Novel 2-thiopyrimidine derivatives as CDK2 inhibitors: Molecular modeling, synthesis, and anti-tumor activity evaluation

Article abstract of DOI:10.1007/s00044-012-0051-9

A novel series of pyrimidine-benzenesulfonamide derivatives as potential cyclin-dependent kinase 2 inhibitors were designed depending upon the molecular docking simulation study. This study was preceded by modification and optimization of the lead compound 4-(2-amino-4-methylthiazol-5-yl)-N-(3- nitrophenyl) pyrimidin-2-amine. The target proposed compounds were synthesized using the derivative 6-(3,4-dimethoxyphenyl)-4-oxo-2-thioxo-1,2,3,4- tetrahydropyrimidine-5-carbonitrile (1) as a key starting compound. Some of the synthesized derivatives were selected as representative examples to evaluate their anti-proliferative activity against cultured human Hela cell line using doxorubicin as a reference drug and the results obtained were correlated with the data of molecular modeling simulation study. The structures of the novel derivatives were confirmed on the bases of micro-analytical and spectral data.

Full text of DOI:10.1007/s00044-012-0051-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:659–673
DOI 10.1007/s00044-012-0051-9
ORIGINAL RESEARCH
Novel 2-thiopyrimidine derivatives as CDK2 inhibitors: molecular
modeling, synthesis, and anti-tumor activity evaluation
•
Omar Abd El-Fattah M. Fathalla Mohamed A. H. Ismail
•
•
Manal M. Anwar Khaled A. M. Abouzid
•
Aisha A. K. Ramadan
Received: 16 September 2011 / Accepted: 3 April 2012 / Published online: 24 April 2012
ꢀ Springer Science+Business Media, LLC 2012
Abstract A novel series of pyrimidine-benzenesulfona-
mide derivatives as potential cyclin-dependent kinase 2
inhibitors were designed depending upon the molecular
docking simulation study. This study was preceded by
modification and optimization of the lead compound 4-(2-
amino-4-methylthiazol-5-yl)-N-(3-nitrophenyl) pyrimidin-
2-amine. The target proposed compounds were synthesized
using the derivative 6-(3,4-dimethoxyphenyl)-4-oxo-2-thi-
oxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1) as a
key starting compound. Some of the synthesized deriva-
tives were selected as representative examples to evaluate
their anti-proliferative activity against cultured human Hela
cell line using doxorubicin as a reference drug and the
results obtained were correlated with the data of molecular
modeling simulation study. The structures of the novel
derivatives were confirmed on the bases of micro-analyti-
cal and spectral data.
encompassing both receptor and non-receptor tyrosine
types and serine/threonine kinases which are players more
downstream in proliferative signaling and involved in the
likes of MAP kinase signaling cascades as well as cell
cycle progression and cell survival pathways (Blume-
Jensen and Hunter, 2001; Levitzki and Gazit, 1995).
Serine/threonine kinases, termed cyclin-dependent
kinases (CDKs), are activated by phosphorylation and
function in complexes with their activating cyclins (pro-
teins that are present at specific stages of the cell cycle) to
drive progression through the different phases of the cell
division cycle (Draetta, 1990; Sherr, 1993; Coleman et al.,
1997). CDK4, CDK6, coupled with their respective cyclin
D partners are responsible for progression through G1,
whereas CDK2 in combination with cyclin E is required for
normal progress from G1 into S-phase, where DNA repli-
cation takes place. CDK1 and CDK2 (in association with
cyclins A and B) are essential for cells to pass through
S-phase into G2 and mitosis (Jhonson et al., 2002; Wang
et al., 2004). CDK/cyclin complexes are kept under control
by nuclear proteins termed CDK inhibitors and exemplified
by p15, p16, p21, and p27, whose loss or inactivation
participates in the development of cancer. CDKs phos-
phorylate and regulate the activity of different cellular
proteins that include tumor suppressors (e.g., pRb, p53),
transcription factors (e.g., E2F-DPI, RNA pol II), replica-
tion factors (e.g., DNA Pol a, replication protein A), and
organizational factors, which influence cellular and chro-
matin structures (e.g., histone H1, lamin A, MAP4)
(Morgan, 1997; Grana and Reddy, 1995; Pines, 1995).
The recent understanding of the role of CDKs in the cell
cycle regulations and the discovery that high rates of
neoplasias are the result of CDK hyperactivation provide
the main impetus to search for their pharmacological
inhibitors.
