Design and synthesis of bis(indolyl)ketohydrazide-hydrazones: Identification of potent and selective novel tubulin inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.04.078

A novel series of ketohydrazide-hydrazones as analogues of naturally occurring coscinamides has been synthesized and evaluated for their anticancer activity against five cancer cell lines. Of the twenty-synthesized ketohydrazide-hydrazones, compounds, 21c, 21f, 21g, 21k and 21o showed cytotoxic effects (less than 50% cell survival) against multiple cancer cell lines when tested at a final concentration of 10?μM. IC₅₀ of three compounds 21f, 21k and 21o was determined to be less than 5?μM for all tested cancer cell lines. Compound 21k exhibited significant anticancer activity against MCF-7, MDA-MB-231, HCT-116 and JURKAT cancer cell lines with IC₅₀ values of 0.8?μM, 0.50?μM, 0.15?μM, and 0.22?μM, respectively. Also, 21k was found to be more selectively cytotoxic against tumor cells when compared to normal cells. Preliminary mechanism of action studies indicated that the most active compound 21k induced caspase-dependent apoptosis in cells. 21k arrests cell cycle in G2/M phase by inhibiting of tubulin polymerization (IC₅₀?=?0.6?μM).

Full text of DOI:10.1016/j.ejmech.2017.04.078

Accepted Manuscript
Design and synthesis of bis(indolyl)ketohydrazide-hydrazones: Identification of potent
and selective novel tubulin inhibitors
Mukund P. Tantak, Linus Klingler, V. Arun, Anil Kumar, Rachna Sadana, Dalip Kumar
PII:
S0223-5234(17)30360-4
10.1016/j.ejmech.2017.04.078
EJMECH 9429
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 December 2016
Revised Date: 25 April 2017
Accepted Date: 30 April 2017
Please cite this article as: M.P. Tantak, L. Klingler, V. Arun, A. Kumar, R. Sadana, D. Kumar, Design and
synthesis of bis(indolyl)ketohydrazide-hydrazones: Identification of potent and selective novel tubulin
inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.078.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Design and synthesis of bis(indolyl)ketohydrazide-hydrazones:
Identification of potent and selective novel tubulin inhibitors
Mukund P. Tantaka, Linus Klinglerb,V. Aruna, Anil Kumara, Rachna Sadana*b, Dalip Kumar*a
aDepartment of Chemistry, Birla Institute of Technology and Science, Pilani 333 031, India. Tel: +91-1596-515238;
E-mail: dalipk@pilani.bits-pilani.ac.in;
bDepartment of Natural Sciences, University of Houston – Downtown, Houston, TX 77002, United States.
Tel: +1-713-226-5562; E-mail: sadanar@uhd.edu
_______________________________________________________________
Abstract: A novel series of ketohydrazide-hydrazones as analogues of naturally occurring
coscinamides has been synthesized and evaluated for their anticancer activity against five cancer
cell lines. Of the twenty-synthesized ketohydrazide-hydrazones, compounds, 21c, 21f, 21g, 21k
and 21o showed cytotoxic effects (less than 50% cell survival) against multiple cancer cell lines
when tested at a final concentration of 10 µM. IC50 of three compounds 21f, 21k and 21o was
determined to be less than 5 µM for all tested cancer cell lines. Compound 21k exhibited
significant anticancer activity against MCF-7, MDA-MB-231, HCT-116 and JURKAT cancer
cell lines with IC50 values of 0.8 µM, 0.50 µM, 0.15 µM, and 0.22 µM, respectively. Also, 21k
was found to be more selectively cytotoxic against tumor cells when compared to normal cells.
Preliminary mechanism of action studies indicated that the most active compound 21k induced
caspase-dependent apoptosis in cells. 21k arrests cell cycle in G2/M phase by inhibiting of
tubulin polymerization (IC50 = 0.6 µM).
Keywords: Coscinamides, Bisindoles, Ketohydrazide-hydrazones, Cytotoxicity, Apoptosis,
Tubulin inhibitor
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1. Introduction
Microtubules, composed of α- and β-tubulin heterodimers, play an important role in various
cellular functions including mitosis, shape maintenance and intracellular transport[1-4]. Due to
the involvement of microtubules, especially in mitosis, the discovery of natural and synthetic
compounds that inhibit tubulin polymerization or interrupt microtubule depolymerization is one
of the useful targets in anticancer drug discovery. Tubulin targeting substances are broadly
categorized into microtubule stabilizing (taxanes, epothilones and discodermolide) and
destabilizing (colchicine, vinca alkaloids, cryptophycins and CA-4P) agents[5-9]. Generally,
anticancer agents that inhibit microtubule assembly are known as antimitotic agents[10, 11].
Indole skeleton is frequently found in many natural and synthetic chemical entities of immense
biological significance[12-14]. In the recent past, several indole containing small molecules have
been identified as potent tubulin inhibitors 1-6, for example, 2-aryl-3-formylindoles (1), 3-
aroylindoles 2 and 3, N-aroylindoles 4, 2-heteroarylthioindoles 5, and 2-indolyl-4-
benzoylimidazoles 6 (Fig. 1)[15-17].
Fig. 1 Indole-based small molecules as tubulin inhibitors (1-6)
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The anticancer activity of these indole-based tubulin inhibitors is due to the blocking of tubulin
polymerization[15]. Particularly, bisindole class of compounds isolated from marine sources
including sponges, tunicates, and bryozoans are reported to possess a broad spectrum of
interesting biological activities[18]. In bisindole compounds, two indole moieties are linked
either through a five/six-membered heterocyclic ring or a linear spacer. For example, Topsentins
(7) having an 2-acylimidazole unit, were isolated from the sponge Topsentia genitrix and found
to inhibit the proliferation of tumor cells (IC50 = 4–40 µM)[19]. Nortopsentins A–C (8) involve
an imidazole moiety, were isolated from the marine sponge Spongosorites ruetzleri and found to
exhibit cytotoxicity against P388 cells (IC50 = 4.5–20.7 µM)[20].
Fig. 2 Structures of selected bisindole alkaloids
Rhopaladin B (9) linking two indole rings via an imidazolinone spacer, was described to show
inhibitory activity against CDK 4 (IC50 = 12.5 µM) and c-erbβ-2 (IC50 = 7.4 µM) kinases[21].
Dragmacidin B (10) with a piperazine spacer, was found to display good anticancer activity (IC50
= 15 µM, P388; 1-10 µM, A-549, HCT-8 and MDA-MB-231)[22]. Hyrtinadine A (11) with a
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pyrimidine moiety was isolated from the extracts of an Okinawan marine sponge Hyrtios sp. and
showed in vitro cytotoxicity against murine leukemia L1210 (IC50= 1 µM) and human
epidermoid carcinoma KB (IC50= 3 µM) cells (Fig. 2)[23]. Jiang et al. have synthesized various
bisindoles with thiazole, pyrazinone, pyrazine and pyridine linkers as potential anticancer
agents[24, 25]. Similarly, Diana and co-workers identified diverse bis(indolyl)heterocycles as
potential antitumor agents by linking two units through thiophene, pyrazole, furan, isoxazole and
pyrrole moieties[26-28]. Bisindole containing linear chain spacers such as 1,2-diketo,
glyoxylamide, enamide, hydrazide-hydrazones, have also been reported to display interesting
biological activities[29, 30].
Fig. 3 Rational design for ketohydrazide-hydrazones
In 2000, Bokesch et al. isolated Coscinamides A-C (12) with linear enamide spacers from an
extract of marine sponge Coscinoderma sp and were reported to exhibit partial cytoprotection
against HIV[31] and antitumor activity against human prostate cancer cell line (IC50 = 7.6
µM)[32]. Another indolic enamide, Igzamide was isolated from the extract of sponge Plocamissa
igzoand exhibited modest cytotoxic activity against L1210 murine leukemia cell line[33].
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Hyrtiosin B (13) bearing 1,2-diketo spacer, was isolated from marine sponge Hyrtios erecta and
possessed in vitro cytotoxicity against epidermoid carcinoma KB cell line[34]. Didemnidine B,
an indole-3-glyoxylamide was isolated from the New Zealand ascidian Didemnum sp with
moderate cytotoxicity towards non malignant L6 cell line[35]. Indibulin (D-24851) 14 with a
glyoxylamide moiety showed good in vitro cytotoxicity towards human cancer cell lines (IC50 =
0.036-0.285 µM)[36]. Further, biological potential of these indole-based ketoamides are largely
unexplored in new drug discovery. Natural products, Coscinamides (12), Hyrtiosin B (13) and
synthetic tubulin inhibitor, Indibulin (14) possessing 1,2-diketo or α-keto amide functionality are
reported to exhibit potent anticancer activity. Recently we have identified bis(indolyl)hydrazide-
hydrazones 15 (IC50 = 1.0 µM; MDA-MB-231) as potent anticancer agents[37, 38]. Structural
units including α-ketoamide, diketone and hydrazide-hydrazone have been shown to be valuable
for the cytotoxicity of existing natural and synthetic bisindoles. In present work, we designed
novel bis(indolyl)ketohydrazide-hydrazones (21) by incorporating α-ketoamide, diketone and
hydrazide-hydrazone as potent tubulin inhibitors (Fig. 3).
2. Results and discussion
2.1. Synthesis
Coscinamide analogues 21a-o and 23a-e were synthesized in high yields by reacting indolyl
glyoxalylhydrazides 18 with appropriate aldehydes 20. Indolyl glyoxalylhydrazides 18 were
synthesized by the initial reaction of indole 16 with oxalyl chloride to produce indolyloxalyl
chlorides 17a-c. Subsequent reaction of 17a-c with hydrazine hydrate led to ketohydrazides 18a-
c in good yields.
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O
O
R1
NHNH2
R1
R1
Cl
(COCl)2, 0 °C
Et2O, 0.5-1 h.
NH2NH2.H2O
EtOH, 0 °C to rt, 1 h.
O
O
N
N
N
R2
R2
R2
16
17a-c
18a-c
O
CHO
R3
R4
H
R3
R4
R1
N
R3
R4
POCl3, DMF
aq. NaOH
18a-c
N
EtOH, reflux
R6
N
R6
O
N
R6
N
5-6 h.
N
R5
R5
20
R5
R2
19
21a-o
21h: R1,R2,R4,R6 = H, R3 = Br, R5 = 4-ClC6H4CH2
21i: R1 = Br, R2,R3,R5,R6 = H, R4 = F
21j: R1 = Br, R2,R3,R5,R6 = H, R4 = OMe
21k: R1,R3,R4,R5,R6 = H, R2 = 4-ClC6H4CH2
21l: R1, R2,R3,R4,R5 = H, R6 = 4-ClC6H4CH2
21m: R1 = Br, R2,R4,R6 = H, R3 = OMe, R5 = 4-ClC6H4CH2
21n: R1 = Br, R2,R4,R6 = H, R3 = OMe, R5 = 4-OMeC6H4CH2
21o: R1,R4,R5,R6 = H, R2 = 4-ClC6H4CH2, R3 = OCH3
21a: R1,R2,R3,R4,R5,R6 = H
21b: R1,R2,R3,R5,R6 = H, R4 = Br
21c: R1,R2,R4,R5,R6 = H, R3 = Br
21d: R1,R2,R4,R5,R6 = H, R3 = F
21e: R1,R2,R3,R5,R6 = H, R4 = F
21f: R1,R2,R4,R5,R6 = H, R3 = OMe
21g: R1,R2,R3,R5,R6 = H, R4 = OMe
O
O
H
N
NHNH2
EtOH
N
Ar
23a: R2 = H; Ar = 3,4,5-(CH3O)3C6H2
23b: R2 = H; Ar = 4-CH3OC6H4
ArCHO
reflux
N
O
O
N
23c: R2 = H; Ar = 3,4-(CH3O)2C6H3
23d: R2 = H; Ar = 3-CH3OC6H4
23e: R2 = 4-ClC6H4CH2; Ar = 4-pyridyl
R2
18
R2
23a-e
22
Scheme 1 Synthesis of ketohydrazide-hydrazones 21a-o and 23a-e
On the other hand, indole-3-carboxaldehydes 20 were achieved from the reaction of indoles 19
with phosphorus oxychloride in presence of dimethylformamide (DMF) followed by
alkylation[39]. In an effort to identify potent compounds, one of the indole rings in hydrazide-
hydrazones 21a-o was protected with p-chlorobenzyl moiety; such structural modification was
found to be good in case of Indibulin[40]. Similarly, Coscinamide analogues 23a-e having
diversely substituted aryl ring were prepared from the reaction of indolyl glyoxalyl-hydrazides
18 and arylaldehydes 22 in ethanol under refluxing conditions (Scheme 1).
The structures of bis(indolyl)ketohydrazide-hydrazones 21a-o and 23a-e were confirmed by
detailed IR, NMR (1H & 13C) and HRMS spectral analysis. Formation of ketohydrazide-
hydrazone 21b was indicative by the presence of bands at 3440 (N-H), 3193 (N-H) and 1658
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(C=N) cm-1 in its IR spectrum. Appearance of duplicate peaks in NMR (1H & 13C) spectra
reveals that bis(indolyl)-ketohydrazide-hydrazones (21) exist as conformational isomers
(rotamers, rotation around N-C(O)) or geometrical isomers) which is in agreement with the
literature reports[41, 42]. The HRMS spectroscopic analysis of 21b displayed a molecular ion
[M + Na]+ at 431.0114 in agreement with the expected mass for C19H13BrN4NaO2 [M + Na]+,
