Structural optimization of indole based compounds for highly promising anti-cancer activities: Structure activity relationship studies and identification of lead molecules

Article abstract of DOI:10.1016/j.ejmech.2013.12.047

Based on the anti-cancer data of previous compounds, 27 more compounds were synthesized and subjected to anti-cancer screening. Compounds were tested over 60 human tumor cell lines of different types of cancer. As per the data available, some compounds ex

Full text of DOI:10.1016/j.ejmech.2013.12.047

European Journal of Medicinal Chemistry 74 (2014) 440e450
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structural optimization of indole based compounds for highly
promising anti-cancer activities: Structure activity relationship studies
and identification of lead molecules
*
Shaveta, Palwinder Singh
UGC Sponsored Centre for Advanced Studies, Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, Punjab, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the anti-cancer data of previous compounds, 27 more compounds were synthesized and
subjected to anti-cancer screening. Compounds were tested over 60 human tumor cell lines of different
types of cancer. As per the data available, some compounds exhibited appreciable anti-cancer properties
over certain cell lines with their GI50 in nM range. With the help of UVevis spectral studies, enzyme
immunoassay and molecular modeling studies, dihydrofolate reductase was found to be the probable
cellular target of the compounds under present investigation.
Received 30 October 2013
Received in revised form
11 December 2013
Accepted 27 December 2013
Available online 8 January 2014
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Indole
Pyrazole
Anti-cancer activity
Dihydrofolate reductase
Interactions
1. Introduction
As a result some highly efficacious drugs like 5-fluorouracil [11],
methotrexate [12], pemetrexed [13] etc appeared in the market
As per the latest information available, it is estimated that if the
spread of cancer continues at its present rate, it may cause over 13.1
million deaths in 2030 worldwide [1]. Irrespective of the un-tired
efforts and screening of myriads of compounds for anti-cancer ac-
tivities [2e4], the uncertainty about the cause of origin of cancer,
limitations in its detection at early stage, its direct connection with
process of cell division, metastatic nature of cancer cells and lack of
drug penetration to cancer tissue are some of the features of this
disease which prove as hurdles in the successful treatment of
cancer [5,6]. Amongst the various approaches to treatment of
cancer, chemotherapy finds a wide use during both pre-operative
and post-operative conditions [7,8]. The main focus of chemo-
therapeutic agents is to block/slow down propagation of cancer
cells for which enzymes associated with cell division are the pri-
mary targets. Ribonucleotide reductase (RNR), thymidylate syn-
thase (TS), thymidylate phosphorylase (TP), dihydrofolate
reductase (DHFR) are primarily concerned with generation of raw
material for cell division [9,10] and hence they were made the
targets during the design and development of anti-cancer agents.
with some hope for the cancer patients. In continuation to earlier
reports for development of anti-cancer agents [14e17], particularly
indole based compounds [14,15], here we report another set of
compounds with appreciable anti-cancer activity over certain hu-
man tumor cell lines.
2. Results and discussion
2.1. Chemistry
A simple synthetic methodology was undertaken to synthesize
conjugates of indole, pyrazole and barbituric acid. Compounds 2a
and 2b were synthesized by condensation of 1a and 1b (1 mmol)
with 1-(3-chlorophenyl)-3-methyl-2-pyrazolin-5-one (1.2 mmol)
under microwave irradiation. Similarly, compounds 4a, b and 5a, b
were prepared by irradiating the mixture of 1a, b with barbituric
acid and 1a, b with oxindole (1,3-dihydroindol-2-one), respectively
under microwaves. Compound 3a and b were procured from re-
action of 1a and b with indolinone (1-[2,6-dichlorophenyl]-1,3-
dihydroindol-2-one) in ethanol-water (1:2) in presence of NaOH
(Scheme 1).
It was also planned to introduce a substituent at N-1 position of
indole in compounds 2e5, for which indoles 1a and b were first
* Corresponding author. Tel.: þ91 183 2258802 09x3495; fax: þ91 183 2258819.
E-mail address: palwinder_singh_2000@yahoo.com (P. Singh).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.047

Shaveta, P. Singh / European Journal of Medicinal Chemistry 74 (2014) 440e450
441
H3C
H3C
N
N
N
N
Cl
Cl
CHO
O
R
NH
N
H
R
R
O
O
O
N
H
1a, b
MWI
MWI
N
H
N
H
5a, b
2a, b
O
O
N
NH
Cl
O
N
H
O
NaOH,
C2H5OH:H2O
Cl
MWI
O
NH
Cl
O
N
NH
R
R
O
O
Cl
3a, b
N
vH
N
H
4a, b
1-5: a: R = Br;
b: R = OCH3
Scheme 1. Synthesis of compounds 2e5.
alkylated/acylated in presence of NaH and thereby compounds 6e
10 were prepared (Scheme 2).
leukemia. LC50 (50% lethal conc of compound) of these compounds
was >100 M indicating their appreciable selectivity indices.
m
Treatment of compound 6e9 with 1-(3-chlorophenyl)-3-
Therefore, results of anti-cancer screening experiments clearly
indicate the potency of these compounds for controlling cancer
growth over certain cell lines.
methyl-2-pyrazolin-5-one/indolinone/barbituric
acid/oxindole
under microwave irradiation provided compounds 11e21 (Scheme
3).
Looking at the structure activity relationship of these com-
pounds, it is quite evident that a small variation in the structure of
the compound made a dramatic change in its biological activity.
Compounds 3b and 23 with OCH3 substituent at C-5 of indole
showed better anti-cancer activity than their analogs 3a and 15
carrying Br at C-5 of indole. 5-Unsubstituted indole-pyrazole
adduct (32, Chart 1) [15] did not show anticancer activity but
with introduction of Br at C-5 position of indole, the resulting
compound 2a exhibited appreciable anticancer activity [16].
Moreover, far better GI50 of compound 12 in comparison to that of
compound 2a indicates the role of substituent at N-1 of indole in
increasing the anti-cancer potency of compound 12. The substitu-
ent specificity was observed by replacing p-chlorobenzoyl group of
compound 12 with 2,6-dichlorobenzoyl in compound 14 which
made compound 14 inactive towards anti-cancer activity. Similarly,
comparison of compounds 11 and 22 indicates that presence of Br
in compound 11 made it selective towards MDA-MB-468 cell line of
breast cancer. It means instead of a single substituent, the biological
activity of the compound is decided by the combination of all the
Similarly, reactions of compound 10 with 1-(3-chlorophenyl)-3-
methyl-2-pyrazolin-5-one/indolinone/barbituric
acid/oxindole
resulted into the formation of compounds 22e25 (Scheme 4).
Since trifluoromethyl group is an active component of a number
of drugs, compounds 26e28 were prepared through condensation
of 2-trifluoromethylbenzaldehyde with oxindole, indolinone and 1-
(3-chlorophenyl)-3-methyl-2-pyrazolin-5-one,
respectively.
Further treatment of compound 26 with p-chlorobenzoyl chloride
and 2-fluorobenzyl bromide in presence of NaH provided com-
pounds 29 and 30 (Scheme 5). Therefore, through a very convenient
synthetic protocol, a library of compounds was procured in almost
quantitative yield.
2.2. Anti-cancer activity
In vitro tumor growth inhibitory activities of compounds 3a, 3b,
4a, 4b, 11, 12, 15, 16, 18, 19, 21, 23, 24, 25, 27, 28 and 29 were
investigated on 60 cell line panel of human cancer cells at National
Cancer Institute (NCI), Bethesda, MD, USA. The compounds were
found to be selective for different cell lines and anti-cancer data of
compounds with significant activity over certain cancer cell lines is
given in Table 1. Compound 3b showed specificity for various cell
lines of non-small cell lung cancer, colon cancer and prostate can-
cer. Compound 11 exhibited high selectivity for MDA-MB-468 cell
line of breast cancer with GI50 120 nM. Compound 12 was identified
as the most potent amongst those screened here. It exhibited
excellent specificity for various cell lines of leukemia along with
non-small cell lung cancer, colon cancer, melanoma, prostate can-
cer and renal cancer. Remarkably, GI50 of compound 12 for NCI-
H522 cell line of non-small cell lung cancer was 1 nM. Interest-
ingly, presence of Br in compound 12 considerably increased its
anti-cancer activity in comparison to its analog 31 (Chart 1), re-
ported earlier [15]. Compounds 16, 18, 19 and 25 showed growth
CHO
CHO
R
Br
NaH, CH3CN
R'
X
N
H
N
R'
R=Br
1
6-9
NaH, CH3CN
R=OCH3
R'
X
6, 10: R' = benzyl
CHO
H3CO
7: R' = p-chlorobenzoyl
8: R' = 2,6-dichlorobenzoyl
9: R' = 2-fluoro benzyl
N
R'
10
inhibition over certain cell lines with GI50 in
mM (Table 1). Com-
pounds 23 and 27 were more specific for various cell lines of
Scheme 2. Synthesis of compounds 6e10.