Keywords Docking ꢀ Pyrimidine-benzenesulfonamides ꢀ
CDK2 ꢀ Anti-proliferative activity ꢀ Hela cell line
Introduction
Protein kinases are generally divided into two classes:
tyrosine kinases which have been identified as the upstream
players in mitogenic signal transduction pathways
O. A. E.-F. M. Fathalla ꢀ M. M. Anwar (&) ꢀ A. A. K. Ramadan
Medicinal Chemistry Department, National Research Centre,
Dokki, Cairo, Egypt
e-mail: manal.hasan52@yahoo.com
M. A. H. Ismail ꢀ K. A. M. Abouzid
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Ain Shams University, Cairo, Egypt
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Med Chem Res (2013) 22:659–673
site with other 4-(substituted pyrazolyl) moieties to study
their effects on the binding modes.
In this study, we aimed to design and synthesize a
number of novel compounds as CDK2 inhibitors having
potential anti-cancer activity. It has been reported that
many pyrimidine-bearing skeletons showed anti-cancer
activity (Arris et al., 2000; Barvian et al., 2000) and dif-
ferent drugs such as purvalanol B (I), olumoucine (IIa), and
its analogue roscovitine (IIb) that contain pyrimidine ring
are potent CDK2 inhibitors (Gray et al., 1998; Schulze-
Gahmen et al., 1995; De Azevedo et al., 1997).
•
•
•
Modification of the core thiazolylpyrimidine ring with
fused imidazo, tetrazolo, and triazolopyrimidines was
also performed aiming to study the effect of such fused
ring systems on the binding modes and their effects on
anti-proliferative activity.
Introduction of a nitrile moiety at C3 of the pyrimidine
ring as this moiety enhances CDK2 inhibition potency
(Wang et al., 2004). Such modification aims to increase
the interaction with amino acids residues in the ribose
and phosphate binding site through the hydrogen bonds.
Introduction of 3,4-dimethoxyphenyl moiety at C6 of
the pyrimidine nucleus to investigate the probability of
enhancing the pharmacokinetic properties and the
interactions of the new derivatives at the binding sites.
Different efforts had resulted in the discovery of a
number of 2-anilino-4-(thiazol-5-yl) pyrimidine derivatives
that were developed as ATP antagonistic CDK2 inhibitors
having anti-cancer activity (Wang et al., 2004), specially
4-(2-amino-4-methylthiazol-5-yl)-N-(3-nitrophenyl) pyr-
imidin-2-amine (III) which showed the best anti-cancer
activity in this series against Hela cell lines.
N
N
H2N
H
N
H
N
N
R2
N
N
Cl
S
N
N
N
N
N
HOOC
NH
NH
R1
O2N
N
H
N
HO
HO
IIa, R1= H, R2= CH3
IIb, R1= C2H5, R2= C3H7
I
III
Thus, according to the above-mentioned points and to
achieve our aim, pyrimidine nucleus was selected as the
main skeleton of the proposed derivatives and the deriva-
tive (III) was selected as our lead compound. Design of the
new CDK2 inhibitors was performed depending upon the
molecular docking simulation study which was preceded
by modification and optimization of the lead compound
(III). This could be illustrated in Fig. 1 according to the
following:
It was an important part in our study to evaluate the anti-
tumor activity of the derivatives, thus some of the newly
synthesized compounds that exhibited promising molecular
docking results were examined as anti-cancer agents
against human cervical carcinoma cell lines.
Materials and methods
Molecular docking simulation study
Aim
•
Maintenance of the pyrimidine nucleus since it forms
hydrophobic and hydrogen bond interactions with the
hinge region residues at ATP binding site of CDK2
(Ibrahim and El-Metwally, 2010).
•
Replacement of m-nitro group with a sulfamoyl moiety
(where the presence of electron-withdrawing group
preserves or enhances the activity) (Wang et al., 2004)
aiming to form hydrogen bonds and electrostatic
interactions with the residues of ATP-binding site of
CDK2. Also, maintaining the NH group is essential as
it is a hydrogen bond donor.
The aim was to study the crystal structure of CDK2 and
explain the possible interactions that might take place
between the proposed tested derivatives and the CDK2
enzyme in comparing to 4-(2-amino-4-methylthiazol-5-yl)-
N-(3-nitrophenyl) pyrimidin-2-amine as a lead compound.
Table 1 demonstrates the data obtained through the
molecular modeling simulation study on all the prepared
compounds.
•
Replacement of the thiazolyl moiety which overlaps
roughly with the space occupied by ribose in ATP-binding
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Med Chem Res (2013) 22:659–673
661
NH2
N
S
R3
Replacement of
the thiazolyl
N
moiety with different
5-membered heterocyclic
rings to be either
in attachment
Introduction of
nitrile group
N
H
N
R4
O2N
III
or fused with
lead
R3= H
R4= H
pyrimidine ring
Maintaining this arrangement
Replacement of
m-nitro with
p-sulfamoyl group
Introduction of
3,4-dimethoxyphenyl
moiety
het
N
CN
O
S
H2N
O
N
H
N
O
O
het: different 5-membered heterocyclic rings
Fig. 1 The design of new CDK2 inhibitors through the modification of the lead compound (III)
Docking studies of the tested derivatives using
Discovery Studio Client V2-5-0-9164
between the indicated amino acids and features of the
tested compounds in comparison with the lead compound.
