One pot synthesis of N-monoalkylated plinabulin derivatives via multicomponent protocol and their application as anticancer agents

Article abstract of DOI:10.1016/j.molstruc.2020.129830

A chemical library of seventeen N-monoalkylated plinabulin derivatives was designed and synthesized in one pot with high atom economy, good yield and operational simplicity in site selective manner with Z-configuration at 3,6-positions (3Z,6Z) which have

Full text of DOI:10.1016/j.molstruc.2020.129830

Journal of Molecular Structure 1229 (2021) 129830
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
One pot synthesis of N-monoalkylated plinabulin derivatives via
multicomponent protocol and their application as anticancer agents
Asha Ganeshera, Priyank Chaturvedib, Bidhu Bhusan Karkaraa, Indranil Chatterjeea,
Dipak Dattab, Gautam Pandaa,∗
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
b Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A chemical library of seventeen N-monoalkylated plinabulin derivatives was designed and synthesized in
one pot with high atom economy, good yield and operational simplicity in site selective manner with Z-
configuration at 3,6-positions (3Z,6Z) which have been explained through 2D-NOESY analysis. Two com-
pounds having very high Selectivity Index (SI), exhibited potent activity against DLD-1 cancer cell with
IC50 of 1.25 μM and 1.20 μM respectively.
Received 11 August 2020
Revised 12 December 2020
Accepted 14 December 2020
Available online 27 December 2020
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
phenylahistin, a fungal metabolite from Aspergillus ustus was dis-
covered during screening for new cell cycle inhibitors. [4] It is a
Cancer is a major public health problem across the world. Glob-
ally in 2018, there were 18.1 million new cancer cases and 9.6
million deaths were reported. [1] Cancer causing agents can be
present in food, water, and air; so to bring cancer under control
is a much more challenging job. Traditional antitumor drugs gen-
erally result in extremely side effects because of missing targeted
therapies. [2] Russo et al. in 2015, showed how marine habitat
is a source of very potent anticancer agents that may inhibit the
growth of human cancer cell. Most of the drugs for cancer treat-
ment are natural products or natural products derived pharma-
cophores which have fewer side effects. Thus, the natural products
have the influence on anticancer drug discovery and design. [3]
new class of colchicines-like microtubule binding agents consisting
of L-phenylalanine and an isoprenylated dehydrohistidine residue
with a quaternary carbon at the 5-position of the imidazole ring.
[5] It exhibits cytotoxic activity against a wide variety of tumor cell
lines. Plinabulin is a synthetic analogue of the natural diketopiper-
azine phenylahistin and acts as a potent vascular disrupting agent
(VDA) against tubulin depolymerisation (IC50= 9.8~18 nM) and cur-
rently is in phase III clinical trial for treatment of non-small cell
lung cancer. [6]
Some recent studies have shown the good promising activity of
plinabulin derivatives with modification at the phenyl ring. [7,8]
On the basis of this information, and to better understand the
2,5-diketopiperazine is an important pharmacophore in various
natural products and biologically active entities. In this respect,
pharmacophore of plinabulin, we thought it worthwhile to pur-
sue modification on the imidazole part (Fig. 1). For further ratio-
nal drug design, we focused on the design, synthesis and biologi-
cal evaluation of some analogues of plinabulin based on the struc-
ture activity relationship studies of earlier known libraries in one
∗
Corresponding author.
E-mail address: gautam_panda@cdri.res.in (G. Panda).
https://doi.org/10.1016/j.molstruc.2020.129830
0022-2860/© 2020 Elsevier B.V. All rights reserved.

A. Ganesher, P. Chaturvedi, B.B. Karkara et al.
Journal of Molecular Structure 1229 (2021) 129830
Fig 1. Approach towards designing of plinabulin analogs.
ing the order of the aldehyde addition, highly selective geometri-
cal isomers (3Z,6Z) could be achieved in 2,5-DKPs through one-pot
multicomponent protocol.
The result exposed that equiv. of aldehydes used and the tem-
perature had a prominent effect on the yield of the desired product
(Table 1). Firstly, 1 equiv. of benzaldehyde and 1 equiv. of hetero-
aromatic aldehyde were used but we got the two products 4d and
4d́ (Table 1, entry 1). So, in order to maintain the site selectivity
at 3- and 6-positions (3Z,6Z), 2 equiv. of hetero-aromatic aldehyde
and only 1 equiv. of benzaldehyde were used to avoid the conden-
sation of same aldehyde (benzaldehyde) at both 3-and 6-position.
The order of addition was also revised by adding the second alde-
hyde i.e. hetero-aromatic aldehyde after the complete condensa-
tion of first aldehyde (benzaldehyde) with 1,4-diacetyl-2,5-DKP fol-
lowed by methylation (Table 1, entry 2). The first aldol condensa-
tion and methylation were done at -10 ˚C and then heated at 95˚C
for 4 h for second aldol condensation but the yield was very low,
perhaps due to the slow attack of hetero-aromatic aldehyde for
second condensation reaction (Table 1, entry 2). Varying the reac-
tion temperature from 95˚C to 110˚C indicated that 110 ˚C was best
for the second aldol condensation (Table 1, entry 3, 4). With the
optimized conditions in hand, the scope to generate various func-
tionalized plinabulin derivatives was explored (Figure 2). A library
of 17 derivatives was prepared from this one pot protocol.
