Synthesis and Biological Evaluation of Pyrazoline and Pyrrolidine-2,5-dione Hybrids as Potential Antitumor Agents

Accepted Article

Title: Synthesis and biological evaluation of pyrazoline and

pyrrolidine-2,5-dione hybrids as potential antitumor agents.

Authors: Kalpana Tilekar, Neha Upadhyay, Franz-Josef Meyer-Almes,

Fulvio Loiodice, Anisimova Natalia Yu, Spirina Tatiana, Darina

V Sokolova, Galina B Smirnova, Jun-yong Choe, Vadim S

Pokrovsky, Antonio Lavecchia, and Ramaa C S

This manuscript has been accepted after peer review and appears as an

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using the Digital Object Identifier (DOI) given below. The VoR will be

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content of this Accepted Article.

To be cited as: ChemMedChem 10.1002/cmdc.202000458

Link to VoR: https://doi.org/10.1002/cmdc.202000458

01/2020

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Synthesis and biological evaluation of pyrazoline and pyrrolidine-

2,5-dione hybrids as potential antitumor agents.

Kalpana Tilekar^a,Neha Upadhyay^a, Franz-Josef Meyer-Almes^b, Fulvio Loiodice^c, Anisimova Natalia Yu^d.,

Spirina Tatiana S^d., Sokolova Darina V^d., Smirnova Galina B.^d, Jun-yong Choe^e, Pokrovsky Vadim S.*^d,f,

Antonio Lavecchia*^g, C S Ramaa*^a

[a]

Title(s), Initial(s), Surname(s) of Author(s) including Corresponding Author(s)

[a]

Kalpana Tilekar^,, Ms. Neha Upadhyay, Prof. C S Ramaa*

Department of Pharmaceutical Chemistry,

Bharati Vidyapeeth’s College of Pharmacy,

Sector 8, CBD Belapur, Navi Mumbai, India.

E-mail: sinharamaa@yahoo.in

[b]

[c]

[d]

[e]

[f]

Prof. Franz-Josef Meyer-Almes

Department of Chemical Engineering and Biotechnology,

University of Applied Science,

Darmstadt, Germany.

Dr. Fulvio Loiodice

Department of Pharmacy-Drug Science,

University of Bari “Aldo Moro”,

Via E. Orabona, 4, 70126 Bari, Italy

Anisimova Natalia Yu., Spirina Tatiana S., Sokolova Darina V., Smirnova Galina B., Dr. Pokrovsky Vadim S*

Laboratory of combined therapy,

N.N. Blokhin Cancer Research Center,

Moscow, Russia.

Dr. Jun-yong Choe

East Carolina Diabetes and Obesity Institute,

Department of Chemistry, East Carolina University,

Greenville, North Carolina, USA.

Dr. Pokrovsky Vadim S*.

Department of biochemistry,

People’s Friendship University,

Moscow, Russia.

E-mail: v.pokrovsky@ronc.ru

[g]

Prof. Antonio Lavecchia*

Department of Pharmacy, “Drug Discovery” Laboratory,

University of Napoli “Federico II”,

Via D. Montesano, 49, 80131 Napoli, Italy.

E-mail: antonio.lavecchia@unina.it

Supporting information for this article is given via a link at the end of the document.

Abstract: In search of novel and effective antitumor agents,

pyrazoline-substituted pyrrolidine-2,5-dione hybrids were designed,

synthesized and evaluated in silico, in vitro and in vivo for anticancer

efficacy. All the compounds exhibited remarkable cytotoxic effects in

MCF7 and HT29 cells. The excellent antiproliferative activity towards

MCF-7 (IC₅₀= 0.78±0.01 µM), HT29 (IC₅₀= 0.92±0.15 µM) and K562

(IC₅₀= 47.25±1.24 µM) cell lines, prompted us to further investigate

the antitumor effects of the best compound S2. In cell cycle analysis,

S2 was found to disrupt the growth phases with increased cell

population in G1/G0 phase and decreased cell population in G2/M

phase. The excellent in vitro effects were also supported by inhibition

of anti-apoptotic protein Bcl-2. In vivo tumor regression studies of S2

in HT29 xenograft nude mice, exhibited equivalent and promising

tumor regression with maximum TGI, 66 % (i.p. route) and 60 % (oral

route) at 50 mg/kg dose by both the routes, indicating oral

bioavailability and anti-tumor efficacy. These findings advocate that

hybridization of pyrazoline and pyrrolidine-2,5-dioes holds promise for

the development of more potent and less toxic anticancer agents.

1. Introduction

Despite the advancement of cancer therapies, current cancer

therapeutics suffers due to toxicity and drug resistance issues^[1]–

^[3], specifying the need of continued search of newer anticancer

agents. Scaffold hybridization approach is a commonly used but

effective way in the era of modern medicinal chemistry. It has

been reported several times, that combining two scaffolds

together to form a hybrid molecule exhibits enhanced activity

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Figure 1. Potential anti-proliferative compounds with pyrrolidine-2,5-dione (red) and substituted pyrazoline (blue) moieties.

Figure 2. Designing of the hybrid molecules.

compared to agents with single scaffold^[4]–[7]. In line with this

approach, we designed and synthesised a hybrid series of novel

molecules combining two pharmacologically attractive

heterocycles, diaryl Pyrazoline and Pyrrodidine-2,5-dione.

(O=C-C(R)-C=O), which includes but not limited to 1,3-

indanediones, thaizolidine-2,4-dione (TZD), phthalimides and

pyrrolidine-2,5-diones (succinimides) have appealed to biologists

and medicinal chemists since a long time. Pyrrolidine-2,5-dione is

a privileged scaffold seen in bioactive compounds making it one

of the most promising structural unit in drug discovery^[18][19]. The

antiproliferative effects of N-substituted pyrrolidine-2,5-diones

have been reported against few cell lines such as hepatoma, lung,

renal, breast cancer and leukaemia^[20]–[25](Fig. 1). Even though

the primary antiproliferative activity has been explored to some

extent, the overall antitumor mechanisms may be the most

important and yet unexplored aspect.

Anti-cancer activity of pyrazolines reviewed by various authors

revealed that N-substituted pyrazolines have exhibited

antiproliferative and antitumor effects in vivo and in vitro^[8][9](Fig.

1). Recent literature showcases the potent cytotoxic activity of3,5-

diaryl pyrazolines in different cancer cell lines^[10][11]. They have

been found to induce apoptosis, caspase activation, cause DNA

intercalation, inhibit tubulin polymerization and autophagy in

various cancer cells^[12]–[17]. Moreover, these compounds have

exerted anti-cancer effects through inhibition of various receptors/

enzymes/proteins involved in cancer cell division and growth^[8].

Thus, it is evident that 3,5- diaryl pyrazolines can provide

structural outlines for further anticancer drug development.

Substituted 1,3-cyclopentadiones having structural framework

Thus, hybrid molecules containing these two scaffolds were

designed, synthesised, purified structurally characterised and

evaluated for in vitro and in vivo antitumor activity.

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attached to carbonyl group was observed at 4.5-4.7 ppm. A

multiplet or doublet of methylene of pyrazole ring was observed

in the range of 3.8-3.9 ppm. ¹³C-NMR exhibited signals for C=O

carbon in the region of 160-180 ppm. Methylene carbons showed

a peak at 40-50 ppm. Pyrrolidine-2,5-dione ethylene carbons

found at around 27-28 ppm, methylene carbon of pyrazole ring

were displayed at 40-45 ppm. Mass spectra in positive ionization

scanning displayed the characteristic [M+H⁺] peak at 100% m/z

intensity for all the compounds.

2. Results and Discussion

2.1 Designing of the hybrid molecules

Earlier we had reported diaryl pyrazolines with remarkable

anticancer activity against MCF-7 (Breast), HOP62 (lung), A498

(Renal) and MIAPACA2 (pancreatic)cell lines^[26](Fig. 2). Thus,

based on our past experience with this scaffold and literature

evidences cited above, we selected diaryl pyrazoline as one

portion of hybrid. In continuing efforts to discover novel

antiproliferative agents, we have also reported TZD and its bio-

isosteres with excellent activity against various leukemic and solid

tumor cell lines^[27]–[29]. In one such attempt we have reported

anthraquinone based N-substituted pyrrolidine-2,5-diones as

anticancer agents with cell line based antiproliferative effects in

HL60 and K562 cell lines^[30](Fig. 2). For this reason, in current

series we selected pyrrolidine-2,5-dione as other portion of the

hybrid, since it is

a bioisostere of TZD, it possesses

antiproliferative potential and has not been much explored with

respect to anticancer mechanism per se. In addition, the bio-

isosteric replacement of TZD ring with pyrrolidine-2,5-dione has

been reported to reduce toxicity and better the anticancer

properties of thiazolidinediones^[31]

.

Based on these observations, it was of interest to design hybrids

incorporating 3,5-diaryl pyrazoline ring system onto nitrogen atom

of pyrrolidine-2,5-dione (Fig. 2) in an attempt to obtain a novel

anticancer agent.

Scheme 1: Synthetic route followed to synthesize S1-S13.

Reagents and conditions (a) Aqueous NaOH, EtOH, rt, 5-6 h; (b)

NH₂NH₂.H₂O, CHCl₃, 80 °C, 12 h; (c) K₂CO₃, ClCH₂COCl, rt, 12

h; (d) K₂CO₃, DMF, rt, 6- 12 h.

2.2 Chemistry

Keeping in mind these designing considerations, we started with

the synthesis of a series of compounds in which, we retained one

or two fluorine atoms in a benzene ring and substituents with

different electronic properties (halogen, methyl, methoxy) in the

other one; in some cases, this last phenyl ring was replaced by a

furan or a thiophene. The synthetic scheme to obtain the final

derivatives S1-S13 is outlined in Scheme 1. The procedure

involves three steps: first step is the formation of chalcones,

Schiff’s bases A1-A13, by condensation of ketones such as

acetophenone/4-fluoroacetophenone/2,4-difluoroacetophenone

(1) and variously substituted aldehydes (2) under basic conditions.

In second step, these chalcones are reacted with hydrazine

hydrate in organic solvent to form the pyrazole ring; to this

reaction mixture chloroacetyl chloride and potassium carbonate

was added to obtain the formation of chloroacetyl derivatives of

pyrazole B1-B13. The third step is an easy condensation of B1-

B13 with pyrrolidine-2,5-dione (succinimide, 3) in DMF, by

catalysis with a mild base to give the final compounds S1-S13. All

2.3 In silico screening to determine physicochemical and

pharmacokinetic profile of S1-S13 by using SwissADME

platform

Apart from issues related to efficacy and toxicity, many times drug

development failures are due to poor pharmacokinetic profile and

bioavailability. Hence current trends of drug discovery have been

observed making use of virtual in vitro testing methods and in

silico property predictions to optimize lead compounds.

swissADME is

a

freely accessible webtool to foresee

pharmacokinetic and physicochemical properties of the new small

molecules. This tool predicts the range of the physicochemical

properties such as Molecular weight, solubility, partition

coefficient, topological surface area etc., and pharmacokinetic

properties such as absorption, distribution, metabolism,

elimination of molecules, which are key parameters for lead

molecule to progress to in vivo preclinical and clinical level^[32]. In

light of these predictions lead molecules can be modified or

improvised such that it can exhibit all the desirable properties to

be considered as lead-like or drug-like.

