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SYNTHESIS AND BIOLOGICAL ACTIVITY
OF SEVERAL ETHYL l-ACYLINDOLE-B-ACETATES

By
ROGER WILLIAM RITZERT

AN ABSTRACT
Submitted to the School of Advanced Graduate Studies
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

MASTER OF SCIENCE

Department of Biochemistry
1961

Approved W

Roger William Ritzert
ABSTRACT

Derivatives of indole-B-acetic acid (1AA), with the
exception of ethyl indole-B-acetate (IAE), are generally
equal to or less active than the parent compound. IAB is
an exception in that greater activity than IAA.has been
demonstrated in several physiological systems. Several
ethyl l-acylindole-B-acetates were synthesized and the
effects of substitution on biological activity were
determined.

Equimolecular amounts or IAE and the appropriate acyl
chloride were refluxed with.benzene or toluene for 24 to 48
hours. The products were obtained by crystallization.from
various solvents. The.£ollowing acyl groups were substi-
tuted in.this.manner: chloroacetyl, dichloroaeetyl, 2-
chloropropionyl, and 3-chloropropionyl. AcetylplAE was
obtained by condensation of l-acetylindole and ethyl
diazoacetate. These derivatives, and 4—nitrobenzoyl and

4-aminobenzoyl-IAE obtained from another source, were as-
sayed for their biological activity.

Comparative biological activity was determined in the
promotion of parthenocarpic tomato ovary growth, bean
petiole abscission, and A123; coleoptile straight growth.
In the promotion of parthenocarpic growth of tomato ovaries,
at 10"3 M in lanolin, dichloroacetyl-IAE was as active as
IAE, and both were more active than IAA. Acetyl-IAE and

chloroacetyl-IAE were less active than IAE, but equal to
IAA. The 2- and 3-chloropropionyl, 4-nitrobenzoy1, and 4-
aminobenzoyl-IAE did not promote parthenocarpic growth of
tomato ovaries. Dichloroacetyl-IAE was the most active
compound in delaying bean petiole abscission. Acetyl,
chloroacetyl, and 4-nitrobenzoyl-IAB were slightly more
active than either IAB or 1AA. However, 2- and B-chloro-
propionyl, and 4-aminobenzoyl-IAE did not significantly
delay petiole abscission. All of the IAE derivatives
synthesized were active in the'gzgga,coleoptile straight
growth test over a wide concentration range. There was no
marked effect due to substitution on the l-indole position
on biological activity of IAE as measured by the 512;;
straight growth assay.

SYNTHESIS AND BIOLOGICAL ACTIVITY
OF SEVERAL ETHYL l-ACYLINDOLE—3-ACETATES

By
ROGER WILLIAM RITZERT

A THESIS
Submitted to the School of Advanced Graduate Studies
Michigan State University of Agriculture and

Applied Science in partial fulfillment of
the requirements for the degree of

MASTER OF SCIENCE

Department of Biochemistry
1961

4/5195"

0/20/91

INTRODUCTION .

HISTORICAL . .

EXPERIMENTAL .

TABLE OF CONTENTS

0 0 O O O O O O O 0

Synthesis of Compounds . . . .

Ethyl
Ethyl
Ethyl
Ethyl
Ethyl
Ethyl
Ethyl

Indole-B-acetate .

l-Acetylindole-B-acetate
l-Chloroacetylindole-B-acetate
l-Dichloroacetylindole-3-acetate

l-(2-Chloropropionyl)indole-B-acetate

l-(B-Chloropropionyl)indole-S-acetate

l-Triphenylmethylindole-B-acetate

Characterization of Compounds
Biological Assays . . . . . .
Tomato Ovary Growth . . .

Bean Petiole Abscission .
m Straight Growth . .

RESULTS AND DISCUSSION . . . . . .

SUMMARY . . . .

BIBLIOGRAPHY .

11

10
12
13
14
14
15
l6
l7
18
18
19
20

22

38

LIST or TABLES
TABLE _ ' PAGE

I. Melting points and elemental analysis
of indole compounds . . . . . . . . . . 24

II. Ultraviolet absorption characteristics
of indole compounds . . . . . . . . . . 25

III. Infrared absorption characteristics
of indole compounds . . . . . . . . . . 32

IV. Results of tomato ovary growth and
bean petiole abscission assays .

34

111

LIST OF FIGURES
FIGURE ' PAGE

1. Infrared absorption spectra in toluene of
ethyl indole-B-acetate, ethyl 1-chloro-
acetylindole-3-acetate, and ethyl l-
dichloroacetylindole-3-acetate . . . . . . . 26

2. Infrared absorption Spectra in toluene of
ethyl l- 2-chloropropiony1 indole-B-acetate,
ethyl l— 3-chloropropionyl -indole-3-acetate,
and ethyl l-triphenylmethylindole-S-acetate 28

3. Infrared absorption spectrum in toluene of
ethyl l-acetylindole-3-acetate . . . . . . . 30

4. Growth curves of Avgna coleoptile sections
in various treating solutions . . . . . . . . 35

iv

ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation
to Professor H. H. Sell for’his guidance and encouragement
throughout the course of this work. Words of appreciation
are also due Dr. H. J. Bukovac for his help with the bio-
logical assays and his many useful suggestions. To Mrs.
Richard Titus for her technical assistance go many kind
words of thanks. The financial assistance granted by the
National Science Foundation to carry on this work is also
appreciated.

