Figure IV-6).

123, 124

Compounds IV-1 – IV-4

have similarities to the analogues discussed above, however lack the azepine ring that is
present in II-2 and II-70. In addition, the glycocyamidine ring is bonded to indole via an
3

sp hybridized quaternary center. This differs from the azepine ring of II-2 and II-70
2

which are bonded to the glycocyamidine ring by an sp bond. The synthesis of IV-1 – IV4 were described previously, and will not be discussed here.

199

123, 124

Figure IV-6. Structures of IV-1 – IV-4.
NF-кB-mediated gene transcription was evaluated by using a luciferase reporter
assay in human cervical epithelial (HeLa) cells containing a stably transfected NF-кB-luc
gene (Figure IV-7). The cells were pretreated for thirty minutes with II-70, II-84, or II-99
followed by activation with TNF-α (25 ng/mL). The proteasome inhibitor MG-132

125

was

used as a positive control, and DMSO (vehicle) was used as a negative control. After 8
hours of incubation, luciferase production was evaluated. The samples were normalized
to the TNF-α activation control. The results of the luciferase assay are summarized in
Table IV-1. HeLa-NF-кB-luc cells pretreated with the analogs without activation by TNF-α
did not induce a significant amount of luciferase activity. This was an indication that
these compounds did not stimulate the NF-кB pathway alone. Compounds II-84, II-99
and IV-1 – IV-4 (Table IV-1) resulted in EC50 values greater than 80 µM for NF-кB
mediated gene transcription. Compound IV-4 showed a slight inhibition with an EC50
value of 51.5 µM. II-70 displayed inhibition of NF-кB mediated gene transcription with an
EC50 of 6.88 µM . II-2 showed potent inhibition of NF-кB-mediated gene transcription
with an EC50 of 0.64 µM. The related analogues were screened for inhibition of the
kinases listed in Table IV-1.Compounds II-70, II-84, II-99, IV-1 – IV-4 were also
evaluated for inhibition of other kinases involved in the NF-кB pathway. None of these
200

compounds showed significant inhibition of the kinases tested with most IC 50 values >
10,000 nM. In addition to inhibition of gene transcription compound II-70 also inhibited
GSK-3β (86 nM).
Table IV-1. Kinase inhibition by hymenialdisine-derived analogs.

II-2
HeLa NF-кB-luc
(µM)

II-70

II-84

II-99

IV-1

IV-2

IV-3

IV-4

0.64

6.88

> 80

> 80

> 80

> 80

> 80

51.5

GSK-3β (µM)

0.073

120, 126

0.086

NA

NA

> 50

> 50

> 50

NA

Chk1 (µM)

1.950

127

0.237

> 10

> 10

> 10

> 10

> 10

> 10

Chk2 (µM)

0.042

127

0.008

> 10

> 10

> 10

> 10

> 10

> 10

CK1δ(h) (µM)

0.035

120, 126

1.352

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

> 10

0.089

NA

NA

NA

NA

NA

NA

2.539

NA

NA

NA

NA

NA

NA

3,381

NA

NA

NA

NA

NA

NA

CK2 (h) (µM)
IKKα(h) (µM)
IKKβ(h) (µM)
MEK1(h) (µM)

7

120, 126

> 10
>5

120, 126

120, 126

0.006

128

120, 126

PKCα(h) (µM)

0.7

PKCβII(h) (nM)

1200

120, 126

The lack of inhibition with compounds II-84, II-99, IV-1 – IV-4 could give an
indication of key structural requirements for biological activity. II-84 has a hydantoin ring
lacking a hydrogen donor for hydrogen bonding. II-99 contains a dimethyl amine that
may prevent efficient binding due to the bulky steric interactions resulting in a poor
inhibitor of NF-кB. II-70 possesses a free amine on the glycocyamidine ring offering a
hydrogen donor and possibly contributing to the inhibitor activity. IV-1 – IV-4 were also
unable to inhibit any of the kinases evaluated in Table IV-1. The azepine ring present in
II-70 was absent in IV-1 – IV-4 suggesting the importance of the amide in the binding
201

pocket. Although compound IV-2 contained the glycocyamidine ring, no inhibition of
kinase activity was found. This could indicate that the free amine is not the only
requirement for binding and activity. Compound IV-4 has the glycocyamidine ring as well
as an amide nitrogen at the 2-indole position. With the free amine on the glycocyamidine
ring and the methyl amide, IV-4 gains back some inhibition activity for NF-кB-mediated
gene transcription with an EC50 of 51.5µM.