442
Shaveta, P. Singh / European Journal of Medicinal Chemistry 74 (2014) 440e450
H3C
H3C
N
N
N
N
Cl
Cl
CHO
O
Br
Br
N
H
MWI
O
NH
O
Br
N
O
MWI
N
R
R
11-14
N
R
6-9
O
19-21
O
11, R = benzyl
N
12, R = p-chlorobenzoyl
13, R = 2-fluoro benzyl
14, R = 2,6-dichlorobenzoyl
Cl
NH
19, R = benzyl
Cl
MWI
MWI
O
N
H
O
20, R = p-chlorobenzoyl
21, R = 2, 6-dichlorobenzoyl
Cl
O
N
NH
Br
O
O
Cl
Br
NH
N
R
O
N
15-17
R
15, R = benzyl;
16, R = p-chlorobenzoyl
17, R = 2-fluorobenzyl
18, R = 2-fluorobenzyl
Scheme 3. Synthesis of compounds 11e21.
fragments of the molecule. Trifluorotolyl substituent showed its
effect for anti-cancer activity only in combination with indolinone
moiety (compound 27). Also, by comparing the anti-cancer activity
of compounds 23 and 25, it was observed that presence of
2,6-dichlorophenyl moiety in compound 23 made it more
potent against various cell lines of non-small cell lung cancer and
colon cancer. In comparison to the anti-cancer activity of one of our
previous compounds (33, Chart 1) [14], presence of OCH3 group in
compound 24 made it inactive for cancer growth inhibition.
Appreciable anti-cancer activity of compounds 12 and 16
amongst their respective groups (11e14 and 15e17) points towards
the suitability of indoleepyrazole and indoleeindolinone in com-
bination with p-chlorobenzoyl group for anti-cancer activity.
Therefore, considerable success was attained in optimizing the
structure of compound 12 and getting a promising lead molecule
for anti-cancer drug.
2.3. Mechanistic investigations of mode of action of test compounds
Based on the anti-cancer activities of the compounds, it was
desirable to investigate their probable cellular target and hence
mode of action. Interactions of the compounds with RNR, TS, TP and
DHFR, the enzymes involved in the process of propagation of can-
cer, were investigated with the help of UVevis spectral studies,
enzyme immunoassay and molecular modeling though the other
cellular target/s of these compounds cannot be ruled out. It was
envisaged that these studies may help in further refinement of the
compounds for increasing their drug efficacy and also to check if
the compounds exhibit promiscuity or not.
2.3.1. UVevisible spectral studies
UVevisible spectral studies were performed to analyze the
interaction of the compounds with enzymes. The compounds were
H3C
CHO
H3C
N
N
H3CO
O
N
N
Cl
Cl
N
H3CO
N
H
O
O
NH
H3CO
MWI
N
O
MWI
N
O
10
O
22
NH
N
Cl
O
N
H
O
25
Cl
MWI
MWI
O
O
NH
NH
O
Cl
N
H3CO
H3CO
O
N
Cl
N
24
23
Scheme 4. Synthesis of compounds 22e25.

Shaveta, P. Singh / European Journal of Medicinal Chemistry 74 (2014) 440e450
443
F3C
O
CHO
NH
F3C
O
CH3
NH
26
N
MWI
N
O
N
O
MWI
Cl
N
,
C
H
a
N
R
X
Cl
MWI
F3C
CH3
Cl
H3C
F3C
F3C
N
N
N
R
O
Cl
O
Cl
N
29, R = p-chlorobenzoyl
30, R = 2-fluorobenzyl
O
Cl
28
27
Scheme 5. Synthesis of compounds 26e30.
evaluated at a concentration varying from 1
mM to 5
m
M by pre-
(50 mL), the peak at 260 nm showed a bathochromic shift to 268 nm
paring the stock solutions in DMSO. The enzyme (0.1 units) was
used as supplied. For studying the enzymeecompound in-
along with hyperchromic shift at both the absorption maxima
(Fig. S45). Similar observations were noticed with compounds 3b
and 27.
Therefore, a simple assay indicates the non-promiscuous na-
ture of the compounds amongst the four enzymatic targets (RNS,
TS, TP and DHFR) and hence they may not be the frequent hitters.
Rather the active compounds seem to be targeting a particular
enzyme.
teractions, 50
The compound solution was added to the enzyme solution by
varying the concentration from 1 M to 5 M. To our surprise,
mL of the enzyme was used and diluted to 1000 mL.
m
m
addition of compounds to solution of RNR, TS and TP did not make
change in the UV spectra of these enzymes. Checking in another
way, addition of enzyme RNR, TS and TP to the solution of com-
pounds also did not change UVevis spectrum of the compound.
However, appreciable change in the UVevis spectrum of compound
12 was observed when it was added to solution of DHFR. Com-
2.3.2. Enzyme inhibition assay
Further, to confirm the interaction of compounds with DHFR
and probably the inhibition in enzymatic activity of DHFR in
presence of compounds, DHFR enzyme immunoassay was
pound 12 (5 mM solution in DMSO) showed two lmax at 260 nm and
397 nm in its UVevis spectrum (Fig. S45). In presence of DHFR
Table 1
GI50 (50% growth inhibitory conc) of compounds 3b, 11, 12, 16, 18, 19, 23, 25 and 27 over various cancer cell lines.
Panel/cell line
GI50 (mM)
3b
11
12
16
18
19
23
25
27
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
22
18
25
25
30
50
57
48
35
>100
>100
0.03
1.5
1.5
0.04
0.01
0.02
0.11
>100
60
62
>100
>100
>100
>100
>100
>100
>100
>100
>100
82
75
>100
>100
>100
0.13
>100
0.10
>100
>100
>100
>100
>100
>100
58
1.1
1.4
0.10
1.4
2.0
1.5
0.14
1.4
>100
Non-small cell lung cancer
HOP-62
HOP-92
NCl-H322M
NCl-H460
NCl-H522
Colon cancer
HCT-116
>100
1.4
>100
>100
>100
>100
>100
>100
48
45
1.4
>100
0.001
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.23
>100
>100
1.41
1.6
>100
>100
>100
>100
1.5
>100
1.6
>100
0.20
>100
>100
>100
>100
>100
>100
>100
1.6
1.10
1.80
1.42
3.0
>100
>100
>100
>100
64
0.02
0.01
1.5
>100
1.5
1.5
1.22
>100
>100
>100
>100
1.4
2.5
>100
HCT-15
HT29
KM12
3.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.4
>100
Melanoma
MALME-3M
Renal cancer
CAKI-1
UO-31
Prostate cancer
PC-3
Breast cancer
[MDA-MB-231/ATCC]
T-47D
[MDA-MB-468]
MCF7
>100
>100
0.02
0.12
>100
0.01
>100
0.12
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.4
>100
>100
>100
>100
>100
>100
>100
>100
2.4
1.53
>100
>100
1.2
>100
>100
1.34
>100
>100
>100
>100
2.10
>100
>100
>100
>100
>100
>100
0.12
>100
0.12
1.22
>100
>100
>100
>100
>100
0.01
>100
>100
>100
>100
>100
>100
1.42
>100
>100
>100
42
>100
>100
>100
0.02

444
Shaveta, P. Singh / European Journal of Medicinal Chemistry 74 (2014) 440e450
H3C
N
Cl
N
H3C
H3C
N
N
N
N
O
Cl
Cl
N
Br
O
O
O
N
H
N
H
Cl
32
2a
31
GI50 3.9μ M
H3C
O
N
N
O
Cl
NH
NH
NH
O
O
Br
O
H3CO
NH
O
O
N
N
N
O
Cl
33
GI50 7.5μ M
12
GI50 0.41 μ M
24
Chart 1. Structural comparison of compounds 12 and 24 with previously reported compounds 2a and 31e33. GI50 is the average over all the cancer cell lines. GI50 for compounds 31
and 32 were not available.
performed using dihydrofolate reductase inhibition assay kit [18].
Some of the compounds showed excellent inhibitory activity with
cell line of non-small cell lung cancer where it exhibited GI50 1 nM.
Compound 12 along with some other compounds also showed
appreciable inhibition of tumor growth over different cell lines of
leukemia, non-small cell lung cancer, colon cancer and prostate
cancer. Over all, modification of previously reported compounds
led to the development of highly potent compounds against certain
types of cancer. Mechanistic investigations of mode of action of
these compounds point towards the possibility of DHFR as their
cellular target. Therefore, stepwise modification of the compounds
led to considerable success in the search of some highly promising
leads for anti-cancer drugs.
IC50 ranging from
standard drug methotrexate. Compounds 3b and 12 showed IC80
0.10 M and 0.28 M, respectively for DHFR while IC50 of com-
mM to nM (Table 2) and comparable to the
m
m
pounds 25 and 27 was calculated as 54 nM and 68 nM, respectively.
