Identification of new 3-phenyl-1H-indole-2-carbohydrazide derivatives and their structure–activity relationships as potent tubulin inhibitors and anticancer agents: A combined in silico, in vitro and synthetic study

Article abstract of DOI:10.1016/j.bioorg.2021.104795

Virtual screening of commercially available molecular entities by using CDRUG, structure-based virtual screening, and similarity identified eight new derivatives of 3-phenyl-1H-indole-2-carbohydrazide with anti-proliferative activities. The molecules were tested experimentally for inhibition of tubulin polymerisation, which revealed furan-3-ylmethylene-3-phenyl-1H-indole-2-carbohydrazide (27a) as the most potent candidate. Molecule 27a was able to induce G2/M phase arrest in A549 cell line, similar to other tubulin inhibitors. Synthetic modifications of 27a were focussed on small substitutions on the furan ring, halogenation at R₁ position and alteration of furyl connectivity. Derivatives 27b, 27d and 27i exhibited the strongest tubulin inhibition activities and were comparable to 27a. Bromine substitution at R₁ position showed most prominent anticancer activities; derivatives 27b-27d displayed the strongest activities against HuCCA-1 cell line and were more potent than doxorubicin and the parent molecule 27a with IC₅₀ values <0.5 μM. Notably, 27b with a 5-methoxy substitution on furan displayed the strongest activity against HepG2 cell line (IC₅₀ = 0.34 μM), while 27d displayed stronger activity against A549 cell line (IC₅₀ = 0.43 μM) compared to doxorubicin and 27a. Fluorine substitutions at the R₁ position tended to show more modest anti-tubulin and anticancer activities, and change of 2-furyl to 3-furyl was tolerable. The new derivatives, thiophenyl 26, displayed the strongest activity against A549 cell line (IC₅₀ = 0.19 μM), while 1-phenylethylidene 21b and 21c exhibited more modest anticancer activities with unclear mechanisms of action; 26 and 21c demonstrated G2/M phase arrest, but showed weak tubulin inhibitory properties. Molecular docking suggests the series inhibit tubulin at the colchicine site, in agreement with the experimental findings. The calculated molecular descriptors indicated that the molecules obey Lipinski's rule which suggests the molecules are drug-like structures.

Full text of DOI:10.1016/j.bioorg.2021.104795

Bioorganic Chemistry 110 (2021) 104795
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Identification of new 3-phenyl-1H-indole-2-carbohydrazide derivatives and
their structure–activity relationships as potent tubulin inhibitors and
anticancer agents: A combined in silico, in vitro and synthetic study
,
Rungroj Saruengkhanphasita, Chutikarn Butkinareeb c, Narittira Ornnorkd,
Kriengsak Lirdprapamongkold, Worawat Niwetmarina, Jisnuson Svastid,
,
e
,f,*
Somsak Ruchirawata ,f, Chatchakorn Eurtivonga
a Program in Chemical Sciences, Chulabhorn Graduate Institute, Chulabhorn Royal Academy, Bangkok 10210, Thailand
b Program in Applied Biological Sciences, Chulabhorn Graduate Institute, Chulabhorn Royal Academy, Bangkok 10210, Thailand
c National Omics Center, National Science and Technology Development Agency, Pathum Thani 12120, Thailand
d Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand
e Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand
f Center of Excellence on Environmental Health and Toxicology (EHT), Commission on Higher Education (CHE), Ministry of Education, Bangkok 10400, Thailand
A R T I C L E I N F O
A B S T R A C T
Keywords:
Virtual screening of commercially available molecular entities by using CDRUG, structure-based virtual
screening, and similarity identified eight new derivatives of 3-phenyl-1H-indole-2-carbohydrazide with anti-
proliferative activities. The molecules were tested experimentally for inhibition of tubulin polymerisation,
which revealed furan-3-ylmethylene-3-phenyl-1H-indole-2-carbohydrazide (27a) as the most potent candidate.
Molecule 27a was able to induce G2/M phase arrest in A549 cell line, similar to other tubulin inhibitors. Syn-
thetic modifications of 27a were focussed on small substitutions on the furan ring, halogenation at R1 position
and alteration of furyl connectivity. Derivatives 27b, 27d and 27i exhibited the strongest tubulin inhibition
activities and were comparable to 27a. Bromine substitution at R1 position showed most prominent anticancer
activities; derivatives 27b-27d displayed the strongest activities against HuCCA-1 cell line and were more potent
Virtual screening
CDRUG
3-phenyl-1H-indole-2-carbohydrazide
Anticancer agents
Tubulin polymerisation
Molecular docking
than doxorubicin and the parent molecule 27a with IC50 values <0.5 μM. Notably, 27b with a 5-methoxy
substitution on furan displayed the strongest activity against HepG2 cell line (IC50 = 0.34 µM), while 27d dis-
played stronger activity against A549 cell line (IC50 = 0.43 µM) compared to doxorubicin and 27a. Fluorine
substitutions at the R1 position tended to show more modest anti-tubulin and anticancer activities, and change of
2-furyl to 3-furyl was tolerable. The new derivatives, thiophenyl 26, displayed the strongest activity against A549
cell line (IC50 = 0.19 µM), while 1-phenylethylidene 21b and 21c exhibited more modest anticancer activities
with unclear mechanisms of action; 26 and 21c demonstrated G2/M phase arrest, but showed weak tubulin
inhibitory properties. Molecular docking suggests the series inhibit tubulin at the colchicine site, in agreement
with the experimental findings. The calculated molecular descriptors indicated that the molecules obey Lipinski’s
rule which suggests the molecules are drug-like structures.
1. Introduction
towards opposing poles along the mitotic spindle fibres before the two
daughter cells are formed [2]. Due to its key role in mitosis, tubulin has
Tubulin is a protein monomer of polymeric microtubules that
contribute to key functions in cell physiology: formation of mitotic
spindle fibres, intracellular transport, maintenance of cell shape, and
cell motility [1]. During mitosis, duplicated chromosomes separate
been designated as a key biomolecular target for an array of anticancer
agents [1,2]. These agents typically modulate microtubule dynamics
and are broadly characterised as either microtubule-destabilising or
stabilising agents, which inhibit and promote tubulin polymerisation,
* Corresponding author at: Program in Chemical Sciences, Chulabhorn Graduate Institute, Chulabhorn Royal Academy, 906 Kamphaeng Phet 6, Talat Bang Khen,
Lak Si, Bangkok 10210, Thailand.
E-mail address: chatchakorn@cgi.ac.th (C. Eurtivong).
https://doi.org/10.1016/j.bioorg.2021.104795
Received 30 December 2020; Received in revised form 22 February 2021; Accepted 2 March 2021
Available online 4 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