INTRODUCTION

INTRODUCTION

Anxins are defined as "compounds characterized by
their capacity to induce elongation in shoot cells" (24).
Auxins are these compounds which resemble the principal
naturally occurring auxin, indole-j-acetic acid (1AA), in
physiological action. It should be emphasized that cell
elongation is not the only reaponse observed. Auxins are
also reaponeible for the control of cell differentiation,
reproduction, abscission of leaves and fruit, and apical
dominance. They are widely used in agriculture as herbi-
cides, for fruit thinning, for control of pre-harvest drop,
for setting of parthenocarpic fruit, for control of flower-
ing, and for induction of rooting.

The majority of the auxins known are not structurally
related to IAA. Many compounds structurally related to the
common auxins have been tested for their ability to induce
cell elongation, but few were as active as IAA. Of the
several IAA derivatives assayed, only a few were substi-
tuted at the one position. From the many compounds tested
for cell elongation properties, theories as to the struc-
tural requirements necessary for activity have been proposed.

This present study was initiated to determine if sub-
stitution on the l-indole position of ethyl indole-B-
acetate (IAE) alters the observed activity of the parent
compound. The effects of different substituents were also

of interest. IAE was used as the parent compound because

2

3
it is more active than IAA and because fewer complications
in the acylation reaction were observed.

The problem was divided into two parts: (l) to find
suitable methods for the acylation of ethyl indole-3-acetate
on the l-indole position and to determine the physical prop-
erties of the compounds synthesized; (2) to assay the com-
pounds for their biological activity in three different
physiological systems.

HISTORICAL

HISTORICAL

The first observation of the growth regulating properb
ties of IAA.waS made by KBgl,‘gt‘§;. (13) after their iso-
lation of the compound from.human urine. It was observed
that at low concentrations, IAA was capable of inducing cell
elongation in,51gng coleoptile sections. The first isola-
tion of IAA from plant material, namely corn kernels, was
accomplished by Haagen-Smit and co-workers (5), to suggest
that IAA is one of the important naturally occurring growth
hormones. Its presence in other plant tissues is now well
documented (2). Although IAE has been shown to be more
active than IAA in the tomato ovary test (19, 20) and in
the A1933 straight growth assay (18), it has been positively
identified only in grape seeds and coconut milk (17).

Many synthetic compounds have been tested for their
capacity to induce cell elongation. From.the various comp
pounds assayed, Koepfli, Thimann, and Went (12) postulated
the following structural requirements for cell elongation
activity:

(a) a ring system as nucleus

(b) a double bond in this ring

(c) a side chain

(d) a carboxyl group (or a structure
readily converted to a carboxyl)
on this side chain at least one
carbon atom removed from the ring

(e) a particular space relationship

between the ring and the carboxyl
group.

6
Veldstra and B001: (26) condensed these five require-
ments into two. They proposed that in order to induce cell
elongation, a compound must have the following structural
characteristics:

(a) Basal ring system (non-polar part)
with high interface activity.

(b) Carboxyl group (polar part) - in
general a group of acidic charac-
ter - in such a Spatial position
with respect to the ring system,
that on absorption of the active
molecule to a boundary:(thenon-
polar part playing the most imp
portant role) this functional
group will be situated as periph-
erally as possible.

Muir, Hansch, and co-workera (7, 8, 14, 15) have sug-
gested that maximum cell elongation inducing properties are.
obtained when the positions ortho to the side chain (2- and
4-indole positions) are unsubstituted. Such an effect was
observed for various phenoxyacetic acids and indole-S-acetic
acids. This phenomenon has been called the "ortho effect.”
The same workers (8) have thus postulated a two-point
attachment of auxin to the enzyme. However, this effect
is not observed if 4-chlcroindole-3-acetic acid is used, as
it shows more activity than IAA (14).

It has also been observed (4, 10) that substitution on
the phenylene nucleus often enhances activity in cell elonga-
tion assays and that substitution on the pyrrolene radical
tends to inactivate the molecule for cell elongation proper-

ties. It has been shown that 1- and 2-methylindole-3-acetic

7
acids are inactive in the cell elongation assays (25).

Sell, gtmgl. (20) found that l-benzoylindole-B-acetic

acid is less active than IAA in the tomato fruit set assay.

Substitution for or alteration of the carboxyl group
of IAA also appears to affect cell elongation activity.
Nitsch and Nitsch (18) have shown that in the 51233 straight
growth test, IAE and indole-3-acetonitrile (IAN) are more
active than IAA. Hamilton,ԤtԤ1. (6) observed a weak
auxin effect when a tetrazole ring is substituted for the
carboxyl group of IAA. Hofert and Sell (9) assayed several
steryl esters of IAA for their capacity to set partheno-
carpic tomato fruit and found them to be inactive.

