Synthesis and biological activity of novel bis-indole inhibitors of bacterial transcription initiation complex formation

Article abstract of DOI:10.1039/c4ob00460d

The increasing resistance of bacteria against clinically approved antibiotics is resulting in an alarming decrease in therapeutic options for today's clinicians. We have targeted the essential interaction between bacterial RNA polymerase and σ⁷⁰/σ^A for the development of lead molecules exhibiting a novel mechanism of antibacterial activity. Several classes of structurally related bis-indole inhibitors of bacterial transcription initiation complex formation were synthesized and their antimicrobial activities were evaluated. Condensation of indole-7- and indole-2-carbohydrazides with 7- and 2-trichloroacetylindoles or indole-7- and indole-2-glyoxyloyl chlorides resulted in the successful synthesis of 7,7′-, 2,2′-, 2,7′- and 3,2′-linked bis-indole derivatives with -CO-NH-NH-CO- and -CO-CO-NH-NH-CO- linkers. Indole-7-glyoxyloyl chlorides were reacted with hydrazine hydrate in different ratios to afford respective -CO-CO-NH-NH-CO-CO- bis-indole or hydrazide derivatives. The resulting compounds were found to be active against the β′-CH- σ⁷⁰/σA2.2 interaction in ELISA assays and inhibited the growth of both Gram-positive and Gram-negative bacteria. Structure-activity relationship (SAR) studies were performed in order to identify the structural features of the synthesized inhibitors required for biological activity. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00460d

Organic &
Biomolecular Chemistry
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PAPER
Synthesis and biological activity of novel bis-
indole inhibitors of bacterial transcription
initiation complex formation†
Cite this: Org. Biomol. Chem., 2014,
12, 2882
Marcin Mielczarek,a Ruth V. Devakaram,a Cong Ma,b Xiao Yang,b Hakan Kandemir,a
Bambang Purwono,a David StC. Black,a Renate Griffith,c Peter J. Lewis*b and
Naresh Kumar*a
The increasing resistance of bacteria against clinically approved antibiotics is resulting in an alarming
decrease in therapeutic options for today’s clinicians. We have targeted the essential interaction between
bacterial RNA polymerase and σ70/σA for the development of lead molecules exhibiting a novel mechan-
ism of antibacterial activity. Several classes of structurally related bis-indole inhibitors of bacterial tran-
scription initiation complex formation were synthesized and their antimicrobial activities were evaluated.
Condensation of indole-7- and indole-2-carbohydrazides with 7- and 2-trichloroacetylindoles or indole-
7- and indole-2-glyoxyloyl chlorides resulted in the successful synthesis of 7,7’-, 2,2’-, 2,7’- and 3,2’-
linked bis-indole derivatives with –CO–NH–NH–CO– and –CO–CO–NH–NH–CO– linkers. Indole-7-
glyoxyloyl chlorides were reacted with hydrazine hydrate in different ratios to afford respective –CO–
CO–NH–NH–CO–CO– bis-indole or hydrazide derivatives. The resulting compounds were found to be
active against the β’-CH-σ70/σ2A.2 interaction in ELISA assays and inhibited the growth of both Gram-posi-
tive and Gram-negative bacteria. Structure–activity relationship (SAR) studies were performed in order to
identify the structural features of the synthesized inhibitors required for biological activity.
Received 28th February 2014,
Accepted 17th March 2014
DOI: 10.1039/c4ob00460d
www.rsc.org/obc
threatens to pose a grave threat to human health in the
coming years.8,9 Compounding this problem, many new com-
Introduction
Since Alexander Fleming’s serendipitous discovery of the anti- pounds being released to the market are derivatives of existing
bacterial activity of the fungus Penicillium notatum in 1928, drugs to which resistance has already appeared. Consequently,
antibiotics have emerged as the primary treatment option for there is a clear and urgent need for the discovery and develop-
infectious diseases.1 Unfortunately, the over-use of antibiotics ment of novel classes of antibiotics and drug targets that can
has resulted in the rapid development of drug-resistant bac- slow the rate of appearance of resistance.6
terial strains.2–6 Most of the antibiotic classes in common use
Bacterial RNA polymerase (RNAP) and transcription are
today were discovered between 1945 and 1960, during the promising targets that have been exploited for the develop-
“Golden Era” of antibiotics, but since that time only a few ment of novel antimicrobials. There are two forms of bacterial
more new classes of antibiotics have been developed.6 More- RNAP called core and holoenzyme, and transcription consists
over, the discovery and development of new antibacterial of three main sequential stages: initiation, elongation and ter-
agents has decreased at an alarming rate in recent years.1,7 In mination.10 The RNAP core enzyme is catalytically active, but
the absence of new antibacterial drugs, antibiotic resistance is unable to initiate transcription with appropriate specificity
and efficiency when transcription initiation factor σ is not
present.10,11 In order to form a holoenzyme recognizing DNA
aSchool of Chemistry, The University of New South Wales, Sydney, NSW 2052,
promoters and initiating the transcription cycle, the core
Australia. E-mail: n.kumar@unsw.edu.au; Fax: +61 2 9385 6141;
enzyme must associate with σ factors.10,12 σ Factors are unique
Tel: +61 2 9385 4698
bSchool of Environmental and Life Sciences, University of Newcastle, Callaghan,
NSW 2308, Australia. E-mail: peter.lewis@newcastle.edu.au; Fax: +61 2 4921 5472;
Tel: +61 2 4921 5701
to bacteria and are essential for correct gene expression and
viability, with the σ70 factor in Gram-negative bacteria and the
σA factor in Gram-positive bacteria being the most important
proteins required for direct contact between RNAP and promo-
ter DNA sequences.11–14 The interaction between region 2.2 of
σ70/σA and the β′-CH region of the RNAP core is essential for
cSchool of Medical Sciences, Department of Pharmacology, The University of New
South Wales, Sydney, NSW 2052, Australia. E-mail: r.griffith@unsw.edu.au
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ob00460d
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the formation of the RNAP holoenzyme.15 Domains of σ70/σA act against both Gram-positive and Gram-negative bacteria,
factors that interact with the core enzyme are highly con- and present our structure–activity relationship studies (SAR)
served,10,12,16,17 and so molecules capable of disrupting the on this class of novel RNAP-σ70/σA inhibitors.
interaction between σ70/σA and the core enzyme would be
expected to exhibit broad spectrum activity. Therefore, the
interaction between σ70/σ2A.2 and the β′-CH region represents an
excellent target for the design and development of new anti-
Results and discussion
biotic leads with novel scaffolds and a completely unique Variation of substituents at position 3 of the indole ring, types
mechanism of antimicrobial activity.
of linkage between the two indole units (7,7′, 2,2′, 2,7′ and 3,2′)
There are numerous natural and synthetic compounds that and types of linkers (–CO–NH–NH–CO–, –CO–CO–NH–NH–
inhibit transcription by targeting RNAP.12,18–22 Rifampicin and CO– and –CO–CO–NH–NH–CO–CO–) were considered to
lipiarmycin target the active site of RNAP and are especially produce analogues of GKL001 (16) and GKL003 (10a).
active against Gram-positive bacteria, such as Mycobacterium
The well-established five-step synthesis of 3-aryl-4,6-
tuberculosis and Clostridium difficile, but resistance can rapidly dimethoxyindoles 1a–e and the one-pot synthesis of 4,6-
develop through mutation at multiple locations within the dimethoxy-3-methylindole 1f (Scheme 1) were previously
active site.12,23 Sorangicin and streptolydigin, antibiotics not reported by our group.27 Owing to the nature of the activating
approved in the clinic, also exhibit selective activity against substituents, 3-aryl-4,6-dimethoxyindoles 1a–e and 4,6-
Gram-positive bacteria.12,24 Recently, smaller molecules such dimethoxy-3-methylindole 1f are able to undergo electrophilic
as SB2, SB4, SB5, SB7, SB8 and SB12 have been successfully substitution reactions without the use of a catalyst. Therefore,
identified as potential inhibitors of the interaction between indoles 1a–f were reacted two reagents, trichloroacetyl chloride
σ70/σA and RNAP.19
or oxalyl chloride to yield varieties of indoles such as 7-tri-
As the majority of the antibiotics targeting transcription chloroacetylindoles 2a–e, 2-trichloroacetylindoles 3a–f, indole-
exhibit significant activity against only Gram-positive bacteria, 7-glyoxyloylchlorides 6a–d and indole-2-glyoxyloyl chlorides
novel compounds to combat Gram-negative bacteria are 7a–d,28 respectively (Scheme 1).
urgently required due to the rapid increase in resistant
strains.12,23
Anhydrous 1,2-dichloroethane was found to be the most
suitable solvent for the reaction between indoles 1a–f and tri-
In our preliminary research, bis-indole compounds GKL001 chloroacetyl chloride, as it provided reasonable yields of the
(16) and GKL003 (10a) (Fig. 1) were found to inhibit the essen- products (20–37% for the 7-trichloroacetyl derivatives 2a–e and
tial interaction between σ70/σ2A.2 and the β′-CH region of the 10–23% for the 2-trichloroacetyl derivatives 3a–f after chroma-
core RNAP in ELISA assays. Moreover, modeling studies tographic separation) and minimal production of a third iso-
employing a Bacillus subtilis RNAP homology model25 and iso- meric
side
product,
N-trichloroacetylindole
(<1%).
thermal titration calorimetry experiments26 indicated that Interestingly, the 7-trichloroacetyl derivatives were not pro-
GKL003 binds to the β′-CH region of RNAP, thereby inhibiting duced when 4,6-dimethoxy-3-methylindole was reacted with
transcription initiation. Both molecules inhibited the growth trichloroacetyl chloride. Treatment of a suspension of the tri-
of Bacillus subtilis and Escherichia coli.
chloroacetyl derivatives 2a–e and 3a–f in acetonitrile with
The aim of this study was to elaborate our bis-indole library excess hydrazine hydrate (5–9 equivalents) resulted in for-
and to develop novel inhibitors of the interaction between mation of the corresponding indole-7-carbohydrazides 4a–e
RNAP and σ70/σA, and to evaluate the antibacterial activity of
the compounds by ELISA and bacterial growth inhibition
assay. In this paper, we report the discovery of molecules that
Scheme 1 Reagents and conditions: (a) CCl3COCl (3 equiv.), 1,2-
dichloroethane, 80 °C, 3.5 h, 20–37% (2a–e), 10–23% (3a–f); (b)
NH2NH2·H2O (5–9 equiv.), MeCN, room temp., 3.5 h, 74–80% (4a–e),
Fig. 1 Bis-indoles as potential inhibitors of interaction between RNAP
core and σ70/σA in bacteria.
73–81% (5a–f); (c) oxalyl chloride (3 equiv.), diethyl ether, 0 °C → room
temp., 3 h, 30–33% (6a–d), 35–43% (7a–d).
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and indole-2-carbohydrazides 5a–f in very good yields (74–80%
and 73–81%, respectively) and with no chromatography
required. The indoles 1a–f were found to be more reactive with
oxalyl chloride compared to trichloroacetyl chloride, and so
the reactions could be performed in diethyl ether at 0 °C28 in
order to minimize formation of side products. Similarly, two
isomeric products were obtained: indole-7-glyoxyloyl chlorides
6a–d (30–33%) and indole-2-glyoxyloyl chlorides 7a–d
(35–43%). Following a well-established procedure, indole-3-
glyoxyloyl chloride 9 was synthesized from indole 8.29
Condensations of the trichloroacetyl derivatives 2a–e and
3a–f with the hydrazides derivatives 4a–e and 5a–f in anhy-
drous acetonitrile with triethylamine as a catalyst resulted in
the formation of a variety of novel bis-indoles 10a–f, 11a–c and
12a–g comprising the –CO–NH–NH–CO– motif (Scheme 2).