Scheme 1. Standard protocol for synthesis of Plinabulin derivatives.
pot via multicomponent approach with highly selective geometri-
cal isomers.
2. Results and discussion
2.1. Synthesis
The literature survey revealed that most of the libraries in-
volved the modification in the left-hand unit of the structure
like phenyl group replacement by the ferrocenyl moiety, [9] ben-
zophenone moiety, [10,11] phenoxy [12] and various substituted
phenyl ring [13] and all the modifications was well tolerated
with good efficacy. But the right-hand unit was kept intact as
imidazole moieties in most of the cases. So, our strategy was
to replace the imidazole group by the various 5-membered, 6-
membered and bicyclic hetero-aromatic moieties to synthesize the
various plinabulin analogues. Synthesis of the designed compounds
was performed by standard procedure (Scheme1). [14] The com-
mercially available glycine anhydride was heated under reflux in
acetic anhydride overnight afforded intermediate 1, which under-
goes first condensation reaction with benzaldehyde in the pres-
ence of Cs2CO3 as a base in dry DMF at room temperature fur-
nished compound 2 in 81% yield. Compound 2 was then treated
with CH3I in the presence of K2CO3 in dry DMF to yield methyl
protected compound 3 in 65% yields. Next, the compound 3 was
used for the second condensation reaction with a variety of hetero-
aromatic aldehydes and ketones in the presence of Cs2CO3 as a
base in dry DMF under an argon atmosphere to afford our de-
sired compounds in 31- 40% yield. But there were some problems
regarding this protocol, firstly, the purification of the intermedi-
ate 2 was troublesome due to the enolization of the amide group
and additionally, the overall yield of the target compounds was
very low. So, with the above questions and the previous litera-
ture report in mind, [15] the reaction was performed in one pot
by using 1,4- diacetyl-2,5-DKP (1.0 eq.), Cs2CO3 (2.5 eq.), methyl
iodide (2.5 eq.), benzaldehyde (1.0 eq.), and after hetero-aromatic
aldehyde (2.0 eq.) in dry DMF is added into reaction with the in-
termediate formed which is not isolated from the reaction mix-
ture (Scheme 2). By controlling the reaction temperature and alter-
2.2. Standard protocol
2.2.1. One-pot multicomponent reaction
2.2.1.1. Structural elucidation of representative compound 4d. The
NOESY analysis gave observed NOE between the N-Me pro-
ton in the ꢀPhe residue (δ 2.92) and the phenyl proton (δ
7.19), and not between the N-Me proton and the olefinic pro-
ton, indicating the geometry of the double bond in the ꢀPhe
residue to be Z. On the other hand, the N-H proton in the
ꢀThiophene residue (δ 7.84) gave NOE with the thiophene pro-
ton (δ 6.97) and not with the olefinic proton (δ 7.04). This data
indicates the geometry of the double bond in the ꢀThiophene
residue to be Z. The representative compound 4d was identified
from these data as (Z)-6-((Z)-benzylidene)-3-((5-bromothiophen-
2-yl)methylene)-1-methylpiperazine 2,5-dione (Fig. 3). The 2D-
NOESY data is mentioned in the supplementary material.
2.3. Cytotoxic activity
The methyl protected plinabulin derivatives 4a-q were synthe-
sized and their anti-proliferative activities were evaluated against
DLD-1 colon cancer cell line. Detailed results of these investiga-
tions in terms of percentage inhibitions of cell growth at 10 μM of
each compound are given in Table 2.
2.3.1. Structure-activity relationship (SAR) study
In the literature, [14] it was mentioned that methyl protection
improves the solubility and phenyl group was present in almost all
2

A. Ganesher, P. Chaturvedi, B.B. Karkara et al.
Journal of Molecular Structure 1229 (2021) 129830
Scheme 2. One-pot multicomponent protocol for the synthesis of Plinabulin derivatives.
Table 1
Optimization of reaction conditionsa.
Entry
Temp (˚C)/ Time (h)
B/D (equiv.)
Yield (%)b
4d / 4d
́
1
2
3
4
-10˚C /4h; 95˚C /4h
-10˚C /4h; 95˚C /4h
-10˚C /4h; 100˚C /4h
-10˚C /4h; 110˚C /4h
1.0/ 1.0
1.0/ 2.0
1.0/ 2.0
1.0/ 2.0
7/19
0/30
0/38
0/46
a
Reactions conditions: A (1 equiv.), C (2.5 equiv.), base (Cs2CO3(2.5 equiv.)), solvent (DMF), B, D, temperature and time
according to the described conditions.
b
Isolated Yields
the derivatives synthesized and to retain the activity, the free N-H
Table 2
% Inhibition at 10 μM solutions in DLD-1 colon
cancer cell line.
group was required for H-bonding. Thus we kept these three parts
intact in our designed scaffold. From the biological evaluation, we
observed that when the Ar group was picoline, quinoline and fu-
ran group, the derivatives showed no activities, indicating that they
were not suitable for biological activities. But, when the Ar group
was thiophene and pyridine, two derivatives 4b and 4d had high
activity against DLD-1 colon cancer cell line. Also, when R group
was hydrogen, the derivatives showed moderate activity but when
hydrogen was replaced by methyl group, derivatives lost their ac-
tivity Fig. 4.
