Synthesis, in vitro anticancer activity and in silico studies of certain pyrazole-based derivatives as potential inhibitors of cyclin dependent kinases (CDKs)

Article abstract of DOI:10.1016/j.bioorg.2021.105347

New diphenyl-1H-pyrazoles were synthesized and screened for CDK2 inhibition where 8d, 9b, 9c, and 9e exhibited promising activity (IC₅₀ = 51.21, 41.36, 29.31, and 40.54 nM respectively) compared to R-Roscovitine (IC₅₀ = 43.25 nM). Furthermore, preliminary anti-proliferative activity screening of some selected compounds on 60 cancer cell lines was performed at the (NCI/USA). Compounds 8a-c displayed promising growth inhibitory activity (mean %GI; 73.74, 94.32 and 74.19, respectively). Additionally, they were further selected by the NCI for five-dose assay, exhibiting pronounced activity against almost the full panel (GI₅₀ ranges; 0.181–5.19, 1.07–4.12 and 1.07–4.82 μM, respectively) and (Full panel GI₅₀ (MG-MID); 2.838, 2.306 and 2.770 μM, respectively). Screening the synthesized compounds 8a-c for inhibition of CDK isoforms revealed that compound 8a exhibited nearly equal inhibition to all the tested CDK isoforms, while compound 8b inhibits CDK4/D1 preferentially than the other isoforms and compound 8c inhibits CDK1, CDK2 and CDK4 more than CDK7. Flow cytometry cell cycle assay of 8a-c on Non-small cell lung carcinoma (NSCL HOP-92) cell line revealed S phase arrest by 8a and G1/S phase arrest by 8b and 8c. Apoptotic induction in HOP-92 cell line was also observed upon treatment with compounds 8a-c. Docking to CDK2 ATP binding site revealed similar interactions as the co-crystallized ligand R-Roscovitine (PDB code; 3ddq). These findings present compounds 8a-c as promising anti-proliferative agents.

Full text of DOI:10.1016/j.bioorg.2021.105347

Journal Pre-proofs
Synthesis, in vitro anticancer activity and in silico studies of certain pyrazole-
based derivatives as potential inhibitors of cyclin dependent kinases (CDKs)
Esraa Z. Mohammed, Walaa R. Mahmoud, Riham F. George, Ghaneya S.
Hassan, Farghaly A. Omar, Hanan H. Georgey
PII:
S0045-2068(21)00724-0
DOI:
https://doi.org/10.1016/j.bioorg.2021.105347
YBIOO 105347
Reference:
To appear in:
Bioorganic Chemistry
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Accepted Date:
6 July 2021
15 August 2021
7 September 2021
Please cite this article as: E.Z. Mohammed, W.R. Mahmoud, R.F. George, G.S. Hassan, F.A. Omar, H.H.
Georgey, Synthesis, in vitro anticancer activity and in silico studies of certain pyrazole-based derivatives as
potential inhibitors of cyclin dependent kinases (CDKs), Bioorganic Chemistry (2021), doi: https://doi.org/
10.1016/j.bioorg.2021.105347
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Synthesis, in vitro anticancer activity and in silico studies of
certain pyrazole-based derivatives as potential inhibitors of
cyclin dependent kinases (CDKs).
Esraa Z. Mohammeda*, Walaa R. Mahmoudb, Riham F. Georgeb*, Ghaneya S.
Hassanb,c, Farghaly A. Omard and Hanan H. Georgeyb,e
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, October 6 University, Giza 12585,
Egypt.
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562,
Egypt.
c Pharmaceutical Chemistry Department, School of Pharmacy, Badr University in Cairo (BUC),
Badr City, Cairo,11829, Egypt.
d Medicinal Chemistry Department, Faculty of Pharmacy, Assuit University, 71526 Assuit/ Egypt.
e Pharmaceutical Chemistry Department, Faculty of Pharmacy and Drug Technology, Egyptian
Chinese University, 11786, Cairo, Egypt.
* Corresponding authors;
Riham F. George: e-mail: riham.eskandar@pharma.cu.edu.eg
Esraa Z. Mohammed: e-mail: dr.esraazakaria@o6u.edu.eg
Conflict of interests: The authors declare no conflict of interest.
Abstract
New diphenyl-1H-pyrazoles were synthesized and screened for CDK2 inhibition
where 8d, 9b, 9c, and 9e exhibited promising activity (IC50 = 51.21, 41.36, 29.31,
and 40.54 nM respectively) compared to R-Roscovitine (IC50 = 43.25 nM).
Furthermore, preliminary anti-proliferative activity screening of some selected
compounds on 60 cancer cell lines was performed at the (NCI/USA). Compounds
8a-c displayed promising growth inhibitory activity (mean %GI; 73.74, 94.32 and
74.19, respectively). Additionally, they were further selected by the NCI for five-
dose assay, exhibiting pronounced activity against almost the full panel (GI50
ranges; 0.181–5.19, 1.07-4.12 and 1.07-4.82 µM, respectively) and (Full panel
GI50 (MG-MID); 2.838, 2.306 and 2.770 µM, respectively). Screening the
synthesized compounds 8a-c for inhibition of CDK isoforms revealed that
compound 8a exhibited nearly equal inhibition to all the tested CDK isoforms,
while compound 8b inhibits CDK4/D1 preferentially than the other isoforms and
compound 8c inhibits CDK1, CDK2 and CDK4 more than CDK7. Flow cytometry
cell cycle assay of 8a-c on Non-small cell lung carcinoma (NSCL HOP-92) cell
line revealed S phase arrest by 8a and G1/S phase arrest by 8b and 8c.
1

Apoptotic induction in HOP-92 cell line was also observed upon treatment with
compounds 8a-c. Docking to CDK2 ATP binding site revealed similar interactions
as the co-crystallized ligand R-Roscovitine (PDB code; 3ddq). These findings
present compounds 8a-c as promising anti-proliferative agents.
Keywords: Pyrazole based derivatives; Anti-proliferative; CDK inhibitors;
Molecular docking.
1. Introduction.
Over many years, cancer has been envisioned as a global public health problem
[1]. Despite of the indispensable progresses accomplished in the development of
the anticancer agents, innovation of novel potent and selective small molecules
remains a serious challenge [2, 3]. Beside lack of selectivity [4], several
limitations for current cancer treatments still exist, such as undesirable side
effects [5], and the multiple-drug resistance by cancer cells [6]. Cell cycle
deregulation is a common hallmark of cancer that is tightly related to cyclin
dependent kinases (CDKs), which upon binding to their cyclin subunits, play a
key role from initiation, through DNA replication, to mitosis [7-9]. This is often
correlated with cyclin amplification and overexpression or through mutation
and/or silencing of the encoded genes [10]. Several CDKs abnormalities have
been linked to diverse human cancer molecular pathology [11]. CDK2 is a crucial
regulator for G1/S cell cycle transition phase and a guide for G2/M transition
phase which halts cells with damaged DNA from initiating mitosis [12].
Consequently, CDK2 enzyme inhibition could result in G1/S and G2/M cell cycle
phases arrest and apoptosis induction [13]. Literature review revealed that
pyrazole template represents one of the principle pharmacophoric elements for
both anticancer and CDK2 enzyme inhibition activities [14-24]. Representative
examples are listed in Figure 1 [14, 15, 19, 23, 24].
2