R. Saruengkhanphasit et al.
Bioorganic Chemistry 110 (2021) 104795
respectively. Certain classes of microtubule modulators have emerged as
clinically effective drugs, e.g., Vinca alkaloids (destabilising agents) and
taxanes (stabilising agents) [1,2]. Despite clinical approval, the adverse
effects of these agents are apparent: taxanes are associated with several
severe side effects that include neurotoxicity [3], reduced blood cell
counts [3,4], muscle and joint pain [5], and cardiotoxicity [6], whereas
the Vinca alkaloids are associated with neurotoxicity [7,8], hepatotox-
icity [9], decreased white blood cell count [10], and hypertension [11].
Thus, novel microtubule modulators with fewer side effects are highly
desirable. An additional limitation of these agents is their large molec-
ular size making them difficult to be absorbed through oral adminis-
tration. Consequently, the drugs are typically administered by
intravenous injection, thereby making it inconvenient for patients. To
address this limitation, decrease in molecular size with drug-like phys-
icochemical properties are typical traits of orally available drugs.
However, despite tremendous developments of small molecular weight
tubulin inhibitors, clinical approval has yet to be achieved to treat
cancer patients primarily due to lack of effectiveness, pharmacokinetic
barriers and selectivity. Most small molecules, which target tubulin
inhibit, inhibit the colchicine site, thereby destabilising the microtubule
structure. There are numerous drug candidates, which inhibit the
colchicine site, having diverse chemical scaffolds, e.g., colchicine de-
rivatives [12], combretastatin derivatives [13], phenstatins [14],
podophyllotoxins [15], chalcones [16,17], thienopyridines [18], sul-
fonamides [19,20], as well as an array of nitrogen-containing hetero-
cycles [21–23]. A major issue in developing small molecule tubulin
inhibitors is selectivity, as these molecules exhibit narrow therapeutic
indexes, often resulting in toxicity to healthy cells [24]. Therefore, orally
available novel selective small molecule drugs with improved thera-
peutic profiles are highly desirable.
anticancer activities. Twenty molecules were selected (Fig. 1) based on
colour codes and CDRUG scores that indicate medium to high possibility
of anticancer activities (Table S1). Additionally, included in Table S1,
are matched chemical structures from the NCI60 DTP database, and
NCI60 growth inhibition activities. The selected molecules include
moieties with di- and trimethoxy-substituted phenyl rings for 1, 2, 5, 8,
11 and 15. Additionally, 11 contains a pyridine. Other nitrogen-
containing aromatic heterocycles include tetrazoles for 3, piperidine
for 4, quinolines for 6 and 13, pyrimidines for 10, 12 and 14, and in-
doles for 16–20. Molecules 4, 7, 9 and 10 are benzodioxole, dibenza-
zepine, pyridazinone and dihydronaphthalene derivatives, respectively.
2.1.2. Identification of 3-phenyl-1H-indoles as active anticancer scaffolds
To verify their anticancer activities, molecules were tested experi-
mentally against four human cancer cell lines: lung adenocarcinoma
(A549), hepatocellular carcinoma (HepG2), cholangiocarcinoma
(HuCCA-1), and leukaemia (MOLT-3). The results shown in Table 1
indicate that the MOLT-3 cell line was the most sensitive cell line
whereas HepG2 and HuCCA-1 were weakly sensitive, and A549 was
insensitive (IC50 > 50 µM). Molecule 19 exhibited the strongest anti-
cancer activities seen in all four cell lines (IC50 < 33 µM). Molecule 12
was second best demonstrating activities in three cell lines, MOLT-3,
HepG2 and HuCCA-1 (IC50 < 50 µM). Molecule 20 was third best, dis-
playing IC50 ~ 22 µM for two cell lines: MOLT-3 and HepG2. Molecules
1, 7, 10, 11 and 18 showed minimal activities, and were only active
against MOLT-3 (IC50 < 30 µM). Interestingly, 19 matched with a 3-
phenyl-1H-indole, NSC131408365, an anticancer molecule that
exhibited strong growth inhibition with logGI50 = ꢀ 6.58 (see Table S1).
The 3-phenyl-1H-indoles have been established as tubulin inhibitors
with anti-proliferative effects [32,33]. Furthermore, five out of the
twenty molecules predicted using CDRUG contain 1H-indole with three
molecules, 16, 18 and 19, matching with NSC131408365. These find-
ings encouraged us to further explore the SAR based on the chemical
scaffold of 19 to find new inhibitors with improved potencies.
Currently, applications of in silico tools to drug discovery and
development have been pivotal in identifying and developing new
bioactive agents. This is due to cost-effectiveness, ease of use and ability
to deliver quality results, e.g., discovery of tirofiban [25]. Some of the
approaches include structure-based virtual screening and ligand-based
similarity. Structure-based virtual screening revolves screening a 3D
library of molecules against 3D structures of proteins of interest by
molecular docking [26]. Calculations of scores, binding energies, and
binding modes determine ligand–protein complex complementarity
[26]. Similarity approaches are used to find structurally similar mole-
cules from databases, often using molecular fingerprints to find matches
[27]. Its utility is well-known for rapid structure–activity relationship
(SAR) expansion of biologically active scaffolds [28,29]. The Cancer
Drug (CDRUG) web server was established to predict anticancer activ-
ities of chemical collections based on the chemical fingerprints of
molecule datasets from the NCI60 Developmental Therapeutics Program
(DTP) [30]. Applying CDRUG to a chemical collection can establish
structural similarity relationships between the chemical collection and
biologically active molecules from the DTP with anti-proliferative ac-
tivities. Applying CDRUG to a virtual screening campaign can lead to
useful prediction of hit candidates, e.g., identification of anticancer
natural products from biodiverse sources [31]. In this project, the aim is
to use a combination of CDRUG, structure- and similarity-based virtual
screening methods to identify new chemical entities predicted to inhibit
tubulin polymerisation with potent anticancer activities. The hit can-
didates can be tested using in vitro assays to verify their biological action.
The best candidates are followed by preliminary synthetic modifications
to further explore the SAR.
2.1.3. 3-phenyl-1H-indole-2-carbohydrazides as potent anticancer agents
Similarity-based approach was applied using the chemical scaffold of
19 as the search query in the Chembridge online store website. A sim-
ilarity coefficient value of 0.7 was used, since 0.7 is regarded as being
appropriate to expand the SAR of the series, i.e., search outputs result in
molecules that are not too structurally similar or different [28]. Two
hundred and forty-seven molecules resulted and were downloaded in 3D
format, and energy minimised to prepare for virtual screening by mo-
lecular docking.
The docking protocol was first validated. The co-crystallised
colchicine was removed, energy minimised, and re-docked back to the
colchicine site to observe for the reproducibility of the binding orien-
tation of colchicine (see experimental section). The root mean squared
deviations (RMSDs) including only heavy atoms were measured for the
co-crystallised colchicine and the highest scoring conformation for each
scoring function. The RMSD values were calculated: RMSD = 0.53, 0.49,
0.31 and 0.31 Å for scoring functions GoldScore (GS) [34], Chemscore
(CS) [35], ChemPLP [36] and Astex Statistical Potential (ASP) [37],
respectively. Low RMSD values confirmed comparable binding orien-
tations between the re-docked colchicine and the co-crystallised
colchicine.
The molecules were subjected to molecular docking to the colchicine
site of tubulin in a virtual screening campaign to predict the best can-
didates for tubulin inhibition (scores are shown in Table S2). The filter
criteria used were GS > 65, CS > 30, ChemPLP > 70 and ASP > 26. A
total of twenty-seven molecules resulted. Benzylidene-3-phenyl-1H-
indole-2-carbohydrazide derivatives were removed as these were re-
ported structures [32,33]. A reported tubulin inhibitor, (E)-5-chloro-N’-
(4-methoxybenzylidene)-3-phenyl-1H-indole-2-carbohydrazide (21a)
[33], that belongs to the benzylidene-3-phenyl-1H-indole-2-carbohy-
drazide class was purchased from InterBioScreen (IBS) to be used as
2. Results and discussion
2.1. Screening
2.1.1. Identification of potential anticancer candidates using CDRUG
A collection of ~100,000 molecular entities was used as the input
query and filtered using CDRUG to identify potential candidates with
2

R. Saruengkhanphasit et al.
Bioorganic Chemistry 110 (2021) 104795
Fig. 1. The chemical structures purchased from Chembridge based on screening using the CDRUG web server.
future experimental control. Three 3-phenyl-1H-indole-2-carbohy-
drazides were obtained from Chembridge: 21b and 21c with 1-phenyl-
ethylidene, and 22 has a novel N-cyclopentylidene of hydrazide
(structures in Table 2). All three molecules contain the carbohydrazide
functionality, which was presumed to be important for strong anti-
proliferative activities [32,33,38–40]. Furthermore, the 3-phenyl-1H-
indole-2-carboxamide molecular scaffold was used as the query for a
substructure in the IBS database. Five more 3-phenyl-1H-indole-2-
carbohydrazides, 23-27a were identified and purchased from IBS for
further experimental testing (structures in Table 2). Molecules 23, 24
and 25 have cyclohexylidene, hydrogen and acetyl groups substituted at
the R2 position, respectively. Molecules 26 and 27a are heterocyclic
derivatives and contain unsubstituted thiophenyl and furyl moieties,
respectively.
3