TNF- + II-70

Fold Induction (%)

100
80
60
40
20

M

G

-1 TN
32 F- V
(5  + ehi
0
v cle
40  M eh
) i
 M + cle
T
II- NF
70 
on
40 ly

20 M

10 M
M
5

2. M
5
M
1

0. M
5
M

0

Figure IV-7. Dose-response activity of II-70 in inhibition of luciferase production in HeLaNF-кB-luc cells.
IV.D

Evaluation of II-70 as an inhibitor of IL-1β induced TNF-α production in
human whole blood
The results of the luciferase reporter assay lead to further analysis of II-70 for its

anti-inflammatory potential in human whole blood. II-70 was evaluated for its ability to
202

inhibit an NF-кB mediated cytokine response in IL-1β stimulated human blood (Theresa
Landsell). Human whole blood was pretreated with II-70 for 2 hours and was stimulated
by IL-1β. Plasma was harvested 22 hours after stimulation. IL-1β induced TNF-α
production was measured using a human TNF-α ELISA assay. The amount of IL-1β
induced TNF-α production was significantly higher than the level of TNF-α in the
unstimulated vehicle treated blood (Figure IV-8). Dexamethasone (dex) is an antiinflammatory drug known to inhibit TNF-α production

129

and was used here as a

positive control. II-70 inhibited TNF-α production in a dose-dependent manner (EC50 = 4
µM) as compared to the vehicle treated control. Due to the lack of inhibition against any
kinases examined in Table IV-1, II-84, II-99, IV-1 – IV-4 were not evaluated in human
whole blood.

IL-1 and II-70

pg/mL TNF

200

100

IL Un
-1 tre

+ ate
v
d
10 eh
i
 M cle
D
e
30 x
M
10
M
3
M
1

0. M
3
M

0

Figure IV-8. Dose-response activity of II-70 in inhibition of TNF-α production in IL-1β
stimulated human blood.
203

IV.E

Evaluation of II-70 as an inhibitor of NF-кB in vitro.
II-70 demonstrated inhibition of NF-кB mediated gene transcription and TNF-α

production. The mechanism of action of II-70 in the NF-кB pathway remained unclear. In
vitro evaluation of NF-кB translocation of the p65 subunit followed. HeLa-NF-кB-luc cells
were pretreated with II-70, DMSO or MG-132 for 30 minutes at varying concentrations.
The cells were then activated with TNF-α (10 ng/mL) for 30 minutes. Nuclear extracts
were collected and western blot analysis ensued as seen in Figure IV-9. Samples that
were not activated with TNF-α did not show increased basal levels of p65. II-70 alone
did not increase levels of p65 indicating that it is not an activator of NF-кB. The
combination of TNF-α and II-70 at high (30 µM) and low (3 µM) doses did not show any
decrease in levels of activated p65. Levels of induced p65 were inhibited by the
proteasome inhibitor MG-132 (Figure IV-9), the positive control. This illustrates that II-70
is not inhibiting nuclear translocation of the p65 subunit of NF-кB.

0

0

0

30 20 10 3

30

0

µM compound II-70

-

+
-

+
-

+
+
-

+
-

+
+
+

TNF-α
Vehicle (DMSO)
MG132 (10 µM)
p65
β-actin

+
+
-

+
+
-

+
+
-

Figure IV-9. Western blot of TNF-α induced p65 after 30 minutes in HeLa-NF-кB-luc
cells.
II-70 was then evaluated for its ability to inhibit phosphorylation of p65. The
nuclear extracts collected above were analyzed with Western blot and probed for p-p65
at phosphorylation sites S276, S468 and S536. GSK-3β is known to phosphorylate p65
at S468 as mentioned above,

103

Although II-70 inhibits GSK-3β at 86 nM, there was no
204

evidence of inhibition of phosphorylation of S468 (Figure IV-10). II-70 did not inhibit
phosphorylation at S276 or at S536 at any concentration.
0
-

