3-Substituted-4-hydroxycoumarin as a new scaffold with potent CDK inhibition and promising anticancer effect: Synthesis, molecular modeling and QSAR studies

Article abstract of DOI:10.1016/j.bioorg.2016.06.005

A new series of 3-substituted-4-hydroxycoumarin derivatives was designed, synthesized, and evaluated for CDK inhibiting and anticancer activities. All the synthesized target compounds showed remarkably high affinity and selectivity towards CDK1B, compared to flavopiridol, with Ki values in the low nanomolar range (Ki?=?0.35–0.88?nM). Most of them elicited considerable inhibiting effect against CDK9T1 (Ki?=?3.26–23.45?nM). Moreover, all the target compounds were tested in vitro against eighteen types of human tumor cell lines. The hydrazone 3a, N-phenylpyrazoline derivative 6b and 2-aminopyridyl-3-carbonitrile derivative 8c were the most potent anticancer agents against MCF-7 breast cancer cell line (IC₅₀?=?0.21, 0.21 and 0.23?nM, respectively). The target compounds 3a, 6b and 8c were further evaluated in MCF-7 breast cancer mouse xenograft model and showed in vivo efficacy at 10?mg/kg dose. The docking study confirmed a unique binding mode in the active site of CDK1B with better score than flavopiridol. Quantitative structure activity relationship study was done and revealed a highly predictive power R² of 0.81.

Full text of DOI:10.1016/j.bioorg.2016.06.005

Accepted Manuscript
3-Substituted-4-Hydroxycoumarin as a New Scaffold with Potent CDK Inhib-
ition and Promising Anticancer Effect: Synthesis, Molecular Modeling and
QSAR Studies
Nehad A. Abdel Latif, Rasha Z. Batran, Mohammed A. Khedr, Mohamed M.
Abdalla
PII:
S0045-2068(16)30093-1
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.06.005
YBIOO 1919
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 May 2016
17 June 2016
23 June 2016
Please cite this article as: N.A. Abdel Latif, R.Z. Batran, M.A. Khedr, M.M. Abdalla, 3-Substituted-4-
Hydroxycoumarin as a New Scaffold with Potent CDK Inhibition and Promising Anticancer Effect: Synthesis,
Molecular Modeling and QSAR Studies, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.
2016.06.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

3-Substituted-4-Hydroxycoumarin as a New Scaffold with Potent
CDK Inhibition and Promising Anticancer Effect: Synthesis,
Molecular Modeling and QSAR Studies
Nehad A. Abdel Latif,a,b* Rasha Z. Batran,a Mohammed A. Khedr,c
Mohamed M. Abdallad
a
Chemistry of Natural Compounds Department, Pharmaceutical Industries Division, National
Research Center, 33 El bohouth st. ( former El tahrir st.) Dokki, Giza, p. o. box 12622, Egypt
b Chemistry Department, Faculty of Science, Taif University, Taif, Kingdom of Saudi Arabia
c Pharmaceutical Chemistry Department, Faculty of Pharmacy, Helwan University, Ein Helwan,
Cairo, p.o.box 11795, Egypt
d Research Unit, Saco Pharm. Co., 6th October City, Egypt
* Corresponding author
E-mail address: nehad_km@yahoo.com
Abstract
A new series of 3-substituted-4-hydroxycoumarin derivatives was designed,
synthesized, and evaluated for CDK inhibiting and anticancer activities. All the
synthesized target compounds showed remarkably high affinity and selectivity
towards CDK1B, compared to flavopiridol, with Ki values in the low nanomolar
range (Ki= 0.35-0.88 nM). Most of them elicited considerable inhibiting effect
against CDK9T1 (Ki= 3.26- 23.45 nM). Moreover, all the target compounds were
tested in vitro against eighteen types of human tumor cell lines. The hydrazone 3a,
N-phenylpyrazoline derivative 6b and 2-aminopyridyl-3-carbonitrile derivative 8c
were the most potent anticancer agents against MCF-7 breast cancer cell line
(IC50=0.21, 0.21 and 0.23 nM, respectively). The target compounds 3a, 6b and 8c
were further evaluated in MCF-7 breast cancer mouse xenograft model and showed
in vivo efficacy at 10 mg/kg dose. The docking study confirmed a unique binding
mode in the active site of CDK1B with better score than flavopiridol. Quantitative
1

structure activity relationship study was done and revealed a highly predictive
power R2 of 0.81.
Keywords; 4-Hydroxycoumarin, pyrazolines, 2-aminopyridine-3-carbonitriles,
CDK, anticancer, molecular docking, and QSAR.
1. Introduction
Cyclin-dependent kinases (CDKs) constitute a family of serine/ threonine
protein kinases involved in cell cycle and transcription. They include two main
groups; cell cycle CDKs and transcriptional CDKs [1]. The functionality of CDKs
depends on specific interactions with regulatory proteins named cyclins which
form heterodimeric complexes with their partners [2-5]. These complexes are
important regulators of the cellular processes, especially in the cell cycle
progression. Cyclins D-CDK4/6 and E-CDK2 promote the cell cycle transition
from G1 to S phase by sequential phosphorylation of the retinoblastoma protein
(Rb). Cyclin A-CDK1/2 and Cyclin B-CDK1 are important for the progression of
S-phase and G2/M transition, respectively [6]. While, CDK5 is involved in the
regulation of glucose uptake [7], CDK7-cyclin H and CDK9-cyclin T are crucial
for transcription regulation by phosphorylating RNA polymerase II [8].
CDK-specific inhibitors are important validated targets in drug discovery; they
induce apoptosis by perturbing the cell cycle and/or by repressing transcription [9].
CDK-cyclin complexes govern the regulation of the cell cycle and their level
depends on the balance between protein synthesis and proteasomal degradation
[10].
The enzymatic activity of CDKs is affected by endogenous protein inhibitors
and modulated by their phosphorylation state [11]. However, CDK-cyclin activity
is deregulated in the cancerous cells [12]; with diverse mechanisms including
overexpression of cyclins, upstream activation of signal transduction [13], in
2

addition to amplification of CDKs, and inactivation or loss of endogenous protein
inhibitors, especially p16 [14].
Since 1990, several CDK inhibitors have been clinically developed.
Flavopiridol (Figure 1) and R-roscovitine (seliciclib), were the first generation of
pan-CDKs inhibitors with anti-tumor activity attributed to down-regulation of
CDK9-mediated anti-apoptotic proteins, especially Mcl-1 [15-17]. Recently, a new
generation of CDK has been developed and advanced to phase III trials for certain
types of cancer [18,19]. Dinaciclib, a selective inhibitor of CDK1, CDK2, CDK5,
and CDK9 [20,21], was directed towards refractory chronic lymphocytic leukemia
while palbociclib was tested against advanced estrogen receptor (ER)-positive
breast cancer as a selective inhibitor of CDK4 and CDK6 [22].
Figure 1
Moreover, various naturally occurring flavones and chromone alkaloids were
reported for their CDK inhibiting properties [23]. Eupatilin, a natural flavone
compound extracted from Artemisia asiatica Nakai (Figure1), down-regulated the
expression of cyclin D1 in MCF-10A-ras cells. It caused a reduction in c-Jun
expression and DNA binding activity of AP-l transcription factor which is an
important factor in the cell cycle arrest of ras-transformed breast epithelial cells
[24]. Moreover, several flavopiridol analogues were synthesized and evaluated as
potent CDK inhibitors [25-27].
On the other hand, the isosteric coumarin nucleus revealed wide spectrum and
diversity of biological effects especially cytotoxic activity against various types of
cancers [28-32]. It had a considerable anti-CDKs effect as exemplified by RKS262
(Figure 1) which is a specific cyclin/CDK inhibitor. It affected the viability of
multiple cancer cell-lines, particularly ovarian cancer cells, by delaying the
progression of OVCAR-3 cell cycle and up-regulating p27 with cyclin D1 and
CDK6 down regulation [33].
3