R. Saruengkhanphasit et al.
Bioorganic Chemistry 110 (2021) 104795
2.1.4. Tubulin polymerisation inhibition
Table 1
Selected molecular structures predicted from the CDRUG web server and their
anticancer effects in four cancer cell lines: HuCCA-1, A549, MOLT-3 and HepG2.
The assay was conducted as three independent experiments and the mean IC50
values are shown.
The eight 3-phenyl-1H-indole-2-carbohydrazides purchased from
Chembridge and IBS were further evaluated for tubulin polymerisation
inhibition (see Fig. 2A). The molecules showed tubulin inhibition ac-
tivities but were not as potent compared to the known inhibitor, 21a.
Among the eight molecules, furan-3-ylmethylene-3-phenyl-1H-indole-2-
carbohydrazide (27a) was clearly the best inhibitor with RFU ~ ꢀ 200
arbitrary units. Molecules 21c and 21b were second and third best
candidates with RFU ~ 400 and 500 arbitrary units, respectively. Mol-
ecules 22, 23, and 26 lacked anti-tubulin activities with RFU ~ 600.
From the data, 24 and 25 slightly promoted tubulin assembly (RFU ~
600 to 700). However, this finding seemed doubtful as indole-based
molecules have been suggested to inhibit the colchicine site that ac-
commodates small molecules in favour of stabilising site, e.g., taxane.
The taxane site is by far more spacious making it suitable to accom-
modate larger molecules suggesting the colchicine site as a more plau-
sible inhibition site.
Molecule
HuCCA-1 (µM)
A549 (µM)
MOLT-3 (µM)
HepG2 (µM)
1
>50
>50
16.6 ± 1.4
>50
>50
2
>50
>50
>50
3
>50
>50
>50
>50
4
>50
>50
>50
>50
5
>50
>50
>50
>50
6
>50
>50
>50
>50
7
>50
>50
25.5 ± 0.9
>50
>50
8
>50
>50
>50
9
>50
>50
>50
>50
10
>50
>50
20.2 ± 1.1
13.6 ± 0.3
24.3 ± 0.5
>50
>50
11
>50
>50
>50
12
43.6
>50
16.5 ± 1.1
>50
13
>50
>50
14
>50
>50
>50
>50
The results from this assay prompted us to synthesise derivatives to
explore the SAR based on the chemical scaffold of 27a. Two positions on
the scaffold were explored: halogen substitutions at R1, and halogens
and methoxy substitution at R2 positions. Incorporation of fluorine
groups have been suggested to enhance pharmacokinetic properties
such as membrane penetration and metabolic stability. This encouraged
us to explore fluorine groups at R1 position. Furthermore, derivatives
with halogen and methoxy substitutions at R2 position (27b–27h) and
derivatives with 2- (27i) and 3-furanyl (27j) were synthesised. The
15
>50
>50
>50
>50
16
>50
>50
>50
>50
17
>50
>50
>50
>50
18
>50
>50
29.3 ± 0.3
12.7 ± 0.5
23.1 ± 0.7
0.009 ± 0.002
>50
19
32.4 ± 8.9
>50
26.5 ± 1.4
>50
15.4 ± 0.6
20.8 ± 0.8
0.57 ± 0.04
20
Doxorubicina
0.92 ± 0.03
0.44 ± 0.10
a
Positive control.
Table 2
Anti-proliferative activities of 3-phenyl-1H-indole-carbohydrazides. The assay was conducted as three independent experiments and the mean IC50 values are
shown.
Molecule
R1
R2
R3
X
HuCCA-1 (µM)
A549 (µM)
MOLT-3 (µM)
HepG2 (µM)
21aa
21b
21c
22
Cl
Br
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
F
OCH3
H
CH3
CH3
–
–
>50
>50
0.10 ± 0.02
0.87 ± 0.13
0.60 ± 0.02
10.1 ± 2.4
39.0 ± 5.0
9.3 ± 0.9
0.82 ± 0.20
3.6 ± 0.4
2.3 ± 0.3
>50
CH2CH3
–
>50
23.6 ± 2.8
6.7 ± 0.4
>50
CH2CH3
–
8.3 ± 1.2
>50
c-C5H8
–
23
c-C6H10
–
–
>50
>50
>50
24
H
–
–
>50
>50
31.5 ± 0.1
>50
25
COCH3
–
–
>50
>50
35.2 ± 6.4
0.07 ± 0.02
0.47 ± 0.05
0.09 ± 0.01
0.23 ± 0.05
0.32 ± 0.02
0.25 ± 0.06
0.03 ± 0.01
0.15 ± 0.01
0.17 ± 0.01
0.33 ± 0.05
0.51 ± 0.13
0.024 ± 0.006
26
–
–
–
S
3.6 ± 0.1
33.6 ± 1.2
0.06 ± 0.01
0.46 ± 0.04
0.38 ± 0.03
13.6 ± 2.6
15.5 ± 1.1
1.6 ± 0.1
1.3 ± 0.2
13.0 ± 0.4
1.6 ± 0.1
1.2 ± 0.4
0.19 ± 0.01
23.8 ± 3.9
47.4 ± 0.9
14.9 ± 2.2
0.43 ± 0.09
16.2 ± 1.4
20.6 ± 1.9
18.6 ± 0.1
13.8 ± 0.6
25.7 ± 0.5
21.3 ± 0.8
0.72 ± 0.03
0.73 ± 0.09
1.1 ± 0.3
0.34 ± 0.01
1.6 ± 0.1
1.7 ± 0.1
13.8 ± 2.3
2.0 ± 0.2
1.8 ± 0.1
3.5 ± 1.7
9.3 ± 1.3
10.6 ± 3.5
0.49 ± 0.02
27a
27b
27c
27d
27e
27f
–
O
O
O
O
O
O
O
O
–
OCH3
–
Cl
Br
H
–
–
–
F
OCH3
Cl
Br
–
–
27g
27h
27i
F
–
F
–
Br
F
–
27j
–
–
–
–
–
Doxorubicinb
–
–
a
b
Reported tubulin inhibitor from Ref. [33].
Positive control.
4

R. Saruengkhanphasit et al.
Bioorganic Chemistry 110 (2021) 104795
Fig. 2. Effect of 3-phenyl-1H-indole-2-carbohy-
drazide derivatives at 10 µM on tubulin poly-
merisation at 37 ◦C with molecule 21a as the
known inhibitor and 10 µM of paclitaxel as the
known microtubule-stabilising agent. (A) Effect
of tubulin polymerisation by molecules 21b, 21c
and 22-27a. The curve is the average from two
experimental runs (B) Tubulin polymerisation
inhibition activities of furan-3-ylmethylene-3-
phenyl-1H-indole-2-carbohydrazide derivatives
27b-27j. The curves are the averages of three
experimental runs.
A
B
control molecule, 21a remained the most potent with RFU ~ ꢀ 300
arbitrary units. Molecules with bromine at R1 exhibited strong tubulin
inhibition (27a, 27b and 27i) with RFU ~ ꢀ 200 arbitrary units, whilst
dual halogen substitutions at R1 and R2 demonstrated partial tubulin
inhibition with RFU ~ 50 to 200 arbitrary units (27c and 27d). Unfor-
tunately, most derivatives with fluorine substitution at R1 were less
potent (27e, 27g, 27h, and 27j) than other brominated derivatives.
Nevertheless, an exception was seen for 27f that demonstrated compa-
rable tubulin inhibition to 27a, 27b and 27i. This was evident that
methoxy at R2 position can enhance tubulin inhibition independent of
R1 substitutions. Altering from 2-furyl to 3-furyl position was tolerable
as seen for molecules 27a vs. 27i, and 27e vs. 27j.
line was the most sensitive followed by HepG2, HuCCA-1 and A459,
respectively. Generally, furyl derivatives with bromine at R1 with halo-
and methoxy substitutions at R2 (27b-27d) seemed to demonstrate
strongest anti-proliferative effects, i.e., IC50 < 0.5 μM in HuCCA-1 and
MOLT-3 cell lines, and IC50 < 2 µM for HepG2. Notably, 27b-27d dis-
played stronger anticancer effects against the HuCCA-1 cell line than
parent molecule, 27a, and the clinically approved doxorubicin (IC50
<
0.5 µM). In particular, 27d with dual bromine substitutions at R1 and R2
also showed improved anti-proliferative activity against the A549 cell
line with IC50 = 0.43 µM compared to doxorubicin (IC50 = 0.72 µM). In
general, the derivatives with fluorine substitutions at R1 were weaker
than their bromo-substituted counterparts, which was apparent in all
four cell lines. The results correlate relatively well with tubulin poly-
merisation inhibition, suggesting tubulin inhibition as the key mecha-
nism of action, i.e., bromo-substituted derivatives tended to display
stronger anticancer and anti-tubulin effects than fluoro-substituted de-
rivatives. The 3-furyl derivative seen for 27i did not significantly in-
crease activity when compared to 27a for A549 and MOLT-3, whilst
2.1.5. Biological evaluation of 3-phenyl-1H-indole-2-carbohydrazides
The 3-phenyl-1H-indole-2-carbohydrazides were further subjected to
biological evaluation against four cancer cell lines: HuCCA-1, A459,
MOLT-3 and HepG2 (Table 2). Analysis of the data in Table 2 suggested
the derivatives displayed anti-proliferative properties. The MOLT-3 cell
5