1

final compounds were structurally characterized by IR, H-NMR,

¹³C-NMR, and mass spectrometry; purity was higher than 95%

and was established by HPLC.

2D structures of the compounds, S1-S13, were sketched utilizing

chemdraw ultra version 12.0 software, and were imported in the

swissADME environment (http://www.swissadme.ch/) where

SMILES of the structures were produced and used to run the

swissADME tool. The readings for various physicochemical and

pharmacokinetic parameters were generated, which were

exported as CSV file and data was extracted. Bioavailability radar

FT-IR spectra of S1-S13 displayed the characteristic absorption

bands corresponding to stretching vibrations of pyrrolidine-2,5-

dione in the region of 1699 to 1720 cm^-1, and tertiary in the region

of 1666-1689 cm^-1. Proton NMR data of S1-S13 displayed the four

protons singlet of ethylene of pyrrolidine-2,5-dione in the region of

2.70-2.76 ppm. A singlet of 2 protons corresponding to methylene

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Table 1. SwissADME predictions of physicochemical and pharmacokinetic properties for S1-S13.

ESOL

Fraction

BBB^I

permeant

P-gp^j

substrate

Code

MW^a

RBs^b

HBA^c

HBD^d

TPSA^e

iLOGP^f

GIA^h

Class^g

Soluble

Csp3

0.27

0.26

0.24

0.26

0.3

S1

375.42

393.41

411.4

5

7

4

5

6

7

4

5

4

5

6

8

0

70.05

83.19

70.05

98.29

83.19

88.51

3.16

3.19

2.85

3.11

2.93

2.96

3

High

Yes

No

S2

S3

S4

387.34

395.84

361.39

379.38

367.42

351.36

369.35

457.43

S5

Yes

No

S6

S7

Yes

No

S8

S9

2.94

2.86

2.93

3.3

S10

S11

S12

No

Moderately

soluble

S13

430.28

0.24

5

4

0

70.05

3.12

High

Yes

No

^a-Molecular weight, ^b-Rotatable bonds, ^c- Hydrogen bond acceptors, ^d-Hydrogen bond donors, ^e- Topological surface area, ^f-octanol–

water partition coefficient, ^g-Solubility class, ^h-Gastro-intestinal absorption, ⁱ-Blood brain barrier, ^j- P‐glycoprotein

Figure 3. Left, Bioavailability radar plot of compound S2. Right, BOILED-Egg plot of S1-S13 generated in SwissADME

environment

plots displayed by SwissADME tool were straight taken for the

interpretation.

hydrogen bond donors, (HBD) were <5. Out of 13 compounds, 12

compounds were predicted to be soluble and one was found to

be moderately soluble. Gastrointestinal (GI) absorption for the all

the compounds was predicted to be high. 55% of the compounds

were found penetrating to BBA (blood–brain barrier). Also,

compounds were predicted to non‐substrates for P‐glycoprotein,

Data analysis revealed that, the molecular weights of the

compounds S1-S13 falls between 361.39 to 557.43 g/mol, ilogP

values^[33]were found in the range of 2.85 to 3.47, topological polar

surface area, (TPSA) was ranging from 7.05 to 115.87 Å. The

count of hydrogen bond acceptors, (HBA) were <10, and of

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Table 2. 50% Inhibitory concentration (IC₅₀) values of active S-series compounds in MCF7, HT29 and K562 cell lines obtained

by MTT assay.

MCF-7

HT-29

K562

Code

X₁

X₂

R

(IC₅₀μM)

S1

H

F

H

F

H

F

H

-

H

F

H

F

H

-

4-CH₃

4.55±0.37

0.78±0.01

7.31±0.24

8.62±0.07

15.62±0.73

0.42±0.13

0.74±0.19

8.90±0.36

21.64±0.37

4.12±0.60

20.12±0.08

2.02±0.12

1.21±0.22

0.07±0.04

0.35±0.004

2.43±0.38

0.92±0.15

2.84±0.43

1.99±0.34

1.44±0.24

3.99±0.44

2.52±0.42

3.91±0.38

1.72±0.32

1.66±0.45

3.90±0.79

2.57±0.39

0.39±0.12

19.94±1.28

0.32±0.007

24.74±0.86

47.25±1.24

53.99±1.68

40.85±1.84

40.47±1.57

40.4±1.30

S2

4-CH₃

S3

4-CH₃

S4

2-Furyl in place of phenyl

S5

2-Cl

S6

H

S7

4-F

31.56±1.22

46.06±1.21

45.22±1.06

39.42±1.91

71.73±1.56

48.75±0.01

61.92±0.03

40.30±0.73

0.29±0.004

S8

H

S9

Thiophen-2-yl in place of phenyl

S10

2-Furyl in place of phenyl

S11

2-Furyl in place of phenyl

S12

3,4-Dimethoxy

S13

3,4-Dichloro

Pioglitazone

Paclitaxel

-

Experiments were performed in duplicates and values are presented as Mean±SD.

(P‐gp). P‐gp is found in various tissues and acts as efflux

transporters, pushing the xenobiotics out from the cells, leading

to enhanced clearance of these xenobiotics^[34]. The bioavailability

score was found to be 0.55 and compounds were predicted to

small molecules, thus the BOILED-Egg can be of reliable support

in the process of lead optimization^[40]. Observation of the

placement of our newly synthesized derivatives on BOILED-Egg

plot (Fig. 3, Right), found that all the compounds were present

either in yellow (yolk) or white zone, and none was deviating in

grey area, indicating that will be highly passively absorbed in GI

tract, thus can exhibit oral bioavailability. Out of 13 compounds, 7

compounds were found in yellow (yolk) zone, and are predicted

to penetrant to Brain. This indicates both, chances of CNS

adverse effects as well as possibility of usefulness to treat brain

tumors. For this series of compounds, from in vivo efficacy studies

(presented in section 2.8) it was observed that any of the treated

animal did not exhibited any visible behavioral changes, which

indicates that this series of compounds may not display any CNS

drug-like, as indicted by Lipinski’s rule of five^[35], Muegge’s filter^[36]

Veber’s rule^[37], Egan rule^[38], and Ghose filter^[39]

,

.

The Brain Or IntestinaL EstimateD permeation method (BOILED-

Egg) is projected as a precise predictive model that give

predictions for both brain and intestinal permeation and works by

calculating the lipophilicity (WLOP) and polarity (TPSA) of small

molecules. The BOILED-Egg model delivers a fast, reproducible,

experimentally validated, intuitive, yet statistically robust method

to forecast the passive GI absorption and brain penetration of

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concentrations up to IC₅₀value, increase in number of cells in

G0/G1 phase was observed in comparison with control cells (56%

at IC₅₀concentration compared to 47% in control) (Supporting

data, Supplementary Table. ST3, Fig. S4). Also, decrease in the

number of cells in the G2/M phase relative to the control cells was

observed (21.5% at IC₅₀concentration compared to 30% in

control).

adverse effects, and also exhibited promising antitumor efficacy

after oral administration.

The swissADME also generates a bioavailability radar plot for

each compound, which gives likely contour of oral bioavailability

of the compounds, the pink zone of the radar plot is the ideal zone

indicating, place where the lead-like molecules are found. The six

physicochemical properties measured to generate this ideal zone,

are lipophilicity-LIPO, size-SIZE, solubility -INSOLU, polarity-

POLAR, saturation-INSATU and flexibility-FLEX. Bioavailability

radar plot of compounds, S2 has been presented in Fig. 3, Left.

The compounds S1-S13 were observed to be situated in pink area

of the radar plot. Thus, prediction data indicates that these series

of compounds may display favorable pharmacokinetic and

physicochemical profile to be drug-like molecules.

2.6 Effect on Bcl-2 expression.

Bcl-2 alters the activity of a variety of cell signaling proteins

involved in apoptosis, proliferation, and cell survival.

Overexpression of Вcl-2 mediates resistance to apoptosis

through alteration of other cell signaling pathways, such as cyclin

D1/p27, p53/mdm2, and NF-κB^[43], thus considered as anti-

apoptotic protein. Hence, inactivation of this protein leads to

unavailability of activated/phosphorylated form, offers a way to

make tumor cells to undergo apoptosis. Effects of S2 on

expression levels of Bcl-2 protein were studied in K562 cells (Fig.

4). The cells were seeded in 96-well plates and incubated at 37

ºС in a 5% СО₂atmosphere during 18 h, thereafter S2 was added

to the cells, and the incubation was continued for 48 or 72 h more

(Supporting data, Supplementary Table. ST4). The experiments

were performed as per the manufacturer’s protocol using Muse^®

Bcl-2 Activation Dual Detection Kit (MCH200105 EMD Millipore

Corp., USA). SPSS 21 software was used for statistical analysis.

One-way ANOVA and post hoc Dunnett`s test was performed to

assess differences between the individual groups against one

control. Data are presented as mean±SD. P value <0.05 was

considered statistically significant. Dose-dependent decrease

was observed in the expression levels of phosphorylated Bcl-2 as

compared to control cells (55.0% at IC₅₀concentration (p=0.008)

or 64.0% at ½ IC₅₀concentration (p=0.034) compared to 85.0%in

control). Thus, significant cytotoxic/antiproliferative effects can be

attributed to inhibition of Bcl-2 protein.

2.4 In vitro antiproliferative effects by MTT assay

The intriguing cytotoxic/antiproliferative effects in various tumor

cells shown by both pyrrolidine-2,5-dione- and pyrazoline-based

derivatives prompted us to investigate the activity of S1-S13 in

solid and hematological tumor cell lines. In vitro cytotoxicity of the

compounds was determined by MTT assay utilizing three cell

lines, myeloid leukemia cell line K562, breast cancer cell line

MCF7, and colon cancer cell line HT29, as described

previously^[41][42]. Each cell line was exposed to test compounds for

48 hrs. Paclitaxel and Pioglitazone were used as standard

references and untreated cells were used as negative control.