Both symmetrical and unsymmetrical molecules were syn-
thesized using three different types of linkages: the 7,7′
linkage (10a–f), the 2,2′ linkage (11a–c) and the 2,7′ linkage
(12a–g). Bis-indoles 12a–g could be obtained from the reaction
of the trichloroacetyl derivatives 2a–e with hydrazides 5a–f, or
from the reaction of the trichloroacetyl derivatives 3a–f with
hydrazides 4a–e, based on the availability of the starting
materials. Depending on the reactivity of the building blocks
2a–e, 3a–f, 4a–e and 5a–f, reactions were carried out at room
temperature or at reflux for 5–24 h.
When glyoxyloyl chlorides 6a–b and 7a–c were condensed
with hydrazides 4a–c, 4e and 5f, 7,7′-linked bis-indoles 15a–d,
2,2′-linked bis-indole 13 and 2,7′-linked bis-indoles 14a–c con-
taining an unsymmetrical –CO–CO–NH–NH–CO– linker were
produced (Scheme 3). Because glyoxyloyl chlorides were found
to decompose at higher temperatures and were readily suscep-
tible to hydrolysis into glyoxylic acids in the presence of moist-
ure, all reactions were carried out at room temperature in
anhydrous acetonitrile with triethylamine as a catalyst.
Scheme 3 Reagents and conditions: (a) Et3N, MeCN, room temp.,
5–26 h; (b) NH2NH2·H2O (0.5 equiv.), Et3N, MeCN, room temp., 1.5 h; (c)
NH2NH2·H2O (excess), Et3N, MeCN, room temp., 5 h.
Two different products were obtained when glyoxyloyl chlor-
ides 6a–b were reacted with hydrazine hydrate. When a
2 : 1 molar ratio of the glyoxyloyl chloride 6a to hydrazine
hydrate was used, the symmetrical bis-indole 16 was produced.
However, when excess hydrazine hydrate was used in the
glyoxyloyl chloride 6b, the hydrazide 17 was generated instead.
Similarly, when 3-glyoxyloyl chloride 9 was reacted with
hydrazine hydrate in a 2 : 1 molar ratio, the symmetrical 3,3′-
linked bis-indole 18 was produced.30 In addition, when glyoxy-
loyl chloride 9 and hydrazides 5a–b and 5e were reacted, it pro-
duced 3,2′-linked bis-indoles 19a–c (Scheme 4).
A library of thirty synthesized compounds was evaluated for
antibacterial activity. Inhibition of the interaction between σ70/
σA2.2 and the β′-CH region of the core RNAP was examined by
ELISA at 15 μM compound concentration and expressed as a
% of the negative control. Bacterial growth inhibition was eval-
Scheme 2 Reagents and conditions: (a) Et3N, MeCN, room temp. or
reflux, 5–24 h, 40–81%.
Scheme 4 Reagents and conditions: (a) NH2NH2·H2O (0.5 equiv.), Et3N,
MeCN, room temp., 1.5 h; (b) Et3N, MeCN, room temp., 5 h.
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uated at ca. 200 μM compound concentration using two repre-
Based on the biological activity of the synthesized com-
sentative bacterial species, B. subtilis (Gram-positive) and pounds, structure–activity relationship (SAR) studies could be
E. coli (Gram-negative), and expressed as a % of the negative performed. The compounds synthesized in this study varied in
control (Table 1). Negative control was related to either the the nature of the linkage between the two indole units and in
interaction between σ70/σ2A.2 and the β′-CH region of the core the type of substituent at position 3 of the indole ring. Three
RNAP or bacterial growth in the absence of the inhibitor of different types of linkers (–CO–CO–NH–NH–CO–CO–, –CO–
transcription initiation complex formation.
CO–NH–NH–CO– and –CO–NH–NH–CO–) in combination with
The compounds generally showed significant activity four types of linkage (7,7′, 2,2′, 2,7′ and 3,2′) were represented
against the β′-CH-σ70/σ2A.2 binding interaction at 15 μM, with 21 in the library of compounds.
of the 30 novel compounds exhibiting ≥50% inhibition at this
For 7,7′-linked compounds with a –CO–NH–NH–CO–
concentration. Surprisingly, the molecules were found to be linkage compound 10a with a 4-ClC6H4 substituent at position
more active against Gram-negative bacteria than Gram-positive 3 of the indole rings showed 63% inhibition of the β′-CH-σ70/
bacteria, with 14 of the 30 molecules exclusively inhibiting σA2.2 interaction at 15 μM, compared to 40% for 10b containing
E. coli growth at ca. 200 μM compound concentration. Com- a 4-BrC6H4 substituent. Furthermore, 10a was over twice as
pounds 10f, 12g, 15c and 19c showed ≥35% exclusive inhi- potent against B. subtilis growth compared to 10b. Interest-
bition of E. coli growth. Moreover, molecule 12g being the ingly, when the 7-CO–NH–NH–CO-7′ linkage 10b was replaced
most potent inhibitor of E. coli growth, was found to affect by the 7-CO–CO–NH–NH–CO–CO-7′ linkage 16, a two-fold
exponential phase of bacterial growth (Table 1). Exponential increase in inhibition of B. subtilis growth was observed, which
phase of bacterial growth is a phase of growth observed in a was accompanied by a more than two-fold decrease in inhi-
bacterial population where the growth of cells increases by bition of E. coli growth. However, there was no significant
a multiplicative factor per unit of time. Compound 18 could be difference in the ability of the compounds to inhibit the β′-CH-
a good broad spectrum candidate as it exhibited 47% inhi- σ70/σ2A.2 interaction. This suggested that the variation in anti-
bition of B. subtilis growth and 33% inhibition of E. coli bacterial activity of 10b and 16 in the two types of bacteria
growth, respectively. Many of the molecules precipitated out of could be due to differences in permeability, since Gram-posi-
the solution when diluted to ca. 200 μM concentration with tive and Gram-negative bacteria have substantial differences in
the media (Table 1).
the nature of their cell walls. Interestingly, incorporation of the
7-CO–CO–NH–NH–CO-7′ linkage 15a–d completely abolished
the ability of the molecules to inhibit B. subtilis growth, but
had no effect on the ability of these molecules to inhibit the
β′-CH-σ70/σ2A.2 interaction. These differences might be due to
the ability of these compounds to cross cell walls and mem-
branes of the Gram-positive and Gram-negative bacteria.
Notably, molecules 10f and 15b–d showed activity against the
β′-CH-σ70/σ2A.2 interaction and were also exclusive inhibitors of
E. coli growth. Good correlation between inhibition of the
β′-CH-σ70/σ2A.2 interaction and inhibition of E. coli growth for
molecules 10f and 15c suggested that inhibition of the inter-
action between σ70/σ2A.2 and the β′-CH region of the core RNAP
is the mechanism of observed antibacterial activity.
Compounds with 2,2′ linkage (11b–c and 13) showed low
inhibition of E. coli growth. Moreover, they did not inhibit
B. subtilis growth regardless of the type of linkage and the
nature of the substituent at position 3 of the indole ring. Given
that these molecules exhibited relatively high activities against
the β′-CH-σ70/σ2A.2 interaction, their low antibacterial activity
against E. coli and no activity against B. subtilis might be due
to their limited ability to cross the outer membrane of the cell
wall in Gram-negative bacteria and the cell wall in Gram-posi-
tive bacteria.
Table 1 Evaluation of antibacterial activity of the synthesized
compounds
Bacterial growth
β′-CH-σ70/σ2A.2 binding inhibition at 200 μM [%]
inhibition at 15 μM
Compound by ELISA [%]
Bacillus subtilis Escherichia coli
10a
10b
10d
10e
10f
11b
11c
12a
12b
12d
12f
12g
13
14a
14b
14c
15a
15b
15c
15d
16
63
40
73
68
60
50
86
62
58
60
61
72
66
71
72
61
72
63
52
68
39
25
50
57
16
41
30a
ND
14a
37a
13a
23a
11a
21a
No activitya
No activity
No activity
No activitya
No activitya
No activitya
No activity
No activity
No activity
No activitya
No activitya
No activitya
No activitya
No activitya
No activitya
No activitya
31a,b
43a
16
16
21a
No activitya
No activitya
23
45b
16
25a
23a
23a
No activitya
6a
35a
22a
15a
The 2,7′-linked compounds were found not to inhibit
B. subtilis growth. An increase in β′-CH-σ70/σ2A.2 inhibitory
activity accompanied by a two-fold increase in antibacterial
activity against E. coli was observed when a 4-BrC6H4 substitu-
ent 12f was replaced with a 4-ClC6H4 substituent 12g, which
was consistent with the observation made for the 7,7′-linked
molecules.
17
18
19a
19b
19c
87a,b
No activitya
47b
33
15
11
39
25b
3
No activity
a Precipitation at ca. 200 μM. b Affects exponential phase of bacterial
growth, ND no data.
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Among the 3,2′-linked bis-indoles, replacement of
a
and for their antibacterial activity against both Gram-positive
4-BrC6H4 substituent 19b by a 4-ClC6H4 substituent 19a also and Gram-negative bacteria in culture. Most of the molecules
resulted in a significant increase in β′-CH-σ70/σ2A.2 inhibitory were found to efficiently inhibit the interaction between σ70/
activity and inhibition of B. subtilis growth, and a slight σA2.2 and the β′-CH region of the core RNAP, and showed moder-
increase in inhibition of E. coli growth was also observed. ate inhibition of E. coli growth. However, the majority of the
Compound 19c exhibited good activity against the β′-CH-σ70/ compounds exhibited no antibacterial activity against B. subti-
σA2.2 interaction and exclusive inhibition of E. coli growth.
lis. Structure–activity relationship studies suggested that
The only 3,3′-linked bis-indole, 18, exhibited good activity aspects of molecular size and electronic nature may result in
against the β′-CH-σ70/σ2A.2 interaction as well as inhibition of differences in cellular permeability, thereby influencing anti-
both B. subtilis growth and E. coli growth.
bacterial activity. This research also furnished molecules
The only mono-indole derivative, 17, was found to be the capable of inhibiting the growth of Gram-negative bacteria,
most active inhibitor of B. subtilis growth. However, the mole- which is significant as discovering new compounds active
cule showed relatively low potency in the ELISA, suggesting against Gram-negative bacteria is much more challenging than
that it may possess a different mechanism of antibacterial for Gram-positive bacteria.9 Moreover, antibiotic-resistant
activity unrelated to the inhibition of the interaction between infections related to Gram-negative bacteria such as Entero-
σ70/σA2.2 and the β′-CH region of the core RNAP.
bacteriaceae sp., Salmonella sp., Klebsiella sp., Shigella sp.,
Considering all of the data, preliminary structure–activity Pseudomonas sp. and Acinetobacter sp. are currently a major
relationships can be drawn for this set of compounds. Firstly, cause of concern within the clinical environment.3,4,7,9,31
the nature of the linkage between indole units has a critical Therefore, the identification of molecules such as 10f, 12g, 15c
effect on the biological activity of the molecules. The 7,7′- and 19c showing potent activity against E. coli (Gram-negative)
linked compounds containing the –CO–NH–NH–CO– linker growth while having no effect against B. subtilis (Gram-posi-
and the –CO–CO–NH–NH–CO–CO– linker inhibited both tive) growth is a significant outcome in terms of both identify-
B. subtilis growth and E. coli growth. It was found that incor- ing novel drug compounds as well as for combating bacterial
poration of the –CO–NH–NH–CO– linker into the 2,2′- and the resistance. Future research will focus on the identification and
2,7′-linked molecules and the –CO–CO–NH–NH–CO– linker in synthesis of smaller, lower molecular weight inhibitors of bac-
to the 7,7′-, the 2,2′- and the 2,7′-linked compounds completely terial transcription initiation complex formation in order to
abolished their ability to inhibit B. subtilis growth. Interest- overcome solubility problems and the limited permeability of
ingly, the 3,2′-linked molecules having the –CO–CO–NH–NH– the molecules through the outer membrane of the cell wall in
CO– linkage were still active against B. subtilis growth. Further- Gram-negative bacteria.