While pan-specific CDK inhibitors affect multiple pathways which are
deregulated in malignancy yielding positive outcomes, it was found that targeting a
single specific CDK improves selectivity of cancer therapy and prevents damage of
healthy cells. As most of the currently available CDK inhibitors affect two or more
kinases, it was challenging to discover new mono-specific inhibitors [34].
Encouraged by the aforementioned research information, coupled with the hope of
developing new selective CDK inhibitors, we have identified a new class of
chemical compounds for studying their anti CDK inhibitory and anticancer effects.
The present investigation particularly concentrate on the 4-hydroxycoumarin
and pyrazole [35-37] /2-aminopyridine-3-carbonitrile derivatives [38,39] (Figure
2) which have been pointed out as new scaffolds with significant therapeutic
efficacy against CDKs. The synthesized target compounds shared common
structure features of a 4-hydroxycoumarin nucleus substituted at position 3 with
either a 5-aryl substituted pyrazoline group (5a-d, 6a-c and 7a-c) or 2-
aminopyridine-3-carbonitrile moiety (8a-c). A noncyclic bioisosteric modification
of the cyclic pyrazolines 5a-d was adopted to get the noncyclic arylidene-
ethylidene hydrazine bioisosteres (3a-d) (Figure 2) (Schemes 1 and 2).
Figure 2
The synthesized compounds were evaluated for their CDK inhibition and in
vitro anticancer activity against eighteen types of human tumor cell lines. In
addition, in vivo antitumor activity of compounds 3a, 6b and 8c was validated in
MCF-7 breast cancer xenograft mouse model. Molecular docking study was
performed to interpret and explain the possible mode of binding of the active
compounds. Quantitative structure activity relationship (QSAR) of the active set of
compounds was done to find out the descriptors that may be correlated to the
activity.
4

2. Results and Discussion
2.1. Chemistry
The synthesis of the target compounds 3a-d, 5a-d, 6a-c, 7a-c and 8a-c was
depicted in Schemes 1 and 2. 3- Acetyl-4-hydroxycoumarin 1 was prepared by
acetylation of the commercially available 4-hydroxycoumarin with acetic acid in
the presence of POCl3 [40]. Reaction of 1 with hydrazine hydrate at room
temperature afforded the corresponding hydrazone 2 [40]. Condensation of the
hydrazone 2 with the appropriate aldehydes; namely; benzaldehydes, 2-chloro-
benzaldehyde, 3,4-dimethoxybenzaldehyde and 4-dimethylaminobenzaldehyde
yielded the arylidene-ethylidenehydrazines 3a-d (Scheme 1).
Scheme 1
The intermediate chalcone compounds 4a-d were prepared by conventional
Claisen-Schmidt condensation reaction of 1 with the previously mentioned aryl
aldehydes in the presence of piperidine drops in ethanol [41-43] (Scheme 2). The
coumarin-pyrazoline hybrids 5-7 were synthesized through 1,4-addition of
hydrazine hydrate or phenyl hydrazine to the α,ß unsaturated carbonyl moiety of
the chalcones 4a-d, followed by dehydration and rearrangement (Scheme 2).
Condensation of the precursor chalcones 4a-d with hydrazine hydrate or
phenylhydrazine in absolute ethanol furnished the N1- unsubstituted and N1-
phenylpyrazolines 5a-d and 6a-c, respectively. Moreover, cyclocondensation of
4b-d with hydrazine hydrate in glacial acetic acid produced the N-acetylpyrazoline
derivatives 7a-c due to in situ acetylation of the initially formed pyrazolines 5b-d.
In addition, one pot, four component synthesis of the target 2-amino-3-
carbonitriles 8a-c was achieved through cyclization reaction of the acetyl
compound 1 with the appropriate aldehydes and malononitrile in the presence of
ammonium acetate in ethanol.
Scheme 2
5

The structure assignment of the newly synthesized compounds was confirmed
by analytical and spectral data (IR, 1HNMR, and mass spectroscopy).
2.2. Biology
2.2.1. CDK inhibiting properties
Anti- CDKs effect of the target compounds was determined radiometrically
using the Millipore Kinase Profiler services [44]. All the synthesized target
compounds selectively inhibited CDK1B in a low nanomolar range and displayed
higher inhibitory activity than flavopiridol (Ki= 0.35-0.88 nM vs Kiflav=13 nM).
Most of them displayed remarkable inhibiting effect against CDK9T1 compared to
the reference standard (Ki= 3.26- 23.45 nM vs Kiflav= 3 nM) (Table 1). The N-
acetylpyrazoline compound 7a was the most potent CDK1B inhibitor (Ki= 0.35
nM), it displayed higher inhibition activity than the reference flavopiridol. The
hydrazone derivative 3d and N-phenylpyrazoline compounds 6c displayed
comparable CDK9T1 inhibiting effects to flavopiridol (Ki= 3.26, 3.46 nM,
respectively).
The 4-dimethylaminophenyl hydrazone compound 3d showed the highest
CDK1B inhibiting activities within the synthesized hydrazone series (Ki= 0.46
nM). A slight decrease in activity was observed with the unsubstituted phenyl
hydrazone 3a (Ki= 0.56 nM). It was the most potent CDK9T1 inhibitor of all the
synthesized compounds displaying a comparable Ki value to that of the reference
flavopiridol and being 8 fold more active than the unsubstituted phenyl hydrazone
compound 3a ( Ki3d= 3.26 nM vs Ki3a= 23.45 nM). A non significant decrease in
CDK9T1 inhibiting properties was observed for the 3,4-dimethoxyphenyl
derivative 3c (Ki= 4.56 nM) with an improvement in CDK2E inhibiting activity
(Ki= 15.45 nM). Phenyl hydrazone compound 3a was the most active CDK7H
inhibitor being 2.5 more active than the reference flavopiridol (Ki3a= 44.56 nM vs
Kiflav= 113 nM).
6

The cyclized pyrazoline compounds containing the electron donating
substituents 5c and 5d exerted a decrease in both CDK9T1 and CDK1B inhibiting
properties than the open hydrazone congeners 3d and 3c (Ki (CDK9T1)5c, 5d = 7.89,
7.09 nM vs Ki (CDK9T1)3c, 3d = 4.56, 3.26 nM) and (Ki (CDK1B)5c, 5d = 0.88, 0.76
nM vs Ki (CDK1B)3c, 3d = 0.66, 0.46 nM).
On the other hand, The pyrazoline derivatives 5b, 6a, 7a with the electron
withdrawing Cl atom showed a remarkable increase in CDK9, 2, 1 inhibiting effect
than the open hydrazone form 3b (Ki (CDK9T1)5b,6a,7a= 5.98, 7.65, 6.49 nM vs Ki
(CDK9T1)3b= 9.87 nM), (Ki (CDK2E)5b,6a,7a= 25.77, 14.14,19.57 nM vs Ki
(CDK2E)3b= 43.63 nM) and (Ki (CDK1B)5b,6a,7a= 0.59, 0.77, 0.35 nM vs Ki
(CDK1B)3b= 0.84 nM, respectively).
N-acetyl pyrazoline derivative 7b containing 3,4-dimethoxyphenyl group
exhibited better CDK1, 2 and 9 than the N-phenylpyrazoline 5c and N-
unsubstituted compound 6b. (Ki (CDK9,1,2)7b= 4.59, 0.40, 10.68 nM vs Ki
(CDK9,1,2)5c= 7.89, 0.88, 16.56 nM and Ki (CDK9,1,2)6b= 6.78, 0.63, 16.25 nM,
respectively).
Moreover, N-phenylpyrazoline derivative 6c containing 4-dimethylamino-
phenyl moiety displayed higher inhibiting CDK1B and CDK9T1 activities (Ki =
0.54, 3.46 nM, respectively) than the N-acetylpyrazoline 7c (Ki (CDK1, 9) = 0.59
and 6.79 nM, respectively) and N-unsubstituted congener 5d (Ki (CDK1,9)= 0.76,
7.09 nM, respectively).
N-acetyl pyrazoline series 7a-c generally displayed the best CDK1B inhibiting
effect (Ki= 0.35, 0.40 and 0.59 nM respectively) with compound 7a revealing the
most potent CDK1B inhibiting activity (Ki= 0.35 nM). 3,4-Dimethoxyphenyl-N-
acetyl pyrazoline 7b showed exceptionally the highest CDK2E inhibiting
properties (Ki= 10.68 nM) while the 2-chlorophenyl 7a and 4-
7

