Synthesis and biological evaluation of 2,5-di(7-indolyl)-1,3,4-oxadiazoles, and 2- and 7-indolyl 2-(1,3,4-thiadiazolyl)ketones as antimicrobials

Article abstract of DOI:10.1016/j.bmc.2014.01.025

A range of novel hydrazine bridged bis-indoles was prepared from readily available indole-7-glyoxyloylchlorides and 7-trichloroacetylindoles and underwent cyclodehydration to produce 2,5-di(7-indolyl)-1,3,4-oxadiazoles and a 2,2′-bi-1,3,4-oxadiazolyl with

Full text of DOI:10.1016/j.bmc.2014.01.025

Bioorganic & Medicinal Chemistry 22 (2014) 1672–1679
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 2,5-di(7-indolyl)-1,3,
4-oxadiazoles, and 2- and 7-indolyl 2-(1,3,4-thiadiazolyl)ketones
as antimicrobials
Hakan Kandemir a,d, Cong Ma b, Samuel K. Kutty a, David StC. Black a, Renate Griffith c, Peter J. Lewis b,
Naresh Kumar a,
⇑
a School of Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia
b School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia
c School of Medical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia
d School of Chemistry, Faculty of Art and Science, Namık Kemal University, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
A range of novel hydrazine bridged bis-indoles was prepared from readily available indole-7-glyoxyloyl-
chlorides and 7-trichloroacetylindoles and underwent cyclodehydration to produce 2,5-di(7-indolyl)-
1,3,4-oxadiazoles and a 2,20-bi-1,3,4-oxadiazolyl with phosphoryl chloride in ethyl acetate. This efficient
protocol was subsequently used for the synthesis of 2- and 7-indolyl 2-(1,3,4-thiadiazolyl)ketones from
related indolyl-hydrazine carbothioamides. The synthesised bis-indoles were evaluated for their antimicro-
bial properties, particularly the inhibition of protein–protein complex formation between RNA polymerase
Received 24 November 2013
Revised 9 January 2014
Accepted 17 January 2014
Available online 31 January 2014
Keywords:
Bis-indoles
Oxadiazoles
and
r
factor and their bactericidal effect on Gram positive Bacillus subtilis and Gram negative Escherichia coli.
Ó 2014 Elsevier Ltd. All rights reserved.
Thiadiazoles
Indole hydrazides
Antimicrobial
RNA polymerase
1. Introduction
pounds such as the 3-indolylglyoxylamide 3 have been shown to
possess strong antibacterial activity against Gram-negative and
The 1,3,4-oxadiazoles and 1,3,4-thiadiazoles are unique hetero-
cyclic systems with importance in synthetic, medicinal and materi-
Gram-positive bacteria.12 We have also reported that a 7,70-bis-
indolylcarbohydrazide inhibit transcription initiation complex for-
als chemistry.1 These five-membered heterocycles play
a
mation by preventing the unique bacterial r initiation factor from
particularly vital role in medicinal chemistry because they display
a variety of biological activities,2–5 possess a favourable metabolic
profile, and have a propensity to form hydrogen bonds. In particu-
lar, these ring systems have been found in marketed antihyperten-
sive agents such as tiodazosin and nesapidil, antibiotics such as
furamizole, and the carbonic anhydrase inhibitor acetazolamide.6–9
Indole linked oxadiazoles and thiadiazoles are novel classes of
compounds that also exhibit a range of interesting biological activ-
ities. For example, the recently developed 2-(3-indolyl)-1,3,4-oxa-
diazole 1 has been screened as a potent anticancer agent and
showed inhibitory activity against prostate and pancreatic cancer
cell lines,10 while the 2-(3-indolyl)-1,3,4-thiadiazole 2 displays sig-
nificant cytotoxic activity against pancreatic cancer cell lines.11
There has been much recent interest in bis-indole amides and
glyoxylamides as potent antibacterial agents. For example, com-
binding to RNA polymerase.13 It was therefore of interest to inves-
tigate the nature of the amide groups attached to the indole units
and subsequent cyclisation of diamides and thiosemicarbazides to
their bioisosters oxadiazoles and thiadiazoles respectively.
OBn
N
OMe
N
N
N
O
N
S
Br
N
H
N
H
2
1
HN
Br
O
N
Br
O
N
N
H
H
⇑
Corresponding author. Tel.: +61 2 9385 4698; fax: +61 2 9385 6141.
E-mail address: n.kumar@unsw.edu.au (N. Kumar).
3
http://dx.doi.org/10.1016/j.bmc.2014.01.025
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

H. Kandemir et al. / Bioorg. Med. Chem. 22 (2014) 1672–1679
1673
R1
R1
Given the importance of oxadiazoles and thiadiazoles, and the
extensive scope available for the development of novel indole
linked oxadiazoles and thiadiazoles, we were interested in the
development of 2,5-di(7-indolyl)-1,3,4-oxadiazoles, and 2- and 7-
indolyl 2-(1,3,4-thiadiazolyl)ketones. A range of synthetic ap-
proaches to 1,3,4-oxadiazoles have been reported in the literature,
with many of these protocols involving the cyclization of acylhyd-
razides using harsh reagents such as thionyl chloride,14 triflic
anhydride15 and phosphoryl chloride.16 Mild cyclodehydration re-
agents such as Burgess reagent,17 4-methylbenzenesulfonyl chlo-
ride (TsCl)18 and propylphosphonic anhydride (T3P)19 have also
been used. It was therefore anticipated that the target structures
could be prepared via cyclization of indole-7-acylhydrazines,
which in turn could be synthesized from methoxy activated in-
doles which are capable of undergoing reaction at the otherwise
unreactive C7 position.20,21
MeO
MeO
a
R2
R2
27-69%
N
H
N
H
MeO
MeO
Cl3C
O
4
5
R1 = H, R2 = Ph
R1 = Cl, R2 = H
4-7a
4 7b
-
b
49-81%
R1
4-7c
R
1 = Br, R2 = H
R1
MeO
MeO
O
R2
N
H
c
MeO
HN
R2
MeO
MeO
N
H
O
O
71-82%
N
N
HN
2. Results and discussion
H
H
N
MeO
N
R2
R2
2.1. Synthesis of 2,5-di(7-indolyl)-1,3,4-oxadiazoles
MeO
MeO
The preparation of 2,5-di(7-indolyl)-1,3,4-oxadiazoles was
achieved over a convenient three-step process. The first step in-
volved the reaction of activated indoles 4a–c with trichloroacetyl
chloride at reflux for 3 h, which afforded 7-trichloroacetylindoles
5a–c in 27–69% yields (Scheme 1).22 Following this, 7-trichloro-
acetylindoles 5a–c were heated at reflux for 24 h with half an
equivalent of hydrazine hydrate in the presence of triethylamine
in acetonitrile to afford the 7,70-bis-indoles 6a–c in 49–81% yield.
Initial attempts to generate the 2,5-di(7-indolyl)-1,3,4-oxadiaz-
oles 7a–c under mild conditions were unsuccessful. Heating bis-in-
doles 6a–c at reflux with 1.1 equiv of T3P in the presence of
triethylamine in ethyl acetate for 12 h resulted in recovery of the
starting materials. Increasing the amount of T3P or base gave the
same result. Similarly, no reaction was observed upon use of stron-
ger bases such as DBU. It was therefore concluded that T3P was not
strong enough to induce this dehydrative cyclization reaction.
The alternative stronger cyclodehydration reagent phosphoryl
chloride was subsequently investigated. Treatment of bis-indoles
6a–c with neat phosphoryl chloride at either room temperature
or 50 °C resulted in formation of a black tar. Dilution of the reaction
mixture with ethyl acetate as a solvent and heating bis-indoles 6a–
cat reflux for 2 h in the presence of excess phosphoryl chloride
afforded the desired bis-indolyl-1,3,4-oxadiazoles 7a–c in 71–82%
yields after elimination of some baseline impurities by column
chromatography.