The newly proposed compounds including the intermediate
ones were docked into the active site of CDK2 using
CDOCKER protocol, which was carried out by down-
loading the 1PXO PDB file that images the bioactive
conformer of the lead compound co-crystallized with the
CDK2. The lead compound and the tested molecules were
drawn, and underwent energy minimization using the
prepared ligand protocol. The test set was docked into the
active site of the enzyme using CDOCKER protocol. Then
the resulting poses of each molecule were examined.
Finally the retained poses were sorted by CHARMm
energy (CDOCKER interaction energy) and the top scoring
(most negative, thus favorable to binding) poses were
selected and arranged in Table 2 which exhibits the tested
molecules CDOCKER interaction energy indicating their
affinities to the binding site and the hydrogen bonding
Chemistry
All melting points were uncorrected and measured using an
Electro-thermal IA 9100 apparatus (Shimadzu, Japan).
Micro-analytical data were performed by Vario El-Mentar
apparatus (Shimadzu, Japan), National Research Centre
(NRC), Cairo, Egypt. The found values were within
0.4 % of the theoretical values. IR spectra (KBr) were
recorded on a Perkin-Elmer 1650 spectrophotometer, NRC.
1H NMR and 13C NMR spectra were determined on a
Varian Mercury (300 MHz) spectrometer (Varian, UK) and
the chemical shifts were expressed in d ppm relative to
TMS as an internal reference, Faculty of science, Cairo
University, Egypt. Mass spectra were recorded at 70 eV on
EI Ms-QP 1000 EX (Shimadzu, Japan), NRC. TLC was
performed on silica gel 60 254F plates (Merck) using a
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Med Chem Res (2013) 22:659–673
from DMF/water. Yield: 65 %; mp (ꢁC): 89–91. IR (KBr,
Table 1 Molecular modeling results of the newly designed
derivatives
cm-1): 3343 (NH), 3005 (CH, aromatic), 2930 (CH, ali-
1
cyclic), 2210 (CN), 1139 (C=S). H NMR (DMSO-d6, d
Compounds
-CDOCKER_interaction_energy
(negative values)
ppm): 3.69, 3.71 (2s, 6H, 2OCH3), 6.9–7.2 (m, 3H, aro-
matic-H) and 8.20 (s, 1H, NH exchangeable with D2O).
MS m/z: M? 314 (20 %), 274 (32 %), 177 (20 %), 111
(100 %). Anal. calcd for C13H10N6O2S (314.34): C, 49.67;
H, 3.20; N, 26.73; S, 10.20. Found: C, 49.31; H, 3.00; N,
26.41; S, 10.42.
Lead (III)
45.81
38.72
42.83
76.86
45.64
72.73
41.69
42.56
68.22
44.40
74.49
49.64
85.07
45.80
76.12
43.23
70.25
45.25
67.67
2
3
4
5
6
4-(8-Cyano-7-(3,4-dimethoxyphenyl) tetrazolo[1,5-
c]pyrimidin-5-ylamino)benzene sulfonamide (4)
7
8
9
A mixture of compound 3 (0.5 g, 1.6 mmol) and sulfanil-
amide (0.27 g, 1.6 mmol) in DMF (5 mL) containing few
drops of TEA was refluxed for 15 h. Then the mixture was
cooled and poured gradually onto ice/water. The obtained
precipitate was filtered off, dried and re-crystallized from
DMF/water. Yield: 65 %; mp (ꢁC): 199–210. IR (KBr,
cm-1): 3420, 3235 (NH, NH2), 2229 (CN), 1327, 1155
10
11
12a
13a
12b
13b
14
15
16
17
1
(SO2NH2). H NMR (DMSO-d6, d ppm): 3.70, 3.84 (2s,
6H, 2OCH3), 5.20 (1s, 2H, NH2 exchangeable with D2O),
7.01–7.99 (m, 7H, aromatic-H) and 7.99 (s, 1H, NH
exchangeable with D2O). MS m/z: (M ? 1)? 453 (30 %),
436 (10 %),79 (100 %). Anal. calcd for C19H16N8O4S
(452.46): C, 50.43; H, 3.56; N, 24.76; S, 7.08. Found: C,
50.05; H, 3.32; N, 24.43; S, 7.42.
mixture of chloroform and ethanol (15:1, v/v) as an eluent.
UV light at k 254 and iodine accomplished visualization.