0
+

0 30 20 10 3 30
+ + + + + + + + + + +

0
+
+

µM II-70

-

-

-

+

Mg132 (10 µM)
p-p65 S276
β-actin

-

-

-

-

-

TNF-α
Vehicle (DMSO)

p-p65 S468
β-actin
p-p65 S536
β-actin

Figure IV-10. Western blot of HeLa cells with TNF-α induced p-p65 after 30 minutes.
Inhibition of NF-кB-DNA binding was evaluated next with II-70. The cellular nuclei
were isolated and evaluated for NF-кB-DNA binding utilizing the fluorescent-labeled NFкB-DNA consensus sequence (Figure IV-11). Nuclear extracts stimulated with TNF-α
and the NF-кB consensus oligo resulted in DNA binding to NF-кB (Figure IV-11, lanes
3-7). As a negative control, cells were left unactivated and were also exposed to the NFкB consensus sequence, resulting in background NF-кB/DNA binding (Figure IV-11,
lanes 1-2). Treatment of cells with II-70 followed by TNF-α did not result in inhibition of
NF-кB/DNA (Figure IV-11, lanes 4-7). II-70, at 30 µM without TNF-α stimulation did not
accumulate NF-кB/DNA binding (Figure IV-11, lane 8). NF-кB-mediated gene
transcription in vitro in addition to TNF-α production in whole human blood was inhibited
by II-70. Cellular studies indicated that II-70 was did not inhibit translocation of p65 into
the nucleus. The EMSA assay shows that NF-кB/DNA binding is also not inhibited by II70.

205

0

0 0 30 20 10 3 30 0

0

µM II-70

+
-

+ +
- +

+
+

+
+

+
+

+
+

+
-

+
+

+
+

DNA
TNF-α

1

2 3

4

5

6

7

8

9

10
NF-кB/DNA
Non-specific
binding

DNA

Figure IV-11. EMSA Assay for NF-кB Activation by TNF-α (10 ng/mL).
In summary, a series of indole analogs inspired by hymenialdisine and the
marine alkaloid from Dendrodoa grossularia were prepared and evaluated for their
biological activity. Most of the synthesized compounds were not biologically active with
the exception of II-70. A kinase inhibition panel revealed that II-70 inhibited GSK-3β (86
nM). Further evaluation of II-70 demonstrated its ability to inhibit NF-кB mediated gene
transcription (6.88 µM) as well as TNF-α production in human whole blood (4 µM). The
analogs synthesized (II-84 and II-99) lost activity when the free amine was replaced with
dimethyls or an oxygen.. Further cellular studies concluded that II-70 does not inhibit
p65 translocation into the nucleus, nor does it inhibit phosphorylation of p65. An EMSA
binding assay revealed that II-70 does not inhibit NF-кB/DNA binding.

206

IV.F

General experimental information
All cell lines were cultured at 37°C with 5% bone dry CO2 atmosphere, 97%

humidity and ambient O2 in a Napco water-jacketed incubaor. Adherent cells were
routinely passaged by 0.25% trypsin-EDTA (Invitrogen, Fredrick, MD) using sterile
technique and materials in a Nuaire biological safety cabinet Class II type A2. For all
lysate and whole cell experiments, the inhibitors were dissolved in DMSO and the final
DMSO concentration of the experiments was maintained at ≤ 0.1% in the reaction buffer
or culture medium. Cells were observed using a 1200CMI series spectrum inverted
microscope. Cells were counted with a hemacytometer or using the Beckman Coulter Z1
Coulter® Particle Counter. Centrifugation was performed using the Beckman Coulter
Allegra X-15R Centrifuge or Spectrafuge 16M microcentrifuge. Buffers, solutions and
media were made with water purified with the Millipore Quantum® Ex filtration system.
Buffers requiring pH adjustments were monitored with a Mettler Toledo SevenEasy pH
e

meter. Protein assays were read using the Molecular Devices SpectraMax M5 plate
reader. Western blots were run on hand-poured SDS PAGE gels using the BioRad mini
protean pouring and electrophoresis system with the BioRad PowerPac HC. Gels were
transferred to polyvinylidene fluoride membrane (BioRad) with the BioRad Criterion
blotter wet transfer system. Blots and gels were quantified using the BioRad Molecular
Imager FX. Luminescence in the luciferase assays were monitored and quantified using
the Turner Biosystems Veritas Microplate Luminometer.
Cell Culture. The human cell line HeLa-NF-кB-luc was purchased from
Panomics, Inc. (Fremont, CA).