R1
R1
7
6
Scheme 1. Reagents and conditions: (a) CCl3COCl, 1,2-dichloroethane, reflux, 3 h;
(b) NH2NH2ꢀH2O, Et3N, CH3CN, reflux, 24 h; (c) POCl3, EtOAc, reflux, 2 h.
Preparation of the symmetrical bis-oxalohydrazide 9a, which
possesses an extended linker between the two indole moieties,
was subsequently carried out by addition of oxalyl chloride to
hydrazide 8a in dichloromethane at room temperature for 1 h.
Triturating the crude product with hot methanol gave the pure
compound 9a in 53% yield.
The tandem cyclodehydration reaction was performed using the
optimized conditions described above. Notably, a prolonged reac-
tion time was required, with the reaction reaching completion
after 24 h. Formation of bi-2,20-(1,3,4-oxadiazolyl) 10 was indi-
cated by the disappearance of the amide nitrogen protons of com-
pound 9a at 9.94 ppm in the 1H NMR spectrum and a shift of the
indole NH protons from 11.11 to 10.79 ppm. The presence of a
molecular ion in the high resolution mass spectrum further con-
firmed the structure.
Similar treatment of 7-trichloroacetylindoles 5b and 5c affor-
ded the corresponding indole-7-carbohydrazides 8b,c and bis-
oxalohydrazides 9b,c. However, the dehydrative cyclization of
these 3-substituted analogues proved to be problematic, resulting
in a mixture of inseparable products.
The next objective of interest was the development of glyoxyl
derivatives related to the di(7-indolyl)-1,3,4-oxadiazoles 7. 4,6-
Dimethoxyindole 4a undergoes exclusive C7-substitution with
oxalyl chloride to afford the 7-glyoxyloyl chloride 11 which can
readily react with a range of amines to produce the corresponding
glyoxyl amides.23 Therefore, treatment of acid chloride 11 with a
half equivalent of hydrazine hydrate in acetonitrile gave the bis-in-
dole glyoxyloyl hyrazide 12 in 83% yield (Scheme 3).
Both bis-indoles 6a–c and 7a–c were poorly soluble in organic
solvents, had high melting points and good stability under nor-
mal laboratory conditions. The 1H NMR spectrum of the com-
pound 7b was characteristic for the oxadiazole compounds,
showing the disappearance of hydrazide nitrogen protons of
compound 6b at 10.54 ppm, and a shift of the indole NH protons
from 11.54 to 11.17 ppm. A high resolution mass spectrum
further confirmed the structure, revealing the anticipated
molecular ion.
2.2. Synthesis of 5,50-di(7-indolyl)bi-2,20-(1,3,4-oxadiazolyl)
compounds
Cyclodehydration of the bis-indole 12 was subsequently at-
tempted under the optimized conditions, however, only recovery
of the starting material was observed. The use of different solvents
such as chloroform, dichloromethane, tetrahydrofuran and neat
phosphoryl chloride at either room temperature or reflux, and
the use of T3P in different solvents were examined but similarly
gave no reaction. Two possible reasons causing the inhibition of
the dehydrative cyclization in the 7,70-linked system 12 are postu-
lated. Firstly, there is potentially a greater steric restriction in the
With the 2,5-di(7-indolyl)-1,3,4-oxadiazoles successfully in
hand, it was of interest to extend the methodology to a related
bi-2,20-(1,3,4-oxadiazolyl) system 10. Treatment of 7-trichloro-
acetylindole 5a at room temperature for 1 h with 1.5 equiv of
hydrazine hydrate in the presence of triethylamine gave the simple
7-carbohydrazide 8a in high yield (Scheme 2).

1674
H. Kandemir et al. / Bioorg. Med. Chem. 22 (2014) 1672–1679
R1
R1
the procedure developed for the 1,3,4-oxadiazoles described
above. Reaction of 7-glyoxylchlorides 11 and 13 with 4,4-di-
methyl-3-thiosemicarbazide in the presence of triethylamine in
acetonitrile gave intermediates 14a and 14b in 75–79% yields
(Scheme 4).
Subsequent cyclization of compounds 14a and 14b with phos-
phoryl chloride in ethyl acetate led to the formation of 7-indolyl
2-(1,3,4-thiadiazolyl)ketones 15a and 15b in 59% and 57% yields,
respectively. The mass spectrum of the products 15a and 15b re-
vealed molecular ions, while their 1H NMR spectra showed the dis-
appearance of the amide NH signals at 10.38 and 9.82 ppm from
the respective precursors.
Similarly, the 2-indolylglyoxyloyl chlorides 16a and 16b reacted
with 4,4-dimethyl-3-thiosemicarbazide to give 55% and 62% yields,
respectively of the intermediates 17a and 17b, which were con-
verted into the 2-indolyl-(1,3,4-thiadiazolyl) ketones 18a and
18b respectively in 33% and 36% yields (Scheme 5).
MeO
MeO
R2
R2
a
N
H
N
H
MeO
Cl3C
MeO
HN
74-85%
O
O
NH2
5
8
5a, 8-9a R1 = H, R2 = Ph
5b, 8 9b
5c, 8 9c
b
53-92%
R1
-
-
R1 = Cl, R2 = H
R1 = Br, R2 = H
MeO
MeO
Ph
MeO
R2
N
H
Ph
N
H
N
N
MeO
HN
2.4. Biological properties of selected compounds
O
O
c
O
O
HN
Significantly, in our previous study compound 6b was found to
inhibit the key protein–protein interaction between bacterial RNA
polymerase (RNAP) and its cognate sigma factor at low micromolar
concentration by specifically binding to the b0-CH region of the
RNAP core.13 Furthermore the peptidomimetic nature of the inter-
mediate bis-amides 6, 9 and 12 and their cyclic bioisosteres 7 and
10 makes them attractive for investigation as inhibitors of bacterial
protein–protein interactions. Therefore, the synthesized com-
pounds were evaluated for their biological properties particularly
N
N
82%
H
N
Ph
HN
HN
O
O
MeO
Ph
H
N
MeO
MeO
R2
MeO
10
R1
9
the inhibition of RNAP–r initiation complex formation via an ELI-
SA assay. Additionally, the antimicrobial properties of these com-
pounds were tested against the Gram positive Bacillus subtilis and
Gram negative Escherichia coli (Table 1). The 2,3-diphenylindole
analogues were not tested due to their poor solubility in the assay
conditions.
The results of the biology assay indicated that the bis-indolyl
systems attached via diamides such as in compounds 6b,c and in-
dole thiosemicarbazides 17a,b were most active in inhibiting
Scheme 2. Reagents and conditions: (a) NH2NH2ꢀH2O, Et3N, CH3CN, rt, 1 h; (b)
oxalyl chloride, Et3N, DCM; rt, 1 h; (c) POCl3, EtOAc, reflux, 24 h.
formation of the 1,3,4-oxadiazole due to the neighbouring glyoxyl
carbonyl group. Alternatively, the strong hydrogen bonding be-
tween the carbonyl groups and NH protons could result in the for-
mation of five or six membered rings which are too stable to be
broken even under vigorous conditions.
RNAP-
tion at late exponential phase. Particularly, compound 6b was most
active with 63% inhibition of RNAP- initiation complex formation
r initiation complex formation and bacterial growth inhibi-
2.3. Synthesis of 2- and 7-indolyl 2-(1,3,4-thiadiazolyl)ketones
r
via an ELISA assay and compound 17b was most active in bacterial
inhibition studies with 70% inhibition of both Gram positive B. sub-
With the apparent difficulties in achieving the cyclodehydration
of the extended glyoxyl system 12, attention turned to the simpli-
fied target system 15. To the best of our knowledge, there are only
limited reports relating to the preparation of indolyl-1,3,4-thi-
adiazoles or indolyl 2-(1,3,4-thiadiazolyl)ketones in the literature.