dimethylaminophenyl 7c derivatives, showed the best CDK6D3 inhibiting
properties (Ki= 13.40 and 13.63 nM, respectively).
The effect of 4-dimethylaminophenyl substituent on improving the CDK9T1
inhibiting properties was clearly observed for the most potent CDK9T1 inhibitor
hydrazone compound 3d (Ki= 3.26 nM), the N-phenylpyrazoline 6c (Ki= 3.46 nM)
and the aminopyridylcarbonitrile compound 8c (Ki= 4.68 nM).
3,4-Dimethoxyphenyhydrazone 3c and its N- acetylpyrazoline congener 7b in
addition to 2-chlorophenyl-aminopyridinecarbonitrile 8a demonstrated comparable
high CDK9T1 activity (Ki= 4.56, 4.59 and 4.38nM, respectively).
Table 1
2.2.2. In vitro anticancer activity:
All the target compounds were screened for their anticancer activity on
eighteen different types of human cancer cell lines using MTT assay according to
the Mosmann’s method [45] (Tables 2a,b).
Generally ten compounds showed remarkable activity when tested against
MCF-7 breast cancer cell line (IC50=0.21-0.56 nM). The hydrazone 3a (IC50=0.21
nM), N-phenylpyrazoline 6b (IC50=0.21 nM) and aminopyridine carbonitrile 8c
(IC50=0.23 nM) were the most potent anticancer agents; they exhibited significant
potency on a low nanomolar value. The hydrazone 3a was about 2.5 fold more
active than 2-chloro derivative 3b (IC50=0.54 nM) while a dramatic drop in the
activity was observed for the 3,4-dimethoxyphenyl 3c and 4-dimethylaminophenyl
3d derivatives as well as the cyclized pyrazoline congener 5a (IC50= 14, 67 and 23
nM, respectively). Moreover, the 3,4-dimethoxyphenyl substituted N-
phenylpyrazoline 6b showed a comparable potency to that of 4-
dimethylaminophenyl substituted derivative 6c (IC50=0.21 and 0.34 nM,
respectively). It was about 2.5 fold more active than its N-acetylpyrazolyl
8

derivative 7b (IC50=0.56 nM) while a sharp decrease in activity was demonstrated
by the N-unsubstituted pyrazoline compound 5c (IC50=54 nM).
Furthermore, the 3,4-dimethoxyphenyl hydrazone compound 3c exhibited an
outstanding potency on a low nanomolar value when tested against K562
Leukemia cell line being the most active agent among all the synthesized
compounds (IC50=0.07nM). It was about 10 fold more active than the 2-chloro-
phenylhydrazone 3b (IC50=0.8 nM). The 4-dimethylaminophenyl hydrazone 3d
showed a single digit nanomolar IC50 value towards the two subtypes of Leukemia
cell lines K562and HL60 (IC50= 4.0 and 7.6 nM, respectively). Furthermore, the
pyrazoline compounds 5b-d and 6a demonstrated a significant anticancer effect
against K562 Leukemia cell line (IC50=0.83, 0.55, 0.44 and 0.72 nM, respectively).
Both N-acetylpyrazolines 7a-c and 2-aminopyridine-3-carbonitriles 8a-c
displayed a pronounced anticancer activity against A549 lung and MCF-7 breast
carcinoma cell lines (IC50 (A549)7,8a-c= 0.35-0.55 nM and IC50 (MCF-7)7,8a-c= 0.23-
0.56 nM, respectively).
While the 4-dimethylaminophenyl-N-phenylpyrazoline 6c exerted the best
anticancer effect against A549 lung carcinoma cell line (IC50=0.33nM), the N-
unsubstituted pyrazoline 5a and the 2-aminopyridine-3-carbonitriles 8b and 8c
displayed a remarkable anticancer activity against the other subtype of lung
carcinoma cell line H460 (IC50=0.69, 0.44 and 0.54 nM, respectively).
The effect of the hydrazone moiety on anticancer activity was clearly observed
against the HeLa cell line where the hydrazone series 3a-d demonstrated the
lowest IC50 values among all the synthesized compounds (IC50= 2.3-11.0 nM).
The 3,4-dimethoxyphenyl-2-aminopyridine-3-carbonitrile derivative 8b
demonstrated a single digit nanomolar IC50 value when tested against HT29 colon
cancer cell line (IC50= 3.0 nM). While, the N- unsubstitued pyrazolines 5b and 5c
and the 2-aminopyridine-3-carbonitrile derivative 8c showed the best anticolon
9

cancer effects against HCT-116 p53wtRb cell line (IC50= 4.4, 3.7 and 3.4 nM,
respectively), the N- acetylpyrazoline 7c and the 2-aminopyridine-3-carbonitrile 8a
displayed remarkable equipotent effects against HCT-116 p53null Rb cell line
(IC50= 6.2 and 7.1 nM, respectively).
All the tested compounds presented exceptionally high anticancer potency
against SKOV-3 ovarian cell line (IC50=0.24- 5.4 nM) with the 3,4-
dimethoxyphenyl and 4-dimethylaminophenylhydrazones 3c and 3d giving rise to
IC50 values in the low nanomolar range (IC50= 0.24, 0.34 nM, respectively). On the
other hand the phenylhydrazone 3a and 2-chlorophenylhydrazone 3b were the best
anti-A2780 ovarian cancer agents (IC50= 94.0, 52.0 nM, respectively).
Additionally, all the synthesized compounds showed anticancer activity
against two subtypes of prostate cancer cell lines DU145 (IC50= 0.33- 0.76 nM)
and LANCaP (IC50= 0.07- 0.98 nM) in low nanomolar concentrations. The 3,4-
dimethoxyphenyl-N-acetylpyrazoline 7b displayed the highest potency against
LANCaP (IC50= 0.07 nM), while pyrazoline 5d and N-phenylpyrazoline 6c
carrying the 4-dimethylaminophenyl group were the most active anticancer
compounds against DU145 cell line (IC50= 0.33 and 0.34 nM, respectively).
All the synthesized compounds showed anticancer activity > 1000 nM when
tested against Hep G2 hepatic cell line and Miapaca-2 pancreatic cell line and all
of them were completely inactive agents both renal carcinoma cell lines CAK1-1
and IGROV-1.
Table 2a
Table 2b
2.2.3. In vivo anticancer activity:
Lead compounds 3a, 6b and 8c were evaluated in MCF-7 breast cancer mouse
xenograft model [46]. Significant inhibition of tumor growth in vivo was observed
with compounds 3a, 6b and 8c. Representative testing results are shown in
10

figure3. Selected compounds were shown to inhibit tumor growth in mice bearing
MCF-7 xenograft tumor with high potency and efficacy at10 mg/kg dose.
Figure3
2.3. Molecular Modeling Study:
Cyclin dependent kinases are overactive in many types of cancer. In fact, this
depends on the overexpression of some of them according to the cancer type [47-
53]. To date, there is no inhibitor with broad-spectrum activity against all types of
CDKs that may refer to the difference in binding site geometry and/or sequence
identity. In a trial to find out the percentage of identity between the sequences of
CDK1B and CDK2E that were reported to be overexpressed in breast cancer [49],
sequence alignment was used and 64.90% identity was indicated (Figure 4).
Figure 4
An alignment between the sequences of CDK9, CDK7, CDK1B, CDK2 and
CDK6 was done in order to identify the percentage of similarity and/or identity.
All sequences were downloaded as fasta format from uniprot.org with uniprot IDs;
P50750, P50613, P24033, P24941 and Q00534 for CDK9, CDK7, CDK1B, CDK2
and CDK6 respectively. As a result, the percentage of identity was found 17.31 %
which is very low. Low percentage of identity means that these CDKs have
dissimilar pockets and will not respond to inhibitors in a similar way. So, when an
inhibitor is highly active and selective towards one of the previously mentioned
CDKs, this is not a guarantee that it will display activity on the other CDKs as
well. For example, compound 7a showed the highest activity in inhibition of
CDK1B (Ki = 0.35 nM) but, was not the best inhibitor of the other CDKs.
Figure 5
11