Therefore, it seems that the compounds under present investiga-
tion probably target DHFR for exhibiting anti-cancer activities.
However, compounds 19, 23 and 24 did not show inhibition of
DHFR enzymatic activity.
2.3.3. Molecular docking studies
In order to have an insight into the molecular interactions of the
compounds with DHFR, their docking in the active site of the
enzyme was performed. The crystal coordinates of DHFR in
complexation with an anti-cancer agent were taken from protein
data bank (www.rcsb.org) (PDB ID 3GHW) [19]. Compounds 12, 3b
and 27 were docked in the active site of DHFR. Compound 12 sits in
the active site of the enzyme interacting through H-bonding be-
4. Experimental data
4.1General remarks
Melting points were determined in capillaries and are uncor-
rected. IR spectra were recorded on PerkinElmer Fourier Transform
Infrared spectrometer. 1H and 13C NMR spectra were recorded on
JEOL 300 MHz and 75 MHz NMR spectrometer, respectively using
CDCl3 and/or DMSO-d6 as solvent. Chemical shifts are given in ppm
with TMS as an internal reference. J values are given in Hertz.
Signals are abbreviated as singlet, s; doublet, d; double-doublet, dd;
tween pyrazolic N and Gln35 residue of
enzyme (Fig. 1, Fig. S47). The indole part of the compound aligns in
parallel with one -sheet (yellow in color) while its p-chlor-
obenzoyl fragment overlaps with another -sheet (blue in color)
a-helical part of the
b
b
indicating the favorable hydrophobic interactions of the indole
moiety and polar interactions from the p-chlorobenzoyl group.
CPK model of the DHFR active site also clearly shows the
compatibility of compound 12 in the active site of the enzyme
(Fig. 2). Similar mode of interaction of compound 3b (Figs. 3 and 4,
Fig. S46) and 27 (Figs. S48 and S49) was observed when docked in
the active site of DHFR. Therefore, the crystal coordinates of the
enzyme in association with compound 12, 3b and 27 support the
experimental results of interactions and enzyme inhibitory activ-
ities of these compounds.
Table 2
IC50 (50% inhibitory concentration) for compounds 3b,
11, 12, 16, 18, 19, 23, 24, 25 and 27 for dihydrofolate
reductase (DHFR).
Compound
IC50 (mM)
3b
11
12
16
18
19
0.10a
1.25
0.28a
0.69
0.30
e
23
24
e
3. Conclusions
e
25
27
53.9b
67.7b
1.39
A number of compounds, significantly important for structure
activity relationship studies, were synthesized using a simple
synthetic methodology. Anti-cancer screening of these compounds
identified compound 12 with considerable specificity for NCI-H522
Methotrexate
a
Corresponds to IC80
IC50 in nM.
.
b

Shaveta, P. Singh / European Journal of Medicinal Chemistry 74 (2014) 440e450
445
4.1.1. General procedure for synthesis of compounds 2e5 and 11e
28 (Procedure A)
finely ground mixture of 5-substituted indole-3-
A
carboxaldehyde (1 mmol) and active methylene compound (indo-
linone/oxindole/barbituric acid/1-(3-chlorophenyl)-3-methyl-2-
pyrazolin-5-one)/2-trifluoromethylbenzaldehyde (1.2 mmol) was
irradiated in microwave oven for 1e5 min. The solid product was
washed with diethyl ether and purified by column chromatography
to get pure products 2e5 and 11e28 except compound 3. Com-
pound 3 was prepared by treatment of 5-substituted indole-3-
carboxaldehyde with indolinone in ethanol-water in presence of
NaOH (1.5 mmol).
4.1.2General procedure for synthesis of compounds 6e10 (Procedure
B)
Sodium hydride (NaH, 1.5 mmol) was washed with hexane
several times to remove all the paraffin and then suspended in
acetonitrile (ACN). 5-Br/OCH3 indole-3-carboxaldehyde (1, 1 mmol)
was added to the suspension of NaH in ACN and kept stirred at 0 ꢁC.
Reaction progress was monitored by TLC. Reaction was complete in
30 min. The reaction mixture was extracted with ethyl acetate
followed by washing of organic layer with water. The organic layer
was passed through anhydrous Na2SO4 and concentrated to give
crude products 6e10 which were later purified by washing with
hexane.
Fig. 1. Crystal coordinates of DHFR in complexation with compound 12 (pink)
(generated in pymol). H-Bond between pyrazole N of compound 12 and Gln35 is
visible. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
4.1.3. General procedure for synthesis of compounds 29 and 30
Compound 29 and 30 were prepared by following the same
procedure as (B) starting with compound 26 (1 mmol).
triplet, t; multiplet, m. In 13C NMR spectral data, þve, ꢀve terms
correspond to CH3, CH, CH2 signals in DEPT-135 NMR spectra. Mass
spectra were recorded on Bruker MicroTOF QII mass spectrometer
in þve ESI mode with capillary voltage 4500 V except for com-
pound 4a and 12 (recorded in APCI mode, in solid state). Chroma-
tography was performed with silica 100e200 mesh and reactions
were monitored by thin layer chromatography (TLC) with silica
plates coated with silica gel GF-254. Reactions under microwaves
were performed using microwave oven (INALSA model 1MW17EG)
with microwave power 700 W and operating frequency 2450 MHz.
UVevisible spectral studies were performed on Biotek Power-
waveXS spectrophotometer to analyze the interaction of the com-
pounds with enzymes.
4.1.4. 4-(5-Bromo-1H-indol-3-ylmethylene)-2-(3-chlorophenyl)-5-
methyl-2,4-dihydropyrazol-3-one, 2a
Pure product was obtained as an orange solid in a yield of 65%,
mp 280 ꢁC (decomposed); IR nmax (KBr, cmꢀ1): 1645 (C]O), 3164
(NH); 1H NMR (300 MHz, DMSO-d6)
d ppm: 2.40 (s, 3H, CH3), 7.20
(d, J ¼ 9.0 Hz, 1H, ArH), 7.43 (t, J ¼ 9.0 Hz, 2H, ArH), 7.53 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.94 (d, J ¼ 8.1 Hz, 1H, ArH), 8.13 (d,
J ¼ 9.0 Hz, 2H, ArH), 8.49 (s, 1H, bridged ]CH), 9.70 (s, 1H, indole
2-H), 12.7 (NH, D2O exchange); 13C NMR (normal/DEPT-135)
(DMSO þ CDCl3)
d
ppm: 13.02 (þve, CH3), 111.779 (C), 114.507
(þve, CH), 115.142 (C), 115.81 (þve, CH), 117.19 (þve, CH), 118.61
(C), 121.23 (þve, CH), 123.22 (þve, CH), 125.86 (þve, CH), 129.96
(þve, CH), 133.29 (C), 135.13 (C), 137.29 (þve, CH), 139.02 (þve,
CH), 139.93 (C), 151.43 (C), 162.85 (C]O); HRMS (ESI) Calcd for
C
19H13BrClON3: 435.9823, 437.9802. Found: m/z 435.9808,
437.9788 ([M þ Na])þ.
4.1.5. 2-(3-Chlorophenyl)-4-(5-methoxy-1H-indol-3-ylmethylene)-
5-methyl-2,4-dihydro-pyrazol-3-one, 2b
Pure product was obtained as an orange solid in a yield of
80%, mp 239e240 ꢁC; IR nmax (KBr, cmꢀ1): 1649 (C]O), 3169
(NH); 1H NMR (300 MHz, DMSO-d6)
d ppm: 2.45 (s, 3H, CH3),
3.93 (s, 3H, OCH3), 6.92 (d, J ¼ 8.4 Hz, 1H, ArH), 7.11 (d, J ¼ 7.5 Hz,
1H, ArH), 7.32e7.43 (m, 2H, ArH), 7.75 (s, 1H, ArH), 7.89 (s, 1H,
ArH), 7.89 (s, 1H, ArH), 8.01 (d, J ¼ 8.1 Hz, 1H, ArH), 8.15 (s, 1H,
bridged ]CH), 9.82 (s, 1H, indole 2-H), 12.26 (s, 1H, NH); HRMS
(ESI) Calcd for C20H16ClO2N3: 388.0823. Found: m/z 388.0839
([M þ Na])þ.
4.1.6. 3-(5-Bromo-1H-indol-3-ylmethylene)-1-(2,6-
dichlorophenyl)-1,3-dihydroindol-2-one, 3a
Pure product was obtained as a yellow solid in a yield of 77%, mp
275e277 ꢁC; IR nmax (KBr, cmꢀ1): 1676 (C]O), 3268 (NH); 1H NMR
(300 MHz, CDCl3)
J ¼ 7.2 Hz, 3H, ArH), 7.35e7.45 (m, 2H, ArH), 7.51e7.58 (m, 2H, ArH),
d
ppm: 6.48 (d, J ¼ 7.2 Hz, 1H, ArH), 7.19 (d,
Fig. 2. CPK model of active site of DHFR with docking of compound 12.