431.0120. Purity of all the newly synthesized keto-hydrazide hydrazones was > 98% as analyzed
by using HPLC.
2.2. Anticancer activity
Synthesized ketohydrazide-hydrazones 21a-o and 23a-e were assessed in vitro for their
cytotoxicity against a panel of cancer cell lines; human breast cell lines (BT474, MCF-7 and
MDA-MB-231), human colon carcinoma cell lines (HCT-116), leukemia cell line (Jurkat).
Firstly, we screened all the ketohydrazide-hydrazones at 10 µM concentration against a panel of
five different cancer cell lines (Table 1). Compounds 21c, 21f, 21g, 21k and 21o showed
cytotoxic effects by inhibiting cell survival by 50% against the tested cancer cell lines.
Replacement of one indole ring in 21 with an aryl or pyridyl group resulted in compounds 23
with percentage cell survival more than 90% except for Jurkat cancer cell line. These results
suggested that both the indole rings are necessary for the activity of ketohydrazide-hydrazones.
Compounds 21c, 21f, 21g, 21k and 21o with less than 50% cell survival were further
investigated for their cytotoxicity (Table 2).
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Table 1. In vitro screening of bis(indolyl)ketohydrazide-hydrazones and indolyl ketohydrazide-
hydrazones
Percentage cell survival (@ 10 µM)
compd
BT-474
MCF-7
MDA-MB-231
98.6 ± 0.35
95.1 ± 5.4
34 ± 5.2
HCT-116
88.4 ± 2.7
94.5 ± 5.5
26.3 ± 5.1
85.1 ± 5.9
Jurkat
83 ± 8.5
99.2 ± 4.7
92.7 ± 8.1
35.0 ± 5.0
105 ± 9.0
40.2 ± 2.1
17.2 ± 3.1
34.6 ± 5.1
107.2 ± 5.1
97.5 ± 5.3
91.4 ± 9.9
15.0 ± 4.6
107.1 ± 2.5
33.6 ± 7.7
75.4 + 8.3
25.5 ± 3.2
56.7 ± 4.7
21a
21b
86.8 ± 0.6
82.4 ± 7.7
112 ± 5.5
88.2 ± 0.5
20.3 ± 2.1
95.6 ± 2.9
94.7 ± 5.2
94.8 ± 1.6
92.0 ± 3.1
22.5 ± 2.5
94.2 ± 7.1
21c
102 ± 8.7
21d
105.5 ± 8.2
21.6 ± 3.5
103.4 ± 9.6
88.8 ± 2.3
111.1 ± 4.7
111.5 ± 5.5
17.1 ± 4.4
98.8 ± 10.1
103.9 ± 4.4 69.9 ± 3.9
21e
17.6 ± 2.5
38.8 ± 4.3
72.6 ± 2.5
84.2 ± 5.4
71.4 ± 6.1
13.3 ± 1.5
71.8 ± 8.1
86.1 ± 7.2
65.7 ± 7.4
38.2 ± 1.2
99.1 ± 2.9
19.3 ± 1.5
52.1 ± 8.5
93.2 ± 1.3
95.8 ± 2.9
74.1 ± 2.2
20.6 ± 2.8
81.6 ± 1.4
46.6 ± 3.2
81.9 ± 1.3
36.1 ± 1.6
69.6 ± 3.5
21f
21g
21h
21i
21j
21k
21l
21m
21n
99.1± 3.5
91.1 ± 9.4
96.1± 4.6
103.2 ± 5.8
102.2 ± 4.0
112.2 ± 9.9
21.2 ± 2.3
101.1 ± 4.1
97.5 ± 1.7
103.4 ± 7.7
95.1 ± 4.6
78.7 ± 6.5
17.6 ± 1.1
33.7 ± 1.8
110.2 ± 1.5
106.2 ± 5.4
99.8 ± 4.9
96.8 ± 2.2
82.2 ± 5.8
27 ± 1.4
29.4 ± 2.2
102.8 ± 6.1
102.4 ± 3.2
94.6 ± 9.7
97.2 ± 2.5
71.2 ± 4.3
22.5 ± 3.5
21o
23a
101.3 ± 6.3 61.9 ± 1.5
23b
92.8 ± 2.3
72.4 ± 5.5
69.1 ± 5.5
25 ± 4.2
78.9 ± 1.1
59.7 ± 6.4
66.7 ± 5.7
21 ± 1.5
23c
23d
23e
Doxorubicin
The activity data represents mean values ± SD of experiments conducted in triplicates at three
independent times. N.D = not determined.
Introduction of a 5-bromo substituent in the indole ring of 21a led to 21c that showed moderate
activity against the tested cancer cells. Interestingly, replacement of bromine in 21c with
methoxy group resulted analogue 21f which showed enhanced cytotoxicity against tested cancer
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cell lines with 16 and 10-fold selective cytotoxicity towards HCT-116 (IC50 = 0.4 µM) and
Jurkat (IC50 = 0.5 µM) cell lines (21c vs 21f).
Table 2. In vitro cytotoxic activity of the selected bis(indolyl)ketohydrazide-hydrazones
Cancer cell lines (IC50, µM)
compd
BT-474
MCF-7
MDA-MB-231 HCT-116
Jurkat
N.D.
3.3 ± 1.2
5.6 ± 0.9
1.7 ± 0.3
6.6 ± 1.1
0.4 ±0.1
2.5 ± 0.4
5.0 ± 0.9
21c
21f
1.1 ± 0.3
1.8 ± 0.4
0.5 ± 0.2
N.D.
6.1 ± 1.2
0.8 ± 0.11
3.8 ± 0.7
1.1[29]
N.D.
10.0 ± 1.2
21g
2.1 ± 0.3
2.7 ± 0.5
-
21k
0.5 ± 0.1
2.9 ± 0.35
0.074[40]
0.55 ± 0.22
0.15 ± 0.03 0.22 ± 0.04
4.2 ± 0.9
-
3.6 ± 0.7
-
21o
Indibulin
Doxorubicin
0.3 ± 0.12
0.39 ± 0.16
0.5 ± 0.11
0.26 ± 0.12
Replacement of fluorine with a methoxy group (compound 21g) was found to be unfavourable
for the activity as compound 21g showed ~ 2.5-fold decrease in activity against MCF-7 cells
(IC50 = 6.1 µM). Protection of indole nitrogen with p-chlorobenzyl moiety led to the compound
21k with significantly enhanced cytotoxicity against the tested cell lines (IC50 = 0.15-2.1 µM).
Compound 21k was found to be comparable to lead anticancer agent, indibulin and standard
drug doxorubicin. Furthermore, to improve the activity of ketohydrazide-hydrazones, we
combined these two key substituents of potent compounds 21f and 21k in a single molecule and
prepared derivative 21o but the cytotoxicity (IC50 = 2.7-4.2 µM) was not improved. SAR studies
revealed that C-5 methoxy, bromo and N-(4-chlorobenzyl) groups in indole ring are beneficial
for the activity. Both the indole rings are necessary for the potency of coscinamide analogues
(Fig. 4). Compound 21f with 5-methoxyindole is more potent than the corresponding 6-methoxy
analogue 21g. Also, it was observed that the identified bis(indolyl)ketohydrazide-hydrazones
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21a-o are superior or comparable to reported bisindoles having a five/six membered heterocyclic
system or a linear spacer as a linker between two indole rings[18-20,28,29].
Fig. 4 Structure-activity relationship for bis(indolyl)ketohydrazide-hydrazones
2.3. Toxicity study
The lactate dehydrogenase (LDH) assay is a commonly-used tool for assessing toxicity in
vitro[43]. The toxicity of potent compounds 21k and 21o was evaluated using LDH assay as
illustrated in Figure 5. The LDH activity result shows that the tested compounds 21k and 21o are
very less (20-25%) toxic as compared to LDH (+ve control; 140%) and their toxicity is similar to
untreated cells.
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Fig. 5 Toxicity induced by 21k and 21o in terms of LDH release
2.4. Apoptosis induction
Apoptosis is a programmed cell death which plays a key role in the maintenance of tissue
homeostasis and cell survival. Thus, inducing apoptosis in cancer cells has emerged as an
attractive strategy in cancer therapy[44].
Fig. 6 Caspase activation-induced by 21c, 21f, 21k and 21o in Jurkat cells
Caspase-3, one of the main executioner caspases, is activated in the apoptotic cells in both
extrinsic and intrinsic pathways. In order to find out whether compounds 21c, 21f, 21k and 21o
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induced caspase-3 dependent apoptosis, JURKAT cells were treated with the compounds and
activity of caspase-3/7 activity was measured. As shown in Figure 6, Compounds 21c, 21f, 21k
and 21o showed 2-5-folds enhancement in fluorescence indicative of increase in caspase 3/7
activity compared to the control cells.
2.5. Microtubule instability
To investigate whether the anti-proliferative activities of potent compounds 21k and 21o are
related to the interaction with tubulin, both the compounds were evaluated for tubulin inhibition
activity in a cell-free in vitro assay. Addition of 21k and 21o caused a decrease in tubulin
polymerization in a dose dependent manner as shown in Figure 7. Colchicine, compounds 21k
and 21o inhibited microtubule formation by 50% at 0.3 µM, 0.6 µM and 3.2 µM, respectively.
These results indicated that analogues 21k and 21o inhibit microtubule formation, a critical part
of cell division.
Fig. 7 Dose-dependent inhibition of tubulin polymerization by 21k and 21o
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2.6. Molecular modeling and docking studies on 21k
Tubulin polymerization assay suggests that bis(indolyl)ketohydrazide-hydrazones 21 exhibits
cytotoxicity through the inhibition of tubulin polymerization. To better understand how
ketohydrazide-hydrazones interact with tubulin, we investigated in silico binding modes of the
most potent compound 21k of the series at colchicine binding site in tubulin dimer using
Schrodinger 2011 molecular modeling suite (Schrodinger, Inc., New York, NY). Docking studies
were performed on reported high-resolution crystal structure of tubulin-DAMA-colchicine (CN-
2) complex (PDB ID: 1SA0)[45] using Glide module of Schrodinger. Compound 21k
demonstrated good binding in this model with a glide score of -6.54. The close view of potential
binding pos is illustrated in Figure 8.
CSY241
O
LYS352
H
N
N
O
N
H
N
Cl
Fig. 8 Binding modes of 21k in the colchicine binding site of tubulin showed hydrogen bonding
interactions (pink dotted lines)
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Interactions of 21k (green tube model) are strongly stabilized by two hydrogen bonds: first one
between C=O (linked to indole) and CYS241; the second one between N-H of glyoxalic amide
and LYS352. Similar hydrogen bonding interactions were also observed between the oxygen of
one methoxy group in colchicine[45] and -SH of β-CYS241. Apart from hydrogen bonding
interactions, compound 21k also strongly stabilized by hydrophobic contacts with ALA354,
LEU248, ALA316, MET259 and LEU255 (ESI Fig. S1).
2.7 Competitive experiment
Fig. 9 Inhibition of tubulin-colchicine fluorescence by 21k in a dose dependent manner.
Colchicine (a non-fluorescent molecule) become fluorescent (Excitation 350 nm, Emission 430
nm) upon binding to tubulin. As described in experimental section, we used fluorescence based
assay[46] to determine if 21k competes for the colchicine binding site on tubulin. In this study it
was found that, 21k displaced colchicine from tubulin in a dose dependent manner as shown in
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Figure 9. This fluorescence based assay results are well agreement with our molecular docking
studies.
2.8. Cell cycle progression analysis
Antimitotic drugs induced cell cycle arrest at G2/M phase in various cancer cell lines and led to
an increment of the relative peak in the DNA histogram. To gain further insight into the mode of
action, the effect of compound 21k on the cell cycle was measured by flow cytometry against
MCF-7 human cancer cells. To test this effect of 21k on cell cycle progression of MCF-7 cells
Fig. 10. Cell cycle progression analysis in 21k treated MCF-7cells: (a1) cells treated with
vehicle, (a2) cells treated with colchicine (3.0 µM) and (a3) cells treated with 21k (3.0 µM) for
24 h. After incubation, cells were processed with RNase A and stained with PI for detection
through flow cytometer. Data are representative of two experiments. (b1) A graph representing
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% of cells in G1, S and G2/M phase was analyzed using flow cytometry (Fig. 10). Nearly 45 ±
5.6% of the cells were arrested at G2/M phase of the cell cycle upon treatment with 21k (3.0
µM) for 24 h., whereas, only 15 ± 1.5% untreated cells were arrested in G2/M phase. Cells
treated with colchicine (3.0 µM) were used as a positive control in the experiment.
3. Conclusions
A new series of bis(indolyl)ketohydrazide-hydrazones was synthesized from the reaction of
indolyl glyoxalylhydrazides 18 with appropriate aldehydes and evaluated their in vitro anticancer
activity. Compound 21k exhibited the most potent anticancer activity against five tested cancer
cell lines with IC50 values ranging 0.15-0.8 µM. The most potent compound 21k was selectively
more cytotoxic to cancerous cells compared to non-cancerous cells. SAR studies revealed that
both side indole rings are critical for the excellent in vitro anticancer activity of ketohydrazide-
hydrazones. Mechanism of the action studies suggested that bis(indolyl)ketohydrazide-
hydrazones induced apoptosis via caspase 3/7 activation but was not toxic. Compound 21k
arrested cell cycle in G2/M phase by binding to tubulin and inhibiting tubulin polymerization
(IC50 = 0.6 µM). Moreover, molecular docking studies indicate that 21k tubulin binding is
similar to colchicine/tubulin binds which supports inhibition of tubulin polymerization by 21k.
4. Experimental section
General remarks
The laboratory grade reagents were obtained commercially either from Spectrochem or Aldrich
and used without further purification otherwise noted. Organic solvents were distilled off using
Buchi rotary evaporator. The reactions were monitored by thin layer chromatography (TLC),
16