2.3.1. Molecular docking
In order to interpret the biological results, a molecular modeling study was
done starting from the previous alignment that confirms the high identity between
CDK1B and CDK2E. The second step of this study was a molecular docking study
in which the crystal structure of CDK1B was downloaded from protein data bank
with pdb code = 4y72 which possesses a {[(2,6-difluorophenyl) carbonyl]
amino}-N-(4-fluorophenyl)-1H-pyrazole-3-carboxamide inhibitor in complex
with the active site of CDK1B (Figure 6).
Figure 6
The structure of this inhibitor shared common features with compound 7a.
Therefore, it gave some insights into the activity of our synthesized compounds
especially those with substituted pyrazole ring and having potent activity against
CDK1B like compound 7a. The binding mode of this inhibitor was compared to
that of compound 7a (the most active CDK1B inhibitor in the series) to find out the
difference in the mode of binding of both compounds.
The molecular docking results showed that both compounds shared an
intramolecular hydrogen bond that may be a reason for their stability within the
active site. In addition, both have a halo substituted phenyl ring with the reported
inhibitor having two flouro atoms while 7a had only one chloro atom, which
means that increasing the halo atoms in this ring may increase the activity. Both
compounds interacted with Leu 83 residue and both of them have another aromatic
ring important for hydrophobic interactions (Figure 7).
Figure 7
12

The best binding poses of the active compounds 7a, 3a, 6b and 8c are shown in
Figure 8. It was confirmed that orientation of the halo substituted ring and position
of pyrazole ring in both the reported inhibitor and compound 7a was the same.
Compounds 3a, and 8c shared a hydrogen bond formed by their hydroxyl groups
with Gln 132. The binding mode of the other compounds was also predicted by the
docking in which, compounds 3a, 3b, 3c and 3d that have a shared conjugated two
imine groups showed common interactions with Ile 10, Lys 33 and Gln 132.
Compounds 5a, 5b, 5c, and 5d had a common hydrogen bond between their NH of
the pyrazole ring and Asn 133 and a hydrogen bond between –OH of the coumarin
ring and Leu 83. Compounds 6a, 6b, and 6c showed hydrophobic interactions by
their phenyl rings and Ala 145 in addition to other hydrophobic interactions
between the coumarin ring and Val 18. Compounds 7a, 7b, and 7c with acetylated
pyrazole ring showed common hydrophobic interactions with Phe 80. Compound
7a had the best binding and achieved the best docking score. Compounds 8a, 8b
and 8c with substituted pyridine ring showed common interactions by forming
hydrogen bond with Leu 83 and –OH, in addition to hydrophobic interaction with
Phe 82. The docking scores of 7a, 7b, and 7c (acetylated pyrazole derivatives)
were -13.75, -13.45, and -13.25 (kcal/mol), respectively, that are very close to that
of the reported pyrazole inhibitor (-14.50 kcal/mol).The docking results are
summarized in table 3.The binding mode of flavopiridol as well was identified and
was found to bind by different interactions in which it showed two hydrogen bonds
with Lys 143 and Glu 81 by its β-hydroxyl group (Figure 8).
Figure 8
Table 3
13

2.4. QSAR study
The main aim of the quantitative structure activity relationship study was to
identify the properties to which the biological activity may be correlated. The best
way was to calculate different types of descriptors either 2D or 3D. Upon
measuring each set of descriptors (a sum of 151 descriptors of 2D type and 85 of
3D descriptors) a correlation coefficient (R2) is calculated. This correlation
coefficient (R2) is an indication of the predictive quality of the model. A QSAR
model with (R2) more than 0.6 is considered of high accuracy. If a model is created
with such good (R2) value, it means that this set of descriptors is responsible for the
biological activity. In addition, a QSAR model with a high predictive value can be
used in further prediction of the activity of unknown compounds in the future.
In this work the subdivided surface area 2D descriptors showed the highest R2
value (0.81) and the lowest RMSE (0.01) compared to the other types of
descriptors. The predictive power of the subdivided surface area descriptors was
high enough to get a precise predicted PKi values with minimal residuals (Figure
9). The correlation between the PKi (biological value) and the predicted PKi was a
linear correlation. Partial least square analysis was done during this process to
ensure the accuracy of the model.
Figure 9
3. Conclusion
A new series of 3-substituted-4-hydroxycoumarin derivatives was designed,
synthesized, and evaluated for CDK inhibiting and anticancer activities. All the
synthesized target compounds selectively inhibited CDK1B in a low nanomolar
range (Ki= 0.35-0.88 nM vs Kiflav=13 nM). Most of them displayed remarkable
inhibiting effect against CDK9T1 compared to the reference standard (Ki= 3.26-
14

23.45 nM vs Kifalv= 3 nM). Moreover, all the synthesized compounds were tested
in vitro against eighteen types of human cancer cell lines. The hydrazone 3a, N-
phenylpyrazoline 6b and 2-aminopyridine-3-carbonitrile 8c were the most potent
anticancer agents against MCF-7 breast cancer cell line (IC50=0.21, 0.21 and 0.23
nM, respectively). Moreover, the lead compounds 3a, 6b and 8c showed in vivo
efficacy in mice bearing MCF-7 xenograft tumors at 10 mg/kg dose. Molecular
docking study identified the best predicted binding mode for all the active
compounds. While QSAR study revealed that the subdivided surface area
descriptors were directly correlated with the inhibitory activity with high predictive
power (R2 = 0.81).
4. Experimental
4.1. Chemistry
Melting points were determined on Electrothermal IA 9000 apparatus and were
uncorrected. Elemental analyses were carried out at the Micro-analytical
Laboratory, Central Services Laboratory, Faculty of Science, and Cairo University,
Egypt. Infrared (IR) spectra were recorded using Nexus 670 FT-IR FT-Raman
spectrometer as potassium bromide discs at the National Research Center, Dokki,
Cairo, Egypt. The proton nuclear magnetic resonance (1H NMR) spectra were
determined on Bruker 600 MHz at King Abdulaziz University, Jedaha, KSA and
on Varian Mercury 300 MHz spectrometer at Faculty of Science, Cairo University,
Cairo, Egypt, using tetramethylsilane (TMS) as the internal standard. The mass
spectra were performed on GCMS-QP 2010 Plus Shimadzu Gas Chromatograph
Mass Spectrometer at Faculty of Science, Cairo University, Egypt. The reactions
were followed by TLC (silica gel, aluminum sheets 60 F254, Merck) using
chloroform-methanol (9.5:0.5 v/v) as eluent and sprayed with iodine-potassium
15