Thimann (23) recently reported the biological activi-
ties of twenty indole compounds in the 532;; straight growth
test. He concluded that it is necessary to define the
Specific bioassay in which an auxin functions. Thimann
also suggested that the empirical rules relating structure
to activity in the aromatic series are also valid for the
indole series, but that the requirements set forth in these
theories are perhaps not as well described as they should
be. It is further suggested that attempts to correlate
structure with activity should perhaps include the physical
effects of substitution on the nucleus.

This thesis reports the synthesis and biological activ-
ity of several ethyl 1-acylindole-3-acetates and the biologi-
cal activity of two ethyl 1-aroylindole-3-acetates.

EXPERIMEN TAL

EXPERIMENTAL

Synthesis 2; Cgmpgunds

The methods for the synthesis of the ethyl l-acylindole-
3-acetates and ethyl l—triphenylmethylindole-3-acetate were
chosen for their Simplicity. The reaction used is the stan-
dard reaction of an acyl chloride with a secondary amine to
give a tertiary amide, and in the case of the triphenylmethyl
derivative, the reaction of triphenylmethyl chloride with a
secondary amine to give a tertiary amine. In most cases,
the reactions gave only fair yields. Crude oils were usu-
ally obtained initially and the desired crystalline product
acquired from.this crude mixture by use of various solvents.
The reactions were made by refluxing in benzene or toluene
for 24 to 48 hours using equimolecular amounts of IAE and
the acyl chloride .1: triphenylmethyl chloride. Acetyl IAE
was prepared from l-acetylindole and ethyl diazoacetate.

Two methods for the preparation of IAE were employed:
one involved the condensation of indole with ethyl diazo-
acetate; and the other, the esterification of IAA with
ethanol catalyzed by anhydrous hydrogen chloride.

The procedures are outlined below.

10
Ethyl Indole-3-acetate

Procedure A.

HOl'NHgCHQCOOOEHE + NaN02 —“‘* NQCHCOOCQHB

,\

’/ ~11 + 112030000235 @2912 * /\/£ Jcnzcoocsz
K: /'« l ‘ ' I
H

H

Ethyl diazoacetate (13). A cold solution of 210 g. (1.5
moles) of glycine ethyl ester hydrochloride and 1.05 g.
of sodium acetate in 220 ml. of water was poured into a
large beaker cooled in an ice bath. While this mixture
was agitated with a motor stirrer, 157.5 g. (2.3 moles)
of sodium nitrite in 220 ml. of water was added, followed
by 125 ml. of ether. The bath was kept at 20°C. or lower
by the addition of cracked ice throughout the remainder of
the reaction. Twenty-five m1. of a 10% solution.of sul-
furic acid was slowly added and the mixture stirred until
the reaction subsided (about 25 minutes). The complete
mixture was then drawn into a large separatory funnel by
application of a vacuum from a water aspirator at the top
of the funnel. The aqueous layer was returned to the re-
action vessel and the ether layer transferred to a smaller
separatory funnel and washed immediately with a cold 10%
solution of sodium carbonate.

The same cycle of operations was repeated using 100
ml. of ether and 25 ml. of a 10% solution of sulfuric acid.
This was followed by a third cycle using 100 ml. of ether
and 75 m1. of a 10% solution of sulfuric acid. The combined

11
ether solutions were then washed with a saturated sodium
chloride solution. The ether solution was dried over
anhydrous sodium sulfate, and the ether distilled off in
‘zgggg. The last traces of ether were removed by passing
a stream of dry nitrogen through the oil and 144.3 g. (84%)
of a light yellow oil obtained. The product was stored in
a dark bottle in the cold until used.
_§3§y} Tndole-§:ag§§g§e (16). To a solution of 46.8 g.
(0.4 mole) of indole in 100 m1. of dry benzene in a three-
necked fla3k was added 0.2 g. of cuprous chloride. The
mixture was brought to reflux and the drcpwise addition
of 54.6 g. (0.48 mole) of ethyl diazoacetate in 320 ml. of
dry benzene begun. The addition of ethyl diazoacetate was
continued at such a rate that the evolution of nitrogen gas
was constant. The mixture was refluxed for one hour after
the addition was complete. The mixture was cooled, filtered,
and the benzene removed under reduced pressure. The remain-
ing oil was distilled in, mg, and the product collected at
154-155°0. and 0.8 mm. pressure.1 The slightly yellow oil
weighed 37.5 g. (46%) and crystallized upon cooling.
Procedure B (11).

I- 01120003 + 01130112011 Ml» E r- 1' 0320000255
537$“ ' N / A '~\/"'\N
H H

I. Jackson (11) reports b.p. 180° and 2 mm. pressure.

12

To one liter of absolute ethanol saturated with 80 g. of_
anhydrous hydrogen chloride was added 60 g. (0.34 mole)
of IAA. This mixture was allowed to stand at room tempera-
ture for five days after which the ethanol and hydrogen
chloride were removed in mg. The crude oil that re-
mained was distilled at 1543155°c. and 0.08 mm. pressure1
to give 44 g. (64%) of IAE, which crystallized upon stand-
ing. The infrared Spectrum showed 7\;;§?f3§ 2.90 (NH)
and 5.76 (ester 0 = 0).