OH
HN
Cl
O
O
N
H
N
HN
N
H2N
N
Cl
H
O
O
N
N
NH2
N
H
N
N
H
H
AT-7519 (I) [14]
PHA-533533 (II) [15]
CAN508 (III) [19]
OCH3
Cl
HO
S
H3CO
HN
O
NH
HN
O
O
N
N
N
O
N
N
N
O
(IV) [20]
(V) [23]
(VI) [24]
O
N
OCH3
N
H
N
(VII) [25]
Figure 1: Structures of some pyrazole derivatives with CDK2 inhibition and
anti-proliferative activities.
Wyatt et al. presented AT-7519 (I) as a CDK2 inhibitor with IC50 = 47 nM [14]. Its
inhibitory activity relied on binding to the CDK2 ATP binding site (Figure 2). The
binding mode disclosed the importance of the pyrazole core as a pharmacophoric
feature for occupying the adenine region of the ATP binding pocket. AT-7519 (I)
was anchored into the CDK2 hinge region via H-bonding interactions mediated
by the pyrazole core NH with Glu82. Additional H-bondings were also observed
between the carboxamide side chain N atom, and the piperidinyl NH with Leu83,
and His84 residues, respectively. Hydrophobic interactions with the binding site
were exhibited via the 2,6-dichlorobenzamide moiety [14] (Figure 2).
3

H
N
His84
O
R
O
R
H
Asp145
N
Cl
Cl
HN
N
O
H
N
N
O
R
H
H
N
Leu
83
R
O
NH
R
Glu82
Figure 2: Schematic illustration of the binding mode of the lead compound
AT7519 (I) to CDK2 ATP binding site.
Interestingly, compound II displayed potent CDK2 inhibition (IC50 = 37 nM) and
anticancer activity against A2780 and HT-29 cells with an IC50 values = 0.744
and 0.639 M, respectively [15]. Moreover, the diamino pyrazole derivative
CAN508 (III) explored a CDK2 inhibitory activity with IC50 value of 3.5 M [19].
Subsequently, numerous related pyrazoles were synthesized and inspected for
their growth inhibition activity against different cancer cell lines as compound IV
that exhibited promising CDK2 inhibition (IC50 = 25 nM) and anti-proliferative
activity against H460, MCF-7 and A549 cell lines at a micromolar range (0.75-
4.21 µM) [20]. Besides, the pyrazolyl chromenone derivative V displayed high
activity against SGC-7901 gastric cancer cell with an IC50 value of 8.09 nM [23].
Moreover, the diphenyl pyrazole VI revealed promising activity against AS49 cell
line (IC50 = 1.91 M) [24].
Recently, we published the results of CDK2 inhibitory activity of a series of
diphenyl-1H-pyrazoles demonstrating promising lead compounds [25], compound
VII was the most promising, and it displayed a promising selective profile for
CDK2 inhibition (IC50 = 42 nM) versus CDK1, CDK4, and CDK7 isoforms (IC50 =
955, 16681, and 326 nM, respectively). Consequently, we report herein novel
CDK2 inhibitors aiming to offer a tolerable strategy option in cancer treatment,
taking in consideration the reported findings and structure activity relationships
4

(SAR) studies of AT7519 (I) as CDK2 ATP competitive inhibitor [14]. Two
approaches were encompassed.
The first one is represented by 1,3-diphenylpyrazole derivatives; 4, 8a-e, and
9a-f (Figure 3) comprising the following structural characteristics:
i)
The pharmacophoric pyrazole core in the lead compounds (I - VII) which
fills the adenine region of the ATP binding pocket is preserved.
H-bondings interaction will be furnished via the extended side chains at
position 5 of the pyrazole ring as represented by NH, CO, SO2, and
hydrazone groups. Furthermore, the extended side chains were linked to
variably substituted aryl moieties to impart both electronic and lipophilic
diversity.
ii)
iii)
iv)
1,3-Diphenyl substitution attains additional hydrophobic interaction within
the ATP-binding site of CDK enzyme.
It is worth mentioning that a nitrile group has been implemented in the side
chain of series 8a-e and 9a-f (Figure 3); to afford hydrogen bond acceptor
functionality and forms non-specific dipole interactions with the amino acid
residues promoting the binding with the sterically occluded protein kinases
and consequently would enhance the activity [26].
The second approach represented by compounds 10a,b (Figure 3); is mainly
directed towards studying the effect of the more rigid hydrophobic analogs on the
inhibitory activity. This will be accomplished via rigidification of the extended side
chain of structures 9a-f into a chromene-2-imine moiety which is well known for
its diverse biological activity notably the anticancer one [23, 27-29].
5

(3B)
(3A)
O
O
S
O
CN
O
N
N
N
N
H
N
N
NH
N
H
R
8a-e
4
(3D)
(3C)
R
O
O
CN
N
N
N
N
N
H
N
O
H
HN
R
10a,b
9a-f
Figure 3: General structures of the newly designed pyrazole derivatives.
All the target compounds were tested for in vitro CDK2 enzyme inhibition and the
selected compounds by the NCI-USA were screened for their anticancer activity
versus 60 human cancer cell lines. Selectivity profile versus other CDK isoforms;
CDK1, CDK4, and CDK7 was performed for the most potent compounds in
addition to cell cycle analysis and apoptotic assay. A molecular docking study
was performed to explore the possible binding modes within the CDK2 ATP
binding site. Ultimately, ADMET computational parameters were assessed to
predict drug-likeness characteristics of the targeted compounds.
2. Results and Discussion
2.1. Chemistry
The synthetic routes for preparation of the target compounds are depicted in
schemes 1 and 2. Reacting methyl benzoate with acetonitrile in dry toluene using
sodium
hydride
resulted
in
3-oxo-3-phenylpropanenitrile
(1)
[30].
Cyclocondensation of compound 1 with phenyl hydrazine yielded 1,3-diphenyl-
1H-pyrazol-5-amine (2) [31]. N-chloroacetylation of the latter with chloroacetyl
6

