Mechanistic selectivity investigation and 2D-QSAR study of some new antiproliferative pyrazoles and pyrazolopyridines as potential CDK2 inhibitors

European Journal of Medicinal Chemistry 218 (2021) 113389

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Mechanistic selectivity investigation and 2D-QSAR study of some new

antiproliferative pyrazoles and pyrazolopyridines as potential CDK2

inhibitors

Ghaneya S. Hassan ^b, Hanan H. Georgey , Esraa Z. Mohammed

a

,

a

,

c

d, **

,

a,

*

a

e

Riham F. George , Walaa R. Mahmoud , Farghaly A. Omar

a

Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt

b

Pharmaceutical Chemistry Department,School of Pharmacy, Badr University in Cairo (BUC), Badr City, Cairo, 11829, Egypt

Pharmaceutical Chemistry Department, Faculty of Pharmacy, Heliopolis University for Sustainable Development, Cairo, 11777, Egypt

Pharmaceutical Chemistry Department, Faculty of Pharmacy, October 6 University, Giza, 12585, Egypt

Medicinal Chemistry Department, Faculty of Pharmacy, Assuit University, 71526, Egypt

c

d

e

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 23 December 2020

Received in revised form

Novel series of diphenyl-1H-pyrazoles (4a-g) and pyrazolo[3,4-b]pyridines (5a-g and 7a-i) were syn-

thesized and evaluated for their antiproliferative activity against breast cancer cell line (MCF7) and

Hepatocellular carcinoma cell line (HepG2). The highest MCF7 growth inhibition activity was attained via

12 March 2021

compounds 4f and 7e (IC50 ¼ 1.29 and 0.93

active ones against HepG2 (IC50 ¼ 1.57 and 1.33

and 7.30 M, respectively). Cell cycle analysis showed arrest at S and G2-M phases in MCF7 cells treated

m

M, respectively), while compounds 5b and 7f were the most

Accepted 14 March 2021

Available online 18 March 2021

m

M, respectively) compared to doxorubicin (IC50 ¼ 1.88

m

with 4f and 7e, and at G2-M and G1/S phases in HepG2 cells treated with 5b and 7f, respectively.

Apoptotic effect of compounds 4f, 5b, 7e, and 7f was indicated via their pre-G1 early and late apoptotic

effects and augmented levels of caspase-9/MCF7 and caspase-3/HepG2. A worthy safety proﬁle was

assessed for compounds 4f and 7e on MCF10A and compounds 5b and 7f on THLE2 treated normal cells.

Furthermore, compounds 4f, 5b and 7f displayed a promising selective proﬁle for CDK2 inhibition vs.

CDK1, CDK4, and CDK7 isoforms as proved from their selectivity index. Docking in CDK2 ATP binding site,

co-crystallized with R-Roscovitine, demonstrated analogous interactions and comparable binding energy

with the native ligand. 2D QSAR sighted the possible structural features governing the CDK2 inhibition

activity elicited by the studied pyrazolo[3,4-b]pyridines. These ﬁndings present compounds 4f, 5b, and 7f

as selective CDK2 inhibitors with promising antiproliferative activity against MCF7 and HepG2 cancer

cells.

Keywords:

Diphenyl-1H-pyrazoles

pyrazolo[3,4-b]pyridines

Antiproliferative

Apoptosis

CDK2

2D QSAR

©

1

. Introduction

small molecules that are both potent and selective remains a

serious challenge for medicinal chemists [5,6]. WHO ranked breast

and liver cancers among the most prevailing cancers [1]. Moreover,

in developing countries, about 50% of breast cancer cases and 58%

of deaths occur [7], besides, about 83% of liver cancer cases are

diagnosed with the highest incidence in Asia and Africa [8]. Higher

proliferation rate and evading the programmed cell death

“apoptosis”, which is intimately correlated to cell cycle, are outlined

among the approved cancer hallmarks [9]. Great attention has been

given to Cyclin-Dependent Kinases (CDKs) as the cell cycle key

regulators [10e12]. CDKs become activated upon association with

their cyclins, they catalyze the transfer of phosphate group from

adenosine triphosphate (ATP) to serine or threonine amino-acid

Cancer represents one of the main death causes worldwide [1].

Despite the indispensable progress accomplished in cancer treat-

ments, several limitations still exist. Examples are the selectivity for

cancer tissues [2], side effects [3], in addition to the multiple-drug

resistance acquisition by cancer cells, rendering them unresponsive

to the standard therapy [4]. Subsequently, the innovation of novel

*

Corresponding author.

*

* Corresponding author.

E-mail addresses: dr.esraazakaria@o6u.edu.eg (E.Z. Mohammed), riham.

eskandar@pharma.cu.edu.eg , rihamfgeorge@gmail.com (R.F. George).

https://doi.org/10.1016/j.ejmech.2021.113389

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

residues in the protein substrates. These phosphorylated proteins

could modulate different cellular functions [13,14]. Rigorous regu-

lation of CDKs activity is very critical for cell growth and division

and any CDKs abnormality has been strongly linked to the molec-

ular pathology of diverse human cancers [11,12,15]. Cyclin-

Dependent Kinase 2 (CDK2) is the cell cycle key component that

guides the G1/S cell cycle transition phase and regulates the G2/M

transition phase [16e19]. Besides, CDK2 was reported to be over-

expressed in breast and hepatocellular carcinoma [12,20,21].

Consequently, CDK2 inhibitors could induce cell cycle arrest at G1/S

and G2/M phases in cancer cells offering a tolerable strategy option

for cancer treatment [22].

Literature review infers that pyrazole and pyrazole-azine het-

erocyclic scaffolds have gotten an exceptional interest for their

organic and biological impact [23e25]. In particular, they represent

dominant pharmacophore elements for antitumor and CDK2

enzyme inhibition activities [26e34]. Fig. 1 illustrates some

representative examples of CDK2 inhibitors with promising anti-

tumor activity. Wyatt et al. identiﬁed a pyrazole-carboxamide de-

pyrazolopyridines as potential antiproliferative agents against

breast cancer and hepatocellular carcinoma with expected CDK2

inhibitory activity and less toxicity. The anticipated design com-

prises three approaches: the ﬁrst one is represented by the general

structure A (Fig. 3) aiming to i) Maintain the pyrazole core to ﬁll the

adenine region of the ATP binding pocket. ii) Introduce two phenyl

groups at positions 1 and 3 of the pyrazole core as a lipophilic

moiety to augment the hydrophobic contact within the binding

site. iii) Introduction of an amino propenone linker connected to a

substituted aryl moiety that might attain hydrophilic/hydrophobic

optimizing segment through electronic and lipophilic diversity of

the substituents. The NH and CO groups of the added linker might

furnish an extra H-bonding with the binding site essential residues.

The second approach (Structure B) (Fig. 3) involves the bioisosteric

replacement of the purine moiety with pyrazolopyridine through

rigidiﬁcation of the previous open-chain analogs (Fig. 3A) to

explore the impact of the new rigidiﬁed analogs (Fig. 3B) on the

inhibitory activity. Finally, the third approach (Structure C) (Fig. 3),

is designed to identify optimal groups for the hydrophilic region of

the ATP-binding site. Hence, different heterocyclic amines were

introduced to the aromatic linker at position 4 of the main core.

Moreover, a nitrile group was introduced at position 5 as a

hydrogen bond acceptor and non-speciﬁc dipole interactions which

promotes binding with sterically occluded protein kinases

enhancing the activity [39].

rivative, AT-7519 (I) as

(

a

CDK2 small molecule inhibitor

IC50 ¼ 47.0 nM) [26] that binds to the ATP binding site of CDK2

Fig. 2). The interaction disclosed the prominence of the pyrazole

nucleus as a dominating pharmacophore element to occupy the

adenine region of the ATP binding pocket. Besides, the molecule is

anchored to the hinge region of CDK2 through H-bonding in-

teractions via NH of the pyrazole core, N atom of the carboxamide

We screened the anticipated compounds for their growth

inhibitory activity against breast cancer cell line (MCF7) and he-

patocellular carcinoma cell line (HepG2). We investigated the most

active compounds for cell cycle analysis, apoptotic assay, activation

assay of caspase-9/MCF7 and caspase-3/HepG2, and safety proﬁle

assessment on normal cell lines. Furthermore, we carried out

in vitro CDK2 enzyme inhibition assay for all the proposed com-

pounds 4a-g, 5a-g, and 7a-i. We investigated the selectivity proﬁle

against a panel of other CDKs isoforms (CDK1, CDK4, and CDK7) for

the most promising compounds. This study validated the selection

of CDK2 among the aforementioned CDKs isoforms that are asso-

ciated with breast cancer and hepatocellular carcinoma progression

as previously reported [40e44]. We scheduled a molecular docking

study to the ATP-binding site of CDK2 to gain insight into the

possible binding modes of the investigated compounds. Moreover,

we performed a 2D QSAR study to discover the structural charac-

teristics governing the CDK2 inhibition activity. Finally, ADMET

computational study predicted the pharmacokinetic aspects and

toxicity risks for the studied compounds.

þ

side chain, and at the physiological pH, the protonated NH

2

of the

piperidinyl substituent with the amino acid residues Glu82, Leu83,

and His84, respectively. Hydrophobic contacts with the binding site

attained through 2,6-dichlorobenzamide [26] (Fig. 2). Interestingly,

lead optimization of AT-7519 (I) was illustrated by its analog II, with

enhanced CDK2 inhibition (IC50 ¼ 25.0 nM) and antiproliferative

activity against MCF7 cells in a micromolar IC50 value [31].

Alternatively, the purine analog R-Roscovitine (III), a CDK2 in-

hibitor, showed antitumor activity against MCF7 and HepG2 cancer

cells (IC50 ¼ 7.8, 25.9

mM, respectively) [29]. R-Roscovitine occupies

nearly the same region of ATP binding pocket in CDK2, (Fig. 2). The

purine ring suits the adenine binding pocket and the two essential

H-bondings with Leu83 residue were also attained via the imid-

azole core N-atom and the side chain NH. The hydroxylated alky-

lamino substitution in Roscovitine (III) is involved in a hydrophilic

interaction in the ribose binding site. Moreover, Roscovitine ben-

zylamino substituent provides

p-p interaction with Ile10, Phe82,

and His84 residues [28]. However, the R-Roscovitine phase-I study

revealed unwanted adverse effects as hypokalemia and raising liver

enzymes [37]. Consequently, several purine isosteres have been

developed aiming to afford more effective and less toxic anticancer

agents. The pyrazolo[1,5-a]pyrimidine analog 4K(BS-194) (IV), a

purine isostere of Roscovitine, revealed enhanced cytotoxic activity

2. Results and discussion

2.1. Chemistry

against MCF7 and HepG2 (IC50 ¼ 0.3, 1.0

CDK2 inhibitory activity (IC50 ¼ 3.0 nM) [28]. Moreover, Dinaciclib

V) which showed promising CDK2 inhibition (IC50 ¼ 1.0 nM) [30],

has been evaluated for numerous cancer indications in clinical trials

38]. Consequently, several related pyrazolopyridines and pyr-

azolopyrimidines were investigated for their potential growth

inhibitory activity against various cancer cell lines. Interestingly,

the pyrazolpyrimidinone VI showed antitumor activity against

m

M, respectively) with

The synthetic pathways for the preparation of the target com-

pounds are illustrated in Schemes 1e3. The synthesis was initiated

by reacting a dry toluene solution of the appropriate methyl ben-

zoate ester with acetonitrile in presence of sodium hydride to

afford 3-oxo-3-phenyl-propanenitriles (1a-c) [45]. The key inter-

mediate 1,3-diphenyl-1H-pyrazol-5-amine (2) was attained

through reacting 3-oxo-3-phenylpropanenitrile (1a) with phenyl-

hydrazine in a neat condition reaction [46] (Scheme 1).

(

[

HepG2 cell line (IC50

¼

0.4

m

M), CDK2 inhibitory activity

The targeted 3-(1,3-diphenyl-1H-pyrazol-5-ylamino)-1-(4-un/

substituted phenyl)prop-2-en-1-ones (4a-g) and 4-(4-un/

substituted phenyl)-1,3-diphenyl-1H-pyrazolo[3,4-b]pyridines (5a-

g) were prepared as displayed in Scheme 2. The synthesis of the

ﬁrst series (4a-g) has been achieved through treatment of the key

intermediate, 1,3-diphenyl-1H-pyrazol-5-amine (2), with the

respective 3-(dimethylamino)-1-(aryl)prop-2-en-1-one (3a-g) in

glacial acetic acid:ethanol (1:2) at ambient temperature. The

(

IC50 ¼ 0.19 M), in addition to apoptosis induction and cell cycle

m

arrest at S and G2/M phases [35]. Bioisosteric replacement of the

common pyrazolopyrimidine motif with pyrazolopyridine as in

compound VII presented a promising CDK2 inhibition at a nano-

molar level (IC50 ¼ 9.0 nM) [36].

Motivated by these previous ﬁndings, our design strategy aimed

at the synthesis and investigation of some pyrazoles and

2

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Fig. 1. Structure of some lead antiumor pyrazole and pyrazole-azine derivatives.

reagents (3a-g) are easily accessible via condensation of methyl-

arylketones with dimethylformamide-dimethylacetal [47]. Mean-

while, the rigidiﬁed analogs, 1,3-diphenyl-1H-pyrazolo[3,4-b]

pyridines (5a-g), were obtained through intramolecular cyclization

of the corresponding pyrazole derivatives 4a-g in boiling glacial

acetic acid.

3-oxo-3-un/substituted phenylpropanenitriles (1a-c) and the

appropriate 4-(un/substituted-1-yl)benzaldehydes (6a-c) [48], us-

ing a catalytic amount of triethylamine, in absolute ethanol.

Structures of the ﬁnal compounds 4a-g, 5a-g, and 7a-i were in

agreement with their spectral data (Supporting data) and

1

elemental analyses. H NMR spectra of compounds 4a-g revealed

Scheme 3 demonstrates the adopted synthetic pathways for the

targeted 1,3-diphenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile

derivatives (7a-i). It involves a simple and efﬁcient one-pot three-

components reaction of the key intermediate (2) with subsequent

their existence as Z/E geometrical isomers around the exocyclic

double bond in 70:30% ratio, respectively. A pair of doublet signals

attributed to eCOeCH- at

isomers, respectively were observed. The D

d

¼ 6.20e6.31 and 6.44e6.54 ppm for Z/E

2

O exchangeable NH

3

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Fig. 2. Graphical illustration of AT7519 (I) and R-Roscovitine (III) binding mode to CDK2 ATP binding site.