Colon
Sr. No.
Compound Code
DLD-1
1
(4a)
22.03
69.27
14.26
70.97
22.75
2.93
2
(4b)
3
(4c)
4
(4d)
5
(4e)
6
(4f)
7
(4g)
1.11
8
(4h)
11.32
3.87
9
(4i)
10
11
12
13
14
15
16
17
18
(4j)
4.72
2.3.2. Nonetheless, our two compounds 4b and 4d were selected as a
lead compound for further studies
(4k)
1.50
(4l)
45.12
2.08
Further, we examined cytotoxicity and selectivity of our two
lead compounds (4b & 4d) and standard compound (Plinabulin) on
the DLD-1 colon cancer cell line and the normal Vero cell using
SRB assay. Results are expressed as IC50 and SI value of the DLD-
1 colon cancer cell line and the normal Vero cell and shown in
Table 3.
(4m)
(4n)
11.66
0.86
(4o)
(4p)
3.24
(4q)
25.08
77.62
Doxorubicine (10μM)
3

A. Ganesher, P. Chaturvedi, B.B. Karkara et al.
Journal of Molecular Structure 1229 (2021) 129830
Fig. 2. Substrate scope.
Table 3
Cytotoxic activity as expressed as IC50 (μM) in DLD-1 Colon cancer
cell line and in Vero cell and SI value.
Compound No.
DLD-1 IC50 (μM)
Vero IC50 (μM)
SI∗
4b
1.25
1.20
0.09
2.44
>20.0
>20.0
0.07
>16.0
>16.6
0.77
4d
Plinabulin
Doxorubicin
7.15
2.93
SI∗ (Selectivity Index) = IC50 Vero Cell / IC50 Cancer Cell line.
cer cells and the Vero cells respectively. The IC50 values were used
to determine the selectivity indexes (SI) which represent the over-
all activity. The degree of selectivity of the compounds can be ex-
pressed by its Selectivity Index (SI) value. The SI values were cal-
culated as follows: SI = IC50 normal cell/IC50 cancer cell. High SI
value (>2) of a compound gives a selective toxicity towards can-
cer cells.[16] Our both synthesized compounds showed excellent
SI values, which was >16.0 for both whereas, SI value for the stan-
dard inhibitor (Plinabulin) was very low (only 0.77). Hence, 4b and
4d display potential to be further exploited in the discovery and
development of new anticancer agents.
Fig 3. Structure elucidation of compound 4d.
Our both compounds exhibited a substantial anti-proliferative
effect on the DLD-1 cell line. Dose-dependent studies revealed IC50
of 1.25 μM and IC50 of >20.0 μM for compound 4b and IC50 of
1.20 μM and IC50 of >20.0 μM for compound 4d and IC50 of 0.09
μM and IC50 of 0.07 μM for plinabulin on the DLD-1 colon can-
Fig 4. Structure-Activity relationship of designed scaffold.
4

A. Ganesher, P. Chaturvedi, B.B. Karkara et al.
Journal of Molecular Structure 1229 (2021) 129830
2.3.3. Experimental procedure
(Z)-6-((Z)-benzylidene)-1-methyl-3-((4-methylthiazol-2-
yl)methylene)piperazine-2,5-dione (4c):
Organic solvents were dried by standard methods. All the prod-
ucts were characterized by 1H, 13C, ESI-MS, HRMS. Analytical TLC
was performed using 2.5 × 5 cm plates coated with a 0.25 mmol
thickness of silica gel (60F-254); visualization was accomplished
with iodine and under UV lamp. Column chromatography was per-
formed using silica gel (60–120 and 100–200 mesh). NMR spec-
tra were recorded on BrukerAvance DPX 200FT, Bruker Robotics,
Bruker DRX 300 and 400 Spectrometers at 400 MHz, 500 MHz (1H)
and 100 MHz (13C). Experiments were recorded in CDCl3 at 25 °C.
Chemical shifts are given on the δscale and are referenced to the
TMS at 0.00 ppm for proton. J values were expressed in hertz (Hz).
For 13C NMR reference CDCl3 appeared at 77.16 ppm. Mass spectra
were recorded on a JEOL JMS-600H high resolution spectrometer
using EI mode at 70 eV.