chloride in glacial acetic acid afforded 2-chloro-N-(1,3-diphenyl-1H-pyrazol-5-
yl)acetamide (3) [32]. The final pyrazole derivative 4 was achieved through
condensation of compound 3 with sodium benzenesulfinate in absolute ethanol
(Scheme 1). IR spectrum of compound 4 revealed the expected new SO2 group
1
at 1328 and 1157 cm-1. The H and 13C NMR spectra were in accordance with
their expected signals (supporting data), in addition to the extra protons of the
additional aromatic ring.
O
O
(b)
(a)
N
O
CN
NH2
N
2
1
(c)
O
S
O
O
O
Cl
NH
N
N
(d)
N
N
N
H
4
3
Reagents and reaction conditions: (a) Dry toluene, acetonitrile, NaH, reflux, 4h (b)
Phenylhydrazine, fusion, 130 °C, 15 min. (c) ClCH2COCl, anhy. sod. acetate, gl. acetic acid,
stirring, r.t., 1 h. (d) PhSO2 Na, absolute ethanol, reflux, 12 h.
Scheme 1. Synthetic route for the target compound 4.
Scheme 2 involves three steps synthetic pathway of the key intermediate 7,
Cyanoacetic acid hydrazide (5) was first prepared through condensation of ethyl
cyanoacetate and hydrazine hydrate. Subsequent cyclocondensation of (5) and
acetylacetone afforded 1-cyanoacetyl-3,5-dimethyl pyrazole (6). Finally, the key
intermediate, 1,3-diphenylpyrazol-5-yl-cyanoacetamide (7) was obtained in high
yield via transcyanoacetylation of the intermediate amine (2) and the dimethyl
pyrazole (6). It is worth mentioning that compound 7 was first tried to be obtained
via reacting compound (3) with potassium cyanide in absolute ethanol; but
unfortunately, a very low yield was obtained from this synthetic method.
7

The target phenylacetohydrazonoyl cyanides (8a-e) were then obtained via
coupling of compound (7) with the respective diazonium salts of different
aromatic amines. Alternatively, the cyanoacrylamide derivatives (9a-e) were
attained by treatment of 7 with aromatic aldehydes. Remarkably, condensation of
compound
7
with 2-hydroxybenzaldehydes e.g. salicylaldehyde and
bromosalicylaldehyde produced the chromene-2-imines 10a,b, respectively. It is
evident that the resulting cyanoacrylamides undergo intramolecular cyclization
(Scheme 2).
The spectroscopic characteristics of compounds 8a-e, 9a-f and 10a,b confirmed
their structures. In the later case, compounds 10a-b, the IR spectra revealed an
1
extra NH band with the disappearance of the CN band. H NMR spectra
(supporting data) showed singlet signals at δ = 2.30 and 3.78 ppm for CH3 and
OCH3 groups in compounds 8d and 8e, and at δ = 3.88 and 3.87 for -OCH3 and -
(OCH3)2 groups in compounds 9e and 9f, respectively. In addition, a
characteristic singlet signal assigned to the (=CH) proton appeared at δ = 8.17-
8.36 ppm in compounds 9a-f. Finally, 13C NMR spectra of all the compounds
confirmed their proposed carbon skeleton (refer to the experimental part).
8

H
O
(b)
(a)
N
CN
NC
N
NH2
NC
N
N
O
N
N
O
H
6
5
7
(c)
(e)
(d)
O
CN
HN
O
CN
O
O
NH N NH
N
NH
N
N
NH
R
N
R
N
N
R
8a-f
9a-f
10a-b
R
H
4-F
4-Cl
9
a
b
c
R
10
a
b
R
H
4-F
4-Cl
4-CH3
4-OCH3
8
H
a
b
c
d
e
Br
d 2,4-diCl
e
f
4-OCH3
2,6-diOCH3
Reagents and reaction conditions: (a) Acetylacetone, H2O (25 mL): HCl (1mL), r.t., 1h, (b)
Compound 2, Dry toluene, reflux, 6h. (c); (i) Ar-NH2, HCl, sodium nitrite solution, 0-5 Cº, 15 min,
+
(ii) Ar-N2 Cl-, ethanol, sod. acetate, stirring, 0-5 oC, 1 h. (d) Ar-CHO, absolute ethanol/ piperidine,
reflux, 2 h. (e) Ar-CHO(OH), absolute ethanol/ piperidine, reflux, 2 h.
Scheme 2. Synthetic route for the target compounds 8a-e, 9a-f and 10a,b.
2.2. Biological screening
2.2.1. In vitro cyclin dependent kinase 2 /Cycline 2A enzyme inhibition assay.
The studied pyrazoles 4, 8a-e, 9a-f, and 10a, b were assessed for their in vitro
CDK2/CyclineA2 enzyme inhibition assay using the well-established ADP-Glo
assays [33, 34]. R-Roscovitine has been chosen as a reference for comparison,
since it is considered as a validated CDK2 ATP competitive inhibitor. The
obtained dose response curves were used for calculation of IC50 values (nM)
(Table 1). The results revealed that the 4-chlorophenyl substituted pyrazole
derivative 9c exhibited the highest CDK2 inhibition activity (IC50 = 29.31 nM),
9

which was non-significantly different from the reference R-Roscovitine (at p <
0.05). Also, compounds 8d, 9b, and 9e were proved to have statistically non-
significant (at p < 0.05) CDK2 inhibition activity (IC50 = 51.21, 41.36, and 40.54
nM, respectively) compared to the reference. The remaining tested compounds
displayed significant (at p < 0.05) lower inhibitory activity (IC50 range; 101.6-323.6
nM) in comparison with R-Roscovitine.
Structural exploration of compounds 8a-e showed that the presence of CH3
group at the para position of the side chain phenyl moiety was substantial for the
CDK2 inhibition activity, as demonstrated by compound 8d (IC50 = 51.21 nM).
Alternatively, the results of compounds 9a-f showed that mono-substitution either
with an electron-withdrawing (4-Cl) or donating groups (4-OCH3) was more
promising for the CDK2 inhibitory activity than di-substitution (2,4-diCl) or (2,6-
diOCH3). This was evident in compound 9c versus 9d (IC50; 29.31 versus 175.8
nM) and compound 9e versus 9f (IC50; 40.54 versus 91.4 nM).
Table 1. CDK2/cyclinA2 enzyme inhibition assay of pyrazole derivatives and
R-Roscovitine.
Compound ID
4
R
-
IC50 a ±SD (nM)
219.20 ± 12.00b
323.60 ± 18.00 b
141.20 ± 7.90 b
77.94 ± 4.30 b
51.21 ± 2.90c
284.70 ± 16.00 b
158.20 ± 8.80 b
41.36 ± 2.30c
29.31 ± 1.60c
175.80 ± 9.80 b
40.54 ± 2.10c
91.40 ± 5.10b
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
9f
H
4-F
4-Cl
4-CH3
4-OCH3
H
4-F
4-Cl
2,4-diCl
4-OCH3
2,6-diOCH3
10