Recently, the synthesis of 5-(3-indolyl)-1,3,4-thiadiazoles has been
described.11 It was anticipated that the indolyl 2-(1,3,4-oxothi-
adiazolyl)ketones could be synthesized through modification of
MeO
MeO
Cl
Ph
Ph
R
R
Ph
Ph
a
N
MeO
O
N
MeO
O
H
H
75-79%
O
O
HN
NH
11, 13
MeO
Ph
N
S
14
Ph
N
11 14 15a
MeO
O
,
-
R = H
MeO
Cl
b
57-59%
Ph
Ph
H
13 14 15b
R = OMe
,
-
O
O
Ph
a
MeO
NH
NH
N
MeO
O
H
R
83%
O
Ph
N
H
MeO
O
H
N
MeO
N
N
O
11
Ph
S
N
Ph
MeO
15
12
Scheme 3. Reagents and conditions: (a) NH2NH2ꢀH2O, CH3CN, rt, 1 h.
Scheme 4. Reagents and conditions: (a) 4,4-dimethyl-3-thiosemicarbazide, Et3N,
CH3CN, rt, 1.5 h; (b) POCl3, ethyl acetate, reflux, 2 h.

H. Kandemir et al. / Bioorg. Med. Chem. 22 (2014) 1672–1679
1675
R
R
indolyl bis-amides and indole-thiosemicarbazides showed promis-
ing antimicrobial properties. The cyclised derivatives tend to show
decreased activity and this highlights the need for flexible linkers
in order to retain the protein-protein inhibition activity. These
indolyl peptidomimetics will be more biologically stable and con-
siderably cheaper than synthesized peptides. Additionally, there is
the potential for significant chemical modification to enhance their
drug-like and antimicrobial properties (e.g. solubility, uptake). We
are currently investigating the use of these compounds and the
synthesis of further analogues as inhibitors of essential bacterial
protein-protein interactions involved in nucleic acid synthesis
and cell division that are unique to the eubacterial kingdom and
historically have been underutilized for the development of clini-
cally effective antibiotics.
MeO
MeO
a
R
R
O
O
55-62%
N
H
N
H
NH
HN
S
Cl
MeO
MeO
O
O
N
17
16
b
33-36%
R
16 18a
16-18b R = Br
-
R = Cl
MeO
O
N
4. Experimental
N
MeO
S
H
N
4.1. General methods
N
18
Melting points were measured using a Mel-Temp melting point
apparatus and are uncorrected. Infrared spectra were recorded on a
Mattson Genesis Series FTIR spectrophotometer as KBr disks. Ultra-
violet spectra were measured using a Varian Cary 100 spectropho-
tometer. 1H and 13C NMR spectra were obtained in the designated
solvents on a Bruker DPX 300. High-resolution mass spectrometry
was performed by the Mass Spectrometry unit at the Bioanalytical
Mass Spectrometry unit in the school of chemistry, UNSW. Micro-
analysis was performed on a Carlo Erba Elemental Analyzer EA
1108 at the Campbell Microanalytical Laboratory, University of
Otago, New Zealand. Anhydrous solvents were obtained using a
PureSolv MD Solvent Purification System. Ajax Finechem Silica
200–325 mesh was used for column chromatography and Merck
silica gel 60H was used for flash chromatography.
Scheme 5. Reagents and conditions: (a) 4,4-dimethyl-3-thiosemicarbazide, Et3N,
CH3CN, rt, 1.5 h; (b) POCl3, ethyl acetate, reflux, 2 h.
Table 1
Biological screening against RNAP-
inhibition
r protein–protein interaction and bacterial growth
Compd % In vitro binding inhibition
% In vivo bacterial growth
ELISA (15
lM)
inhibition at late exponential phase
(200
lM)
B. subtilis (Gram
E. coli (Gram
positive)
negative)
6b
6c
7b
7c
17a
17b
18a
18b
62.63
39.54
17.83
16.75
20.44
20.83
17.23
18.98
29.80
14.29
NA
NA
37.37
NA
NA
NA
4.1.1. N,N0-Bis(4,6-dimethoxy-2,3-diphenyl-1H-indole-7-
carbonyl) hydrazide (6a)
32.48
69.87
NA
46.96
70.01
10.59
16.80
To a solution of 7-trichloroacetylindole 5a (0.5 g, 1.05 mmol) in
acetonitrile (20 mL), hydrazine hydrate (0.028 mL, 0.57 mmol) fol-
lowed by triethylamine (5 drops) was added and the mixture was
heated under reflux for 24 h. The resulting precipitate was filtered,
washed with acetonitrile and water to afford the title compound
(0.22 g, 57%) as a brown solid. Mp >320 °C. 1H NMR (300 MHz,
CDCl3): d 3.80 (s, 6H, OMe), 4.19 (s, 6H, OMe), 6.28 (s, 2H, H5),
7.20–7.42 (m, 20H, aryl H), 10.94 (s, 2H, NH), 11.12 (br s, 2H,
NH). IR (KBr): mmax 3392, 2359, 1594, 1453, 1346, 1226, 1173,
NA
NA-no activity.
tilis and Gram negative E. coli. These results, in general, suggest
that cyclisation of diamides 6 and thiosemicarbazides 17 to their
bioisosteric oxadiazoles 7 and thiadiazoles 18 respectively de-
creases protein–protein inhibition activity. This trend in activity
suggests that the need for hydrogen bond donors and a degree of
flexibility in the linker are crucial for activity. It is also interesting
to note that compounds 6b,c have high in vitro (ELISA) inhibition
but low in vivo inhibition activity, whereas compound 17b has
high in vivo inhibition activity but low in vitro (ELISA) inhibition.
This could be attributed to low cell permeation of 6b,c and possible
non-specific interaction with other cellular targets of 17b. Further
structural modification and biological studies will be required to
establish the correlation between the ELISA binding assay and
the bacterial inhibition assay of these bis-indolyl and indole-thio-
semicarbazide systems.
1143, 991, 752, 699 cmꢁ1
102,850 cmꢁ1 Mꢁ1), 250 (120,100), 323 (109,600). HRMS (+ESI):
. UV–vis (CH2Cl2): kmax 227 nm (e
C
48H38N4O6 [M]+ requires 742.2791, found 742.2782. The sample
was not soluble enough for 13C NMR measurement.
4.1.2. N,N0-Bis(3-(4-chlorophenyl)-4,6-dimethoxy-1H-indole-7-
carbonyl) hydrazide (6b)
To a solution of 7-trichloroacetylindole 5b (0.372 g, 0.86 mmol) in
acetonitrile (25 mL), hydrazine hydrate (0.021 mL, 0.43 mmol) fol-
lowed by triethylamine (5 drops) was added and the mixture was
heated under reflux for 24 h. The resulting precipitate was filtered,
washed with acetonitrile and water to afford the title compound
(0.23 g, 81%) as a brown solid. Mp >300 °C. 1H NMR (300 MHz,
DMSO-d6): d 3.95 (s, 6H, OMe), 4.15 (s, 6H, OMe), 6.59 (s, 2H, H5),
7.28 (d, J = 2.1 Hz, 2H, H2), 7.41, 7.56 (2d, J = 8.5 Hz, 8H, aryl H),
10.54 (s, 2H, NH), 11.54 (br s, 2H, NH). IR (KBr): mmax 3388, 1592,
3. Conclusion
An efficient methodology for the synthesis of 2,5-di(7-indolyl)-
1,3,4-oxadiazoles was developed and the tandem ring closure of a
di(7-indolyl)-oxalohydrazide to a 5,50-di(7-indolyl)-bi-2,20-(1,3,4-
oxadiazolyl) compound was achieved for a 2,3-diphenyl-substi-
tuted indole, but was not general. Adaptation of this synthetic
methodology also led to the successful synthesis of monomeric
2- and 7-indolyl 2-(1,3,4-thiadiazolyl)ketones. The peptidomimetic
1448, 1340, 1213, 1151, 792 cmꢁ1. UV–vis (THF): kmax 243 nm (
e
86,634 cmꢁ1 Mꢁ1), 279 (45,830), 348 (57,198), 364 (42,599). HRMS
(+ESI): C34H28Cl2N4O6 [M+Na]+ requires 681.1284, found 681.1273.