Yield 48%; mp 186°C; yellowish solid; Rf (30% EtOAc/Hexane)
0.68; 1H NMR (400 MHz, CDCl3) 11.76 (1H, s, -NH), 7.33-7.29 (2H,
m, Ar-H), 7.27-7.25 (1H, m, Ar-H, 1H, m, Ar-H), 7.21-7.20 (1H, m,
Ar-H, 1H, m, =C-H), 6.87-6.87 (1H, m, Ar-H), 6.81 (1H, s, =C-H),
2.94 (3H, s, -NCH3), 2.45 (3H, s, -CH3); 13C NMR (100 MHz, CDCl3)
162.4, 158.9, 158.6, 134.2, 130.3, 129.4, 129.4, 129.1, 128.4, 128.4,
128.2, 121.3, 114.6, 103.6, 36.9, 17.2; HRMS (ESI) m/z: 326.0955
(M+H)+ (calcd for C17 H15N3O2S 326.0958); m/z (ESI) 326.04 (100
%, MH+), 357.81 (13%, MCH3OH+).
(Z)-6-((Z)-benzylidene)-3-((5-bromothiophen-2-yl)methylene)-
1-methylpiperazine 2,5-dione (4d):
Yield 47%; mp 224°C; light yellowish solid; Rf (30%
EtOAc/Hexane) 0.65; 1H NMR (400 MHz, CDCl3) 7.84 (1H, s,
-NH, f), 7.33-7.26 (3H, m, Ar-H, b,c), 7.24-7.19 (3H, m, Ar-H, =C-H,
a,d), 7.04-7.03 (1H, m, =C-H, g), 6.99 (1H, s, Ar-H, i), 6.97-6.96 (1H,
m, =C-H, h), 2.92 (3H, s, -NCH3, e); 13C NMR (100 MHz, CDCl3)
159.2 (C=O, C10), 159.1 (C=O, C8), 137.3 (ArC,C12), 133.8 (ArC, C4),
131.1 (ArC-H, C14 ), 130.1 (>C=C, C6), 129.7 (ArC-H, C13), 129.4
(ArC-H, C1), 128.6 (ArC-H, C3), 128.3 (ArC-H, C2), 124.4 (>C=C, C9),
121.5 (=C-H, C5), 115.1 (ArC, C15), 109.3 (=C-H, C11 ), 36.7 (-NCH3,
C7); HRMS (ESI) m/z: 388.9951 (M+H)+ (calcd for C17 H13BrN2O2S:
388.9954).
1,4-diacetylpiperazine - 2,5-dione (1):
2,5 diketopiperazine (5 gm, 43.8 mmol) in acetic anhydride
(200 ml, 2117.7 mmol) was refluxed for 10h. After completion of
reaction, the acetic anhydride was evaporated to obtain solid prod-
uct and used for the next step. Yield 95%, as whitish solid, Rf (50%
EtOAc/Hexane) 0.48; 1H NMR (400 MHz, CD3OD) 4.53(s, 4H, -CH2),
2.43(s, 6H, -CH3); 13C NMR (100 MHz, CD3OD) 170.7, 165.9, 47.1,
+
26.6; HRMS (ESI) m/z: 199.0713 (M+H)
(calcd for C8H10N2O4
199.0713)
General procedure for the synthesis of (4a-4q):
(Z)-6-((Z)-benzylidene)-1-methyl-3-(thiazol-2-ylmethylene)
piperazine-2,5-dione (4e):
In a 25 ml two neck round bottomed flask, 1,4- diacetyl-2,5-
diketopiperazine (100 mg, 0.50 mmol, 1.0 eq.), benzaldehyde (0.50
mmol, 1.0 eq.), methyl iodide (1.26 mmol, 2.5 eq.), Cs2CO3 (1.26
Yield 49%; mp 177°C; light yellowish solid; Rf (30%
EtOAc/Hexane) 0.64; 1H NMR (400 MHz, CDCl3) 11.89 (1H, s,
-NH), 7.95-7.94 (1H, d, J= 3.28Hz, Ar-H), 7.40-7.34 (5H, m, Ar-H,
1H, m, =C-H), 7.33-7.29 (1H, m, Ar-H), 6.96 (1H, s, =C-H), 3.02
(3H, s, -NCH3); 13C NMR (100 MHz, CDCl3) 163.3, 158.9, 158.5,
143.8, 134.1, 130.2, 129.6, 129.4, 128.5, 128.2, 121.5, 119.6, 103.1,
36.9; HRMS ESI m/z: 312.0796 (M+H)+ (calcd for C16 H13N3O2S:
312.0801).
˚
mmol, 2.5 eq.) and 4 A MS (400 mg) in 5 ml dry DMF were added
under inert atmosphere. The reaction was allowed to stir at -10
˚C until the completion of the first aldol condensation and methy-
lation. Then, the second aldehyde/ketone (hetero-aromatic alde-
hyde/ketone, 1.0 mmol, 2.0 eq.) was added and the reaction was al-
lowed to stir at 110 ˚C until the completion of the reaction. The re-
action was monitored by TLC, after the completion of the reaction,
solvent was removed under vaccum. The reaction mixture was ex-
tracted with ethyl acetate thrice. The organic layer was dried under
anhydrous Na2SO4, filtered and concentrated under reduced pres-
sure. The crude product thus obtained was then purified over col-
umn chromatography to give desired product.