446
Shaveta, P. Singh / European Journal of Medicinal Chemistry 74 (2014) 440e450
J ¼ 8.1 Hz, 1H, ArH), 6.91 (d, J ¼ 8.7 Hz, 1H, ArH), 7.14e7.16 (m, 2H,
ArH), 7.35e7.41 (m, 3H, ArH), 7.53 (d, J ¼ 8.1 Hz, 2H, ArH), 7.75 (d,
J ¼ 5.7 Hz, 1H, ArH), 8.05 (s, 1H, bridged ]CH), 9.02 (s, 1H, NH),
9.50 (s, 1H, indole 2-H); 13C NMR (DEPT-135) (CDCl3)
d ppm:
56.01 (þve, OCH3), 100.45 (þve, CH), 108.75 (þve, CH), 110.14 (C),
112.59 (þve, CH), 112.76 (þve, CH), 115.60 (C), 118.16 (þve, CH),
122.26 (C), 123.34 (C), 128.17 (þve, CH), 128.95 (C), 130.50 (þve,
CH), 130.69 (C), 131.23 (þve, CH), 133.83 (C), 136.05 (C), 136.82
(C), 138.85 (C), 155.73 (C]O); HRMS (ESI) Calcd for
C
24H16Cl2O2N2: 435.0662, 437.0634. Found: m/z 435.0396,
437.0376 ([M þ H])þ.
4.1.8. 5-(5-Bromo-1H-indol-3-ylmethylene)-pyrimidine-2,4,6-
trione, 4a
Pure product was obtained as a yellow solid in a yield of 77%, mp
>300 ꢁC; IR nmax (KBr, cmꢀ1): 1650, 1689, 1728 (C]O), 3170 (NH);
1H NMR (300 MHz, DMSO-d6)
d
ppm: 7.40 (d, J ¼ 8.4 Hz, 1H, ArH),
7.52 (d, J ¼ 8.1 Hz, 1H, ArH), 7.98 (s, 1H, bridged ]CH), 8.64 (s, 1H,
ArH), 9.51 (s, 1H, indole 2-H), 11.08 (s, 1H, NH), 11.16 (s, 1H, NH),
12.77 (s, 1H, NH); 13C NMR (CDCl3 þ DMSO-d6)
d ppm: 101.85,
109.13, 110.78, 113.42, 114.77, 130.05, 130.97, 135.07, 140.26, 143.46,
150.28, 156.35 (C]O), 162.49 (C]O), 164.32 (C]O); HRMS (APCI)
Calcd for C13H8BrO3N3: 332.9744, 334.9723. Found: m/z 332.9465,
334.9465 ([M])þ.
Fig. 3. Crystal coordinates of DHFR with compound 3b (pink). H-Bond between 5-
methoxy O of compound 3b and Gln35 is visible. NADPH is also visible. (For inter-
pretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)
4.1.9. 5-(5-Methoxy-1H-indol-3-ylmethylene) pyrimidine-2,4,6-
trione, 4b
7.76 (d, J ¼ 6.0 Hz, 1H, ArH), 8.01 (s, 1H, bridged ]CH), 8.07 (s, 1H,
ArH), 9.31 (s, 1H, NH), 9.55 (s, 1H, indole 2-H); 13C NMR (CDCl3)
Pure product was obtained as a yellow solid in a yield of 60%, mp
d
ppm: 108.87, 110.52, 112.48, 113.27, 115.06, 118.50, 121.60, 122.50,
>300 ꢁC; IR nmax (KBr, cmꢀ1): 1650, 1685 (C]O), 3181, 3344, 3480
125.96, 127.65, 128.98, 130.64, 130.67, 133.30, 134.60, 135.95, 166.42
(C]O); HRMS (ESI) Calcd for C23H13BrCl2ON2: 504.9481, 506.9459,
508.9431. Found: m/z 504.9472, 506.9453, 508.9445 ([M þ Na])þ.
(NH); 1H NMR (300 MHz, DMSO-d6)
d ppm: 3.89 (s, 3H, OCH3), 6.90
(d, J ¼ 8.7 Hz, 1H, ArH), 7.42 (d, J ¼ 8.7 Hz, 1H, ArH), 8.04 (s, 1H,
bridged ]CH), 8.74 (s,1H, ArH), 9.52 (s,1H, indole 2-H),10.95 (s,1H,
NH), 11.02 (s, 1H, NH), 12.51 (s, 1H, NH); 13C NMR (CDCl3 þ DMSO-
4.1.7. 1-(2,6-Dichlorophenyl)-3-(5-methoxy-1H-indol-3-
ylmethylene)-1,3-dihydroindol-2-one, 3b
d6) d ppm: 55.23 (OCH3), 99.71, 107.23, 111.77, 112.85,113.50, 130.20,
130.95, 131.29, 140.08, 144.21, 150.30, 155.95 (C]O), 163.08 (C]O),
164.68 (C]O); HRMS (ESI) Calcd for C14H11O4N3: 308.0642, Found:
m/z 308.0658 ([M þ Na])þ.
Pure product was obtained as a yellow solid in a yield of 69%,
mp 246e247 ꢁC; IR nmax (KBr, cmꢀ1): 1680 (C]O), 3260 (NH); 1H
NMR (300 MHz, CDCl3)
d ppm: 3.94 (s, 3H, OCH3), 6.45 (d,
4.1.10. 3-(5-Bromo-1H-indol-3-ylmethylene)-1,3-dihydroindol-2-
one, 5a
Pure product was obtained as a pale yellow solid in a yield of
76%, mp 270 ꢁC (decomposed); IR nmax (KBr, cmꢀ1): 1678 (C]O),
3210, 3408 (NH); 1H NMR (300 MHz, DMSO-d6)
d ppm: 6.83e
6.99 (m, 2H, ArH), 7.09e7.19 (m, 1H, ArH), 7.29e7.34 (m, 1H,
ArH), 7.42e7.49 (m, 1H, ArH), 7.72e7.86 (m, 2H, ArH), 8.12 (s, 1H,
bridged ]CH), 9.47 (s, 1H, indole 2-H), 10.42 (s, 1H, NH), 12.04
(s, 1H, NH); 13C NMR (DEPT-135) (CDCl3 þ DMSO-d6)
d ppm:
108.77 (þve, CH), 110.73 (C), 113.65 (þve, CH), 120.63 (þve,
CH), 124.61 (þve, CH), 124.93 (C), 125.38 (C), 126.13 (þve, CH),
126.53 (þve, CH), 128.08 (C), 129.80 (C), 134.25 (þve, CH),
139.09 (C), 141.63 (C), 168.05 (C]O); HRMS (ESI) Calcd for
C
17H13BrON2: 363.0103, 365.0084. Found: m/z 363.0131,
365.0089 ([M þ Na])þ.
4.1.11. 3-(5-Methoxy-1H-indol-3-ylmethylene)-1,3-dihydroindol-2-
one, 5b
Pure product was obtained as a yellow solid in a yield of 74%, mp
240 ꢁC (decomposed); IR nmax (KBr, cmꢀ1): 1678 (C]O), 3221, 3361
(NH); 1H NMR (300 MHz, DMSO-d6)
d ppm: 3.93 (s, 3H, OCH3),
6.87e7.12 (m, 4H, ArH), 7.37 (d, J ¼ 9.0 Hz, 2H, ArH), 7.68 (s, 1H,
ArH), 7.94 (s, 1H, bridged ]CH), 9.47 (s, 1H, indole 2-H), 10.13 (s, 1H,
NH), 11.52 (s, 1H, NH); HRMS (ESI) Calcd for C18H14O2N2: 291.1128.
Found: m/z 291.1146 ([M þ H])þ.
Fig. 4. CPK model of active site of DHFR with docking of compound 3b.

Shaveta, P. Singh / European Journal of Medicinal Chemistry 74 (2014) 440e450
447
4.1.12. 4-(1-Benzyl-5-bromo-1H-indol-3-ylmethylene)-2-(3-
chlorophenyl)-5-methyl-2,4-dihydropyrazol-3-one, 11
CH), 122.36 (þve, CH), 125.88 (þve, CH), 126.62 (þve, CH), 126.93
(C), 127.24 (þve, CH), 128.03 (þve, CH), 128.94 (þve, CH), 129.10
(C), 130.52 (þve, CH), 134.93 (C), 135.75 (C), 135.96 (C), 138.38 (C),
154.73 (C), 165.20 (C]O); HRMS (ESI) Calcd for C30H19BrCl2ON2:
573.0131, 575.0110, 577.0082 Found: m/z 573.0141, 575.0036,
576.9989 ([M þ H])þ.