The sample was not soluble enough for 13C NMR measurement.

1676
H. Kandemir et al. / Bioorg. Med. Chem. 22 (2014) 1672–1679
4.1.3. N,N0-Bis(3-(4-bromophenyl)-4,6-dimethoxy-1H-indole-7-
carbonyl) hydrazide (6c)
(0.068 g, 71%) as a
brown solid. Mp 299–301 °C. 1H NMR
(300 MHz, CDCl3): d 3.93 (s, 6H, OMe), 4.07 (s, 6H, OMe), 6.63 (s,
2H, H5), 7.37 (d, J = 2.5 Hz, 1H, H2), 7.50, 7.53 (2d, J = 8.9 Hz, 8H,
aryl H), 11.18 (d, J = 2.2 Hz, 2H, NH). IR (KBr): mmax 3353, 1596,
1537, 1484, 1411, 1332, 1214, 1084, 985 cmꢁ1. UV–vis (THF): kmax
To a solution of 7-trichloroacetylindole 5c (0.6 g, 1.25 mmol) in
acetonitrile (20 mL), hydrazine hydrate (0.03 mL, 0.43 mmol) fol-
lowed by triethylamine (5 drops) was added and the mixture
was heated under reflux for 24 h. The resulting precipitate was fil-
tered, washed with acetonitrile and water to afford the title com-
pound (0.23 g, 49%) as a pale brown solid. Mp >300 °C. 1H NMR
(300 MHz, DMSO-d6): d 3.92 (s, 6H, OMe), 4.11 (s, 6H, OMe), 6.56
(s, 2H, H5), 7.24 (d, J = 2.5 Hz, 2H, H2), 7.46, 7.50 (2d, J = 8.8 Hz,
8H, aryl H), 10.50 (s, 2H, NH), 11.51 (d, J = 2.3 Hz, 2H, NH). IR
239 nm (
(34,550). HRMS (+ESI):
e
56,900 cmꢁ1 Mꢁ1), 284 (30,900), 358 (43,800), 375
C
34H2679/81Br2N4O5 [M+H]+ requires
731.0328, found 731.0324.The sample was not soluble enough
for 13C NMR measurement.
4.1.7. 4,6-Dimethoxy-2,3-diphenyl-1H-indole-7-carbohydrazide
(8a)
(KBr):
242 nm (
m
max 3389, 1589, 1448, 1340, 1212 cmꢁ1. UV–vis (THF): kmax
e
65,200 cmꢁ1 Mꢁ1), 281 (34,450), 348 (42,600), 365
To a solution of 7-trichloroacetylindole 5a (1.06 g, 2.46 mmol)
in anhydrous acetonitrile (50 mL), hydrazine hydrate (0.167 mL,
3.44 mmol) was added followed by triethylamine (5 drops) and
the mixture was stirred at room temperature for 1 h. Water was
added to quench the reaction, the resulting precipitate was filtered
and recrystallised from ethanol to afford the title compound (0.72 g,
85%) as a light brown solid. Mp 224–226 °C. 1H NMR (300 MHz,
CDCl3): d 3.79 (s, 3H, OMe), 4.04 (s, 3H, OMe), 6.23 (s, 1H, H5),
7.24–7.45 (m, 10H, aryl H), 9.09 (s, 1H, NH), 11.21 (br s, 1H, NH).
13C NMR (75 MHz, CDCl3): d 55.71, 57.12 (OMe), 87.82 (C5),
126.48, 127.52, 127.83, 128.15, 128.34, 128.50, 128.83, 131.90
(aryl CH), 96.24, 114.17, 114.22, 133.00, 133.61, 136.26, 138.78,
157.07, 158.21 (aryl C), 169.10 (C=O). IR (KBr): mmax 3392, 1620,
1596, 1494, 1462, 1235, 1112, 991, 700 cmꢁ1. UV–vis (MeOH): kmax
249 (36,050 cmꢁ1 Mꢁ1), 319 (23,300). HRMS (+ESI): C23H22N3O3
[M+H]+ requires 388.1661, found 388.1656.
(31,550). HRMS (+ESI): C34H28Br2N4O6 [M+Na]+ requires
769.0273, found 769.0265. The sample was not soluble enough
for 13C NMR measurement.
4.1.4. 2,5-Di(4,6-dimethoxy-2,3-diphenyl-1H-indol-7-yl)-1,3,4-
oxadiazole (7a)
To a solution of bis-indole 6a (0.11 g, 0.14 mmol) in ethyl ace-
tate (30 mL), POCl3 (15 mL) was added and the solution was heated
under reflux for 2 h. The solvent was evaporated under reduced
pressure and the remaining residue was quenched with water
and made alkaline by the addition of 5 N NaOH (30 mL). The result-
ing precipitate was collected by filtration, washed with water,
dried and recrystallised from methanol to afford the title compound
(0.09 g, 82%) as a white solid. Mp >320 °C. 1H NMR (300 MHz,
CDCl3): d 3.82 (s, 6H, OMe), 4.04 (s, 6H, OMe), 6.38 (s, 2H, H5),
7.24–7.45 (m, 20H, aryl H), 10.79 (br s, 2H, NH). IR (KBr): mmax
3380, 3353, 1598, 1489, 1351, 1328, 1220, 1147, 692 cmꢁ1. UV–
4.1.8. 3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indole-7-
carbohydrazide (8b)
vis (CH2Cl2): kmax 247 nm (
380 (65,425). HRMS (+ESI):
e
105,018 cmꢁ1 Mꢁ1), 343 (90,292),
C
46H36N4O5 [M+H]+ requires
To a solution of 7-trichloroacetylindole 5b (1.06 g, 2.46 mmol)
in anhydrous acetonitrile (50 mL), hydrazine hydrate (0.167 mL,
3.44 mmol) was added followed by triethylamine (5 drops) and
the mixture was stirred at room temperature for 1 h. Water was
added to quench the reaction, the resulting precipitate was filtered
and recrystallised from methanol to yield the title compound
(0.72 g, 85%) as a light brown solid. Mp 203–205 °C. 1H NMR
(300 MHz, CDCl3): d 3.91 (s, 3H, OMe), 4.06 (s, 3H, OMe), 6.28 (s,
1H, H5), 7.16 (d, J = 2.4 Hz, 1H, H2), 7.33, 7.53 (2d, J = 8.5 Hz, 4H,
aryl H), 9.08 (s, 1H, NH), 11.11 (br s, 2H, NH). 13C NMR (75 MHz,
CDCl3): d 55.64, 57.16 (OMe), 87.65 (C5), 122.48 (C2), 128.13,
131.12 (aryl CH), 96.55, 111.31, 117.21, 131.99, 134.72, 139.69,
157.17, 157.85 (aryl C), 168.96 (C=O). IR (KBr): mmax 3372, 3347,
1621, 1590, 1462, 1346, 1214, 1093 cmꢁ1. UV–vis (MeOH): kmax
238 (39,400 cmꢁ1 Mꢁ1), 309 (18,200). HRMS (+ESI): C17H16ClN3O3
[M+H]+ requires 346.0958, found 346.0953.