163.20 ±9.00 b
101.60 ± 5.70b
43.20 ± 3.00
10a
10b
H
Br
R-Roscovitine
-
a IC50: The inhibitory concentration of the compounds required to
produce 50% inhibition, IC50 value was interpolated from dose–
response 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
c
test. b Statistically significant from R-Roscovitine at p < 0.05.
Statistically non-significant from R-Roscovitine at p < 0.05.
2.2.2. In vitro anti-proliferative activity against 60 cell lines
The structures of the synthesized 1,3-diphenylpyrazoles 4, 8a-e, 9a-f, and 10a, b
were submitted to the National Cancer Institute (NCI) Developmental Therapeutic
Program (www.dtp.nci.nih. gov) for evaluation of potential anti-proliferative
activity against a panel of NCI 60 cancer cell lines. Only the aryl
cyanoacetohydrazonoyl pyrazoles 8a-e were approved for screening at 10 µM
against the approved 60 cancer cell lines.
The human cancer cell lines were derived from 9 variant cancer types: leukemia,
melanoma, lung, colon, CNS, ovarian, renal, prostate and breast cancers.
Results are displayed as percentages of growth inhibition (GI %) of the screened
compounds against the full panel of cell lines (Supporting data). The mean GI %
of the screened compounds against the NCI 60 cell lines indicated superior
activity of three compounds 8a, 8b and 8c with GI % mean of 73.74, 94.32, and,
74.19 respectively. The results (supporting data) demonstrated that compound 8a
exhibited selective growth inhibition against some 2 subpanels renal cancer cell
lines namely: A498, and SN12C (GI % = 98.38 and 96.59, respectively); While
the 8b showed pronounced growth inhibition on non-small cell lung carcinoma
(NSCLC): NCI-H23, and NCI-H322M (GI % = 99.54, 99.28, respectively), as well
as breast cancer cells MCF7 with GI % of 99.88. For compound 8c, leukemia
cells RPMI-8226 (GI % of 98.19); CNS cancer cells SF-295 (GI % = 98.38), and
renal cancer cells SN12C (GI % = 99.86) were the most susceptible subpanels.
Furthermore, NSCL cells HOP-92 and breast cancer cells MDA-MB-468 were the
11

most inhibited ones by compounds 8d and 8e (GI % = 83.65, and 85.09,
respectively).
The attained results revealed the impact of the electronic characteristics of the
side chain substituents on the anticancer activity among the hydrazonoyl-
un/substituted phenyl pyrazoles 8a-e. Whereby, the electron-withdrawing
substituted derivatives 8b and 8c and the unsubstituted derivative 8a exhibited
pronounced anticancer activity against all the tested cell lines with GI % mean;
73.74 for 8a, 94.32 for 8b and 74.19 for 8c. Changing the electronic environment
of the substituents to electron donating ones resulted in deacrease in the growth
inhibitory activity (GI % mean; 8d: 30.15 and 8e: 34.91). Nevertheless,
compounds 8d and 8e showed broad spectrum anticancer activity against 57 and
55 cell lines. (Supporting data).
2.2.2.2. Five dose full NCI 60 cell panel assay.
Compounds 8a-c that displayed the most promising results in the preliminary
screening were chosen for further assessment at five doses concentrations (0.01,
0.1, 1, 10, 100 µM). The calculated response parameters: GI50, TGI and LC50
against several cell lines are shown in Table 2, whereby, GI50 indicates the
compound concentration that causes 50% decrease in the net cell growth, TGI
(Total growth inhibition) represents cytostatic activity, while LC50 (Lethal dose 50)
represents the compound concentration that causes net 50% loss of the initial
cells [35]. Besides, subpanel and full panel mean graph midpoints (MG-MID)
were calculated for the GI50 to indicate the average activity parameter over the
subpanels and full panel cell lines for each compound (Table 3).
Generally, compounds 8a-c displayed noticeable anticancer activity against
almost the whole panel of cancer cell lines with GI50 range 0.18–5.19, 0.14-4.54
and 1.07-4.82 M, respectively (Table 2). Additionally, compound 8a revealed
marked cytostatic activity against non-small cell lung cancer (HOP-92; TGI = 2.58
M), colon cancer (COLO 205; TGI = 4.77 M), and melanoma (SK-MEL-5; TGI
= 4.86 M) (Table 2). Compound 8b, revealed promising cytostatic effect against
leukemia cells (RPMI-8226; TGI = 4.47 M), NSCL (HOP-92; TGI = 3.84 M),
12

colon cancer (COLO 205; TGI = 3.60 M), melanoma (SK-MEL-5; TGI = 3.16
M), renal cancer (A498; TGI = 2.86 M). On the other hand, compound 8c had
cytostatic effect against non-small cell lung cancer (HOP-92; TGI = 4.49 M),
colon cancer (COLO 205; TGI = 4.51 M), melanoma (SK-MEL-5; TGI = 4.27
M), and breast cancer (MDA-MB 468; TGI = 5.93 M). Fortunately, LC50 values
of compounds 8a and 8c surpassed 100 M against all the tested cell lines which
reflected their non-lethal effects. While, LC50 values of compound 8b exceeded
100 M against most of the tested cell lines with some exceptions (Table 2).
Table 2. NCI in vitro testing of compounds 8a-c at five dose level (GI50, TGI,
and LC50 in M).
GI50 (M)
TGI (M)
LC50 (M)
Panel line.
Leukemia
CCRF-CEM
8a
8b
8c
8a
8b
8c
8a
8b
8c
2.5
2.94
2.43
3.75
1.8
1.21
1.89
1.45
2.16
0.75
2.27
2.27
3.05
1.72
3.5
> 100
22.1
9.31
5.43
12.8
9.92
4.47
8.74
> 100.00
9.38
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
>100.00
HL-60(TB)
K-562
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100
> 100
39.2
> 100.00
> 100.00
6.67
MOLT-4
RPMI-8226
SR
1.77
3.59
5.19
> 100
> 100.00
Non small cell lung cancer
A549/ATCC
2.95
2.93
2.84
0.18
2.45
2.84
2.69
0.43
3.20
> 100.00 15.60
> 100.00 15.30
> 100.00 > 100.00
> 100.00 > 100.00
> 100.00 > 100.00
> 100.00 > 100.00
> 100.00 > 100.00
EKVX
HOP-62
HOP-92
2.64
3.31
1.07
49.90
2.58
11.20
3.84
> 100
4.49
-
> 100.00
> 100.00
> 100.00
39.90
> 100.00
NCI-H226
NCI-H23
2.80
2.73
2.92
2.84
1.75
2.79
2.51
2.65
3.00
> 100.00 13.70
72.90
65.40
> 100.00
> 100.00
60.40
22.40
10.60
12.90
> 100.00 > 100.00
> 100.00 > 100.00
NCI-H322M
> 100.00 > 100.00
68.00 > 100.00
> 100.00 > 100.00
NCI-H460
NCI-H522
2.72
2.06
2.02
-
2.51
2.38
22.10
12.60
10.20
-
> 100.00 > 100.00
> 100.00 > 100.00
Colon cancer
13