more, the nature of the substituent at position 3 of the indole
ring could also influence the biological activity of the com-
pounds. Changing from a 4-BrC6H4 substituent to a 4-ClC6H4
substituent enhanced biological potency across three classes
of molecules: 7,7′-, 2,7′- and 3,2′-linked bis-indoles. As PhCl is
Materials and methods
General chemistry details
smaller but more electron-withdrawing compared to PhBr, this Trichloroacetyl chloride was synthesized from trichloroacetic
suggests that the size and/or electronic character of this substi- acid according to a slightly modified literature procedure.32 All
tuent may play a role in the biological activity for these types commercially available reagents and solvents were purchased
of compounds, possibly as a consequence of influencing the from Sigma-Aldrich and Alfa Aesar. No further purification
cellular permeability of the molecules.
was performed for commercial chemicals. Anhydrous aceto-
Differences between inhibition of the β′-CH-σ70/σ2A.2 inter- nitrile and anhydrous diethyl ether were obtained from a Pure-
action and inhibition of bacterial growth observed for some Solv MD Solvent Purification System available in the School of
compounds could be explained in two ways. Firstly, since Chemistry at UNSW.
many molecules were partly insoluble at ca. 200 μM, bacterial
1H NMR and 13C NMR spectra were recorded in CDCl3 and
growth inhibitory activities need to be treated as qualitative DMSO-d6 on a Bruker Avance III 300 MHz spectrometer in the
data only. Secondly, inhibition of bacterial growth could be Nuclear Magnetic Resonance Facility in the Mark Wainwright
dependent on the ability of the novel compounds to cross the Analytical Centre at UNSW and were internally calibrated to
outer membrane in Gram-negative bacteria and the cell wall in the solvent peaks. Chemical shifts (δ) were reported in parts
Gram-positive bacteria.
per million (ppm). Splitting patterns were reported as singlet
(s), broad singlet (bs), doublet (d) and multiplet (m), and the
observed coupling constants (J) provided in hertz (Hz).
Infrared spectra were acquired on a Thermo Nicolet Avatar
370 FT-IR spectrophotometer using KBr discs or on a Perkin
Conclusion
A library of thirty novel molecules was synthesized, fully Elmer 298 IR spectrometer using paraffin mulls. The wave
characterized and evaluated as potential inhibitors of tran- numbers (νmax) related to the transmittance minima were
scription initiation complex formation. The compounds were reported in cm−1. Ultraviolet-visible light spectra were recorded
tested for their ability to inhibit the β′-CH-σ70/σ2A.2 interaction using a Varian Cary 100 Bio UV-visible spectrophotometer or a
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Hitachi U-3200 spectrometer. The absorption maxima (λmax) in ArH), 8.96 (s, 1H, NH); 13C NMR (75 MHz, CDCl3): δ 55.4, 55.8
nm together with the molar absorptivities (ε) were reported. (OMe), 85.7 (C7), 94.1 (C5), 96.3 (CCl3), 113.3 (C3), 120.8 (C2),
Absolute methanol and HPLC quality tetrahydrofuran were 127.5, 131.8 (ArCH), 132.5, 133.0, 133.4, 139.3, 156.7, 162.6
used as the solvents.
(ArC), 170.8 (CvO); MS (EI): m/z 435 (M + 2, Cl37/37, 10%), 433
High resolution mass spectra were acquired using
a
(M, Cl35/35, 25), 314 (35), 279 (90), 264 (50), 150 (100).
Thermo Scientific LTQ Orbitrap XL LC-MS mass spectrometer
(electrospray ionization (ESI) mode) in the Bioanalytical Mass Spec-
trometry Facility in the Mark Wainwright Analytical Centre at
UNSW. Masses found for hydrogen adducts ([M + H]+) and To
General procedure for the synthesis of indole-
7-carbohydrazides and indole-2-carbohydrazides
a
suspension of 3-(4-chlorophenyl)-4,6-dimethoxy-7-tri-
sodium adducts ([M + Na]+) were reported with accuracy to chloroacetyl-1H-indole 2b (0.94 g, 2.2 mmol) in anhydrous
four decimal places. Required masses were calculated using acetonitrile (25 mL) or 3-(4-chlorophenyl)-4,6-dimethoxy-2-tri-
Xcalibur software installed on the spectrometer. The EI mass chloroacetyl-1H-indole 3b (1.76 g, 4.1 mmol) in anhydrous
spectra were recorded on a VG Quattro mass spectrometer at acetonitrile (45 mL) was added hydrazine hydrate (excess,
70 eV ionization voltage and 200 °C ion source temperature.
1 mL, 20.5 mmol) and the reaction mixture was stirred at room
Melting points were measured using a Mel-Temp melting temperature for 3.5 h. The resulting precipitate was filtered,
point apparatus and are uncorrected. Temperature quoted as washed with acetonitrile (3 × 10 mL), water (1 × 10 mL) and
0 °C was achieved with a cooling bath of ice-water.
air-dried to yield the respective hydrazide in 74 and 81% yield.
Both gravity column chromatography and flash column
3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indole-7-carbohydrazide
chromatography were performed using Grace Davisil LC60A (4b). White solid; mp 210–212 °C (water–acetonitrile); UV-vis:
40-63 micron silica gel. Reaction progress was monitored by λmax(THF)/nm 212 (ε/dm3 mol−1 cm−1 18 500), 238 (27 500),
thin-layer chromatography using Merck TLC Silica gel 60 F254 302 (13 300); IR (KBr): νmax/cm−1 3370 (NH), 3345 (NH), 3012,
aluminium sheets and detection by short and long wavelength 2939, 2841, 1622 (CvO), 1591 (CvO), 1535, 1463, 1346, 1264,
ultraviolet light.
1215, 1151, 1094, 1012, 983, 950, 895, 841, 796; 1H NMR
(300 MHz, DMSO-d6): δ 3.88 (s, 3H, OMe), 4.02 (s, 3H, OMe),
4.59 (bs, 2H, NH2), 6.48 (s, 1H, H5), 7.24 (d, J = 2.5 Hz, 1H,
H2), 7.34–7.41 (m, 2H, ArH), 7.48–7.57 (m, 2H, ArH), 9.16
General procedure for the synthesis of 7-trichloroacetylindoles
and 2-trichloro-acetylindoles
To a solution of 3-(4-chlorophenyl)-4,6-dimethoxy-1H-indole (s, 1H, NH–NH2), 11.44 (d, J = 1.6 Hz, 1H, NH); 13C NMR
1b (2.93 g, 10.2 mmol) in anhydrous 1,2-dichloroethane (75 MHz, DMSO-d6): δ 55.3, 56.8 (OMe), 88.1 (C7), 96.6 (C5),
(50 mL) was added trichloroacetyl chloride (3.5 mL, 31 mmol) 110.0 (C3), 115.2 (ArC), 123.4 (C2), 127.5 (ArCH), 130.0 (ArC),
dropwise and the reaction mixture was heated at reflux for 130.6 (ArCH), 134.8, 138.1, 156.0, 156.4 (ArC), 165.8 (CvO);
3.5 h. After cooling to room temperature the reaction mixture HRMS (+ESI): found m/z 368.0771 ([M
+
Na]+),
was washed with water (1 × 50 mL), dried over anhydrous [C17H16ClN3O3Na]+ requires m/z 368.0772 (monoisotopic
sodium sulfate, mixed with silica gel and the solvent was mass).
removed in vacuo. Gravity column chromatography (30 cm ×
3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indole-2-carbohydrazide
3.5 cm, dichloromethane–n-hexane, 10%/90% (v/v) → 35%/ (5b). White solid; mp 238–240 °C (water–acetonitrile); UV-vis:
65% (v/v)) afforded the respective trichloroacetylindole deriva- λmax(THF)/nm 212 (ε/dm3 mol−1 cm−1 33 200), 251 (28 500),
tives in 37 and 23% yield.
306 (16 100); IR (KBr): νmax/cm−1 3418 (NH), 3313 (NH), 3016,
3-(4-Chlorophenyl)-4,6-dimethoxy-7-trichloroacetyl-1H-indole 2936, 2841, 1576 (CvO), 1503, 1462, 1278, 1202, 1139, 1091,
1
(2b). Yellow solid; mp 178 °C (dichloromethane–n-hexane); 1041, 997, 925, 836, 814; H NMR (300 MHz, DMSO-d6): δ 3.59
UV-vis: λmax(MeOH)/nm 212 (ε/dm3 mol−1 cm−1 14 000), 256 (s, 3H, OMe), 3.77 (s, 3H, OMe), 4.35 (bs, 2H, NH2), 6.15 (d, J =
(12 600), 343 (8000); IR (KBr): νmax/cm−1 3380 (NH), 1610 2.0 Hz, 1H, H5), 6.50 (d, J = 2.0 Hz, 1H, H7), 7.33–7.41 (m, 4H,
(CvO), 1580, 1560, 1340, 1245, 1215, 1080;1H NMR (300 MHz, ArH), 8.25 (s, 1H, NH–NH2), 11.49 (s, 1H, NH); 13C NMR
CDCl3): δ 3.93 (s, 3H, OMe), 4.00 (s, 3H, OMe), 6.23 (s, 1H, (75 MHz, DMSO-d6): δ 55.0, 55.3 (OMe), 86.7 (C7), 92.4 (C5),
H5), 7.08 (d, J = 2.4 Hz, H2), 7.30–7.37 (m, 2H, ArH), 7.43–7.51 111.2 (C3), 116.5 (ArC), 125.1 (C2), 127.0 (ArCH), 131.0 (ArC),
(m, 2H, ArH), 10.29 (s, 1H, NH); 13C NMR (75 MHz, CDCl3): 132.7 (ArCH), 133.9, 137.2, 154.9, 158.2 (ArC), 161.6 (CvO);
δ 55.6, 55.8 (OMe), 87.8 (C7), 98.6 (CCl3), 98.8 (C5), 110.9 (C3), HRMS (+ESI): found m/z 368.0779 ([M
+
Na]+),
118.7 (ArC), 121.7 (C2), 127.9, 130.9 (ArCH), 132.2, 133.9, [C17H16ClN3O3Na]+ requires m/z 368.0772 (monoisotopic
139.9, 160.5, 161.5 (ArC), 182.5 (CvO); MS (EI): m/z 435 mass).
(M + 2, Cl37/37, 7%), 433 (M, Cl35/35, 15), 316 (33), 314 (100).
Trichloroacetyl and hydrazide derivatives of indoles with
3-(4-Chlorophenyl)-4,6-dimethoxy-2-trichloroacetyl-1H-indole other aryl or methyl substituents at position 3 of the indole
(3b). Yellow solid; mp 214 °C (dichloromethane–n-hexane); ring were synthesized following the above procedures.
UV-vis: λmax(MeOH)/nm 210 (ε/dm3 mol−1 cm−1 21 200), 281 However, the reaction of 4,6-dimethoxy-3-methyl-1H-indole
(14 300), 360 (9600); IR (KBr): νmax/cm−1 3400 (NH), 1670 with trichloroacetyl chloride did not produce the corres-
(CvO), 1615, 1570, 1380, 1350, 1250, 1210, 1150; 1H NMR ponding 7-trichloroacetyl derivative. Gravity column chromato-
(300 MHz, CDCl3): δ 3.63 (s, 3H, OMe), 3.88 (s, 3H, OMe), 6.13 graphy
afforded
only
4,6-dimethoxy-3-methyl-2,7-
(d, J = 1.7 Hz, 1H, H5), 6.45 (d, J = 1.7 Hz, 1H, H7), 7.36 (s, 4H, trichloroacetyl-1H-indole as the faster moving batch (Rf = 0.54)
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and 4,6-dimethoxy-3-methyl-2-trichloroacetyl-1H-indole as the mixture was stirred at room temperature (reactions involving
slower moving batch (Rf = 0.42).
glyoxyloyl chlorides or trichloroacetylindole derivatives) or at
reflux (reactions involving trichloroacetylindole derivatives)
until the TLC showed the completion of the reaction (5–26 h).