the cells were maintained in Dulbecco’s Modified

Eagle’s Medium (DMEM, Invitrogen, Fredrick, MD) containing 4.5 g/L glucose, 3.7 g/L
207

bicarbonate, and supplemented with 5% fetal bovine serum, 100 U/ml penicillin, 100
µg/mL streptomycin, 1 mM sodium pyruvate, 0.2 mM L-glutamine, and 100 µg/mL of
hygromycin B. Cells were cultured at 37°C, 5% CO2 atmosphere, 97% relative humidity
and were routinely passaged by 0.25% trypsin-EDTA (Invitrogen, Fredrick, MD)
treatment according to ATCC protocol.
NF-kB-luc reporter assay. HeLa NF-кB-luc cells (5.0 x 10

5

cells/ml) were

seeded into a 96-well white opaque plate using DMEM medium supplemented with 5%
fetal bovine serum, 100 U/ml penicillin, 100 µg/mL streptomycin, 1 mM sodium pyruvate,
0.2 mM L-glutamine, and 100 µg/mL of hygromycin B (Roche). After 24 h the cell culture
medium was replaced with DMEM medium supplemented with 100 U/ml penicillin, 100
µg/mL streptomycin, 1 mM sodium pyruvate, and 0.2 mM L-glutamine. Cell cultures were
pretreated with vehicle (1% DMSO), 50 µM Mg132 (EMD Biosciences, San Diego, CA)
or analogues II-70, II-84, and II-99 (final concentrations were 40, 20, 10, 5, 2.5, 1, 0.5 µM
(II-70) and 80, 40, 20, 10, 5, 2,5, 1.25 µM (II-84, and II-99) for 30 min at 37°C in 5% CO2.
TNF-α (Invitrogen Cytokines & Signaling, Camarillo, CA) was added to a final
concentration of 25 ng/mL and the samples were further incubated for 8 hours at 37°C in
5% CO2. The plate and Steady-Glo assay reagent (Promega Corporation, Madison, WI)
were equilibrated to room temperature. An equal volume of Steady-Glo assay reagent
was added to each well. The contents of the plate were gently mixed for 5 min and the
luminescence of each well was measured using a Veritas microplate luminometer
(Turner Biosystems, CA).
experiments.

All reported data are the average of two independent

The data were analyzed using GraphPad Prism 5.0.

208

The data was

normalized to TNF-α activation and the EC50 values were calculated using the equation
for the sigmoidal curve for variable slope.
GSK-3β, Chk1, Chk2, CK1δ, CK2, IKKα, IKKβ, MEK1, PKCα, PKCβ in vitro
kinase assay. Compounds evaluated were tested in vitro against the kinases indicated
by Millipore UK limited, Dundee, UK using a Kinase Profiler Assay according to the
manufacture’s protocol. Briefly, in a final volume of 25 µL, the kinase was incubated with
the desired buffer and the required polypeptide substrate, in presence of 10 mM
33

magnesium acetate and γ- P-ATP (10 µM). After incubation for 40 min at room
temperature, the reaction was stopped by the addition of 3% H3PO4 (5µL). A 10µL
aliquot of the reaction was then spotted on a P30 filtermat and washed 3 X in 75 mM
H3PO4 and finally in methanol. Samples were then dried and signals counted on a
scintillation counter.
Human whole blood IL-1β challenge. 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 human volunteer and was
collected in glass citrated tubes by venipuncture. Only samples with a white blood count
falling within normal range (4,800-10,800 white blood cells per liter) were used. To
support the viability of white blood cells, blood was diluted 1:10 in RPMI-1640 media
supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL
streptomycin. Aliquots of diluted blood (1 mL) were preincubated with vehicle (0.1%
DMSO, final concentration) or II-70 (final concentrations were 30, 10, 3, 1, 0.3 µM) for 2
hours at 37°C in 5% CO2. IL-1β (Roche) was added to a final concentration of 200 U/mL
and the samples were further incubated for 18 hours at 37°C in 5% CO2. At the end of
209