725.2764, found 725.2761. The sample was not soluble enough
for 13C NMR measurement.
4.1.5. 2,5-Di(3-(4-chlorophenyl)-4,6-dimethoxy-1H-indol-7-yl)-
1,3,4-oxadiazole (7b)
To a solution of bis-indole 6b (0.109 g, 0.16 mmol) in ethyl ace-
tate (30 mL), POCl3 (10 mL) was added and the solution was heated
under reflux for 2 h. The solvent was evaporated under reduced
pressure and the remaining residue was quenched with water
and made alkaline by the addition of 5 N NaOH (30 mL). The result-
ing precipitate was collected by filtration, washed with water,
dried and purified by flash chromatography using dichlorometh-
ane/ethyl acetate (90:10) as eluent to afford the title compound
(0.079 g, 74%) as a pale yellow solid. Mp 303–304 °C. (Found: C,
62.32; H, 4.10; N, 8.35; C34H26Cl2N4O5 0.2 CH2Cl2 requires C,
62.38; H, 4.04; N, 8.51). 1H NMR (300 MHz, CDCl3): d 3.93 (s, 6H,
OMe), 4.06 (s, 6H, OMe), 6.63 (s, 2H, H5), 7.37 (d, J = 2.5 Hz, 1H,
H2), 7.40, 7.57 (2d, J = 8.6 Hz, 8H, aryl H), 11.17 (d, J = 2.5 Hz, 2H,
NH). IR (KBr): mmax 3352, 1596, 1411, 1332, 1214, 1149, 1084,
4.1.9. 3-(4-Bromophenyl)-4,6-dimethoxy-1H-indole-7-
carbohydrazide (8c)
A solution of 7-trichloroacetylindole 5c (0.499 g, 1.05 mmol) in
anhydrous acetonitrile (20 mL), hydrazine hydrate (0.064 mL,
1.31 mmol) was added followed by triethylamine (3 drops) and
the mixture was stirred at room temperature for 1 h. Water was
added to quench the reaction, the resulting precipitate was filtered
and recrystallised from ethanol to afford the title compound (0.3 g,
74%) as a pale yellow solid. Mp 215–217 °C. 1H NMR (300 MHz,
CDCl3): d 3.87 (s, 3H, OMe), 4.02 (s, 3H, OMe), 6.25 (s, 1H, H5),
7.12 (d, J = 2.4 Hz, 1H, H2), 7.43, 7.47 (2d, J = 8.9 Hz, 4H, aryl H),
9.04 (s, 1H, NH), 11.07 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3):
d 55.13, 56.67 (OMe), 87.19 (C5), 121.95 (C2), 130.56, 130.98 (aryl
CH), 96.07, 110.78, 116.72, 119.63, 134.69, 139.21, 156.68, 157.35
985 cmꢁ1. UV–vis (THF): kmax 247 nm ( 64,600 cmꢁ1 Mꢁ1), 282
e
(35,000), 358 (51,300), 375 (40,400). HRMS (+ESI): C34H26Cl2N4O5
[M+H]+ requires 641.1359, found 641.1348. The sample was not
soluble enough for 13C NMR measurement.
4.1.6. 2,5-Di(3-(4-bromophenyl)-4,6-dimethoxy-1H-indol-7-yl)-
1,3,4-oxadiazole (7c)
To a solution of bis-indole 6c (0.101 g, 0.13 mmol) in ethyl ace-
tate (30 mL), POCl3 (10 mL) was added and the solution was heated
under reflux for 2 h. The solvent was evaporated under reduced
pressure and the remaining residue was quenched with water
and made alkaline by the addition of 5 N NaOH (30 mL). The result-
ing precipitate was collected by filtration, washed with water,
dried and purified by flash chromatography using dichlorometh-
ane/ethyl acetate (90:10) as eluent to afford the title compound
(aryl C), 168.44 (C=O). IR (KBr): mmax 3363, 3344, 1621, 1588, 1463,
1344, 1214, 1093 cmꢁ1. UV–vis (MeOH): kmax 237 (30,100 cmꢁ1
-
Mꢁ1), 306 (14,200). HRMS (+ESI): C17H1679BrN3O3 [M+Na]+ requires
412.0273, found 412.0267.

H. Kandemir et al. / Bioorg. Med. Chem. 22 (2014) 1672–1679
1677
4.1.10. N,N0-Bis(4,6-dimethoxy-2,3-diphenyl-1H-indole-7-
carbonyl)oxalohydrazide (9a)
4.1.13. 5,50-Bis(4,6-dimethoxy-2,3-diphenyl-1H-indol-7-yl)-2,20-
bi-(1,3,4-oxadiazolyl) (10)
Oxalyl chloride (0.04 mL, 0.46 mmol) in dry dichloromethane
(5 mL) was added dropwise to a solution of 7-carbohydrazide 8a
(0.34 g, 0.87 mmol) in dry dichloromethane (10 mL) containing tri-
ethylamine (0.12 mL, 0.87 mmol). The reaction mixture stirred at
room temperature for 1.5 h. The solvent was then evaporated and
the residue was quenched with water. The resulting precipitate
was filtered, dried and recrystallised from methanol to yield the title
compound (0.191 g, 53%) as a pale yellow solid. Mp >300 °C. (Found:
C, 68.65; H, 4.93; N, 9.91; C48H40N6O8. 0.2 CH2Cl2 requires C, 68.44;
H, 4.81; N, 9.94). 1H NMR (300 MHz, DMSO-d6): d 3.74 (s, 6H, OMe),
4.05 (s, 6H, OMe), 6.45 (s, 2H, H5), 7.21–7.28 (m, 20H, aryl H), 9.94 (s,
2H, NH), 11.11 (s, 2H, NH). 13C NMR (75 MHz, DMSO-d6): d 55.86,
57.15 (OMe), 88.76 (C5), 126.67, 127.59, 127.72, 127.85, 128.98,
131.45 (aryl CH), 95.84, 112.99, 114.00, 132.30, 132.34, 135.85,
137.93, 157.34, 158.08 (aryl C), 158.95, 165.90 (C=O). IR (KBr): mmax
3379, 1629, 1594, 1430, 1351, 1238, 1144, 696 cmꢁ1. UV–vis (THF):
kmax 248 (67,800 cmꢁ1 Mꢁ1), 332 (60,250). HRMS (+ESI):
To a solution of bis-indole 9a (0.094 g, 0.11 mmol) in ethyl ace-
tate (30 mL), POCl3 (15 mL) was added and the solution was heated
under reflux for 24 h. The solution was poured slowly into ice-
water and made alkaline by the addition of 5 N NaOH (30 mL).
The resulting precipitate was collected by filtration, washed with
water, dried and recrystallised from methanol to afford the title
compound (0.058 g, 64%) as a yellow solid. Mp >300 °C. 1H NMR
(300 MHz, CDCl3): d 3.82 (s, 6H, OMe), 4.13 (s, 6H, OMe), 6.32 (s,
2H, H5), 7.26–7.43 (m, 20H, aryl H), 10.79 (br s, 2H, NH). IR
(KBr): mmax 1597, 1485, 1425, 1358, 1219, 1149, 698 cmꢁ1. UV–
vis (THF): kmax 229 nm (e
68,000 cmꢁ1 Mꢁ1), 248 (78,750), 334
(79,400). HRMS (+ESI): C48H36N6O6 [M+H]+ requires 793.2775,
found 793.2771. The sample was not soluble enough for 13C NMR
measurement.
4.1.14. N,N0-Bis(4,6-dimethoxy-2,3-diphenyl-1H-indole-7-
glyoxyloyl)hydrazide (12)
C
48H40N6O8 [M+H]+ requires 829.2986, found 829.2978.