The solvent was evaporated and water was added to the
General procedure for the synthesis of indol-2-yl-glyoxyloyl
chlorides and indol-7-yl-glyoxyloyl chlorides
The synthesis followed an established general procedure.28 To residue. The resulting precipitate was filtered, dried and recrys-
a solution of 3-(4-chlorophenyl)-4,6-dimethoxy-1H-indole 1b tallized from methanol giving the product as a pale brown,
(1.75 g, 6.08 mmol) in anhydrous diethyl ether (35 mL) at 0 °C grey, yellow or orange solid in the range of 40–65% yield.
was added with oxalyl chloride (1.6 mL, 18 mmol) and the
reaction mixture was warmed from 0 °C to room temperature, indole-7-carbonyl)-4,6-dimethoxy-1H-indole-7-carbohydrazide
and stirred for 3 h and resulted in 33, 43 and 92% yield. (10a). Brown solid; mp >300 °C (methanol); UV-vis: λmax(THF)/
3-(4-Chlorophenyl)-N′-(3-(4-chlorophenyl)-4,6-dimethoxy-1H-
3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-7-yl-glyoxyloyl nm 243 (ε/dm3 mol−1 cm−1 86 600), 279 (45 800), 348 (57 200),
chloride (6b). The filtrate was mixed with silica gel, solidified 364 (42 600); IR (KBr): νmax/cm−1 3388 (NH), 1592 (CvO),
in vacuo and purified by gravity column chromatography 1448, 1340, 1213, 1151, 792; 1H NMR (300 MHz, DMSO-d6):
(dichloromethane–n-hexane, 80%/20% (v/v)) to afford the title δ 3.93 (s, 6H, OMe), 4.13 (s, 6H, OMe), 6.58 (s, 2H, H5), 7.26
1
product 6b as a yellow-orange solid, lit.28; H NMR (300 MHz, (d, J = 2.5 Hz, 2H, H2), 7.34–7.43 (m, 4H, ArH), 7.49–7.59 (m,
CDCl3): δ 3.95 (s, 3H, OMe), 4.00 (s, 3H, OMe), 6.19 (s, 1H, 4H, ArH), 10.52 (s, 2H, NH), 11.52 (d, J = 2.0 Hz, 2H, NH);
H5), 7.10 (d, J = 2.3 Hz, 1H, H2), 7.31–7.38 (m, 2H, ArH), 13C NMR (75 MHz, DMSO-d6): δ 55.5, 57.2 (OMe), 88.3 (C5),
7.43–7.50 (m, 2H, ArH), 10.36 (bs, 1H, NH); HRMS (+ESI): 95.8 (C7), 110.2 (C3), 115.4 (ArC), 123.6 (C2), 127.5 (ArCH),
found m/z 378.0311 ([M + H]+), [C18H13Cl2NO4H]+ requires 130.1 (ArC), 130.7 (ArCH), 134.6, 138.0, 156.6, 157.2 (ArC),
378.0294 (monoisotopic mass).
163.1 (CvO); HRMS (+ESI): found m/z 681.1273 ([M + Na]+),
3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-2-yl-glyoxyloyl [C34H28Cl2N4O6Na]+ requires m/z 681.1278 (monoisotopic
chloride (7b). The resulting precipitate was filtered out from mass).
the reaction mixture, washed with anhydrous diethyl ether (1 ×
3-(4-Bromophenyl)-N′-(3-(4-bromophenyl)-4,6-dimethoxy-1H-
30 mL) and dried to yield the title product 7b as a dark red indole-7-carbonyl)-4,6-dimethoxy-1H-indole-7-carbohydrazide
solid, lit.28; 1H NMR (300 MHz, DMSO-d6): δ 3.58 (s, 3H, OMe), (10b). Pale brown solid; mp >300 °C (methanol); UV-vis:
3.80 (s, 3H, OMe), 6.15 (d, J = 1.9 Hz, 1H, H5), 6.52 (d, J = λmax(THF)/nm 242 (ε/dm3 mol−1 cm−1 65 200), 281 (34 400),
1.9 Hz, 1H, H7), 7.29–7.39 (m, 4H, ArH), 11.97 (s, 1H, NH); 348 (42 600), 365 (31 600); IR (KBr): νmax/cm−1 3377 (NH), 1614
HRMS (+ESI): found m/z 378.0311 ([M
+
H]+), (CvO), 1593 (CvO), 1444, 1335, 1218, 1150, 1109, 979, 795;
1H NMR (300 MHz, DMSO-d6): δ 3.93 (s, 6H, OMe), 4.13 (s, 6H,
[C18H13Cl2NO4H]+ requires 378.0294 (monoisotopic mass).
Glyoxyloyl chlorides of indoles with other aryl or methyl OMe), 6.58 (s, 2H, H5), 7.26 (d, J = 2.4 Hz, 2H, H2), 7.42–7.59
substituents at position 3 of the indole ring were synthesized (m, 8H, ArH), 10.51 (s, 2H, NH), 11.53 (d, J = 1.6 Hz, 2H, NH);
following the above procedures. However, only trace amounts HRMS (+ESI): found m/z 747.0440 ([M
+
H]+),
(<0.5%) of the 7-glyoxyloyl chloride were produced in reaction [C34H28Br2N4O6H]+ requires m/z 747.0448 (monoisotopic
of 4,6-dimethoxy-3-methyl-1H-indole and oxalyl chloride. mass). The compound was not soluble enough in DMSO-d6 for
Additional recrystallization from hot ethyl acetate was also 13C NMR measurement.
required to purify the corresponding 2-glyoxyloyl chloride.
4,6-Dimethoxy-N′-(4,6-dimethoxy-3-(4-fluorophenyl)-1H-indole-
1H-Indol-3-yl-glyoxyloyl chloride (9). To a solution of indole 7-carbonyl)-3-(4-fluorophenyl)-1H-indole-7-carbohydrazide (10c).
8 (2.67 g, 22.8 mmol) in anhydrous diethyl ether (50 mL) at Light brown solid; mp >300 °C (methanol); IR (KBr): νmax/cm−1
0 °C was added oxalyl chloride (6.1 mL, 69 mmol) in one go 3370 (NH), 1616 (CvO), 1592 (CvO), 1444, 1339, 1215, 1150,
and the reaction mixture was warmed from 0 °C to room temp- 1105, 979, 793; 1H NMR (300 MHz, DMSO-d6): δ 3.79 (s, 6H,
erature, and stirred for further 3 h. The resulting precipitate OMe), 3.94 (s, 6H, OMe), 6.41 (s, 2H, H5), 7.32 (d, J = 2.2 Hz,
was filtered out from the reaction mixture, washed with anhy- 2H, H2), 7.29–7.49 (m, 8H, ArH), 10.58 (s, 2H, NH), 11.48 (bs,
drous diethyl ether and dried to yield the title product 9 as a 2H, NH); HRMS (+ESI): found m/z 627.2055 ([M + H]+),
dark red solid, lit.29; 1H NMR (300 MHz, DMSO-d6): δ [C34H28F2N4O6H]+ requires m/z 627.2050 (monoisotopic mass).
7.23–7.31 (m, 2H, H5, H6), 7.52–7.58 (m, 1H, H7), 8.16–8.21 The compound was not soluble enough in DMSO-d6 for 13C
(m, 1H, H4), 8.43 (d, J = 3.3 Hz, 1H, H2), 12.37 (s, 1H, NH).
NMR and in THF for UV-vis measurements.
4,6-Dimethoxy-N′-(4,6-dimethoxy-3-(4-methoxyphenyl)-1H-
indole-7-carbonyl)-3-(4-methoxyphenyl)-1H-indole-7-carbohy-
drazide (10d). Brown solid; mp >300 °C (methanol); IR (KBr):
General procedure for the synthesis of 10a–f, 11a–c, 12a–g, 13,
14a–c, 15a–d and 19a–c
To a solution or suspension of the appropriate 2-trichloroace- νmax/cm−1 3376 (NH), 1620 (CvO), 1590 (CvO), 1443, 1339,
tylindole 3a–f, 7-trichloroacetylindole 2a–e, 2-glyoxyloyl chlor- 1216, 1150, 1105, 978, 790; 1H NMR (300 MHz, DMSO-d6):
ide 7a–d, 7-glyoxyloyl chloride 6a–c or 3-glyoxyloyl chloride 9 δ 3.79 (s, 3H, OMe), 3.85 (s, 3H, OMe), 3.91 (s, 6H, OMe), 4.03
(1.0 equiv.) in acetonitrile was added the appropriate indole-7- (s, 6H, OMe), 6.47 (s, 2H, H5), 7.35 (d, J = 2.2 Hz, 2H, H2),
carbohydrazide 4a–e or indole-2-carbohydrazide 5a–f (1.0 7.40–7.59 (m, 8H, ArH), 10.44 (s, 2H, NH), 11.51 (bs, 2H, NH);
equiv.) followed by triethylamine (5 drops) and the reaction HRMS (+ESI): found m/z 651.2449 ([M + H]+), [C36H34N4O8H]+
2888 | Org. Biomol. Chem., 2014, 12, 2882–2894
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requires m/z 651.2449 (monoisotopic mass). The compound (s, 3H, OMe), 3.76 (s, 3H, OMe), 3.80 (s, 3H, OMe), 3.82 (s, 3H,
was not soluble enough in DMSO-d6 for 13C NMR and in THF OMe), 6.12 (d, J = 1.7 Hz, 1H, H5), 6.19 (d, J = 1.6 Hz, 1H, H5),
for UV-vis measurements.
4,6-Dimethoxy-N′-(4,6-dimethoxy-3-(4-tolyl)-1H-indole-7-car- (d, J = 8.4 Hz, 2H, ArH), 7.46 (d, J = 8.4 Hz, 2H, ArH), 9.21 (s,
bonyl)-3-(4-tolyl)-1H-indole-7-carbohydrazide (10e). Pale 1H, NH), 9.70 (s, 1H, NH), 11.01 (s, 1H, NH), 11.66 (s, 1H,
6.41 (d, J = 1.7 Hz, 1H, H7), 6.56 (d, J = 1.6 Hz, 1H, H7), 7.38
brown solid; mp >300 °C (methanol); UV-vis: λmax(THF)/nm NH); 13C NMR (75 MHz, DMSO-d6): δ 12.0 (Me), 55.0, 55.2,
212 (ε/dm3 mol−1 cm−1 68 400), 245 (72 200), 269 (46 400), 348 55.2, 55.3 (OMe), 86.4, 86.7 (C7), 91.7, 92.6 (C5), 111.3, 112.6
(47 200), 365 (36 400); IR (KBr): νmax/cm−1 3366 (NH), 1615 (C3), 116.6, 118.2, 123.1, 123.9 (ArC), 127.0 (ArCH), 131.2
(CvO), 1591 (CvO), 1448, 1338, 1216, 1151, 1107, 979, 790; (ArC), 132.9 (ArCH), 133.6, 137.6, 137.7, 155.1, 156.0, 158.6,
1H NMR (300 MHz, DMSO-d6): δ 2.31 (s, 6H, Me), 3.89 (s, 6H, 158.7 (ArC), 160.7, 161.1 (CvO); HRMS (+ESI): found m/z
OMe), 4.05 (s, 6H, OMe), 6.51 (s, 2H, H5), 7.22 (d, J = 2.3 Hz, 585.1492 ([M + Na]+), [C29H27ClN4O6Na]+ requires m/z 585.1511
2H, H2), 7.38–7.51 (m, 8H, ArH), 10.65 (s, 2H, NH), 11.56 (bs, (monoisotopic mass).