COLO 205
HCC-2998
HCT-116
HCT-15
HT29
2.17
3.81
2.14
2.98
3.20
2.97
3.15
1.82
3.62
2.16
2.34
3.07
2.75
3.16
2.07
3.74
2.70
2.14
3.32
2.92
3.11
4.77
> 100
> 100
> 100
17.30
> 100
> 100
3.60
4.51
> 100.00 7.12
> 100.00
> 100.00
> 100.00
19.30
11.50
45.40
11.60
33.40
20.40
> 100.00 > 100.00 95.80
> 100.00 > 100.00 59.90
> 100.00 > 100.00 > 100.00 > 100.00
> 100.00 > 100.00 51.50 > 100.00
KM12
> 100.00 > 100.00 > 100.00 > 100.00
> 100.00 > 100.00 > 100.00 > 100.00
SW-620
CNS cancer
SF-268
3.85
3.14
3.30
3.60
-
2.37
1.66
3.26
2.99
1.93
3.04
2.65
2.86
3.29
3.17
-
> 100.00 25.90
> 100.00 5.75
> 100.00
> 100.00
> 100.00
> 100.00
-
> 100.00
> 100.00
> 100.00
> 100.00
-
> 100.00 > 100.00
SF-295
SF-539
SNB-19
SNB-75
U251
38.60
> 100.00
> 100.00
> 100.00
-
19.70
12.80
64.50
> 100.00 13.40
49.60
-
8.45
> 100.00
49.80
3.14
3.08
16.00
13.30
> 100.00
> 100.00
> 100.00
Melanoma
LOX-IMVI
MALME-3M
M14
-
4.54
1.85
2.84
2.55
1.22
3.35
0.82
1.54
0.85
-
-
17.70
-
-
52.50
-
2.84
2.68
3.06
2.20
3.78
1.54
2.47
1.65
2.34
3.70
2.79
2.39
3.62
1.63
2.35
2.03
> 100.00 13.40
> 100.00 100.00
> 100.00 100.00
-
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00 > 100.00
> 100.00 > 100.00
> 100.00 > 100.00
> 100.00
> 100.00
8.89
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
9.94
5.01
53.60
> 100.00 > 100.00
13.90 > 100.00
> 100.00 > 100.00
> 100.00
> 100.00 77.20
> 100.00
4.27
4.86
9.93
3.16
8.69
8.62
> 100.00 17.00
> 100.00
94.00
> 100.00
Ovarian cancer
IGROV1
4.49
3.24
2.01
3.39
3.69
2.40
2.39
3.52
4.12
2.71
2.25
2.99
> 100
33.20
8.95
18.30
15.60
21.80
17.20
> 100.00 > 100.00 77.90
> 100.00 > 100.00 80.60
> 100.00
> 100.00
OVCAR-3
OVCAR-4
OVCAR-5
7.62
> 100.00 > 100.00 > 100.00
> 100
> 100.00 > 100.00 > 100.00 > 100.00
14

OVCAR-8
NCI/ADR-RES
SK-OV-3
3.25
2.83
2.25
2.61
2.85
1.61
2.96
3.09
2.28
> 100
30.20
63.80
12.10
6.39
> 100.00 > 100.00 > 100.00 > 100.00
> 100.00 > 100.00 > 100.00 > 100.00
6.66
> 100.00 > 100.00 > 100.00
-
Renal cancer
786-0
3.04
-
3.41
0.14
2.55
1.56
2.24
1.83
3.43
2.50
4.05
-
16.30
-
14.00
2.86
> 100.00
-
> 100.00
-
48.70
25.60
65.30
43.50
56.50
> 100.00
-
A498
ACHN
2.98
2.88
2.26
2.74
4.27
2.58
2.84
2.44
2.06
2.44
4.82
2.94
> 100.00 10.50
> 100.00 8.72
> 100
-
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
CAKI-1
RXF 393
SN12C
TK-10
7.69
7.00
6.32
> 100.00 8.45
> 100.00 16.20
> 100.00 13.40
> 100.00
> 100.00
> 100.00
> 100.00 > 100.00
62.60 > 100.00
UO-31
> 100.00 > 100.00
Prostate cancer
2.13
1.77
3.62
2.40
3.90
9.48
8.70
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00 > 100.00
> 100.00 > 100.00
PC-3
4.08
> 100
20.80
DU-145
Breast cancer
MCF7
2.46
3.34
3.5
2.11
1.99
3.39
2.11
1.63
1.46
2.31
2.86
3.00
2.52
2.01
1.84
> 100.00 11.40
> 100.00 5.60
> 100.00 53.80
-
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00
> 100.00 > 100.00
MDA-MB-231/ATCC
HS 578T
> 100.00
> 100.00
7.18
28.20
> 100.00
> 100.00 > 100.00
> 100.00 > 100.00
> 100.00 > 100.00
BT-549
2.20
2.03
1.42
6.47
8.11
5.88
11.20
100.00
6.01
T-47D
7.12
MDA-MB-468
5.93
40.80
> 100.00
Regarding the sensitivity towards different cell lines, compound 8a exerted anti-
proliferative effect against almost the whole NCI panel with full panel GI50 (MG-
MID) value of 2.838 µM and subpanels GI50 (MG-MID) range of 2.459-3.406 µM
(Table 3). Among the tested cancer subpanels; NSCL, breast cancer, melanoma,
colon cancer and renal cancer were the most susceptible subpanels to
compound 8a (GI50 (MG-MID) = 2.459, 2.492, 2.528, 2.927 and 2.964 µM,
respectively) (Table 3). For compound 8b, the full panel GI50 (MG-MID) value
was 2.306 µM and the subpanels GI50 (MG-MID) range was 1.622 -2.724 µM
15

(Table 3). leukemia, breast, melanoma, renal and NSCL cancer subpanels were
the most sensitive cell lines for 8b with GI50 (MG-MID) values of 1.622, 2.115,
2.174, 2.208 and 2.226 µM, respectively. Results of compound 8c revealed that
the full panel GI50 (MG-MID) value was 2.770 µM and the subpanels GI50 (MG-
MID) range was 2.423-3.150 µM (Table 3). Breast cancer, NSCL, melanoma,
leukemia and colon cancer subpanels were the most sensitive cell lines for 8c
with GI50 (MG-MID) values of 2.423, 2.586, 2.606, 2.650 and 2.857 µM,
respectively. Moreover, the selectivity index (SI) was calculated by dividing the
full panel MG-MID (µM) for each tested compound by its individual subpanel MG-
MID (µM), it is considered as a measuring parameter for compound selectivity
towards the subpanels. Compounds 8a-c were found to be non-selective with
broad spectrum anticancer activity against all cancer subpanels, with selectivity
ratios ranging from 0.833–1.154 for 8a, 0.847-1.422 for 8b and 0.879–1.143 for
8c (Table 3).
Table 3. Mean graph mid points values (MG-MIDa) for GI50 parameter (uM) of
the subpanel tumor cell lines.
Subpanel type.
8a
MG-MID Selectivity MG-MID Selectivity MG-MID Selectivity
indexc indexc
index
8b
8c
3.102
2.459
2.917
3.406
2.528
3.066
2.964
3.105
2.492
1.622
2.226
2.703
2.542
2.174
2.724
2.208
2.695
2.115
2.650
2.586
2.857
3.010
2.606
2.914
3.084
3.150
2.423
Leukemia
0.915
1.422
1.045
NSCL cancer
Colon cancer
CNS cancer
Melanoma
1.154
0.973
0.833
1.123
0.926
0.957
0.914
1.139
1.036
0.853
0.907
1.061
0.847
1.044
0.856
1.090
1.071
0.970
0.920
1.063
0.951
0.898
0.879
1.143
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
Full panel MG-MIDb; for 8a = 2.838 µM, for 8b = 2.306 µM and for 8c =.2.770 µM.
16