ACCEPTED MANUSCRIPT
which was performed on commercially available Merck pre-coated plates (silica gel 60 F254, 0.25
mm). Melting points (mps) were determined on E-Z melting apparatus. NMR (1H & 13C) spectra
were recorded on a Bruker advance II (400/500 & 100/125 MHz, respectively) spectrometer
using DMSO-d6 as a solvent and the chemical shifts are expressed in δ units (ppm) from
tetramethylsilane. HRMS spectra were obtained on a Bruker microTOF-Q II 10348 (ESI)
spectrometer. Purity of all the synthesized compounds was >98% as determined by WATERS
515 HPLC system with a Sunfire C-18 column (5 µm, 4.6 × 250 mm) and PDA detector using a
flow rate of 1 mL/min. and a gradient of acetonitrile.
General procedure for synthesis of indolyl oxoacetyl chlorides (17)[29, 47]: To a solution of
indole 16 (8.5 mmol) in diethyl ether (10 mL) was added oxalyl chloride (1.3 g, 10.25 mmol)
while maintaining the temperature at 0 °C. The resulting yellowish mixture was allowed to stir at
same temperature for 30 min. After completion of the reaction, suspension was filtered, washed
with cold diethyl ether to afford 17 as yellow solids in 81-90% yields.
General procedure for synthesis of indolyl oxoacetohydrazides (18): To a solution of
glyoxalyl chloride 17 (2.40 mmol) in dry methanol (5 mL) was added hydrazine hydrate (0.36 g,
7.20 mmol) at 0 °C and the contents were allowed to stir at same temperature for 30 min. Upon
completion of the reaction, suspension obtained was filtered and washed with methanol (10 mL)
to afford a solid product which was recrystallized from methanol to obtain pure glyoxalyl-
hydrazides 18 in 80-85% yields as light yellow solids.
2-(1H-Indol-3-yl)-2-oxoacetohydrazide (18a)
Yield 85%; Light yellow solid; mp 220–222 °C; IR (KBr, ν, cm-1): 3256, 3109, 1680, 1660,
1607, 1583, 1523, 1433, 1240, 736; 1H NMR (400 MHz, DMSO-d6): δ 8.74–8.72 (m, 1H), 8.28–
8.18 (m, 1H), 7.55 (d, J = 7.5 Hz, 1H), 7.29–7.23 (m, 2H), 4.61 (s, 2H); 13C NMR (100 MHz,
17