2H, NH); HRMS (+ESI): found m/z 619.2554 ([M + H]+),
[C36H34N4O6H]+ requires m/z 619.2551 (monoisotopic mass). bonyl)-3-methyl-1H-indole-2-carbohydrazide
The compound was not soluble enough in DMSO-d6 for 13C brown solid; mp 288–290 °C (methanol); UV-vis: λmax(THF)/nm
4,6-Dimethoxy-N’-(4,6-dimethoxy-3-methyl-1H-indole-2-car-
(11c). Light
NMR measurement.
215 (ε/dm3 mol−1 cm−1 23 300), 246 (27 000), 312 (33 600);
3-(4-Bromophenyl)-N′-(3-(4-chlorophenyl)-4,6-dimethoxy-1H- IR (KBr): νmax/cm−1 3288 (NH), 3185, 2932 (NH), 2837 (NH),
indole-7-carbonyl)-4,6-dimethoxy-1H-indole-7-carbohydrazide 2135, 2069, 1651 (CvO), 1423, 1380, 1098, 1060, 998, 938, 876,
1
(10f). Pale yellow solid; mp >300 °C (methanol); UV-vis: 806; H NMR (300 MHz, DMSO-d6): δ 2.67 (s, 6H, Me), 3.77 (s,
λmax(THF)/nm 210 (ε/dm3 mol−1 cm−1 65 100), 239 (70 400), 6H, OMe), 3.83 (s, 6H, OMe), 6.13 (d, J = 1.8 Hz, 2H, H5), 6.44
275 (41 000), 329 (38 900); IR (KBr): νmax/cm−1 3415 (NH), 1626 (d, J = 1.8 Hz, 2H, H7), 9.69 (s, 2H, NH), 11.05 (s, 2H, NH); 13C
(CvO), 1580 (CvO), 1560, 1536, 1465, 1351, 1323, 1214, 1183, NMR (75 MHz, DMSO-d6): δ 12.1 (Me), 55.2 (2 × OMe), 86.5
1
1150, 1089, 980, 796; H NMR (300 MHz, DMSO-d6): δ 3.93 (s, (C7), 91.5 (C5), 112.6 (C3), 116.4 (C3a), 123.4 (C2), 137.7 (C7a),
3H, OMe), 3.96 (s, 3H, OMe), 3.98 (s, 3H, OMe), 4.12 (s, 3H, 156.0, 158.7 (ArC), 161.5 (CvO); HRMS (+ESI): found m/z
OMe), 6.51 (s, 1H, H5), 6.57 (s, 1H, H5), 7.25 (d, J = 2.3 Hz, 1H, 489.1745 ([M + Na]+), [C24H26N4O6Na]+ requires m/z 489.1745
H2), 7.29 (d, J = 2.3 Hz, 1H, H2), 7.35–7.67 (m, 8H, ArH), 9.94 (monoisotopic mass).
(s, 1H, NH), 10.89 (s, 1H, NH), 11.44 (d, J = 1.8 Hz, 1H, NH),
3-(4-Chlorophenyl)-4,6-dimethoxy-N′-(4,6-dimethoxy-3-(4-
11.56 (d, J = 1.8 Hz, 1H, NH); 13C NMR (75 MHz, DMSO-d6): methoxyphenyl)-1H-indole-7-carbonyl)-1H-indole-2-carbohy-
δ 55.5, 55.9, 57.2, 57.3 (OMe), 88.4, 88.7 (C5), 96.2, 101.6 (C7), drazide (12a). Grey-brown solid; mp 174–176 °C (methanol);
109.9, 110.2 (C3), 115.4, 116.4, 118.9 (ArC), 123.6, 123.7 (C2), UV-vis: λmax(THF)/nm 213 (ε/dm3 mol−1 cm−1 67 800), 252
127.5 (ArCH), 130.1 (ArC), 130.5, 130.7, 131.1 (ArCH), 134.7, (68 400), 315 (36 100), 339 (38 100); IR (KBr): νmax/cm−1 3388
137.1, 138.1, 156.6, 157.2, 161.0, 162.0 (ArC), 164.6, 165.9 (NH), 1620 (CvO), 1590 (CvO), 1539, 1455, 1344, 1244, 1213,
1
(CvO); HRMS (+ESI): found m/z 703.0948 ([M
+
H]+), 1152, 1138, 805; H NMR (300 MHz, DMSO-d6): δ 3.60 (s, 3H,
[C34H28BrClN4O6H]+ requires m/z 703.0954 (monoisotopic OMe), 3.77 (s, 3H, OMe), 3.80 (s, 3H, OMe), 3.88 (s, 3H, OMe),
mass). 4.05 (s, 3H, OMe), 6.18 (d, J = 1.5 Hz, 1H, H5), 6.49 (s, 1H, H5),
3-(4-Chlorophenyl)-N′-(3-(4-chlorophenyl)-4,6-dimethoxy-1H- 6.56 (d, J = 1.5 Hz, 1H, H7), 6.82–7.02 (m, 2H, ArH), 7.12 (d, J =
indole-2-carbonyl)-4,6-dimethoxy-1H-indole-2-carbohydrazide 2.3 Hz, 1H, H2), 7.27–7.62 (m, 6H, ArH), 9.36 (s, 1H, NH), 9.99
(11a). White solid; mp 292–294 °C (methanol); UV-vis: (s, 1H, NH), 11.25 (s, 1H, NH), 11.70 (s, 1H, NH); 13C NMR
λmax(THF)/nm 214 (ε/dm3 mol−1 cm−1 64 500), 254 (49 600), (75 MHz, DMSO-d6): δ 55.0, 55.0, 55.3, 55.4, 57.0 (OMe), 86.6,
315 (26 100); IR (KBr): νmax/cm−1 3354 (NH), 1626 (CvO), 87.9 (C5), 92.6, 95.6 (C7), 110.4, 111.5 (C3), 113.1 (ArCH),
1
1536, 1259, 1210, 1132, 1089, 811; H NMR (300 MHz, CDCl3): 116.4, 118.5 (ArC), 122.5, 123.5 (C2), 127.0 (ArCH), 128.1 (ArC),
δ 3.60 (s, 6H, OMe), 3.83 (s, 6H, OMe), 6.12 (d, J = 1.7 Hz, 2H, 130.1 (ArCH), 131.3 (ArC), 132.8 (ArCH), 133.6, 137.6, 138.0,
H5), 6.41 (d, J = 1.7 Hz, 2H, H7), 7.45 (s, 8H, ArH), 7.77 (bs, 155.2, 156.5, 157.4, 157.4, 158.7 (ArC), 159.7, 164.5 (CvO);
2H, NH), 9.13 (bs, 2H, NH); 13C NMR (75 MHz, CDCl3): δ 55.2, HRMS (+ESI): found m/z 655.1955 ([M
+
H]+),
55.8 (OMe), 86.0 (C5), 93.3 (C7), 113.0 (C3), 119.0 (ArC), 122.2 [C35H31ClN4O7H]+ requires m/z 655.1954 (monoisotopic mass).
(C2), 128.8, 132.2 (ArCH), 132.9, 134.3, 137.5, 156.1, 159.1 3-(4-Chlorophenyl)-4,6-dimethoxy-N′-(4,6-dimethoxy-3-(4-tolyl)-
(ArC), 160.3 (CvO); HRMS (+ESI): found m/z 681.1292 ([M + 1H-indole-7-carbonyl)-1H-indole-2-carbohydrazide (12b). Grey-
Na]+), [C34H28Cl2N4O6Na]+ requires m/z 681.1278 (monoisoto- brown solid; mp 176–178 °C (methanol); UV-vis: λmax(THF)/nm
pic mass).
213 (ε/dm3 mol−1 cm−1 102 700), 248 (94 600), 312 (51 800);
3-(4-Chlorophenyl)-4,6-dimethoxy-N’-(4,6-dimethoxy-3-methyl- IR (KBr): νmax/cm−1 3372 (NH), 1622 (CvO), 1593 (CvO),
1H-indole-2-carbonyl)-1H-indole-2-carbohydrazide (11b). Grey 1539, 1456, 1342, 1248, 1213, 1152, 1137, 808; 1H NMR
solid; mp 274–276 °C (methanol); UV-vis: λmax(THF)/nm 214 (300 MHz, DMSO-d6): δ 2.32 (s, 3H, Me), 3.60 (s, 3H, OMe),
(ε/dm3 mol−1 cm−1 46 400), 250 (40 000), 313 (33 600); IR (KBr): 3.80 (s, 3H, OMe), 3.89 (s, 3H, OMe), 4.05 (s, 3H, OMe), 6.18
νmax/cm−1 3319 (NH), 2933 (NH), 2840 (NH), 1644 (CvO), 1584 (d, J = 1.6 Hz, 1H, H5), 6.50 (s, 1H, H5), 6.56 (d, J = 1.6 Hz, 1H,
(CvO), 1533, 1463, 1322, 1258, 1216, 1153, 1043, 991, 941, H7), 7.13 (s, 1H, H2), 7.13–7.56 (m, 8H, ArH), 9.35 (d, J = 2.1 Hz,
820; 1H NMR (300 MHz, DMSO-d6): δ 2.63 (s, 3H, Me), 3.61 1H, NH), 9.99 (d, J = 1.8 Hz, 1H, NH), 11.27 (d, J = 1.7 Hz, 1H,
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NH), 11.68 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): δ 21.9 11.63 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): δ 20.9 (Me),
(Me), 55.4, 55.8, 55.8, 57.4 (OMe), 87.1, 88.3 (C5), 93.2, 96.1 55.0, 55.2, 55.5, 57.0 (OMe), 86.6, 88.1 (C5), 92.5, 95.7 (C7),
(C7), 110.9, 112.0 (C3), 116.9 (ArC), 122.9 (C2), 127.6, 128.5 110.0, 111.9 (C3), 115.4, 118.5 (ArC), 123.2, 123.5 (C2), 128.3,
(ArCH), 128.7 (ArC), 130.6 (ArCH), 131.8, 132.3 (ArC), 133.3 130.4 (ArCH), 130.6 (ArC), 130.7, 131.0 (ArCH), 131.7, 135.0,
(ArCH), 134.1, 136.3, 136.9, 138.0, 138.4, 157.0, 157.8, 159.2, 136.1, 137.7, 138.0, 155.4, 156.7, 157.3, 158.7 (ArC), 160.0,
159.9 (ArC), 160.2, 165.1 (CvO); HRMS (+ESI): found m/z 164.4 (CvO); HRMS (+ESI): found m/z 683.1518 ([M + H]+),
661.1847 ([M + Na]+), [C35H31ClN4O6Na]+ requires m/z 661.1824 [C35H31BrN4O6H]+ requires m/z 683.1500 (monoisotopic mass).
(monoisotopic mass).
N‘-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indole-7-carbonyl)-
(12f).