(Z)-6-((Z)-benzylidene)-3-((2-chloroquinolin-3-yl)methylene)-1-
methylpiperazine-2,5-dione (4f):
Yield 50%; mp 220°C; yellowish solid; Rf (30% EtOAc/Hexane)
0.54; 1H NMR (400 MHz, CDCl3) 9.45 (1H, s, -NH), 8.27 (1H, s, Ar-
H), 7.97 (1H, d, J= 8.4Hz, Ar-H), 7.82 (1H, d, J= 7.8Hz, Ar-H) 7.65-
7.61 (1H, m, Ar-H), 7.52-7.49 (1H, m, Ar-H), 7.40-7.31 (3H, m, Ar-H),
7.19 (1H, s, =C-H), 7.13 (2H, d, J= 6.6Hz, Ar-H), 6.64 (1H, s, =C-
H), 2.94 (3H, s, -NCH3); 13C NMR (100 MHz, CDCl3) 160.1, 158.6,
150.1, 147.0, 138.2, 133.6, 131.1, 129.6, 129.4, 128.6, 128.4, 128.3,
128.2, 127.7, 127.6, 126.7, 125.8, 121.6, 112.4, 36.7; HRMS (ESI) m/z:
390.0999 (M+H)+(calcd for C22H16 ClN3O2 390.1009).
(Z)-6-((Z)-benzylidene)-3-(isoquinoline-3-ylmethylene)-1-
methylpiperazine-2,5-dione (4a):
Yield 47%; mp 243°C; yellowish solid; Rf (30% EtOAc/Hexane)
0.66; 1H NMR (400 MHz, CDCl3) 7.97 (1H, s, -NH), 7.86-7.79 (2H,
m, Ar-H), 7.55 (1H, s, Ar-H), 7.43-7.39 (2H, m, Ar-H), 7.38-7.35 (3H,
m, Ar-H), 7.34-7.23 (3H, m, Ar-H, 2H, m, =C-H), 2.97 (3H, s, -NCH3);
13C NMR (100 MHz, CDCl3) 159.2, 159.1, 154.7, 146.8, 136.9, 134.0,
131.3, 130.3, 130.1, 129.3, 128.7, 128.4, 128.2, 127.4, 127.1, 126.7,
123.8, 121.5, 109.8, 36.9; HRMS (ESI) m/z: 356.1382 (M+H)+ (calcd
for C22H17 N3O2: 356.1394); m/z (ESI) 356.10 (100 %, MH+), 387.85
(10%, MCH3OH+).
(Z)-6-((Z)-benzylidene)-1-methyl-3-(pyridin-2-
ylmethylene)piperazine-2,5-dione (4g):
Yield 51%; mp 203°C; yellowish solid; Rf (30% EtOAc/Hexane)
0.59; 1H NMR (400 MHz, CDCl3) 12.7 (1H, s, -NH), 8.64 (1H, d,
J= 4.1 Hz, Ar-H), 7.75-7.71 (1H, m, Ar-H), 7.40-7.34 (5H, m, Ar-H),
7.32-7.29 (1H, m, Ar-H), 7.27-7.23 (1H, m, =C-H), 7.21-7.20 (1H,
m, Ar-H), 6.81 (1H, s, =C-H), 3.02 (3H, s, -NCH3); 13C NMR (100
MHz, CDCl3) 159.1, 158.9, 154.9, 148.5, 137.0, 134.3, 130.7, 130.5,
129.4, 128.3, 128.2, 126.1, 122.1, 120.8, 109.8, 36.9; HRMS (ESI)
m/z: 306.1230 (M+H)+ (calcd for C18 H15N3O2 306.1237); m/z (ESI)
306.09 (100 %, MH+).
(Z)-3-(benzo[b]thiophen-3-ylmethylene)-6-((Z)-benzylidene)-1-
methylpiperazine-2,5-dione (4b):
Yield 49%; mp 215°C; yellowish solid; Rf (30% EtOAc/Hexane)
0.64; 1H NMR (400 MHz, CDCl3+CD3OD) 9.43-9.38 (1H, m, -NH,
1H, m, Ar-H), 9.04-8.95 (2H, m, Ar-H) 8.80-8.75 (1H, m, Ar-H),
8.70-8.66 (1H, m, Ar-H), 8.63-8.58 (3H, m, Ar-H), 8.56-8.54 (1H,
m, Ar-H), 8.53-8.50 (1H, m, Ar-H, 1H, m, =C-H), 8.14 (1H, s,
=C-H), 4.25 (3H, s, -NCH3); 13C NMR (100 MHz, CDCl3+CD3OD)
159.2 (>C=O), 154.7 (>C=O), 146.8 (Ar-C), 136.9 (Ar-C), 134.0
(Ar-C), 131.3 (Ar-C), 130.3 (Ar-C-H), 130.1 (>C=C), 129.3 (Ar-C-H),
128.7 (Ar-C-H), 128.4 (Ar-C-H), 128.2 (Ar-C-H), 127.4 (Ar-C-H), 127.1
(Ar-C-H), 126.7 (>C=C), 123.8 (=C-H), 121.5 (Ar-C-H), 109.8 (=C-
H), 36.9 (-NCH3); HRMS (ESI) m/z: 361.0999 (M+H)+ (calcd for
C21H16 N2O2S 361.1005); m/z (ESI) 359.79 (53%).