Ethyl l-Acetylindole-3-acetate

.c.’ 1' \ G // ‘ “ —~———-
,« r— 1% + NQCHCOOCZHS 9E211 , “I /A\ H01120000235
,2 ‘~‘ . N /.

0:0 0:0
CH3 CH3

- A solution of 15.9 g. (0.1 mole) of l-acetylindole2
in 30 ml. of dry benzene was brought to reflux. A small
amount of cuprous chloride was added and the dropwise ad-
dition of 11.4 g. (0.1 mole) of ethyl diazoacetate in 60
ml. of dry benzene begun and continued at a rate necessary
for a steady evolution of nitrogen gas. After three hours,
the mixture was cooled, filtered, and the benzene removed
inwzgggg. The residual oil was distilled at 164-167°0.
and 0.6 mm. pressure. The distillate upon redistillation
gave 2.7 g. of a yellow oil of b.p. 142-147°c. and 0.3 mm.
pressure. The product was dissolved in ethyl'ether-hexane
and upon cooling an orange oil soon separated out. The

I. Uackson (11) reports b.p. 1800 and 2 mm. pressure.
2. Obtained from.Regis Chemical Company.

13

supernatant mother liquor upon cooling at 0°C. gave 1 g. of
colorless crystals melting at 90°C. The compound showed
7\ toluefie 5. 81 (ester C = O). 5. 93 (amide C = 0), and no
band near 2.90 (NH); aggaagi)‘95%> 258 (108:: 4.174).
and shoulder at 287-288 (loge: 3.498).

4951. Calcd. for 014H15N03: C, 68.55; H, 6.16; N
5.71. Found: C, 68.50; H, 6.12; N, 5.73.

Ethyl 1-Chloroacetylindole-3-acetate

//\‘“\ -l__. -t. , ~
N“~JCH20006235 + 010320001 , ‘ 1-:103200002H5
Lay/NH / N .
H C = 0
03201

To a hot solution of 4.06 g. (0.02 mole) of IAE in
15 ml. of dry benzene was added dropwise a solution of
2.3 g. (0.02 mole) of chloracetyl chloride in 10 ml. of
benzene. The mixture was refluxed for 24 hours, cooled,
and the benzene removed in m. The residue was dis-
solved in 95% ethanol, treated with Norite A, filtered,
and the ethanol removed under reduced pressure. The
residue was dissolved in 95% ethanol-ethyl ether and upon
cooling gave colorless needles. Recrystallization from
95% ethanol gave 2.15 g. of colorless needles, m.p. 118-
119°c. The absorption spectra showed \“meng 5. 78 (ester
0 = 0). 5.86 (amide C = 0), and no band at about 2.90 (NH);
2x gmgfifififl 245 (loge 4.090), 290 (log 6 3.738), and
300 (loge 3.633).

14
Anal. Calcd. for 014H1501N03: C, 60.11; H, 5.04;
N, 5.00. Found: C, 59.66; H, 5.19; N, 4.87.
Ethyl 1-Dichloroacetylindole-3-acetate

1
I
\l

l éjcfiz ZCOOC2H5 + 015110001 - CH20000235

/i 1
N/J
C: O
CH012

A solution of 4. 06 g. (0. 02 mole) of IAE was dissolved
in 25 ml. of dry toluene and the solution brought to reflux.

The dropwise addition of 2.95 g. (0.02 mole) of dichloro-

acetyl chloride was begun and the mixture refluxed for 30

hours. The mixture was cooled and the toluene removed in

12223. The residue was dissolved in 95% ethanol, treated

with Norite A, and filtered. The product was precipitated

by the addition of water, and cooling. Recrystallization
from.95% ethanol gave 2.65 g. of colorless needles, mtp.
74°C. Absorption Spectra showed 7’Egiufgfi 5.79 (ester

0 = O). 5.88 (amide C = 0), and no band at about 2.90 (NH);

72 ethanol (95$) 250 (log 6 4.228), 291 (loge: 3.691), and

max (mu)

304 (loge: 3.733).

Anal. Calcd. for 014H13012N03: 0. 53.52; H, 4.17;

N, 4.45. Found: C, 53.91; H, 4.28; N, 4.32.

Ethyl l-(2-Chloropropionyl)indole-3-acetate

f 1“”10320000235 + 03303010001 > . J~-§CH200002H5
‘X A / >3- ‘\ /
“” N ‘ N
H C = 0
CHOl
CH

3

15

A solution of 4.06 g. (0.02 mole) of IAE in 15 ml. of
dry toluene was brought to reflux and the dropwise addition
of 2.6 g. (0.02 mole) of 2-chloropropionyl chloride in 10
m1. of dry toluene begun. The mixture was refluxed for
48 hours, cooled, and the toluene removed in m. The
resulting oil was dissolved in ethanol, treated with
Norite A, and filtered. The ethanol was removed under
reduced pressure, the residue dissolved in hexane-ethanol,
and the solution placed in the cold for three weeks. The
desired product upon recrystallization from ethanol gave
1.04 g. of light yellow platelets, m.p. 63°C. The absorp-
tion spectra showed '2 “1“ n? 5. 75 (ester 0:0), 5.86
(amide c = 0), and no band at about 2.90 (NH); 713§§39355955)
241 (10315 4.421), 292 (10863 3.954). and 301 (loge: 3.988).