aMG-MID is the average activity parameter over individual subpanels for each tested compound.
bFull panel MG-MIDb is the average sensitivity over all cell lines (full panel) for each tesed compound.
cSelectivity index was attained by dividing the full panel MG-MID (µM) for each compound by its individual
subpanel MG-MID (µM)
2.2.3. Selectivity profile of compounds 8a-c versus different CDK isoforms.
The most active compounds 8a-c were further subjected to investigate their
selectivity profile against three CDK isoforms; CDK1-cyclin B, CDK4-cyclin D1
and CDK7-cyclin H to determine their preferential selectivity compared to CDK2
isoform. Results in Table 4 revealed that compound 8a displayed its highest
inhibition activity against CDK1/cyclin B and CDK7/cyclin H with almost the same
IC50 values for both isoforms; 265 nM. On the other hand, compounds 8b and 8c
exhibited the uppermost inhibition activity versus CDK4/cyclinD1 with IC50 values
of 77 and 48 nM, respectively. It can be concluded that compound 8a inhibits all
tested CDK isoforms nearly in an equal manner. Whereas, compound 8b inhibits
CDK4/D1 preferentially than the other isoforms and compound 8c inhibits CDK1,
CDK2 and CDK4 more than CDK7. Previously, we recognized compound VII as
a promising selective inhibitor for CDK2 isoform (IC50 = 42 nM) [25].
Interestengly, our recent study identifies promising inhibitors for CDK1, CDK2,
CDK4 and CDK7 isoforms.
Table 4. Selectivity profile for compounds 8a-c against different CDKs
isoforms.
CDKs inhibitory activity IC50±SD (nM) and Ki (nM)
CDK2/A2
(Ki)
CDK1/B
(Ki)
CDK4/D1
(Ki)
CDK7/H
(Ki)
ID
8a
8b
8c
323.6±18.
(91.3)
265.0±14.0
(120)
342.0±20.0
(170)
265.0±14.0
(160)
141.2±7.9
(41.6)
223.0±12.0
(100)
77.0±4.0
(40)
393.0±20.0
(240)
77.94±4.3
(19.2)
56.0±3.0
(20)
48.0±3.0
(20)
130±7.0
(80)
The IC50 value was interpolated from dose–response data with 4 variable
concentrations which are triplicatally repeated. The Ki Kinetic parameter was
calculated by Graph Pad Prism software.
17

2.2.4. Cell cycle analysis and apoptosis detection
2.2.4.1. Cell cycle analysis
The in-vitro anti-proliferative screening results pointed towards non small lung
cancer cell line HOP-92 as a common susceptible cell line for compounds 8a-c
(Table 2). Moreover, their inhibition results for different CDK isoforms motivated
us to investigate their effect on the cell cycle arrest and apoptosis in HOP-92 cell
line. Therefore, treatment of the HOP-92 with the aforementioned compounds at
their GI50 concentrations and incubation for 48 h was performed and the results
are presented in Table 5 and Figure 4. Great pre-G1 apoptosis percentages
(26.85%, 30.49%, and 39.21%) were observed after treatment of HOP-92 with
compounds 8a-c, respectively; (Control; 1.95%). Moreover, a higher cell
accumulation (44.54%) was noticed at the S phase for compound 8a (Control;
38.74%). Besides, accumulations were observed at G0-G1 and S phases; (51.94
and 44.54%) for compound 8b, and (56.19 and 41.61%) for compound 8c;
(Controls; 51.63 and 38.74%), respectively. The attained results signified that
compound 8a halted HOP-92 cell cycle at S phase, while compounds 8b and 8c
induced arrest at G1/S phase.
18

8a
HOP92 Control
8c
8b
Figure 4. Cell cycle analysis of compounds 8a-c on HOP-92 cell line.
Table 5. Cell cycle analysis of compounds 8a-c on HOP-92 cell line.
Sample
%G0-G1
%S
%G2-M
% pre G1 apoptosis
51.63
38.74
9.63
1.95
HOP-92 Control
44.62
51.94
56.19
44.54
48.02
41.61
8.99
0.04
2.2
26.85
30.49
39.21
8a/ HOP-92
8b/ HOP-92
8c/ HOP-92
*Control: Un treated cells
2.2.4.2. Apoptosis assay
Results of HOP-92 cell cycle analysis, after treatment with compounds 8a-c,
revealed a pre-G1 peak (Table 5) which is a sign for apoptosis induction. To
validate the ability of the tested compounds to induce apoptosis, HOP-92 cells
were stained with Annexin V/PI, incubated for 48 h with 8a-c and analyzed. Early
and late apoptosis analysis illustrated the ability of compounds 8a-c to induce
significant levels of late apoptosis in HOP-92 treated cells compared to the
19

control cells. Early apoptosis effect is minor. The results are summarized in
Table 6 and graphically represented in Figure 5.
8a
HOP-92
Control
8b
8c
Figure 5. Apoptosis induction analysis for compounds 8a-c on HOP-92
cells.
Table 6. Apoptosis induction analysis for compounds 8a-c in HOP-92 cells.
Apoptosis induction analysis
Sample
Total
Early
0.61
2.17
2.55
1.81
Late
HOP-92 control
8a/ HOP-92
8b/ HOP-92
8c/ HOP-92
1.95
0.17
26.85
30.49
39.21
15.42
8.57
30.52
2.3. Molecular modeling study
2.3.1. Molecular docking study
Docking patterns of compounds 8d, 9b, 9c, 9e that displayed the uppermost
CDK2 inhibition activity (IC50 = 51.21, 41.36, 29.31 and 40.54 nM, respectively)
were studied in detail. Consequently, the X-ray crystal structure of the CDK2 co-
crystallized with R-Roscovitine, one of the well-known CDK2 ATP competitive
20

inhibitors [16], was downloaded from PDB (PDB code: 3ddq)
(http://www.rcsb.org/).
The docking protocol involved an initial validation step by redocking of the co-
crystallized ligand to assess validation parameters e.g. RMSD = 0.494 Aº,
docking score (S) = -14.915 Kcal.mol-1. Moreover, identification of binding
patterns of the native ligand was performed. These results ensured the validity of
the applied docking protocol (Figure 6). R-Roscovitine occupied almost the ATP
binding region whereby the purine moiety accommodated the adenine region.
Two H bonds with the essential Leu83 residue were mediated via the pyrazole
core N atom and side chain NH, in addition, an extra H bond with Glu81 residue
was formed. Furthermore, π-π hydrophobic interaction was observed between
the benzyl amino substituent of R-Roscovitine and Ile10, Phe82 and His84
residues [16] (Figure 6).
A
B
Figure 6. 2D ligand interaction diagram (A) and 3D representation (B) of
superimposition of the native ligand (green) and its redocked pose (red) in
CDK2 ATP binding site.
Subsequently, the same docking protocol has been applied for the studied
compounds. The results showed that most of the docked compounds displayed
related binding mode to that of the native ligand R-Roscovitine (Supporting data).
21