(Z)-6-((Z)-benzylidene)-1-methyl-3-((5-methylthiophen-2-
yl)methylene)-1-piperazine 2,5-dione (4h):
Yield 53%; mp 186°C; yellowish solid; Rf (30% EtOAc/Hexane)
0.59; 1H NMR (400 MHz, CDCl3) 8.02 (1H, s, -NH), 7.40-7.29 (6H,
m, Ar-H, =C-H), 7.13 (1H, s, =C-H), 7.09-7.08 (1H, m, Ar-H), 6.80-
6.79 (1H, m, Ar-H), 2.98 (3H, s, -NCH3), 2.54 (3H, s,-Ar-CH3); 13C
NMR (100 MHz, CDCl3) 159.6 (C=O), 159.2 (C=O), 143.3 (ArC),
134.0 (ArC), 133.6 (ArC), 130.6 (=C-H), 130.4 (>C=C), 129.4 (ArC-
H), 128.4 (ArC-H), 128.2 (ArC-H), 126.6 (ArC-H), 122.7 (>C=C),
5

A. Ganesher, P. Chaturvedi, B.B. Karkara et al.
Journal of Molecular Structure 1229 (2021) 129830
120.8 (ArC-H), 111.0 (=C-H), 36.7 (-NCH3), 15.5 (-Ar-CH3); HRMS
(ESI) m/z: 325.1000 (M+H)+ (calcd for C18 H16 N2O2S 325.1011); m/z
(ESI) 324.84 (30 %), 290.92 (100%).
Yield 43%, mp186 °C, as light yellowish solid; Rf (30%
EtOAc/Hexane) 0.59; 1H NMR (400 MHz, CDCl3) 7.79 (1H), s, -NH),
7.38-7.23 (4H, m, Ar-H), 7.19 (1H, s, Ar-H), 7.12 (1H, s, =C-H), 6.98
(1H, d, J=3.7 Hz, Ar-H), 6.83 (1H, d, J=3.7 Hz, Ar-H), 2.90 (3H, s, -
NCH3), 2.47 (3H, s, -CH3); 13C NMR (100 MHz, CDCl3) 160.3, 159.9,
142.7, 133.6, 130.6, 130.4, 129.5, 128.6, 128.3, 127.6, 123.7, 123.0,
120.8, 114.6, 36.3, 21.4; HRMS (ESI) m/z: 404.0119 (M+H)+ (calcd
for C18 H15N2O2S 404.2929); m/z (ESI) 402.84 (22 %), 323.84 (13%).
(Z)-6-((Z)-benzylidene)-1-methyl-3-(1-(pyridine-2-
(Z)-6-((Z)-benzylidene)-3-(furan-2-ylmethylene)-1-
methylpiperazine-2,5-dione (4i):
Yield 48%; mp 174°C; brown solid; Rf (30% EtOAc/Hexane) 0.59;
1H NMR (400 MHz, CDCl3) 9.12 (1H, s, -NH), 7.51-7.50 (1H, m, Ar-
H), 7.32-7.22 (3H, m, Ar-H), 7.20-7.18 (2H, m, Ar-H), 6.71 (1H, s,
=C-H), 6.51 (1H, d, J=3.4Hz, Ar-H), 6.46-6.45 (1H, d, J= 1.8Hz, =C-
H), 2.91 (3H, s, -NCH3); 13C NMR (100 MHz, CDCl3) 159.2, 158.8,
150.7, 144.0, 134.2, 130.3, 129.4, 128.3, 128.2, 123.5, 120.7, 114.4,
112.4, 103.4, 36.8; HRMS (ESI) m/z: 295.1077 (M+H)+ (calcd for
C17 H14 N2O3 295.1083).
yl)ethylidene)piperazines-2,5-dione(4p):
Yield 47%; mp 160 °C; light yellowish solid; Rf (30%
EtOAc/Hexane) 0.61; 1H NMR (400 MHz, CDCl3) 13.57 (1H, s, -NH),
8.60 (1H, d, J=8.6Hz, Ar-H), 7.76-7.72 (1H, m, Ar-H), 7.59 (1H, d,
J=8.2 Hz, Ar-H), 7.32-7.19 (6H, m, Ar-H), 7.18-7.13 (1H, m, =C-H),
2.93 (3H, s, -NCH3), 2.57 (3H, s, -CH3); 13C NMR (100 MHz, CDCl3)
160.9, 158.3, 147.2, 137.4, 134.0, 131.3, 129.5, 128.4, 128.3, 127.6,
123.0, 122.1, 120.6, 119.9, 36.4, 16.5; HRMS (ESI) m/z: 320.1392
(M+H)+ (calcd for C19 H17 N3O2 320.1394)
(Z)-3-(benzo[b]thiophen-2-ylmethylene)-6-((Z)-benzylidene)-1-
methylpiperazine-2,5-dione (4j):
Yield 49%; mp 208°C; light yellowish solid; Rf (30%
EtOAc/Hexane) 0.59; 1H NMR (500 MHz, CDCl3) 8.28 (1H, s, -NH),
7.85-7.80 (2H, m, Ar-H), 7.49 (1H, s, Ar-H), 7.39-7.25 (7H, m, Ar-H,
2H, m, =C-H), 3.01 (3H, s, -NCH3); 13C NMR (100 MHz, CDCl3)
159.2, 159.2, 139.9, 139.2, 135.3, 133.8, 130.1, 129.4, 128.6, 128.3,
126.7, 125.7, 125.5, 125.3, 124.1, 122.3, 121.5, 110.4, 36.8; HRMS
(ESI) m/z: 361.1005(M+H)+ (calcd for C21H16 N2O2S 361.1011); m/z
(ESI) 392.03 (37 %, MCH3OH+), 377.83 (34%, MH2O+), 227.73 (20%).