Pure product was obtained as an orange solid in a yield of 69%,
mp 217e219 ꢁC; IR nmax (KBr, cmꢀ1): 1683 (C]O); 1H NMR
(300 MHz, CDCl3)
d ppm: 2.35 (s, 3H, CH3), 5.39 (s, 2H, CH2),
7.05e7.16 (m, 4H, ArH), 7.19 (s, 1H, ArH), 7.24e7.31 (m, 4H, ArH),
7.63 (s, 1H, ArH), 7.88 (d, J ¼ 8.1 Hz, 1H, ArH), 7.93 (s, 1H, ArH),
8.03 (s, 1H, bridged ]CH), 9.87 (s, 1H, indole 2-H); 13C NMR
4.1.17. 3-[5-Bromo-1-(4-chlorobenzoyl)-1H-indol-3-ylmethylene]-
1-(2,6-dichlorophenyl)-1,3-dihydroindol-2-one, 16
(DEPT-135) (CDCl3)
d
ppm: 13.15 (þve, CH3), 51.85 (ꢀve, CH2),
111.40 (C), 112.90 (þve, CH), 116.45 (C), 116.70 (þve, CH), 118.85
(þve, CH), 120.53 (C), 121.03 (þve, CH), 124.31 (þve, CH), 126.73
(þve, CH), 128.38 (þve, CH), 129.09 (þve, CH), 129.73 (þve, CH),
134.49 (C), 134.66 (þve, CH), 134.94 (C), 135.38 (C), 139.91 (C),
141.48 (C), 150.78 (C), 163.31 (C]O); HRMS (ESI) Calcd for
Pure product was obtained as a yellow solid in a yield of 70%,
mp 240 ꢁC (decomposed); IR nmax (KBr, cmꢀ1): 1677 (C]O); 1H
NMR (300 MHz, DMSO-d6)
d
ppm: 6.43 (d, J ¼ 6.6 Hz, 1H, ArH),
7.15 (s, 2H, ArH), 7.32 (d, J ¼ 8.1 Hz, 2H, ArH), 7.41 (d, J ¼ 8.4 Hz,
2H, ArH), 7.53 (d, J ¼ 7.5 Hz, 1H, ArH), 7.62 (d, J ¼ 7.8 Hz, 3H, ArH),
7.91e7.96 (m, 2H, ArH), 8.21 (s, 1H, bridged ]CH), 8.29 (s, 1H,
ArH), 9.43 (s, 1H, indole 2-H); 13C NMR (CDCl3 þ DMSO-d6)
C
26H19BrClON3: 526.0292, 528.0272, Found: m/z 526.0339,
528.0326 ([M þ Na])þ.
d
ppm: 107.85, 110.40, 113.28, 113.77, 116.89, 117.67, 119.82, 121.52,
4.1.13. 4-[5-Bromo-1-(4-chlorobenzoyl)-1H-indol-3-ylmethylene]-
2-(3-chlorophenyl)-5-methyl-2,4-dihydropyrazol-3-one, 12
124.42, 124.63, 126.22, 127.35, 128.23, 129.30, 129.45, 129.99,
130.08, 130.30, 134.27, 134.74, 134.92, 134.98, 138.03, 140.27,
164.98 (C]O), 167.00 (C]O); HRMS (ESI) Calcd for
Pure product was obtained as a reddish orange solid in a yield of
79%, mp >300 ꢁC; IR nmax (KBr, cmꢀ1): 1679, 1700 (C]O); 1H NMR
C
30H16BrCl3O2N2: 620.9533, 622.9513, 624.9485. Found m/z
(300 MHz, TFA þ CDCl3)
d ppm: 2.78 (s, 3H, CH3), 7.27e7.81 (m,10H,
ArH), 8.11e8.28 (m, 2H, ArH), 8.47 (s, 1H, ArH); HRMS (APCI) Calcd
617.0165, 619.0143, 621.0115.
for
C26H16N3O2BrCl2: 551.9876, 553.9855, 555.9827. Found:
4.1.18. 3-[5-Bromo-1-(2-fluorobenzyl)-1H-indol-3-ylmethylene]-1-
(2,6-dichlorophenyl)-1,3-dihydroindol-2-one, 17
551.9850, 553.9824, 555.9807 ([M þ H])þ.
Pure product was obtained as a yellow solid in a yield of 82%, mp
4.1.14. 4-[5-Bromo-1-(2-fluorobenzyl)-1H-indol-3-ylmethylene]-2-
(3-chlorophenyl)-5-methyl-2,4-dihydropyrazol-3-one, 13
280 ꢁC (decomposed); IR nmax (KBr, cmꢀ1): 1703 (C]O); 1H NMR
(300 MHz, CDCl3)
d ppm: 5.42 (s, 2H, CH2), 6.45e6.48 (dd,
Pure product was obtained as an orange solid in a yield of 85%,
J ¼ 2.4 Hz, J ¼ 6.6 Hz, 1H, ArH), 6.90 (t, J ¼ 7.2 Hz, 1H, ArH), 7.00 (t,
J ¼ 7.5 Hz, 1H, ArH), 7.04e7.10 (m, 1H, ArH), 7.15e7.25 (m, 4H, ArH),
7.34e7.41 (m, 2H, ArH), 7.54 (d, J ¼ 8.1 Hz, 2H, ArH), 7.74e7.76 (dd,
J ¼ 1.8 Hz, J ¼ 5.4 Hz, 1H, ArH), 7.97 (s, 1H, ArH), 8.08 (s, 1H, bridged
]CH), 9.58 (s, 1H, indole 2-H); HRMS (ESI) Calcd for
mp 238e240 ꢁC; IR nmax (KBr, cmꢀ1): 1673 (C]O); 1H NMR
(300 MHz, CDCl3)
d ppm: 2.43 (s, 3H, CH3), 5.51 (s, 2H, CH2),
7.00e7.15 (m, 4H, ArH), 7.24e7.42 (m, 4H, ArH), 7.70 (s, 1H, ArH),
7.96 (d, J ¼ 8.4 Hz, 1H, ArH), 8.01 (s, 1H, bridged ]CH), 8.09 (s,
1H, ArH), 9.96 (s, 1H, indole 2-H); 13C NMR (DEPT-135) (CDCl3)
C
30H18BrCl2FON2: 612.9856, 614.9835, 616.9807 Found: m/z
d
ppm: 13.16 (þve, CH3), 50.62 (ꢀve, CH2), 111.57 (C), 112.63 (þve,
612.9858, 614.9840, 616.9815 ([M þ Na])þ.
CH), 116.60 (C), 116.95 (þve, CH), 119.09 (þve, CH), 120.60 (C),
121.09 (þve, CH), 122.52 (C), 124.53 (C), 124.83 (þve, CH), 126.94
(þve, CH), 128.86 (C), 129.79 (þve, CH), 130.36 (C), 130.90 (C),
134.51 (C), 134.94 (þve, CH), 135.29 (C), 138.05 (C), 139.76 (C),
141.55 (þve, CH), 150.92 (þve, CH), 163.26 (C]O); HRMS (ESI)
4.1.19. 5-[5-Bromo-1-(2-fluorobenzyl)-1H-indol-3-ylmethylene]
pyrimidine-2,4,6-trione, 18
Pure product was obtained as a yellow solid in a yield of 80%, mp
>300 ꢁC; IR nmax (KBr, cmꢀ1): 1654, 1685, 1724 (C]O), 3189, 3431
Calcd for
C26H18BrClFON3: 544.0198, 546.0178, Found: m/z
(NH); 1H NMR (300 MHz, DMSO-d6)
d ppm: 5.67 (s, 2H, CH2), 7.14e
543.9566, 545.9546 ([M þ Na])þ.
7.23 (m, 2H, ArH), 7.29e7.39 (m, 2H, ArH), 7.45 (d, J ¼ 8.4 Hz, 1H,
ArH), 7.65 (d, J ¼ 8.7 Hz, 1H, ArH), 8.02 (s, 1H, bridged ]CH), 8.60 (s,
1H, ArH), 9.60 (s, 1H, indole 2-H), 11.10 (s, 1H, NH), 11,18 (s, 1H, NH);
HRMS (ESI) Calcd for C20H13BrFO3N3: 442.0197, 444.0178. Found:
m/z 442.0379, 444.0354 ([M þ H])þ.