4,6-Dimethoxy-N′-(4,6-dimethoxy-3-(4-tolyl)-1H-indole-2-car- 4,6-dimethoxy-3-methyl-1H-indole-2-carbohydrazide
bonyl)-3-(4-tolyl)-1H-indole-7-carbohydrazide (12c). Grey solid; Grey solid; mp 280–282 °C (methanol); UV-vis: λmax(THF)/nm
mp 170–172 °C (methanol); UV-vis: λmax(THF)/nm 212 (ε/dm3 212 (ε/dm3 mol−1 cm−1 38 200), 243 (43 600), 334 (33 100);
mol−1 cm−1 52 800), 250 (52 200), 313 (26 800), 332 (26 600); IR (KBr): νmax/cm−1 3442 (NH), 3406 (NH), 2936 (NH), 2838
IR (KBr): νmax/cm−1 3409 (NH), 1616 (CvO), 1589 (CvO), (NH), 1590 (CvO), 1534, 1464, 1344, 1212, 1154, 1010, 981,
1542, 1460, 1344, 1247, 1213, 1151, 1137, 807; 1H NMR 809, 794; 1H NMR (300 MHz, DMSO-d6): δ 2.71 (s, 3H, Me),
(300 MHz, DMSO-d6): δ 2.32 (s, 3H, Me), 2.37 (s, 3H, Me), 3.57 3.78 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.92 (s, 3H, OMe), 4.10 (s,
(s, 3H, OMe), 3.79 (s, 3H, OMe), 3.88 (s, 3H, OMe), 4.04 (s, 3H, 3H, OMe), 6.14 (d, J = 1.8 Hz, 1H, H5), 6.45 (d, J = 1.8 Hz, 1H,
OMe), 6.15 (d, J = 1.5 Hz, 1H, H5), 6.48 (s, 1H, H5), 6.56 (d, J = H7), 6.55 (s, 1H, H5), 7.26 (d, J = 2.5 Hz, 1H, H2), 7.43–7.57 (m,
1.5 Hz, 1H, H7), 7.13 (s, 1H, H2), 7.13–7.49 (m, 8H, ArH), 8.57 4H, ArH), 9.99 (d, J = 1.9 Hz, 1H, NH), 10.11 (d, J = 2.4 Hz, 1H,
(d, J = 2.4 Hz, 1H, NH), 9.98 (d, J = 2.0 Hz, 1H, NH), 11.28 (s, NH), 11.13 (s, 1H, NH), 11.49 (s, 1H, NH); 13C NMR (75 MHz,
1H, NH), 11.64 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): DMSO-d6): δ 12.1 (Me), 55.2, 55.2, 55.5, 57.1 (OMe), 86.5 (C7),
δ 20.7, 21.0 (Me), 55.0, 55.2, 55.4, 56.9 (OMe), 86.6, 87.9 (C5), 88.3, 91.8 (C5), 96.2 (C7), 110.1, 112.6 (C3), 115.4, 116.4, 118.6,
92.5, 95.5 (C7), 110.3, 111.9 (C3),116.6, 119.3 (ArC), 122.8, 123.2 123.3 (ArC), 123.6 (C2), 130.4, 131.1 (ArCH), 135.1, 137.7,
(C2), 128.2, 128.3, 129.0, 130.7 (ArCH), 131.7, 132.8, 134.3, 138.2, 156.0, 156.7, 157.2, 158.7 (ArC), 160.8, 164.9 (CvO);
136.1, 137.7, 138.0, 155.4, 156.6, 157.4, 158.7 (ArC), 160.0, 164.5 HRMS (+ESI): found m/z 629.0984 ([M
+
Na]+),
H]+), [C29H27BrN4O6Na]+ requires m/z 629.1006 (monoisotopic
mass).
N’-(3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indole-7-carbonyl)-
(12g).
solid; mp 168–170 °C (methanol); UV-vis: λmax(THF)/nm 212 Grey solid; mp 282–284 °C (methanol); UV-vis: λmax(THF)/nm
(CvO); HRMS (+ESI): found m/z 619.2550 ([M
+
[C36H34N4O6H]+ requires m/z 619.2551 (monoisotopic mass).
4,6-Dimethoxy-N’-(4,6-dimethoxy-3-(4-fluorophenyl)-1H-indole-
7-carbonyl)-3-(4-tolyl)-1H-indole-2-carbohydrazide
(12d). Grey 4,6-dimethoxy-3-methyl-1H-indole-2-carbohydrazide
(ε/dm3 mol−1 cm−1 62 200), 249 (57 800), 313 (28 500), 336 243 (ε/dm3 mol−1 cm−1 43 800), 334 (36 400); IR (KBr): νmax
/
(30 700); IR (KBr): νmax/cm−1 3390 (NH), 1615 (CvO), 1594 cm−1 3449 (NH), 3405 (NH), 2937 (NH), 2839 (NH), 1595
(CvO), 1542, 1461, 1344, 1256, 1213, 1151, 1136, 804; 1H NMR (CvO), 1537, 1464, 1345, 1214, 1154, 1137, 982, 806, 794; H
1
(300 MHz, DMSO-d6): δ 2.39 (s, 3H, Me), 3.58 (s, 3H, OMe), NMR (300 MHz, DMSO-d6): δ 2.71 (s, 3H, Me), 3.78 (s, 3H,
3.81 (s, 3H, OMe), 3.92 (s, 3H, OMe), 4.06 (s, 3H, OMe), 6.17 OMe), 3.84 (s, 3H, OMe), 3.92 (s, 3H, OMe), 4.10 (s, 3H, OMe),
(d, J = 1.5 Hz, 1H, H5), 6.52 (s, 1H, H5), 6.58 (d, J = 1.5 Hz, 1H, 6.14 (d, J = 1.7 Hz, 1H, H5), 6.45 (d, J = 1.7 Hz, 1H, H7), 6.55 (s,
H7), 7.23 (d, J = 2.7 Hz, 1H, H2), 7.25–7.52 (m, 8H, ArH), 8.58 1H, H5), 7.26 (d, J = 2.4 Hz, 1H, H2), 7.38 (d, J = 8.6 Hz, 2H,
(d, J = 2.7 Hz, 1H, NH), 10.00 (d, J = 2.7 Hz, 1H, NH), 11.40 (s, ArH), 7.54 (d, J = 8.6 Hz, 2H, ArH), 10.00 (d, J = 2.0 Hz, 1H,
1H, NH), 11.65 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): NH), 10.11 (d, J = 2.6 Hz, 1H, NH), 11.13 (s, 1H, NH), 11.49 (d,
δ 21.4 (Me), 55.5, 55.7, 55.8, 57.4 (OMe), 87.0, 88.4 (C5), 93.0, J = 1.5 Hz, 1H, NH); 13C NMR (75 MHz, DMSO-d6): δ 12.1 (Me),
96.0 (C7), 110.8, 112.4 (C3), 116.6 (ArCH), 117.1 (ArC), 123.3 55.2, 55.2, 55.5, 57.0 (OMe), 86.5 (C7), 88.3, 91.7 (C5), 96.2
(C2), 128.7, 129.4, 131.2 (ArCH), 132.1, 133.3, 134.8, 136.1, (C7), 110.2, 112.6 (C3), 115.4, 116.4, 123.3 (ArC), 123.5 (C2),
136.6, 138.1, 138.4, 155.8, 157.1, 157.9, 159.2, 162.0 (ArC), 127.5 (ArCH), 130.1 (ArC), 130.7 (ArCH), 134.7, 137.7, 138.2,
160.4, 164.9 (CvO); HRMS (+ESI): found m/z 623.2301 ([M + 156.0, 156.7, 157.2, 158.7 (ArC), 160.8, 164.9 (CvO); HRMS
H]+), [C35H31FN4O6H]+ requires m/z 623.2300 (monoisotopic (+ESI): found m/z 585.1514 ([M + Na]+), [C29H27ClN4O6Na]+
mass).
N′-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indole-7-carbonyl)-
4,6-dimethoxy-3-(4-tolyl)-1H-indole-2-carbohydrazide
requires m/z 585.1511 (monoisotopic mass).
N‘-(2-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indol-2-yl)-2-
(12e). oxoacetyl)-4,6-dimethoxy-3-methyl-1H-indole-2-carbohydrazide
Creamy white solid; mp 238–240 °C (methanol); UV-vis: (13). Orange solid; mp 286–288 °C (methanol); UV-vis:
λmax(THF)/nm 213 (ε/dm3 mol−1 cm−1 113 400), 248 (117 600), λmax(THF)/nm 214 (ε/dm3 mol−1 cm−1 97 100), 246 (58 600),
309 (67 500), 332 (68 700); IR (KBr): νmax/cm−1 3377 (NH), 1622 307 (42 500); IR (KBr): νmax/cm−1 3405 (NH), 2936 (NH), 2839
(CvO), 1586 (CvO), 1540, 1461, 1346, 1257, 1213, 1152, 1139, (NH), 1620 (CvO), 1522, 1463, 1383, 1318, 1267, 1210, 1155,
1
805; H NMR (300 MHz, DMSO-d6): δ 2.37 (s, 3H, Me), 3.56 (s, 1133, 812; 1H NMR (300 MHz, DMSO-d6): δ 2.65 (s, 3H, Me),
3H, OMe), 3.79 (s, 3H, OMe), 3.90 (s, 3H, OMe), 4.04 (s, 3H, 3.60 (s, 3H, OMe), 3.76 (s, 3H, OMe), 3.81 (s, 3H, OMe), 3.83 (s,
OMe), 6.15 (d, J = 1.7 Hz, 1H, H5), 6.50 (s, 1H, H5), 6.55 (d, J = 3H, OMe), 6.13 (d, J = 1.9 Hz, 1H, H5), 6.17 (d, J = 1.9 Hz, 1H,
1.7 Hz, 1H, H7), 7.06–7.60 (m, 9H, H2, ArH), 8.56 (d, J = 2.8 H5), 6.42 (d, J = 1.9 Hz, 1H, H7), 6.68 (d, J = 1.9 Hz, 1H, H7),
Hz, 1H, NH), 9.98 (d, J = 2.3 Hz, 1H, NH), 11.39 (s, 1H, NH), 7.29–7.37 (m, 2H, ArH), 7.45–7.52 (m, 2H, ArH), 9.58 (s, 1H,
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NH), 10.90 (bs, 1H, NH), 11.08 (s, 1H, NH), 11.89 (s, 1H, NH); NH); 13C NMR (75 MHz, DMSO-d6): δ 55.3, 55.5, 55.5, 57.2
13C NMR (75 MHz, DMSO-d6): δ 12.0 (Me), 55.2 (3 × OMe), 55.4 (OMe), 86.6, 88.2 (C5), 93.4, 95.4 (C7), 110.4, 112.4 (C3), 114.2,
(OMe), 86.5, 86.7 (C7), 91.8, 93.5 (C5), 112.2, 112.6 (C3), 116.6, 114.5 (ArCH), 115.7 (ArC), 123.3 (C2), 126.3 (ArC), 126.6
120.4, 123.0, 126.4, 126.7 (ArC), 129.6, 132.8 (ArCH), 133.5, (ArCH), 127.4 (ArC), 130.8, 130.9 (ArCH), 131.9, 132.1 (ArC),
137.8, 139.9, 156.0, 156.0, 158.8, 160.6 (ArC), 160.9, 162.9, 132.7 (ArCH), 132.9, 138.0, 139.9, 156.2, 156.8, 157.5, 161.0
178.1 (CvO); HRMS (+ESI): found m/z 657.0940 ([M + Na]+), (ArC), 162.5, 164.1, 179.6 (CvO); HRMS (+ESI): found m/z
[C30H27BrN4O7Na]+ requires m/z 657.0955 (monoisotopic 671.1712 ([M + H]+), [C35H28ClFN4O7H]+ requires m/z 671.1703
mass).
N′-(2-(3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-2-yl)-2-
(monoisotopic mass).