Paclitaxel was selected as one of the standards as it is a clinically

used anticancer agent, whereas Pioglitazone was chosen as

second standard as it is a PPARγ agonist containing the TZD

group bio-isostere of pyrrolidine-2,5-dione. The IC₅₀values are

shown in Table 2 and mean cell viability plots are presented in

Supplementary section, Supplementary Fig.S3-S5. All the

compounds were found several times more active against MCF7

and HT29 as compared to K562. In MCF7 cell lines, compounds

S2, S6 and S7 exhibited IC₅₀0.78±0.01 μM, 0.42±0.13 μM and

0.74±0.19 μM, respectively. Against HT29, compounds S2 and

S13 exhibited activity in sub-micromolar range with IC₅₀0.92±0.15

μM and 0.39±0.12 μM, respectively. All remaining compounds

exhibited IC₅₀values between 4 to 22 μM for MCF-7 cell lines and

< 4 µM in HT29 cells. Almost all compounds exhibited moderate

activity against K562 cell line in mid-micromolar range. When

compared to standard Pioglitazone, most of the compounds

exhibited much better antiproliferative effects towards HT29 and

K562 cells. In addition, S2 and S13 displayed excellent

antiproliferative effects in MCF7 and HT29 comparable to the

clinical anticancer agent Paclitaxel. Thus, our newly synthesized

pyrazoline-substituted pyrrolidine-2,5-dione series prove to be a

promising series of compounds with outstanding antiproliferative

profile in solid tumors.

2.5 Cell Cycle Analysis.

Compound S2 was selected for further in vitro and in vivo study

because of its cytotoxicity (IC₅₀= 0.92 μM) towards this tumor cell

line that is about 20-fold higher than reference compound

Pioglitazone and only 3-fold lower than Paclitaxel. The effect of

treatment of S2 on cell cycle distribution in K562 cells was

performed on the basis of DNA content in K562 cells, after

treatment with increasing concentrations of S2 for 48 h by using

Muse® Cell Cycle Kit (MCH100106 EMD Millipore Corp., USA).

After 48 h exposure of K562 cells to S2 at three increasing

Figure 4. Graphical presentation of dose dependent decrease

in expression of activated Bcl-2 after treatment with S2 for 48

h and 72 h in K562 cells. Values are presented as Mean of 3

experiments; error bars represent standard error of mean.

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2.7 Docking at binding site of Bcl-2.

compounds (Supporting data, Supporting Fig. S1). To rule out

any potential bond with the receptor, we also evaluated a possible

antagonist activity by conducting a competitive binding assay in

which PPARγ activity at a fixed concentration of the reference

agonist Pioglitazone (2 μM) was measured in cells treated with

increasing concentrations of representative compounds S1-S7,

but also in this case we did not observe any significant activity

(Supporting data, Supporting Fig. S2). Thus, this series of

compounds do not interact with PPARγ receptor as agonists or

antagonists, and antitumor effects exhibited are independent of

PPARγ.

Bcl-2 is a pro-survival member of the Bcl-2 protein family

characterized by a conserved hydrophobic groove on the surface.

The α-helical BH3 domain of proapoptotic members of the Bcl-2

protein family (e.g. Bim, Bid, Puma, Bad) can bind to the

hydrophobic groove of Bcl-2, thereby initiating the apoptosis

cascade. It has been shown that small-molecule BH3 mimics,

[44][45]

such as ABT-737

or navitoclax ^[45][46], efficiently induce cell

death in tumors. As dose dependent decrease was observed in

levels of Bcl-2, we undertook docking analysis of S2 at binding

groove of Bcl-2. Docking was performed using Autodock Vina. At

first, the docking procedure was validated by redocking the

respective ligand into the crystal structure of complexes between

Bcl-2 and two different small molecule ligands. Both redocked

ligands showed excellent overlap with the corresponding

crystallized ligand showing RMSD-values of 0.69 Å (PDB-ID

4AQ3) and 0.37 Å (PDB-ID 6GL8), respectively (Supporting data,

Supplementary Fig. S5). Therefore, docking parameters were

optimally suited for docking unknown compound S2. To

rationalize the putative molecular interaction between of S2 and

Bcl-2, the two stereoisomers R-S2 and S-S2 with chiral carbon in

the pyrazole ring were docked into two different crystal structures

of Bcl-2 (PDB-IDs 4AQ3 and 6GL8). Differences in the crystal

structures of Bcl-2 were attributed to a certain degree of flexibility

around the binding groove and considered by an energy

minimization of the original best docking pose within a radius of

10 Å around the ligand using MOE 2019 and AMBER14 force field.

The prolonged ligand in the crystal structure PDB-ID 4AQ3

occupies most of the hydrophobic groove on the surface of Bcl-2,

similar to the BH3-α-helix of proapoptotic proteins (Fig. 5- A, B).

In contrast, the much smaller ligand from PDB-ID 6GL8 and R-S2

both occupy the deeper hydrophobic subpocket defined by F71,

F63, M74, V92, L96 and do not cover the shallower distant second

site (Fig. 5- B, C, D). R-S2 forms a hydrogen bond to R105

through a carbonyl oxygen next to the pyrazoline group (Fig. 5-C,

D). In addition, R-S2 forms an intramolecular hydrogen bond,

which fixes the ligand in a favorable pose, thereby reducing

potential entropy losses. The aromatic groups of R-S2 show pi-

stacking interactions with F71 and numerous alkyl-aryl

interactions with surrounding hydrophilic amino acid side chains

flanking the lower section of the deeper subpocket (Fig. 5- C, D).

Thus, our experimental results corelates well with docking studies

indicative of possible Bcl-2 inhibition potential of S2.

2.9 HDAC activity assay

Pyrazoline ring has been identified as an appropriate surface

recognition motif in designing of HDAC inhibitors ^[49]–[51], to

ascertain HDAC inhibitory potential, we undertook preliminary

HDAC screening of S1-S13 on two isoforms of HDACs, HDAC 4

(Class II HDAC) and HDAC 8 (Class I HDAC) belonging to two

different HDAC classes. A single dose of 50 μM of each test

compound was used in preliminary screening, and residual

Figure 6. Percent relative activity of HDAC4 and HDAC8 after

treatment with S1-S13 at 50 μM concentration. n = 3, values

are presented as Mean and error bars represents standard error

of mean.

percentage HDAC activity observed is presented in Fig. 6 and

details have been presented in Supplementary data,

Supplementary Tables-ST1 and ST2. For HDAC4, the relative

HDAC activity after treatment with all compounds, was >0.6 (60%)

hence IC₅₀values were not determined for any of the compounds

as high HDAC4 activity was retained. Only S4 and S13 showed

higher activities and were taken forward to determine IC₅₀values.

The IC₅₀values for S4 were >50 μM against HDAC4 and 24.9μM

against HDAC8, respectively, while S13 turned out to be to have

IC₅₀values >50 µM on both HDAC isoenzymes. Thus, our results

indicate that this series of compounds is presently does not exhibit

significant HDAC inhibition but could be modified extensively

further possibly to obtain class I HDAC inhibitors. Details of

experimental method are included in Supplementary data.

2.8 PPARγ transactivation assay

Compounds S1-S13, containing a pyrrolidine-2,5-dione moiety

which resembles the thiazolidinedione ring of PPARγ agonists

Rosiglitazone and Pioglitazone, were evaluated for both agonist

and antagonist activity on the human PPARγ (hPPARγ) subtype

to ascertain effects of bio isosteric replacement of TZD ring with

pyrrolidine-2,5-dione, at binding site of PPARγ. For this purpose,

GAL-4 PPARγ chimeric receptor was expressed in transiently

transfected HepG2 cells according to a previously reported

procedure^[47][48]. The agonist activity of these compounds was

evaluated at three concentrations (1 μM, 5 μM and 25 μM) and

compared with that of the corresponding reference agonist

Pioglitazone, but no significant activity was observed for all

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Figure 5. Binding poses of small molecule BH3 mimetics within the binding groove of Bcl-2. A) Sideview and B) Top view of

superposed crystal structures of Bcl-2 (PDB-ID 4AQ3 (F-chain) in complex with its native ligand (green) and docked to R-S2 (orange).

Surface of binding site is shown in gray. C) Site view of best binding pose of R-S2 within the deep main pocket of Bcl-2. Hydrogen

bonds are shown as green dashed lines. The dashed dark blue line denotes an edge-to-face Pi-interactions between F71 and one

phenyl rings of R-S2. The transparent surface of the binding site varies from hydrophobic (green) to polar (magenta). D) 2D protein-

ligand interactions. Green dashed arrow denotes the key hydrogen bond to R105.

2.8 In vivo tumor regression in HT29 xenografts

identification of safe starting doses and potential human toxicity.

Therefore, first acute toxicity study was carried out which

determined maximum non-toxic single dose of compound S2, as

100 mg/kg (details in supplementary data) and half of this dose

was selected for efficacy study. None of the animals showed the

changes in behavioral or physical activity. Also, no significant

changes were observed in weight of the treated animals as

compared to control group. The autopsy of the animals revealed

no visible changes in the organs such as liver, heart and spleen

Various literature reports revealed the in vivo efficacy of diaryl

pyrazoline derivatives^[52]–[54], but to the best of our knowledge,

there were no reports found about the in vivo antitumor efficacy

of pyrrolidine-2,5-diones, hence it was of interest to pursue the in

vivo effects of these novel diaryl pyrazoline and pyrrolidine-2,5-

dione hybrids in tumor xenografts. Pre-clinical toxicology studies

in mice permits for optimization of treatment cycles as well as

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and tissue damage. Anticancer effect of S2 was evaluated in HT-

29 colorectal adenocarcinoma xenografts in Balb/c nude mice.

Male Balb/c nude mice 6–8 weeks of age were obtained from

N.N. Blokhin Cancer Research Center. The mice were housed in

a pathogen-free environment under controlled conditions of light

and humidity and received food and water ad libitum. HT-29 cells

(5×10⁶) were implanted subcutaneously with Matrigel^®(1:1;

Matrix) into left flank of mice. Once the mean tumor volume

reached ∼65 mm³, animals were randomized into treatment

groups (n=6–10), and dosing with S2 was initiated (day 1): 50

mg/kg i.p. (intraperitoneally) or per os (orally) daily during 20 days

(cumulative dose 1 g/kg).Tumor sizes and body weights were

measured twice weekly. Tumor volume was calculated by the

formula: V=π/6_*(L_*W_*H), where L is length, W is width and H is

height of tumor. Efficiency assessment was carried out using TGI

(tumor growth inhibition). Statistical analyses of in vivo research

were performed using SPSS V. 22.0 comparing continuous

variables by non-parametrical Mann-Whitney U. Data are

expressed as mean ± standard deviation (SD). Effect of S2 on

tumor progression in HT-29 xenografts is presented in fig. 7. S2

demonstrated significant antitumor effect after both i.p. and per os

(oral route) administration. On day 12 of treatment, maximum TGI

66 and 60% was observed with average tumor volume

167.7±28.6 mm³(p=0.002) and 193.1±15.2 mm³(p=0.002) vs

488.0±95.7 mm³in S2 i.p., S2 per os (orally) and control groups,

respectively. On next day after the end of treatment (day 21) TGI

49 and 48% were detected, no significant differences between i.p.

and per os groups were observed. Statistical analysis revealed

the significant differences between the group receiving compound

S2 and the control group (details in supplementary data,

Supplementary Table ST5 to ST7), suggesting that S2 has good

availability when absorbed from the gastrointestinal tract.