4.1.15. 4-{5-Bromo-1-[1-(2,6-dichlorophenyl)-vinyl]-1H-indol-3-
ylmethylene}-2-(3-chloro-phenyl)-5-methyl-2,4-dihydropyrazol-3-
one, 14
Pure product was obtained as an orange solid in a yield of 73%,
mp 265 ꢁC; IR nmax (KBr, cmꢀ1): 1687, 1714 (C]O); 1H NMR
(300 MHz, CDCl3)
d
ppm: 2.39 (s, 3H, CH3), 7.12 (d, J ¼ 7.5 Hz, 1H,
4.1.20. 3-(1-Benzyl-5-bromo-1H-indol-3-ylmethylene)-1,3-
dihydroindol-2-one, 19
ArH), 7.30 (d, J ¼ 8.1 Hz, 1H, ArH), 7.52e7.56 (m, 4H, ArH), 7.66 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.78 (d, J ¼ 7.5 Hz, 1H, ArH), 7.96 (s, 2H, bridged
]CH, ArH), 8.59 (d, J ¼ 8.4 Hz, 1H, ArH), 9.53 (s, 1H, indole 2-H);
Pure product was obtained as a yellow solid in a yield of 77%,
mp 280e281 ꢁC; IR nmax (KBr, cmꢀ1): 1678 (C]O), 3200 (NH); 1
H
HRMS (ESI) Calcd for
C
26H15BrCl3O2N3: 585.9486, 587.9465,
NMR (300 MHz, DMSO-d6) d ppm: 5.56 (s, 2H, CH2), 6.84 (d,
589.9437. Found: m/z 585.9502, 587.9491, 589.9482 ([M þ H])þ.
J ¼ 7.5 Hz, 1H, ArH), 6.97 (t, J ¼ 7.5 Hz, 1H, ArH), 7.13 (t,
J ¼ 7.2 Hz, 1H, ArH), 7.23e7.34 (m, 6H, ArH), 7.49 (d, J ¼ 8.4 Hz,
1H, ArH), 7.92 (d, J ¼ 7.2 Hz, 1H, ArH), 8.09 (s, 1H, bridged ]CH),
8.46 (s, 1H, ArH) 9.55 (s, 1H, indole 2-H), 10.48 (s, 1H, NH); 13C
4.1.16. 3-(1-Benzyl-5-bromo-1H-indol-3-ylmethylene)-1-(2,6-
dichlorophenyl)-1,3-dihydro-indol-2-one, 15
Pure product was obtained as a yellow solid in a yield of 73%,
NMR (DEPT-135) (CDCl3 þ DMSO-d6)
d
ppm: 50.06 (ꢀve, CH2),
mp 140 ꢁC; IR nmax (KBr, cmꢀ1): 1701 (C]O); 1H NMR (300 MHz,
108.86 (þve, CH), 110.55 (þve, CH), 112.65 (þve, CH), 114.16 (C),
118.98 (þve, CH), 120.35 (þve, CH), 121.33 (C), 125.04 (C), 125.36
(þve, CH), 125.64 (þve, CH), 126.86 (þve, CH), 127.55 (þve, CH),
128.53 (þve, CH), 130.63 (C), 134.51 (C), 136.57 (C), 136.94 (þve,
CH), 139.32 (C), 167.94 (C]O); HRMS (ESI) Calcd for
CDCl3)
d
ppm: 5.37 (s, 1H, CH2), 6.47 (d, J ¼ 7.2 Hz, 1H, ArH), 7.12e
7.18 (m, 6H, ArH), 7.28e7.41 (m, 4H, ArH), 7.55 (d, J ¼ 7.8 Hz, 2H,
ArH), 7.76 (d, J ¼ 6.3 Hz, 1H, ArH), 7.99 (s, 1H, ArH), 8.08 (s, 1H,
bridged ]CH), 9.58 (s, 1H, indole 2-H); 13C NMR (DEPT-135)
(CDCl3)
d
ppm: 51.44 (ꢀve, CH2), 108.79 (C), 110.91 (C), 112.41
C24H17BrON2: 429.0597, 431.0578. Found: m/z 429.0659,
(þve, CH), 115.23 (C), 118.38 (þve, CH), 118.66 (C), 121.01 (þve,
431.0641 ([M þ H])þ.

448
Shaveta, P. Singh / European Journal of Medicinal Chemistry 74 (2014) 440e450
4.1.21. 3-[5-Bromo-1-(4-chlorobenzoyl)-1H-indol-3-ylmethylene]-
1,3-dihydroindol-2-one, 20
128.82, 131.03, 136.10, 142.05, 143.08, 150.36, 156.48 (C]O),
163.18 (C]O), 164.50 (C]O); HRMS (ESI) Calcd for C21H17O4N3:
398.1111. Found: m/z 398.1128 ([M þ Na])þ.
Pure product was obtained as a yellow solid in a yield of 65%, mp
280 ꢁC (decomposed); IR nmax (KBr, cmꢀ1): 1683 (C]O), 3254 (NH);
1H NMR (300 MHz, DMSO-d6)
d
ppm: 6.87 (t, J ¼ 7.2 Hz, 1H, ArH),
4.1.26. 3-(1-Benzyl-5-methoxy-1H-indol-3-ylmethylene)-1,3-
dihydroindol-2-one, 25
6.98 (t, J ¼ 7.8 Hz, 1H, ArH), 7.12 (t, J ¼ 7.5 Hz, 1H, ArH), 7.30 (s, 1H,
ArH), 7.39 (d, J ¼ 8.4 Hz, 1H, ArH), 7.67e7.89 (m, 4H, ArH), 8.01 (s,
1H, bridged ]CH), 8.10 (s, 1H, ArH), 9.47 (s, 1H, indole 2-H), 10.15 (s,
1H, ArH), 11.77 (s, 1H, NH); HRMS (ESI) Calcd for C24H14BrClO2N2:
498.9819, 500.9799 Found: m/z 498.9612, 500.9597 ([M þ Na])þ.
Pure product was obtained as a yellow solid in a yield of 67%, mp
246 ꢁC; IR nmax (KBr, cmꢀ1): 1680 (C]O), 3200 (NH); 1H NMR
(300 MHz, CDCl3) d ppm: 3.91 (s, 3H, OCH3), 5.38 (s, 2H, CH2), 6.85 (d,
J ¼ 7.5 Hz, 2H, ArH), 7.05 (t, J ¼ 7.5 Hz, 1H, ArH), 7.13e7.33 (m, 8H,
ArH), 7.62 (d, J ¼ 7.2 Hz,1H, ArH), 7.70 (s,1H, ArH), 7.90 (s,1H, bridged
]CH), 9.49 (s,1H, indole 2-H); 13C NMR (DEPT-135) (CDCl3 þ DMSO-
4.1.22. 3-[5-Bromo-1-(2,6-dichlorobenzoyl)-1H-indol-3-
ylmethylene]-1,3-dihydroindol-2-one, 21
d6)
d
ppm: 50.61 (ꢀve, CH2), 55.37 (þve, CH3), 100.08 (þve, CH),
Pure product was obtained as a yellow solid in a yield of 86%, mp
108.82 (þve, CH),110.54 (C),111.03 (þve, CH), 111.96 (þve, CH),117.40
(þve, CH), 118.76 (C), 120.27 (þve, CH), 125.42 (C), 125.64 (þve, CH),
126.19 (þve, CH), 126.28 (þve, CH), 127.29 (þve, CH), 128.26 (þve,
CH), 129.63 (C), 130.70 (C), 135.82 (C), 136.76 (þve, CH), 138.59 (C),
155.08 (C), 168.34 (C]O); HRMS (ESI) Calcd for C25H20O2N2:
403.1417. Found: m/z 403.1439 ([M þ Na])þ.
270 ꢁC (decomposed); IR nmax (KBr, cmꢀ1): 1709 (C]O), 3190 (NH);
1H NMR (400 MHz, DMSO-d6)
d
ppm: 6.76 (t, J ¼ 7.6 Hz, 1H, ArH),
6.84 (t, J ¼ 8.0 Hz, 1H, ArH), 7.16e7.22 (m, 1H, ArH), 7.39 (d,
J ¼ 7.6 Hz, 1H, ArH), 7.58 (s, 1H, ArH), 7.64e7.78 (m, 4H, ArH), 7.93e
8.05 (m, 1H, ArH), 8.47e8.52 (m, 1H, ArH), 9.25 (s, 1H, indole 2-H),
10.55 (s, 1H, NH); HRMS (ESI) Calcd for C24H13BrCl2O2N2: 510.9610,
512.9589, 514.9561 Found: m/z 510.9830, 512.9607, 514.9832
([M þ H])þ.