ACCEPTED MANUSCRIPT
DMSO-d6): δ 182.6, 162.8, 138.7, 136.8, 126.5, 123.9, 122.9, 121.7, 113.1, 113.0; (ESI-MS) [M
+ H]+ m/z 204.2.
2-(5-Bromo-1H-indol-3-yl)-2-oxoacetohydrazide (18b)
Yield 83%; Light yellow solid; mp 249–251 °C; IR (KBr, ν, cm-1): 3340, 3290, 3050, 2920,
1
1686, 1646, 1605, 1545, 724; H NMR (400 MHz, DMSO-d6): δ 8.77 (s, 1H), 8.34 (d, J = 1.8
13
Hz, 1H), 7.52 (d, J = 8.6 Hz, 1H), 7.41 (dd, J = 8.6, 2.0 Hz, 1H), 4.63 (s, 2H); C NMR (100
MHz, DMSO-d6): δ 182.5, 162.2, 139.8, 135.6, 128.3, 126.5, 123.8, 115.8, 115.1, 112.4; (ESI-
MS) [M + H]+ m/z 282.2 and 284.1 [M + H + 2]+.
2-(1-(4-Chlorobenzyl)-1H-indol-3-yl)-2-oxoaceto-hydrazide (18c)
Yield 80%; Light yellow solid; mp 155–157 °C; IR (KBr, ν, cm-1): 3310, 3163, 2931, 1682,
1
1620, 1542, 1442, 1218, 1141, 778, 724; H NMR (400 MHz, DMSO-d6): δ 10.08 (s, 1H), 8.94
(s, 1H), 8.31–8.16 (m, 1H), 7.62–7.52 (m, 1H), 7.44–7.40 (m, 2H), 7.34–7.27 (m, 4H), 5.61 (s,
13
2H), 4.60 (s, 2H); C NMR (100 MHz, DMSO-d6): δ 182.3, 162.4, 141.3, 136.6, 136.2, 132.9,
129.7, 129.2, 127.2, 124.1, 123.5, 122.0, 112.4, 111.9, 49.5; (ESI-MS) [M + H]+ m/z 328.0.
General procedure for the synthesis of indole-3-carboxaldehydes (20)[37, 48]: A round-
bottomed flask containing (28 mL, 370 mmol) freshly distilled dimethylformamide (DMF) was
cooled to 0 °C for about 30 min and freshly distilled phosphorus oxychloride (8.41 mL, 90
mmol) was subsequently added with stirring to the DMF over a period of 30 min. A solution of
indole 19 (85.47 mmol) in DMF (10 mL, 130 mmol) was added to the yellow solution over a
period of 1 h. The solution was stirred at room temperature till it become a yellow paste. At the
end of the reaction, 30 g of crushed ice was added to the paste with stirring to make a clear
cherry-red aqueous solution. Sodium hydroxide solution (1N, 100 mL) was added dropwise with
stirring to this cherry-red solution. The resulting suspension was heated rapidly to 90 °C and
allowed to cool at room temperature, after which it was refrigerated for overnight. The product
was filtered, washed with water (2 × 100 mL) and air-dried to afford pure indole-3-
18