Moreover, in the mice group treated with S2, no significant body

weight loss and no evident toxic signs in liver and spleen were

observed.

absorption post oral administration. Apart from the oral

bioavailability, the fact that S2 did not induce any toxic effects in

vivo, strongly supports the potential of this molecule as a lead for

further considerations. Thus, our finding about the in vivo

antitumor efficacy of pyrrolidine-2,5-dione derivatives is kind of

amongst first few prototype examples and thus, the significant

contribution to the scientific community. The in-silico studies

indicated desirable physicochemical and pharmacokinetic

properties of molecules making the drug like molecules. Thus, our

current results advocate that this series of compounds holds

promise for the development of more potent, less toxic and

specific anticancer agents.

Figure 7. Tumor regression by S2 in HT29 xenografts. Graph

of tumor volume vs. time in HT29 xenografts treated with S2, 50

mg/kg i.p. (rectangle), S2, 50 mg/kg per os. (up triangle), and

saline i.p. control (diamond). Error bars represent SEM, n=6.

p<0.002.

3. Conclusion

4. Material and Methods

In conclusion, a series of novel pyrazoline-substituted pyrrolidine-

2,5-diones were synthesized. As both pyrazoline and pyrrolidine-

2,5-dione moieties are present in numerous antiproliferative

molecules, all the derivatives were screened to determine their in

vitro cytotoxic activity against three human tumor cell lines,

namely MCF7, HT29, and K562. The results showed that most

compounds displayed excellent cytotoxic activities towards MCF-

4.1 Chemistry

Commercial Grade reagents of make S D Fine, Sigma Aldrich or

Research Lab were acquired from dealers in India. Thin layer

chromatography (TLC) was done on pre-coated Merck Silica Gel

60 F254. Melting points were determined by open capillary

method using VEEGO melting point device and are uncorrected.

Infrared (FTIR) spectra were obtained using Schimadzu FT/IR-

8400S with use of direct sampling technique. ¹H and ¹³C NMR

spectra were recorded on a Bruker instrument at 400 MHz, TMS

as internal standard and chemical shift values (δ) are described

in ppm. J values (Coupling constants) are stated in hertz (Hz).

Abbreviations in NMR interpretation are, s- singlet, d-doublet, dd

- doublet of doublet, t-triplet, m- multiplet, and q - quartet. Mass

spectrums were recorded with LC-MS Agilent Technologies 1260

Infinity instrument. The percentage purity (≥95 %) of final products

S1-S13, were established by using HPLC (high-performance

liquid chromatography) Agilent 1100 instrument. The conditions

of HPLC chromatography: column - Hemochrome C18 (15cm),

detector- UV visible detector, detection wavelength- 300 nm, flow

rate-1mL/min, sample concentration- 10 ppm, oven temperature

- 30 °C; technique - gradient elution with a run time of 15 min using

7

and HT29 cells and moderate effects on K562 cells.

Compounds S2, S6, S7, and S13 were found to be the most

effective antiproliferative agents. Particularly, S2 was found to be

the most potent, on MCF7 and HT29 with IC₅₀values 0.78 μM and

0.92 μM, respectively. S2 also caused disruption in cell cycle

phases leading to decreased population of cells in G2/M phase

and increased cell population in G0/G1 phase. The excellent

antiproliferative effects of S2 can be correlated with the dose

dependent inhibition of anti-apoptotic protein Bcl-2 and

experimental results found to corelate well with docking studies.

Even with the similar structural features to PPARγ and HDAC

inhibitors, current series of compounds does not affect both the

targets, and thus antitumor effects can be attributed to targeting

of Bcl-2. S2, also, exhibited equivalent and promising in vivo

tumor regression by both i.p. and oral route in nude mice,

indicating bioavailability and anti-tumor efficacy through GI

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mobile phase consisting of Methanol: Formic acid (0.1%) in 70:30

ratio.

7.39–7.42 (m, 3H), 7.50 (d, J=15.6 Hz, 1H), 7.62–7.64 (m, 2H),

7.81 (d, J=15.6 Hz, 1H), 8.03–8.07 (m, 2H).

4.2 Synthesis

1-phenyl-3-(thiophen-2-yl)prop-2-en-1-one

(A9).

Yellow

crystals. Yield 6.9 g (82%). M.P. 68-70 °C. FTIR (cm^-1) 3107, 3066,

1660, 1597, 1552. ¹H-NMR (400 MHz, CDCl₃) δ ppm 7.44 – 7.46

(m, 2H), 7.60 – 7.64 (m, 2H), 7.67 (s, 1H), 7.84 (s, 1H), 7.92 –

7.93 (m, 1H), 8.03 – 8.07 (m, 2H), 8.42 – 8.43 (m, 1H).

4.2.1 Synthetic procedure for A1-A13

Various chalcones were prepared as reported previously. Briefly,

A solution of 1 (acetophenone or 4-Flouroacetophenone or 2,4-

Diflouroacetophenone) and 2 (variously substituted aldehydes),

in equimolar proportion, were stirred in hydroalcoholic solution of

sodium hydroxide in cold conditions. The solid obtained was

filtered at pump and recrystallized from ethanol to obtain

chalcones, A1-A13.

3-(furan-2-yl)-1-phenylprop-2-en-1-one (A10). Pale yellow

crystals. Yield 5.6 g (85%). M.P. 72-74 °C. FTIR (cm^-1) 3107, 3066,

1

1660, 1597. H-NMR (400 MHz, CDCl₃) δ ppm 7.15 – 7.21 (m,

2H), 7.29 – 7.33 (m, 1H), 7.44 (s, 1H), 7.46 (s, 1H), 7.66 – 7.68

(m, 1H), 8.03 – 8.06 (m, 2H), 8.07 – 8.11 (m, 2H).

1-phenyl-3-(p-tolyl)prop-2-en-1-one (A1) Yellow crystals. Yield

6.8 g (83%). M.P. 88-89 °C. FTIR (cm^-1) 3057, 3022, 2912, 1656,

1591, 1566. ¹H-NMR (400 MHz, CDCl₃) δ ppm 2.39 (s, 3H), 7.13–

7.18 (m, 2H), 7.19 – 7.26 (m, 3H), 7.53 – 7.55 (m, 2H), 7.78 (s,

1H), 7.81 (s, 1H), 8.02 – 8.07 (m, 2H).

1-(4-fluorophenyl)-3-(furan-2-yl)prop-2-en-1-one

(A11).

Yellow crystals. Yield 8.6 g (88%). M.P. 100-101 °C. FTIR (cm^-1)

3109, 3066, 1658, 1597, 1587, 1217. ¹H-NMR (400 MHz, CDCl₃)

δ ppm 6.50–6.51 (m, 1H), 6.71 (d, 1H), 7.13–7.18 (m, 2H), 7.42

(d, J=15.3 Hz, 1 H), 7.52 (d, 1H), 7.59 (d, J=15.3 Hz, 1H), 8.04–

8.08 (m, 2H).

1-(4-fluorophenyl)-3-(p-tolyl)prop-2-en-1-one (A2). Yellow

crystals. Yield 8.4 g (80%). M.P. 140.5-141 °C. FTIR (cm^-1) 3074,

2918, 1660, 1593, 1523, 1215. ¹H-NMR (400 MHz, CDCl₃) δ ppm

2.39 (s, 3H), 7.15 – 7.19 (m, 2H), 7.22 – 7.26 (m, 2H), 7.47 (s,

1H), 7.53 – 7.55 (m, 2H), 7.77 (s, 1H), 8.03 – 8.07 (m, 2H).

1-(2,4-difluorophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-

one (A12). Off-white colour solid. Yield 5.6 g (78 %). M.P. 111-

113 °C. FTIR (cm^-1) 3072, 2843, 1654, 1591, 1508, 1220. ¹H-NMR

(400 MHz, CDCl₃) δ ppm 3.93 (s, 6H), 6.87 – 6.93 (m, 2H), 6.96

– 7.01 (m, 1H), 7.13 (m, 1H), 7.20 – 7.23 (m, 2H), 7.69-7.73 (m,

1H), 7.84 – 7.90 (m, 1H).

1-(2,4-difluorophenyl)-3-(p-tolyl)prop-2-en-1-one (A3). Yellow

crystals. Yield 7 g (80%). M.P. 66-68 °C. FTIR (cm^-1) 3302, 2902,

1662, 1595, 1568, 1286, 1265, 1234. ¹H-NMR (400 MHz, CDCl₃)

δ ppm 2.35 (s, 3H), 7.14 – 7.19 (m, 2H), 7.23 – 7.27 (m, 2H), 7.48

(s, 1H), 7.53 – 7.55 (m, 1H), 7.77 – 7.81 (m, 1H), 8.03 – 8.07 (m,

2H).

3-(3,4-dichlorophenyl)-1-phenylprop-2-en-1-one (A13). Yellow

crystals. Yield 6.7 g (83%). M.P. 140-142 °C. FTIR (cm^-1) 3169,

3043, 1664, 1579, 688. ¹H-NMR (400 MHz, CDCl₃) δ ppm 6.86 –

6.97 (m, 2H), 7.49 – 7.53 (m, 2H), 7.57 – 7.58 (m, 1H), 7.59 –

7.66 (m, 2H), 7.82 (s, 1H), 8.00 – 8.03 (m, 2H).

1-(2,4-difluorophenyl)-3-(furan-2-yl)prop-2-en-1-one

(A4).

Yellow crystals. Yield 8.8 g (89%). M.P. 67-68 °C. FTIR (cm^-1)

3078, 1668, 1545, 1251, 1234. ¹H-NMR (400 MHz, CDCl₃) δ ppm

6.51–6.52 (m, 1H), 6.74 (d, J=3.4 Hz, 1H), 6.96–7.01 (m, 1H),

7.29 (m, 1H), 7.57 (t, J=7.6 Hz, 2H), 7.86–7.92 (m, 1H).

4.2.2 Synthetic procedure for B1-B13

A1-A13 (5g, 0.02mol) were dissolved in chloroform and hydrazine

hydrate (2.5mL, 0.04mol) was added dropwise. The reaction

mixture was refluxed at 80°C for 12h. The reaction mixture was

then cooled to the room temperature and K₂CO₃(8g, 0.05mol)

was added and stirred for 15min in ice bath. To this ice-cold

solution chloroacetyl chloride (4mL, 0.03mol) was added

dropwise using dropping funnel and stirred for 12h at room

temperature. Chloroform layer was washed with water and

evaporated under vacuum. Crude solid was purified by extracting

with diethyl ether to get white solid of intermediates B1-B13.

3-(2-chlorophenyl)-1-phenylprop-2-en-1-one (A5). Pale yellow

crystals. Yield 5 g (88%). M.P. 67-68 °C. FTIR (cm^-1) 3074, 1664,

1579, 686. ¹H-NMR (400 MHz, CDCl₃) δ ppm 6.97 – 7.01 (m, 1H),

7.09 – 7.13 (m, 1H), 7.57 – 7.60 (m, 2H), 7.61 – 7.65 (m, 3H),

7.76 – 7.79 (m, 1H), 8.03 – 8.17 (m, 3H).