As shown in Figures 7-10, the most potent CDK2 inhibitors 8d, 9b, 9c, 9e could
accommodate for the essential interactions with the enzyme binding site in
particular Leu83; that observation might rationalize their obtained activity.
Docking pattern of compound 8d (IC50 = 51.21 nM) is illustrated in Figure 7,
whereby H-bonding was mediated via the C≡N group with the Leu83 residue.
Extra H-bonding interactions with His84 and Glue8 amino acids were also
observed, in addition to π-H hydrophobic interactions with Gln85, Val18 and
Lys89 residues. The aforementioned binding pattern for compound 8d might
explain its observed inhibitory activity.
Figure 7. 2D ligand interaction diagram of compound 8d with CDK2 ATP
binding site (S = -13.05 Kcal.mol-1).
Figure 8 illustrated the binding mode of compound 9b (IC50 = 41.36 nM),
whereby H-bonding interaction with Leu83 essential residue was mediated.
Besides, other H-bonding and π-H hydrophobic interactions were formed with
His84 and Ile10 residues, respectively.
22

Figure 8. 2D ligand interaction diagram of compound 9b with CDK2 ATP
binding site (S = -14.14 Kcal.mol-1).
Binding mode of compound 9c to CDK2 ATP region (Figure 9) might explain its
remarkable CDK2 inhibitory activity (IC50 = 29.31 nM). As shown, the essential H-
bonding interaction with Leu83 residue was mediated via the Cl substituent on
the side chain phenyl moiety. In addition, other H-bondings were formed with
Glu81 and Gly11 residues via the same Cl atom and the side chain C=O group,
respectively. Extra π-H and π-cation hydrophobic interactions with Gln131 and
Lys89 via the aromatic moieties might stabilize the binding of compound 9c.
Figure 9. 2D ligand interaction diagram of compound 9c with CDK2 ATP
binding site (S = -13.82 Kcal.mol-1).
23

As shown in Figure 10, compound 9e (IC50 = 40.54 nM) could mediate H-
bonding interaction with Leu83 essential residue, in addition to π-cation
hydrophobic interaction with Lys89 amino acid.
Figure 10. 2D ligand interaction diagram of compound 9e with CDK2 ATP
binding site (S = -13.67 Kcal.mol-1).
Table 7. Docking energy scores (S) “Kcal.mol-1”.and Ki values “nM”.
Compound
(S)
Ki
Binding Interacions Amino acids involved
R-Roscovitine
-14.91
20.0
H-bonding
H-Bonding
H-Bonding
π-H
Leu83 ─ N (Pyrazole)
Leu83 ─ NH (Benzylamino)
Glu81 ─ C (Pyrazole)
Ile10 ─ (Benzyl)
8d
-13.05
12.8
H-Bonding
H-Bonding
H-Bonding
π-H
Leu83 ─ C≡N
His84 ─ C (pyrazole)
Glue8─ C (N-phenyl)
Gln85─ (phenyl)
π-H
π-H
Val18─ (N-phenyl)
Lys89 ─ (pyrazole)
9b
9c
-14.14
-13.82
15.2
8.63
H-Bonding
H-Bonding
π-H
Leu83 ─ CH
His84 ─ C (pyrazole)
Ile10 ─ (N-phenyl)
H-Bonding
H-Bonding
H-Bonding
π-H
Leu83 ─ Cl
Glue81─ Cl
Gly11─ C=O
Gln131─ (phenyl)
Lys89─ (N-phenyl)
π-Cation
24

9e
-13.67
11.9
H-Bonding
Leu83 ─ CH
π-Cation
Lys89─ (pyrazole)
2.3.2. In silico ADMET study
2.3.2.1. Pharmacokinetic and drug-likeness prediction
Pharmacokinetics and drug-likeness aspects prediction of the studied
compounds 4, 8a-e, 9a-f and 10a, b was accomplished using the freely available
web server Swiss ADME (http://www.swissadme.ch/). This study depends on the
chemical structure of the compounds which are involved in the calculation of
certain parameters including human gastrointestinal absorption (HIA), blood-brain
barrier (BBB) permeation, substrate, or non-substrate for the permeability
glycoprotein (P-gp), Logkp and interaction of molecules with cytochromes P450
isomers (CYP).
Results are presented as a 2D plot drawn using the calculated TPSA and LogP
properties of the target molecules “BOILED-EGG chart” (Supporting data).
Whereby, white area indicates GIT passive absorption probability, yellow region
predicts BBB penetration [36], blue dots predict effluxing by P-gp (PGP+) and red
dot predicts no effluxing via P gp (PGP-). The BOILED-Egg chart for the target
compounds (Supporting data) displayed their GIT passive absorption probability
with no BBB permeability predicting minimal CNS adverse effects. Besides, the
target compounds might not be substrates for the P-glycoprotein (PGP-) and thus
eliminating the possibility of tumor cell lines resistance through efflux mechanism
[37] (Table 8). Furthermore, most of the studied compounds are predicted to
have no activity on Cytochrome P450 isomers (CYP3A2 and CYP2D6) and
consequently are expected to have no drug-drug interactions upon administration
[38]. Additionally, good bioavailability score (Score = 0.55) was predicted for the
target molecules based on their compliance to the five rule-based filters [39]:
Lipinski [40], Ghose [41], Veber [42], Egan [43] and Muegge [44] rules (Table 8).
25

Table 8. Computer aided ADME assessment of the studied compounds.
Cpd.
Pharmaco-kinetics aspects
Drug likeness (#violations)
4
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
9f
High
High
High
High
High
High
High
High
High
High
High
High
High
High
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
Yes
No
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
10a
10b
2.3.2.2. Toxicity Prediction
The studied compounds were further subjected to another virtual filter (Osiris
Property Explorer (http://www.organic-chemistry.org/prog/peo/)) for prediction of
their probable toxicities like mutagenicity, carcinogenicity, tumorigenicity and
teratogenicity. This online program predicts on basis of functional group similarity
of the investigated compound with in vitro and in vivo studied compounds within
its database. The results are color coded such as red, green and yellow. Green
color suggests low toxic potential, yellow means mild toxicity and red color
indicates high probability of toxicity [45] [46]. The results (Supporting data)
26