iodide reagent. The purity of the newly synthesized compounds was assessed by
TLC and elemental analysis.
3-Acetyl-4-hydroxy-2H-chromen-2-one 1
Compound 1 was prepared according to the reported method [40].
1-(1-(4-Hydroxy-2-oxo-2H-chromen-3-yl)ethylidene) hydrazine 2
Compound 2 was prepared according to the reported method [40].
Preparation of 2-arylidene-1-(1-(4-hydroxy-2-oxo-2H-chromen-3-yl) ethyl-
idene hydrazine 3a-d
To a solution of 3-acetyl-4-hydroxycoumarin 1(1 g, 5 mmol) in ethyl alcohol
(10 ml) the appropriate aromatic aldehyde, namely, benzaldehyde, 2-chloro-
benzaldehyde, 3,4-dimethoxybenzaldehyde and/ or 4-dimethylaminobenzaldehyde
(5 mmol) was added in the presence of acetic acid (1 mL) and the reaction mixture
was refluxed for 4-6 hours. The solution was cooled and the formed precipitate
was filtered washed with ethanol and recrystallized from acetic acid to yield the
target compounds 3a [40], 3b, 3c and 3d [40].
2-(2-Chlorobenzylidene)-1-(1-(4-hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)
hydrazine 3b
Yield 90%; mp 230-2°C; mol wt 340.76; IR (cm-1, KBr): 3424 (OH), 3074 (CH
aromatic stretching), 2923 (CH aliphatic stretching), 1707 (C=O, α-pyrone), 1606
(C=N). 1H NMR (DMSO-d6, δ, ppm): 3.01 (3H, s, CH3), 7.32−8.14 (9H, m, Ar−H
and OH), 8.95 (1H, s, CH=N). MS m/z (RA %): 340/342 (M+/ M++2) (19.9/ 6.98
%), 202 (100%). Anal.calcd. for C18H13ClN2O3: C, 63.44; H, 3.85; N, 8.22, found:
C, 64.04; H, 4.05; N, 8.29.
2-(3,4-Dimethoxybenzylidene)-1-(1-(4-hydroxy-2-oxo-2H-chromen-3-yl)
ethylidene) hydrazine 3c
Yield 93%; mp 220-2°C; mol wt 366.37; IR (cm-1, KBr): 3436 (OH), 3078 (CH
aromatic stretching), 2924, 2841 (CH aliphatic stretching), 1695 (C=O, α-pyrone),
16

1
1606 (C=N). H NMR (DMSO-d6, δ, ppm): 2.99 (3H, s, CH3), 3.85 (3H, s,
OCH3), 3.86 (3H, s, OCH3), 7.11−8.10 (8H, m, Ar−H and OH), 8.68 (1H, s,
CH=N). MS m/z (RA %): 366(M+) (100%). Anal. calcd. for C20H18N2O5: C,
65.57; H, 4.95; N, 7.65, found: C, 66.01; H, 4.99; N, 7.77.
Preparation of 1-(4-hydroxy-2-oxo-2H-chromen-3-yl)-3-aryl-2-propen-1-ones
4a-d
The chalcone compounds 4a, 4b, 4c and 4d were prepared according to the
reported methods [41-43].
Preparation of 3-(5-aryl-4,5-dihydro-1H-pyrazol-3-yl)-4-hydroxy-2H-chromen-
2-one derivatives 5a-d
A mixture of the appropriate chalcones 4a-d (1 mmol) and hydrazine hydrate
99% (2 mmol) in ethyl alcohol (30 mL) was refluxed for 1 hour. The reaction
mixture was cooled and the formed precipitate was filtered off, washed with
ethanol and recrystallized from methyl alcohol to give the target compounds 5a
[41], 5b, 5c and 5d.
3-(5-(2-Chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)-4-hydroxy-2H-chromen-
2-one 5b
Yield 88%; mp 268-70°C; mol wt 340.76; IR (cm-1, KBr): 3429 (OH), 3201
1
(NH), 3089 (CH aromatic stretching), 1663 (C=O, α-pyrone), 1609 (C=N). H
NMR (DMSO-d6, δ, ppm): 3.41-3.43 (1H, dd, H-pyrazoline), 4.13- 4.21 (1H, dd,
H-pyrazoline), 5.12-5.18 (1H, dd, H-pyrazoline), 7.19−8.11 (9H, m, Ar−H+ NH-
pyrazoline), 13.7 (1H, s, OH, D2O exchangeable). MS m/z (RA %): 340/ 342(M+/
M++2) (19.13/ 6.83 %), 235 (100%). Anal. calcd. for C18H13ClN2O3: C, 63.44; H,
3.85; N, 8.22, found: C, 62.99; H, 3.79; N, 8.20.
17

3-(4,5-Dihydro-5-(3,4-dimethoxyphenyl)-1H-pyrazol-3-yl)-4-hydroxy-2H-
chromen-2-one 5c
Yield 87%; mp 224-6 °C; mol wt 366.37; IR (cm-1, KBr): 3426 (OH), 3181 (NH),
3002 (CH aromatic stretching), 2933 (CH aliphatic stretching), 1675 (C=O, α-
1
pyrone), 1606 (C=N). H NMR (DMSO-d6, δ, ppm): 3.72 (3H, s, OCH3), 3.75
(3H, s, OCH3), 3.46-3.48 (1H, dd, H-pyrazoline), 4.02-4.07 (1H, dd, H-
pyrazoline), 4.76-4.81 (1H, dd, H-pyrazoline), 7.12 (1H, s, NH, D2O
exchangeable), 6.92−7.93 (7H, m, Ar−H), 13.34 (1H, s, OH, D2O exchangeable).
MS m/z (RA %): 366 (M+) (82%), 229(100%). Anal. calcd. for C20H18N2O5: C,
65.57; H, 4.95; N, 7.65, found: C, 65.87; H, 5.01; N, 7.88.
3-(5-(4-(Dimethylamino)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)-4-hydroxy-2H-
chromen-2-one 5d
Yield 88%; mp 216-8 °C; mol wt 349.38; IR (cm-1, KBr): 3423(OH), 3195 (NH),
3089 (CH aromatic stretching), 2917(CH aliphatic stretching), 1670 (C=O, α-
1
pyrone), 1608 (C=N). H NMR (DMSO-d6, δ, ppm): 2.85 (6H, s, 2CH3), 3.32-
3.41 (1H, dd, H-pyrazoline), 3.95-4.04 (1H, dd, H-pyrazoline), 4.72- 4.78 (1H, dd,
H-pyrazoline), 7.12 (1H, s, NH, D2O exchangeable), 6.69−7.95 (8H, m, Ar−H),
13.34 (1H, s, OH, D2O exchangeable). MS m/z (RA %): 349 (M+) (100%). Anal.
calcd. for C20H19N3O3: C, 68.75; H, 5.48; N, 12.03, found: C, 68.99; H, 5.78; N,
12.10
Preparation of 3-(5-aryl-4,5-dihydro-1-phenyl-1H-pyrazol-3-yl)-4-hydroxy-
2H-chromen-2-one derivatives 6a-c
A mixture of the selected chalcones 4b-d (1 mmol) and phenylhydrazine (0.1
mL, 1 mmol) in ethyl alcohol (30 mL) was refluxed for 2 hours. The reaction
mixture was cooled and the formed precipitate was filtered off, washed with
ethanol and recrystallized from acetic acid to give the desired compounds 6a, 6b
and 6c [43].
18