Chalcone (A6)

Yellow shiny crystals. Yield 7 g (93%). M.P. 56-58 °C. FTIR (cm^-

¹) 3084, 3053, 1660, 1573. ¹H-NMR (400 MHz, CDCl₃) δ ppm

7.35–7.37 (m, 3H), 7.40–7.49 (m, 2H), 7.51–7.55 (m, 2H), 7.58–

7.61 (m, 2H), 7.78 (d, J=15.7 Hz, 1H), 7.99 (q, 2H).

2-chloro-1-(3-phenyl-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-

yl)ethanone (B1). Cream colour solid. Yield 5.3 g (52%). M.P.

117-118 °C. FTIR (cm^-1) 2951, 1666, 1599, 1514, 1489, 1429, 688.

¹H-NMR (400 MHz, CDCl₃) δ ppm 2.31 (s, 3H), 3.21 (dd, J=17.8

Hz, 1H), 3.73-3.80 (m, 1H), 4.57 (m, 2H), 5.56 (dd, J=11.8 Hz,

1H), 7.13 (s, 4H), 7.43-7.46 (m, 3H), 7.74-7.76 (m, 2H).

3-(4-fluorophenyl)-1-phenylprop-2-en-1-one (A7). Yellow crystals.

Yield 7 g (87%). M.P. 65-66 °C. FTIR (cm^-1) 3078, 1662, 1575,

1321. ¹H-NMR (400 MHz, CDCl₃) δ ppm 7.09 – 7.13 (m, 2H), 7.48

– 7.52 (m, 3H), 7.57 – 7.60 (m, 1H), 7.61 – 7.65 (m, 3H), 8.00 –

8.02 (m, 2H).

2-chloro-1-(3-(4-fluorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-

pyrazol-1-yl)ethanone (B2). Off white colour. Yield 4.8 g (56%).

M.P. 119-121°C. FTIR (cm^-1) 2835, 1649, 1602, 1512, 1236, 831.

¹H-NMR (400 MHz, CDCl₃) δ ppm 3.20 (dd, J=17.8 Hz, 1H), 3.70-

3.74 (m, 1H), 3.77 (s, 3H), 4.54 (s, 2H), 5.55 (dd, J=11.8 Hz, 1H),

6.83-6.86 (m, 2H), 7.10-7.18 (m, 4H), 7.73-7.77 (m, 2H).

1-(4-fluorophenyl)-3-phenylprop-2-en-1-one (A8). Yellow

crystals. Yield 5 g (61%). M.P. 73-74 °C. FTIR (cm^-1) 3059, 1658,

1585, 1209 ¹H-NMR (400 MHz, CDCl₃) δ ppm 7.14–7.19 (m, 2H),

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5.72 (m, 1H), 6.31-6.33 (m, 1H), 6.39-6.40 (m, 1H), 7.26-7.30 (m,

1H), 7.42-7.49 (m, 3H), 7.75-7.78 (m, 2H).

2-chloro-1-(3-(2,4-difluorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-

pyrazol-1-yl)ethanone (B3). White colour. Yield 5 g (67%). M.P.

120-122 °C. FTIR (cm^-1) 1681, 1600, 1514, 1502, 1257, 1207, 813. M.P. 139-141 °C. FTIR (cm^-1) 1672, 1600, 1514, 1487, 1222, 808.

2-chloro-1-(3-(4-fluorophenyl)-5-(furan-2-yl)-4,5-dihydro-1H-

pyrazol-1-yl)ethanone (B11). Grey colour. Yield 6.9g (78.5%).

¹H-NMR (400 MHz, CDCl₃) δ ppm 2.31 (s, 3H), 3.27 – 3.33 (m,

1H), 3.80 – 3.88 (m, 1H), 4.49– 4.56 (m, 2H), 5.53 (dd, J=11.6 Hz,

1H), 6.84 – 6.90 (m, 1H), 6.95 – 7.00 (m, 1H), 7.11 – 7.15 (m, 4H),

7.95 – 8.01 (m, 1H).

¹H-NMR (400 MHz, CDCl₃) δ ppm 3.47 (dd, J=17.6 Hz, 1H), 3.60

(dd, J=17.6 Hz, 1H), 4.52 (q, 2H), 5.68 (dd, J=11.5 Hz, 1H), 6.30

(q, 1H), 6.37 (d, J=3.2 Hz, 1H), 7.10–7.15 (m, 2H), 7.29 (t, J=3.7

Hz, 1H), 7.73–7.77 (m, 2H).

2-chloro-1-(3-(2,4-difluorophenyl)-5-(furan-2-yl)-4,5-dihydro-

2-chloro-1-(3-(2,4-difluorophenyl)-5-(3,4-dimethoxyphenyl)-

1H-pyrazol-1-yl)ethanone (S4). Cream colour. Yield 4.5 g (51%). 4,5-dihydro-1H-pyrazol-1-yl)ethanone (B12). Off-white colour

M.P. 109.5-110.5 °C. FTIR (cm^-1) 1672, 1602, 1494, 1263, 1217,

783. ¹H-NMR (400 MHz, CDCl₃) δ ppm 3.55–3.61 (m, 1H), 3.66–

3.74 (m, 1H), 4.52 (q, 2H), 5.67 (m, 1H), 6.32 (q, 1H), 6.39 (d,

J=3.2 Hz, 1H), 6.86–6.94 (m, 1H), 6.95-7.00 (m, 1H), 7.31 (t,

J=0.86 Hz, 1H), 7.95–8.01 (m, 1H).

solid. Yield 5 g (74 %). M.P.145-148 °C. FTIR (cm^-1) 3076, 3007,

2939, 1670, 1604, 1514, 1464, 1253, 1234, 846. ¹H-NMR (400

MHz, CDCl₃) δ ppm 3.29 – 3.35 (m, 1H), 3.79 – 3.88 (m, 7H),

4.45– 4.59 (m, 2H), 5.52 (dd, J=11.6Hz, 1H), 6.76 – 6.83 (m, 3H),

6.85 – 6.91 (m, 1H), 6.95 – 7.00 (m, 1H), 7.95 – 8.01 (m, 1H).

2-chloro-1-(5-(2-chlorophenyl)-3-phenyl-4,5-dihydro-1H-

2-chloro-1-(5-(3,4-dichlorophenyl)-3-phenyl-4,5-dihydro-1H-

pyrazol-1-yl)ethanone (B5). White colour solid. Yield 5.2 g (56%). pyrazol-1-yl)ethanone(B13). Off white colour solid. Yield 5.4 g

1

M.P. 151-152 °C. FTIR (cm^-1) 1672, 1593, 1566, 758. H-NMR

(400 MHz, CDCl₃) δ ppm 3.13 (dd, J=17.8 Hz, 1H), 3.84-3.92 (m,

1H), 4.60 (q, 2H), 5.91-5.95 (m, 1H), 7.08–7.12 (m, 1H), 7.20–

7.26 (m, 2H), 7.39–7.45 (m, 4H), 7.72–7.75 (m, 2H).

(50%). M.P. 159-161 °C. FTIR (cm^-1) 1681, 1668, 1600, 1568,

1541, 783. ¹H-NMR (400 MHz, CDCl₃) δ ppm 3.17-3.23 (m, 1H),

3.76-3.84 (m, 1H), 4.52-4.64 (m, 2H), 5.74 (dd, J=12 Hz, 1H),

6.79-6.86 (m, 2H), 7.14-7.18 (m, 1H), 7.41-7.48 (m, 3H), 7.49-

7.73 (m, 2H).

2-chloro-1-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-

yl)ethanone (B6). White colour solid. Yield 4.6 g (51%). M.P.

117-118 °C. FTIR (cm^-1) 2955, 1660, 1597, 1568, 1542, 1521, 756.

¹H-NMR (400 MHz, CDCl₃) δ ppm 3.20 (dd, J=17.8 Hz, 1H), 3.75

(dd, J=17.8 Hz, 1H), 4.57 (q, 2H), 5.57 (dd, J=11.7 Hz, 1H), 7.22–

7.27 (m, 3H), 7.29–7.33 (m, 2H), 7.40–7.45 (m, 3H), 7.72–7.75

(m, 3H).

4.2.3 Synthetic procedure for derivatives S1-S13

Finally, the intermediates B1-B13 (1g, 0.01 mol) were condensed

with pyrrolidine-2,5-dione, 3 (1g, 0.03-0.04 mol), in the presence

of mild base potassium hydroxide (2.5 g, 0.03 moles) in DMF by

stirring at room temperature for 6-12 hrs. The crude product was

precipitated by adding water to the reaction mixture, which was

filtered at pump, washed with water and recrystallized either from

ethanol, chloroform or (3:1) combination of methanol and

2-chloro-1-(5-(4-fluorophenyl)-3-phenyl-4,5-dihydro-1H-

pyrazol-1-yl)ethanone (B7). Buff white colour solid. Yield 5 g

(57%). M.P. 119-121 °C. FTIR (cm^-1) 1656, 1602, 1509, 1330, 719. chloroform to obtain white needle shaped crystals of S1-S13.

¹H-NMR (400 MHz, CDCl₃) δ ppm 3.20 (dd, J=18 Hz, 1H), 3.75-

3.83 (m, 1H), 4.52-4.61 (m, 2H), 5.57 (dd, J=11.6 Hz, 1H), 6.99-

7.04 (m, 2H), 7.21-7.26 (m, 2H), 7.42-7.49 (m, 3H), 7.74-7.76 (m,

2H).

1-(2-oxo-2-(3-phenyl-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-

yl)ethyl)pyrrolidine-2,5-dione (S1) Yield 1.25 g (62.5%); M.P.

148.1°C; White crystalline solid; IR (cm^-1) 2866, 1705, 1674,

1602; ¹H NMR (400 MHz, DMSO-d₆, δ ppm) 2.28(s, 3H), 2.74(s,

4H), 3.11-3.27 (dd, J = 18.4 Hz, 1H), 3.69-3.76 (m, 1H), 4.67-

4.71(d, J = 16 Hz, 1H), 4.83-4.87(d, J = 16 Hz, 1H), 5.48-5.52 (dd,

J = 11.6 Hz, 1H), 7.08-7.14 (m, 6H), 7.71-7.74 (m, 3H); ¹³C NMR

(400 MHz, DMSO-d₆) 20.57, 27.97, 42.00, 59.53, 115.7-138.6,

155, 177, 162.7, 162.19; UV Spectrum (10 ppm) λ_max299.2 nm;

Theoretical mass 375.4, LC-MS (m/z, I %), 376.2 [(M+H)⁺, 100%];

HPLC Purity % Area 99.3 %, RT 6.52 mins.