ACCEPTED MANUSCRIPT
carboxaldehydes 20 in 80-90% yields. Indole-3-carboxaldehyde, pale yellow solid; mp 194-196
°C (Lit.[37]196-197 °C).
1-(4-Chlorobenzyl)-indole-3-carboxaldehyde, mp 123-124 °C (Lit.[37]123-125 °C).
1-(4-Methoxybenzyl)-indole-3-carboxaldehyde, mp 126-128 °C (Lit.[37]127-129 °C).
5-Bromo-1-(4-chlorobenzyl)-indole-3-carboxaldehyde, mp 193-196
5-Methoxy-1-(4-chlorbenzyl)-indole-3-carboxaldehyde, mp 135-137 °C
6-Fluoro-1-(4-chlorobenzyl)-indole-3-carboxaldehyde, mp 140-142 °C
5-Methoxy-1-(4-methoxybenzyl)-indole-3-carboxaldehyde, mp 85-88 °C
6-Fluoro-1-(4-methoxybenzyl)-indole-3-carboxaldehyde, mp 146-148 °C
General procedure for the synthesis of bis(indolyl)ketohydrazide-hydrazones (21a-o) and
indolyl ketohydrazide-hydrazones (23a-e): A mixture of glyoxalylhydrazide 18 (1.47 mmol)
and aldehyde 20 or 22 (1.47 mmol) in ethanol (5 mL) was refluxed for 6 h. After completion of
the reaction, ethanol was distilled off and the residue was taken into water (10 mL) and allowed
to stir at room temperature for 30 min. The obtained suspension was filtered, washed with water
and dried to obtain yellow solid ketohydrazide-hydrazones 21a-o and 23a-e in 88-96% yields.
N'-((1H-Indol-3-yl)methylene)-2-(1H-indol-3-yl)-2-oxoacetohydrazide (21a)
Yield 90%; Yellow solid; mp 248–250 °C; IR (KBr, ν, cm-1): 3440, 3193, 1658, 1603, 1496,
1
1434, 1234, 1157, 1126, 740; H NMR (500 MHz, DMSO-d6): (rotamers ratio 1:0.28) δ 12.34
(s, 1H), 11.87 (s, 1H), 11.66 (s, 1H), 8.83 (s, 1H), 8.68 (s, 1H), 8.29–8.33 (m, 2H), 7.85 (s, 1H),
7.56–7.58 (m, 1H), 7.47 (d, J = 7.9 Hz, 1H), 7.29–7.31 (m, 2H), 7.25–7.37 (m, 2H); 13C NMR
(100 MHz, DMSO-d6): δ 187.2, 183.0, 169.2, 159.9, 147.4, 141.9, 138.9, 137.5, 137.3, 137.2,
19

ACCEPTED MANUSCRIPT
136.8, 136.3, 131.4, 130.6, 126.6, 125.6, 124.8, 124.3, 124.0, 123.7, 123.2, 123.1, 122.8, 122.7,
122.5, 122.2, 121.7, 121.3, 121.0, 120.3, 113.8, 113.1, 113.0, 112.4, 112.1, 111.8, 111.7; Anal.
RP-HPLC tR = 2.699 min, purity 99.73%; (ESI-MS) [M + H]+ m/z 331.20; HRMS (ESI+): m/z
calcd. for C19H14N4O [M + H]+: 331.1195, found: 331.1191.
N'-((6-Bromo-1H-indol-3-yl)methylene)-2-(1H-indol-3-yl)-2-oxoacetohydrazide (21b)
Yield 88%; Yellow solid; mp 279–281 °C; IR (KBr, ν, cm-1): 3302, 1689, 1604, 1488, 1427,
1
1242, 1126, 810, 740; H NMR (500 MHz, DMSO-d6): (rotamers ratio 1:0.28) δ 12.34 (s, 1H),
11.93 (s, 1H), 11.76 (s, 1H), 8.82 (s, 1H), 8.66 (s, 1H), 8.24–8.29 (m, 2H), 7.88 (d, J = 2.2 Hz,
1H), 7.65 (s, 1H), 7.55–7.57 (m, 1H), 7.34 (d, J = 8.2 Hz, 1H), 7.29–7.31 (m, 2H); 13C NMR
(100 MHz, DMSO-d6): δ 182.9, 160.0, 146.8, 139.1, 138.4, 136.8, 132.3, 126.6, 124.2, 124.0,
123.9, 123.8, 123.8, 123.6, 123.1, 123.0, 121.7, 115.9, 115.0, 113.1, 112.9, 112.3; Anal. RP-
HPLC tR = 2.676 min, purity 98.65%; (ESI-MS) [M + H]+ m/z 409.35; HRMS (ESI+): m/z calcd.
for C19H13BrNaN4O2 [M + Na]+: 431.0120, found: 431.0114 [M + Na]+ and 433.0128 [M + 2 +
Na]+.
N'-((5-Bromo-1H-indol-3-yl)methylene)-2-(1H-indol-3-yl)-2-oxoacetohydrazide (21c)
Yield 89%; Yellow solid; mp 239–240 °C; IR (KBr, ν, cm-1): 3440, 3255, 1658, 1597, 1496,
1427, 1226, 1118, 794, 740; 1H NMR (400 MHz, DMSO-d6): (rotamers ratio 1:0.13) δ 12.12 (s,
1H), 11.72 (s, 1H), 11.60 (s, 1H), 8.87 (d, J = 3.3 Hz, 1H), 8.65 (s, 1H), 8.45 (d, J = 1.8 Hz, 1H),
8.27–8.30 (m, 1H), 7.65 (d, J = 2.8 Hz, 1H), 7.46–7.49 (m, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.24–
7.26 (m, 1H), 7.20–7.23 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ 182.8, 160.0, 146.9, 141.7,
139.2, 137.4, 136.8, 136.3, 132.8, 131.9, 126.6, 126.5, 125.7, 125.5, 124.7, 124.4, 124.0, 123.7,
123.1, 122.7, 121.7, 114.4, 113.7, 113.1, 112.9, 111.7, 111.2; Anal. RP-HPLC tR = 2.721 min,
20