3-(5-(2-Chlorophenyl)-4,5-dihydro-1-phenyl-1H-pyrazol-3-yl)-4-hydroxy-2H-
chromen-2-one 6a
Yield 94%; mp 220-2°C; mol wt 416.86; IR (cm-1, KBr): 3399 (OH), 3056 (CH
aromatic stretching), 1711 (C=O, α-pyrone), 1595 (C=N). 1H NMR (DMSO-d6, δ,
ppm): 3.41-3.43 (1H, dd, H-pyrazoline), 4.27-4.35 (1H, dd, H-pyrazoline), 5.66-
5.71 (1H, dd, H-pyrazoline), 6.86−8.05 (13H, m, Ar−H), 13.70 (1H, s, OH, D2O
exchangeable). MS m/z (RA %): 416/418 (M+/ M++2) (41.97/15.42%), 305
(100%). Anal. calcd. for C24H17ClN2O3: C, 69.15; H, 4.11; N, 6.72, found: C,
68.98; H, 4.04; N, 6.60.
3-(4,5-Dihydro-5-(3,4-dimethoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)-4-
hydroxy-2H-chromen-2-one 6b
°
Yield 96%; mp 183-5 C; mol wt 442.46; IR (cm-1, KBr): 3431 (OH), 3065 (CH
aromatic stretching), 2995 (CH aliphatic stretching), 1721 (C=O, α-pyrone), 1607
1
(C=N). H NMR (DMSO-d6, δ, ppm): 3.56-3.59 (1H, dd, H-pyrazoline), 3.85
(3H, s, OCH3), 3.87 (3H, s, OCH3), 4.23-4.26 (1H, dd, H-pyrazoline), 5.12-5.14
(1H, dd, H-pyrazoline), 6.83−8.05 (12H, m, Ar−H), 14.07 (1H, s, OH, D2O
exchangeable). MS m/z (RA%): 442(M+) (32.3%), 80 (100%). Anal. calcd. for
C26H22N2O5: C, 70.58; H, 5.01; N, 6.33, found: C, 70.68; H, 5.04; N, 6.40.
Preparation of 3-(1-acetyl-5-aryl--4,5-dihydro-1H-pyrazol-3-yl)-4-hydroxy-
2H-chromen-2-ones 7a-c
A mixture of chalcones 4b-d (1 mmol) and hydrazine hydrate 99% (2 mmol) in
glacial acetic acid (30 mL) was refluxed for 2-3 hours. The reaction mixture was
cooled and diluted with water; the formed precipitate was filtered off, washed with
water and recrystallized from ethyl alcohol to give compounds 7a-c.
19

3-(1-Acetyl-5-(2-chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)-4-hydroxy-2H-
chromen-2-one 7a
Yield 88%; mp 143-5°C; mol wt 382.8; IR (cm-1, KBr): 3414 (OH), 3066 (CH
aromatic stretching), 2920 (CH aliphatic stretching), 1718 (C=O, α-pyrone), 1670
1
(C=O, acetyl), 1617 (C=N). H NMR (DMSO-d6, δ, ppm): 2.36 (3H, s, COCH3),
3.96-4.01 (1H, dd, H-pyrazoline), 4.07-4.14 (1H, dd, H-pyrazoline), 5.66-5.70 (1H,
dd, H-pyrazoline), 6.74−8.01 (9H, m, Ar−H and OH). MS m/z (RA%): 382/ 384
(M+/ M++2) (15.46/4.24 %), 229 (100%). Anal. calcd. for C20H15ClN2O4: C, 62.75;
H, 3.95; N, 7.32, found: C, 62.68; H, 3.89; N, 7.23.
3-(1-Acetyl-4,5-dihydro-5-(3,4-dimethoxyphenyl)-1H-pyrazol-3-yl)-4-hydroxy-
2H-chromen-2-one 7b
Yield 87%; mp 204-6°C; mol wt 408.4; IR (cm-1, KBr): 3435 (OH), 3067 (CH
aromatic stretching), 2997, 2939 (CH aliphatic stretching), 1719 (C=O, α-pyrone),
1
1632 (C=O, acetyl), 1562 (C=N). H NMR (DMSO-d6, δ, ppm): 2.33 (3H, s,
COCH3), 3.74 (3H, s, OCH3), 3.76 (3H, s, OCH3), 3.40-342 (1H, dd, H-
pyrazoline), 3.96-4.04 (1H, dd, H-pyrazoline), 5.43-5.47 (1H, dd, H-pyrazoline),
6.74−8.04 (8H, m, Ar−H and OH). MS m/z (RA %): 408 (M+) (29.1%),
229(100%). Anal. calcd. for C22H20N2O6: C, 64.70; H, 4.94; N, 6.86, found: C,
64.78; H, 4.99; N, 6.90.
3-(1-Acetyl-5-(4-(dimethylamino)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)-4-
hydroxy-2H-chromen-2-one 7c
Yield 86%; mp 170-2°C; mol wt 391.42; IR (cm-1, KBr): 3415 (OH), 3088 (CH
aromatic stretching), 2923, 2856 (CH aliphatic stretching), 1719 (C=O, α-pyrone),
1
1610 (C=O, acetyl), 1567 (C=N). H NMR (DMSO-d6, δ, ppm):1.89 (3H, s,
COCH3), 2.80 (6H, s, 2CH3), 3.68-3.88 (1H, dd, H-pyrazoline), 4.42-4.55 (1H, dd,
H-pyrazoline), 5.32-5.33 (1H, dd, H-pyrazoline), 6.47−7.98 (9H, m, Ar−H and
20

OH). MS m/z (RA %): 391 (M+) (6.9%), 229(100%). Anal. calcd. for
C22H21N3O4: C, 67.51; H, 5.41; N, 10.74, found: C, 67.58; H, 5.44; N, 10.80.
Preparation of 2-amino-4-aryl-6-(4-hydroxy-2-oxo-2H-chromen-3-yl)-pyridin-
3-carbonitriles (8a-c)
To a mixture of 3-acetyl-4-hydroxycoumarin 1 (1 g, 5 mmol) and the
appropriate aldehyde, namely; 2-chlorobenzaldehyde, 3,4-dimethoxybenzaldehyde
or 4-dimethylaminobenzaldehyde (5 mmol) in ethyl alcohol (20 mL), malononitrile
(0.33 g, 5 mmol) and ammonium acetate (0.75 g, 10 mmol) were added. The
reaction mixture was refluxed for 7–9 hours. The obtained solid was filtered off,
washed with ethyl alcohol and recrystallized from DMF/H2O to give the desired
compounds 8a, 8b and 8c [54].
2-Amino-4-(2-chlorophenyl)-6-(4-hydroxy-2-oxo-2H-chromen-3-yl) pyridine-
3-carbonitrile 8a
Yield 90%; mp ˃300°C; mol wt 389.79; IR (KBr, cm-1): 3343 (OH), 3229, 3182
(NH2), 3062 (CH aromatic stretching), 2212 (C≡N), 1694 (C=O, α-pyrone), 1630
(C=N).1HNMR (DMSO-d6, d, ppm): 7.32–8.01 (8H, m, Ar-H), 8.03 (1H, s, 5H-
pyridine), 8.63 (2H, s, NH2, D2O exchangeable), 16.77 (1H, s, OH,D2O
exchangeable). MS m/z (RA %): M+/ M++2 (389/ 391) (89.5/ 32.3%), 121(100%).
Anal. calcd. for C21H12ClN3O3: C, 64.71; H, 3.10; N, 10.78, found: C, 64.79; H,
3.15; N, 10.82.
2-Amino-6-(4-hydroxy-2-oxo-2H-chromen-3-yl)-4-(3,4-dimethoxyphenyl)
pyridine-3-carbonitrile 8b
Yield 96%; mp ˃300°C; mol wt 415.40; IR (KBr, cm-1): 3467(OH), 3324,3227
(NH2), 3088 (CH aromatic stretching), 2923 (CH aliphatic stretching), 2202
(C≡N), 1634 (C=O, α-pyrone), 1603 (C=N).1HNMR (DMSO-d6, d, ppm): 3.83
(3H, s, OCH3), 3.85 (3H, s, OCH3),7.16–8.02 (7H, m, Ar-H), 8.21 (1H, s, 5H-
pyridine), 8.46 (2H, s, NH2,D2O exchangeable), 17.7 (1H, s, OH, D2O
21

exchangeable). MS m/z (RA %): M+ (415) (100%). Anal. calcd. for C23H17N3O5:
C, 66.50; H, 4.12; N, 10.12, found: C, 66.44; H, 4.04; N, 10.05.
4.2. Biology
4.2.1. Kinase assay
Inhibition of CDKs was measured by radiometric assay using the Millipore
Kinase Profiler services. Half-maximal inhibition (IC50) values were calculated
from 10-point dose–response curves and apparent inhibition constants (Ki) were
calculated from the IC50 values and appropriate Km (ATP) values for the kinases in
question [44].
4.2.2. MTT toxicity assays
The cytotoxicity of the newly synthesized compounds against cancer cell lines in
vitro was performed with the MTT assay according to the Mosmann’s method
[45]. The MTT assay is based on the reduction of the soluble 3-(4,5-methyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) into a blue-purple
formazan product, mainly by mitochondrial reductase activity inside living cells.
The cells used in cytotoxicity assay were cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum. Cells suspended in the medium (2 ×
104/mL) were plated in 96-well culture plates and incubated at 37◦C in a 5% CO2
incubator. After 12 hours, the test sample (2 μL) was added to the cells (2Ý 104) in
96-well plates and cultured at 37◦C for 3 days. The cultured cells were mixed with
20 μL of MTT solution and incubated for 4 hours at 37◦C. The supernatant was
carefully removed from each well and 100 μL of DMSO were added to each well
to dissolve the formazan crystals which were formed by the cellular reduction of
MTT. After mixing with a mechanical plate mixer, the absorbance of each well
was measured by a microplate reader using a test wavelength of 570 nm.
22