2

.2. Biological screening

.2.1. Antiproliferative activity

The synthesized pyrazoles 4a-g, pyrazolo[3,4-b]pyridines 5a-g

and pyrazolo[3,4-b]pyridine-5-carbonitrile analogs 7a-i were

assessed for their antiproliferative activity towards two cancer cell

lines: breast adenocarcinoma (MCF7) and hepatocellular carcinoma

(HepG2). As for compounds that might exist as Z/E mixture, i.e. the

substituted pyrazole derivatives 4a-g, the screening was performed

using the obtained isomeric mixture as their separation was not

accessible. Doxorubicin was used as a control for comparison and

the viability of the cells was measured using the MTT colorimetric

assay [49].

Fig. 3. Core structures of the designed pyrazole and pyrazolo[3,4-b]pyridines.

2

.2.1.1. Antiproliferative activity against MCF7 cell line. The

observed growth inhibitory activity of the tested compounds 4a-g,

a-g, and 7a-i against MCF7 (Table 1) demonstrates IC50 values in

the micromolar range (1.29e30.20, 1.57e61.14, and 0.93e27.01 M,

respectively). Interestingly, compound 7e is the most potent

growth inhibitor (IC50 ¼ 0.93 M) compared to doxorubicin

⁽^I^C50 ¼ 1.88 M). Also, compounds 4f as well as 5b exhibit non-

signiﬁcant enhanced growth inhibitory activity (IC50 ¼ 1.29 and

.57 M, respectively) in comparison to doxorubicin (at p < 0.05).

Moreover, compounds 7b, d, f, h, and i (IC50 ¼ 3.34, 2.76, 2.65, 3.65,

and 2.71 M, respectively) did not show a statistically signiﬁcant

difference in the IC50 values (at p < 0.05) compared to doxorubicin.

5

proton displayed also an E/Z pattern as pair of doublets at

m

d

¼ 9.66e9.83 and 11.94e12.35 ppm, respectively. For compounds

_a_-_g_,_t_h_e_i_r1H NMR spectra revealed a downﬁeld doublet signal

5

m

assigned to pyridine H-2 at

doublet signal at

¼ 7.98e8.04 ppm of the neighboring H-3.

NMR spectra of compounds 7a-i revealed signals at 24.5e49.1,

8.2e66.3, and 47.8e54.7 ppm attributed to the piperidine, mor-

pholine, and piperazine carbons for (7a, d, and g), (7b, e, and h) and

7c, f, and i), respectively (see the experimental section).

d

¼ 8.69e8.75 ppm, and an upﬁeld

13

m

d

C

1

m

4

m

(

Scheme 1. Synthesis of 1,3-diphenyl-1H-pyrazol-5-amine (2).

4

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Scheme 2. Synthesis of the target pyrazoles (4a-g) and 1,3-diphenyl-1H-pyrazolo[3,4-b]pyridines (5a-g).

Scheme 3. Synthetic pathway for the target 1,3-diphenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (7a-i).

The rest of the tested compounds were less active than doxorubicin

IC50 range; 5.59e61.14 M), as shown by the statistical signiﬁcance

differences (at p < 0.05).

Fig. 4 summarizes the most favorable vs. unfavorable sub-

stituents for optimum growth inhibitory activity of the tested

compounds against HepG2 and MCF7 cell lines. Further, it

summarizes the signiﬁcant observations regarding the detailed

structure-activity relationships (SAR) of each series. Whereby, the

rigidiﬁed analogs 5b, d, and e (IC50 ¼ 1.57, 13.30, 5.59 M, respec-

tively) surpass the growth inhibitory activity of the corresponding

opened analogs 4b, d, and e (IC50 ¼ 9.18, 21.70, 30.20 M, respec-

tively). Meanwhile, compounds 5a, c, f, and g (IC50 ¼ 28.20, 61.14,

(

m

5

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Table 1

revealed lower activity with an IC50 range of 10.50e61.10 mM, as

Growth inhibitory activity (IC50

against MCF7 and HepG2 cancer cells.

mM) of the tested compounds and doxorubicin

evidenced by the statistical signiﬁcant differences (at p < 0.05). The

impact of the structural features and substituents on the observed

activity against HepG2 cancer cell line is also depicted in Fig. 4.

2

.2.2. Cell cycle analysis and apoptosis detection

.2.2.1. Cell cycle analysis. Compounds 4f and 7e showing the

highest antiproliferative activity against MCF7, as well as 5b and 7f,

the most superior inhibitors for HepG2 cell line, were chosen for

further mechanistic investigation. Their effect on cell cycle progress

and apoptosis induction was examined. Analysis by ﬂow cytometry

using BD FACSCalibur was applied. This provides a measure for the

cellular DNA content revealing the distribution of cells in the three

major phases of the cycle and gives a chance to discover apoptotic

cells with fractional DNA content [50]. Cells were treated with the

aforementioned compounds at their IC50 concentrations and

incubated for 48 h.

As presented in Table 2 and Fig. 5, a high percentage of pre-G1

apoptosis (36.51% and 24.36%) was observed after treatment of

MCF7 with compounds 4f and 7e, respectively (Control; 1.84%).

Besides, a higher cell accumulation (59.24%) was noticed at the S

phase for compound 4f (Control; 39.15%) and G2-M phase (21.2%)

for compound 7e (Control; 5.14%) indicating cell cycle arrest at S

and G2-M phases for 4f and 7e, respectively. Likewise, the treat-

ment of HepG2 cells with compounds 5b and 7f resulted in pre G1

apoptotic peaks (32.19% and 22.15%, respectively) compared to the

control; 1.52%. Cells accumulation was also observed at the G2-M

phase for compound 5b (37.64% vs. control; 7.34%) and %G0-G1

and %S phases for 7f (48.02, 48.36, respectively) indicating cell cycle

arrest at the G2-M and G1/S phases. The Results demonstrated that

compounds (4f and 7e) and (5b and 7f) inhibit the proliferation of

MCF7 and HepG2 cancer cells, respectively, and cause apoptotic

DNA fragmentation.

a

Compound ID Aryl

R X

IC50 ±SD (

m

M)

MCF7

HepG2

Doxorubicin

e

CH

O

1.88 ± 0.07

9.65 ± 0.36

9.18 ± 0.35

8.07 ± 0.30

7.30 ± 0.32

2.66 ± 0.12

b

4

5

7

a

b

c

d

e

f

g

a

b

c

d

e

f

g

a

b

c

d

e

f

g

h

i

C

6

H

5

e

b

59.90 ± 2.66^b

4-F-C

4-Cl-C

4-Br-C

4-CH -C

4-OCH -C

6

H

4

e

b

6

H

4

e

41.20 ± 1.83

b

c

6

H

4

e

21.70 ± 0.82 8.81 ± 0.39

b

c

3

6

H

4

e

30.20 ± 1.14 7.32 ± 0.33

1.29 ± 0.05^c

2.03 ± 0.09^b

3

6

H

4

e

b

Thiophene-2-yl

C

e

15.40 ± 0.58 2.42 ± 0.11

b

₃_.₂₁_±₀_.₁₄b

1.57 ± 0.07

6

H

5

e

28.20 ± 1.06

1.57 ± 0.06^c

b

4-F-C

4-Cl-C

4-Br-C

4-CH -C

4-OCH -C

6

H

4

e

61.14 ± 2.29 61.10 ± 2.71^b

b

6

H

4

e

13.30 ± 0.50 49.90 ± 2.22^b

b

6

H

4

e

b

3

6

H

4

e

5.59 ± 0.21

10.70 ± 0.48

b

c

3

6

H

4

e

28.70 ± 1.08 6.59 ± 0.29

39.31 ± 1.46 39.30 ± 1.73^b

b

Thiophene-2-yl

e

27.01 ± 1.02 30.90 ± 1.37^b

b

e

H

OCH

Cl

2

3.34 ± 0.13^c

10.50 ± 0.47

b

NeCH

CH

O

NeCH

CH

O

3

14.30 ± 0.54 3.14 ± 0.14^b

₂_.₇₆_±₀_.₁₀c

b

3

2

12.50 ± 0.56

c

0.93 ± 0.03

8.13 ± 0.36

2.65 ± 0.10^c

1.33 ± 0.06^b

10.60 ± 0.40 3.21 ± 0.14^b

b

2

c

b

3.65 ± 0.14

21.50 ± 0.96

NeCH

2.71 ± 0.10^c

14.60 ± 0.65^b

a

2.2.2.2. Apoptosis assay. Results of MCF7 and HepG2 cell cycle

analysis after treatment with compounds (4f and 7e) and (5b and

7f), respectively revealed a pre-G1 peak (Table 2) which is a sign for

apoptosis induction. To validate the ability of the tested compounds

to induce apoptosis, MCF7 and HepG2 cells were stained with

Annexin V/PI, incubated for 48 h with (4f, 7e) and (5b, 7f),

respectively, and analyzed. Analysis of early and late apoptosis il-

lustrates the ability of compounds (4f, 7e) and (5b, 7f) to induce

signiﬁcant levels of apoptosis in MCF7 and HepG2 treated cells

compared to the control cells. The results are summarized in Table 3

and represented graphically in Fig. 6.

IC50: The inhibitory concentration of the compounds required to produce 50%

inhibition of cell growth. Data are displayed as mean ± SD from triplicates. Statistical

analysis was performed using one-way ANOVA followed by Dunnett post hoc test.

b

Statistically signiﬁcant from doxorubicin at p < 0.05.

Statistically non-signiﬁcant from doxorubicin at p < 0.05.

c

2

8.70, 39.31, respectively) displayed decreased growth inhibitory

activity on MCF7 cells compared to the open-chain analogs 4a, c, f,

M, respectively). A plausible

and g (IC50 ¼ 9.65, 8.07, 1.29, 15.40

m

explanation for the variation in the growth inhibition activity of the

two series 4a-g and 5a-g might be attributed to the molecular

ﬂexibility vs. rigidity, respectively. In the ﬂexible series 4a-g, the

molecules might be able to adopt the most preferable orientation

within the active site. On the other hand, the rigidiﬁed analogs 5a-

g, attend limited conformations for binding to the active site.

2

.2.3. Caspase-9 and -3 activation assay (apoptosis induction

hallmarks)

Caspases (cysteinyl aspartate-speciﬁc proteinases) are crucial in

apoptosis regulation. Based on the previously detected activity of

caspase-9 in MCF7 cells [51] and caspase-3 within HepG2 cells [52],

compounds 4f and 7e were evaluated for caspase-9 activation in

MCF7 treated cells, while 5b and 7f for caspase-3 activation in

HepG2 cells. Compounds were tested at their IC50 values; the ELISA

technique was applied and results were expressed in terms of

caspase-9 and -3 concentration (ng/ml) in the treated cell lines. The

results revealed that compounds 4f and 7e increase caspase-9

concentration in MCF7 by ~9 and 10 folds, respectively compared

to the control. Likewise, compounds 5b and 7f induce caspase-3

activation in HepG2 by ~10 folds compared to the control

(Table 3). The results demonstrate that the tested compounds could

be considered as promising apoptotic inducers in MCF7 and HepG2

cancer cells via activation of caspase-9 and -3, respectively.

2

.2.1.2. Antiproliferative activity against HepG2 cell line. The tested

compounds 4a-g, 5a-g, and 7a-i displayed comparable growth

inhibitory activities against HepG2 cancer cells (Table 1); (IC50

ranges; 2.03e59.90, 1.57e61.10 and 1.33e30.90

In particular, compound 7f shows the highest growth inhibition

activity (IC50 ¼ 1.33 M), which is signiﬁcantly different than

doxorubicin (IC50 ¼ 7.30 M) (at p < 0.05). Also, compounds 4a, f, g,

a, b, and 7c, g display signiﬁcant superior growth inhibitory ac-

tivity (IC50¼ 2.66, 2.03, 2.42, 3.21, 1.57, 3.14, and 3.21 M, respec-

tively) than that of the reference drug. Whereas compounds 4e, d,

f and 7e did not display a statistically signiﬁcant difference in the

IC50 values (at p < 0.05) (IC50 ¼ 7.32, 8.81, 6.59, and 8.13 M)

compared to doxorubicin. The rest of the examined compounds

mM, respectively).

m

5

m

5

m

6

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Fig. 4. Overall summary of SAR study for MCF7 and HepG2 growth inhibition activity.

Table 2

assuring their tolerable cytotoxicity proﬁle.

Cell cycle analysis after 48 h incubation with the tested compounds.

Sample

%G0-G1

%S

%G2-M

% pre G1 apoptosis

2

.2.5. Cyclin-dependent kinase 2/Cycline A2 enzyme inhibition

In the light of cell cycle analysis results, the target compounds

a-g, 5a-g, and 7a-i were assessed for their in vitro CDK2/Cycline

MCF7 Control*

55.71

37.29

44.38

47.29

38.47

48.02

39.15

59.24

34.42

45.37

23.89

48.36

5.14

3.47

21.20

7.34

37.64

3.62

1.84

4

7

f/MCF7

e/MCF7

36.51

24.36

1.52

32.19

22.15

4

HepG2 Control*

A2 enzyme inhibition assay. Kinase inhibitory activity was assayed

by using the well-established ADP-Glo assays [55,56]. The investi-

gational CDK2 ATP competitive inhibitor, R-Roscovitine (III), has

been chosen for comparison as a positive control. The attained

5

7

b/HepG2

f/HepG2

*Control: Untreated cells

dose-response curves were used to calculate IC50 values (mM) and

are listed in Table 5. The observed CDK2 inhibitory activity (IC50

values) showed a good correlation with the corresponding value for

the antiproliferative activity of the tested compounds (Table 1).