3-(4-Bromophenyl)-N′-(2-(3-(4-bromophenyl)-4,6-dimethoxy-
oxoacetyl)-4,6-dimethoxy-3-(4-methoxyphenyl)-1H-indole-7-car- 1H-indol-7-yl)-2-oxoacetyl)-4,6-dimethoxy-1H-indole-7-carbohy-
bohydrazide (14a). Orange solid; mp 288–290 °C (methanol); drazide (15a). Pale yellow solid; mp >300 °C (methanol);
UV-vis: λmax(THF)/nm 214 (ε/dm3 mol−1 cm−1 62 100), 245 UV-vis: λmax(THF)/nm 211(ε/dm3 mol−1 cm−1 113 200), 239
(50 400), 263 (42 700), 343 (29 800); IR (KBr): νmax/cm−1 3362 (133 600), 276 (76 400), 330 (75 400); IR (KBr): νmax/cm−1 3416
(NH), 1617 (CvO), 1595 (CvO), 1541, 1451, 1350, 1259, 1211, (NH), 1626 (CvO), 1588 (CvO), 1560, 1536, 1466, 1351, 1324,
1
1152, 1137, 805; H NMR (300 MHz, DMSO-d6): δ 3.57 (s, 3H, 1215, 1183, 1151, 980, 796; 1H NMR (300 MHz, DMSO-d6):
OMe), 3.86 (s, 3H, OMe), 3.93 (s, 3H, OMe), 3.96 (s, 3H, OMe), δ 3.93 (s, 3H, OMe), 3.96 (s, 3H, OMe),3.98 (s, 3H, OMe), 4.12
4.05 (s, 3H, OMe), 6.13 (d, J = 1.5 Hz, 1H, H5), 6.36 (d, J = 1.5 (s, 3H, OMe), 6.51 (s, 1H, H5), 6.57 (s, 1H, H5), 7.25 (d, J = 2.2
Hz, 1H, H7), 6.58 (s, 1H, H5), 7.07–7.49 (m, 9H, H2, ArH), 9.57 Hz, 1H, H2), 7.29 (d, J = 2.2 Hz, 1H, H2), 7.44–7.64 (m, 8H,
(s, 1H, NH), 11.33 (d, J = 1.5 Hz, 1H, NH), 11.82 (s, 1H, NH); ArH), 9.93 (s, 1H, NH), 10.88 (s, 1H, NH), 11.44 (s, 1H, NH),
13C NMR (75 MHz, DMSO-d6): δ 55.6, 55.6, 55.8, 56.0, 57.7 11.55 (d, J = 1.5 Hz, 1H, NH); 13C NMR (75 MHz, DMSO-d6): δ
(OMe), 87.0, 88.7 (C5), 93.6, 96.0 (C7), 110.9, 112.9 (C3), 114.6 55.5, 55.9, 57.2, 57.3 (OMe), 88.4, 88.7 (C5), 96.2, 101.6 (C7),
(ArCH), 116.3 (ArC), 123.7 (C2), 127.4 (ArC), 127.5 (ArCH), 109.9, 110.2 (C3), 115.4, 116.4, 118.6, 118.8 (ArC), 123.6, 123.7
128.9 (ArC), 131.2, 131.3 (ArCH), 132.5, 132.6, 136.5, 138.4, (C2), 130.4, 130.5, 131.1, 131.1 (ArCH), 134.7, 135.0, 137.1,
140.4, 156.8, 157.1, 157.9, 159.6, 159.8 (ArC), 163.1, 164.4, 138.1, 156.6, 157.2, 161.0, 162.0 (ArC), 164.6, 165.8, 187.7
180.2 (CvO); HRMS (+ESI): found m/z 683.1900 ([M + H]+), (CvO); HRMS (+ESI): found m/z 775.0406 ([M
[C36H31ClN4O8H]+ requires m/z 683.1903 (monoisotopic mass). [C35H28Br2N4O7H]+ requires m/z 775.0398 (monoisotopic mass).
4,6-Dimethoxy-3-(4-fluorophenyl)-N′-(2-(4,6-dimethoxy-3-(4- 3-(4-Chlorophenyl)-N′-(2-(3-(4-chlorophenyl)-4,6-dimethoxy-
+
H]+),
tolyl)-1H-indol-2-yl)-2-oxoacetyl)-1H-indole-7-carbohydrazide 1H-indol-7-yl)-2-oxoacetyl)-4,6-dimethoxy-1H-indole-7-carbohy-
(14b). Yellow solid; mp 282–284 °C (methanol); UV-vis: drazide (15b). Dark yellow solid; mp >300 °C (methanol);
λmax(THF)/nm 212 (ε/dm3 mol−1 cm−1 60 000), 242 (48 200), UV-vis: λmax(THF)/nm 212 (ε/dm3 mol−1 cm−1 82 500), 237
343 (24 500); IR (KBr): νmax/cm−1 3380 (NH), 1615 (CvO), 1578 (95 700), 275 (59 900); IR (KBr): νmax/cm−1 3418 (NH), 1626
(CvO), 1544, 1455, 1352, 1257, 1211, 1159, 1138, 806; 1H NMR (CvO), 1583 (CvO), 1560, 1536, 1465, 1354, 1324, 1215, 1183,
1
(300 MHz, DMSO-d6): δ 2.27 (s, 3H, Me), 3.57 (s, 3H, OMe), 1152, 1090, 980, 796; H NMR (300 MHz, DMSO-d6): δ 3.93 (s,
3.81 (s, 3H, OMe), 3.90 (s, 3H, OMe), 4.06 (s, 3H, OMe), 6.15 3H, OMe), 3.95 (s, 3H, OMe), 3.98 (s, 3H, OMe), 4.12 (s, 3H,
(d, J = 1.8 Hz, 1H, H5), 6.53 (s, 1H, H5), 6.62 (d, J = 1.8 Hz, 1H, OMe), 6.51 (s, 1H, H5), 6.57 (s, 1H, H5), 7.24 (d, J = 2.1 Hz, 1H,
H7), 7.00–7.36 (m, 7H, H2, ArH), 7.44–7.62 (m, 2H, ArH), 9.42 H2), 7.29 (d, J = 2.3 Hz, 1H, H2), 7.32–7.72 (m, 8H, ArH), 9.94
(s, 1H, NH), 11.06 (bs, 1H, NH),11.36 (d, J = 1.5 Hz, 1H, NH), (s, 1H, NH), 10.89 (s, 1H, NH), 11.44 (s, 1H, NH), 11.55 (s, 1H,
11.84 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): δ 20.9 (Me), NH); 13C NMR (75 MHz, DMSO-d6): δ 55.5, 55.8, 57.2, 57.3
55.2, 55.4, 55.5, 57.3 (OMe), 86.5, 88.3 (C5), 93.2, 95.6 (C7), (OMe), 88.4, 88.7 (C5), 96.2, 101.6 (C7), 109.9, 110.2 (C3),
110.5, 112.5 (C3), 114.2, 114.5 (ArCH), 115.7 (ArC), 123.4 (C2), 115.4, 116.3 (ArC), 123.6, 123.7 (C2), 127.5, 127.6 (ArCH),
127.1 (ArCH), 127.5, 128.1 (ArC), 130.9 (ArCH), 132.1, 132.1, 130.1, 130.4 (ArC), 130.7, 130.7 (ArCH),134.3, 134.7, 137.0,
136.0, 137.9, 140.0, 156.4, 156.7, 157.5, 160.9 (ArC), 162.6, 138.1, 156.6, 157.2, 161.0, 162.0 (ArC), 164.6, 165.9, 187.7
164.0, 179.8 (CvO); HRMS (+ESI): found m/z 651.2255 ([M + (CvO); HRMS (+ESI): found m/z 687.1406 ([M
H]+), [C36H31FN4O7H]+ requires m/z 651.2250 (monoisotopic [C35H28Cl2N4O7H]+ requires m/z 687.1408 (monoisotopic
mass). mass).
N′-(2-(3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-2-yl)-2- N′-(2-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indol-7-yl)-2-
+
H]+),
oxoacetyl)-4,6-dimethoxy-3-(4-fluorophenyl)-1H-indole-7-carbo- oxoacetyl)-3-(4-chlorophenyl)-4,6-dimethoxy-1H-indole-7-carbo-
hydrazide (14c). Yellow-orange solid; mp 270–272 °C (metha- hydrazide (15c). Pale yellow solid; mp >300 °C (methanol); UV-
nol); UV-vis: λmax(THF)/nm 214 (ε/dm3 mol−1 cm−1 66 200), vis: λmax(THF)/nm 212 (ε/dm3 mol−1 cm−1 201 800), 237
243 (60 600), 263 (48 300), 342 (34 600); IR (KBr): νmax/cm−1 (223 700), 275 (126 900), 330 (120 100); IR (KBr): νmax/cm−1
3381 (NH), 1615 (CvO), 1578 (CvO), 1544, 1454, 1350, 1257, 3418 (NH), 1627 (CvO), 1589 (CvO), 1560, 1537, 1465, 1352,
1218, 1158, 1135, 805; 1H NMR (300 MHz, DMSO-d6): δ 3.59 (s, 1324, 1215, 1184, 1151, 1089, 980, 796;1H NMR (300 MHz,
3H, OMe), 3.82 (s, 3H, OMe), 3.91 (s, 3H, OMe), 4.09 (s, 3H, DMSO-d6): δ 3.93 (s, 3H, OMe), 3.96 (s, 3H, OMe), 3.98 (s, 3H,
OMe), 6.17 (d, J = 1.7 Hz, 1H, H5), 6.53 (s, 1H, H5), 6.63 (d, J = OMe), 4.12 (s, 3H, OMe), 6.51 (s, 1H, H5), 6.57 (s, 1H, H5),
1.7 Hz, 1H, H7), 7.08–7.60 (m, 9H, H2, ArH), 9.52 (s, 1H, NH), 7.24 (d, J = 2.4 Hz, 1H, H2), 7.28 (d, J = 2.4 Hz, 1H, H2),
11.14 (bs, 1H, NH), 11.37 (d, J = 1.5 Hz, 1H, NH), 11.95 (s, 1H, 7.35–7.62 (m, 8H, ArH), 9.93 (s, 1H, NH), 10.88 (s, 1H, NH),
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11.43 (d, J = 1.6 Hz, 1H, NH), 11.55 (d, J = 2.0 Hz, 1H, NH); 13
C
7.23–7.33 (m, 2H, H5, H6), 7.36–7.43 (m, 2H, ArH phenyl),
NMR (75 MHz, DMSO-d6): δ 55.5, 55.9, 57.2, 57.3 (OMe), 88.4, 7.47–7.53 (m, 2H, ArH phenyl), 7.53–7.59 (m, 1H, H7),
88.7 (C5), 96.2, 101.6 (C7), 109.9, 110.2 (C3), 115.4, 116.4, 8.16–8.25 (m, 1H, H4), 8.64 (d, J = 3.3 Hz, 1H, H2), 9.42 (s, 1H,
118.8 (ArC), 123.6, 123.7 (C2), 127.5 (ArCH), 130.1 (ArC), 130.5, NH indole), 10.60 (s, 1H, NH indole), 11.68 (s, 1H, NH hydra-
130.7, 131.1 (ArCH), 134.7, 137.1, 138.1, 156.6, 157.2, 161.0, zide), 12.30 (d, J = 3.0 Hz, 1H, NH hydrazide); 13C NMR
162.0 (ArC), 164.6, 165.8, 187.7 (CvO); HRMS (+ESI): found (75 MHz, DMSO-d6): δ 55.0, 55.3 (OMe), 86.7, 92.7 (ArCH),
m/z 731.0900 ([M + H]+), [C35H28BrClN4O7H]+ requires m/z 111.1, 112.6 (ArC), 112.7 (ArCH), 118.5, 119.8 (ArC), 121.2,
731.0903 (monoisotopic mass).
122.7, 123.6 (ArCH), 123.8, 125.7 (ArC), 129.8, 133.3 (ArCH),
3-(4-Bromophenyl)-N′-(2-(3-(4-chlorophenyl)-4,6-dimethoxy- 133.8, 136.5, 137.6 (ArC), 138.4 (ArCH), 155.1, 158.6 (ArC),
1H-indol-7-yl)-2-oxoacetyl)-4,6-dimethoxy-1H-indole-7-carbo- 160.7, 163.4, 182.3 (CvO); HRMS (+ESI): found m/z 583.0604
hydrazide (15d). Pale yellow solid; mp >300 °C (methanol); ([M + Na]+), [C27H21BrN4O5Na]+ requires m/z 583.0588 (mono-
UV-vis: λmax(THF)/nm 212 (ε/dm3 mol−1 cm−1 201 200), 238 isotopic mass).