The results were expressed as the IC50, which induced a 50% inhibition of
treated cell growth when compared to the growth of control cells. Each experiment
was performed at least 3 times. There was a good reproducibility between replicate
wells with standard errors below 10%.
4.2.3. Human breast cancer xenograft models and animal treatment
To establish MCF-7 human breast cancer xenografts, female athymic
pathogen-free nude mice (nu/nu, 4-6 weeks) were first implanted with a 60-day sc
slow release estrogen pellet (SE-121, 1.7mg 17β-estradiol/pellet; Innovative
Research of America, Sarasota, FL). The next day, cultured MCF-7 cells were
harvested from confluent monolayer cultures, washed twice with serum-free
medium, resuspended and injected subcutaneously (s.c.) (5 × 106 cells, total
volume 0.2mL) into the left inguinal area of the mice. All animals were monitored
for activity, physical condition, body weight, and tumor growth. Tumor size was
determined by caliper measurement in two perpendicular diameters of the implant
every other day. Tumor weight (in g) was calculated by the formula, 1/2a × b2
where “a” is the long diameter and “b” is the short diameter (in cm). The animals
bearing human cancer xenografts were randomly divided into various treatment
groups and a control group (7-10 mice/group). The untreated control group
received the vehicle only. For the MCF-7 xenograft model, tested compounds was
dissolved in PEG400: ethanol: saline (57.1: 14.3: 28.6, v/v/v), and was
administered by intraperitoneal (i.p.) injection at a dose of 10 mg/kg/d, 3d/wk for 3
weeks [46].
4.3. Molecular docking
Molecular Operating Environment (MOE) 2015.10 was used for all modeling
studies [55]. The synthesized compounds were built by MOE builder and saved as
23

moe. The docking protocol was set as rigid receptor. Both receptor-solvent were
kept as a ‘’receptor’’. The placement method was selected to be ‘’Triangle
matcher’’. Two rescoring were computed, rescoring 1 was selected as London dG.
Rescoring 2 was selected as affinity. Force field was used as a refinement.
4.4. QSAR study
In the QSAR study, a total 151 2D descriptors and 85 3D descriptors supplied
by MOE 2015.10 were computed for each compound in the training set. In order to
validate the predictive power of the generated models this set was used. The used
2D descriptors included physical properties, subdivided surface area, atom counts
and bon counts, Kier & Hall connectivity and Kapper shape indices,
pharmacophore feature descriptors and Partial charges. The 3D descriptors
included potential energy descriptors, MOPAC, surface area, volume and shape
descriptors and conformation dependent charge descriptors. Partial least square
analysis (PLS) was used as a statistical method for deriving the significance of the
models. pKi values were calculated for all of the Ki values using the following
equation:
pKi = -log (Ki)
The correlation coefficient (R2) and root mean square error (RMSE) values
were calculated for each set of descriptors to validate the model power. MSE was
calculated using the following equation for linear model:
5. Abbreviations used
CDK; cyclin dependent kinase, QSAR; quantitative structure activity
relationship, AP-1; activator protein-1, RMSE; root mean square error, TLC; thin
24

layer chromatography, DMSO; dimethylsulfoxide, MOPAC; molecular orbital
PACkage.
6. References
[1]Lapenna, S., Giordano, A., Cell cycle kinases as therapeutic targets for
cancer, Nat. Rev. Drug. Discov. 8 (2009) 547-566.
[2]Grana, X., Reddy, E. P., Cell cycle control in mammalian cells: role of
cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and
cyclin-dependent kinase inhibitors (CKIs), Oncogene 11 (1995) 211-219.
[3]Morgan, D. O., Principles of CDK regulation, Nature 374 (1995) 131-134.
[4]Sherr, C. J., Cancer cell cycles, Science 274 (1996) 1672-1777.
[5]Golsteyn, R. M., Cdk1 and Cdk2 complexes (cyclin dependent kinases) in
apoptosis: a role beyond the cell cycle, Cancer Lett. 217 (2005) 129-138.
[6] Shapiro, G.I., Cyclin-dependent kinase pathways as targets for cancer
treatment, J. Clin. Oncol. 24 (2006) 1770-1783.
[7] Lalioti, V., Muruais, G., Dinarina, A., van Damme, J., Vandekerckhove, J.,
Sandoval, I.V., The atypical kinase Cdk5 is activated by insulin, regulates
the association between GLUT4 and E-Syt1, and modulates glucose
transport in 3T3-L1 adipocytes, Proc. Natl. Acad. Sci. U. S. A. 106 (2009)
4249-4253.
[8]MacCallum, D. E., Melville, J., Frame, S., Watt, K., Anderson, S., Gianella-
Borradori, A., Lane, D. P., Green, S. R., Seliciclib (CYC202, R-Roscovitine)
induces cell death in multiple myeloma cells by inhibition of RNA
polymerase II-dependent transcription and down-regulation of Mcl-1,
Cancer Res. 65 (2005) 5399-5407.
[9]Fu, W., Ma, L., Chu, B., Wang, X., Bui, M. M., Gemmer, J., Altiok, S.,
Pledger, W. J., The cyclin-dependent kinase inhibitor SCH 727965
25

(dinacliclib) induces the apoptosis of osteosarcoma cells, Mol. Cancer Ther.
10 (2011) 1018-1027.
[10] Nakayama, K. I., Nakayama, K., Ubiquitin ligases: cell-cycle control and
cancer, Nat. Rev. Cancer 6 (2006) 369-381.
[11] Rudolph, J., Inhibiting transient protein-protein interactions: lessons from
the Cdc25 protein tyrosine phosphatases, Nat. Rev. Cancer 7 (2007) 202-
211.
[12] Sherr, C. J., The Pezcoller lecture: cancer cell cycles revisited, Cancer Res.
60 (2000) 3689-3695.
[13] Ogino, S., Nosho, K., Irahara, N., Kure, S., Shima, K., Baba, Y., Toyoda,
S., Chen, L., Giovannucci, E. L., Meyerhardt, J. A., Fuchs, C. S., A cohort
study of cyclin D1 expression and prognosis in 602 colon cancer cases, Clin.
Cancer Res. 15 (2009) 4431-4438.
[14] Malumbres, M., Barbacid, M., Cell cycle, CDKs and cancer: a changing
paradigm, Nat. Rev. Cancer 9 (2009) 153-166.
[15] Chen, R., Keating, M. J., Gandhi,V., Plunkett, W., Transcription inhibition
by flavopiridol: mechanism of chronic lymphocytic leukemia cell death,
Blood 106 (2005) 2513-2519.
[16] Meijer, L., Raymond, E., Roscovitine and other purines as kinase
inhibitors. From starfish oocytes to clinical trials, Accounts Chem. Res. 36
(2003) 417-425.
[17] McClue, S. J., Blake, D., Clarke, R., Cowan, A., Cummings, L., Fischer,
P.M., MacKenzie, M., Melville, J., Stewart, K., Wang, S., Zhelev, N.,
Zheleva, D., Lane, D. P., In vitro and in vivo antitumor properties of the
cyclin dependent kinase inhibitor CYC202 (R-roscovitine), Int. J. Cancer
102 (2002) 463-468.
26