R. Saruengkhanphasit et al.
Bioorganic Chemistry 110 (2021) 104795
reducing activities by ~ 9 folds against HepG2, and ~ 3 folds against
HuCCA-1. Fluoro-substituted derivative 27j displayed comparable
anticancer activities to its bromo-substituted counterpart, 27i, for A549,
MOLT-3 and HepG2 but was ~ 8 fold stronger against the HuCCA-1 cell
line.
indole-2-carbohydrazide series demonstrated stronger anti-proliferative
activities than the predecessed benzylidene-3-phenyl-1H-indole-2-
carbohydrazides.
2.2. Cell cycle analysis
Cycloalkyl derivatives 22 and 23, non-substituted 24, and 25 acetyl
substitutions displayed detrimental anti-proliferative activities, i.e., IC50
> 50 µM in two to three cell lines: HuCCA-1, A549 and HepG2. Molecule
26 with a thiophenyl moiety displayed the strongest anti-proliferative
effects against A549 with IC50 = 0.19 µM which was ~3 fold more
potent than doxorubicin, and was second best against HepG2 with IC50
= 0.73 µM. Regardless, 26 showed poor tubulin inhibitory effect
implying a different mode of action that needs further investigation.
Molecules 21b and 21c with 1-phenylethylidene moieties, showed
respectable anti-proliferative effects particularly against MOLT-3 (IC50
< 1 µM) and HepG2 (IC50 < 4 µM). However, the tubulin polymerisation
assay results cannot fully verify the key mechanisms of action of 21b and
21c.
Abolishment of microtubule function and arrest in the at G2/M phase
of cell cycle by tubulin inhibitors are well-documented [41]. Therefore,
the three molecules, 21c, 26, and 27a, with differential effects on
tubulin polymerisation were selected for further study on cell cycle
progression. After treatment with the three molecules at 10 µM con-
centration for 16 hrs on A549 lung cancer cells, spherical cell shapes
were observed (Fig. 3A) concomitant with increasing proportion of cells
in G2/M phase (Fig. 3B and C), indicating all the three molecules were
able to induce cell cycle arrest at the G2/M phase. Treatment with 27a, a
pre-established potent inhibitor of tubulin polymerisation, and 21c, a
mild inhibitor, elevated the proportion of cells in the G2/M phase by
81.1 ± 2.4% and 79.1 ± 8.6%, respectively, compared with 33.4 ± 2.0%
in vehicle-treated cells (Fig. 3C). Interestingly, 26 which possessed
strong anti-proliferative effects but did not inhibit tubulin polymerisa-
tion also increased the number of cells in the G2/M phase by 57.8 ±
4.3% (Fig. 3C). The results suggested that molecules 21c and 26 induced
G2/M cell cycle arrest by mechanisms other than the tubulin inhibition.
Several reports have shown that the arrest of G2/M phase in A549 cells
could be achieved by inhibition of targeted proteins other than tubulin,
e.g., topoisomerase II [42], aurora kinase-B [43], and DNA methyl-
transferase [44]. The mechanisms underlying the G2/M phase arrest by
21c and 26 are to be investigated further.
We further compared the anticancer activities of unsubstituted furyl-
(27a) and thiophenyl- (26) 3-phenyl-1H-indole-2-carbohydrazide series
to the predecessor benzylidene-3-phenyl-1H-indole-2-carbohydrazides;
the known tubulin inhibitor 21a showed adequate anti-proliferative
activities against MOLT-3 (IC50 = 0.87 µM) and HepG2 (IC50 = 3.6
µM) and detrimental activities against HuCCA-1 and A549 (IC50 > 50
µM), and ten additional benzylidene-3-phenyl-1H-indole-2-carbohy-
drazide derivatives (21d–21m) which were purchased from IBS and
tested experimentally against the same four cancer cell lines (Table S3).
From the data, it was clear that the furyl- and thiophenyl-3-phenyl-1H-
Fig. 3. Effect of molecule 27a, 21c, and 26 on cell cycle phase distribution of A549 lung cancer cells. The cells were treated with vehicle or 10 µM of each compound
for 16 hrs. The percentages of cells in G0/G1, S, and G2/M phases were analyzed by using Muse Cell Analyzer with Muse Cell Cycle Assay kit. (A) Representative
images of cell morphology after treatment (×200 magnification). (B) Histograms of DNA content in treated cells from a representative experiment. (C) The per-
centages of cells in G0/G1, S, and G2/M phases after treatment. Data are expressed as average with SD from three independent experiments. a, significant difference
from control (p < 0.05); b, significant difference from molecule 27a and 21c (p < 0.05).
6

R. Saruengkhanphasit et al.
Bioorganic Chemistry 110 (2021) 104795
2.3. Synthesis of furan-3-ylmethylene-3-phenyl-1H-indole-2-
carbohydrazides
activities [33] suggesting decreased binding affinity probably as a result
of decreased hydrophobic contacts. Hydrophobic interactions were
formed between the inhibitors and hydrophobic residues, e.g., Val181,
Ala180 and Leu255. Additionally, observations of hydrophobic contacts
to Lys254, Asn258, Asn349, Lys352 and Thr179 were seen with non-
polar parts of the residue structures. The hydrazide arm linked to
The synthesis of the derivatives 27a-27j is described in Scheme 1.
Ethyl acetoacetate 28 was used as the starting material to prepare
ketobutanoate 29 by alkylation with benzylbromide. By applying the
Japp-Klingemann reaction [45–48], ketobutanoate 29 was treated with
p-bromo- and chloro-benzene diazonium salts 30a and 30b to give in-
termediate 31. Without further purification, 31 was subjected to Fischer
indole synthesis [49,50] to afford indole 32 with high yields for both
bromo- and chloro-derivatives. The carbohydrazine functionality of in-
termediate 33 was formed by the treatment of indole 32 with hydrazine.
Condensations of hydrazine with various substituted furfurals obtained
derivatives 27a-27j as the final products; the yields are shown in
Table S4. It should be noted that hydrazine condensation with aldehydes
was expected to give E- and Z-isomeric mixtures, but only the more
chemically stable E-isomer products were isolated [51–53].
furan orientate towards α-tubulin. Hydrogen bonding interactions were
predicted between indole and Thr179, and occasionally between furan
and residue side chains of Ser178 and Lys243. The methylene and furan
ring were predicted to form hydrophobic contacts with Leu248, Tyr224,
Gln11, Gln247, Asn249, and Lys254. The amino acid residues
mentioned have been reported to contribute to tubulin inhibition by the
theinopyridine [18], indolizine [54], and cyclohexanedione [55]
derivatives.
2.4.2. Chemical space
On grounds that the 3-phenyl-1H-indole-2-carbohydrazides were
investigated to be developed as anticancer agents, their physicochemical
properties were evaluated against benchmarks such as the Lipinski’s
filters [56] and Veber’s rule [57] established to assess the drug-like
properties. The molecular descriptors are molecular weight, hydrogen
bond donor and acceptor, rotatable bond, topological polar surface area,
and lastly, Moriguchi log P (MlogP) [58] that calculates lipophilicity.
The calculated molecular descriptors are shown in Table S5 revealing
the derivatives to be within the drug-like chemical space, i.e., the nu-
merical figures did not exceed the Lipinski’s or Veber’s limit. This
determined the derivatives as pharmacokinetically favourable scaffolds
for drug development.
2.4. Molecular modelling
2.4.1. Molecular docking analysis
To gain further insight into the mode of binding of the furan-3-
ylmethylene-3-phenyl-1H-indole-2-carbohydrazides, the molecules
were docked to the colchicine binding site of tubulin located at the
interface between α- and β- subunits. The scores are shown in Table S2.
The predicted binding orientations of the series were more or less
similar, and can be represented by molecules 27a and 27b shown in
Fig. 4. Hydrophobic indole and 3-phenyl groups occupied the hydro-
phobic pockets of the β-subunit. This was seen to be favourable as indole
is well-known for its role in tubulin inhibition [21], and 3-methyl sub-
stitution on the indole scaffold have been reported to lower anticancer
Scheme 1. Synthesis of furan-3-ylmethylene-3-phenyl-1H-indole-2-carbohydrazide derivatives reagents and conditions: (i) NaH, THF, 0_5 ◦C, 10 min, rt, BnBr then
reflux 17 hrs, 64%; (ii) KOH, EtOH, H2O, 30a or 30b, 0 ◦C, 10 min then refrigerated 17 hrs; (iii) HCl, 80 ◦C, 4 hrs, 32a 88% 32b 79%; (iv) N2H4, EtOH, 80 ◦C, 17 hrs;
(v) ArCHO, EtOH, 80 ◦C, 3 hrs.
7

R. Saruengkhanphasit et al.
Bioorganic Chemistry 110 (2021) 104795
Fig. 4. The binding modes and interactions of
furan-3-ylmethylene-3-phenyl-1H-indole-2-carbo-
hydrazides to the colchicine site of tubulin using
molecular docking based on the top scoring
conformation from GoldScore. The hydrogen bonds
are represented as green dotted lines. Binding mode
depictions of molecules 27a (A) and 27b (C) to the
colchicine site. Green ribbons represent β-tubulin
whereas pink ribbons represent α-tubulin. Hydrogen
bonding residues Thr179, Lys254 and Ser178 are
shown in orange, blue and red sticks, respectively.
Two-dimensional illustrations of intermolecular in-
teractions between tubulin, and molecules 27a (B)
and 27b (D). The hydrophobic interactions are
represented as red lashes. Hydrogen bond distances
in angstrom units are shown.
3. Conclusion
4. Experimental section
In this study, a combination of in silico screening, in vitro testing and
synthesis have led to the identification of new biologically active de-
rivatives of 3-phenyl-1H-indole-2-carbohydrazides with anti-
proliferative properties: furan-3-ylmethylene-3-phenyl-1H-indole-2-
carbohydrazides (27a-27j) that inhibit tubulin polymerisation, a highly
potent thiophenyl derivative 26, and phenylethylidene derivatives 21b
and 21c. Tubulin polymerisation assay results revealed derivatives with
bromine were more favourable than fluorine substitutions at C5.
Furthermore, methoxy substitutions on the furan ring showed promi-
nent anti-tubulin activities, whereas change from 3- to 2-furyl was
tolerable. The study was unable to clarify the key mechanisms of action
of thiophenyl 26 and phenylethylidene 21c although they clearly
demonstrated potent anti-proliferative properties and G2/M phase ar-
rest in A549 cell line. Thus, further investigation into their mechanisms
of action is needed. The phenylethylidene, furyl and thiophenyl de-
rivatives showed improved and competent anticancer effects in com-
parison to the predecessed benzylidene-3-phenyl-1H-indole-2-
carbohydrazides. Additionally, thiophenyl 26, and furyl 27b-27d de-
rivatives were more potent than the clinically approved doxorubicin
drug in some cell lines. Molecular docking predicted inhibition of the
colchicine site by furyl derivatives 27a-27j, and formed hydrogen
bonding and hydrophobic interactions with important amino acid resi-
dues that contribute to perturbation of microtubule dynamics.
Furthermore, the calculated physicochemical properties showed that the
derivatives are pharmacokinetically favourable scaffolds for drug
development.
4.1. In silico screening
4.1.1. Ligand preparation
A commercially available library of ~100,000 small molecules was
downloaded from ChemBridge Corporation website in 3D format. The
company is a leading global provider of small molecule drug discovery
with a portfolio that includes >1.3 million diverse compounds. The
quality and reliability of their products adhere to collaborations with
leading global pharmaceutical companies. The Scigress software version
2.8.1 was used to remove any counter ions and salts. Hydrogen atoms
were added to the molecules followed by energy minimisation using the
MM2 [59] force field.
4.1.2. Screening for candidates with anticancer properties using CDRUG
The CDRUG webserver can be used as an online tool to predict for the
anticancer activity of molecular structures. An active dataset of 8565
anticancer compounds and an inactive dataset consisting of 9804 com-
pounds from the NCI60 Developmental Therapeutics Program (DTP)
project were used for benchmarking purposes. Molecular fingerprints
and Tanimoto similarity methods were incorporated to identify mole-
cules with potential anticancer properties, in which a final score is
calculated as a quantitative measurement.
4.1.3. Structure-based virtual screening
Ligands were docked to the crystal structure of tubulin (PDB ID:
4O2B, resolution 2.3 Å) which was obtained from the Protein Data Bank
(PDB) [60]. The co-crystallised colchicine ligand and spectator ligands
8