Illustrative examples are compounds 4f, 5b, and 7f. Analysis of the

CDK2 inhibitory assay results showed that the pyrazolopyridine

2

.2.4. Cytotoxicity screening on normal cell lines

Observing the antiproliferative activities (Table 1), compounds

f, 5b, and 7e were found to be the most promising growth in-

4

hibitors against MCF7 tumor cells. While, compounds 4a, 4f, 4g, 5a,

b and 7c, 7f, 7g were the most potent growth inhibitors against

5

analog 5b is the most potent inhibitor (IC50 ¼ 0.0415

mM) compared

M). Also, compounds 4f, e, 5g,

HepG2 tumor cells. Consequently, the selectivity proﬁle of the

above-mentioned compounds towards cancer cells rather than

normal cells was worthy to be assessed. Whereby, 4f, 5b, and 7e

were subjected to cytotoxicity study on Human Breast Epithelial

normal cell line (MCF10A), and 4a, 4f, 4g, 5a, 5b, 7c, 7f, and 7g were

exposed to cytotoxicity study on Transformed Human Liver

Epithelial-2 normal cell line (THLE2). Doxorubicin has been used as

a reference compound and MTT assay was applied for measuring

the viability of the cells [53]. Cytotoxic concentration (CC50) of the

tested compounds was calculated as a measure for their safety

proﬁle.

The results displayed signiﬁcant higher CC50 values (at p < 0.05)

for compounds 5b, and 7e on MCF10A and 4a, 4f, 4g, 5a, 5b, and 7f

on THLE2 normal cell lines in comparison to doxorubicin. While,

compounds “4f/MCF10A” and “7c, g/THLE2” showed non-

signiﬁcant difference in the CC50 value compared doxorubicin (at

p < 0.05) (Table 4). The selectivity index (SI) of the tested com-

pounds was calculated as a measure for their selectivity towards

MCF7 and HepG2 tumor cells rather than MCF10A and THLE2

normal cells, respectively (selectivity index ¼ CC50 on normal cells/

IC50 on cancer cells) [54]. Compounds (4f, 5b, and 7e) and (4a, 4f,

to R-Roscovitine (III) (IC50 ¼ 0.0432

m

and 7f were revealed to be statistically non-signiﬁcant different

CDK2 inhibitors (at p < 0.05) (IC50 ¼ 0.042, 0.0632, 0.0568, and

0.0665 mM, respectively) compared to compound (III). The

remaining tested compounds displayed signiﬁcant lower inhibitory

activity (IC50 range; 0.0807e0.644

Roscovitine (III).

mM) in comparison with R-

Structural exploration of compounds 4a-g showed that the

presence of electron-donating substituents (CH or OCH ) seems to

3

be more promising for the CDK2 inhibitory activity than both the

un-substitution or the electron-withdrawing substituents (F, Cl,

and Br). This could be exampliﬁed via compounds 4e and 4f

(

IC50 ¼ 0.0632, and 0.042

m

M, respectively) versus compounds 4a

IC50 ¼ 0.318

m

M) and 4b-d (IC50 ¼ 0.133, 0.122 and 0.517

mM,

respectively). On contrary, in the rigidiﬁed analogs 5a-g, com-

pounds with an electron-withdrawing substituent (F, Br) as 5b and

d (IC50 ¼ 0.0415, and 0.0807

inhibitory activity than others with an electron-donating substit-

uent (OCH M), except for 5c. Moreover, in both

mM, respectively) showed higher CDK2

3

) as 5f (IC50 ¼ 0.27

m

series 4a-g and 5a-g, it has been noticed that replacing the phenyl

moiety with the thiophene moiety led to an increase in the CDK2

4

g, 5a, 5b, 7c, 7f, and 7g) revealed selectivity index values superior

inhibitory activity (4a vs. 4g; IC50 ¼ 0.318 vs. 0.201

mM) and (5a vs.

to doxorubicin towards MCF7 and HepG2 tumor cells, respectively

5

g; IC50 ¼ 0.348 vs. 0.0568 M).

m

7

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Fig. 5. Cell cycle analysis after 48 h incubation with the tested compounds.

8

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Table 3

demonstrate two H bondings between Leu83 residue and both

imidazole core N atom and side-chain NH. Additionally, another H-

bond was formed with Glu81 residue. Moreover, the benzyl moiety

of R-Roscovitine demonstrates p-p interaction with the amino acid

residues Ile10, Phe82, and His84 [28] (Fig. 7).

Apoptosis induction analysis and caspases concentrations within MCF7 and HepG2

treated cells.

Sample

Apoptosis induction

analysis

Caspases conc. (ng/ml)

Total

Early

Late

Caspase-9

Caspase-3

Consequently, docking procedures have been completed for the

target compounds. The results demonstrate that most of the

studied compounds have similar binding modes with comparable

docking scores to that of the lead compound R-Roscovitine (III)

MCF7 control

1.84

0.57

2.69

9.36

0.38

6.15

6.07

0.28

20.64

9.86

0.18

17.43

11.17

2.018 ± 0.14

18.96 ± 0.70

20.32 ± 0.55

e

4

7

f/MCF7

e/MCF7

36.51

24.40

1.52

32.19

22.20

e

HepG2 control

0.03822 ± 0.0029

0.38722 ± 0.0154

0.3714 ± 0.0174

(Supporting data). Docking study of compounds 4a-g was ﬁrstly

5

7

b/HepG2

f/HepG2

performed for both E and Z conﬁgurations. Remarkably, results

analysis showed that Z forms demonstrate the essential in-

teractions with the enzyme binding site and their docking scores

agree with the attained IC50 values which might rationalize their

observed activity; (Supporting data for more details of E and Z

conﬁgurations). Whereby, compounds 4b, c, e, and f “Z conﬁgura-

Caspases meausrements were performed in triplicate.

Relating the observed CDK2 inhibitory activity of the pyrazolo

3,4-b]pyridine-5-carbonitriles (7a-i) to their structural features

revealed the signiﬁcance of the piperazine moiety for CDK2 inhi-

bition than piperidine or morpholine moieties. This can be illus-

[

tions”; (IC50 ¼ 0.133, 0.122, 0.0632, and 0.042

mM, respectively)

could mediate a hydrogen bond interaction with Leu83 residue in

the hinge region which is essential for CDK2 inhibitory activity

trated by compounds 7c vs. 7a (IC50 ¼ 0.161 vs. 0.329

mM) and

[

28,57]. This binding pattern might explain the most promising

compounds 7f vs. 7d and 7e (IC50 ¼ 0.0665 vs. 0.0851 and

CDK2 inhibition activity attained by the previously mentioned

compounds. Meanwhile, compounds 4a, d, and g “Z conﬁgurations”

with lower CDK2 inhibitory activity (IC50 ¼ 0.318, 0.517, and

0

.221

.644

m

M), and 7i vs. 7g and 7h (IC50 ¼ 0.129 vs. 0.195 and

M), respectively, except for 7b. Moreover, compounds

) at the para posi-

bearing an electron-donating substituent (OCH

3

0.201 mM, respectively), were not bound to Leu83 residue. Also, the

tion of the phenyl ring showed better CDK2 inhibition activity than

those have electron-withdrawing one (Cl). As evident in Table 5

compounds (7d vs. 7g: IC50 values, 0.0851 vs. 0.195

binding afﬁnity of the docked compounds towards CDK2 was

represented with their energy score (S, Kcal/mol) whereby a lower

docking score indicates better afﬁnity. Docking scores (Table 7) are

well correlated with CDK2 inhibitory activity, additionally, the Ki

values are shown in Table 7. Fig. 8 shows the docking pattern of the

mM), (7e vs.

7

0

h: IC50 values, 0.221 vs. 0.644

.0665 vs. 0.129 M), respectively.

mM), and (7f vs. 7i: IC50 values,

m

most active pyrazole derivative 4f (IC50 ¼ 0.042

mM) with the

2

.2.6. Selectivity proﬁle vs.CDK isoforms

The most promising compounds amongst our study 4f, 5b, 7e,

lowest docking score (S) ¼ ꢁ15.97 kcal/mol. Its binding to the CDK2

ATP site involves H-bonding to Leu83, in addition to two

drophobic interactions with Ile10 and Gly13 residues.

Amongst the pyrazolopyridine series (5a-g), the displayed

docking score of compound 5b (ꢁ15.07 kcal/mol) together with its

binding modes to the CDK2 ATP region (Fig. 9) might justify its

p-H hy-

and 7f were subjected to further investigations for their selectivity

proﬁle against a panel of the other three kinases; CDK1-cyclin B,

CDK4-cyclin D1 and CDK7-cyclin H, in addition to CDK2-cyclinA2

inhibition activity. Results (Table 6) revealed that the most signif-

icant inhibition was exerted by compounds 4f, 5b, 7e, and 7f

remarkable CDK2 inhibitory activity (IC50 ¼ 0.0415

mM). whereby,

against CDK2-cyclinA2 isoform with IC50 range: 0.042e0.221

Lower inhibition activity was displayed against other CDKs iso-

forms; for CDK1-cyclin B (IC50 range: 0.209e1.353 M), for CDK4-

cyclinD1 (IC50 range: 6.9183e104.02 M) and for CDK7-cyclin H

IC50 range: 0.225e1.991 M). The studied compounds are

3.1e22.7 folds more selective for CDK2 versus CDK1, whereas the

mM.

H-bonding with the essential residue Leu83, besides

phobic interactions were observed.

p-H hydro-

m

Similarly, the docking pattern of the homologous series 7a-i

provides further evidence for the impact of binding to Leu83 res-

idue for the CDK2 inhibitory activity (supporting data). Fig. 10 il-

lustrates the binding pattern of compound 7f, the most potent

inhibitor of CDK2 within this series. Compound 7f could mediate

two H-bonding interactions with Leu83 via the CN group and ar-

omatic C atom in addition to another one with Lys89 through the

pyrazole N atom. Unlike the previously displayed binding modes,

the remarkable involvement of the CN group demonstrates its

implication for ligand-target interactions. Additional hydrophobic

interactions with Phe80, Val18, Gln131, and Lys89 residues were

also displayed.

m

(

~

m

selectivity of CDK2 versus CDK4 is the ultimate (~95.8e1564.2

folds). Also, for CDK2 versus CDK7, compounds 4f, 5b, and 7f

showed ~3.4e48 folds, while compound 7e displayed near equal

activity for both isoforms.

2

.3. Molecular modeling study

.3.1. Molecular docking study

The observed CDK2 inhibition activity elicited by the target

compounds, as well as their antiproliferative activity inspired the

exploration of the docking pattern in the CDK2 ATP binding pocket.

Crystal structure of CDK2 co-crystallized with R-Roscovitine was

downloaded from PDB (http://www.rcsb.org/) (PDB code: 3DDQ)

and used in the current docking investigation. Docking protocol

using MOE 2014.0901 molecular modeling program has been

applied for this study. A validation step comprising redocking of the

native ligand into the binding site is primarily achieved. Validation

2.3.2. 2D QSAR studies

QSAR is a signiﬁcant tool to explore the relationship between

chemical structure and biological activities [58]. To investigate the

structural requirements contributing to CDK2 inhibition activity of

the studied pyrazolopyridines 5a-g and 7a-i, a 2D QSAR model (1)

was generated using multiple linear regression (MLR) analysis. The

16 pyrazolopyridines were splitted into a training set of 13 com-

pounds and a test set of 3 compounds. Compounds 5c, 7c, and 7f

were selected as a test set keeping the activity value distribution of

the original dataset. CDK2 inhibition activity was expressed in a

logarithmic scale (pIC50 in Molar concentration) “pIC50 ¼ -log(I-

ꢀ

parameters e.g. RMSD ¼ 0.494 A , together with a similar binding

mode to that of the co-crystallized ligand supported the validity of

the used docking protocol (Fig. 7). The binding modes revealed that

R-Roscovitine occupies almost the ATP binding region, where the

purine nucleus ﬁlls the adenine region. The interactions

ꢁ

6

C

50*10 )”. A wide range of descriptors (total #: 1103) available on

ChemDes web-based platform (http://www.scbdd.com/chemdes)

9

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Fig. 6. Apoptosis induction analysis after 48 h incubation with the tested compounds.

were calculated for training and test compounds. The calculated

descriptors include a variety of 2D and 3D descriptors: constitu-

tional, topological, connectivity indices, kappa shape, basak,

electro-topological state indices, burden, autocorrelation, charge,

molecular properties, MOE-type, geometric, CPSA, WHIM, MoRSE,

and RDF descriptors. The obtained training set “descriptors/CDK2

inhibitory activity” matrix was then transferred to RapidMiner

9.0.003 Edition for 2D QSAR model construction.

First, descriptors ﬁltration was done by excluding the low-

variance descriptors which add no further information to the

10

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Table 4

Cytotoxicity (CC50,

and doxorubicin.

m

M)^aand Selectivity index (SI)^bvalues of the tested compounds

pIC₅₀¼ ꢁ 0:078 * RDFU10 þ 1:860 * GATSe8

þ 0:009 * EstateVSA7 þ 7:858

(1)

Sample

CC50^a

SI^b

2

r ¼ 0.918 r.

MCF10A

¼

0.844 RMSE ¼ 0.134.

Doxorubicin

13.9 ± 0.90

16.9 ± 0.64^c

7.39

4

5

7

f

b

e

13.10

18.02

59.89

RDFU10: 3D unweighted radial distribution function (RDF)

descriptor.

d

28.3 ± 1.25

d

55.7 ± 3.70

GATSe8: 2D Geary autocorrelation descriptor based on atomic

Sanderson electronegativity.

EstateVSA7: 2D MOE-type descriptor using Estate indices and

surface area contributions.

THLE2

Doxorubicin

12.7 ± 0.80

1.73

d

4

5

7

a

f

53.4 ± 2.53

20.07

17.33

8.22

10.65

12.54

4.80

d

35.2 ± 1.67

d

g

a

b

c

f

19.9 ± 0.94

The generated model demonstrates a good performance as

represented by its statistical evaluation parameters and predictions

d

34.2 ± 1.62

19.7 ± 0.88

d

2

₁₅_.₁_±₀_.₇₂c

(Tables 8 and 9) whereby the coefﬁcient of determination R for

d

37.5 ± 2.50

28.19

3.89

training set prediction and L10%O cross-validation are 0.844, 0.740,

with RMSE of 0.134 and 0.176, respectively. The closeness between

the cross-validation and training set prediction results shows the

robustness of the generated model and its lack of over-ﬁtting.