[18] Guha, M., Cyclin-dependent kinase inhibitors move into Phase III, Nature
Rev. Drug Discovery 11 (2012) 892-894.
[19] Guha, M., Blockbuster dreams for Pfizer’s CDK inhibitor, Nature
Biotechnol. 31 (2013) 187.
[20] Paruch, K., Dwyer, M. P., Alvarez, C., Brown, C., Chan, T.-Y., Doll, R.
J., Keertikar, K., Knutson, C., McKittrick, B., Rivera, J., Rossman, R.,
Tucker, G., Fischmann, T., Hruza, A., Madison, V., Nomeir, A. A., Wang,
Y., Kirschmeier, P., Lees, E., Parry, D., Sgambellone, N., Seghezzi, W.,
Schultz, L., Shanahan, F., Wiswell, D., Xu, X., Zhou, Q., James, R. A.,
Paradkar, V. M., Park, H., Rokosz, L. R., Stauffer, T. M., Guzi, T. J.,
Discovery of Dinaciclib (SCH 727965): A Potent and Selective Inhibitor of
Cyclin-Dependent Kinases, ACS Med. Chem. Lett. 1 (2010) 204-208.
[21] Parry, D., Guzi, T., Shanahan, F., Davis, N., Prabhavalkar, D., Wiswell,
D., Seghezzi, W., Paruch, K., Dwyer, M. P., Doll, R., Nomeir, A., Windsor,
W., Fischmann, T., Wang, Y., Oft, M., Chen, T., Kirschmeier, P., Lees, E.
M., Dinaciclib (SCH 727965), a novel and potent cyclin-dependent kinase
inhibitor, Mol. Cancer Ther. 9 (2010) 2344-2353.
[22] Toogood, P. L., Harvey, P. J., Repine, J. T., Sheehan, D. J., Vander Wel, S.
N., Zhou, H., Keller, P. R., McNamara, D. J., Sherry, D., Zhu, T.,
Brodfuehrer, J., Choi, C., Barvian, M. R., Fry, D. W., Discovery of a potent
and selective inhibitor of cyclin-dependent kinase 4/6, J. Med. Chem. 48
(2005) 2388-2406.
[23] Jain, S. K., Bharate, S. B., Vishwakarma, R. A., Cyclin-dependent kinase
inhibition by flavoalkaloids, Mini Rev Med Chem. 12 (2012) 632-649.
[24] Kim, D. H., Na, H. K., Oh, T. Y., Shin, C. Y., Surh, Y. J., Eupatilin
inhibits proliferation of ras-transformed human breast epithelial (MCF-10A-
ras) cells, J. Environ. Pathol. Toxicol. Oncol. 24 (2005) 251-259.
27

[25] Kim, K. S., Sack, J. S., Tokarski, J. S., Qian, L., Chao, S. T., Leith, L.,
Kelly, Y. F., Misra, R. N., Hunt, J. T., Kimball, S. D., Humphreys, W. G.,
Wautlet, B. S., Mulheron, J. G., Webster, K. R., Thio- and oxoflavopiridols,
cyclin-dependent kinase 1 selective inhibitors: synthesis and biological
effects, J. Med. Chem. 43 (2000) 4126-41234.
[26] Murthi, K. K., Dubay, M., McClure, C., Brizuela, L., Boisclair, M. D.,
Worland, P. J., Mansuri, M. M., Pal, K., Structure-activity relationship
studies of flavopiridol analogues, Bioorg. Med. Chem. Lett. 10 (2000) 1037-
1041.
[27] Ahn, Y. M., Vogeti, L., Liu, C. J., Santhapuram, H. K., White, J. M.,
Vasandani, V., Mitscher, L. A., Lushington, G. H., Hanson, P. R., Powell,
D. R., Himes, R. H., Roby, K. F., Ye, Q., Georg, G. I., Design, synthesis,
and antiproliferative and CDK2-cyclin A inhibitory activity of novel
flavopiridol analogues, Bioorg. Med. Chem. 15 (2007) 702-713.
[28] Budzisz, E., Brzezinska, E., Krajewska, U., Rozalski, M., Cytotoxic
effects, alkylating properties and molecular modelling of coumarin
derivatives and their phosphonic analogues, Eur. J. Med. Chem. 38 (2003)
597-603.
[29] Chimenti, F., Bizzarri, B., Bolasco, A., Secci, D., Chimenti, P., Granese,
A., Carradori, S., Rivanera, D., Zicari, A., Scaltrito, M. M., Sisto, F.,
Synthesis, selective anti-Helicobacter pylori activity, and cytotoxicity of
novel N-substituted-2-oxo-2H-1-benzopyran-3-carboxamides, Bioorg. Med.
Chem. Lett. 20 (2010) 4922-4926.
[30] Kostova, I., Synthetic and natural coumarins as cytotoxic agents, Curr.
Med. Chem. Anticancer Agents 5 (2005) 29-46.
28

[31] Ishihara, M., Yokote, Y., Sakagami, H., Quantitative structure-cytotoxicity
relationship analysis of coumarin and its derivatives by semiempirical
molecular orbital method, Anticancer Res. 26 (2006) 2883-2886.
[32] Liu, X. H., Liu, H. F., Chen, J., Yang, Y., Song, B. A., Bai, L. S., Liu, J.
X., Zhu, H. L., Qi, X. B., Synthesis and molecular docking study of novel
coumarin derivatives containing 4,5-dihydropyrazole moiety as potential
antitumor agents, Bioorg. Med. Chem. Lett. 20 (2010) 5705-5708.
[33] Singh, R. K., Lange, T. S., Kim, K. K., Brard, L. A., Coumarin derivative
(RKS262) inhibits cell-cycle progression, causes pro-apoptotic signaling and
cytotoxicity in ovarian cancer cells, Invest. New Drugs 29 (2011) 63-72.
[34] Węsierska-Gądek, J., Maurer, M., Zulehner, N., Komina, O.,Whether to
target single or multiple CDKs for therapy? That is the question, J. Cell
Physiol. 226 (2011) 341-349.
[35] Krystof, V., Cankar, P., Frysová, I., Slouka, J., Kontopidis, G., Dzubák, P.,
Hajdúch, M., Srovnal, J., de Azevedo Jr., W. F., Orság, M., Paprskárová, M.,
Rolcík, J., Látr, A., Fischer, P. M., Strnad, M., 4-Arylazo-3,5-diamino-1H-
pyrazole CDK inhibitors: SAR study, crystal structure in complex with
CDK2, selectivity, and cellular effects, J. Med. Chem. 49 (2006) 6500-6509.
[36] Lin, R., Chiu, G., Yu, Y., Connolly, P. J., Li, S., Lu, Y., Adams, M.,
Fuentes-Pesquera, A. R., Emanuel, S. L., Greenberger, L. M., Design,
synthesis, and evaluation of 3,4-disubstituted pyrazole analogues as anti-
tumor CDK inhibitors, Bioorg. Med. Chem. Lett. 17 (2007) 4557-4561.
[37] Lin, R., Connolly, P. J., Lu, Y., Chiu, G., Li, S., Yu, Y., Huang, S., Li, X.,
Emanuel, S. L., Middleton, S. A., Gruninger, R. H., Adams, M., Fuentes-
Pesquera, A. R., Greenberger, L. M., Synthesis and evaluation of pyrazolo
[3,4-b] pyridine CDK1 inhibitors as anti-tumor agents, Bioorg. Med. Chem.
Lett. 17 (2007) 4297-302.
29