7-(3,4-Dimethoxyphenyl)-3-oxo-5-thioxo-2,3,5,6-
tetrahydro imidazo[1,2-c]pyrimidine-8-carbonitrile (5)
4-Chloro-6-(3,4-dimethoxyphenyl)-2-thioxo-1,2-dihydro-
pyrimidine-5-carbonitrile (2)
A mixture of compound 2 (3 g, 10 mmol) and glycine
(0.75 g, 10 mmol) in DMF (20 mL) was heated under
reflux for 9 h. Then the mixture was poured onto ice/water.
The solid obtained was filtered off and dried. Then the
separated solid was refluxed with acetic anhydride (20 mL)
for 3 h. The product obtained after cooling was filtered off,
dried and crystallized from DMF/water. Yield: 65 %; mp
(ꢁC): 220–222. IR (KBr, cm-1): 3374 (NH), 2212 (CN),
1652 (CO), 1140 (C=S). 1H NMR (DMSO-d6, d ppm): 2.29
(s, 2H, CH2, imidazoline), 3.78, 3.82 (2s, 6H, 2OCH3),
7.01–7.40 (m, 3H, aromatic-H), 7.92 (1s, 1H, NH
exchangeable with D2O). MS m/z: M? 328 (30 %), 327
(20 %), 228 (100 %), 217 (20 %). Anal. calcd for
C15H12N4O3S (328.36): C, 54.86; H, 3.68; N, 17.06; S,
9.76. Found: C, 54.43; H, 3.51; N, 17.32; S, 9.90.
A mixture of compound 1 (10 g, 30 mmol), phosphorous
oxychloride (40 mL) and phosphorous pentachloride (5 g)
was heated on boiling water bath for 8 h. After the reaction
was completed, the mixture was cooled and poured grad-
ually onto crushed ice. The obtained precipitate was fil-
tered off and dried. Yield: 75 %; mp (ꢁC): 180–182. IR
(KBr, cm-1): 3373 (NH), 2934 (CH3 alicyclic), 2219 (CN),
1
1142 (C=S). H NMR (DMSO-d6, d ppm): 3.75, 3.80 (2s,
6H, 2OCH3), 6.98–7.20 (m, 3H, aromatic-H), 8.20 (s, 1H,
NH exchangeable with D2O). MS m/z: M? 308, 310 (25,
9 %), 61 (100 %). Anal. calcd for C13H10ClN3O2S
(307.77): C, 50.73; H, 3.27; N, 13.65; S, 10.42. Found: C,
50.50; H, 3.11; N, 13.32; S, 10.22.
7-(3,4-Dimethoxyphenyl)-5-thioxo-5,6-
dihydrotetrazolo[1,5-c]pyrimidine-8-carbo nitrile (3)
4-(8-Cyano-7-(3,4-dimethoxyphenyl)-3-oxo-2,3-
dihydroimidazo [1,2-c] pyrimidin-5-
ylamino)benzenesulfonamide (6)
A mixture of compound 2 (3 g, 10 mmol) and sodium
azide (0.65 g, 10 mmol) in glacial acetic acid (30 mL) was
refluxed for 8 h. Then, the mixture was cooled and the
formed precipitate was filtered off, dried and re-crystallized
A mixture of imidazolo derivative 5 (0.5 g, 1.5 mmol)
and sulfanilamide (0.26 g, 1.5 mmol) in DMF (5 mL)
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Med Chem Res (2013) 22:659–673
663
Table 2 Results summary of
the molecular modeling studies
of compounds 4, 6, 9, 11, 13a,
b, 15 and 17
Compd.
H-Bonds
3D-Docking Results
76.86
Asp86
4
{2.20 Aº}
Lys33
{2.49 Aº}
Glu162
{2.38 Aº}
Lys129
{2.30 Aº}
72.73
Lys129
{2.31 Aº}
Lys33
6
{2.26 Aº}
Ile10
{2.41 Aº}
containing few drops of TEA was refluxed for 15 h. Then
the mixture was cooled and poured gradually onto ice/
water. The formed precipitate was filtered off, dried and re-
crystallized from DMF/water. Yield: 66 %; mp (ꢁC): 260–
262. IR (KBr, cm-1): 3372, 3278 (NH, NH2), 2209 (CN),
1655 (C=O) and 1332, 1146 (SO2NH2). 1H NMR (DMSO-
d6, d ppm): 2.30 (s, 2H, CH2, imidazoline ring), 3.75, 3.85
(2s, 6H, 2OCH3), 6.80–7.70 (m, 7H, aromatic-H) and 5.62,
7.91 (2s, 3H, NH2, NH exchangeable with D2O). 13C NMR
(DMSO-d6, d ppm): 56.98 (2OCH3), 63.43, 159.23, 173
(imidazoline-C), 113.92 (CN), 98.21, 162.31, 172.00
(pyrimidine-C), 111.12, 122.21, 127.41, 129.34, 131.10,
142.12, 149.11 (aromatic-C). MS m/z: M? 466 (20 %), 311
(20 %), 295 (30 %), 79 (100 %). Anal. calcd for
C21H18N6O5S (466.48): C, 54.07; H, 3.88; N, 18.01; S,
6.87. Found: C, 54.15; H, 4.23; N, 17.68; S, 6.53.
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Med Chem Res (2013) 22:659–673
Table 2 continued
Compd.