To a solution of 7-glyoxyloyl chloride 11 (0.504 g, 1.2 mmol) in
anhydrous acetonitrile (30 mL), hydrazine hydrate (0.031 mL,
0.64 mmol) was added followed by triethylamine (10 drops) and
the solution was stirred at room temperature for 1.5 h. The reac-
tion was quenched with ice-water and the resulting precipitate
was filtered, dried and recrystallised from ethanol to yield the title
compound (0.503 g, 100%) as a yellow solid. Mp 284–286 °C. 1H
NMR (300 MHz, DMSO-d6): d 3.83 (s, 6H, OMe), 4.02 (s, 6H,
OMe), 6.49 (s, 2H, H5), 7.28–7.32 (m, 20H, aryl H), 10.64 (br s,
2H, NH), 11.01 (br s, 2H, NH). 13C NMR (75 MHz, DMSO-d6): d
56.24, 57.33 (OMe), 89.14 (C5), 126.80, 127.77, 127.90, 128.19,
128.33, 128.46, 128.69, 128.82, 131.40 (aryl CH), 101.33, 112.76,
114.87, 132.03, 133.18, 135.51, 137.10, 161.85, 162.45 (aryl C),
166.81, 188.53 (C=O). IR (KBr): mmax 3414, 1643, 1583, 1466,
1361, 1348, 1242, 1219, 1140, 756, 699 cmꢁ1. UV–vis (CH2Cl2):
4.1.11. N,N0-Bis(3-(4-chlorophenyl)-4,6-dimethoxy-1H-indole-7-
carbonyl)oxalohydrazide (9b)
Oxalyl chloride (0.04 mL, 0.46 mmol) in dry dichloromethane
(5 mL) was added dropwise to a solution of 7-carbohydrazide 8b
(0.21 g, 0.61 mmol) in dry dichloromethane (10 mL) containing tri-
ethylamine (0.084 mL, 0.61 mmol). The reaction mixture was stir-
red at room temperature for 1.5 h. The solvent was then
evaporated and the residue was quenched with water. The result-
ing precipitate was filtered, dried and recrystallised from methanol
to yield the title compound (0.208 g, 92%) as a pale yellow solid. Mp
>300 °C. (Found: C, 56.49; H, 4.13; N, 10.92; C36H30Cl2N6O8 0.3
CH2Cl2 requires C, 56.55; H, 4.0; N, 10.9). 1H NMR (300 MHz,
DMSO-d6): d 3.89 (s, 6H, OMe), 4.05 (s, 6H, OMe), 6.49 (s, 2H,
H5), 7.23 (d, J = 2.5 Hz, 2H, H2), 7.36 (d, J = 8.6 Hz, 4H, aryl H),
7.51 (d, J = 8.4 Hz, 4H, aryl H), 9.84 (br s, NH, 2H), 10.80 (br s,
NH, 2H), 11.41 (d, J = 1.2 Hz, 2H, NH) 13C NMR (75 MHz, DMSO-
d6): d 55.80, 57.19 (OMe), 88.37 (C5), 123.81 (C2), 127.88, 131.04
(aryl CH), 96.23, 110.41, 115.78, 130.43, 135.01, 138.59, 157.20,
157.67 (aryl C), 158.69, 165.39 (C=O). IR (KBr): mmax 3396, 1623,
kmax 230 nm (
HRMS (+ESI):
821.2582.
e
C
83,200 cmꢁ1 Mꢁ1), 272 (73,100), 337 (52,700).
48H38N4O8 [M+Na]+ requires 821.2587, found
4.1.15. N,N-Dimethyl(4,6-dimethoxy-2,3-diphenyl-1H-indole-7-
glyoxyloyl))hydrazinecarbothioamide (14a)
1591, 1454, 1347, 1215 cmꢁ1
.
UV–vis (THF): kmax 241
To a solution of 7-glyoxyloyl chloride 1123 (1.03 g, 2.46 mmol)
(67,550 cmꢁ1 Mꢁ1), 281 (34,850), 340 (35,700). HRMS (+ESI): C36-
H30Cl2N6O8 [M+H]+ requires 745.1580, found 745.1572.
in
acetonitrile
(30 mL),
4,4-dimethyl-3-thiosemicarbazide
(0.306 g, 2.56 mmol) was added followed by triethylamine (7
drops). The mixture was stirred at room temperature for 1.5 h after
which water was added to quench the reaction. The resulting pre-
cipitate was filtered, dried and recrystallised from dichlorometh-
ane/n-hexane to afford the title compound (0.975 g, 79%) as a
yellow solid. Mp 153–155 °C. 1H NMR (300 MHz, DMSO-d6): d
3.24 (s, 6H, Me), 3.77 (s, 3H, OMe), 3.92 (s, 3H, OMe), 6.41 (s, 1H,
H5), 7.24–7.29 (m, 10H, aryl H), 9.33 (s, 1H, NH), 10.38 (s, 1H,
NH), 10.91 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): d 41.25
(Me), 56.18, 57.51 (OMe), 89.14 (C5), 126.74, 127.68, 127.86,
128.33, 128.76, 131.40 (aryl CH), 101.47, 112.67, 114.80, 132.06,
133.07, 135.57, 137.14, 161.54, 162.29 (aryl C), 183.38 (C=S),
165.95, 188.79 (C=O). IR (KBr): mmax 3421, 1675, 1588, 1387,
1360, 1320, 1219, 1162, 696 cmꢁ1. UV–vis (MeOH): kmax 247
(27,250 cmꢁ1 Mꢁ1), 331 (13,250). HRMS (+ESI): C27H26N4O4S
[M+H]+ requires 503.1753, found 503.1748.
4.1.12. N,N0-Bis(3-(4-bromophenyl)-4,6-dimethoxy-1H-indole-
7-carbonyl)oxalohydrazide (9c)
Oxalyl chloride (0.04 mL, 0.46 mmol) in dry dichloromethane
(5 mL) was added dropwise to a solution of 7-carbohydrazide 8c
(0.202 g, 0.52 mmol) in dry dichloromethane (10 mL) containing
triethylamine (0.07 mL, 0.52 mmol). The reaction mixture was stir-
red at room temperature for 1.5 h. The solvent was then evapo-
rated and the residue was quenched with water. The resulting
precipitate was filtered, dried and recrystallised from methanol
to yield the title compound (0.18 g, 83%) as a pale yellow solid.
Mp >300 °C. (Found: C, 50.66; H, 3.71; N, 9.75; C36H30Br2N6O8 0.3
CH2Cl2 requires C, 50.70; H, 3.59; N, 9.77). 1H NMR (300 MHz,
DMSO-d6): d 3.89 (s, 6H, OMe), 4.05 (s, 6H, OMe), 6.49 (s, 2H,
H5), 7.23 (d, J = 2.3 Hz, 2H, H2), 7.45 (d, J = 8.77 Hz, 4H, aryl H),
7.50 (d, J = 9.1 Hz, 4H, aryl H), 9.84 (s, NH, 2H), 10.78 (br s, NH,
2H), 11.41 (d, J = 1.6 Hz, 2H, NH). 13C NMR (75 MHz, DMSO-d6): d
55.80, 57.19 (OMe), 88.39 (C5), 123.80 (C2), 130.79, 131.42 (aryl
CH), 96.23, 110.35, 115.80, 118.92, 135.39, 138.60, 157.20,
157.67 (aryl C), 158.79, 165.38 (C=O). IR (KBr): mmax 3395, 1621,
4.1.16. N,N-Dimethyl(4,5,6-trimethoxy-2,3-diphenyl-1H-indole-
7-glyoxyloyl))hydrazinecarbothioamide (14b)
To a solution of 7-glyoxyloyl chloride 1321 (0.185 g, 0.41 mmol)
in acetonitrile (10 mL), 4,4-dimethyl-3-thiosemicarbazide (0.054 g,
0.45 mmol) was added followed by triethylamine (3 drops). The
mixture was stirred at room temperature for 1.5 h after which
water was added to quench the reaction. The resulting precipitate
1589, 1457, 1347, 1215 cmꢁ1
.