Ana, Calcd. for 015H1601N03: C, 61.33; H, 5.49;
N, 4.76. Found: C, 61.21; H, 5.42; N, 4.77.

Ethyl l-(3-Chloropropiony1)indole-3-acetate

;7”\; ~~30H2C0002H5 + 010320320001 --~e [*' H —~:0320000235
J~:..;\.//J’J\ J ~\ //\ /|J
' N ‘ N
H C = O
032
CH201

A solution of 4.06 g. (0.02 mole) of IAE in 15 m1. of
dry toluene was brought to reflux and a solution of 2.6 g.
(0.02 mole) of 3-chloropropiony1 chloride in 10 m1. of
toluene added dropwise. The mixture was refluxed for 48

hours, cooled, and the toluene removed in m. The residue

16.
waa.dissolved.in.ethanol,.treated with.Norite A,.filtered,
.and the.ethanol.removed under reduced.pressnre. The.reaip-
due was.crystallized.frem.ethanolpwater at 0°C. The product
.upon recrystallization from.ethanol.gaxe 1.6 g. of colorless
:needles, map. 75°C. The absorption.Spectra.showed 8*32iu?§§
=5e74 (ester C = 0), 5.85 (amide C = 0), and no band at about
2.90 (NH); 72:2:a?;i)(95%) 248 (log 8 4.445), 266 (log 5
4.589). 292 (loge; 3.955). and 300 (logcs 3.964).

Anal. Calcd. for 015H1501N03: C, 61.33; H, 5.49;
N, 4.76. Found: 0, 61.89; H, 5.28; N, 4.97.

Ethyl l-Triphenylmethylindole-3-acetate

L,\J___ECH2COOCQHS + (06H5)301 .l__; [;a\fi;/UCHQGOOCQH5
.9/ \N’ \V, N
H 0(0635)3
To a solution of 4.06 g. (0.02 mole) of IAE in 15 ml.
of dry toluene was added 5.6 g. (0.02 mole) of triphenyl-
methyl chloride. The mixture was refluxed for 18 hours,
cooled, and the toluene removed in m. The residue was
crystallized from ethyl acetate-ethanol at 0°C. Recrystal-
lization of the product from ethyl acetate-ethanol gave
1.16 g. of colorless platelets, m.p. l79-180°C. The ab-
sorption Spectra showed hrgziu?:§ 5.75 (ester 0 = 0), and
no band at about 2.90 (NH); %\;2§a?gi)(95%) 277 (loge:
3.853). 283 (loge: 3.881), and 294 (loge: 3.831).
Anal. Calcd. for C31H27N02: C, 83.56; H, 6.11;
N, 3.14. Found: C, 83.28; H, 6.01; N, 3.46.

l7
Characterization‘gf Compounds

All melting points were obtained on the FisherAJohns
melting point block and are uncorrected. Carbon and hydro-
gen analyses were determined by Spang Microanalytical Lab-
oratory of Ann Arbor, Michigan. Nitrogen analyses were
made using the micro-Dumas method (22) or by Spang Labora-
tory.

Ultraviolet absorption spectra of the compounds, in
95% ethanol, were determined with the Beckman model DK-2
ratio-recording spectrophotometer using matched silica
cells of one centimeter path length.

Infrared Spectra of the compounds, in spectrograde
toluene, were obtained with the Beckman model IRPS double-
beam recording spectrophotometer. A sodium chloride cell
of 0.1 mm. path length was used for the sample and a varie
able path cell, adjusted to cancel out absorption by tolu-

ene, employed in the reference beam.

18
Bi ical Assays
Tomato Ovary Growth

Langlin sglutigg . Known quantities of each compound
were dissolved in 100 m1. of peroxide-free ethyl ether to
give a solution of l x 10"3 molar concentration. One milli-
liter of this solution was mixed with one milliliter of
lanolin in test tubes and the ether removed from the mix-
ture by heating the tubes in a hot water bath. The solu-
tions were stored in the cold until ready for use.

§1§n§,mgteri§ . Tomato plants (var. Michigan-Ohio
hybrid) of comparable size and nutritional status were
grown in the greenhouse. Three ovaries from the first
flower cluster of each plant were emasculated 24 hours
before anthesis and the remaining ovaries were removed
from that cluster. Three replications were utilized for
each treatment.

Applicgtign‘gf 1§nglin sglutions. Approximately 15
mg. of the lanolin solution of the appropriate compound
was applied to each ovary and pure lanolin was used as a
control. The diameter, in millimeters, of each ovary was
measured four days after treatment, and this diameter was
used as a measure of every growth. The mean of the three
ovaries was used as the ovary diameter for one replication.

The means were analyzed using the analysis-of-variance

19
method described by Snedecor (21). The means were further
compared for significant difference using Duncan's (3)
multiple range test.