H-Bonds
3D-Docking Results
68.22
Lys129
9
{2.00 Aº}
Glu162
{2.01 Aº}
Lys33
{2.09 Aº}
Asp86
{2.00 Aº}
74.49
Lys33
11
{1.88 Aº}
Glu162
{1.91 Aº}
85.07
Asp86
13a
{2.40 Aº}
Lys89
{1.71 Aº}
Glu162
{2.05 Aº}
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Med Chem Res (2013) 22:659–673
665
Table 2 continued
Compd.
H-Bonds
3D-Docking Results
76.12
Lys33
13b
{1.92 Aº}
Lys89
{2.43 Aº}
Glu8
{1.99 Aº}
Glu8
{2.40 Aº}
Lys33
70.25
15
{1.80 Aº}
Lys89
{1.86 Aº}
Glu162
{2.29 Aº}
Glu12
{1.89 Aº}
65.67
Lys33
17
{2.19 Aº}
Lys129
{2.17 Aº}
Asp145
{2.04 Aº}
Glu162
{2.02 Aº}
123

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Med Chem Res (2013) 22:659–673
6-(3,4-Dimethoxyphenyl)-4-hydrazinyl-2-thioxo-1,2-
dihydropyrimidine-5-carbonitrile (7)
6-(3,4-Dimethoxyphenyl)-4-(3,5-dimethyl-1H-pyrazol-1-
yl)-2-thioxo-1,2-dihydro-pyrimidine-5-carbonitrile (10)
A mixture of chloro-derivative 2 (3 g, 10 mmol) and
hydrazine hydrate 99 % (1.6 mL, 50 mmol) in methanol
(10 mL) was stirred for 8 h at r.t. The formed precipitate
was filtered off, dried, and re-crystallized from DMF/water.
Yield: 70 %; mp (ꢁC): 220–222. IR (KBr, cm-1): 3420,
3390, 3190 (2NH, NH2), 3004 (CH, aromatic), 2220 (CN),
A mixture of hydrazinyl compound 7 (1 g, 3 mmol) and
acetylacetone (10 mL, 15 mmol) was heated under reflux
for 4 h. The solid obtained was filtered off and crystallized
from DMF/water. Yield: 73 %; mp (ꢁC): 220–222. IR
1
(KBr, cm-1): 3340 (NH), 2224 (CN) and 1143 (C=S). H
NMR (DMSO-d6, d ppm): 2.15, 2.16 (2s, 6H, 2CH3), 3.79,
3.84 (2s, 6H, 2OCH3), 6.83–7.40 (m, 3H, aromatic-H and
1H, pyrazole ring) and 8.20 (s, 1H, NH exchangeable with
D2O). MS m/z: (M ? 2)? 369 (40 %), 310 (20 %), 217
(10 %), 151 (60 %), 79 (100 %). Anal. calcd for
C18H17N5O2S (367.43): C, 58.84; H, 4.66; N, 19.05; S,
8.72. Found: C, 54.69; H, 4.45; N, 21.00; S, 6.21.
1
1140 (C=S). H NMR (DMSO-d6, d ppm): 3.75, 3.80 (2s,
6H, 2OCH3), 6.50 (s, 2H, NH2 exchangeable with D2O),
6.90–7.30 (m, 3H, aromatic-H), 8.21, 9.82 (2s, 2H, 2NH
exchangeable with D2O). MS m/z: (M - 2)? 301 (30 %),
270 (25 %), 182 (30 %), 78 (100 %). Anal. calcd for
C13H13N5O2S (303.34): C, 51.47; H, 4.31; N, 23.08; S,
10.57. Found: C, 51.61; H, 4.02; N, 23.32; S, 10.21.
4-(5-Cyano-(3,4-dimethoxyphenyl)-4-(3,4-dimethyl-1H-
pyrazol-1-yl) pyrimidin-2-ylamino)benzenesulfonamide
(11)
7-(3,4-Dimethoxyphenyl)-3-methyl-5-thioxo-5,6-dihydro-
[1,2,4] triazolo[4,3-c] pyrimidine-8-carbonitrile (8)
A mixture of pyrazolo derivative 10 (0.5 g, 1.3 mmol) and
sulfanilamide (0.23 g, 1.3 mmol) in DMF (5 mL) con-
taining few drops of TEA was refluxed for 15 h. Then the
mixture was cooled and poured gradually onto ice/water.