4.1.27. 1-(2,6-Dichlorophenyl)-3-(2-trifluoromethylbenzylidene)-
1,3-dihydroindol-2-one, 27
Pure product was obtained as a light yellow solid in a yield of
4.1.23. 4-(1-Benzyl-5-methoxy-1H-indol-3-ylmethylene)-2-(3-
chlorophenyl)-5-methyl-2,4-dihydropyrazol-3-one, 22
84%, mp 134e135 ꢁC; IR nmax (KBr, cmꢀ1): 1658 (C]O); 1H NMR
(300 MHz, CDCl3)
d
ppm: 6.41 (d, J ¼ 7.8 Hz, 1H, ArH), 6.85 (t,
Pure product was obtained as a yellow solid in a yield of 70%, mp
J ¼ 7.5 Hz, 1H, ArH), 7.07 (d, J ¼ 7.8 Hz, 1H, ArH), 7.18 (t, J ¼ 7.8 Hz,
1H, ArH), 7.37e7.42 (m, 1H, ArH), 7.52e7.67 (m, 4H, ArH), 7.82 (t,
J ¼ 7.8 Hz, 2H, ArH), 8.09 (s, 1H, bridged ]CH); HRMS (ESI) Calcd for
181e183 ꢁC; IR nmax (KBr, cmꢀ1): 1676 (C]O); 1H NMR (300 MHz,
CDCl3) d ppm: 2.35 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 5.43 (s, 2H, CH2),
7.17e7.21 (m, 4H, ArH), 7.25 (s, 1H, ArH), 7.29e7.37 (m, 4H,
ArH), 7.75 (s, 1H, ArH), 7.87 (d, J ¼ 8.1 Hz, 1H, ArH), 7.99 (s, 1H,
bridged ]CH), 8.10 (s, 1H, ArH), 9.93 (s, 1H, indole 2-H); 13C NMR
C22H12Cl2F3ON: 456.0140, 458.0112. Found: m/z 456.0136, 458.0108
([M þ Na])þ.
(DEPT-135) (CDCl3)
d
ppm: 13.15 (þve, CH3), 51.85 (ꢀve, CH2), 55.75
4.1.28. 2-(3-Chlorophenyl)-5-methyl-4-(2-
(þve, CH3), 110.84 (C), 112.90 (þve, CH), 116.45 (C), 116.66 (þve, CH),
118.82 (þve, CH), 120.90 (þve, CH), 121.03 (C), 124.29 (þve, CH),
126.66 (þve, CH), 127.48 (C), 128.33 (þve, CH), 129.09 (þve, CH),
134.49 (C),134.68 (þve, CH),134.94 (C),136.14 (þve, CH),139.75 (C),
141.48 (C), 150.33 (C), 162.91 (C]O); HRMS (ESI) Calcd for
trifluoromethylbenzylidene)-2,4-dihydropyrazol-3-one, 28
Pure product was obtained as an orange solid in a yield of 81%,
mp 103e104 ꢁC; IR nmax (KBr, cmꢀ1): 1701 (C]O); 1H NMR
(300 MHz, CDCl3)
d
ppm: 2.36 (s, 3H, CH3), 7.14 (d, J ¼ 7.5 Hz, 1H,
ArH), 7.32 (d, J ¼ 7.8 Hz, 1H, ArH), 7.61e7.70 (m, 2H, ArH), 7.77e7.86
C
27H22ClO2N3: 478.1293, 480.1266. Found: m/z 478.1300, 480.1280
(m, 3H, ArH), 8.00 (s,1H, bridged ]CH), 8.43 (d, J ¼ 7.8 Hz,1H, ArH);
([M þ Na])þ.
13C NMR (DEPT-135) (CDCl3)
d
ppm: 13.53 (þve, CH3), 107.64 (C),
116.89 (þve, CH), 119.04 (þve, CH), 125.31 (þve, CH), 126.52 (þve,
CH), 129.94 (þve, CH), 130.29 (C), 131.87 (þve, CH), 131.95 (C),
132.99 (þve, CH),135.02 (C),139.49 (C),142.37 (þve, CH), 151.30 (C),
161.51 (C]O); HRMS (ESI) Calcd for C18H12ClF3ON2: 387.0482
Found: m/z 387.0491 ([M þ Na])þ.
4.1.24. 3-(1-Benzyl-5-methoxy-1H-indol-3-ylmethylene)-1-(2,6-
dichlorophenyl)-1,3-dihydro-indol-2-one, 23
Pure product was obtained as a yellow solid in a yield of 60%, mp
153e155 ꢁC; nmax (KBr, cmꢀ1): 1688 (C]O); 1H NMR (300 MHz,
CDCl3)
d ppm: 3.94 (s, 3H, OCH3), 5.36 (s, 2H, CH2), 6.46e6.49 (m,
1H, ArH), 6.85e6.89 (dd, J ¼ 2.1 Hz, J ¼ 8.7 Hz, 1H, ArH), 7.14e7.18
(m, 6H, ArH), 7.23e7.30 (m, 2H, ArH), 7.38e7.40 (m, 2H, ArH), 7.52
(s, 1H, ArH), 7.55 (s, 1H, ArH), 7.75e7.78 (m, 1H, ArH), 8.06 (s, 1H,
4.1.29. 1-(4-Chlorobenzoyl)-3-(2-trifluoromethylbenzylidene)-1,3-
dihydroindol-2-one, 29
Pure product was obtained as a light yellow solid in a yield of
bridged ]CH), 9.58 (s, 1H, indole 2-H); 13C NMR (CDCl3)
d ppm:
61%, mp 199e200 ꢁC; IR nmax (KBr, cmꢀ1): 1682, 1722 (C]O); 1H
50.99 (CH2), 55.50 (CH3), 100.20, 108.22, 110.79, 111.33, 112.10,
116.62, 117.60, 121.70, 125.01, 126.21, 126.26, 127.40, 127.55, 128.37,
128.47, 129.99, 130.80, 130.87, 135.53, 135.69, 137.84, 138.36, 155.35,
165.63 (C]O); HRMS (ESI) Calcd for C31H22Cl2O2N2: 547.0951,
549.0923 Found: m/z 547.1022, 549.0966 ([M þ Na])þ.
NMR (300 MHz, CDCl3)
d
ppm: 6.98 (t, J ¼ 7.5 Hz, 1H, ArH), 7.09 (d,
J ¼ 7.8 Hz, 1H, ArH), 7.36 (t, J ¼ 7.8 Hz, 1H, ArH), 7.49 (d, J ¼ 8.1 Hz,
2H, ArH), 7.57e7.76 (m, 5H, ArH), 7.83 (d, J ¼ 7.5 Hz, 1H, ArH), 7.91
(d, J ¼ 8.1 Hz, 1H, ArH), 7.96 (s, 1H, bridged ]CH); 13C NMR (DEPT-
135) (CDCl3)
d
ppm: 106.53 (C), 115.54 (þve, CH), 121.60 (C), 122.90
(þve, CH), 124.57 (þve, CH), 126.60 (þve, CH), 128.12 (C), 128.54
(þve, CH), 129.60 (C), 129.68 (þve, CH), 130.67 (þve, CH), 130.86
(þve, CH), 132.02 (þve, CH), 132.68 (C), 133.10 (C), 134.88 (þve, CH),
139.24 (C), 140.58 (C), 166.74 (C]O), 168.22 (C]O); HRMS (ESI)
Calcd for C23H13ClF3O2N: 450.0479, 452.0451. Found: m/z 450.0913,
452.0892 ([M þ Na])þ.
4.1.25. 5-(1-Benzyl-5-methoxy-1H-indol-3-ylmethylene)
pyrimidine-2,4,6-trione, 24
Pure product was obtained as a yellow solid in a yield of 82%,
mp >300 ꢁC; IR nmax (KBr, cmꢀ1): 1649, 1678, 1719 (C]O), 3167,
3199 (NH); 1H NMR (300 MHz, DMSO-d6)
d ppm: 3.85 (s, 3H,
OCH3), 5.61 (s, 2H, CH2), 6.92 (dd, J ¼ 2.4 Hz, J ¼ 9.0 Hz, 1H, ArH),
7.28e7.37 (m, 6H, ArH), 7.55 (d, J ¼ 8.7 Hz, 1H, ArH), 8.62 (s, 1H,
bridged ]CH), 9.53 (s, 1H, indole 2-H), 10.99 (s, 1H, NH), 11.05 (s,
4.1.30. 1-(2-Fluorobenzyl)-3-(2-trifluoromethylbenzylidene)-1,3-
dihydroindol-2-one, 30
1H, NH); 13C NMR (DMSO-d6)
d
ppm: 50.53 (CH2), 55.57
Pure product was obtained as a yellow solid in a yield of 73%, mp
(CH3), 100.29, 108.21, 110.85, 113.04, 113.35, 127.52, 127.99,
135e136 ꢁC; IR nmax (KBr, cmꢀ1): 1649 (C]O); 1H NMR (300 MHz,

Shaveta, P. Singh / European Journal of Medicinal Chemistry 74 (2014) 440e450
449
CDCl3)
d
ppm: 5.05 (s, 2H, CH2), 6.79 (t, J ¼ 8.1 Hz, 2H, ArH), 6.96 (d,
parameters if the level of activity was reached; however, if the ef-
fect was not reached or exceeded, the value for that parameter was
expressed as greater or less than the maximum or minimum con-
centration tested.
J ¼ 7.2 Hz, 1H, ArH), 7.06e7.19 (m, 3H, ArH), 7.23 (s, 1H, ArH), 7.34 (t,
J ¼ 7.2 Hz, 1H, ArH), 7.53e7.64 (m, 2H, ArH), 7.70 (d, J ¼ 6.9 Hz, 1H,
ArH), 7.81 (d, J ¼ 7.5 Hz, 1H, ArH), 8.04 (s, 1H, bridged ]CH); HRMS
(ESI) Calcd for
C23H15F3ON: 420.0982. Found: m/z 420.1032
([M þ Na])þ.