R. Saruengkhanphasit et al.
Bioorganic Chemistry 110 (2021) 104795
including crystallographic waters were removed. The centre of the
binding pocket was defined at coordinates (x = 13.222, y = 8.371, z =
–23.331) with 10 Å radius. Fifty docking runs were allowed for each
ligand with default search efficiency (100%). The basic amino acids,
lysine and arginine, were defined as protonated and acidic residues,
aspartic and glutamic acids were deprotonated. The Goldscore (GS),
ChemScore (CS), ChemPLP and Astex Statistical Potential (ASP) scoring
functions were implemented to validate the predicted binding modes
and relative energies of the ligands using the GOLD version 5.8.1 soft-
ware suite. Three-dimensional illustrative depictions of binding modes
were constructed using Discovery Studio, and two-dimensional diagram
plots depicting intermolecular interactions were constructed using Lig-
Plot [61].
containing the same volume of complete culture medium) was sub-
tracted from either A550 or A492 to get the absolute absorbance. The
average value from the duplicate wells, which had been treated with
each concentration of the test molecules, was then compared with that
of the untreated wells to yield the percentage of surviving cells. The IC50
value was finally calculated from the dose–response curve as the con-
centration that inhibits the cell growth by 50% in comparison with the
negative control following 48 hrs of exposure to each test molecule.
This assay protocol can be applied for use to test the anti-
proliferative activities of anti-tubulin agents as shown in Table S6,
nocodazole was tested using the same protocol.
4.2.2. Tubulin polymerization assay
Inhibitory effect of test molecules on tubulin polymerization was
analyzed using a commercial fluorescence-based tubulin polymerization
assay kit (Cytoskeleton, catalog no. BK011P) according to the manu-
facturer’s protocol. Briefly, 5 µL of test molecules at indicated concen-
tration was mixed with 50 µL of tubulin reaction mixture containing free
porcine brain tubulin (2 mg/ml) in buffer supplemented with 20%
glycerol and 1 mM GTP on ice. The fluorescence intensity was measured
every 30 s for 90 min at 37 ◦C using a fluorescence microplate reader
(BioTek Instruments). The excitation and emission wavelengths were
360 and 450 nm, respectively.
4.2. In vitro screening
4.2.1. Anti-proliferative assay
The cell lines used in this study were either purchased from the
American Type Culture Collection (ATCC, Manassas, VA, USA) or
received as gifts from other sources. Dulbecco’s Modified Eagle Medium
(DMEM), as well as Ham’s F12 and RPMI 1640 media, were supplied in
powder form by HyClone Laboratories (Logan, UT, USA), while fetal
bovine serum (FBS) and 0.25% trypsin-EDTA were obtained from J R
Scientific, Inc. (Woodland, CA, USA) and Gibco (Grand Island, NY, USA),
respectively. In addition, bovine insulin, DMSO, doxorubicin, etoposide,
glucose, l-glutamine, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazoliumbromide), penicillin–streptomycin, phenazine metho-
sulfate (PMS), and sodium pyruvate were supplied by Sigma–Aldrich (St.
Louis, MO, USA), whereas XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfo-
phenyl)-2H-tetrazolium-5-carboxanilide) was from Fluka Chemie (St.
Louis, MO, USA).
4.3. Cell cycle
Effect of test molecules on cell cycle phase distribution was investi-
gated by flow cytometric technique using Muse Cell Analyzer with the
Muse Cell Cycle Assay kit (Luminex, USA). Briefly, A549 cells (1.5 × 105
cells/well) in 6-well plate were treated with test molecules at 10 µM or
vehicle (0.2% DMSO) for 16 hrs. After treatment, the cells were har-
vested by trypsinization and prepared for cell cycle analysis according to
the kit’s protocol. Results were expressed as percentages of cell pop-
ulations in G0/G1, S, and G2/M phases.
HuCCA-1, A549 and HepG2 cell lines were adherent to the culture
wells, whereas only MOLT-3 was grown in suspension. Each cell line was
maintained in an appropriate culture medium supplemented with
essential nutrients and maintained using standard procedures at 37 ◦C
with 95% humidity and 5% CO2. All the test molecules and doxorubicin
were prepared as 10 mg/mL stock solutions in DMSO and freshly diluted
with the corresponding cell culture medium for each cell line on the day
of analysis.
4.4. Molecular descriptor calculations
The Dragon software package was used to calculate the molecular
descriptors of the compounds.
Prior to the assay, the cells were inoculated as a suspension in the
corresponding cell culture medium (100
μL for adherent cells and 75
μL
4.5. Synthesis
for suspended cells) into 96-well microtiter plates (Costar No. 3599,
Corning Incorporated, Corning, NY, USA) at a density of 5000–20,000
cells per well, depending on their growth rates. Adherent and suspended
cells were then allowed to grow at 37 ◦C with 95% humidity and 5% CO2
for 24 hrs and 30 min, respectively. The cytotoxicity assay was initiated
by adding an equal volume of cell culture medium containing either
each test compound, positive control, or DMSO, at predetermined con-
centrations. Following 48 hrs of exposure to various treatments, cell
viability was determined using MTT assay for adherent cells or XTT
assay for suspended cells, as described below.
All chemical reagents and solvents were brought from commercial
suppliers and used directly unless specific stated. Thin layer chroma-
tography was performed on Merck silica gel 60 F254 plates and visu-
alized by UV irradiation at 254 nm. Flash column chromatography was
performed using Silicycle SilicaFlash® F60 (40–63
μ
m). 1H NMR spectra
were recorded on a Bruker AVANCE-300 MHz. Chemical Shifts (δ) are
reported in parts-per-million (ppm) with respect to the solvent peaks and
coupling constants (J) are in Hertz. NMR peak multiplicities are given
the abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, br = broad. 13C NMR spectra were recorded on a Bruker
AVANCE-75 MHz. Chemical Shifts (δ) are reported in parts-per-million
(ppm) with respect to the solvent peaks and coupling constants (J) are in
Hertz. 19F NMR spectra were recorded on a Bruker AVANCE-282 MHz.
Chemical Shifts (δ) are reported in parts-per-million (ppm) with respect
to the solvent peaks. Infrared (IR) spectra were recorded on Thermo
Scientific Nicolet iS 5 Fourier Transform IR spectrometer. Only selected
peaks are reported and absorption maxima are given in cmꢀ 1. High
Resolution mass spectra (HR-MS) were obtained on a Thermo Scientific
orbitrap Q Exactive Focus mass spectrometer. Melting points were
recorded using a Stuart SMP30 capillary melting point apparatus.
For adherent cells, 100 μL of the MTT reagent (0.5 mg/mL in serum-
free cell culture medium) was added to each well, and the microtiter
plates were further incubated for 2.5–4 hrs at 37 ◦C with 95% humidity
and 5% CO2. The medium was subsequently replaced with 100 μL of
DMSO to dissolve the purple formazan before the absorbance at 550 nm
was measured using a Spectra-Max Plus 384 microplate reader (Molec-
ular Devices, Sunnyvale, CA, USA) with a reference wavelength of 650
nm.
For suspended cells, 75 μL of the XTT reagent (prepared from 5 mL of
1 mg/mL XTT sodium in water and 100 mL of 0.383 mg/mL PMS in
water) was added to each well, and the cells were further incubated for 4
hrs at 37 ◦C with 95% humidity and 5% CO2. Afterwards, the absorbance
of orange formazan at 492 nm was measured with a reference wave-
length of 690 nm using a SpectraMax Plus 384 microplate reader. For
each well, the background absorbance (averaged from the wells
4.5.1. Ethyl 2-benzyl-3-oxobutanoate (29)
Under Argon atmosphere, anhydrous THF (20 mL) was added to NaH
(60% in oil, 0.69 g, 17.43 mmol), and the mixture was cooled to 0 ◦C.
9