The precipitated material was filtered off, dried and re-
crystallized from DMF/water. Yield: 73 %; mp (ꢁC): 220–
222. IR (KBr, cm-1): 3440, 3240 (NH, NH2), 2220 (CN),
1327, 1160 (SO2NH2). 1H NMR (DMSO-d6, d ppm): 2.20,
2.21 (2s, 6H, 2CH3), 3.75, 3.81 (2s, 6H, 2OCH3), 5.81 (s,
2H, NH2 exchangeable with D2O), 6.81–7.90 (m, 7H,
aromatic-H and 1H of pyrazole ring), 8.50 (s, 1H, NH
exchangeable with D2O). MS m/z: (M ? 2)? 507 (20 %),
350 (20 %), 156 (40 %), 80 (100 %). Anal. calcd for
C24H23N7O4S (505.56): C, 57.01; H, 4.58; N, 19.39; S,
6.34. Found: C, 57.31; H, 4.34; N, 19.30; S, 6.11.
A mixture of hydrazinyl derivative 7 (1 g, 3 mmol) and
acetic anhydride (10 mL) was heated under reflux for 4 h.
Then the reaction mixture was cooled and poured onto ice/
water. The formed precipitate was filtered off, dried and re-
crystallized from methanol. Yield: 70 %; mp (ꢁC): 268–
270. IR (KBr, cm-1): 3429 (NH), 2220 (CN), 1141 (C=S).
1H NMR (DMSO-d6, d ppm): 2.13 (s, 3H, CH3), 3.75, 3.85
(2s, 6H, 2OCH3), 6.92–7.03 (m, 3H, aromatic-H) and 8.00
(s, 1H, NH exchangeable with D2O). MS m/z: M? 327
(40 %), 247 (20 %), 202 (40 %), 78 (100 %). Anal. calcd
for C15H13N5O2S (327.37): C, 55.03; H, 4.00; N, 21.39; S,
9.79. Found: C, 55.33; H, 4.34; N, 21.25; S, 9.61.
4-(8-Cyano-7-(3,4-dimethoxyphenyl)-3-methyl-
[1,2,4]triazolo[4,3-c] pyrimidin-5-
ylamino)benzenesulfonamide (9)
6-(3,4-Dimethoxyphenyl)-4-(3-ethoxy/methyl-5-oxo-4,5-
dihydro-1H-pyrazol-1-yl)-2-thioxo-1,2-dihydropyrimidine-
5-carbonitrile (12a, b)
A mixture of triazolo derivative 8 (0.5 g, 1.5 mmol) and
sulfanilamide (0.26 g, 1.5 mmol) in DMF (5 mL) con-
taining few drops of TEA was refluxed for 15 h. Then the
mixture was cooled and poured gradually onto ice/water.
The formed precipitate was filtered off, dried and re-
crystallized from DMF/water. Yield: 70 %; mp (ꢁC): 268–
270. IR (KBr, cm-1): 3430, 3270 (NH, NH2), 2224 (CN),
A mixture of hydrazinyl derivative 7 (1 g, 3 mmol) and
diethylmalonate (0.55 mL, 3 mmol) or ethylacetoacetate
(0.42 mL, 3 mmol) in DMF (10 mL) was heated under
reflux for 4 h. Then, the reaction mixture was cooled and
poured gradually onto ice/water. The formed precipitate
was filtered off, dried, and recrystallized from DMF/water.
1
1360, 1160 (SO2NH2). H NMR (DMSO-d6, d ppm): 2.13
(s, 2H, CH3), 3.70, 3.88 (2s, 6H, 2OCH3), 5.92 (s, 2H,
NH2 exchangeable with D2O), 6.70–7.80 (m, 7H, aro-
matic-H) and 9.80 (s, H, NH exchangeable with D2O).
MS m/z: (M - 1)? 464 (20 %), 296 (40 %), 78 (100 %).
Anal. calcd for C21H19N7O4S (465.50): C, 54.18; H, 4.11;
N, 21.06; S, 6.88. Found: C, 54.42; H, 4.23; N, 21.26; S,
6.51.