(229 000), 275 (134 700), 330 (133 000); IR (KBr): νmax/cm−1
N′-(2-(1H-indol-3-yl)-2-oxoacetyl)-4,6-dimethoxy-3-(4-methoxy-
3416 (NH), 1627 (CvO), 1589 (CvO), 1562, 1537, 1465, 1353, phenyl)-1H-indole-2-carbohydrazide (19c). Pale brown solid;
1324, 1215, 1184, 1151, 1089, 980, 796; 1H NMR (300 MHz, mp >300 °C (methanol); UV-vis: λmax(THF)/nm 212 (ε/dm3
DMSO-d6): δ3.93 (s, 3H, OMe), 3.96 (s, 3H, OMe),3.98 (s, 3H, mol−1 cm−1 60 300), 251 (39 800), 317 (26 400); IR (KBr): νmax
/
OMe), 4.12 (s, 3H, OMe), 6.51 (s, 1H, H5), 6.57 (s, 1H, H5), cm−1 3324 (NH), 3271 (NH), 1709 (CvO), 1614 (CvO), 1582
7.24 (d, J = 2.4 Hz, 1H, H2), 7.29 (d, J = 2.5 Hz, 1H, H2), (CvO), 1542, 1482, 1426, 1287, 1239, 1211, 1150, 1138, 813,
7.35–7.63 (m, 8H, ArH), 9.93 (d, J = 1.8 Hz, 1H, NH), 10.88 (d, 746; 1H NMR (300 MHz, DMSO-d6): δ3.59 (s, 3H, OMe), 3.79 (s,
J = 2.0 Hz, 1H, NH), 11.44 (d, J = 1.7 Hz, 1H, NH), 11.55 (d, J = 3H, OMe), 3.80 (s, 3H, OMe), 6.15 (d, J = 1.9 Hz, H5′), 6.54 (d,
1.9 Hz, 1H, NH); 13C NMR (75 MHz, DMSO-d6): δ 55.5, 55.9, J = 1.9 Hz, H7′), 6.90–6.99 (m, 2H, ArH phenyl), 7.23–7.33 (m,
57.2, 57.3 (OMe), 88.4, 88.7 (C5), 96.2, 101.6 (C7), 109.9, 110.2 2H, H5, H6), 7.34–7.43 (m, 2H, ArH phenyl), 7.51–7.60 (m, 1H,
(C3), 115.4, 116.3, 118.6 (ArC), 123.6, 123.7 (C2), 127.6 (ArCH), H7), 8.15–8.25 (m, 1H, H4), 8.64 (d, J = 3.2 Hz, 1H, H2), 8.68 (s,
130.4 (ArC), 130.4, 130.7, 131.1 (ArCH), 134.3, 135.0, 137.0, 1H, NH indole), 10.60 (bs, 1H, NH indole), 11.59 (s, 1H, NH
138.1, 156.6, 157.2, 161.0, 161.9 (ArC), 164.6, 165.9, 187.7 hydrazide), 12.32 (d, J = 2.9 Hz, 1H, NH hydrazide); 13C NMR
(CvO); HRMS (+ESI): found m/z 731.0902 ([M
[C35H28BrClN4O7H]+ requires m/z 731.0903 (monoisotopic (ArCH), 111.8, 112.5 (ArC), 112.6, 112.8 (ArCH), 119.5 (ArC),
mass). 121.2, 122.7 (ArCH), 123.2 (ArC), 123.6 (ArCH), 125.7, 126.5
N′-(2-(1H-indol-3-yl)-2-oxoacetyl)-3-(4-chlorophenyl)-4,6- (ArC), 132.1 (ArCH), 136.5, 137.7 (ArC), 138.4 (ArCH), 155.4,
+
H]+), (75 MHz, DMSO-d6): δ 55.0, 55.0, 55.2 (OMe), 86.6, 92.4
dimethoxy-1H-indole-2-carbohydrazide
(19a). Pale
brown 158.2, 158.6 (ArC), 160.8, 163.2, 182.0 (CvO); HRMS (+ESI):
solid; mp >300 °C (methanol); UV-vis: λmax(THF)/nm 212 found m/z 535.1601 ([M + Na]+), [C28H24N4O6Na]+ requires m/z
(ε/dm3 mol−1 cm−1 70 000), 253 (41 400), 323 (28 600); IR (KBr): 535.1588 (monoisotopic mass).
νmax/cm−1 3325 (NH), 3278 (NH), 1712 (CvO), 1620 (CvO),
1
1580 (CvO), 1537, 1483, 1425, 1262, 1213, 1138, 816, 747; H
NMR (300 MHz, DMSO-d6): δ 3.62 (s, 3H, OMe), 3.80 (s, 3H,
OMe), 6.19 (d, J = 1.6 Hz, H5′), 6.56 (d, J = 1.6 Hz, H7′),
General procedure for the synthesis of indolyl-
oxoacetohydrazides 16–18
7.22–7.33 (m, 2H, H5, H6), 7.34–7.42 (m, 2H, ArH phenyl), The 7-glyoxyloyl chloride 6a–b or 3-glyoxyloyl chloride 9
7.42–7.50 (m, 2H, ArH phenyl), 7.51–7.62 (m, 1H, H7), (1.0 equiv.) was dissolved in anhydrous acetonitrile. Hydrazine
8.16–8.27 (m, 1H, H4), 8.64 (d, J = 3.1 Hz, 1H, H2), 9.41 (s, 1H, hydrate (0.5 equiv. for 16 and 18, excess for 17) was added fol-
NH indole), 10.60 (bs, 1H, NH indole), 11.69 (s, 1H, NH hydra- lowed by triethylamine (10 drops) and the solution was stirred
zide), 12.31 (d, J = 2.6 Hz, 1H, NH hydrazide); 13C NMR at room temperature for 1.5 h (16 and 18) or 5 h (17). The reac-
(75 MHz, DMSO-d6): δ 55.0, 55.3 (OMe), 86.7, 92.6 (ArCH), tion mixture was quenched with ice-water and the resulting
111.2, 112.6 (ArC), 112.7 (ArCH), 118.5 (ArC), 121.2, 122.7, precipitate was filtered, dried and recrystallized from methanol
123.6 (ArCH), 123.8, 125.7 (ArC), 126.9 (ArCH), 131.1 (ArC), to yield the product as a yellow solid at 50–76% yield.
132.9 (ArCH), 133.4, 136.5, 137.6 (ArC), 138.4 (ArCH), 155.1,
2-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indol-7-yl)-N′-(2-(3-
158.6 (ArC), 160.7, 163.4, 182.2 (CvO); HRMS (+ESI): found (4-bromophenyl)-4,6-dimethoxy-1H-indol-7-yl)-2-oxoacetyl)-2-
m/z 539.1110 ([M + Na]+), [C27H21ClN4O5Na]+ requires m/z oxoacetohydrazide (16). Yellow solid; mp 277–279 °C (metha-
539.1093 (monoisotopic mass).
nol); UV-vis: λmax(THF)/nm 212 (ε/dm3 mol−1 cm−1 50 700),
N′-(2-(1H-indol-3-yl)-2-oxoacetyl)-3-(4-bromophenyl)-4,6- 331 (18 800); IR (KBr): νmax/cm−1 3396 (NH), 1610 (CvO), 1581
1
dimethoxy-1H-indole-2-carbohydrazide (19b). Creamy white (CvO), 1557, 1534, 1323, 1217; H NMR (300 MHz, DMSO-d6):
solid; mp >300 °C (methanol); UV-vis: λmax(THF)/nm 212 δ 3.95 (s, 6H, OMe), 3.97 (s, 6H, OMe), 6.49 (s, 2H, H5), 7.24
(ε/dm3 mol−1 cm−1 100 400), 254 (62 300), 321 (43 200); IR (d, J = 2.5 Hz, 2H, H2), 7.46–7.57 (m, 8H, ArH), 10.52 (s, 2H,
(KBr): νmax/cm−1 3325 (NH), 3270(NH), 1708 (CvO), 1619 NH), 11.56 (d, J = 2.4 Hz, 2H, NH); 13C NMR (75 MHz, DMSO-
(CvO), 1578 (CvO), 1537, 1483, 1425, 1262, 1212, 1138, 817, d6): δ 56.2, 57.3 (OMe), 88.9 (C5), 101.7 (C7), 110.2 (C3), 116.7,
1
746; H NMR (300 MHz, DMSO-d6): δ 3.62 (s, 3H, OMe), 3.80 119.2 (ArC), 124.0 (C2), 130.9, 131.5 (ArCH), 135.0, 137.5,
(s, 3H, OMe), 6.19 (d, J = 1.9 Hz, H5′), 6.55 (d, J = 1.9 Hz, H7′), 161.4, 162.3, 167.0 (ArC), 179.5, 188.3 (CvO); HRMS (+ESI):
2892 | Org. Biomol. Chem., 2014, 12, 2882–2894
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found m/z 803.0347 ([M + H]+), [C36H28Br2N4O8H]+ requires compared to the control without exposure to compounds to
m/z 803.0347 (monoisotopic mass).
calculate absolute inhibition percentages.
2-(3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-7-yl)-2-oxoace-
tohydrazide (17). Pale yellow solid; mp 200–202 °C (metha-
nol); UV-vis: λmax(THF)/nm 213 (ε/dm3 mol−1 cm−1 87 400),
230 (82 200), 254 (85 900), 330 (52 600); IR (KBr): νmax/cm−1
3325 (NH), 1612 (CvO), 1580 (CvO), 1560, 1537, 1326, 1257,
Growth inhibition assays
Compounds were dissolved at 50 mM in DMSO and then
diluted to 200 µM in 100 µl of Luria-Bertani (LB) medium into
individual wells in a 96-well plate. E. coli DH5α or B. subtilis
168 cells were grown at 37 °C in 5 ml LB with shaking until the
OD600 reached 0.6–0.7, and 5 µl of the culture was added to
each well. The plate was incubated in a FLUOstar Optima plate
reader (BMG Labtech) at 37 °C shaking at 600 rpm. The OD600
of the culture was taken every 10 min over a 16 h period using
LB as the blank. Samples were tested in triplicate and the
growth pattern of each sample was compared to cells exposed
to equal amounts of DMSO.
1
1220, 1093, 799; H NMR (300 MHz, DMSO-d6): δ 3.90 (s, 3H,
OMe), 3.93 (s, 3H, OMe), 4.34 (bs, 2H, NH2), 6.46 (s, 1H, H5),
7.20 (d, J = 2.5 Hz, 1H, H2), 7.35–7.44 (m, 2H, ArH), 7.48–7.58
(m, 2H, ArH), 9.40 (s, 1H, NH), 11.51 (d, J = 1.8 Hz, 1H, NH);
HRMS (+ESI): found m/z 396.0734 ([M
+
Na]+),
[C18H16ClN3O4Na]+ requires m/z 396.0722 (monoisotopic
mass). The compound was not soluble enough in DMSO-d6 for
13C NMR measurement.
N′-(2-(1H-indol-3-yl)-2-oxoacetyl)-2-(1H-indol-3-yl)-2-oxoace-
tohydrazide (18). Pale yellow solid; mp (decomp) > 350 °C
(methanol), lit.30; IR (KBr): νmax/cm−1 3229 (NH), 1597 (CvO),
1433, 1237, 1144, 928, 743; 1H NMR (300 MHz, DMSO-d6):
δ 7.22–7.38 (m, 4H, H5, H6), 7.52–7.64 (m, 2H, H7), 8.19–8.33
(m, 2H, H4), 8.71 (s, 2H, H2), 10.75 (s, 2H, NH indole), 12.37
(s, 2H, NH hydrazide); 13C NMR (75 MHz, DMSO-d6): δ 112.5
(ArC), 112.7, 121.3, 122.8, 123.7 (ArCH), 125.8, 136.5 (ArC),
138.5 (ArCH), 163.1, 181.8 (CvO); HRMS (+ESI): found m/z
397.0916 ([M + Na]+), [C20H14N4O4Na]+ requires m/z 397.0907
(monoisotopic mass). The compound was not soluble enough
in THF for UV-vis measurement.
Acknowledgements
This work was supported by a Project Grant from the National
Health and Medical Research Council (NHMRC) Australia to
N.K., P.L. and R.G. [Grant 1008014]. M.M. is thankful to the
University of New South Wales for a Tuition Fee Scholarship
(TFS) and to N.K. for a Living Allowance Scholarship.
Notes and references
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