ACCEPTED MANUSCRIPT
purity 98.96%; HRMS (ESI+): m/z calcd. for C19H13BrNaN4O2 [M + Na]+: 431.0120, found:
431.0115 [M + Na]+ and 433.0109 [M + 2 + Na]+.
N'-((5-Fluoro-1H-indol-3-yl)methylene)-2-(1H-indol-3-yl)-2-oxoacetohydrazide (21d)
Yield 90%; Yellow solid; mp 267–269 °C; IR (KBr, ν, cm-1): 3448, 3217, 1658, 1596, 1488,
1
1442, 1242, 1157, 1126, 794, 740; H NMR (500 MHz, DMSO-d6): (rotamers ratio 1:0.25) δ
8.84 (s, 1H), 8.67 (s, 1H), 8.27 (d, J = 3.8 Hz, 1H), 8.05–8.07 (m, 1H), 7.93 (s, 1H), 7.54–7.58
(m, 1H), 7.48 (s, 1H), 7.28–7.28 (m, 3H), 7.07–7.31 (m, 1H); 13C NMR (100 MHz, DMSO-d6): δ
186.9, 181.9, 160.4, 159.4, 157.1, 146.6, 141.7, 141.2, 134.2, 133.8, 133.0, 127.4, 123.5, 123.4,
122.6, 122.5, 121.6, 113.8, 113.1, 113.0, 112.3, 112.2, 111.4, 111.1, 107.4, 107.1, 79.6; Anal.
RP-HPLC tR = 2.710 min, purity 98.50%; (ESI-MS) [M + H]+ m/z 349.12; HRMS (ESI+): m/z
calcd. for C19H13FNaN4O2 [M + Na]+: 371.0920, found: 371.0925.
N'-((6-Fluoro-1H-indol-3-yl)methylene)-2-(1H-indol-3-yl)-2-oxoacetohydrazide (21e)
Yield 91%; Yellow solid; mp 240–242 °C; IR (KBr, ν, cm-1): 3440, 3224, 1666, 1596, 1496,
1442, 1234, 1126, 810, 740; 1H NMR (500 MHz, DMSO-d6): (rotamers ratio 1:0.27) δ 8.80 (s,
1H), 8.65 (s, 1H), 8.31–8.33 (m, 1H), 8.26–8.27 (m, 1H), 7.85 (s, 1H), 7.53–7.56 (m, 1H), 7.31–
7.36 (m, 1H), 7.25–7.26 (m, 3H), 7.05–7.08 (m, 1H); 13C NMR (100 MHz, DMSO-d6): δ 189.1,
186.7, 182.5, 169.3, 161.1, 160.2, 158.7, 146.8, 141.3, 139.9, 137.7, 137.5, 136.6, 132.1, 126.9,
125.6, 123.8, 123.7, 123.6, 122.9, 122.7, 121.7, 121.2, 113.8, 113.4, 113.2, 113.0, 112.2, 111.8,
109.5, 109.2, 108.2; Anal. RP-HPLC tR = 2.707 min, purity 99.31%; (ESI-MS) [M + H]+ m/z
349.13; HRMS (ESI+): m/z calcd. for C19H14FN4O2 [M + H]+: 349.1101, found: 349.1091.
N'-((5-Methoxy-1H-indol-3-yl)methylene)-2-(1H-indol-3-yl)-2-oxoacetohydrazide
(21f)
Yield 89%; Yellow solid; mp 236–238 °C; IR (KBr, ν, cm-1): 3286, 3085, 1681, 1604, 1488,
21

ACCEPTED MANUSCRIPT
1
1434, 1242, 1157, 1126, 802, 740; H NMR (500 MHz, DMSO-d6): (rotamers ratio 1:0.50) δ
11.62 (s, 1H), 8.84 (s, 1H), 8.67 (s, 1H), 8.28–8.30 (m, 1H), 8.33–8.37 (m, 1H), 7.87 (d, J = 2.3
Hz, 1H), 7.79 (s, 1H), 7.55–7.57 (m, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.27–7.29 (m, 3H), 6.88 (dd,
J = 8.8, 2.4 Hz, 1H), 3.83 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 186.9, 182.8, 169.0, 159.9,
155.0, 154.6, 147.5, 142.9, 139.6, 137.5, 137.3, 137.0, 132.6, 132.1, 131.8, 131.2, 126.8, 125.6,
125.5, 124.7, 123.8, 123.7, 123.0, 122.6, 121.7, 121.3, 113.6, 113.3, 113.1, 113.0, 112.9, 112.7,
111.9, 111.4, 104.9, 102.6, 55.9, 54.6; Anal. RP-HPLC tR = 2.702 min, purity 98.31%; (ESI-MS)
[M + H]+ m/z 361.18; HRMS (ESI+): m/z calcd. for C20H17N4O3 [M + H]+: 361.1301, found:
361.1295.
N'-((6-Methoxy-1H-indol-3-yl)methylene)-2-(1H-indol-3-yl)-2-oxoacetohydrazide
(21g)
Yield 90%; Yellow solid; mp 238–240 °C; IR (KBr, ν, cm-1): 3394, 3271, 1674, 1612, 1496,
1442, 1357, 1242, 1157, 1126, 810, 748; 1H NMR (400 MHz, DMSO-d6): (rotamers ratio 1:0.09)
δ 12.05 (s, 1H), 11.58 (s, 1H), 11.15 (s, 1H), 8.94 (d, J = 2.3 Hz, 1H), 8.66 (s, 1H), 8.35–8.37 (m,
1H), 8.23 (d, J = 8.7 Hz, 1H), 7.49–7.52 (m, 2H), 7.25–7.29 (m, 2H), 6.92 (d, J = 2.2 Hz, 1H),
6.79 (dd, J = 8.7, 2.3 Hz, 1H), 3.83 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 187.3, 182.8,
169.2, 160.0, 156.9, 156.5, 147.2, 139.5, 138.4, 137.2, 130.4, 126.8, 123.9, 123.1, 123.0, 121.7,
118.9, 118.4, 113.8, 113.3, 113.0, 112.2, 111.8, 110.9, 110.3, 95.4, 55.7; Anal. RP-HPLC tR =
2.683 min, purity 98.94%; HRMS (ESI+): m/z calcd. for C20H16NaN4O3 [M + Na]+: 383.1120,
found: 383.1115.
N'-((1-(4-Chlorobenzyl)-5-bromo-1H-indol-3-yl)methylene)-2-(1H-indol-3-yl)-
2-oxoacetohydrazide (21h)
Yield 95%; Yellow solid; mp 233–235 °C; IR (KBr, ν, cm-1): 3178, 1674, 1612, 1496, 1442,
1
1388, 1242, 1157, 1126, 802, 748; H NMR (400 MHz, DMSO-d6): (rotamers ratio 1:0.15) δ
22

ACCEPTED MANUSCRIPT
12.19 (s, 1H), 11.85 (s, 1H), 8.87 (d, J = 3.1 Hz, 1H), 8.66 (s, 1H), 8.49 (d, J = 1.4 Hz, 1H),
8.28–8.30 (m, 1H), 7.87 (s, 1H), 7.48–7.50 (m, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.30–7.28 (m, 3H),
7.20–7.24 (m, 4H), 5.41 (s, 2H); 13C NMR (100 MHz, DMSO-d6): δ 182.7, 159.9, 146.3, 139.3,
136.8, 136.7, 136.1, 135.6, 132.8, 129.6, 129.3, 129.2, 129.1, 127.0, 126.6, 126.0, 125.0, 124.0,
123.2, 121.7, 114.4, 114.1, 113.6, 113.4, 113.1, 113.0, 112.9, 111.5, 111.0, 49.3; Anal. RP-
HPLC tR = 3.173 min, purity 98.74%; HRMS (ESI+): m/z calcd. for C26H18BrClNaN4O2 [M +
Na]+: 555.0199, found: 555.0184 [M + Na]+ and 557.0174 [M + 2 + Na]+.
N'-((6-Fluoro-1H-indol-3-yl)methylene)-2-(5-bromo-1H-indol-3-yl)-2-oxoaceto-hydrazide
(21i)
Yield 96%; Yellow solid; mp 290–292 °C; IR (KBr, ν, cm-1): 3348, 3301, 1689, 1604, 1489,
1
1434, 1226, 1134, 1126, 825, 601; H NMR (400 MHz, DMSO-d6): (rotamers ratio 1:0.09) δ
12.17 (s, 1H), 11.57 (s, 1H), 11.35 (s, 1H), 8.97 (d, J = 3.4 Hz, 1H), 8.69 (s, 1H), 8.51 (d, J = 1.8
Hz, 1H), 8.33–8.37 (m, 1H), 7.60 (d, J = 2.7 Hz, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.35 (dd, J = 8.6,
1.9 Hz 1H), 7.14 (dd, J = 9.0, 2.3 Hz, 1H), 6.90–6.95 (m, 1H); 13C NMR (100 MHz, DMSO-d6):
δ 187.4, 182.8, 168.8, 161.1, 159.6, 158.7, 147.2, 141.6, 140.1, 137.6, 137.5, 137.4, 136.1, 135.6,
132.2, 131.4, 128.4, 127.3, 126.6, 126.4, 123.9, 123.7, 123.6, 123.4, 121.5, 121.1, 115.9, 115.6,
115.2, 113.3, 112.4, 112.2, 111.8, 109.5, 109.3, 98.7, 98.5, 98.2; Anal. RP-HPLC tR = 2.903 min,
purity 98.70%; HRMS (ESI+): m/z calcd. for C19H12BrFNaN4O2 [M + Na]+: 449.0025, found:
449.0022 [M + Na]+ and 451.0009 [M + 2 + Na]+.
23