Bean Petiole Abscission

'glggy mgtgyig . Bean plants (var. Contender) were
grown in the greenhouse with four plants per pet, from
which two of equal size were selected for the experiment.
Pour replications were used for each treatment. One pri-
mary leaf blade of each plant was removed when the first
trifoliate leaf was beginning to unfold from the terminal
bud, leaving about one centimeter of the petiole.

Method 2; treatment. The lanolin solutions of l x 10‘"3

 

u from the previous assay were used. The cut end of the
petiole was covered with the lanolin solution immediately
after deblading, and pure lanolin was used for the control.
To determine when the petioles readily abscissed, they

were tapped with a pencil every 12 hours on the upper sur-
face, using approximately the same pressure for all petioles.
The mean time for abscission to occur in each treatment was
calculated. Using the lanolin control as 100%, the percent
activity of the different treatments compared to control

was determined.

20
Avena Straight Growth

Propagation‘gf,sglgtigns. Sufficient quantities of

each compound to give 200 ml. of 10'4 molar concentration
were dissolved in one milliliter of Tween 20 and adjusted
to volume. Aliquots were diluted to give solutions of
10's, 10'6, 10‘7, and 10"”8 molar concentrations. To 100
ml. of each concentration was added 3 ml. of phosphate-
citrate buffer of pH 5.0 containing sufficient sucrose
,to give a 3% solution of sucrose upon dilution. The stock
buffer solution was made accordingly: 11.97 g. of dibasic
potassium phoSphate, 6.80 g. of citric acid monohydrate,
and 200 g. of sucrose were dissolved in water and adjusted
to a volume of 200 ml. A buffer control containing 3% suc-
rose and a Tween 20 control with buffer using a 0.5% solu-
tion of Tween 20 for the 10'4 H concentrations and appro-
priate dilutions for the other concentrations.
glapt_mateyigl. Brighton cats were submerged in dis-
tilled water in a suction flask and soaked under vacuum
for two hours to remove the natural auxins. The super-
natant water was discarded and the seeds placed on paper
toweling over glass plates. The plates were placed in
germinating dishes, to which a small amount of water had
been added, and covered. The seeds were allowed to germin-
ate in the dark at 25°C. After 24 hours, the seeds were
exposed to two hours of red light to inhibit elongation

21
0f the first internodes. After three days, coleoptiles of
uniform length were selected. This and all subsequent
steps were carried out under red light. Five millimeter
sections were cut from the coleoptiles so as to exclude
the apical 3 or 4 mm. The sections were floated on dis-
tilled water for one hour before treatment.

Methgd gfltyggtment. Ten milliliters of solution
containing the growth substance was placed in a 504ml.
flask, using three replications for each. Eight coleoptile
sections were placed in the solution. The sections were
allowed to elongate in the dark at 25°C. for 24 hours and
the length of the sections in millimeters was measured.
The mean values were expressed as percent of the initial
5 mm. length. The control was taken as an average of the
growth observed for the Tween-buffer control at dilute
concentrations of Tween 20. Inhibition with.0.5% Tween 20

was observed, but was not significant to warrant further

correction of the results.

 

RESULTS AND DISCUSSION

 

RESULTS AND DISCUSSION

The melting points and results of the elemental
analyses for the compounds are summarized in Table I.

The molecular weights and ultraviolet absorption maxima
are given in Table II.

In Figures 1, 2, and 3 are shown the infrared absorp-
tion Spectra of the ethyl indole-3-acetate derivatives
which were synthesized. The important absorption bands
are summarized in Table III. That substitution has oc-
curred at the l-indole position is shown by the absence of
the NH band at about 2.90u, which is the wavelength for the
NH of IAE. Bellamy (1) reports the NH absorption for in-
dole to be at 3491 cm."1 or 2.86u. For all of the compounds,
the ester carbonyl band is present and is found between 5.74u
and 5.81u. The ester carbonyl band is reported to be at
1750-1735 cm.’1 or 5.71-5.76u. For those compounds which
are acylated at the l-indole position, the amide carbonyl
band appears between 5.85u and 5.93u. Bellamy reports this
band as being at 1690 cm."1 or 5.9lu for those tertiary
amides in which a phenyl group is substituted on the nitro-
gen atom.

The ability of these derivatives to induce the growth
of parthenocarpic fruit was measured in the tomato ovary
assay. The results of this assay are summarized in Table

IV. A comparison of themean values of ovary diameter

shows that IAE and dichloroacetyl-IAE are more active in
23

 

24

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aa.e mm.m mm.ao oa.e me.m nn.ae ma oeeceoennuoaoeaaAaaaoaaoaaoaoaaounv1H Haaea
ea.s me.m Hm.ao ma.a ma.m nn.ae no oeeeoeeunieaoesa“Haaoaaoaaoaoaaoumvua Hanna
«n.e mw.a am.nm as.e aa.e mm.mm as owesooeunnoaoeaaaaecasewoaaoanua Harem
em.e ma.m mo.mm oo.m so.m Ha.oo maaumaa easeooeun-oaoeadaapoosoaoaaoua Hanan
ma.m mm.o om.me Ha.m ea.e mm.mo om oeeeooounuoaoeaaaaeooqua Heaps
IIFLIIIIcII :llbll H.wwmwm.mwou unsoaaoo
roach .uoamo wsdpao:
meadoa800 oHouaH no mamhaosm HaemoSoHo was mpaaoa wqapaoz .H capes