2-chloro-1-(3-(4-fluorophenyl)-5-phenyl-4,5-dihydro-1H-

pyrazol-1-yl)ethanone (B8). Off white colour solid. Yield 4.9 g

(53%). M.P. 125-12 7 °C. FTIR (cm^-1) 1678, 1600, 1514, 1494,

1217, 696. ¹H-NMR (400 MHz, CDCl₃) δ ppm 3.20 (dd, J=17.6 Hz,

1H), 3.73-3.80 (m, 1H), 4.52-4.59 (m, 2H), 5.60 (dd, J=11.6 Hz,

1H), 7.10 – 7.15 (m, 2H), 7.21 – 7.25 (m, 2H), 7.26 – 7.29 (m, 1H),

7.31 – 7.35 (m, 2H), 7.72-7.77 (m, 2H).

2-chloro-1-(3-phenyl-5-(thiophen-2-yl)-4,5-dihydro-1H-

1-(2-(3-(4-fluorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-

pyrazol-1-yl)ethanone (B9). White colour solid. Yield 6.1 g (62%). yl)-2-oxoethyl)pyrrolidine-2,5-dione (S2) Yield 1.11 g (55.5%);

M.P. 120-122 °C. FTIR (cm^-1) 1658, 1580, 1500, 1437, 1423, 690.

¹H-NMR (400 MHz, CDCl₃) δ ppm 3.41 (dd, J= 17.8 Hz, 1H), 3.72-

3.80 (m, 1H), 4.52-2.49 (m, 2H), 5.92 (dd, J=11.2 Hz, 1H), 6.92-

6.94 (m, 1H), 7.06-7.07 (m, 1H), 7.19-7.20 (m, 1H), 7.42-7.49 (m,

3H), 7.75-7.77 (m, 2H).

M.P. 203°C; White crystalline solid; IR (cm^-1) 2903, 1703, 1660,

1600, 1153; H NMR (400 MHz, DMSO-d₆, δ ppm) 2.26(s, 3H),

1

2.70(s, 4H), 3.14-3.19(dd, J = 18.4 Hz, 1H), 3.84-3.91(m, 1H),

4.67-4.71(q, J = 15.2 Hz, 2H), 5.47-5.51(dd, J = 11.6 Hz, 1H),

7.07-7.14 (m, 4H), 7.30-7.35(t, J = 8.8 Hz, 2H), 7.87-7.90(m, 2H);

¹³C NMR (400 MHz, DMSO-d₆) 20.57, 27.97, 42.00, 5 9.53, 115.7-

138.6, 155, 177,162.7, 162.19; UV Spectrum (10ppm) λ_max289.9

nm; Theoretical mass 393.1, LC-MS (m/z, I %), 394.1 [(M+H)⁺,

100%]; HPLC Purity % Area 99.15 %, RT 8.71 mins.

2-chloro-1-(5-(furan-2-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-

yl)ethanone (B10). White colour solid. Yield 5.8 g (60%). M.P.

1

138-140 °C. FTIR (cm^-1) 1664, 1595, 1566, 759. H-NMR (400

MHz, CDCl₃) δ ppm 3.49-3.65 (m, 2H), 4.51-4.60 (m, 2H), 5.68-

11

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1-(2-(3-(4-fluorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-

1-(2-(3-(2,4-difluorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-

pyrazol-1-yl)-2-oxoethyl)pyrrolidine-2,5-dione (S3) Yield 1.3 g

yl)-2-oxoethyl)pyrrolidine-2,5-dione (S8) Yield 0.8g (40 %);

M.P. 184.6°C; White crystalline solid; IR (cm^-1) 2821, 1699, 1672,

(65%); M.P. 181.6°C; White crystalline solid; IR (cm^-1) 2921, 1707, 1602, 1151; H NMR (400 MHz, DMSO-d₆, δ ppm) 2.70(s, 4H),

1

1676, 1599, 1168; ¹H NMR (400 MHz, DMSO-d₆, δ ppm) 2.27(s,

3H), 2.71(s, 4H), 3.16-3.17(dd, J = 18.4 Hz, 1H), 3.90-3.97(m, 1H), = 17.4 Hz, 2H), 5.52-5.57(dd, J = 11.8 Hz, 1H), 7.19-7.91(m, 9H);

3.16-3.22 (dd, J = 18.2 Hz, 1H), 3.87-3.95(m, 1H), 4.54-4.67(q, J

4.52-4.64(q, J = 16.4 Hz, 2H), 5.46-5.50(dd, J = 11.8 Hz, 1H),

7.08-7.16 (m, 4H), 7.18-7.20(m, 1H), 7.27-7.32(m, 1H), 7.80-

7.97(m, 1H); ¹³C NMR (400 MHz, DMSO-d₆) 20.58, 27.95, 43.93-

43.98, 59.58, 104.66-138.39, 151.7-151.27, 176.89, 162.87,

162.12; UV Spectrum (10ppm) λ_max287.1 nm; Theoretical mass

411.1, LC-MS (m/z, I %), 412.1 [(M+H)⁺, 100%]; HPLC Purity %

Area 96.81 %, RT 8.04 mins.

¹³C NMR (400 MHz, DMSO-d₆) 27.97, 42.07, 60.17, 115.57-

141.58, 155.06, 177.12, 162.20, 162.84, 164.68; UV Spectrum

(10ppm) λ_max289.2 nm; Theoretical mass 379.1, LC-MS (m/z, I %),

380.1 [(M+H)⁺, 100%]; HPLC Purity % Area 97.76 %, RT 5.27

mins.

1-(2-oxo-2-(3-phenyl-5-(thiophen-2-yl)-4,5-dihydro-1H-

pyrazol-1-yl)ethyl)pyrrolidine-2,5-dione (S9) Yield 1.0g (50 %);

M.P. 189.9°C; White crystalline solid; IR (cm^-1) 2966, 1703, 1676,

1-(2-(3-(2,4-difluorophenyl)-5-(furan-2-yl)-4,5-dihydro-1H-

pyrazol-1-yl)-2-oxoethyl)pyrrolidine-2,5-dione (S4) Yield 1.15

g (57.5%); M.P. 149.8°C; White crystalline solid; IR (cm^-1) 2929,

1

1602; H NMR (400 MHz, DMSO-d₆, δ ppm) 2.73(s, 4H), 3.37-

3.39(m, 1H), 3.87-3.94(m, 1H), 4.49-4.60 (m, 2H), 5.84-5.88(dd,

J = 11.2 Hz, 1H), 6.94-6.96(t, J= 4.4 Hz, 1H), 7.03-7.04(d, J= 3.2

Hz, 1H), 7.41-7.42(m, 1H), 7.41-7.51(m, 3H), 7.84-7.86(m, 2H);

¹³C NMR (400 MHz, DMSO-d₆) 28, 41.62, 55.57, 124.89-143.91,

156.10, 177.11, 162.8; UV Spectrum (10ppm) λ_max289.1 nm;

Theoretical mass 367.1, LC-MS (m/z, I %), 368.1 [(M+H)⁺, 100%];

HPLC Purity- % Area 98.88 %, RT 4.36 mins.

1

1705, 1660, 1602, 1157; H NMR (400 MHz, DMSO-d₆, δ ppm)

2.73(s, 4H), 3.80-3.87(m, 1H), 3.90-3.97(m, 2H), 5.61-5.65(dd, J

= 11.8 Hz, 1H), 6.33(d, J = 2.8, 1H), 6.39(d, J = 1.6, 1H), 7.21-

7.26 (m, 1H), 7.38-7.44(m, 1H), 7.57(s, 1H), 7.97-8.08(m, 1H); ¹³

C

NMR (400 MHz, DMSO-d₆) 27.98, 40.05, 53.35, 104.87-142.64,

151.55-151.70, 177.15, 163.03; UV Spectrum (10ppm) λ_max

285.5nm; Theoretical mass 387.1, LC-MS (m/z, I %), 388 [(M+H)⁺,

100%]; HPLC Purity % Area 98.44 %, RT 4.56 mins.

1-(2-(5-(furan-2-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-

oxoethyl)pyrrolidine-2,5-dione (S10) Yield 0.9g (45 %); M.P.

216.2°C; White crystalline solid; IR (cm^-1) 2920, 1703, 1674, 1600,

1-(2-(5-(2-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-

yl)-2-oxoethyl)pyrrolidine-2,5-dione (S5) Yield 1.22 g (61 %);

M.P. 222.6 °C; White crystalline solid; IR (cm^-1) 2820, 1703, 1676,

1601, 692; ¹H NMR (400 MHz, DMSO-d₆, δ ppm) 2.70(s, 4H),

3.10-3.16 (dd, J = 18 Hz, 1H), 3.97-4.05 (m, 1H), 4.56-4.60 (d, J

= 16.8 Hz, 1H), 4.69-4.73(d, J = 17.2 Hz, 1H), 5.75-5.79 (dd, J =

12 Hz, 1H), 7.11-7.83 (m, 9H); ¹³C NMR (400 MHz, DMSO-d₆):

27.99, 42.92, 57.86, 129.59-130.85, 156.05, 177.11, 162.99,

129.69; UV Spectrum (10ppm) λ_max288.3 nm; Theoretical mass

395.1, LC-MS (m/z, I %), 396.1 [(M+H)⁺, 100%]; HPLC Purity %

Area 98.14 %, RT 7.20 mins.

1

1168; H NMR (400 MHz, DMSO-d₆, δ ppm) 2.73(s, 4H), 3.38-

3.41(m, 1H), 3.70-3.82(m, 1H), 4.49-4.59(m, 2H), 5.63-5.69(dd, J

= 11.6 Hz, 1H), 6.33(m, 1H), 6.39(m, 1H), 7.50-7.59 (m, 4H), 7.83-

8.85(m, 1H); ¹³C NMR (400 MHz, DMSO-d₆): 27.99, 38.16, 53.64,

107.44-142.59, 151.87, 177.15, 162.84; UV Spectrum (10ppm)

λ_max288.1 nm; Theoretical mass 351.1, LC-MS (m/z, I %), 352.1

[(M+H)⁺, 100%]; HPLC Purity- % Area 98.38 %, RT 3.73 mins.

1-(2-(3-(4-fluorophenyl)-5-(furan-2-yl)-4,5-dihydro-1H-

pyrazol-1-yl)-2-oxoethyl)pyrrolidine-2,5-dione (S11) Yield

1.11 g (55.5%); M.P. 175.4°C; White crystalline solid; IR (cm^-1)

1

1-(2-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-

2966, 1703, 1666, 1602, 1168; H NMR (400 MHz, DMSO-d₆, δ

oxoethyl)pyrrolidine-2,5-dione (S6) Yield 0.9 g (45 %); M.P.