12a Yield: 66 %; mp (ꢁC): 258–260. IR (KBr, cm-1):
3399 (NH), 2210 (CN), 1653 (CO), 1138 (C=S). H NMR
1
(DMSO-d6, d ppm): 1.13 (t, 3H, J = 6.7 Hz, CH3 of ethyl
group), 2.40 (s, 2H, CH2, pyrazoline ring), 3.86, 3.89 (2s,
6H, 2OCH3), 4.03 (q, 2H, J = 7.1 Hz, CH2 of ethyl group),
6.90–7.21 (m, 3H, aromatic-H) and 8.00 (s, 1H, NH
123

Med Chem Res (2013) 22:659–673
667
exchangeable with D2O). MS m/z: M? 399 (20 %), 163
(10 %), 137 (20 %), 78 (100 %). Anal. calcd for
C18H17N5O4S: C, 54.12; H, 4.28; N, 17.53; S, 8.02. Found:
C, 54.43; H, 4.40; N, 17.32; S, 8.11.
172 (10 %), 78 (100 %). Anal. calcd for C23H21N7O5S
(507.54): C, 54.42; H, 4.17; N, 19.31; S, 6.31. Found: C,
54.12; H, 4.38; N, 19.57; S, 6.31.
4-(5-Amino-4-cyano-1H-pyrazol-1-yl)-6-(3,4-
dimethoxyphenyl)-2-thioxo-1,2-dihydropyrimidine-5-
carbonitrile (14)
12b Yield: 72 %; mp (ꢁC): 238–240. IR (KBr, cm-1):
3418 (NH), 2208 (CN), 1660 (CO), 1139 (C=S). H NMR
1
(DMSO-d6, d ppm): 2.21 (s, 3H, CH3), 2.46 (s, 2H, CH2
pyrazoline ring), 3.74, 3.80 (2s, 6H, 2OCH3), 6.91–7.20
(m, 3H, aromatic-H) and 8.00 (s, 1H, NH exchangeable
with D2O). MS m/z: M? 369 (30 %), 355 (50 %), 201
(80 %), 78 (100 %). Anal. calcd for C17H15N5O3S
(369.41): C, 55.27; H, 4.09; N, 18.95; S, 8.68. Found: C,
55.01; H, 4.21; N, 18.82; S, 8.40.
A mixture of hydrazinyl derivative 7 (1 g, 3 mmol) and 2-
ethoxy methylene malononitrile (0.40 g, 3 mmol) in DMF
(10 mL) was heated under reflux for 6 h. Then, the mixture
was cooled and poured gradually onto ice/water. The
formed precipitate was filtered off, dried, and recrystallized
from DMF/water. Yield: 66 %; mp (ꢁC): 230–232. IR
(KBr, cm-1): 3314, 3180 (NH, NH2), 2210 (2CN), 1137
4-(5-Cyano-4-(3,4-dimethoxyphenyl)-6-(3-ethoxy/methyl-
5-oxo-4,5-dihydro-1H-pyrazol-1-yl)pyrimidin-2-
ylamino)benzenesulfonamide (13a, b)
1
(C=S). H NMR (DMSO-d6, d ppm): 3.75, 3.80 (2s, 6H,
2OCH3), 5.23 (s, 2H, NH2 exchangeable with D2O), 6.90–
7.53 (m, 3H, aromatic-H and 1H of pyrazole ring), 8.00 (s,
1H, NH exchangeable with D2O). MS m/z: M? 379 (50 %),
105 (30 %), 78 (100 %). Anal. calcd for C17H13N7O2S:
(379.41): C, 53.81; H, 3.45; N, 25.84; S, 8.45. Found: C,
53.61; H, 3.23; N, 26.01; S, 8.19.
A mixture of 12a, b (1.2 mmol) and sulfanilamide
(1.2 mmol) in DMF (5 mL) containing few drops of TEA
was refluxed for 15 h. Then, the reaction mixture was
cooled and poured gradually onto ice/water. The precipi-
tated product was filtered off, dried, and recrystallized from
DMF/water.
4-(4-(5-Amino-4-cyano-1H-pyrazol-1-yl)-5-cyano-6-(3,4-
dimethoxyphenyl) pyrimidin-2-ylamino)
benzenesulfonamide (15)
13a Yield: 65 %; mp (ꢁC): 279–281. IR (KBr, cm-1):
3420, 3193 (NH, NH2), 2210 (CN), 1656 (CO). H NMR
1
(DMSO-d6, d ppm): 1.21 (t, 3H, J = 6.8 Hz, CH3 of ethyl
group), 2.25 (s, 2H, CH2 pyrazoline ring), 3.78, 3.80 (2s,
6H, 2OCH3), 4.10 (q, 2H, J = 7.2 Hz CH2 of ethyl group),
5.61 (s, 2H, NH2 exchangeable with D2O), 6.71–7.91 (m,
7H, aromatic-H) and 8.51 (s, 1H, NH exchangeable with
A mixture of pyrazole compound 14 (0.5 g, 1 mmol) and
sulfanilamide (0.22 g, 1 mmol) in DMF (5 mL) containing
few drops of TEA was refluxed for 15 h. Then, the mixture
was cooled and poured gradually onto ice/water. The pre-
cipitated product was filtered off, dried, and recrystallized
from DMF/water. Yield: 70 %; mp (ꢁC): 228–230. IR
(KBr, cm-1): 3319, 3195, 3120 (NH, 2NH2), 2209 (2CN),
1330, 1143 (SO2NH2). 1H NMR (DMSO-d6, d ppm): 3.75,
3.80 (2s, 6H, 2OCH3), 5.20, 6.50 (2s, 4H, 2NH2), 6.90–
7.80 (m, 7H, aromatic-H and 1H of pyrazole ring), 10.20
(1s, 1H, NH exchangeable with D2O). MS m/z: (M - 1)?