Scattering plots of the observed versus predicted CDK2 inhibitory

activity values in training set prediction and L10%O cross-validation

are represented graphically in Fig. 11 showing a high agreement

between both the observed and the predicted values.

g

12.5 ± 0.59^c

a

CC50: The concentration of compound that reduce the cell viability by 50%.

SI: The ratio of CC50 on MCF10A and THLE2 normal cells to IC50 on MCF7 and

b

HepG2 tumor cell lines, respectively. All measurements were done in triplicate.

Statistical analysis was performed using one-way ANOVA followed by Dunnett post

hoc test.

c

Statistically non-signiﬁcant from doxorubicin at p < 0.05.

Statistically signiﬁcant from doxorubicin at p < 0.05.

d

For further validation of the obtained model, external validation

was carried out to conﬁrm that the model could predict the data

points and not just ﬁts them, so the constructed model was used to

predict the CDK2 inhibitory activity of the test set compounds

Table 5

CDK2 inhibitory activity (IC50

m

M) of the tested compounds and R-Roscovitine (III).

a

Compound ID

Aryl

R

X

IC50 ±SD (

m

M)

(

Table 10). The constructed model showed good test set prediction

2

III

e

CH

O

0.0432 ± 0.003

0.318 ± 0.019^b

with the following statistical evaluation parameters (R ¼ 0.756

and RMSE ¼ 0.201) indicating the robustness and the predictive

capability of the obtained QSAR model.

4

5

7

a

b

c

d

e

f

g

a

b

c

d

e

f

g

a

b

c

d

e

f

g

h

i

6

C H

5

e

b

4-F-C

4-Cl-C

4-Br-C

4-CH -C

4-OCH -C

6

H

4

e

0.133 ± 0.008

0.122 ± 0.007^b

0.517 ± 0.031^b

0.0632 ± 0.004

6

H

4

e

Analysis of 2D QSAR Model.

6

H

4

e

c

For the pyrazolopyridines generated model (equation (1)), three

descriptors (RDFU10, GATSe8, and EstateVSA7) were selected via

the forward selection method. The ﬁrst descriptor RDFU10 is one of

the 3D radial distribution function (RDF) descriptors [60], these

descriptors could be interpreted as the probability distribution to

ﬁnd an atom in a spherical volume of radius r. Generally, the radial

distribution function noted by “RDFrw” is calculated at several

discrete points with a step size for r ¼ 0.5 Å under ﬁve different

weighting schemes (w), including the unweighted case (u), van der

Waals volume (v), atomic mass (m), atomic polarizability (p) and

Sanderson atomic electronegativity (e) [61]. The negative coefﬁ-

cient of the RDFU10 descriptor indicates its inverse correlation with

the CDK2 inhibitory activity of the target pyrazolopyridines. The

second descriptor GATSe8 is the Geary autocorrelation-lag8/

weighted by atomic Sanderson electronegativities [60]. Geary de-

scriptors are 2D autocorrelation descriptors that are attained from

molecular graphs, by summing the atom weights products of the

terminal atoms for all the paths of the considered path length (the

lag) [62]. The third descriptor EstateVSA7 is the 2D MOE-type

descriptor using Estate indices and surface area contributions

[60]. Electrotopological state (Estate) indices provide fruitful in-

formation of both electronic and topological descriptions of the

atoms within the compounds [63]. The positive coefﬁcients of

GATSe8 and EstateVSA7 descriptors indicate their constructive ef-

fect on the CDK2 inhibitory activity for their pyrazolopyridine

derivatives.

3

6

H

4

e

c

3

6

H

4

e

0.042 ± 0.003

Thiophene-2-yl

e

0.201 ± 0.012^b

0.348 ± 0.021^b

0.0415 ± 0.003

6

C H

5

e

4-F-C

4-Cl-C

4-Br-C

4-CH -C

4-OCH

6

H

4

e

b

6

H

4

e

0.386 ± 0.023

0.0807 ± 0.005^b

6

H

4

e

b

3

6

H

4

e

0.0819 ± 0.005

0.27 ± 0.003^b

3

-C

6

H

4

e

c

Thiophene-2-yl

e

0.0568 ± 0.003

₀_.₃₂₉_±₀_.₀₂₀b

e

H

OCH

Cl

2

b

0.096 ± 0.006

NeCH

CH

O

NeCH

CH

O

3

0.161 ± 0.010^b

b

3

2

0.0851 ± 0.005

0.221 ± 0.013^b

c

0.0665 ± 0.004

₀_.₁₉₅_±₀_.₀₁₂b

2

b

0.644 ± 0.039

NeCH

0.129 ± 0.008^b

a

IC50: The inhibitory concentration of the compounds required to produce 50%

inhibition, IC50 value was interpolated from doseeresponse data with four different

concentrations. Results are displayed as mean ± SD from triplicates. Statistical

analysis was performed using one-way ANOVA followed by Dunnett post hoc test.

b

Statistically signiﬁcant from doxorubicin at p < 0.05.

Statistically non-signiﬁcant from doxorubicin at p < 0.05.

c

model (redundant descriptors) leaving a set of 883 descriptors for

model generation. Descriptors selected for the model were then

performed using the conventional forward selection algorithm

(

FSA) available in RapidMiner [59] and multiple linear regression

MLR) was then utilized as the machine learning algorithm for

2.3.3. In silico ADMET study

building a simple, transparent and predictive linear correlation

model. Root mean square error of a leave-10%-out (L10%O) cross-

validation (RMSECV) was used for the model performance assess-

ment. Based on the FSA descriptor selection, a three descriptors

model (Equation (1)) was constructed.

2

.3.3.1. Pharmacokinetic and drug-likeness aspects prediction.

Prediction of pharmacokinetics and drug-likeness aspects of the

target compounds 4a-g, 5a-g, and 7a-i was done using the freely

available web server Swiss ADME (http://www.swissadme.ch/).

Human gastrointestinal absorption (HIA), blood-brain barrier (BBB)

11

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Table 6

Selectivity proﬁle vs. various CDKs isoforms for the most promising compounds.

Sample CDKs inhibitory activity IC50±SD (

m

M) and Ki (

mM)

CDK2/A2 (Ki) CDK1/B (Ki)

SI^afor CDK2 vs. CDK1 CDK4/D1 (Ki)

SI for CDK2 vs. CDK4 CDK7/H (Ki)

SI for CDK2 vs. CDK7

4

5

7

f

0.042 ± 0.003 (0.025) 0.955 ± 0.05 (0.581) 22.7

0.0415 ± 0.003 (0.025) 1.353 ± 0.07 (0.823) 32.6

0.221 ± 0.013 (0.131) 0.718 ± 0.04 (0.437) 3.2

0.0665 ± 0.004 (0.04) 0.209 ± 0.01 (0.127) 3.1

16.681 ± 0.80 (9.12) 397.2

6.9183 ± 0.33 (3.78) 166.7

21.17 ± 1.02 (11.6) 95.8

104.02 ± 5.01 (56.9) 1564.2

0.326 ± 0.02 (0.208) 7.8

b

e

f

1.991 ± 0.11 (1.272) 48.0

0.318 ± 0.02 (0.203) 1.4

0.225 ± 0.01 (0.144) 3.4

a

SI : Selectivity index for CDK2 is calculated as the ratio of IC50 values on different CDKS isoforms to IC50 on CDK2.

The IC50 value was interpolated from doseeresponse data with four different concentrations that are repeated in triplicate. The Kinetic parameter Ki was calculated via

GraphPad Prism software.

Fig. 7. Validation of the adopted docking protocol; (A) overlay complexes and (B) 3D representation of superimposition of the native ligand (green) and its re-docked pose (red) in

CDK2 ATP binding site).

Table 7

studied compounds (Supporting data). The white region where

compounds 4d, g, 5b-e, g, 7b, c, and f are located, indicates their

GIT passive absorption probability. Compounds 4a-c, e, f, 5a and, f

were found in the yellow region predicting their blood-brain bar-

rier penetration probability. More detailed results of ADME pa-

rameters of the studied compounds are summarized in supporting

data.

.

Docking energy scores (S) in kcal/mol and Ki values (mM) for the target compounds

and R-Roscovitine (III).

Compound

(S)

Ki

Compound

(S)

Ki

III

ꢁ14.91

ꢁ13.16

ꢁ14.03

ꢁ13.56

ꢁ13.01

ꢁ14.98

ꢁ15.97

ꢁ13.13

ꢁ12.30

ꢁ15.07

ꢁ12.28

ꢁ14.00

0.026

0.189

0.079

0.072

0.307

0.038

0.025

0.119

0.207

0.025

0.229

0.048

5e

5f

ꢁ14.31

ꢁ11.50

ꢁ14.66

ꢁ13.15

ꢁ14.48

ꢁ13.25

ꢁ13.27

ꢁ13.12

ꢁ13.53

ꢁ13.63

ꢁ11.16

ꢁ13.63

0.049

0.160

0.034

0.195

0.057

0.096

0.051

0.131

0.040

0.116

0.383

0.077

4

5

a

b

c

d

e

f

g

a

b

c

5g

7a

7b

7c

7d

7e

7f

7g

7h

7i

2.3.3.2. Toxicity prediction. The ﬁnal compounds 4a-g, 5a-g, and

7a-i were additionally passed through one more web server; Osiris

Property Explorer (http://www.organic-chemistry.org/prog/peo/)

to estimate their possible toxicities. Prediction through this pro-

gram is based on the functional group resemblance of the examined

compounds with the widely in vitro and in vivo studied compounds

included in its database. The output data are displayed as color-

coded; red, green, and yellow. Whereby, green color proposes low

toxic potential, yellow suggests mild toxicity, and red color means a

high probability of toxicity [70,71]. The results showed that all the

studied compounds are expected to be safe displaying no toxicity

concerning mutagenicity, tumorigenicity, irritant effect, and effect

on the reproductive system (Supporting data).

d

For 4a-g, S values are for Z conﬁgurations. The Kinetic parameter “Ki” was calculated

via GraphPad Prism software.

penetration, substrate or non-substrate for glycoprotein (P-gp)

permeability and, the interaction of molecules with cytochromes

P450 isomers (CYP) were predicted. Also, bioavailability scores

were calculated based on the following ﬁve rule-based ﬁlters [64];

Lipinski [65], Ghose [66], Veber [67], Egan [68], and Muegge [69]

rules.

3. Conclusion

Results of the ADME study are presented as BOILED-EGG, which

is a 2D plot drawn using calculated TPSA and LogP properties of the

Novel series of diphenyl-1H-pyrazoles and pyrazolo[3,4-b]pyr-

idines were synthesized, structurally elucidated, and evaluated for

12

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Fig. 8. 2D ligand interaction diagram of compound 4f with CDK2 ATP binding site.

Fig. 9. 2D ligand interaction diagram of compound 5b with CDK2 ATP binding site.

their antiproliferative activity against MCF7 and HepG2 cancer

cells. Compounds 4f and 7e displayed the highest MCF7 growth

inhibition activity, while compounds 5b and 7f were the most

active against HepG2 cells compared to doxorubicin. Potential

antiproliferative activity mechanisms were explored for the most

potent compounds 4f, 5b, 7e, and 7f. Cell cycle analysis showed that

4f and 7e arrested the MCF7 cell cycle at S and G2-M phases,

respectively, while 5b and 7f arrested the HepG2 cell cycle at G2-M

and G1/S phases, respectively. Also, compounds 4f and 7e are good

apoptotic inducers within MCF7, while compounds 5b and 7f

13

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Fig. 10. 2D ligand interaction diagram of compound 7f with CDK2 ATP binding site.

Table 8

a

Model performance and L10%OCV parameters for the training set (Obs. and predicted (pIC50) and residual errors).

CDK2 Obs. Activity

IC50 M)

0.348

0.0415

0.0807

0.0819

0.27

0.0568

0.329

0.096

0.0851

0.221

0.195

0.644

0.129

Training set prediction

Pred. pIC50

L10%OCV

b

Residual errors^c

Compound ID

(m

Obs. pIC50 (M)

Pred. pIC50

Residual errors

5

7

a

b

d

e

f

g

a

b

d

e

g

h

i

6.458

7.382

7.093

7.087

6.569

7.246

6.483

7.018

7.070

6.656

6.710

6.191

6.889

6.555

7.351

6.965

7.178

6.551

7.313

6.551

6.620

7.011

6.696

6.785

6.313

6.962

ꢁ0.097

0.031

0.128

6.639

7.371

6.916

7.226

6.537

7.408

6.614

6.569

7.000

6.702

6.797

6.407

6.991

ꢁ0.181

0.011

0.177

ꢁ0.091

ꢁ0.139

0.018

0.032

ꢁ0.067

ꢁ0.068

0.398

ꢁ0.162

ꢁ0.131

0.449

0.059

0.070

ꢁ0.04

ꢁ0.075

ꢁ0.122

ꢁ0.073

ꢁ0.046

ꢁ0.087

ꢁ0.216

ꢁ0.102

a

b

c

Leave (10%) out Cross validation.

ꢁ6

pIC50¼ -log(IC50*10 ).

The difference between the observed and the predicted pIC50

.

Table 9

Statistical performance of 2D QSAR model in training set prediction, leave-10%-out

L10%O) cross-validation and test set prediction.

induce apoptosis within HepG2 treated cells. Promising safety

proﬁles for compounds 4f and 7e on MCF10A and 5b and 7f on

THLE2 normal cell lines were displayed. Moreover, compounds 4f,

5b, and 7f showed selectivity towards CDK2 inhibition vs. CDK1,

CDK4, and CDK7 isoforms. The molecular docking study showed

similar binding modes of the studied compounds to that attained

by the co-crystallized ligand R-Roscovitine (III). A 2D QSAR study

revealed that RDFU10, GATSe8, and EstateVSA7 descriptors might

(

Training set prediction

L10%Ocv prediction^a

Test set prediction

_R2

RMSE

0.844

0.134

0.740

0.176

0.756

0.201

a

Leave (10%) out Cross validation.

14

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

Fig. 11. A) Correlation plot for the training set and test set observed vs. predicted pIC50. B) Training set observed vs. L10%OCV predicted pIC50

.

Table 10

compounds were synthesized according to their reported methods:

External validation for the test set (Obs. and predicted (pIC50) and residual errors.

(1a-c) [45], (2) [46], (3a-g) [47], (6a-c) [48], while compound (5a)

CDK2 Obs. Activity

Test set prediction

[72] was prepared via a novel method.