(Z)-6-((Z)-benzylidene)-1-methyl-3-(thiophen-2-
(Z)-6-((Z)-benzylidene)-1-methyl-3-(1-quinolin-2-
yl)ethylidene)piperazines-2,5-dione(4q):
Yield 46%; mp 173°C; light yellowish solid; Rf (30%
EtOAc/Hexane) 0.61; 1H NMR (400 MHz, CDCl3) 13.95 (1H, s,
-NH), 8.18-8.10 (2H, dd, J1=8.86 Hz, J2=22.96 Hz, Ar-H), 7.74-7.67
(3H, m, Ar-H), 7.50 (1H, t, J=14.58 Hz, Ar-H), 7.30-7.23 (5H, m,
Ar-H), 7.14 (1H, s, =C-H), 2.95 (3H, s, -NCH3), 2.68 (3H, s, -CH3);
13C NMR (100 MHz, CDCl3) 160.8, 160.8, 158.4, 145.7, 137.2, 134.0,
131.3, 130.4, 129.5, 128.9, 128.4, 128.3, 128.2, 127.4, 127.2, 126.4,
120.5, 120.3, 119.9, 36.4, 16.8; HRMS (ESI) m/z: 370.1554 (M+H)+
(calcd for C23H19N3O2 370.1550).
ylmethylene)piperazine-2,5-dione (4k):
Yield 52%; mp 199°C; yellowish solid; Rf (30% EtOAc/Hexane)
0.35, 1H NMR (400 MHz, CDCl3) 7.99 (1H, s, -NH), 7.40 (1H, d,
J= 5.04Hz, Ar-H), 7.32-7.29 (2H, m, Ar-H), 7.27-7.25 (1H, m, Ar-H),
7.22-7.18 (3H, m, Ar-H, 1H, m, =C-H), 7.09 (1H, s, =C-H), 7.08-7.06
(1H, m, Ar-H), 2.92 (3H, s, -NCH3); 13C NMR (100 MHz, CDCl3)
159.4, 159.4, 135.7, 133.9, 130.3, 129.9, 129.4, 128.5, 128.3, 127.7,
123.8, 121.1, 110.3, 36.7; HRMS (ESI) m/z: 311.0860 (M+H)+ (calcd
for C17 H13N3O4S 311.0849)
3. Materials and methods
The test samples/molecules were weighed in micro centrifuge
tubes and stock solutions of 20mmol were made by dissolving the
samples in DMSO. Stocks were stored at -20°C. A working solution
of 20 μM concentration was made by diluting the stock solution in
culture medium (RPMI-1640 with 5 % FBS) prior to the assay.
(Z)-6-((Z)-benzylidene)-3-((5-bromopyridin-3-yl)methylene)-1-
methylpiperazine-2,5-dione(4l):
Yield 46%; mp 214°C; yellowish solid; Rf (30% EtOAc/Hexane)
0.32; 1H NMR (400 MHz, CDCl3) 9.42 (1H, s, -NH), 8.54 (1H, s, Ar-
H), 8.49 (1H, s, Ar-H), 7.88 (1H, s, Ar-H), 7.35-7.31 (3H, m, Ar-H),
7.30-7.19 (1H, m, Ar-H, 1H, m, =C-H), 7.08 (1H, s, =C-H), 6.87(1H,
s, Ar-H), 2.92 (3H, s, -NCH3); 13C NMR (100 MHz, CDCl3) 160.4,
158.8, 150.1, 148.0, 138.2, 133.6, 130.9, 129.8, 129.4, 128.8, 128.5,
128.4, 122.1, 121.0, 111.8, 36.7; HRMS (ESI) m/z: 385.2409 (M+H)+
(calcd for C17 H13N3O4S 385.2405)
3.1. SRB assay
Addition of cells: The human cancer cell lines Colon (DLD-1)
maintained in RPMI-1640 medium. The enumerated cells were dis-
pensed in a 96-well tissue culture plate. Each well receives 100 μL
of the cell suspension containing 10,000-30,000 cells (depending
upon the nature of cell line). The cells were then incubated at 37
°C in 5% CO2 concentration for 24 h before addition of the test
samples/standard drugs.