R. Saruengkhanphasit et al.
Bioorganic Chemistry 110 (2021) 104795
With a magnetic stirring, ethyl acetoacetate 28 (2.00 mL, 15.89 mmol)
was added dropwise. The mixture was stirred until the gas evolution
finished and then warmed to room temperature. Benzyl bromide (2.80
mL, 23.54 mmol) was added dropwise, and the mixture was heated to
72 ◦C. After 16 hrs, the mixture was allowed to cool to room tempera-
ture. The mixture was diluted by saturated aqueous ammonium chloride
(10 mL) and was extracted with Et2O (3 × 20 mL). The combined
organic layers were dried (Na2SO4), filtered, and solvent was evapo-
rated. Purification by flash column chromatography, eluted with EtOAc-
Hexane (0.5:9.5 to 1:9), gave ethyl 2-benzyl-3-oxobutanoate 29 (2.23 g,
10.12 mmol, 64%) as an oil; Rf 0.3 [EtOAc-Hexane (1:9)]; 1H NMR (300
MHz, CDCl3) δ 7.31–7.18 (m, 5H, Ph), 4.17 (q, J = 7.2 Hz, 2H,
OCH2CH3), 3.80 (t, J = 7.6 Hz, 1H, CH), 3.19 (d, J = 7.6 Hz, 2H, CH2),
2.20 (s, 3H, CH3), 1.21 (t, J = 7.2 Hz, 3H, OCH2CH3); 13C NMR (75 MHz,
flash column chromatography, elute with EtOAc-Hexane (0.5:9.5 to
2:8), gave indole 32b (0.51 g, 1.89 mmol, 81%) as a yellow solid; Rf 0.4
[EtOAc-Hexane (1:9)]; m.p. 144–147 ◦C; νmax/cmꢀ 1 3308, 3063, 2991,
2943, 1677, 1248; 1H NMR (300 MHz, DMSO‑d6) δ 12.02 (s, 1H, NH),
7.52–7.30 (m, 6H, ArH), 7.19–7.11 (m, 2H, ArH), 4.20 (q, J = 7.1 Hz,
2H, CH2), 1.14 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (75 MHz, DMSO‑d6) δ
′
–
161.07 (C O), 159.20 (d, J = 234.5 Hz, C5-ind), 133.17 (C1 -Ph),
–
132.90 (C7a-ind), 130.31 (C2′-Ph), 127.86 (C3′-Ph), 127.00 (C4′-Ph),
126.88 (d, J = 9.8 Hz, C3a-ind), 124.42 (C2-ind), 122.34 (d, J = 5.5 Hz,
C3-ind), 114.28 (d, J = 10.1 Hz, C7-ind), 113.93 (d, J = 26.3 Hz, C6-
ind), 104.69 (d, J = 23.6 Hz, C4-ind), 60.43 (CH2), 13.92 (CH3); 19F
NMR (282 MHz, DMSO‑d6) δ ꢀ 122.57; HRMS (ESI) m/z [M+H]+ calcd
for C17H15O2NF 284.1081; found 284.1084.
–
CDCl ) δ 202.50 (C O), 169.14 (CO CH CH ), 138.17 (C-Ph), 128.81
4.5.4. (E)-5-bromo-N’-(furan-2-ylmethylene)-3-phenyl-1H-indole-2-
carbohydrazide (27a)
–
(2 × 3CH-Ph), 128.59 (2 × CH-Ph), 126.69 (C3H-Ph), 61.48 (OCH2CH3),
61.31 (CH), 33.99 (CH2), 29.63 (CH3), 14.03 (OCH2CH3). The data was
consistent with that in the literature [62].
2
2
To a solution of indole 31a (0.106 g, 0.31 mmol) in EtOH (15 mL)
was added hydrazine (50% in H2O, 0.58 mL, 9.3 mmol) and the reaction
mixture was heated to 80 ◦C. After 16 hrs, the mixture was allowed to
cool to room temperature and solvent was evaporated to give a crude
intermediate hydrazide 33a as a solid (used without purification). To a
solution of the hydrazide crude 33a in EtOH (20 mL) was added furfural
(0.03 mL, 0.31 mmol) at room temperature, then the mixture was heated
to 80 ◦C. After 4 hrs, the mixture was allowed to cool to room temper-
ature, and solvent was evaporated. Purification by flash column chro-
matography, eluted with EtOAc-Hexane (1:9 to 2:3), gave hydrazide 27a
(0.099 g, 0.24 mmol, 77%) as a yellow solid; Rf 0.5 [EtOAc-Hexane
(4:6)]; m.p. 231–233 ◦C; νmax/cmꢀ 1 3297, 3173, 3053, 2961, 2926,
1636, 1621, 1264; 1H NMR (300 MHz, DMSO‑d6) δ 12.17 (s, 1H, CONH),
11.37 (s, 1H, NH), 7.94 (br s, 1H, H4“-furan), 7.82 (s, 1H, HC = N), 7.72
(d, J = 1.8 Hz, 1H, H4-ind), 7.48–7.37 (m, 7H, H6-ind, H7-ind, PhH),
6.90 (br s, 1H, H2”-furan), 6.60 (br s, 1H, H3“-furan); 13C NMR (75 MHz,
4.5.2. Ethyl 5-bromo-3-phenyl-1H-indole-2-carboxylate (32a)
To a solution of 4-bromoaniline (0.39 g, 2.27 mmol) in EtOH/H2O
(1:1, 2 mL), were added 0.6 mL conc. HCl and a solution of NaNO2
(0.240 g, 3.48 mmol) in H2O (10 mL) dropwise at 0 ◦C. The resulting
solution of diazonium salt 30a was poured into a mixture of ethyl 2-
benzyl-3-oxobutanoate (29) (0.50 g, 2.27 mmol) and KOH (0.61 g,
◦
10.90 mmol) in EtOH/H2O (1:1, 2 mL) at 0 C. The resulting mixture
was refrigerated at approximately 4 ◦C for 17 hrs. The resulting mixture
was allowed to warm to room temperature, diluted with H2O (10 mL)
and extracted with EtOAc (3 × 20 mL). The combined organic layers
were dried (Na2SO4), filtered, and solvent was evaporated to give a
crude of hydrazone intermediate 31a as an oil (used without purifica-
tion). A solution of the hydrazone crude 31a in conc. HCl (2.3 mL) was
heated to 80 ◦C. After 4 hrs, the mixture was allowed to cool to room
temperature. The mixture was diluted by EtOAc (10 mL) and H2O (10
mL), then extracted with EtOAc (3 × 15 mL). The combined organic
layers were dried (Na2SO4), filtered, and solvent was evaporated. Puri-
fication by flash column chromatography, elute with EtOAc-Hexane
(0.5:9.5 to 2:8), gave indole 32a (0.69 g, 2.00 mmol, 88%) as a yel-
low solid; Rf 0.4 [EtOAc-Hexane (1:9)]; m.p. 181–183 ◦C, lit.[63] m.p.
for 32a 174–175 ◦C; νmax/cmꢀ 1 3304, 3057, 2987, 2940, 1677, 1256; 1H
NMR (300 MHz, DMSO‑d6) δ 12.14 (s, 1H, NH), 7.56 (br d, J = 1.8 Hz,
1H, H4-ind), 7.49–7.34 (m, 7H, H6-ind, H7-ind, PhH), 4.22 (q, J = 7.1
–
DMSO‑d ) δ 158.10 (C O), 149.10 (C1”-furan), 145.38 (C4“-furan),
–
6
′
′
–
–
137.17 (C N), 134.36 (C7a-ind), 132.93 (C1 -Ph), 129.57 (C2 -Ph),
128.67 (C2-ind), 128.55 (C3′-Ph), 127.81 (C3a-ind), 126.90 (C4′-Ph),
126.57 (C6-ind), 121.93 (C4-ind), 117.40 (C3-ind), 114.51 (C7-ind),
114.01 (C2”-furan), 112.99 (C5-ind), 112.24 (C3“-furan); HRMS (ESI)
m/z [M+H]+ calcd for C20H15O2N739Br 408.0342; found 408.0344.
Compounds 27b-27j were synthesized following the procedure
described here.
4.5.5. (E)-5-bromo-N’-((5-methoxyfuran-2-yl)methylene)-3-phenyl-1H-
indole-2-carbohydrazide (27b)
Hz, 2H, CH2), 1.15 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (75 MHz,
′
–
DMSO‑d ) δ 161.45 (C O), 135.20 (C7a-ind), 133.34 (C1 -Ph), 130.83
Yellow solid (0.078 g, 0.18 mmol, 58%); Rf 0.5 [EtOAc-Hexane
(2:3)]; m.p. 215–218 ◦C decomposed; νmax/cmꢀ 1 3286, 3175, 3030,
2934, 2851, 1741, 1622, 1602, 1261; 1H NMR (300 MHz, DMSO‑d6) δ
12.14 (s, 1H, CONH), 11.18 (s, 1H, NH), 7.73–7.71 (m, 2H, HC = N and
H4-ind), 7.51–7.32 (m, 7H, H6-ind, H7-ind, PhH), 6.84 (d, J = 3.0 Hz,
–
6
(C2′-Ph), 128.90 (C2-ind), 128.37 (C3′-Ph), 128.18 (C3a-ind), 127.60
(C4′-Ph), 124.48 (C6-ind), 122.97 (C4-ind), 122.18 (C3-ind), 115.34
(C7-ind), 113.48 (C5-ind), 60.98 (CH2), 14.38 (CH3); HRMS (ESI) m/z
[M+H]+ calcd for C17H15O2N79Br 344.0281; found 344.0280.
1H, H2“-furan), 5.56 (d, J = 3.0 Hz, 1H, H3”-furan), 3.89 (s, 3H, OCH3);
13
–
4.5.3. Ethyl 5-Fluoro-3-phenyl-1H-indole-2-carboxylate (32b)
C NMR (75 MHz, DMSO‑d ) δ 163.65 (C4“-furan), 158.28 (C O),
–
6
–
′
–
To a solution of 4-fluoroaniline (0.22 mL, 2.32 mmol) in EtOH/H2O
(1:1, 2 mL), were added 0.6 mL conc. HCl and a solution of NaNO2
(0.240 g, 3.48 mmol) in H2O (10 mL) dropwise at 0 ◦C. The resulting
solution of diazonium salt 30b was poured into a mixture of ethyl 2-
benzyl-3-oxobutanoate (29) (0.51 g, 2.32 mmol) and KOH (0.61 g,
139.69 (C1”-furan), 137.37 (C N), 134.77 (C7a-ind), 133.43 (C1 -Ph),
130.01 (C2′-Ph), 129.43 (C2-ind), 129.01 (C3′-Ph), 128.32 (C3a-ind),
127.34 (C4′-Ph), 126.90 (C6-ind), 122.32 (C4-ind), 118.47 (C2“-furan),
117.52 (C3-ind), 114.93 (C7-ind), 113.39 (C5-ind), 83.55 (C3”-furan),
58.64 (OCH3); HRMS (ESI) m/z [M+H]+ calcd for C21H17O3N379Br35Cl
438.0448; found 438.0449.
◦
10.90 mmol) in EtOH/H2O (1:1, 2 mL) at 0 C. The resulting mixture
was refrigerated at approximately 4 ◦C for 17 hrs. The resulting mixture
was allowed to warm to room temperature, diluted with H2O (10 mL)
and was extracted with EtOAc (3 × 20 mL). The combined organic layers
were dried (Na2SO4), filtered, and solvent was evaporated to give a
crude intermediate hydrazone 31b as an oil (used without purification).
A solution of the hydrazone crude 31b in conc. HCl (2.3 mL) was heated
to 80 ◦C. After 4 hrs, the mixture was allowed to cool to room temper-
ature. The mixture was diluted by EtOAc (10 mL) and H2O (10 mL), then
extracted with EtOAc (3 × 15 mL). The combined organic layers were
dried (Na2SO4), filtered, and solvent was evaporated. Purification by
4.5.6. (E)-5-bromo-N’-((5-chlorofuran-2-yl)methylene)-3-phenyl-1H-
indole-2-carbohydrazide (27c)
Yellow solid (0.076 g, 0.17 mmol, 59%); Rf 0.5 [EtOAc-Hexane
(2:3)]; m.p. 231–234 ◦C; νmax/cmꢀ 1 3259, 3135, 3052, 2922, 2850,
1741, 1633, 1618, 1248; 1H NMR (300 MHz, DMSO‑d6) δ 12.15 (s, 1H,
CONH), 11.43 (s, 1H, NH), 7.84 (s, 1H, HC = N), 7.70 (d, J = 1.8 Hz, 1H,
H4-ind), 7.46–7.32 (m, 7H, H6-ind, H7-ind, PhH), 6.96 (br s, 1H, H2“-
furan), 6.61 (br s, 1H, H3”-furan); 13C NMR (75 MHz, DMSO‑d6) δ
–
–
–
158.24 (C O), 148.88 (C1“-furan), 137.62 (C N), 135.99 (C4”-furan),
–
10