Compound ID

IC50

(m

M)

Obs. pIC50 (M)

Pred. pIC50

Residual errors

4

.1.1. General method for the synthesis of compounds 4a-g

A mixture of the appropriate enaminone (3a-g) (5 mmol) and

5

7

c

f

0.386

0.161

0.0665

6.413

6.793

7.177

6.755

6.756

7.115

ꢁ0.342

0.037

0.062

1,3-diphenyl-1H-pyrazol-5-amine (2) (1.17 g, 5 mmol) were stirred

at RT for 1.5 h (for 4a, b, f and g) and 4 h (for 4c-e). The mixture was

poured onto crushed ice, and the separated solid was ﬁltered,

washed with water, dried, and crystallized from ethanol.

be key contributors for our target pyrazolo[3,4-b]pyridines CDK2

inhibitory activity. These ﬁndings identify three compounds 4f, 5b,

and 7f with enhanced growth inhibitory activity against two pre-

vailing cancer types. The mechanistic aspects furnish a basis for

subsequent optimization in this respect.

4

2

.1.1.1. (E/Z)-3-(1,3-diphenyl-1H-pyrazol-5-ylamino)-1-phenylprop-

-en-1-one (4a). Yellow crystals; yield: (1.63 g, 89%); m.p.

ꢀ

ꢁ1

144e146 C; R

f

: 0.32; IR ʋ cm : 3051 (NH), 3030 (CH aromatic),

1

1629 (C]O), 1591 (C]N); H NMR

d

ppm: 6.31, 6.53 (2d, 1H,

J ¼ 7.92, 13.00 Hz, COeCH (E/Z forms)), 6.98, 7.04 (2s, 1H, H4 pyr-

azole (E/Z forms)), 7.36e7.39 (m, 1H, aromatic H), 7.43e7.51 (m, 4H,

aromatic H), 7.54e7.59 (m, 2H, aromatic H), 7.61e7.67 (m, 1H, ar-

omatic H), 7.69e7.72 (m, 3H, aromatic H), 7.74e7.78 (m, 1H, NHeCH

oleﬁnic), 7.84e7.88 (m, 2H, aromatic H), 7.92e7.95 (m, 2H, aromatic

4

. Experimental

4.1. Chemistry

ꢀ

Melting points ( C) were carried out by open capillary tube

H), 9.77, 12.35 (2d, 1H, J ¼ 9.32, 11.48 Hz, NH, D

2

O exchangeable (E/Z

ppm: 90.4, 95.6, 124.9, 125.6, 125.7, 127.8, 128.4,

28.6, 128.7, 128.9, 129.0, 129.1, 129.2, 129.9, 130.3, 132.2, 132.7,

33.11, 133.16, 138.0, 138.4, 139.5, 142.4, 147.1, 151.3, 188.4, 190.9; MS

method using Biocote melting point apparatus (BIBBY, scientiﬁc

limited stone, Staffordshire) ST, 150 SA, UK and they are uncorrec-

ted. Elemental microanalyses (C, H, and N) were performed on

FLASH 2000 CHNS/O analyzer, Thermo Scientiﬁc at the Regional

Center for Mycology and Biotechnology, Al-Azhar University, Nasr

City, Cairo. Infrared spectra were determined using Shimadzu

Fourier-transform infrared spectroscopy (IR-470, Shimadzu, Kyoto,

Japan). All spectra were expressed as ʋ cm , using potassium

bromide discs. H NMR and C NMR spectra were carried out using

Bruker high-performance digital FT-NMR spectrometer AVANCE III

13

forms)); C NMR

d

1

þ

(m/z, %): 365 (M , 100); Anal. Calcd. For C24

H

19

N

3

O (365.43): C,

78.88; H, 5.24; N, 11.50. Found: C, 78.62; H, 5.43; N, 11.69.

ꢁ1

4.1.1.2. (E/Z)-3-(1,3-diphenyl-1H-pyrazol-5-ylamino)-1-(4-

1

13

ﬂuorophenyl)prop-2-en-1-one (4b). Canary yellow crystals; yield:

ꢀ

ꢁ1

(1.62 g, 84%); m.p. 161e163 C; R

f

: 0.33; IR ʋ cm : 3142 (NH), 3061

1

4

00 MHz (Bruker Corporation, Germany) at Microanalytical Unit,

(CH aromatic), 1631 (C]O), 1597 (C]N); H NMR

d

ppm: 6.30, 6.50

1

Faculty of Pharmacy, Cairo University. H NMR spectra were run at

00 MHz and ¹³C NMR spectra were run at 100 MHz in DMSO‑d

as

a solvent, chemical Shifts are quoted in as parts per million (ppm)

(2d,1H, J ¼ 7.96,13.00 Hz, COeCH (E/Z forms)), 6.99, 7.05 (2s,1H, H4

pyrazole (E/Z forms)), 7.31 (t, 2H, J ¼ 8.82 Hz, aromatic H), 7.36e7.39

(m, 1H, aromatic H), 7.43e7.50 (m, 2H, aromatic H), 7.54e7.71 (m,

4H, aromatic H), 7.74e7.79 (m, 1H, aromatic H), 7.86 (d, 2H,

J ¼ 7.12 Hz, aromatic H), 7.91e7.94 (m, 1H, NHeCH oleﬁnic),

8.00e8.04 (m, 2H, aromatic H), 9.78, 12.30 (2d, 1H, J ¼ 8.84,

4

6

d

downﬁeld from tetramethylsilane (TMS) as an internal standard.

Mass Spectra were run on Single Quadruple Mass spectrometer ISQ

LT, at the Regional Center for Mycology and Biotechnology, Al-

Azhar University, Nasr City, Cairo. Reactions were monitored by

thin-layer chromatography (TLC) using precoated Aluminium

sheets, Silica gel Kieselgel 60 F254; (Merck, Darmstadt, Germany).

Methylene chloride was used as an eluting solvent and TLC sheets

were visualized at 366, 254 nm by UV VilberLourmat 77202 (Vilber,

Marne La Vallee, France). The following starting and intermediate

13

2

11.52 Hz, NH, D O exchangeable (E/Z forms)); C NMR d ppm: 90.5,

95.3, 115.8, 115,9, 116.0, 116.1, 124.9, 125.6, 128.4, 128.6, 128.7, 128.9,

129.1, 129.2, 129.9, 130.3, 130.5, 130.6, 130.7, 133.0, 135.0, 137.9,

138.5, 142.4, 147.0, 147.3, 151.1, 151.3, 163.7, 166.2, 187.0, 189.4; MS

þ

(m/z, %): 383 (M , 100); Anal. Calcd. For C24

H18FN

3

O (383.42): C,

75.18; H, 4.73; N, 10.96. Found: C, 74.89; H, 4.86; N, 11.24.

15

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

þ

4

.1.1.3. (E/Z)-3-(1,3-diphenyl-1H-pyrazol-5-ylamino)-1-(4-

189.8; MS (m/z, %): 395 (M , 100); Anal. Calcd. For C25

H

21

N

3

O

2

chlorophenyl)prop-2-en-1-one (4c). Canary yellow crystals; yield:

(395.45): C, 75.93; H, 5.35; N, 10.63. Found: C, 75.81; H, 5.46; N,

10.87.

ꢀ

ꢁ1

(

1.75 g, 87%); m.p. 171e173 C; R

CH aromatic), 1631 (C]O), 1581 (C]N); H NMR

2d,1H, J ¼ 7.88,13.00 Hz, COeCH (E/Z forms)), 6.99, 7.05 (2s, 1H, H4

f

: 0.38; IR ʋ cm : 3138 (NH), 3055

1

d

ppm: 6.30, 6.48

4.1.1.7. (E/Z)-3-(1,3-diphenyl-1H-pyrazol-5-ylamino)-1-(thiophen-2-

yl)prop-2-en-1-one (4g). Light brown crystals; yield: (1.00 g, 80%);

pyrazole (E/Z forms)), 7.34e7.39 (m, 1H, aromatic H), 7.43e7.50 (m,

H, aromatic H), 7.54e7.59 (m, 3H, aromatic H), 7.61e7.63 (m, 1H,

ꢀ

ꢁ1

2

m.p. 155e157 C; R

f

: 0.22; IR ʋ cm : 3070 (NH), 1625 (C]O), 1562

1

aromatic H), 7.64e7.71 (m, 3H, aromatic H), 7.76e7.82 (m, 1H,

NHeCH oleﬁnic), 7.86 (d, 2H, J ¼ 8.00 Hz, aromatic H), 7.91e7.97 (m,

(C]N); H NMR

d

ppm: 6.20, 6.44 (2d,1H, J ¼ 7.92,13.00 Hz, COeCH

(E/Z forms)), 6.97, 7.01 (2s, 1H, H4 pyrazole (E/Z forms)), 7.18e7.22

(m, 1H, aromatic H), 7.35e7.39 (m, 1H, aromatic H), 7.43e7.50 (m,

2H, aromatic H), 7.54e7.65 (m, 2H, aromatic H), 7.66e7.71 (m, 3H,

aromatic H), 7.72e7.78 (m, 1H, NHeCH oleﬁnic), 7.85e7.87 (m, 2H,

2

H, aromatic H), 9.82, 12.33 (2d, 1H, J ¼ 8.16, 11.56 Hz, NH, D

2

O

13

exchangeable (E/Z forms)); C NMR

25.7, 128.4, 128.6, 128.7, 128.9, 129.0, 129.13, 129.18, 129.2, 129.7,

29.9, 130.3, 133.11, 133.17, 137.01, 137.09, 137.5, 138.0, 138.2, 138.5,

d ppm: 90.6, 95.3, 124.9, 125.6,

1

aromatic H), 7.91e7.93 (m, 2H, aromatic H), 9.77, 11.94 (2d, 1H,

þ

13

142.3, 147.2, 147.6, 151.1, 151.3, 187.1, 189.4; MS (m/z, %): 399 (M ,

J ¼ 10.00, 11.56 Hz, NH, D

2

O exchangeable (E/Z forms)); C NMR

þ

1

00), 401 (M þ2, 32); Anal. Calcd. For C24

H18ClN

3

O (399.87): C,

d

ppm: 90.3, 95.8,124.8,124.9,125.6,125.7,128.4,128.5,128.6,128.9,

129.1,129.2,129.9,130.3,130.5,131.3,133.1,133.2,134.1,138.0,138.5,

42.4, 145.7, 145.9, 146.4, 146.8, 151.1, 151.3, 181.10, 184.1; MS (m/z,

72.09; H, 4.54; N, 10.51. Found: C, 71.88; H, 4.67; N, 10.69.

1

þ

4

.1.1.4. (E/Z)-3-(1,3-diphenyl-1H-pyrazol-5-ylamino)-1-(4-

%): 371 (M , 100); Anal. Calcd. For C22H N OS (371.45): C, 71.14; H,

17 3

bromophenyl)prop-2-en-1-one (4d). Canary yellow crystals; yield:

4.61; N, 11.31. Found: C, 70.89; H, 4.84; N, 11.58.

ꢀ

ꢁ1

(

1.83 g, 82%); m.p. 157e159 C; R

CH aromatic), 1639 (C]O), 1583 (C]N); H NMR

2d,1H, J ¼ 7.92,13.00 Hz, COeCH (E/Z forms)), 6.99, 7.06 (2s,1H, H4

f

: 0.41; IR ʋ cm : 3053 (NH), 3034

1

d

ppm: 6.29, 6.47

4.1.2. General method for the synthesis of compounds 5a-g

The appropriate 3-(1,3-diphenyl-1H-pyrazol-5-ylamino)-1-

arylprop-2-en-1-one (4a-g) (5 mmol) was reﬂuxed in glacial ace-

tic acid (5 mL) for 1 h. The reaction was cooled, the solid product

was ﬁltered off, washed with ethanol, dried, and crystallized from

ethanol.

pyrazole (E/Z forms)), 7.36e7.39 (m, 1H, aromatic H), 7.43e7.50 (m,

H, aromatic H), 7.54e7.61 (m, 2H, aromatic H), 7.63e7.70 (m, 5H,

2

aromatic H), 7.76e7.81 (m, 2H, aromatic H), 7.85e7.93 (m, 3H,

NHeCH oleﬁnic and aromatic H), 9.83, 12.33 (2d, 1H, J ¼ 9.24,

13

1

2

1.52 Hz, NH, D O exchangeable (E/Z forms)); C NMR d ppm: 90.6,

9

5.3, 124.9,125.6,125.7, 126.6, 128.4, 128.7,128.9,129.1, 129.2,129.9,

4.1.2.1. 1,3,4-Triphenyl-1H-pyrazolo[3,4-b]pyridine

(5a). Orange

ꢀ

ꢁ1

130.3, 132.0, 132.1, 133.11, 133.16, 137.4, 137.9, 138.55, 138.58, 142.3,

crystals; yield: (1.46 g, 84%); m.p. 164e166 C; R

3061 (CH aromatic), 1593 (C]N); H NMR d ppm: 7.40 (t, 1H,

f

: 0.68; IR ʋ cm

:

þ

1

(

9

47.2, 147.6, 151.1, 151.3, 187.2, 189.6; MS (m/z, %): 444 (M , 2), 235

100); Anal. Calcd. For C24 18BrN O (444.32): C, 64.88; H, 4.08; N,

.46. Found: C, 65,17; H, 4.31; N, 9.70.

H

3

J ¼ 7.40 Hz, aromatic H), 7.51e7.57 (m, 2H, aromatic Hs), 7.59e7.67

(m, 6H, aromatic H), 8.04 (d, 1H, J ¼ 8.56 Hz, H3 pyridine), 8.16 (d,

2

H, J ¼ 7.20 Hz, aromatic H), 8.28 (d, 2H, J ¼ 7.12 Hz, aromatic H),

4

2

.1.1.5. (E/Z)-3-(1,3-diphenyl-1H-pyrazol-5-ylamino)-1-p-tolylprop-

-en-1-one (4e). Orange red crystals; yield: (1.70 g, 89%); m.p.