201.1 °C; White crystalline solid; IR (cm^-1) 2951, 1707, 1674,

1601; H NMR (400 MHz, DMSO-d₆, δ ppm) 2.71(s, 4H), 3.16-

3.21 (dd, J = 18.2 Hz, 1H), 3.88-3.95(m, 1H), 4.54-4.68 (q, J =

18.1 Hz, 2H), 5.52-5.56(dd, J = 11.2 Hz, 1H), 7.19-7.84 (m, 10H);

¹³C NMR (400 MHz, DMSO-d₆) 27.99, 42.92, 57.86, 129.59-

130.85, 156.05, 177.11, 162.99, 129.69; UV- Spectrum

(10ppm)λ_max290nm; Theoretical mass 361.1, LC-MS (m/z, I %),

362.1 [(M+H)⁺, 100%]; HPLC Purity % Area 98.59 %, RT 5.11

mins.

ppm) 2.73(s, 4H), 3.44-3.45 (m, 1H), 3.78-3.82 (m, 1H), 4.47-

4.57(m, 2H), 5.63-5.67(dd, J = 11.8 Hz, 1H), 6.32(d, J = 2.8, 1H),

6.39(d, J = 1.6, 1H), 7.32-7.36(t, J= 8.8 2H), 7.57(S, 1H), 7.90-

8.92 (m, 1H), 7.57(s, 2H); ¹³C NMR (400 MHz, DMSO-d₆) 27.99,

38.24, 53.75, 107.46-142.59, 151.83, 162.24-162.84, 177.15,

162.84; UV Spectrum (10ppm) λ_max287.5nm; Theoretical mass

369.1, LC-MS (m/z, I %), 370.1 [(M+H)⁺, 100%]; HPLC Purity- %

Area 98.61 %, RT 3.83 mins.

1

1-(2-(3-(2,4-difluorophenyl)-5-(3,4-dimethoxyphenyl)-4,5-

dihydro-1H-pyrazol-1-yl)-2-oxo

ethyl)pyrrolidine-2,5-dione

1-(2-(5-(4-fluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-

yl)-2-oxoethyl)pyrrolidine-2,5-dione Yield 0.99g (49.5 %); M.P.

181.1°C; White crystalline solid; IR (cm^-1) 2903, 1701, 1674,

(S12) Yield 1.25 g (62.5%); M.P. 116.3 °C; White crystalline

solid; IR (cm^-1) 2913, 1703, 1674, 1602, 1166; ¹H NMR (400 MHz,

DMSO-d₆, δ ppm) 2.71(s, 4H), 3.13-3.20 (dd, J=18.4 Hz, 1H),

3.72-3.73 (s, 1H), 3.89-3.97(m, 1H), 4.50-4.54 (d, J= 16.8 Hz, 1H),

4.61-4.65 (d, J= 16.8 Hz, 1H), 5.45-5.49 (dd, J = 11.6 Hz, 1H),

6.70-6.73 (dd, J = 8.4 Hz, 1H), 6.78-6.79 (d, J = 1.8, 1H), 6.88-

6.90 (d, J = 8.4, 1H), 7.20-7.25 (m, 1H), 7.37-7.43 (m, 1H), 7.92-

8.03 (m, 1H); ¹³C NMR (400 MHz, DMSO-d₆) 27.95, 44.03, 55.36-

55.50, 59.52, 104.82-148.77, 151.51, 148.77, 148.06, 177.13,

163.02; UV Spectrum (10ppm) λ_max286.2 nm; Theoretical mass

1

1602; H NMR (400 MHz, DMSO-d₆, δ ppm) 2.71(s, 4H), 3.18-

3.23(dd, J = 11.6 Hz, 1H), 3.90-3.95 (m, 1H), 4.53-4.66(q, J = 16.9

Hz, 2H), 5.53-5.58 (dd, J = 11.6 Hz, 1H), 7.13-7.84(m, 9H); ¹³C

NMR (400 MHz, DMSO-d₆) 27.97, 41.87, 59.42, 125.54-141.58,

155.97, 177.12, 162.57, 162.91, 160.15; UV Spectrum (10ppm)

λ_max289.2 nm; Theoretical mass 379.1, LC-MS (m/z, I %), 380.1

[(M+H)⁺, 100%]; HPLC Purity % Area 97.52 %, RT 5.29 mins.

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457.1, LC-MS (m/z, I %), 458.1 [(M+H)⁺, 100%]; HPLC Purity- %

Area 97.71 %, RT 4.87 mins.

4.5 Molecular Docking

Two crystal structures of Bcl-2 (PDB-IDs 4AQ3 and 6GL8) were

obtained from RCSB Protein Data Bank and subjected to the

Quickprep procedure of MOE 2019 software (Chemical

Computing Group ULC, Canada) including correcting problems

with the structure and 3D-protonation. Then the native ligands

were removed from the receptor proteins, also using MOE.

1-(2-(5-(3,4-dichlorophenyl)-3-phenyl-4,5-dihydro-1H-

pyrazol-1-yl)-2-oxoethyl)pyrrolidine-2,5-dione (S13) Yield

0.8g (40 %); M.P. 209.1°C; White crystalline solid; IR (cm^-1) 2803,

1

1702, 1676, 1601, 1168, 759; H NMR (400 MHz, DMSO-d₆, δ

ppm) 2.71(s, 4H), 3.12-3.17 (dd, J=18.2 Hz, 1H), 3.93-4.01 (m,

1H), 4.56-4.72 (d, J= 17.2 Hz, 2H), 5.71-5.76(dd, J= 12 Hz, 1H),

7.14-7.16(d, J = 8.4 Hz, 1H), 7.37-7.39 (m, 1H), 7.48-7.49 (m, 3H),

7.64 (d, J= 1.2 Hz, 2H), 7.80-7.82 (m, 1H); ¹³C NMR (400 MHz,

DMSO-d₆) 27.99, 40.63, 57.51, 126.90-132.80, 131.93, 156.09,

177.10, 163.09; UV Spectrum (10ppm) λ_max287.6 nm; Theoretical

mass 429.06, LC-MS (m/z, I %), 430.1 [(M+H)⁺, 100%]; HPLC

Purity- % Area 97.52 %, RT 11.43 mins.

Docking

was

carried

out

using

Autodock

in

Vina

PyRx

(http://vina.scripps.edu/)^[55]

embedded

(https://pyrx.sourceforge.io/)^[56], a virtual screening software for

computational drug discovery. The prepared protein structures

and mol2 files of the ligands were loaded into PyRx and converted

to the required pdbqt file format. The original ligands were docked

back in their binding site (redocking) to validate the docking

setting. Subsequently, two stereo isomers of S2 were docked into

the same receptor proteins. To consider the observed flexibility of

binding groove amino acids, docking was followed by an energy

minimization step within a radius of 10 Å around the respective

ligand using MOE 2019 and AMBER14 force field.

4.3 In vitro antiproliferative effects by MTT assay

K562 cells (Chronic myeloid leukemia), MCF7 (Breast cancer)

and HT29 (Colon cancer), were obtained from National Center for

Cell Sciences (NCCS), Pune. K562 cells were kept as suspension

in RPMI 1640 medium (Thermo fisher scientific), supplied with

10% FBS (fetal bovine serum, Gibco, Invitrogen) and 1% anti-

microbial antimycotic 100X solution (100 units/mL of penicillin,

100 µg/mL of streptomycin, and 0.25 µg/mL of Gibco

Amphotericin B). MCF7 and HT29 cells were maintained under

similar conditions except the medium used was DMEM (Thermo

fisher scientific). All cells were kept up in a humidified 5% CO₂air

environment at 37˚C.

MTT assay was employed to find out cytotoxic potential of S1-S13

on proliferation of K562, MCF7 and HT29 cells. The assay was

conducted as described earlier ^[41]. Quickly, the cells were seeded

at a density of around 5×10³cells/well in a flat 96-well plate and

kept overnight at 37 ºC in 95% humidity and supplied with 5% CO₂.

Increasing concentrations of compounds were made in DMSO

and added to the cells. The maximum concentration of DMSO in

the individual assay, was <1.5% and demonstrated no cell

lethality. The cells were incubated with test compounds for 48 h.

then, plates were centrifuged at 300 g at 4°C for 5 minutes and

were washed twice with phosphate buffer. 20 µL of the MTT

staining reagent (5mg/ml in phosphate buffer) was added to each

well and the plates were incubated at 37ºC. After 4 h, 100 µL of

dimethyl sulfoxide (DMSO) was added to each well so as to

solubilize the formazan crystals, and absorbance was recorded

with a 570 nm utilizing micro plate reader.

4.6 In vivo tumor regression in HT-29 xenografts

Anticancer effect of S2 was evaluated in HT-29 colorectal

adenocarcinoma xenografts in Balb/c nude mice. Male Balb/c

nude mice 6–8 weeks of age were obtained from N.N. Blokhin

Cancer Research Center. The mice were housed in a pathogen-

free environment under controlled conditions of light and humidity

and received food and water ad libitum. HT-29 cells (5×10⁶) were

implanted subcutaneously with Matrigel^®(1:1; Matrix) into left

flank of mice. Once the mean tumor volume reached ∼65 mm³,

animals were randomized into treatment groups (n=6–10), and

dosing with S2 was initiated (day 1): 50 mg/kg i.p.

(intraperitoneally) or per os (oral route) daily during 20 days

(cumulative dose 1 g/kg).Tumor sizes and body weights were

measured twice weekly. Tumor volume was calculated by the

formula: V=π/6_*(L_*W_*H), where L is length, W is width and H is

height of tumor. Efficiency assessment was carried out using TGI

(tumor growth inhibition). Statistical analyses of in vivo research

were performed using SPSS V. 22.0 comparing continuous

variables by non-parametrical Mann-Whitney U. Data are

expressed as mean ± standard deviation (SD).

Ethical statement

All animal studies were performed according to the European

Convention for the Protection of Vertebrate Animals used for

Experimental and Other Scientific Purposes. All animal protocols

were approved by the local ethical committee of N.N. Blokhin

Cancer Research Center.

4.4 Cell Cycle analysis and Bcl-2 expression

Effects of S2 on cell cycle distribution and apoptosis signalling

pathways were studied in K562 cells. The cells were seeded in

o

96-well plates and incubated at 37 С in a 5% СО₂atmosphere

during 18 h, thereafter S2 was added to the cells, and the

Declaration of Competing Interest.

incubation was continued for 48 or 72

h more. Final

concentrations of S2 were 47.25 µM, 23.63 µM, 11.81 µM. HBSS

was used in control group. More than 2000 cells were analysed in

every sample using Muse® Cell Analyser and Muse® Detection

Kits (EMD Millipore Corporation) in triplicate. Muse® Cell Cycle

Kit (MCH100106 EMD Millipore Corp., USA) and Muse® Bcl-2

Activation Dual Detection Kit (MCH200105 EMD Millipore Corp.,

USA) were used in accordance with manufacturer's manual.

Results are presented as mean ± SD.

The authors declare that they have no known competing financial

interests.

Acknowledgements

This study was supported by Indo-Poland joint research program

grant from Department of Science and Technology (DST) with

reference number DST/INT/Pol/P-27/2016 (to CSR). The

13

10.1002/cmdc.202000458

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FULL PAPER

biological studies were supported by Russian Scientific

Foundation RSF grant 18-75-10008(to VPS).

[25] N.P. Milosevic, V. Kojic, J. Curcic, D. Jakimov, N. Milic, N.