ACCEPTED MANUSCRIPT
N'-((6-Methoxy-1H-indol-3-yl)methylene)-2-(5-bromo-1H-indol-3-yl)-2-oxoaceto-hydrazide
(21j)
Yield 90%; Yellow solid; mp > 300 °C; IR (KBr, ν, cm-1): 3417, 3278, 1666, 1604, 1504, 1434,
1
1357, 1234, 1141, 817, 609; H NMR (500 MHz, DMSO-d6): (rotamers ratio 1:0.26) δ 12.49 (s,
1H),11.94 (s, 1H), 11.72 (s, 1H), 8.88 (s, 1H), 8.67 (s, 1H), 8.41 (s, 1H), 8.30–8.33 (m, 1H), 7.87
(s, 1H), 7.5–7.56 (m, 1H), 7.43–7.48 (m, 1H), 7.26–7.24 (m, 1H), 7.04–7.10 (m, 1H), 3.40 (s,
3H); 13C NMR (100 MHz, DMSO-d6): δ 187.5, 182.9, 168.8, 159.5, 156.94, 156.5, 147.5, 141.8,
140.1, 138.4, 138.1, 137.3, 136.1, 135.6, 130.6, 129.6, 128.4, 127.3, 126.6, 126.4, 123.8, 123.4,
123.1, 122.7, 118.9, 118.4, 115.9, 115.5, 115.2, 113.4, 112.5, 112.2, 111.7, 111.0, 110.0, 95.4,
95.1, 55.7; Anal. RP-HPLC tR = 2.870 min, purity 98.13%; HRMS (ESI+): m/z calcd. for
C20H15BrNaN4O3 [M + Na]+: 461.0225, found: 461.0228 [M + Na]+ and 463.0210 [M + 2 +
Na]+.
N'-((1H-Indol-3-yl)methylene)-2-(1-(4-chlorobenzyl)-1H-indol-3-yl)-2-oxoaceto-hydrazide
(21k)
Yield 89%; Yellow solid; mp 225–227 °C; IR (KBr, ν, cm-1): 3186, 3055, 1689, 1627, 1504,
1
1442, 1365, 1234, 1172, 810, 740; H NMR (400 MHz, DMSO-d6): (rotamers ratio 1:0.09) δ
11.60 (s, 1H), 11.30 (s, 1H), 9.02 (s, 1H), 8.66 (s, 1H), 8.29–8.35 (m, 2H), 7.56 (d, J = 2.7 Hz,
1H), 7.36–7.38 (m, 2H), 7.19–7.27 (m, 6H), 7.08–7.14 (m, 2H), 5.43 (s, 2H); 13C NMR (100
MHz, DMSO-d6): δ 187.0, 182.8, 159.9, 159.9, 155.5, 147.4, 141.9, 137.7, 137.5, 137.3, 137.1,
136.7, 136.2, 136.2, 133.0, 132.5, 132.3, 131.4, 129.8, 129.2, 129.1, 128.8, 127.3, 125.2, 124.8,
124.3, 124.2, 124.0, 123.6, 123.2, 123.2, 123.1, 122.8, 122.6, 122.2, 122.1, 121.0, 121.0, 112.6,
112.4, 112.4, 112.4, 112.1, 112.0, 111.9, 111.7, 49.6, 49.3; Anal. RP-HPLC tR = 2.727 min,
24

ACCEPTED MANUSCRIPT
purity 98.04%; (ESI-MS) [M + H]+ m/z 455.6; HRMS (ESI+): m/z calcd. for C26H20ClN4O2 [M +
H]+: 455.1275, found: 455.1269.
N'-((2-(4-Methoxyphenyl)-1H-indol-3-yl)methylene)-2-(1H-indol-3-yl)-2-oxoaceto-
hydrazide (21l)
Yield 91%; Yellow solid; mp >300 °C; IR (KBr, ν, cm-1): 3394, 3271, 1674, 1612, 1496, 1442,
1
1357, 1242, 1157, 1126, 810, 748; H NMR (400 MHz, DMSO-d6): (rotamers ratio 1:0.10) δ
12.04 (s, 1H), 11.74 (s, 1H), 11.54 (s, 1H), 8.86 (d, J = 3.2 Hz, 1H), 8.82 (s, 1H), 8.43 (d, J = 7.1
Hz, 1H), 8.25–8.28 (m, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.43–7.46 (m, 1H), 7.36 (d, J = 7.3 Hz,
1H), 7.17–7.21 (m, 2H), 7.07–7.17 (m, 2H), 7.02 (d, J = 8.8 Hz, 2H), 3.82 (s, 3H); 13C NMR
(100 MHz, DMSO-d6): δ 187.4, 182.9, 169.0, 160.3, 159.7, 147.8, 142.8, 142.1, 141.8, 139.1,
137.3, 136.9, 136.8, 136.5, 131.0, 130.8, 126.6, 126.2, 125.7, 125.6, 124.0, 123.8, 123.3, 123.1,
122.9, 121.8, 121.2, 114.9, 113.9, 113.1, 113.0, 111.8, 111.5, 107.9, 107.4, 55.8; Anal. RP-
HPLC tR = 2.780 min, purity 99.42%; HRMS (ESI+): m/z calcd. for C26H20N4O3 [M + H]+:
437.1614, found: 437.1613.
N'-((1-(4-Chlorobenzyl)-5-methoxy-1H-indol-3-yl)methylene)-2-(5-bromo-1H-indol-3-yl) -2-
oxoacetohydrazide (21m)
Yield 91%; Yellow solid; mp 223–225 °C; IR (KBr, ν, cm-1): 3340, 1689, 1620, 1489, 1411,
1
1350, 1226, 1172, 833, 702; H NMR (400 MHz, DMSO-d6): (rotamers ratio 1:0.30) δ 12.44 (s,
1H), 11.89 (s, 1H), 8.91 (s, 1H), 8.70 (s, 1H), 8.42 (s, 1H), 8.23 (d, J = 7.5 Hz, 1H), 8.0 (s, 1H),
7.88–7.89 (m, 1H), 7.56 (d, J = 8.9 Hz, 1H), 7.39–7.45 (m, 2H), 7.26–7.30 (m, 2H), 7.17 (d, J =
8.2 Hz, 1H), 6.9 (dd, J = 8.8, 2.0 Hz, 1H) 5.45 (s, 2H), 3.82 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 186.7, 182.7, 168.6, 159.4, 155.4, 155.0, 147.1, 142.6, 140.2, 137.9, 137.0, 136.1,
135.7, 134.7, 134.4, 132.7, 132.6, 132.4, 132.1, 129.5, 129.3, 129.1, 129.0, 128.5, 127.2, 126.6,
25