4.3. UVevis spectral studies
UVevisible spectral studies were performed on Biotek power-
waveXS spectrophotometer. The stock solution for both the com-
pounds and the enzyme were prepared in HPLC grade DMSO. The
conc of the compound stock solution was kept at 10ꢀ3 M and was
4.2. Procedure for in vitro anticancer screening
Anticancer screening of compounds was carried out at Na-
tional Cancer Institute, Bethesda, MD, USA as per the standard
procedure [20]. Human tumor cell lines of the cancer screening
panel were grown in RPMI 1640 medium containing 5% fetal
diluted accordingly to get the final conc of 1
mMe5
mM. 50 mL of the
enzyme was diluted to 1000
compound solution.
mL (2.89 mM) for further titration with
bovine serum and 2 mM
96 well microtiter plates in 100
L
-glutamine. Cells were inoculated into
L at plating densities ranging
m
4.4. Procedure for dihydrofolate reductase inhibition assay
from 5000 to 40,000 cells/well depending on the doubling time
of individual cell lines. After cell inoculation, the microtiter plates
were incubated at 37 ꢁC, 5% CO2, 95% air and 100% relative hu-
midity for 24 h prior to addition of experimental drugs. After
24 h, two plates of each cell line were fixed in situ with TCA, to
represent a measurement of the cell population for each cell line
at the time of compound addition (Tz). Compounds were solu-
bilized in dimethyl sulfoxide at 400-fold the desired final
maximum test concentration and stored frozen prior to use. At
the time of compound addition, an aliquot of frozen concentrate
was thawed and diluted to twice the desired final maximum test
The dihydrofolate reductase inhibition assay was performed as
per the manual of DHFR assay kit (SIGMA Product Code CS0340). All
the dilutions were made in assay buffer pH 7.5. 10 mM stock so-
lutions of dihydrofolic acid and NADPH in assay buffer were pre-
pared. Stock solutions of the test compounds with different conc
were prepared in DMSO and 20 mL of each were taken to attain final
conc of 10ꢀ8 M, 10ꢀ7 M, 10ꢀ6 M and 10ꢀ5 M in the respective wells
of 96 well plate containing assay buffer. 0.1 units of DHFR as sup-
plied in the kit were diluted and 20
used in each reaction. Each well of the 96 well plate was charged
m
L of its 3 ꢃ 10ꢀ3 units were
concentration with complete medium containing 50
mg/ml
with 157.8
each well except 1A, 1B (blank well). 20
(including methotrexate as positive control) was added to each well
except 1Ae1H. Reaction was started by the addition of 1 L of
dihydrofolic acid to each well except 1C and 1D. 1G and 1H con-
tained 20 L of DMSO to check any inhibition of enzyme activity
mL assay buffer. 1.2
m
L of NADPH solution was added to
gentamicin. Additional four, 10-fold or ½ log serial dilutions were
made to provide a total of five compound concentrations plus
mL of test compound
control. Aliquots of 100
added to the appropriate microtiter well already containing
100 L of medium, resulting in the required final concentrations.
mL of these different drug dilutions were
m
m
m
Following compound addition, the plates were incubated for an
additional 48 h at 37 ꢁC, 5% CO2, 95% air, and 100% relative hu-
midity. For adherent cells, the assay was terminated by the
addition of cold TCA. Cells were fixed in situ by the gentle
due to DMSO. The change in absorbance at 340 nm was monitored
as a function of time. Percentage inhibition of enzymatic activity
was calculated after nullifying the effects of NADPH, folate and
solvent. IC50 was calculated by plotting a graph between percentage
inhibitions and corresponding conc of the compound using
Graphpad Prism version 6.01.
addition of 50 mL of cold 50% (w/v) TCA (final concentration, 10%
TCA) and incubated for 60 min at 4 ꢁC. The supernatant was
discarded, and the plates were washed five times with tap water
and air dried. Sulforhodamine B (SRB) solution (100 mL) at 0.4%
4.5. Molecular docking
(w/v) in 1% acetic acid was added to each well, and plates were
incubated for 10 min at room temperature. After staining, un-
bound dye was removed by washing five times with 1% acetic
acid and the plates were air dried. Bound stain was subsequently
solubilized with 10 mM trizma base, and the absorbance was
read on an automated plate reader at a wavelength of 515 nm.
Using the absorbance measurements [time zero, (Tz), control
growth, (C), and test growth in the presence of compound at five
concentration levels (Ti)], the percentage growth was calculated
at each of the compound concentrations. Percentage growth in-
hibition was calculated as
Compounds were built using the builder tool kit of the software
package Argus Lab 4.0.1 [21] and energy minimized with semi-
empirical quantum mechanical method PM3. Crystal co-ordinates
of DHFR (PDB ID 3GHW) was downloaded from protein data bank
and in the molecule tree view of the software, the monomeric
structures of the crystal coordinate was selected and the active site
ꢀ
was defined as 15 A around the ligand.
The molecule to be docked in the active site of the enzyme was
pasted in the work space carrying the structure of the enzyme. The
docking programme implements an efficient grid based docking
algorithm which approximates an exhaustive search within the free
volume of the binding site cavity. The conformational space was
explored by the geometry optimization of the flexible ligand (rings
were treated as rigid) in combination with the incremental con-
struction of the ligand torsions. Thus, docking occurs between the
flexible ligand parts of the compound and enzyme. Docking was
repeated several times (approx.10000 iterations) until no change in
position of the ligand and a constant value of binding energy was
observed. The ligand orientation was determined by a shape
scoring function based on Ascore and final positions were ranked
by lowest interaction energy values. H-bonds and hydrophobic
interactions between the respective compound and enzyme were
explored.
½ðTi ꢀ TzÞ=ðC ꢀ TzÞꢂ ꢃ 100 for conc for which Ti ꢄ Tz
½ðTi ꢀ TzÞ=Tzꢂ ꢃ 100 for conc for which Ti < Tz
Growth inhibition of 50% (GI50) was calculated from [(Ti ꢀ Tz)/
(C ꢀ Tz)] ꢃ 100 ¼ 50, which was the compound concentration
resulting in
a 50% reduction in the net protein increase
(as measured by SRB staining) in control cells during the drug in-
cubation. The LC50 (concentration of drug resulting in a 50%
reduction in the measured protein at the end of the drug treatment
as compared to that at the beginning) indicating a net loss of
cells following treatment was calculated from [(Ti
Tz] ꢃ 100 ¼ ꢀ50. Values were calculated for each of these two
ꢀ
Tz)/

450
Shaveta, P. Singh / European Journal of Medicinal Chemistry 74 (2014) 440e450
[5] J.J. Guidry, A. Greisinger, L.A. Aday, R.J. Winn, S. Vernon, T.A. Throckmorton,
Acknowledgments
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[8] P.A. Bunn Jr., K. Kelly, Clin. Cancer Res. 4 (1998) 1087e1100.
[9] P. Singh, A. Bhardwaj, Mini Rev. Med. Chem. 8 (2008) 388e398 (and refer-
ences therein).
[10] W.B. Parker, Chem. Rev. 109 (2009) 2880e2893.
[11] J.L. Grem, Invest. New Drugs 18 (2000) 299e313.
[12] F.M. Huennekens, Adv. Enzyme Regul. 34 (1994) 397e419.
[13] S. Chattopadhyay, R.G. Moran, I.D. Goldman, Mol. Cancer Ther. 6 (2007) 404e
417.
Financial assistance from CSIR, New Delhi and DST, New Delhi is
gratefully acknowledged. Shaveta thanks UGC for fellowship. Na-
tional Cancer Institute, Bethesda USA is acknowledged for anti-
cancer screening of compounds. Authors gratefully acknowledge
the contributions of Prof. A.S. Brar, vice-chancellor, GNDU for
creating state of the art research facilities.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.12.047.
[14] P. Singh, M. Kaur, P. Verma, Bioorg. Med. Chem. Lett. 19 (2009) 3054e
3058.
[15] P. Singh, M. Kaur, W. Holzer, Eur. J. Med. Chem. 45 (2010) 4968e4982.
[16] P. Singh, M. Kaur, Shaveta, J. Med. Chem. 55 (2012) 6381e6390.
[17] P. Singh, A. Bhardwaj, J. Med. Chem. 53 (2010) 3707e3717.
[18] Dihydrofolate Reductase Inhibition Assay Kit. SIGMA Product Code CS0340.
[19] A. Gangjee, W. Li, R.L. Kisliuk, V. Cody, J. Race, J. Piriano, J. Makin, J. Med. Chem.
52 (2009) 4892e4902.
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