R. Saruengkhanphasit et al.
Bioorganic Chemistry 110 (2021) 104795
134.42 (C7a-ind), 132.85 (C1′-Ph), 129.56 (C2′-Ph), 129.40 (C2-ind),
128.55 (C3′-Ph), 127.81 (C3a-ind), 126.94 (C4′-Ph), 126.64 (C6-ind),
121.95 (C4-ind), 117.58 (C3-ind), 116.52 (C2“-furan), 114.55 (C7-ind),
113.01 (C5-ind), 109.50 (C3”-furan); HRMS (ESI) m/z [M+H]+ calcd for
C20H14O2N739Br35Cl 441.9952; found 441.9948.
10.0 Hz, C3a-ind), 118.15 (C3-ind), 116.40 (C2“-furan), 113.82 (d, J =
9.6 Hz, C7-ind), 112.87 (d, J = 26.0 Hz, C6-ind), 109.47 (C3”-furan),
104.39 (d, J = 23.9 Hz, C4-ind); 19F NMR (282 MHz, DMSO‑d6) δ
ꢀ 122.69; HRMS (ESI) m/z [M+H]+ calcd for C20H14O2N335ClF
382.0753; found 382.0753.
4.5.7. (E)-5-bromo-N’-((5-bromofuran-2-yl)methylene)-3-phenyl-1H-
indole-2-carbohydrazide (27d)
4.5.11. (E)-N’-((5-bromofuran-2-yl)methylene)-5-fluoro-3-phenyl-1H-
indole-2-carbohydrazide (27h)
◦
Yellow solid (0.113 g, 0.23 mmol, 79%); Rf 0.5 [EtOAc-Hexane
(2:3)]; m.p. 215–218 ◦C decomposed; νmax/cmꢀ 1 3304, 3222, 3054,
2957, 2852, 1738, 1640, 1619, 1258; 1H NMR (300 MHz, DMSO‑d6) δ
12.19 (s, 1H, CONH), 11.46 (s, 1H, NH), 7.87 (s, 1H, HC = N), 7.73 (br d,
J = 1.8 Hz, 1H, H4-ind), 7.50–7.36 (m, 7H, H6-ind, H7-ind, PhH), 6.96
(br s, 1H, H2“-furan), 6.75 (br s, 1H, H3”-furan); 13C NMR (75 MHz,
Yellow solid (0.098 g, 0.23 mmol, 64%); m.p. 203–207 C decom-
posed; νmax/cmꢀ 1 3319, 3254, 1649, 1623, 1242; 1H NMR (300 MHz,
DMSO‑d6) δ 12.06 (s, 1H, CONH), 11.43 (s, 1H, NH), 7.86 (s, 1H, H-
–
C
–
N), 7.51–7.43 (m, 5H, PhH), 7.33–7.29 (m, 2H, H4-ind, H7-ind),
7.14 (td, J = 9.1, 2.4 Hz, 1H, H6-ind), 6.93 (br s, 1H, H2“-furan), 6.72
(br s, 1H, H3”-furan); 13C NMR (75 MHz, DMSO‑d6) δ 159.27 (d, J =
–
–
–
–
–
DMSO‑d ) δ 158.19 (C O), 151.02 (C1“-furan), 135.89 (C N), 134.39
233.9 Hz, C5-ind), 158.33 (C O), 151.07 (C1“-furan), 135.84 (C N),
–
–
–
6
(C7a-ind), 132.83 (C1′-Ph), 129.55 (C2′-Ph), 128.54 (C3′-Ph), 128.47
(C2-ind), 127.78 (C3a-ind), 126.91 (C4′-Ph), 126.59 (C6-ind), 121.92
(C4-ind), 117.52 (C3-ind), 116.74 (C2”-furan), 114.52 (C7-ind), 114.26
(C3“-furan), 112.96 (C5-ind), C4” not observed; HRMS (ESI) m/z
[M+H]+ calcd for C20H14O2N379Br2 485.9447; found 485.9437.
133.17 (C1′-Ph), 132.46 (C7a-ind), 129.43 (C2′-Ph), 129.02 (C2-ind),
128.49 (C3′-Ph), 126.77 (C4′-Ph), 126.28 (d, J = 9.4 Hz, C3a-ind),
124.86 (C4”-furan), 118.14 (C3-ind), 116.67 (C2“-furan), 114.26 (C3”-
furan), 113.82 (d, J = 9.4 Hz, C7-ind), 112.89 (d, J = 26.0 Hz, C6-ind),
104.40 (d, J = 23.7 Hz, C4-ind); 19F NMR (282 MHz, DMSO‑d6) δ
ꢀ 122.68; HRMS (ESI) m/z [M+H]+ calcd for C20H14O2N379BrF
426.0248; found 426.0247
4.5.8. (E)-5-fluoro-N’-(furan-2-ylmethylene)-3-phenyl-1H-indole-2-
carbohydrazide (27e)
Yellow solid (0.094 g, 0.27 mmol, 75%); Rf 0.5 [EtOAc-Hexane
4.5.12. (E)-5-bromo-N’-(furan-3-ylmethylene)-3-phenyl-1H-indole-2-
carbohydrazide (27i)
(2:3)]; m.p. 218–221 ◦C; νmax/cmꢀ 1 3307, 3262, 3060, 1644, 1608,
1229; 1H NMR (300 MHz, DMSO‑d6) δ 12.05 (s, 1H, CONH), 11.34 (s,
Yellow solid (0.052 g, 0.13 mmol, 76%); Rf 0.5 [EtOAc-Hexane
(2:3)]; m.p. 214–217 ◦C decomposed; νmax/cmꢀ 1 3306, 3238, 3056,
2917, 1643, 1623, 1231; 1H NMR (300 MHz, DMSO‑d6) δ 12.14 (s, 1H,
CONH), 11.29 (s, 1H, NH), 8.11 (br s, 1H, H1“-furan), 8.01 (br s, 1H,
H4”-furan), 7.73–7.71 (m, 2H, HC = N, H4-ind), 7.51–6.34 (m, 7H, H6-
ind, H7-ind, PhH), 6.77 (br s, 1H, H3“-furan); 13C NMR (75 MHz,
–
1H, NH), 7.94 (s, 1H, H4“-furan), 7.81 (s, 1H, H-C N), 7.51–7.43 (m,
–
5H, PhH), 7.33–7.29 (m, 2H, H4-ind, H7-ind), 7.13 (td, J = 9.2, 2.3 Hz,
1H, H6-ind), 6.88 (br s, 1H, H2”-furan), 6.59 (br s, 1H, H3“-furan); 13
C
NMR (75 MHz, DMSO‑d6) δ 159.27 (d, J = 234.0 Hz, C5-ind), 158.26
–
–
–
(C O), 149.14 (C1”-furan), 145.35 (C4“-furan), 137.09 (C N), 133.24
–
(C1′-Ph), 132.42 (C7a-ind), 129.44 (C2′-Ph), 129.16 (C2-ind), 128.50
(C3′-Ph), 126.73 (C4′-Ph), 126.31 (d, J = 8.9 Hz, C3a-ind), 118.02 (C3-
ind), 113.98 (C2”-furan), 113.80 (d, J = 10.1 Hz, C7-ind), 112.83 (d, J =
27.8 Hz, C6-ind), 112.24 (C3“-furan), 104.39 (d, J = 24.1 Hz, C4-ind);
19F NMR (282 MHz, DMSO‑d6) δ ꢀ 122.72; HRMS (ESI) m/z [M+H]+