UV–vis (THF): kmax 241
(60,900 cmꢁ1 Mꢁ1), 284 (31,750), 343 (33,150). HRMS (+ESI): C36-
H30Br2N6O8 [M+Na]+ requires 855.0390, found 855.0385.

1678
H. Kandemir et al. / Bioorg. Med. Chem. 22 (2014) 1672–1679
was filtered, dried and recrystallised from dichloromethane/n-hex-
ane to afford the title compound (0.165 g, 75%) as a yellow solid. Mp
180–182 °C. 1H NMR (300 MHz, CDCl3): d 3.37 (s, 6H, Me), 3.66 (s,
3H, OMe), 3.82 (s, 3H, OMe), 4.02 (s, 3H, OMe), 7.26–7.36 (m, 10H,
aryl H), 8.21 (d, J = 7.1 Hz, 1H, NH), 9.82 (d, J = 6.4 Hz, 1H, NH),
10.28 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3): d 40.76 (Me),
61.28, 61.31, 62.59 (OMe), 126.60, 127.73, 127.98, 128.58, 131.14
(aryl CH), 106.39, 114.74, 118.78, 131.78, 133.37, 134.97, 135.07,
139.25, 155.21, 156.22 (aryl C), 178.68 (C=S), 162.06, 188.02
(C=O). IR (KBr): mmax 3427, 3188, 2936, 1685, 1623, 1572, 1448,
1378, 1324, 1260, 1052, 828, 764, 698 cmꢁ1. UV–vis (MeOH): kmax
249 (51,350 cmꢁ1 Mꢁ1), 363 (21,550). HRMS (+ESI): C28H28N4O5S
[M+H]+ requires 533.1858, found 533.1852.
yellow solid. Mp 219–221 °C. 1H NMR (300 MHz, DMSO-d6): d
3.21 (s, 6H, Me), 3.58 (s, 3H, OMe), 3.79 (s, 3H, OMe), 6.15 (d,
J = 1.9 Hz, 1H, H5), 6.65 (d, J = 1.9 Hz, 1H, H7), 7.36 (s, 4H, aryl
H), 9.35 (br s, 1H, NH), 10.80 (br s, 1H, NH), 11.91 (s, 1H, NH).
13C NMR (75 MHz, DMSO-d6): d 41.25 (Me), 55.57, 55.76 (OMe),
86.89 (C5), 93.98 (C7), 127.06, 132.89 (aryl CH), 112.67, 126.37,
126.69, 131.90, 133.57, 139.99, 156.30, 161.29 (aryl C), 182.96
(C=S), 163.13, 178.05 (C=O). IR (KBr): mmax 3129, 1701, 1631,
1529, 1389, 1309, 1207, 809 cmꢁ1. UV–vis (THF): kmax 226 nm (
e
27,150 cmꢁ1 Mꢁ1), 247 (25,500), 356 (13,950). HRMS (+ESI): C21-
H21ClN4O4S [M+H]+ requires 461.1050, found 461.1047.
4.1.20. N,N-Dimethyl(3-(4-bromophenyl)-4,6-dimethoxy-1H-
indole-2-glyoxyloyl))hydrazinecarbothioamide (17b)
4.1.17. (4,6-Dimethoxy-2,3-diphenyl-1H-indol-7-yl) (5-
To a
solution of 2-glyoxyloyl chloride 16b23 (0.606 g,
(dimethylamino)-1,3,4-thiadiazol-2-yl)ketone (15a)
1.44 mmol) in acetonitrile (25 mL), 4,4-dimethyl-3-thiosemicarba-
zide (0.189 g, 1.58 mmol) was added followed by triethylamine (6
drops). The mixture was stirred at room temperature for 1.5 h after
which water was added to quench the reaction. The resulting pre-
cipitate was filtered, dried and recrystallised from dichlorometh-
ane/n-hexane to afford the title compound (0.45 g, 62%) as a
yellow solid. Mp 217–219 °C. 1H NMR (300 MHz, DMSO-d6): d
3.21 (s, 6H, Me), 3.58 (s, 3H, OMe), 3.79 (s, 3H, OMe), 6.15 (d,
J = 1.9 Hz, 1H, H5), 6.65 (d, J = 1.9 Hz, 1H, H7), 7.30, 7.49 (2d,
J = 8.2 Hz, 4H, aryl H), 9.35 (br s, 1H, NH), 10.80 (br s, 1H, NH),
11.92 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): d 41.22 (Me),
55.59, 55.76 (OMe), 86.89 (C5), 94.0 (C7), 129.97, 133.23 (aryl
CH), 112.60, 126.37, 126.66, 133.97, 140.01, 156.30, 161.30 (aryl
C), 182.94 (C=S), 163.12, 178.08 (C=O). IR (KBr): mmax 3134, 1701,
To a solution of indole 14a (0.121 g, 0.24 mmol) in ethyl acetate
(15 mL), POCl3 (4 mL) was added and the solution was heated un-
der reflux for 2 h. The solvent was evaporated under reduced pres-
sure and the remaining residue was quenched with water and
made alkaline by the addition of 5 N NaOH (12 mL). The resulting
precipitate was collected by filtration, washed with water, dried
and purified by flash chromatography using dichloromethane/
ethyl acetate (70:30) as eluent to afford the title compound
(0.067 g, 59%) as
a
yellow solid. Mp 180–182 °C. 1H NMR
(300 MHz, CDCl3): d 3.26 (s, 6H, Me), 3.79 (s, 3H, OMe), 3.87 (s,
3H, OMe), 6.25 (s, 1H, H5), 7.21–7.39 (m, 10H, aryl H), 10.05 (br
s, 1H, NH). 13C NMR (75 MHz, CDCl3): d 41.54 (Me), 55.32, 57.08
(OMe), 89.02 (C5), 126.07, 127.08, 127.34, 127.82, 128.34, 131.28
(aryl CH), 113.24, 114.86, 132.26, 132.82, 135.49, 137.67, 159.33,
160.48, 161.06, 173.66 (aryl C), 183.74 (C=O). IR (KBr): mmax 3390,
1566, 1452, 1287, 1237, 1219, 1147, 698 cmꢁ1. UV–vis (MeOH):
1638, 1389, 1207, 1156, 809 cmꢁ1. UV–vis (THF): kmax 226 nm (
e
35,850 cmꢁ1 Mꢁ1), 252 (32,250), 356 (17,150). HRMS (+ESI): C21-
H21BrN4O4S [M+H]+ requires 505.0545, found 505.0532.
kmax 283 nm (e
29,900 cmꢁ1 Mꢁ1), 329 (21,000). HRMS (+ESI): C27-
H24N4O3S [M]+ requires 484.1569, found 484.1564.
4.1.21. (3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-2-yl)(5-
(dimethylamino)-1,3,4-thiadiazol-2-yl)ketone (18a)
4.1.18. (4,5,6-Trimethoxy-2,3-diphenyl-1H-indol-7-yl) (5-
(dimethylamino)-1,3,4-thiadiazol-2-yl)ketone (15b)
To a solution of indole 17a (0.186 g, 0.4 mmol) in ethyl acetate
(20 mL), POCl3 (3 mL) was added and the solution was heated un-
der reflux for 2 h. The solution was poured slowly into ice–water
(40 mL) and made alkaline by the addition of 5 N NaOH (10 mL).