8.46 (d, 2H, J ¼ 7.72 Hz, aromatic H), 8.75 (d, 1H, J ¼ 8.56 Hz, H2

13

pyridine); C NMR d ppm: 114.0, 116.1, 121.2, 126.4, 127.4, 127.8,

ꢀ

ꢁ1

159e161 C; R

f

: 0.36; IR ʋ cm : 3055 (NH), 3037 (CH aromatic),

129.42, 129.46, 129.5, 129.7, 130.3, 132.4. 132.5, 138.5, 139.5, 143.9,

1

þ

1

6

1

639 (C]O), 1587 (C]N); H NMR

.51 (2d, 1H, J ¼ 7.96, 13.00 Hz, COeCH (E/Z forms)), 6.95, 7.02 (2s,

d

ppm: 2.37 (s, 3H, CH

3

), 6.28,

151.3, 156.5; MS (m/z, %): 347 (M , 100); Anal. Calcd. For C24

H

17

N

3

(347.41): C, 82.97; H, 4.93; N, 12.10. Found: C, 82.69; H, 5.09; N,

12.34.

H, H4 pyrazole (E/Z forms)), 7.30 (d, 2H, J ¼ 8.00 Hz, aromatic H),

7.35e7.39 (m, 1H, aromatic H), 7.43e7.49 (m, 2H, aromatic H),

7

.54e7.59 (m, 1H, aromatic H), 7.61e7.65 (m, 1H, aromatic H),

.67e7.74 (m, 3H, aromatic H), 7.75e7.76 (m, 1H, aromatic H),

.83e7.87 (m, 3H, aromatic H), 7.92 (d, 1H, J ¼ 7.24 Hz, NHeCH

oleﬁnic), 9.70, 12.33 (2d, 1H, J ¼ 10.24, 11.40 Hz, NH, D

4.1.2.2. 4-(4-Fluorophenyl)-1,3-diphenyl-1H-pyrazolo[3,4-b]pyridine

ꢀ

(5b). Canary yellow crystals; yield: (1.52 g, 83%); m.p. 160e162 C;

ꢁ

1

R

f

: 0.70; IR ʋ cm : 3064 (CH aromatic), 1597 (C]N); H NMR

2

O

d

ppm: 7.38e7.44 (m, 3H, aromatic H), 7.53 (t, 1H, J ¼ 7.34 Hz, ar-

13

exchangeable (E/Z forms)); C NMR

25.6, 125.7, 127.9, 128.3, 128.5, 128.6, 128.9, 129.1, 129.2, 129.5,

29.6, 129.9, 130.3, 133.1, 133.2, 135.8, 136.9, 138.0, 138.6, 142.3,

42.5, 142.9, 146.4, 146.7, 151.1, 151.3, 187.9, 190.6; MS (m/z, %): 379

d

ppm: 21.5, 90.2, 95.5, 124.9,

omatic H), 7.59e7.67 (m, 4H, aromatic H), 8.04 (d, 1H, J ¼ 8.56 Hz,

H3 pyridine), 8.15 (d, 2H, J ¼ 7.28 Hz, aromatic H), 8.35 (m, 2H,

aromatic H), 8.43 (d, 2H, J ¼ 7.76 Hz, aromatic H), 8.75 (d, 1H,

1

3

J ¼ 8.56 Hz, H2-pyridine); C NMR

d ppm: 113.9, 116.0, 116.3, 116.5,

þ

(

M , 100); Anal. Calcd. For C25

H

21

N

3

O (379.45): C, 79.13; H, 5.58; N,

121.3, 126.5, 127.5, 129.4, 129.6, 129.7, 130.0, 130.1, 132.4, 132.7,

11.07. Found: C, 78.96; H, 5.72; N, 11.34.

135.04, 135.07, 139.5, 144.0, 151.3, 155.5, 162.6, 165.0; MS (m/z, %):

þ

3

65 (M , 28), 77 (100); Anal. Calcd. For C24

H

16FN

3

(365.4): C, 78.89;

4

.1.1.6. (E/Z)-3-(1,3-diphenyl-1H-pyrazol-5-ylamino)-1-(4-

H, 4.41; N, 11.50. Found: C, 78.65; H, 4.63; N, 11.69.

methoxyphenyl)prop-2-en-1-one (4f). Orange red crystals; yield:

ꢀ

ꢁ1

(

3

1.63 g, 89%); m.p. 167e169 C; R

f

: 0.29; IR ʋ cm : 3055 (NH), 3008

4.1.2.3. 4-(4-Chlorophenyl)-1,3-diphenyl-1H-pyrazolo[3,4-b]pyridine

1

ꢀ

CH aromatic), 1629 (C]O), 1602 (C]N); H NMR

d

ppm: 3.83 (s,

(5c). Canary yellow crystals; yield: (1.70 g, 89%); m.p. 170e172 C;

ꢁ1

1

H, OCH

3

), 6.27, 6.54 (2d,1H, J ¼ 8.00,13.00 Hz, COeCH (E/Z forms)),

R

f

: 0.74; IR ʋ cm : 3057 (CH aromatic), 1591 (C]N); H NMR

6

.95, 7.00 (2s, 1H, H4 pyrazole (E/Z forms)), 7.01e7.03 (m, 2H, aro-

d

ppm: 7.40 (t, 1H, J ¼ 7.32 Hz, aromatic H), 7.52 (t, 1H, J ¼ 7.18 Hz,

matic H), 7.35e7.39 (m, 1H, aromatic H), 7.43e7.48 (m, 2H, aromatic

H), 7.56e7.62 (m,1H, aromatic H), 7.64e7.70 (m, 5H, aromatic H and

NHeCH oleﬁnic), 7.84e7.87 (m, 2H, aromatic H), 7.93 (d, 2H,

J ¼ 8.76 Hz, aromatic H), 9.66, 12.31 (2d, 1H, J ¼ 10.16, 11.36 Hz, NH,

aromatic H), 7.58e7.65 (m, 6H, aromatic H), 8.02 (d, 1H, J ¼ 8.52 Hz,

H3 pyridine), 8.13 (d, 2H, J ¼ 7.56 Hz, aromatic H), 8.27 (d, 2H,

J ¼ 8.44 Hz, aromatic H), 8.41 (d, 2H, J ¼ 8.04 Hz, aromatic H), 8.75

13

(d, 1H, J ¼ 8.52 Hz, H2 pyridine); C NMR

d ppm: 114.1, 116.0, 121.2,

13

D

9

1

2

O exchangeable (E/Z forms)); C NMR

d

ppm: 55.8, 55.9, 90.0,

126.5, 127.4, 129.4, 129.5, 129.5, 129.7, 132.4, 132.7, 135.2, 137.3,

þ

5.4, 114.2, 114.3, 124.8, 124.9,125.6, 125.7, 128.3, 128.5,128.6,128.8,

29.1, 129.2, 129.9, 130.00, 130.08, 130.3, 131.1, 132.1, 133.1, 133.2,

38.0, 138.6, 142.6, 145.9, 146.2, 151.1, 151.3, 162.6, 163.0, 186.9,

139.4, 143.9, 151.2, 155.1; MS (m/z, %): 381 (M , 100), 383 (M þ2,

22); Anal. Calcd. For C24 16ClN (381.86): C, 75.49; H, 4.22; N, 11.00.

Found: C, 75.21; H, 4.43; N, 10.86.

H

3

16

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

4

.1.2.4. 4-(4-Bromophenyl)-1,3-diphenyl-1H-pyrazolo[3,4-b]pyridine

4.1.3.1. 1,3,6-Triphenyl-4-[4-(piperidin-1-yl)phenyl]-1H-pyrazolo

ꢀ

(

5d). Canary yellow crystals; yield: (1.82 g, 85%); m.p. 196e198 C;

[3,4-b]pyridine-5-carbonitrile (7a). Biege crystals; yield: (2.40 g,

ꢁ

1

ꢀ

ꢁ1

R

d

f

: 0.76; IR ʋ cm : 3066 (CH aromatic), 1593 (C]N); H NMR

90%); m.p. 240e242 C; R

f

: 0.75; IR ʋ cm : 3030 (CH aromatic),

1

ppm: 7.40 (t, 1H, J ¼ 7.38 Hz, aromatic H), 7.52 (t, 1H, J ¼ 7.30 Hz,

2222 (C^N), 1602 (C]N); H NMR

d

ppm: 1.60 (bs, 6H, piperidine

aromatic H), 7.59e7.66 (m, 4H, aromatic H), 7.77 (d, 2H, J ¼ 8.52 Hz,

aromatic H), 8.04 (d, 1H, J ¼ 8.52 Hz, H3 pyridine), 8.15 (d, 2H,

J ¼ 7.28 Hz, aromatic H), 8.21 (d, 2H, J ¼ 8.56 Hz, aromatic H), 8.42

H), 3.23 (bs, 4H, piperidine H), 6.72 (d, 2H, J ¼ 8.40 Hz, aromatic H),

7.15e7.19 (m, 6H, aromatic H), 7.28 (bs, 1H, aromatic H), 7.43 (t, 1H,

J ¼ 7.34 Hz, aromatic H), 7.62 (bs, 5H, aromatic H), 8.00e8.01 (m,

13

(

d, 2H, J ¼ 7.84 Hz, aromatic H), 8.75 (d, 1H, J ¼ 8.56 Hz, H2 pyri-

2H, aromatic H), 8.29 (d, 2H, J ¼ 7.84 Hz, aromatic H); C NMR

13

dine); C NMR

d

ppm: 114.1, 115.9, 121.2, 124.0, 126.4, 127.4, 129.4,

d ppm: 24.6, 25.0, 49.1, 102.1, 114.3, 118.6, 122.1, 127.5, 128.0, 128.9,

1

29.5, 129.6, 129.7, 132.3, 132.4, 132.7, 137.6, 139.4, 143.9, 151.2,

129.3, 129.8, 129.9, 130.5, 131.1, 131.2, 132.0, 134.9, 137.1, 138.7, 143.4,

145.2, 147.5, 152.1, 152.3, 161.1; MS (m/z, %): 531 (M , 100); Anal.

þ

155.2; MS (m/z, %): 425 (M , 6), 77 (100); Anal. Calcd. For

C

24

H16BrN

3

(426.31): C, 67.62; H, 3.78; N, 9.86. Found: C, 67.49; H,

29 5

Calcd. for C36H N (531.65): C, 81.33; H, 5.50; N, 13.17. Found: C,

4

.05; N, 10.13.

81.35; H, 5.59; N, 13.17.

4

.1.3.2. 4-(4-Morpholinophenyl)-1,3,6-triphenyl-1H-pyrazolo[3,4-b]

4

.1.2.5. 1,3-Diphenyl-4-p-tolyl-1H-pyrazolo[3,4-b]pyridine

(5e).

: 0.71;

ꢀ

pyridine-5-carbonitrile (7b). Light yellow crystals; yield: (2.35 g,

88%); m.p. 252e254 C; R : 0.69; IR ʋ cm : 3050 (CH aromatic),

2

Orange red crystals; yield: (1.50 g, 83%); m.p. 174e176 C; R

f

ꢀ

ꢁ1

1

IR ʋ cm : 3034 (CH aromatic), 1595 (C]N); H NMR

H, CH

d

ppm: 2.41 (s,

f

1

223 (C^N), 1604 (C]N); H NMR

d

ppm: 3.14 (t, 4H, J ¼ 4.70 Hz,

3

), 7.39 (d, 3H, J ¼ 7.92 Hz, aromatic Hs), 7.52 (t, 1H,

morpholine H), 3.73 (t, 4H, J ¼ 4.68 Hz, morpholine H), 6.78 (d, 2H,

J ¼ 8.76 Hz, aromatic H), 7.12e7.18 (m, 4H, aromatic H), 7.23 (d, 2H,

J ¼ 8.68 Hz, aromatic H), 7.27e7.31 (m, 1H, aromatic H), 7.44 (t, 1H,

J ¼ 7.40 Hz, aromatic H), 7.61e7.65 (m, 5H, aromatic H), 8.00e.8.02

J ¼ 7.34 Hz, aromatic H), 7.59e7.67 (m, 4H, aromatic H), 8.02 (d, 1H,

J ¼ 8.56 Hz, H3 pyridine), 8.17 (t, 4H, J ¼ 8.46 Hz, aromatic H), 8.46

(

d, 2H, J ¼ 7.84 Hz, aromatic H), 8.73 (d, 1H, J ¼ 8.56 Hz, H2 pyri-

13

dine); C NMR

d ppm: 21.3, 113.8, 116.0, 121.2, 126.4, 127.4, 127.7,

13

(

d

m, 2H, aromatic H), 8.29 (d, 2H, J ¼ 7.68 Hz, aromatic H); C NMR

129.4, 129.6, 129.7, 130.1, 132.5, 132.5, 135.8, 139.6, 140.1, 144.0,

þ

ppm: 48.2, 66.3, 102.1, 112.3, 114.2, 118.4, 122.1, 123.5, 127.4, 128.0,

151.4, 156.6; MS (m/z, %): 361 (M , 62), 183 (100); Anal. Calcd. For

128.5,128.9,129.3,129.8,129.9,130.6,131.1,131.9,138.2,138.7,147.4,

C

5

25 19 3

H N (361.44): C, 83.08; H, 5.30; N, 11.63. Found: C, 82.94; H,

.26; N, 11.86.

þ

1

50.4, 152.4, 153.2, 161.0; MS (m/z, %): 533, (M , 99), 77 (100); Anal.

Calcd. For C35 O (533.62): C, 78.78; H, 5.10; N, 13.12. Found: C,

9.04; H, 5.34; N, 13.28.