(Z)-6-((Z)-benzylidene)-1-methyl-3-(1-(4-methylthiazol-2-
yl)ethylidene)piperazines-2,5-dione(4m):
Yield 45%, mp 137°C, as yellowish solid, Rf (30% EtOAc/Hexane)
0.47; 1H NMR (400 MHz, CDCl3) 13.06 (1H, s), 7.40-7.36 (2H, m, Ar-
H), 7.33-7.30 (3H, m, Ar-H), 7.24 (1H, s, =C-H), 6.99 (1H, s, Ar-H),
3.00 (3H, s, -NCH3), 2.68 (3H, s, -CH3), 2.55 (3H, s, -CH3); 13C NMR
(100 MHz, CDCl3) 169.0, 160.5, 160.1, 154.0, 134.0, 130.9, 129.5,
128.4, 128.3, 125.5, 120.1, 114.6, 36.6, 17.9, 17.3; HRMS (ESI) m/z:
340.1122 (M+H)+ (calcd for C18 H17 N3O2S 340.1120)
Addition of test samples: 100 μL of working solution of the test
sample was added to the cell monolayer to give a final concentra-
tion 10 μM. For each sample, duplicate wells were included. Pos-
itive (Drug) and Negative (Vehicle) Controls: In every assay plate
both positive and vehicle controls were included. In positive con-
trol well, adriamycin (Doxorubicin) was added at 10 μM concentra-
tion. In vehicle control wells, DMSO was added. In all assay wells
the final concentration of DMSO was 0.1%. The plates were then
incubated at 37 °C in 5% CO2 concentration for 48 h.
Addition of SRB and Colorimetric reading: After 48 h incu-
bation, cells attached to substratum of the plate were fixed by
adding cold 50% trichloroacetic acid (TCA, 50 μl/well) on top of the
medium and incubated at 4 °C for 1 h. After that the plate is gen-
tly washed 5 times with slow running tap water via plastic tubing
to remove TCA, culture medium and dead cells. The water stream
should not be injected very fast or directly on to the bottom of
the wells as this can cause cell monolayer to detach. After wash-
ing, the plates were allowed to dry in air (plates can be stored for
long periods at room temperature after fixing and drying step) To
(3Z,6Z)-3-(1-(benzo[b]thiophene-2-yl)ethylidene)-6-
benzylidene-1-methylpiperazine-2,5-dione(4n):
Yield 44%; mp 180°C; light yellowish solid; Rf (30%
EtOAc/Hexane) 0.59; 1H NMR (500 MHz, CDCl3) 8.03 (1H, s,
Ar-H), 7.84-7.78 (2H, m, Ar-H), 7.39-7.36 (4H, m, Ar-H), 7.34-7.31
(4H, m, Ar-H), 7.18 (1H, s, =C-H), 2.99 (3H, s, -NCH3), 2.64 (3H, s,
-CH3); 13C NMR (100 MHz, CDCl3) 160.2, 160.0, 141.1, 140.0, 139.4,
133.7, 130.6, 129.5, 128.6, 128.3, 125.3, 125.0, 124.7, 124.0, 123.9,
123.4, 122.2, 120.7, 36.3, 21.4; HRMS (ESI) m/z: 375.1142 (M+H)+
(calcd for C22H18 N2O2S 375.1167)
(Z)-6-((Z)-benzylidene)-3-(1-(5-bromothiophen-2-
yl)ethylidene)-1-methylpiperazine-2,5-dione(4o):
6

A. Ganesher, P. Chaturvedi, B.B. Karkara et al.
Journal of Molecular Structure 1229 (2021) 129830
dry plates, 50 μL /well of SRB solution was added and left at room
temperature for 30 min. At the end of the staining period, unbound
SRB was removed by quickly rinsing plates 4-5 times with 1 % (v/v)
acetic acid. The Plates were allowed to air-dry at room tempera-
ture (Stained and dried plates can be stored indefinitely at room
temperature). 150 μL of 10 mmol Tris base solution was added to
each well and plate was shaken for 15 min on agyratory shaker to
solubilize the protein-bound dye. Alternatively, if a shaker was un-
available, SRB gets solubilized after 30 min in Tris base solution.
Absorbance was measured at 510 nm in a microplate spectropho-
tometer.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2020.129830.
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The authors declare that they have no known competing finan-
cial interestsor personal relationships that could have appeared to
influence the work reported in this paper.
CRediT authorship contribution statement
Asha Ganesher: Methodology, Data curation, Writing - original
draft. Priyank Chaturvedi: Data curation. Bidhu Bhusan Karkara:
Methodology. Indranil Chatterjee: Methodology. Dipak Datta:
Data curation. Gautam Panda: Conceptualization, Writing - review
& editing, Supervision.
Acknowledgments
A.G. and B.B.K. thank CSIR and P.C. thanks DBT for research fel-
lowship. We also thank Rahul Kumar for some initial preliminary
experiment of this manuscript and SAIF division, CDRI for provid-
ing analytical data. The authors also thank Ministry of Earth Sci-
ences (MoES/09-DS/3/201P5C –IV dated 27.01.2016), New Delhi for
funding this work. This has CDRI communication no 10172.
7