ACCEPTED MANUSCRIPT
126.4, 126.2, 125.4, 123.8, 123.5, 115.9, 115.5, 115.2, 115.1, 113.6, 113.0, 112.9, 112.4, 111.9,
111.5, 111.0, 105.3, 103.2, 55.9, 54.6, 49.3, 49.2; Anal. RP-HPLC tR = 3.328 min, purity
98.67%; HRMS (ESI+): calcd. for C27H21BrClN4O3 [M + H]+: 563.0486; found: 563.0430 and
565.0418 [M + H + 2]+.
N'-((1-(4-Methoxybenzyl)-5-methoxy-1H-indol-3-yl)methylene)-2-(5-bromo-1H-indol-3-yl)-
2-oxoacetohydrazide (21n)
Yield 91%; Yellow solid; mp 156–158 °C; IR (KBr, ν, cm-1): 3340, 3301, 1689, 1612, 1488,
1
1419, 1234, 1172, 1141, 877, 748; H NMR (500 MHz, DMSO-d6): (rotamers ratio 1:0.44) δ
8.80 (d, J = 8.1 Hz, 1H), 8.66 (s, 1H), 8.34 (s, 1H), 8.24 (s, 1H), 7.97 (s, 1H), 7.87 (s, 1H), 7.47–
7.48 (m, 1H), 7.39–7.42 (m, 2H), 7.34 (d, J = 8.4 Hz, 1H), 7.24–7.29 (m, 2H), 7.16 (d, J = 8.4
Hz, 1H), 6.89 (dd, J = 8.9, 2.4 Hz, 1H), 5.44 (s, 2H), 3.81 (s, 6H); 13C NMR (100 MHz, DMSO-
d6): δ 182.4, 159.6, 159.2, 159.1, 155.3, 154.9, 147.1, 142.6, 141.1, 136.6, 134.5, 134.3, 132.4,
129.8, 129.3, 129.0, 128.8, 127.3, 126.3, 126.2, 125.4, 123.8, 123.4, 115.7, 115.5, 115.3, 114.5,
114.4, 113.5, 113.0, 112.7, 112.4, 112.0, 111.2, 111.0, 110.7, 105.2, 103.2, 56.0, 55.5, 54.6;
Anal. RP-HPLC tR = 3.189 min, purity 98.97%; (ESI-MS) [M + H]+ m/z 559.2; HRMS (ESI+):
m/z calcd. for C28H24BrN4O4 [M + H]+: 559.0981, found: 559.0973 and 561.970 [M + 2 + Na]+.
2-(1-(4-Chlorobenzyl)-1H-indol-3-yl)-N'-((5-methoxy-1H-indol-3-yl)methylene)-2-
oxoacetohydrazide (21o)
Yield 86%; Yellow solid; mp 217–219 °C; IR (KBr, ν, cm-1): 3184, 3057, 1688, 1627, 1502,
1446, 1367, 1236, 1175, 811, 740; 1H NMR (400 MHz, DMSO-d6): (rotamers ratio 1:0.5) δ
11.85 (s, 1H), 11.54 (s, 1H), 9.12 (s, 1H), 8.68 (s, 1H), 8.35–8.29 (m, 1H), 7.88 (d, J = 2.2 Hz,
1H), 7.79 (s, 1H), 7.61 (d, J = 2.5 Hz, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.37–7.34 (m, 2H), 7.31 (d,
26

ACCEPTED MANUSCRIPT
J = 5.2 Hz, 2H), 7.08 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 5.63 (s, 2H), 3.82 (s, 3H);
13C NMR (100 MHz, DMSO-d6): δ 186.6, 182.5, 168.5, 159.6, 155.3, 155.0, 147.3, 142.8, 140.0,
137.8, 137.1, 136.0, 135.4, 134.7, 134.3, 132.7, 132.6, 132.4, 132.0, 129.5, 129.2, 129.1, 129.0,
128.5, 127.0, 126.5, 126.4, 126.2, 125.3, 123.7, 123.5, 116.0, 115.5, 115.2, 115.1, 113.5, 113.0,
112.8, 112.3, 111.9, 111.4, 111.0, 105.3, 103.2, 56.0, 54.9, 49.8, 49.2; Anal. RP-HPLC tR =
3.274 min, purity 99.87%; ESI-MS (ESI+): m/z calcd. for C27H22ClN4O3 [M + H]+: 485.14,
found: 485.20.
N'-(3,4,5-Trimethoxybenzylidene)-2-(1H-indol-3-yl)-2-oxoacetohydrazide (23a)
Yield 96%; Yellow solid; mp 155–157 °C; IR (KBr, ν, cm-1): 3232, 2985, 1681, 1610, 1496,
1434, 1234, 1126, 995, 810, 740; 1H NMR (400 MHz, DMSO-d6): (rotamers ratio 1:0.07) δ 8.79
(s, 1H), 8.46 (s, 1H), 8.30–8.32 (m, 1H), 8.12 (s, 1H), 7.52–7.54 (m, 1H), 7.25–7.27 (m, 2H),
13
7.02 (s, 2H), 3.88 (s, 6H), 3.77 (s, 3H); C NMR (100 MHz, DMSO-d6): δ 182.2, 160.9, 153.7,
153.3, 150.1, 140.0, 139.9, 137.8, 130.1, 126.8, 123.8, 123.0, 121.6, 113.5, 112.8, 104.9, 104.2,
60.6, 56.4, 55.8; Anal. RP-HPLC tR = 2.855 min, purity 98.62%; HRMS (ESI+): m/z calcd. for
C20H19N3O5 [M + H]+: 382.1403; found: 382.1395.
N'-(4-Methoxybenzylidene)-2-(1H-indol-3-yl)-2-oxoacetohydrazide (23b)
Yield 91%; Yellow solid; mp 247–249 °C; IR (KBr, ν, cm-1): 3224, 3062, 1674, 1604, 1496,
1
1434, 1249, 1164, 1118, 810, 740; H NMR (400 MHz, DMSO-d6): (rotamers ratio 1:0.06) δ
12.17 (s, 1H), 11.99 (s, 1H), 8.80 (d, J = 3.3 Hz, 1H), 8.50 (s, 1H), 8.31–8.33 (m, 1H), 7.70 (d, J
= 8.8 Hz, 2H), 7.51–7.53 (m, 1H), 7.25–7.27 (m, 2H), 6.96 (d, J = 8.8 Hz, 2H), 3.84 (s, 3H); 13C
NMR (100 MHz, DMSO-d6): δ 182.5, 161.6, 160.4, 150.3, 145.4, 139.1, 136.7, 129.3, 128.8,
127.1, 126.5, 124.1, 123.2, 121.7, 114.9, 113.1, 112.8, 55.8; Anal. RP-HPLC tR = 2.866 min,
27

ACCEPTED MANUSCRIPT
purity 97.77%; HRMS (ESI+): m/z calcd. for C18H15NaN3O3 [M + Na]+: 344.1011, found:
344.1020.
N'-(3,4-Dimethoxybenzylidene)-2-(1H-indol-3-yl)-2-oxoacetohydrazide (23c)
Yield 95%; Yellow solid; mp 198–200 °C; IR (KBr, ν, cm-1): 3309, 1666, 1627, 1504, 1435,
1
1265, 1134, 810, 740; H NMR (400 MHz, DMSO-d6): (rotamers ratio 1:0.09) δ 12.16 (s, 1H),
12.01 (s, 1H), 8.81 (d, J = 3.3 Hz, 1H), 8.48 (s, 1H), 8.33–8.33 (m, 1H), 7.50–7.53 (m, 1H), 7.41
(d, J = 1.8 Hz, 1H), 7.24–7.28 (m, 2H), 7.20 (dd, J = 8.3, 1.8 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H),
3.89 (s, 3H), 3.87 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 182.6, 160.4, 151.5, 150.5, 149.5,
139.1, 136.9, 127.2, 126.5, 124.1, 123.2, 122.7, 121.7, 113.2, 112.8, 111.9, 108.7, 56.1, 55.9;
Anal. RP-HPLC tR = 2.838 min, purity 98.31%; HRMS (ESI+): m/z calcd. for C19H17NaN3O4 [M
+ Na]+: 374.1117; found: 374.1090.
N'-(3-Methoxybenzylidene)-2-(1H-indol-3-yl)-2-oxoacetohydrazide (23d)
Yield 92%; Yellow solid; mp 227–229 °C; IR (KBr, ν, cm-1): 3386, 3247, 1689, 1627, 1504,
1
1427, 1288, 1242, 1149, 1118, 810, 748, 601; H NMR (400 MHz, DMSO-d6): (rotamers ratio
1:0.09) δ 12.06 (s, 2H), 8.83 (d, J = 3.3 Hz, 1H), 8.54 (s, 1H), 8.33–8.35 (m, 1H), 7.50–7.53 (m,
1H), 7.25–7.35 (m, 5H), 6.95–6.98 (m, 1H), 3.85 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ
182.4, 160.6, 160.0, 150.3, 139.2, 136.9, 136.0, 130.5, 126.5, 124.1, 123.2, 121.7, 120.8, 117.2,
113.2, 112.8, 111.7, 55.7; Anal. RP-HPLC tR = 2.922 min, purity 98.84%; HRMS (ESI+): m/z
calcd. for C18H15NaN3O3 [M + Na]+: 344.1011, found: 344.1004.
28