27 5

H N

7

4

.1.2.6. 4-(4-Methoxyphenyl)-1,3-diphenyl-1H-pyrazolo[3,4-b]pyri-

ꢀ

dine (5f). Orange red crystals; yield: (1.61 g, 85%); m.p. 185e187 C;

R

d

4

.1.3.3. 4-(4-(4-Methylpiperazin-1-yl)phenyl)-1,3,6-triphenyl-1H-

ꢁ

1

f

: 0.66; IR ʋ cm : 3070 (CH aromatic), 1593 (C]N); H NMR

ppm: 3.87 (s, 3H, OCH

), 7.15 (d, 2H, J ¼ 8.84 Hz, aromatic H), 7.40

pyrazolo[3,4-b]pyridine-5-carbonitrile (7c). Light yellow crystals;

yield: (2.52 g, 92%); m.p. 222e224 C; R : 0.78; IR ʋ cm : 3062 (CH

f

3

ꢀ

ꢁ1

(

7

8

t, 1H, J ¼ 7.40 Hz, aromatic H), 7.52 (t, 1H, J ¼ 7.34 Hz, aromatic H),

.59e7.67 (m, 4H, aromatic H), 8.00 (d,1H, J ¼ 8.60 Hz, H3 pyridine),

.16 (d, 2H, J ¼ 7.20 Hz, aromatic H), 8.26 (d, 2H, J ¼ 8.88 Hz, aro-

matic H), 8.46 (d, 2H, J ¼ 7.64 Hz, aromatic H), 8.71 (d, 1H,

1

aromatic), 2220 (C^N), 1604 (C]N); H NMR

CH

piprazine H), 6.75 (d, 2H, J ¼ 8.76 Hz, aromatic H), 7.11e7.20 (m, 6H,

aromatic H), 7.26e7.30 (m, 1H, aromatic H), 7.43 (t, 1H, J ¼ 7.40 Hz,

aromatic H), 7.60e7.64 (m, 5H, aromatic H), 7.99e8.01 (m, 2H, ar-

d ppm: 2.24 (s, 3H,

3

), 2.43 (t, 4H, J ¼ 4.72 Hz, piprazine H), 3.18 (t, 4H, J ¼ 4.72 Hz,

13

J ¼ 8.56 Hz, H2 pyridine); C NMR

d ppm: 55.8, 113.4, 114.9, 115.6,

1

21.1, 126.4, 127.4, 129.3, 129.4, 129.6, 129.7, 130.9, 132.4, 132.5,

13

omatic H), 8.29 (d, 2H, J ¼ 7.80 Hz, aromatic H); C NMR

6.2, 47.8, 54.7, 102.1, 112.3, 114.3, 118.4, 122.1, 122.9, 127.3, 128.0,

28.5,128.9,129.3,129.8,129.9,130.6,131.1,131.9,138.2,138.7,147.4,

d ppm:

þ

1

39.6, 144.0, 151.5, 156.3, 161.3; MS (m/z, %): 377 (M , 100); Anal.

Calcd. For C25 O (377.44): C, 79.55; H, 5.07; N, 11.13. Found: C,

9.41; H, 5.28; N, 11.40.

4

1

19 3

H N

7

þ

1

50.4, 152.2, 153.3, 161.0; MS (m/z, %): 546 (M , 42), 71 (100); Anal.

Calcd. for C36 (546.66): C, 79.10; H, 5.53; N, 15.37. Found: C,

8.97.; H, 5.68; N, 15.29.

30 6

H N

4

.1.2.7. 1,3-Diphenyl-4-(thiophen-2-yl)-1H-pyrazolo[3,4-b]pyridine

7

ꢀ

(

5g). Light brown crystals; yield: (1.45 g, 82%); m.p. 180e182 C; R

f

:

ꢁ1

1

0

.64; IR ʋ cm : 3064 (CH aromatic), 1593 (C]N); H NMR

d

ppm:

4

.1.3.4. 6-(4-Methoxyphenyl)-1,3-diphenyl-4-(4-(piperidin-1-yl)

7

.24e7.26 (m, 1H, aromatic H), 7.39 (t, 1H, J ¼ 7.38 Hz, aromatic H),

phenyl)-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile

crystals; yield: (2.61 g, 93%); m.p. 214e216 C; R

3

(7d). Yellow

7

.52 (t, 1H, J ¼ 7.32 Hz, aromatic H), 7.58e7.65 (m, 4H, aromatic H),

ꢀ

ꢁ1

f

: 0.72; IR ʋ cm

:

7

.78 (d, 1H, J ¼ 4.96 Hz, aromatic H), 7.98 (d, 1H, J ¼ 8.56 Hz, H3

032 (CH aromatic), 2958 (CH aliphatic), 2220 (C^N), 1612 (C]N);

pyridine), 8.05 (d, 1H, J ¼ 3.16 Hz, aromatic H), 8.14 (d, 2H,

1

H NMR

piperidine H), 3.88 (s, 3H, OCH

H), 7.13e7.18 (m, 8H, aromatic H), 7.25e7.29 (m, 1H, aromatic H),

.43 (t, 1H, J ¼ 7.36 Hz, aromatic H), 7.62 (t, 2H, J ¼ 7.88 Hz, aromatic

H), 8.00 (d, 2H, J ¼ 8.68 Hz, aromatic H), 8.30 (d, 2H, J ¼ 8.20 Hz,

d

ppm: 1.60 (bs, 6H, piperidine H), 3.22e3.23 (m, 4H,

J ¼ 7.24 Hz, aromatic H), 8.44 (d, 2H, J ¼ 7.76 Hz, aromatic H), 8.69

3

), 6.71 (d, 2H, J ¼ 8.72 Hz, aromatic

13

(

d, 1H, J ¼ 8.56 Hz, H2 pyridine); C NMR

d ppm: 113.8, 114.9, 120.8,

126.4, 127.5, 128.0, 129.3, 129.5, 129.6, 129.7, 130.4, 132.4, 132.6,

7

þ

1

39.5, 144.2, 144.4, 150.8, 152.0; MS (m/z, %): 353 (M , 100); Anal.

Calcd. For C22 S (353.44): C, 74.76; H, 4.28; N, 11.89. Found: C,

H

15

N

3

13

aromatic H); C NMR

14.3,114.4,118.8,122.1,122.3,127.3,128.0,128.4,129.3,129.7,130.5,

31.2, 131.6, 132.0, 138.8, 147.4, 150.5, 152.3, 153.4, 160.6, 161.4; MS

d ppm: 24.6, 25.0, 49.1, 55.9, 101.6, 112.0,

74.49; H, 4.47; N, 12.08.

1

þ

4

.1.3. General method for the synthesis of compounds 7a-i

To a solution of 1,3-diphenyl-1H-pyrazol-5-amine (2) (1.17 g,

mmol) in absolute ethanol (20 mL), the corresponding 3-oxo-3-

31 5

(m/z, %): 561 (M , 100); Anal. Calcd. for C37H N O (561.67): C,

79.12; H, 5.56; N, 12.47. Found: C, 78.98; H, 5.79; N, 12.65.

5

un/substituted phenylpropanenitrile (1a-c) (5 mmol) and the

appropriate 4-(un/substituted-1-yl)benzaldehyde (6a-c) (5 mmol)

were added. 3 Drops of triethylamine were added as a catalyst, then

the mixture was reﬂuxed for 10 h. The reaction mixture was cooled,

and the precipitated crystalline product was ﬁltered, dried, and

recrystallized from ethanol.

4.1.3.5. 6-(4-Methoxyphenyl)-4-(4-morpholinophenyl)-1,3-diphenyl-

1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (7e). Orange crystals;

ꢀ

ꢁ1

yield: (2.63 g, 93%); m.p. 237e239 C; R

f

: 0.66; IR ʋ cm : 3061 (CH

1

aromatic), 2960 (CH aliphatic), 2220 (C^N), 1606 (C]N); H NMR

d

ppm: 3.14 (t, 4H, J ¼ 4.76 Hz, morpholine H), 3.75 (t, 4H,

J ¼ 4.72 Hz, morpholine H), 3.88 (s, 3H, OCH ), 6.78 (d, 2H,

3

17

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

J ¼ 8.84 Hz, aromatic H), 7.12e7.24 (m, 8H, aromatic H), 7.27e7.30

8.03 (d, 2H, J ¼ 8.48 Hz, aromatic H), 8.27 (d, 2H, J ¼ 7.76 Hz, aro-

13

(

m, 1H, aromatic H), 7.43 (t, 1H, J ¼ 7.42 Hz, aromatic H), 7.63 (t, 2H,

matic H); C NMR d ppm: 46.2, 47.8, 54.7, 102.0, 112.4, 114.3, 118.3,

J ¼ 8.00 Hz, aromatic H), 8.00 (d, 2H, J ¼ 8.84 Hz, aromatic H), 8.30

122.1, 122.8, 127.4, 128.0, 128.5, 129.0, 129.3, 129.7, 131.1, 131.81,

1

3

(

1

(

d, 2H, J ¼ 8.68 Hz, aromatic H); C NMR

01.7, 112.0, 114.2, 114.4, 122.1, 123.7, 128.0, 129.3, 129.8, 130.4, 131.0,

31.6, 131.9, 138.7, 147.4, 150.5, 152.4, 161.4; MS (m/z, %): 563

d

ppm: 48.2, 55.9, 66.3,

131.89, 135.7, 137.0, 138.6, 147.4, 150.3, 152.2, 153.2, 159.8; MS (m/z,

þ

%): 581 (M , 5), 71 (100); Anal. Calcd. for C36

H29ClN

6

(581.11): C,

74.41; H, 5.03; N, 14.46. Found: C, 74.68; H, 5.21; N, 14.32.

þ

29 5 2

M ,100); Anal. Calcd. For C36H N O (563.65): C, 76.71; H, 5.19; N,

12.43. Found: C, 76.94; H, 5.08; N, 12.67.

4.2. Biological screening

4

.1.3.6. 6-(4-Methoxyphenyl)-4-(4-(4-methylpiperazin-1-yl)phenyl)-

All biological screenings were performed at the Diagnostic and

Conﬁrmatory lab in the Holding Company for Biological Products

and Vaccines (VACSERA), Giza, Egypt.

1,3-diphenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile

(7f).

: 0.74; IR ʋ

ꢀ

Orange crystals; yield: (2.40 g, 83%); m.p. 223e225 C; R

cm : 3031 (CH aromatic), 2939 (CH aliphatic), 2220 (C^N), 1606

f

ꢁ1

1

(

C]N); H NMR

piprazine H), 3.15 (t, 4H, J ¼ 4.56 Hz, piprazine H), 3.88 (s, 3H,

OCH

d

ppm: 2.24 (s, 3H, CH

3

), 2.43 (t, 4H, J ¼ 4.46 Hz,

4.2.1. Antiproliferative activity

The method deﬁned by T. Mosmann et al. [49] was applied for

the assessment of the antiproliferative activity against MCF7 and

HepG2 cancer cell lines. The cell lines were obtained from American

Type Culture Collection (ATCC, Manassas, Virginia, USA). Brieﬂy,

cells were cultured into 96-well microplates (cells density 1.2e1.8).

10,000 cells/well were used, and culturing was done using Dul-

becco’s modiﬁed Eagle’s medium (DMEM; Invitrogen/Life Tech-

nologies, Waltham, Massachusetts, USA) supplemented with 10%

FBS (Hyclone, Whiteﬁeld Bengaluru Bangalore KA 560067 IN, In-

3

), 6.72 (d, 2H, J ¼ 8.64 Hz, aromatic H), 7.09e7.18 (m, 8H, ar-

omatic H), 7.26 (t, 1H, J ¼ 6.84 Hz, aromatic H), 7.41 (t, 1H,

J ¼ 7.38 Hz, aromatic H), 7.61 (t, 2H, J ¼ 7.82 Hz, aromatic H), 7.99 (d,

2

H, J ¼ 8.64 Hz, aromatic H), 8.29 (d, 2H, J ¼ 8.04 Hz, aromatic H);

1

3

C NMR

18.7, 121.9, 123.1, 127.2, 128.0, 128.4, 129.2, 129.7, 130.4, 131.0, 131.6,

31.9, 138.8, 147.3, 150.5, 152.2, 153.3, 160.5, 161.41; MS (m/z, %): 576

d ppm: 46.2, 47.8, 54.7, 55.8, 101.6, 111.9, 114.33, 114.39,

1

þ

(

M , 79), 70 (100); Anal. Calcd. for C37

32

H N

6

O (576.69): C, 77.06; H,

5

.59; N, 14.57. Found: C, 77.30; H, 5.82; N, 14.39.

dia), 10

m

g/ml of insulin (Sigma, St. Louis, Missouri United States),

ꢀ

and 1% penicillin-streptomycin at 37 C in a 5% CO

then were incubated for 24 h at 37 C in a humidiﬁed 5% CO

2

incubator. Cells

at-

ꢀ

4

.1.3.7. 6-(4-Chlorophenyl)-1,3-diphenyl-4-(4-(piperidin-1-yl)

2

phenyl)-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (7g). Green crys-

mosphere. Cell conﬂuency was 75e80%, then the cells were

exposed to variant concentrations of the tested compounds or

doxorubicin for 48 h. Then the viability of treated cells was deter-

mined using the MTT technique as follows: media were removed,

MTT solution “M-5655” (Sigma, St. Louis, Missouri United States)

was added and incubated for an additional 4 h to allow for

metabolizing the dye into colored-insoluble formazan crystals. The

remaining MTT solution was cast-off from the wells, and formazan

crystals were solubilized and the plate was shaken at room tem-

perature. Absorbance was measured using a ROBONIK P2000 Elisa

Reader at 570 nm. The cell viability was expressed as a concen-

tration that induces 50% of maximum inhibition of cell proliferation

(IC50) and it was determined using Graph Pad Prism version 5

software (Graph Pad Software Inc, CA) [49], each value represents

the mean ± SD from triplicate determinants.

ꢀ

ꢁ1

f

tals; yield: (2.55 g, 90%); m.p. 243e244 C; R : 0.79; IR ʋ cm : 3061

1

(

CH aromatic), 2223 (C^N), 1604 (C]N); H NMR d ppm: 1.58 (s,

6

H, piperidine H), 3.21 (s, 4H, piperidine H), 6.69 (d, 2H, J ¼ 8.64 Hz,

aromatic H), 7.10e7.14 (m, 6H, aromatic H), 7.27 (t, 1H, J ¼ 6.66 Hz,

aromatic H), 7.41 (t, 1H, J ¼ 7.38 Hz, aromatic H), 7.60 (t, 2H,

J ¼ 7.78 Hz, aromatic H), 7.69 (d, 2H, J ¼ 8.32 Hz, aromatic H), 8.02

(

d, 2H, J ¼ 8.36 Hz, aromatic H), 8.26 (d, 2H, J ¼ 8.00 Hz, aromatic

13

H); C NMR

d ppm: 24.5, 25.0, 49.0, 101.8, 112.3, 114.2, 118.4, 121.9,

1

21.9, 127.3, 128.0, 128.4, 129.0, 129.3, 129.7, 131.2, 131.7, 131.9, 135.6,

þ

1

37.0, 138.6, 147.4, 150.3, 152.3, 153.3, 159.7; MS (m/z, %): 565 (M ,

), 77 (100); Anal. Calcd. for C36 28ClN (566.09): C, 76.38; H, 4.99;

N, 12.37. Found: C, 76.19; H, 5.12; N, 12.59.