calcd for C20H15O2N3F 348.1143; found 348.1143.
DMSO‑d ) δ 158.00 (C O), 145.75 (C1”-furan), 144.93 (C N), 140.41
–
–
–
–
6
(C4“-furan), 134.32 (C7a-ind), 132.97 (C1′-Ph), 129.58 (C2′-Ph),
128.86 (C2-ind), 128.55 (C3′-Ph), 127.88 (C3a-ind), 126.92 (C4′-Ph),
126.49 (C6-ind), 122.39 (C4-ind), 121.89 (C2”-furan), 117.23 (C3-ind),
114.50 (C7-ind), 112.95 (C5-ind), 107.14 (C3“-furan); HRMS (ESI) m/z
[M+H]+ calcd for C20H15O2N739Br 408.0342; found 408.0337.
4.5.9. (E)-5-fluoro-N’-((5-methoxyfuran-2-yl)methylene)-3-phenyl-1H-
indole-2-carbohydrazide (27f)
4.5.13. (E)-5-fluoro-N’-(furan-3-ylmethylene)-3-phenyl-1H-indole-2-
carbohydrazide (27j)
Yellow solid (0.078 g, 0.21 mmol, 68%); Rf 0.5 [EtOAc-Hexane
(2:3)]; m.p. 185–187 ◦C; νmax/cmꢀ 1 3235, 3026, 2979, 2938, 1615,
1569, 1252; 1H NMR (300 MHz, DMSO‑d6) δ 12.02 (s, 1H, CONH), 11.16
Yellow solid (0.112 g, 0.32 mmol, 40%); Rf 0.5 [EtOAc-Hexane
(2:3)]; m.p. 198–200 ◦C; νmax/cmꢀ 1 3306, 3238, 3056, 2917, 1643,
1623, 1231; 1H NMR (300 MHz, DMSO‑d6) δ 12.01 (s, 1H, CONH), 11.24
–
(s, 1H, NH), 7.72 (s, 1H, H-C N), 7.49–7.43 (m, 5H, PhH), 7.31–7.28
(s, 1H, NH), 8.09 (br s, 1H, H1“-furan), 8.00 (br s, 1H, H4”-furan), 7.71
–
–
(m, 2H, H4-ind, H7-ind), 7.12 (br t, J = 8.4 Hz, 1H, H6-ind), 6.81 (br s,
(s, 1H, H-C N), 7.50–7.40 (m, 5H, PhH), 7.31–7.27 (m, 2H, H4-ind, H7-
–
1H, H2“-furan), 5.53 (br s, 1H, H3”-furan), 3.87 (s, 3H, OCH3); 13C NMR
ind), 7.11 (td, J = 9.2, 2.3 Hz, 1H, H6-ind), 6.76 (br s, 1H, H3“-furan);
(75 MHz, DMSO‑d6) δ 163.21 (C4“-furan), 159.27 (d, J = 233.5 Hz, C5-
13C NMR (75 MHz, DMSO‑d6) δ 159.26 (d, J = 233.9 Hz, C5-ind), 158.16
′
–
–
–
–
–
ind), 158.02 (C O), 139.28 (C1”-furan), 136.86 (C N), 133.31 (C1 -
(C O), 145.69 (C1”-furan), 144.90 (C N), 140.34 (C4“-furan), 133.33
–
–
–
Ph), 132.38 (C7a-ind), 129.45 (C2′-Ph and C2-ind), 128.53 (C3′-Ph),
126.73 (C4′-Ph), 126.36 (d, J = 9.8 Hz, C3a-ind), 117.98 (C2“-furan),
117.64 (C3-ind), 113.78 (d, J = 9.5 Hz, C7-ind), 112.71 (d, J = 26.4 Hz,
C6-ind), 104.37 (d, J = 23.7 Hz, C4-ind), 83.08 (C3”-furan), 58.19
(OCH3); 19F NMR (282 MHz, DMSO‑d6) δ ꢀ 122.79; HRMS (ESI) m/z
[M+H]+ calcd for C21H17O3N3F 378.1248; found 378.1247.
(C1′-Ph), 132.38 (C7a-ind), 129.46 (C2′-Ph), 129.35 (C2-ind), 128.50
(C3′-Ph), 126.73 (C4′-Ph), 126.38 (d, J = 10.3 Hz, C3a-ind), 122.41
(C2”-furan), 117.84 (d, J = 5.2 Hz, C3-ind), 113.79 (d, J = 9.5 Hz, C7-
ind), 112.75 (d, J = 29.5 Hz, C6-ind), 107.17 (C3“-furan), 104.36 (d, J =
23.3 Hz, C4-ind); 19F NMR (282 MHz, DMSO‑d6) δ ꢀ 122.78; HRMS (ESI)
m/z [M+H]+ calcd for C20H15O2N3F 348.1143; found 348.1140.
4.5.10. (E)-N’-((5-chlorofuran-2-yl)methylene)-5-fluoro-3-phenyl-1H-
indole-2-carbohydrazide (27g)
Declaration of Competing Interest
Yellow solid (0.087 g, 0.23 mmol, 64%); m.p. 204–207 ◦C; νmax
/
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
cmꢀ 1 3221, 3226, 2925, 1617, 1602, 1252; 1H NMR (300 MHz,
DMSO‑d6) δ 12.05 (s, 1H, CONH), 11.43 (s, 1H, NH), 7.86 (s, 1H, H-
–
C
N), 7.51–7.43 (m, 5H, PhH), 7.32–7.28 (m, 2H, H4-ind, H7-ind),
–
7.13 (td, J = 9.2, 2.4 Hz, 1H, H6-ind), 6.97 (br s, 1H, H2“-furan), 6.62
Acknowledgement
(br s, 1H, H3”-furan); 13C NMR (75 MHz, DMSO‑d6) δ 159.26 (d, J =
–
–
–
233.7 Hz, C5-ind), 158.30 (C O), 148.91 (C1“-furan), 137.51 (C N),
–
I am deeply indebted to the Chulabhorn Royal Academy for the
funding support of this project. Also, I would like to express my gratitude
to Dr. Pakamas Intachote, Ms. Suchada Sengsai and Ms. Busakorn
135.87 (C4”-furan), 133.18 (C1′-Ph), 132.45 (C7a-ind), 129.42 (C2′-
Ph), 128.98 (C2-ind), 128.48 (C3′-Ph), 126.76 (C4′-Ph), 126.29 (d, J =
11

R. Saruengkhanphasit et al.
Bioorganic Chemistry 110 (2021) 104795
polymerization inhibitors targeting the colchicine binding site: structural basis and
antitumor efficacy, J. Med. Chem. 61 (4) (2018) 1704–1718.
Saimanee of the Bioactivity Screening Unit, Chulabhorn Research
Institute, for the significant contribution in compound testing in cancer
cell lines.
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pp. 5–21.
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