the incubation period, the blood samples were centrifuged at 3000 rpm for 5 min. The
plasma was removed, snap frozen and stored at -80°C.
Western blot analysis. Cells were cultured in 100 mm plates until ~80%
confluence. Cells were pre-treated with II-70 (in DMSO) for 30 minutes, then activated
with TNF-α (10 ng/ml) for 30 minutes. Cells were then with ice cold PBS (2 ml) twice.
PBS was then removed and lysis buffer (10 mM HEPES (pH =7.9), 1% Igepal-630, 1.5
mM MgCl2, 10 mM KCl2, 20 μM DTT, 2 μM NaVO4, 2 μM NaF, 2 μM PMSF, 0.02 μg/ml
Aprotinin, 0.02 μg/ml Leupeptin) was added and rocked at 4°C for 10 minutes. Samples
were vortexed and centrifuged at 3000 RPM for 3 minutes. Cytosolic extracts were
removed and nuclear extracts were lysed open with high salt buffer (3.4 M glycerol, 2
0mM HEPES (pH=7.9), 1.5 mM MgCl2, 0.42 M NaCl, 0.1 M EDTA, 20 μM DTT, 2 μM
NaVO4, 2 μM NaF, 2 μM PMSF, 0.02 μg/ml Aprotinin, 0.02 μg/ml Leupeptin). The
samples were vortex at 2,000 RPM for 20 minutes and centrifuged at 3000 RPM for 15
minutes. 25 μg of protein (quantified with Bradford assay (BioRad, Hercules, CA)) was
diluted with 2X SDS-PAGE buffer and water to bring to equal volumes, and boiled for 3
minutes. Equal volumes of boiled protein were electrophoresed on SDS-polyacrylamide
gels and transferred to PVDF membranes using wet transfer. Membranes were blocked
with 4% blocking grade non-fat dairy milk (NFDM) for 1 hour at room temperature and
washed with Tris-buffered saline-Tween 20 (TBS-T) containing 10X Tris Buffer and 0.1%
Tween 20 for 10 minutes. Blots were then probed with primary AB NF-κB p65 (Santa
Cruz Biotechnology, CA) 1:1000 dilution in 4% NFDM (1 hour room temperature) and
were washed with TBS-T (3 x 10 minutes). AB NF-κB Phospho-p65 S276, S468, S536
(Cell Signaling Technology, MA) were diluted (1:1000) in TBS-T containing 5% w/v
210

bovine serum albumin (Sigma, WI). They were then incubated with horse radish
peroxidase conjugated to secondary antibody goat anti-rabbit (Cell Signaling
Technology) diluted 1:2000 in TBS-T containing 5% BSA. Actin antibody (1:10,000
dilution) (Amersham) was incubated in 4% NFDM in TBS-T, washed and incubated with
horse radish peroxidase conjugated to secondary antibody mouse (1:3,000 dilution in 4%
NFDM). Complexes were detected by enhanced chemiluminescence (ECL) technique
according to manufacturer’s directions (Denville) on XAR film.
EMSA Assay for NF-кB-DNA binding. HeLa-NF-кB-luc cells were cultured in
100 mm plates until ~80% confluence. Cells were pre-treated with II-70 (in DMSO) for 30
minutes, then activated with TNF-α (10 ng/ml) for 30 minutes. Nuclear extracts were
collected as previously described. Nuclear extracts (20 μg total protein) were incubated
for 20 min at room temperature with a double-stranded Cy3-labelled NF-кB consensus
oligonucleotide, 5’-AGTTGAGGGGACTTTC CCAGGC-3’ (0.16 pmol). The binding
mixture (40 μL) contained 500 mM KCl, 25 mM DTT, 0.5% Igepal CA-630, 1 μg/μL
herring sperm DNA. The mixture was loaded on a 5% TBE gel prepared in 1 X Tris
borate/EDTA buffer and electrophoresed at 120V for 30 min. After electrophoresis, the
gel was analyzed using a phosphorimager (Biorad FX plus) for detection of NF-кB-DNA
binding.

211

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212

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