7

H

5

4

1

.1.3.8. 6-(4-Chlorophenyl)-4-(4-morpholinophenyl)-1,3-diphenyl-

H-pyrazolo[3,4-b]pyridine-5-carbonitrile (7h). Green crystals;

ꢀ

ꢁ1

yield: (2.36 g, 83%); m.p. 239e241 C; R

f

: 0.71; IR ʋ cm : 3061 (CH

4.2.2. Cell cycle analysis and apoptosis detection

1

aromatic), 2223 (C^N), 1606 (C]N); H NMR

J ¼ 4.50 Hz, morpholine H), 3.74 (t, 4H, J ¼ 4.50 Hz, morpholine H),

.75 (d, 2H, J ¼ 8.72 Hz, aromatic H), 7.09e7.20 (m, 6H, aromatic H),

d

ppm: 3.13 (t, 4H,

4.2.2.1. Cell cycle analysis. MCF7 cell line was treated with the most

potent compounds 4f and 7e, while HepG2 was treated with the

most active compounds 5b and 7f at their IC50 concentrations for

48 h. After treatment, the cells were suspended in 0.5 mL of PBS,

centrifuged for collection, and ﬁxed in ice-cold ethanol (70% v/v),

washed with PBS, resuspended with RNase, and stained with pro-

pidium iodide. The analysis was then performed by ﬂow cytometry

using FACSCalibur (Becton Dickinson, Franklin Lakes, NJ

07417e1880, USA) Phoenix Flow Systems and Verity Software

House were used for the calculation of the cell cycle distributions

[73].

6

7

.28 (t, 1H, J ¼ 7.08 Hz, aromatic H), 7.42 (t, 1H, J ¼ 7.38 Hz, aromatic

H), 7.60 (t, 2H, J ¼ 7.86 Hz, aromatic H), 7.69 (d, 2H, J ¼ 8.44 Hz,

aromatic H), 8.03 (d, 2H, J ¼ 8.44 Hz, aromatic H), 8.27 (d, 2H,

13

J ¼ 7.84 Hz, aromatic H); C NMR

d ppm: 48.2, 66.3, 102.1, 112.4,

114.2, 118.3,122.1, 123.4,127.4,128.0,128.5,129.0,129.3,129.8,131.1,

1

31.8, 131.9, 135.7, 137.0, 138.6, 147.4, 150.3, 152.4, 153.2, 159.8; MS

þ

(

m/z, %): 568 (M , 15), 565 (100); Anal. Calcd. For C35

568.07): C, 74.00; H, 4.61; N, 12.33. Found: C, 73.87; H, 4.85; N,

2.60.

5

H26ClN O

(

1

4.2.2.2. Apoptosis assay. MCF7 cell line was treated with com-

4

.1.3.9. 6-(4-Chlorophenyl)-4-(4-(4-methylpiperazin-1-yl)phenyl)-

pounds 4f and 7e, while HepG2 was treated with compounds 5b

and 7f, respectively at their IC50 concentrations for 48 h. After that,

the cells were suspended in PBS, centrifuged, and ﬁxed in ice-cold

ethanol (70% v/v). Then, the ethanol suspended cells were centri-

fuged, suspended PBS, centrifuged again, and resuspended with PE

Annexin V and PI staining solution according to the manufacturer’s

guidelines. In the end, analysis by ﬂow cytometry using FACSCali-

bur (Becton Dickinson, Franklin Lakes, NJ 07417e1880, USA) was

performed. Phoenix Flow Systems and Verity Software House were

1,3-diphenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile

(7i).

: 0.81; IR ʋ

ꢀ

Green crystals; yield: (2.56 g, 88%); m.p. 228e230 C; R

cm : 3062 (CH aromatic), 2970 (CH aliphatic), 2222 (C^N), 1598

f

ꢁ1

1

(

C]N); H NMR

d

ppm: 2.24 (s, 3H, CH

3

), 2.43 (t, 4H, J ¼ 4.74 Hz,

piprazine H), 3.18 (t, 4H, J ¼ 4.72 Hz, piprazine H), 6.75 (d, 2H,

J ¼ 8.80 Hz, aromatic H), 7.11e7.20 (m, 6H, aromatic H), 7.28 (t, 1H,

J ¼ 6.86 Hz, aromatic H), 7.43 (t, 1H, J ¼ 7.42 Hz, aromatic H), 7.62 (t,

2

H, J ¼ 7.92 Hz, aromatic H), 7.70 (d, 2H, J ¼ 8.48 Hz, aromatic H),

18

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

used for the cell cycle distributions calculations [74].

40 min. Afterwards, an amount of Kinase Detection Reagent (10 mL)

was added and incubated for 30 min at room temperature. Lumi-

nescence was then detected. The luminescent signal was directly

correlated with the quantity of ATP present but inversely correlated

with the kinase activity. Graphpad Prism software was used for

analyzing the luminescence data [76]. The IC50 value was interpo-

lated from doseeresponse data with four different concentrations

for each tested compound and ±SD value was recorded from trip-

licate experiment.

4

.2.3. Caspase-9 and -3 activation assay (apoptosis induction

hallmarks)

The Invitrogen human active caspase-3 “Catalog # KHO1091”

Invitrogen Corporation, Camarillo, CA 93012, United States) and

(

DRG® human caspase-9 “EIA-4860” (DRG International Inc., USA)

ELISA kits are used to detect and quantify the level of human active

caspases. In brief, after MCF7 cells were treated with (4f and 7e)

and HepG2 were treated with (5b and 7f) at their IC50 concentra-

tions, they were trypsinized and rinsed with PBS. After centrifu-

gation, the pellet was suspended in PBS (1 mL). Cells were exposed

4.2.6. Selectivity proﬁle vs. CDK isoforms

The in vitro CDK1/cyclin B, CDK4/cyclin D1 and CDK7/cyclin H

enzyme inhibition assays were performed using the following BPS

Bioscience luminescence kinase assay kits as described [77e79];

ꢀ

to freezing at < ꢁ20 C and thawing with mixing. Freeze/thaw cycle

repeated 3 times, before centrifugation at 1500 rpm for 10 min at

ꢀ

00

2

e8 C to remove cellular debris. ELISA “a solid-phase sandwich

CDK1 “Catalog # 79597”, CDK4 “Catalog # 79674”, CDK7 Catalog

Enzyme-Linked Immuno-Sorbent Assay” was used. A monoclonal-

speciﬁc antibody for the human target protein has been wiped onto

the wells of the provided microtiter strips. Samples, containing a

standard including the human target protein control specimens,

and also unknowns, were pipetted into the wells and then a rabbit

antibody speciﬁc for human active target protein was added to

these wells. During the ﬁrst incubation, the human target protein

binds to the immobilized (capture) antibody and the speciﬁc active

protein antibody serves as a detection antibody by binding to the

immobilized active protein. After the ﬁrst incubation step and

washing to remove excess protein and detection antibody, a

horseradish peroxidase-labeled Anti-Rabbit IgG (Anti-Rabbit IgG

HRP) was added. This binds to the detection antibody to complete

the four-member sandwich. After a third incubation and washing to

remove all the excess Anti-Rabbit IgG HRP, a substrate solution is

added, which was acted upon by the bound enzyme to produce

color. ROBONIK P2000 SPECTROPHOTOMETER was used to mea-

sure the color intensity at a wavelength of 450 nm, all compounds

were tested in triplicates per experiment. The intensity of this

colored product is directly proportional to the concentration of

human active protein present in the original specimen [35].

#79603” (BPS Bioscience. Inc. Corporate, 6042 Cornerstone Court

W, Ste B San Diego, CA 92121, United States). The assays were car-

ried out at room temperature. The target compounds were dis-

solved and added to the reaction mixture at variant concentrations.

Continuous kinetic monitoring of the enzyme activity was per-

formed on Tecan espark READER [77e79]. The percent inhibition of

the enzyme activity was calculated for all the compounds at four

different concentrations, IC50 values were calculated via Graphpad

prism, and each value represents the mean ± SD from triplicate

determinants.

4.2.7. Statistical analysis

Results have been expressed as the mean ± S.D for three repli-

cates. All the statistical analysis was performed via Graph Pad Prism

software (Graph Pad Software Inc, CA). One-way ANOVA followed

by a Dunnett post hoc test were applied to check the differences

between the inhibition activities of the tested compounds and the

reference drug, a p-value less than 0.05 (p < 0.05) was considered

statistically signiﬁcant.

4.3. Molecular modeling study

4.2.4. Cytotoxicity study on normal cell lines

4.3.1. Molecular docking study

Two normal cells “MCF10A and THLE2 normal cell Lines”

A Molecular docking study was performed using the Molecular

Operating Environment (MOE 2014.0901) program. All minimiza-

tion in docking procedures was achieved with MOE until an RMSD

attained from American Type Culture Collection were cultured us-

ing DMEM (Invitrogen/Life Technologies, Waltham, Massachusetts,

USA) supplemented with 10% FBS (Hyclone, Whiteﬁeld Bengaluru

ꢁ

1

ꢁ1

gradient of 0.05 kcal mol

Å

with MMFF94 forceﬁeld. Partial

Bangalore KA 560067 IN, India), 10

m

g/ml of insulin (Sigma, St.

charges were automatically calculated. Triangle Matcher placement

method and London dG scoring function were utilized as a docking

protocol. The X-ray crystal structure of the CDK2 enzyme co-

crystallized with R-Roscovitine (PDB code: 3DDQ) was down-

loaded from the protein data bank in PDB format (http://www.rcsb.

org/), and prepared for the docking study: i) removal of chains C, D,

E, water molecules and ligands that are not involved in the binding.

ii) protonate 3D protocol with default options in MOE.

Louis, Missouri United States), and 1% penicillin-streptomycin in

plate cells (cells density: 1.2e1.8 ꢂ 10,000 cells/well). Cytotoxicity

of compounds 4f, 5b and 7e on MCF10A cell line and compounds

4

a, 4f, 4g, 5a, 5b, 7c, 7f and 7g on THLE2 cell line was measured

using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) reagent which is reduced to formazan reﬂecting

the cell viability. Brieﬂy, as previously described [75], grown cells

on a 96-well plate were incubated with the tested compounds at

various concentrations in complete DMEM. Formazan formation

was measured via reading the optical absorbance [75], the experi-

ments were performed in triplicates and the mean value ± SD was

recorded.

The studied compounds were built in a 3D format using MOE

and exposed to the following: i) structure 3D protonation. ii)

Conformational analysis through systemic search. iii) Choosing the

least energetic conformer. iv) Running the same docking protocol

used with the native ligand. Docking setup was ﬁrst validated by re-

docking of the co-crystallized ligand R-Roscovitine into the enzyme

ꢀ

4.2.5. Cyclin dependent kinase 2/Cycline A2 enzyme inhibition

binding site. Redocked pose resulted in an RMSD value of 0.494 A .

The In vitro CDK2 Kinase assay was carried out for the target

The validated setup was then used in predicting the binding modes

of the target ligands into the CDK2 ATP binding region.

compounds using Promega Corporation CDK2 luminescence kinase

assay kit (Promega Corporation, 2800 Woods Hollow Road, Madi-

son, WI 53711-5399 USA). Protocol steps involve diluting the

enzyme and the substrate, ATP and the inhibitors in Kinase Buffer

4.3.2. 2D QSAR studies

4.3.2.1. Data set and target property manipulation. A data set of 16

pyrazolopyridines (5a-g and 7a-i) was ﬁrst checked for outliers

using quartiles and Z score methods [69] and no outliers were

detected; supporting data. All compounds were drawn in 3D format

before the addition of 1

of the substrate/ATP mix. After 10 min incubation at room tem-

perature, 5 L of ADP-Glo™ Reagent was added then incubated for

mL of inhibitor, 2 mL of the enzyme and 2 mL

m

19

G.S. Hassan, H.H. Georgey, E.Z. Mohammed et al.

European Journal of Medicinal Chemistry 218 (2021) 113389

and energy minimized using Molecular Operating Environment

chemotherapeutics: mechanisms and lab approaches, Canc. Lett. 347 (2014)

159e166, https://doi.org/10.1016/j.canlet.2014.03.013 .

(

0

MOE, 2014.0901) software until an RMSD gradient of

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ꢁ

1

ꢁ1

.05 kcal mol

Å

with MMFF94x force ﬁeld and the partial

charges were automatically calculated.

The target property “CDK2 inhibitory activity” was expressed as

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(

IC50*10 )” for mathematical convenience and to have the

1871520615666141210152128 .

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[

4.3.2.2. Descriptors

calculation

and

feature

selection.

[

Descriptors available on ChemDes web-based platform (total: 1103)

were calculated for the training and test data sets http://www.

scbdd.com/chemdes. Rapidminer (9.0.003) software was used to

get rid of the descriptors which show low variance among the

different compounds using “Remove Useless Attributes“ operator.

After that, Forward Selection Algorithm (FSA) was used to select the

descriptors which are highly correlated to our target property. In

FSA, Leave-10%-out (L10%O) cross-validation root mean square er-

ror (RMSECV) was used to evaluate the model performance to

select the best model during the descriptor selection step.

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model internal validation. Furthermore, external validation was

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[

4

.3.3. In silico ADMET study

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was used for estimation of the potential toxicities like mutagenicity,

tumorigenicity irritant, and reproductive effect.

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Declaration of competing interest

There are no interests to declare.

Appendix A. Supplementary data

1

0.1007/s11030-018-9902-8 .

[

24] J. Faria, P. Vegi, A.G. Miguita, M. Santos, N. Boechat, A. Bernardino, Recently

reported biological activities of pyrazole compounds, Bioorg. Med. Chem. 25

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M. Smith, M.S. Squires, G. Trewartha, M.T. Walker, A.J.-A. Woolford, Identiﬁ-

cation of N-(4-Piperidinyl)-4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113389.

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22

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Mechanistic selectivity investigation and 2D-QSAR study of some new antiproliferative pyrazoles and pyrazolopyridines as potential CDK2 inhibitors

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