25

.Hocafianm .m
.vaobaom as cow: was Ammmv Hosanna .H

 

 

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eam.n con

mmm.n mom

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mmm.n Hon

smm.n «mm

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nna.n eon

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nnw.m con

Rafi 8m

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saa.e mmm

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w mag .28 a.“ K unmask

Isiah-man? 8988: 8838

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moddenfioo cacao“ no moapmanoposammo Soavauomnm poacabanpab .HH capes

26

Figure 1

Infrared absorption Spectra in toluene of
ethyl indole-3-acetate (top), ethyl l-
chloroacetylindole-3-acetate (center), and
ethyl l-dichloroacetylindole-3-acetate.

 

 

 

W

 

27

 

28

Figure 2

Infrared absorption spectra in toluene of
ethyl l-(2-chloropropionyl)indole-3-acetate
(top), ethyl l-(3-chloropropiony1)indole-3-
acetate (center), and ethyl l-triphenylmethyl-
indole-3-acetate.

 

29

3;—
80

.— ..,..

9.0.5.! 1. Tact-nu)...» _
a o

2.1::

I. I... l:

2.. .2. Z... I... 1.

g.

8: 8:
3'0 55')"

a".

:1:

I: :2:
88

 

z...
88

 

L2: .2.
89 88

30

Figure 3

Infrared absorption Spectrum in toluene
of ethyl l-acetylindole-3—acetate.

 

 

31

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O— O O

110 is!

g—

8»—

 

32

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em.m me.m 1- compose-n1oaoeaaaHasedaoaaoaoaao-mv1a Hanan
mm.m ma.m - ope»see-n-oaoeaaaapooeoSoaaoaa-H Hanna
em.m ma.m - opusoos-n-oaoeadaaeooeonoaao-H Harem
mm.m Hm.m -1 oversee-n1oaoesHaaeeoa-H Harem

-1 . ma.m om.m oneness-n-oaoeaH Harem

cnSoQSoo

 

 

 

 

 

monsoSSoo oHous« Ho newsmanopomnsno Sodpanonno consnmsH .HHH canoe

33
this system than IAA. Acetyl-IAE and chloroacetyl-IAE possess

the same order of activity as does IAA. However, 2- and 3-
chloropropionyl-IAE, 4-nitrobenzoyl-IAE3, and 4-aminobenzoyl-
IAE3 did not Show ability to set parthenocarpic fruit. These
compounds with larger substituent groups are perhaps inactive
because they are not able to penetrate the ovary tissue.

In the bean petiole abscission assay, the capacity of
the compounds to delay abscission of the debladed petiole
was measured. The results of this assay are shown in
Table IV. Dichloroacetyl-IAE was the most active compound
in this assay. Acetyl-IAE, chloroacetyl-IAE, and 4-nitro-
benzoyl-IAE were slightly more active than both IAA and IAE.
However, 2- and 3-chlcropropionyl-IAE, and 4-aminobenzoyl-
IAE were not more active than IAA or IAE. In this system,
IAE appears to be somewhat more active than IAA, as it is in
the other assays. I

The'Ayggg straight growth test measures the capacity
of the compounds to induce cell elongation. The coleoptile
elongation in solutions of the various compounds is com-
pared in Figure 4. The greater activity of IAE over IAA
in the induction of cell elongation has been reported by
Nitsch and Nitsch (18). All of the other derivatives as-
sayed showed activity greater than that of IAA, and were
as active as the parent compound, IAE. At 10'7H, the two
acyl derivatives with no halogen on the carbon adjacent to

3. Kindly supplied by Mrs. Richard Titus.

34

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.HoSoH am one as psonomwae handsoaudswam pom unsaved no Hopped name he eokoaaon under .N
.pdoSSdoap nevus whee noon scanners unoposdaads ad haspo no noposan .H

 

 

 

 

 

    

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com on a.» oeeeoee-n-oaeeaaHaeoooowoaaean-H Harem
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«ea es a.m essence-n-oaoeaaaaeoea1a Harem
mna he a.» spouses-n-eaoeaH Heaps
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com a n.m oaoz
HOHPfloo .HO mama HO m05dd>

mm hpabapo

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m mamaum1muauq1mmaaqw

abound scammacmns eaoapoa some use mvmeuw humbo 09880» no madamom .>H canoe

 

35

Figure 4

Growth curves of Avena coleoptile sections
in various treating solutions: (1) IAA,
(2) IAE (3) acet yl-IAE, (4) chloroacetyl-
IAE- (55 dichloroacetyl-IAE, (6) 2-chloro-
ropionyl-IAE, (7) 3-chloropro ionyl-IAE,
(8) 4-nitrobenzoy1-IAE, and (9 4-amino-
benzoyl-IAE. Control represents the Tween
20 control as described in the text.