Banjac, G. Uscumlic, R. Kaliszan, Journal of

Pharmaceutical and Biomedical Analysis 2017, 137, 252.

[26] A. Sharma, T. Pathan, R. Mohan, C. S. Ramaa, LDDD

2011, 8, 843.

Keywords

[27] H. Joshi, K. Marulkar, V. Gota, C. S. Ramaa, ACAMC

2017, 17, 524.

[28] T. Pal, H. Joshi, C.S. Ramaa, ACAMC 2014, 14, 872.

[29] V. Patil, K. Tilekar, S. Mehendale-Munj, R. Mohan, C.S.

Ramaa, European Journal of Medicinal Chemistry 2010,

45, 4539.

•Pyrrolidine-2,5-dione • Synthesis • Cytotoxicity • Docking • Antitumor

• Bcl-2 • Pyrazoline • in vivo

References

[30] A. Kabir, K. Tilekar, N. Upadhyay, C.S. Ramaa, ACAMC

2019, 18, 1551.

[31] S. Saha, D.S.Z. Chan, C.Y. Lee, W. Wong, L.S. New,

W.K. Chui, C.W. Yap, E.C.Y. Chan, H.K. Ho, European

Journal of Pharmacology 2012, 697, 13.

[32] A. Daina, O. Michielin, V. Zoete, Sci Rep 2017, 7, 42717.

[33] A. Daina, O. Michielin, V. Zoete, J. Chem. Inf. Model.

2014, 54, 3284.

[34] Md.L. Amin, Drug Target�Insights 2013, 7, DTI.S12519.

[35] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney,

Advanced Drug Delivery Reviews 2001, 46, 3.

[36] I. Muegge, S.L. Heald, D. Brittelli, J. Med. Chem. 2001,

44, 1841.

[37] D.F. Veber, S.R. Johnson, H.-Y. Cheng, B.R. Smith, K.W.

Ward, K.D. Kopple, J. Med. Chem. 2002, 45, 2615.

[38] W.J. Egan, K.M. Merz,, J.J. Baldwin, J. Med. Chem.

2000, 43, 3867.

[39] A.K. Ghose, V.N. Viswanadhan, J.J. Wendoloski, J.

Comb. Chem. 1999, 1, 55.

[40] A. Daina, V. Zoete, ChemMedChem 2016, 11, 1117.

[41] K. Tilekar, N. Upadhyay, N. Jänsch, M. Schweipert, P.

Mrowka, F.J. Meyer-Almes, C.S. Ramaa, Bioorganic

Chemistry 2020, 95, 103522.

[42] N. Upadhyay, K. Tilekar, N. Jänsch, M. Schweipert, J.D.

Hess, L. Henze Macias, P. Mrowka, R.J. Aguilera, J.

Choe, F.-J. Meyer-Almes, C.S. Ramaa, Bioorganic

Chemistry 2020, 100, 103934.

[43] C.A. Tucker, A.I. Kapanen, G. Chikh, B.G. Hoffman, A.H.

Kyle, I.M. Wilson, D. Masin, R.D. Gascoyne, M. Bally,

R.J. Klasa, Molecular Cancer Therapeutics 2008, 7, 749.

[44] C.-M. Park, M. Bruncko, J. Adickes, J. Bauch, H. Ding, A.

Kunzer, K.C. Marsh, P. Nimmer, A.R. Shoemaker, X.

Song, S.K. Tahir, C. Tse, X. Wang, M.D. Wendt, X. Yang,

H. Zhang, S.W. Fesik, S.H. Rosenberg, S.W. Elmore, J.

Med. Chem. 2008, 51, 6902.

[45] C. Tse, A.R. Shoemaker, J. Adickes, M.G. Anderson, J.

Chen, S. Jin, E.F. Johnson, K.C. Marsh, M.J. Mitten, P.

Nimmer, L. Roberts, S.K. Tahir, Y. Xiao, X. Yang, H.

Zhang, S. Fesik, S.H. Rosenberg, S.W. Elmore, Cancer

Research 2008, 68, 3421.

[46] M.D. Wendt, Expert Opinion on Drug Discovery 2008, 3,

1123.

[47] A. Pinelli, C. Godio, A. Laghezza, N. Mitro, G. Fracchiolla,

V. Tortorella, A. Lavecchia, E. Novellino, J.-C. Fruchart,

B. Staels, M. Crestani, F. Loiodice, J. Med. Chem. 2005,

48, 5509.

[48] L. Porcelli, F. Gilardi, A. Laghezza, L. Piemontese, N.

Mitro, A. Azzariti, F. Altieri, L. Cervoni, G. Fracchiolla, M.

Giudici, U. Guerrini, A. Lavecchia, R. Montanari, C. Di

Giovanni, A. Paradiso, G. Pochetti, G.M. Simone, P.

Tortorella, M. Crestani, F. Loiodice, J. Med. Chem. 2012,

55, 37.

[49] J. Wen, Q. Niu, J. Liu, Y. Bao, J. Yang, S. Luan, Y. Fan,

D. Liu, L. Zhao, Bioorganic & Medicinal Chemistry Letters

2016, 26, 375.

[50] J. Yang, G. Cheng, Q. Xu, S. Luan, S. Wang, D. Liu, L.

Zhao, Bioorganic & Medicinal Chemistry 2018, 26, 1418.

[51] C. Zagni, A. Citarella, M. Oussama, A. Rescifina, A.

Maugeri, M. Navarra, A. Scala, A. Piperno, N. Micale,

IJMS 2019, 20, 945.

[1]

[2]

A. Colombo, C. Cipolla, M. Beggiato, D. Cardinale, Curr

Cardiol Rep 2013, 15, 362.

N. Milman, L. Ginini, Z. Gil, Drug Resistance Updates

2019, 45, 1.

B.S.P. Sandor Eckhardt, CMCACA 2002, 2, 419.

A. Ameri, G. Khodarahmi, H. Forootanfar, F.

Hassanzadeh, G.-H. Hakimelahi, Chem. Biodiversity

2018, 15, e1700518.

D. Havrylyuk, B. Zimenkovsky, O. Vasylenko, A. Gzella,

R. Lesyk, J. Med. Chem. 2012, 55, 8630.

A. Saeed, P.A. Mahesar, P.A. Channar, F.A. Larik, Q.

Abbas, M. Hassan, H. Raza, S.-Y. Seo, Chem.

Biodiversity 2017, 14, e1700035.

V.R. Solomon, C. Hu, H. Lee, Bioorganic & Medicinal

Chemistry 2009, 17, 7585.

S. Chauhan, S. Paliwal, R. Chauhan, Synthetic

Communications 2014, 44, 1333.

K.M. Kasiotis, E.N. Tzanetou, S.A. Haroutounian, Front.

Chem. 2014, 2.

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10] F.M. Awadallah, G.A. Piazza, B.D. Gary, A.B. Keeton,

J.C. Canzoneri, European Journal of Medicinal Chemistry

2013, 70, 273.

[11] P. Rathore, S. Yaseen, S. Ovais, R. Bashir, R. Yaseen,

A.D. Hameed, M. Samim, R. Gupta, F. Hussain, K. Javed,

Bioorganic & Medicinal Chemistry Letters 2014, 24, 1685.

[12] H. Fahmy, N. Khalifa, M. Ismail, H. El-Sahrawy, E.

Nossier, Molecules 2016, 21, 271.

[13] C. Fan, J. Zhao, B. Zhao, S. Zhang, J. Miao, Chem. Res.

Toxicol. 2009, 22, 1517.

[14] R.F. George, M.A. Fouad, I.E.O. Gomaa, European

Journal of Medicinal Chemistry 2016, 112, 48.

[15] G. Nitulescu, C. Draghici, O. Olaru, IJMS 2013, 14,

21805.

[16] Y.-J. Qin, Y. Li, A.-Q. Jiang, M.-R. Yang, Q.-Z. Zhu, H.

Dong, H.-L. Zhu, Eur J Med Chem 2015, 94, 447.

[17] L.-W. Zheng, Y. Li, D. Ge, B.-X. Zhao, Y.-R. Liu, H.-S. Lv,

J. Ding, J.-Y. Miao, Bioorganic & Medicinal Chemistry

Letters 2010, 20, 4766.

[18] E.D. Deeks, Drugs 2015, 75, 1393.

[19] F.A. Luzzio, D.Y. Duveau, E.R. Lepper, W.D. Figg, J. Org.

Chem. 2005, 70, 10117.

[20] J. Best, C. Schotten, G. Lohmann, G. Gerken, A.

Dechêne, Expert Opinion on Pharmacotherapy 2017, 18,

727.

[21] K.S. Devi, P. Subramani, S. Parthiban, N.

Sundaraganesan, Journal of Molecular Structure 2020,

1203, 127403.

[22] S.H. Han, S. Kim, U. De, N.K. Mishra, J. Park, S. Sharma,

J.H. Kwak, S. Han, H.S. Kim, I.S. Kim, J. Org. Chem.

2016, 81, 12416.

[23] D.V. Kaminskyy, R.B. Lesyk, Biopolym. Cell 2010, 26,

136.

[24] E.L. Luzina, A.V. Popov, Journal of Fluorine Chemistry

2014, 168, 121.

14

10.1002/cmdc.202000458

ChemMedChem

FULL PAPER

[52] K. Chen, Y.-L. Zhang, J. Fan, X. Ma, Y.-J. Qin, H.-L. Zhu,

European Journal of Medicinal Chemistry 2018, 156, 722.

[53] D. Havrylyuk, B. Zimenkovsky, O. Vasylenko, A. Gzella,

R. Lesyk, J. Med. Chem. 2012, 55, 8630.

[56] S. Dallakyan, A.J. Olson, in Chemical Biology (Eds: J.E.

Hempel, C.H. Williams, C.C. Hong), Springer New York,

New York, NY 2015, 243.

[54] K.M. Amin, A.A.M. Eissa, S.M. Abou-Seri, F.M.

Awadallah, G.S. Hassan, European Journal of Medicinal

Chemistry 2013, 60, 187.

[55] O. Trott, A.J. Olson, J. Comput. Chem. 2009, NA.

15

10.1002/cmdc.202000458

ChemMedChem

FULL PAPER

Entry for the Table of Contents

hybrids of Pyrazoline and Pyrrolidine-2,5-dione as antitumor agents with oral bioavailability and tumor regression efficacy

•

In vitro antiproliferative effects of compound S2 in HT29, MCF-7 and K562 cell lines were in sub-micromolar range.

Compound S2 found to cause in vivo tumor regression by intraperitoneal and Oral route in HT29 xenografts.

Compound S2 exhibited dose dependent inhibition of anti-apoptotic protein, Bcl-2 and corelated well with docking studies.

16

Article Doi

Synthesis and Biological Evaluation of Pyrazoline and Pyrrolidine-2,5-dione Hybrids as Potential Antitumor Agents

DOI: 10.1002/cmdc.202000458

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Article abstract of DOI:10.1002/cmdc.202000458

Full text of DOI:10.1002/cmdc.202000458

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