revealed that most of the studied compounds are predicted to be safe and
expected to display low or no toxicity concerning mutagenicity, tumorigenicity,
irritant effect and effect on reproductive system. Compounds 8a and 9d might
have a high risk of tumorigenicity, while compound 8e was predicted to have mild
tumorigenicity. Also compound 9d might possess a high risk for irritant effect; in
addition, compounds 8c and 10a might produce moderate toxic effects on the
reproductive system.
3. Conclusion
New 1,3-diphenyl-1H-pyrazoles 4, 8a-e, 9a-f and 10a,b were synthesized and
evaluated for their CDK2 inhibition. Compounds 9c, 9e, 9b, and 8d were
promising CDK2 inhibitors (IC50 = 29.31, 40.54, 41.36, and 51.21 nM,
respectively) compared to R-Roscovitine (IC50 = 43.25 nM). Compounds 8a-e
were selected by (NCI/USA) for full panels screening of anticancer activity
against 60 cancer cell lines. Compounds 8a-c displayed promising growth
inhibitory activity (mean %GI; 73.74, 94.32 and 74.19, respectively). NCI five-
dose assay displayed pronounced anticancer activity against almost the full panel
(GI50 ranges; 0.181–5.19, 1.07-4.12 and 1.07-4.82, respectively) and (Full panel
GI50 (MG-MID); 2.838, 2.306 and 2.770 µM, respectively). CDK isoform
selectivity study indicated that compound 8a exerted nearly equal inhibition for all
tested CDK isoforms, compound 8b inhibited CDK4/D1 more preferentially than
the other isoforms, while compound 8c inhibited CDK1, CDK2 and CDK4 more
than CDK7. Flow cytometry cell cycle assay in HOP-92 cells showed S phase
arrest in case of 8a, and G1/S phase arrest for compounds 8b and 8c indicating
the apoptotic induction ability of compounds 8a-c. Comparable binding
interactions with the co-crystallized ligand R-Roscovitine (PDB code; 3ddq) were
noticed for the docked compounds in CDK2 ATP binding site, in particular
binding with Leu83 residue. Finally, the synthesized compounds explored good
ADMET properties and toxicity profiles.
27

4. Experimental
4.1. Chemistry
Melting points (°C) were recorded by Biocote melting point apparatus (BIBBY,
scientific limited stone, staeeordshire) ST, 150 SA, UK using open capillary tube
method and are uncorrected. 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, Schimadzu, Kyoto, Japan). All spectra were expressed as
1
υ cm-1, using potassium bromide discs. H NMR and 13C NMR spectra were
carried out using Bruker high performance digital FT-NMR spectrometer
AVANCE III400 MHz (Bruker Corporation, Germany) at the Microanalytical Unit,
1
Faculty of Pharmacy, Cairo University and Mansoura University. H/ 13C NMR
spectra were run at 400/100 MHz, respectively in DMSO-d6 as a solvent,
chemical shifts are quoted in δ as parts per million (ppm) downfield from
tetramethylsilane (TMS) as internal standard. Mass Spectra were performed 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 aluminum
sheets, Silica gel Kieselgel 60 F254; (Merck, Darmstadt, Germany). Methyelene
chloride was used as the eluting solvent and TLC sheets were visualized at 366,
254 nm by UV VilberLourmat 77202 (Vilber, Marne La Vallee, France).
Compounds (1) [30], (2) [31], (3) [32], (5 and 6) [47] were synthesized according
to the reported methods.
4.1.1. Synthesis of N-(1,3-Diphenyl-1H-pyrazol-5-yl)-2-(phenylsulfonyl)
acetamide (4).
To a solution of 2-chloro-N-(1,3-diphenyl-1H-pyrazol-5-yl)acetamide (3) (1.55 g, 5
mmol) in absolute ethanol (15 mL), sodium benzenesulfinate (0.164 g, 5 mmol)
was added and the reaction mixture was heated under reflux for 12 h. The
mixture was cooled and the separated solid was filtered, washed with ethanol
and dried. The obtained product (4) was crystallized from ethanol.
28

White powder; Yield: (1.82 g, 87%); m.p. 169-171 °C; Rf: 0.27; IR υ cm-1: 3278
(NH), 3066 (CH aromatic), 2991 (CH aliphatic), 1672 (C=O), 1566 (C=N), 1328,
1
1157 (SO2); H NMR δ ppm: 4.56 (s, 2H, CH2), 6.89 (s, 1H, H-4 pyrazole), 7.35
(t, 1H, J = 7.28 Hz, aromatic H), 7.42-7.49 (m, 3H, aromatic Hs), 7.55-7.58 (m,
4H, aromatic Hs), 7.67 (t, 2H, J = 7.68 Hz, aromatic Hs), 7.78 (t, 1H, J = 7.38 Hz,
aromatic H), 7.86 (t, 4H, J = 8.90 Hz, aromatic Hs), 10.49 (s, 1H, NH, D2O
exchangeable); 13C NMR δ ppm: 61.4, 99.1, 124.4, 125.6, 128.3, 128.5, 128.6,
129.2, 129.7, 129.8, 133.0, 134.6, 136.7, 138.5, 139.4, 150.7, 160.4; MS (m/z,
%): 416 (M+-1, 100); Anal. Calcd. for C23H19N3O3S (417.48): C, 66.17; H, 4.59; N,
10.07. Found: C, 66.39; H, 4.66; N, 10.23.
4.1.2. Synthesis of 2-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)acetamide (7).
1-Cyanoacetyl-3,5-dimethyl pyrazole (6) (1.48 g, 10 mmol) was added to a
solution of 1,3-diphenyl-1H-pyrazol-5-amine (2) (2.35 g, 10 mmol) in dry toluene
(10 mL). The reaction mixture was heated for 6 h and allowed to cool, then the
precipated product (7) was filtered off, dried and crystallized from ethanol.
White powder; Yield: (2.90 g, 96%); m.p. 165-167 °C; Rf: 0.12; IR υ cm-1: 3196
(NH), 3020 (CH aromatic), 2964, 2920 (CH aliphatic), 2258 (C≡N), 1678 (C=O),
1593 (C=N); 1H NMR δ ppm: 3.93 (s, 2H, CH2), 6.96 (s,1H, H4 pyrazole), 7.36 (t,
1H, J = 7.24 Hz, aromatic H), 7.43-7.47 (m, 3H, aromatic Hs), 7.53-7,61 (m, 4H,
aromatic Hs), 7.86 (d, 2H, J = 7.68 Hz, aromatic Hs), 10.49 (s, 1H, NH, D2O
exchangeable); 13C NMR δ ppm: 26.3, 99.8, 115.9, 124.2, 125.6, 128.3, 128.6,
129.2, 129.8, 133.0, 136.7, 138.6, 150.7, 162.4; MS (m/z, %): 302 (M+, 8), 68
(100); Anal. Calcd. for C18H14N4O (302.33): C, 71.51; H, 4.67; N, 18.53. Found:
C, 71.59; H, 4.78; N, 18.70.
4.1.3. General method for the synthesis of compounds (8a-e).
A solution of the appropriate primary aromatic amines (5 mmol) in hydrochloric
acid (1 mL) was cooled to 0-5oC in an ice bath, then a solution of sodium nitrite
(0.34 g, 5 mmol) in water (2.5 mL) was added dropwise with stirring for 15 min.
The reaction mixture was then added dropwise to a solution of 2-cyano-N-(1,3-
29