 

 

 

 

 

 

 

 

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L L L L L L L L L L L L L L L L L L L L L
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IkGZML HEEL. to N

10‘7 10"5 10‘51 10‘4 10"8 (0'7 10"5 (0'5 10‘4
MOLAR CONCENTRATION

10"8

37
the amide carbonyl were slightly less active than those with
a halogen at this position. This is demonstrated by acetyl-
IAB and 3-chloropropionyl-IAE. However, such an effect is
not true with the two benzoyl derivatives.

The biological activity of these indole derivatives
indicates that substitution at the l-position does not affect
the activity observed with IAE in the‘Aygng_straight growth
test. However, further experiments must be made to determine
whether the molecule remains intact during the growth period.
Further work is also necessary to decide if other substi-
tuents at the l-indole position may increase or decrease
the auxin activity observed with the cell elongation assay.

It is apparent from the three essays that substitution
on the 1-indole position does not alter the activity ob-
served with IAE, but that the alteration of activity is due

to the Specific group substituted.

SUMMARY

SUMMARY

The methods of synthesis and the biological activity
for several ethyl l-acylindole-3-acetates, and the synthesis
of ethyl l-triphenylmethylindole-3-acetate are given. The
following acyl derivatives were prepared:

Ethyl l-Acetylindole-3-acetate

Ethyl l-Chloroacetylindole-3-acetate

Ethyl l-Dichloroacetylindole-3-acetate

Ethyl l-(2-Chloropropionyl indole-3-acetate

Ethyl 1-(3-Chloropropionyl indole-3-acetate

The melting points, and the ultraviolet and infrared
absorption Spectra were determined for the compounds.
Results of the elemental analysis for carbon, hydrogen,
and nitrogen are also reported.

In addition to the above compounds, ethyl l-(4-nitro-
benzoyl)ind01e-3-acetate and ethyl l-(4-aminobenzoyl)-
indole-3-acetate were assayed for biological activity.

The acetyl, chloroacetyl, and dichloroacetyl derivatives
were active in the tomato ovary growth and the bean petiole
abscission assays, and the nitrobenzoyl derivative showed
activity in the latter. All of the compounds showed
activity in the Ayggg straight growth test, and were as

active as ethyl indole-3-acetate.

39

BIBLIOGRAPHY

BI BLI OGRAPHY
1. Bellamy, L. J., "The Infra-red Spectra of Complex
Molecules," John Wiley & Sons, New York, 1958.
2. Bentley, J. A., Ann. Rev. Plant Physiol. 2, 47 (1958).
3. Duncan, D. B., Biometrics g;, 1 (1955).

4. Findlay, S. P. and G. Dougherty, J. Biol. Chem.
183. 361 (19505.

5. Haagen-Smit, A. J., W. D. Leech, and W. R. Bergen,
Amer. Jour. Bot. 22. 500 (1942).

6. Hamilton, R. H., A. Kivilaan and J. M. McManus,
Plant Physiol.,fifi, 136 (1960).

7. Hansch, 0., and R. M. Muir, Plant Physiol. 25, 389 (1950).

8. Hansch, C., R. M. Muir, and R. L. Metzenberg, Jr.,
Plant Physiol. .26, 812 (1951).

9. Hofert, J. P., and H. M. Sell, J. Org. Chem. 25,
1831 (1960).

10. Hoffman, O. L., S. W. Fox, and M. W. Bullock, J. Biol.
Chem. 1 6, 437 (1952)-

11. Jackson, H. H., J. Biol. Chem. 88, 659 (1930).

12. Koepfli, J. B., K. V. Thimann and F. W. Went,
J. Biol. Chem. 122, 763 (1938)

13. KBgl, P., A. J. Haagen-Smit, and H. Erxleben, '
z. Physiol. Chem. 28, 90 (1934).

14. mlir R. H., and C. Hansch, Plant Physiol. 35.
218 (1953).

15. Muir, R. M., C. H. Hansch, and A. H. Gallup,
Plant Physiol. 24. 359 (1949)

16. Nametkin, S. 8., N. N. Mel'nkov, and K. S. Bokarev,
Zhur. Pricklad. Khim. 22, 459 (1956).

17. NitSCh, J. P. and C. Nitsch, Beitr. Biol. Pflanz.
11. 387 (1955).

18. Nitsch, J. P., and C. Nitsch, Plant Physiol. 11,
94 (1956).

41

42

19. Redemann, C. T., S. H. Wittwer, and H. M. Sell,
Arch. Biochem. Biophys.,fig, 80 (1951). »

20. Sell, H. H., S. H. Wittwer, T. L. Rebstock, and
C. T. Redemann, Plant Physiol. 8, 481 (1953).

21. Snedecor, G. H., "Statistical Methods," 4th ed.,
The Iowa State College Press, Ames, Iowa, 1946.

22. Steyermark, A., "Quantitative Organic Microanalysis,"
The Blakiston Company, New York, 1951.

23. Thimann, K. V., Plant Physiol. 23, 311 (1958).

24. Tukey, H. 3., F. 11. vent, R. x. Muir, and J. van
Overbeek, Plant Physiol. 2.2- 307 (1954)-

25. Veldstra, H., Enzymologia L}.- 97 (1942).

26. Veldstra, H. and H. L. Booij, Biochem. Biophys.
Acta 1, 278 (1949).

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