It was then extracted with ethyl acetate (2 ꢂ 20 mL) and dichloro-
methane (1 ꢂ 20 mL). The organic extracts were combined, dried
over anhydrous sodium sulfate and concentrated under reduced
pressure. The crude mixture was purified by flash chromatography
using dichloromethane/ethyl acetate (90:10) as eluent to afford the
title compound (0.057 g, 33%) as an orange-brown solid. Mp 276–
278 °C. 1H NMR (300 MHz, CDCl3): d 3.27 (s. 6H, Me), 3.64 (s, 3H,
OMe), 3.87 (s, 3H, OMe), 6.10 (d, J = 1.9 Hz, 1H, H5), 6.43 (d,
J = 1.9 Hz, 1H, H7), 7.34, 7.44 (2d, J = 8.7 Hz, 4H, aryl H), 11.50 (br
s, 1H, NH). 13C NMR (75 MHz, CDCl3): d 41.56 (Me), 55.04, 55.46
(OMe), 85.77 (C5), 93.35 (C7), 126.97, 131.98 (aryl CH), 113.07,
126.85, 127.68, 132.74, 133.13, 139.41, 156.64, 161.44 (aryl C),
162.72 (N–C=N), 170.54 (C=O–C=N), 174.03 (C=O). IR (KBr): mmax
3343, 1611, 1484, 1290, 1203, 1154, 1047, 921 cmꢁ1. UV–vis
To a solution of indole 14b (0.109 g, 0.2 mmol) in ethyl acetate
(10 mL), POCl3 (3 mL) was added and the solution was heated un-
der reflux for 2 h. The solvent was evaporated under reduced pres-
sure and the remaining residue was quenched with water and
made alkaline by the addition of 5 N NaOH (10 mL). The resulting
precipitate was collected by filtration, washed with water, dried
and purified by flash chromatography using dichloromethane/
ethyl acetate (70:30) as eluent to afford the title compound
(0.06 g, 57%) as
a
yellow solid. Mp 116–118 °C. 1H NMR
(300 MHz, CDCl3): d 3.29 (s, 6H, Me), 3.60 (s, 3H, OMe), 3.83 (s,
3H, OMe), 3.97 (s, 3H, OMe), 7.23–7.41 (m, 10H, aryl H), 9.61 (br
s, 1H, NH). 13C NMR (75 MHz, CDCl3): d 41.63 (Me), 61.04, 61.17,
62.48 (OMe), 126.31, 127.39, 127.57, 127.89, 128.40, 131.10 (aryl
CH), 109.06, 114.55, 118.61, 132.02, 132.87, 134.41, 135.26,
140.05, 153.28, 153.33, 159.18, 174.10 (aryl C), 184.83 (C=O). IR
(KBr): mmax 3497, 2359, 2341, 1558, 1465, 1281, 1102, 1049,
695 cmꢁ1. UV–vis (MeOH): kmax 231 nm (
e
45,150 cmꢁ1 Mꢁ1),
(THF): kmax 226 nm (e
45,400 cmꢁ1 Mꢁ1), 337 (16,150). HRMS
318 (29,400). HRMS (+ESI):
515.1753, found 515.1744.
C
28H26N4O4S [M+H]+ requires
(+ESI): C21H19ClN4O3S [M+H]+ requires 443.0945, found 443.0936.
4.1.22. (3-(4-Bromophenyl)-4,6-dimethoxy-1H-indol-2-yl)(5-
(dimethylamino)-1,3,4-thiadiazol-2-yl)ketone (18b)
4.1.19. N,N-Dimethyl(3-(4-chlorophenyl)-4,6-dimethoxy-1H-
indole-2-glyoxyloyl))hydrazinecarbothioamide (17a)
To a solution of indole 17b (0.45 g, 0.89 mmol) in ethyl acetate
(20 mL), POCl3 (3 mL) was added and the solution was heated un-
der reflux for 2 h. The solution was poured slowly into ice–water
(40 mL) and made alkaline by the addition of 5 N NaOH (10 mL).
It was then extracted with ethyl acetate (2 ꢂ 20 mL) and dichloro-
methane (1 ꢂ 20 mL). The organic extracts were combined, dried
over anhydrous sodium sulfate and concentrated under reduced
pressure. The crude mixture was purified by flash chromatography
To
a
solution of 2-glyoxyloyl chloride 16a23 (0.535 g,
1.42 mmol) in acetonitrile (25 mL), 4,4-dimethyl-3-thiosemicarba-
zide (0.186 g, 1.56 mmol) was added followed by triethylamine (6
drops). The mixture was stirred at room temperature for 1.5 h after
which water was added to quench the reaction. The resulting pre-
cipitate was filtered, dried and recrystallised from dichlorometh-
ane/n-hexane to afford the title compound (0.552 g, 55%) as a

H. Kandemir et al. / Bioorg. Med. Chem. 22 (2014) 1672–1679
1679
using dichloromethane/ethyl acetate (90:10) as eluent to afford the
title compound (0.153 g, 36%) as an orange-brown solid. Mp 280–
282 °C. (Found: C, 51.28; H, 3.87; N, 11.23; C21H19BrN4O3S0.1 CH2-
Cl2 requires C, 51.11; H, 3.90; N, 11.30). 1H NMR (300 MHz, DMSO-
d6): d 3.20 (s. 6H, Me), 3.57 (s, 3H, OMe), 3.79 (s, 3H, OMe), 6.14 (d,
J = 1.9 Hz, 1H, H5), 6.84 (d, J = 1.9 Hz, 1H, H7), 7.27, 7.45 (2d,
J = 8.8 Hz, 4H, aryl H), 11.83 (br s, 1H, NH). 13C NMR (75 MHz,
DMSO-d6): d 41.66 (Me), 55.28, 55.54 (OMe), 87.62 (C5), 93.65
(C7), 129.88, 133.26 (aryl CH), 112.40, 120.18, 125.61, 127.06,
134.68, 139.84, 156.08 (aryl C), 160.69 (N–C=N), 172.39 (C=O–
C=N), 173.95 (C=O). IR (KBr): mmax 1610, 1540, 1456, 1291, 1242,
DH5
37 °C in 5 mL LB with shaking until the OD reached 0.6–0.7, and
L of the culture was added to each well. The plate was incu-
bated in the FLUOstar Optima plate reader (BMG Labtech) at
37 °C with 600 rpm shaking. The OD of the culture was taken every
10 min using LB as the blank for 16 h at 600 nm. The samples were
tested in triplicate and the growth pattern of each sample was
compared to cells exposed to equal amounts of DMSO.
a as the Gram negative test cells. The cells were grown at
5
l
Acknowledgments
1203, 1154, 813 cmꢁ1. UV–vis (THF): kmax 292 nm (
e
31,350 cmꢁ1
-
We thank the University of New South Wales and the Turkish
Government for their financial support. This work was supported
with funding from the NHMRC (APP1008014).
Mꢁ1), 329 (13,350). HRMS (+ESI): C21H1981BrN4O3S [M+H]+ re-
quires 489.0374, found 489.0408.
4.2. ELISA based assay
References and notes
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Purified full-length
fered saline (PBS) and 100
Maxisorp™ microtitre plate wells. Following overnight incubation
at 4 °C the wells were washed 3 times with 300 L of PBS and
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r
A was diluted to 250 nM in phosphate buf-
L of the solution added into NUNC
l
l
l
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for 2 h. After blocking, plates were washed three times with wash
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l
L PBS was then added to wells fol-
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the addition of 100 L rabbit anti-GST primary antibody (1:2000
l
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in PBS) to each well and incubated for 1 hour at room temperature.
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by the addition of 100
L TMB substrate system (3,30,5,50-Tetra-
lL PBS/Tween-20 washes be-
l
methylbenzidine liquid substrate system for ELISA, Sigma–Aldrich)
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The compounds were dissolved to 50 mM in DMSO and diluted
in 100
lL of LB medium to 200 lM in 96-well NUNC Microwell™
plate. B. subtilis 168 was used as the Gram positive and E. coli