516 (20 %), 501 (10 %), 365 (20 %), 78 (100 %). Anal.
calcd for C23H19N9O4S (517.53): C, 53.37; H, 3.70; N,
24.35; S, 6.20. Found: C, 53.51; H, 3.52; N, 24.54; S, 6.53.
D2O). 13C NMR (DMSO,
d ppm): 15.29, 60.11
(OCH2CH3), 55.12 (2OCH3), 36.34, 158.34, 172.10 (pyr-
azoline-C), 113.11 (CN), 98.29, 159.54, 164.31, 172.90
(pyrimidine-C), 111.02, 122.21, 127.61, 129.34, 131.90,
142.12, 149.11 (aromatic-C). MS m/z: M? 537 (30 %), 381
(15 %), 156 (20 %), 137 (20 %), 78 (100 %). Anal. calcd
for C24H23N7O6S (537.56): C, 53.62; H, 4.31; N, 18.23; S,
5.96. Found: C, 53.81; H, 4.10; N, 18.49; S, 5.96.
13b Yield: 65 %; mp (ꢁC): 270–272. IR (KBr, cm-1):
3343, 3190 (NH, NH2), 2208 (CN), 1657 (CO), 1330, 1148
(SO2NH2). 1H NMR (DMSO-d6, d ppm): 2.10 (s, 3H,
CH3), 2.40 (s, 2H, CH2 pyrazoline ring), 3.75, 3.81 (2s, 6H,
2OCH3), 5.61 (s, 2H, NH2 exchangeable with D2O), 6.71–
7.91 (m, 7H, aromatic-H) and 8.61 (s, 1H, NH exchange-
able with D2O). 13C NMR (DMSO, d ppm): 16.31 (CH3),
55.98 (2OCH3), 40.91, 152.41, 165.00 (pyrazoline-C),
113.32 (CN), 98.21, 159.11, 162.31, 172.00 (pyrimidine-
C), 111.12, 122.21, 127.41, 129.34, 131.10, 142.12, 149.11
(aromatic-C). MS m/z: (M - 2)? 505 (40 %), 426 (10 %),
7-(3,4-Dimethoxyphenyl)-3-oxo-5-thioxo-2,3,5,6-
tetrahydro-[1,2,4]triazolo [4,3-c]pyrimidine-8-
carbonitrile (16)
A mixture of hydrazinyl compound 7 (1 g, 3 mmol) and
ethylchloroformate (0.31 mL, 3 mmol) in pyridine
(10 mL) was heated under reflux for 6 h. Then, the mixture
was cooled, poured gradually onto ice/water and neutral-
ized with HCl. The formed precipitate was filtered off,
dried, and recrystallized from DMF/water. Yield: 70 %;
123

668
Med Chem Res (2013) 22:659–673
mp (ꢁC): 250–252. IR (KBr, cm-1): 3320, 3211 (2NH),
2211 (CN), 1657 (CO), 1130 (C=S). H NMR (DMSO-d6,
Table 3 Variation correlation of the anti-cancer evaluation data
against human cervix carcinoma cell line (Hela)
1
d ppm): 3.75, 3.80 (2s, 6H, 2OCH3), 6.80–7.80 (m, 3H,
aromatic-H), 8.00, 10.11 (2s, 2H, 2NH exchangeable with
D2O). MS m/z: (M - 1)? 328 (20 %), 301 (10 %), 191
(40 %), 137 (10 %), 78 (100 %). Anal. calcd for
C14H11N5O3S (329.34): C, 51.05; H, 3.36; N, 21.26; S,
9.73. Found: C, 51.35; H, 3.52; N, 21.01; S, 9.92.
Compound no. Concentration Mean
(Ug/mL)
V
SD
SEM Mean
4
0
1
0.000 0.000 0.000 1.000
5
1.079 0.007 0.088 0.036 1.079
0.676 0.033 0.181 0.074 0.676
0.244 0.002 0.049 0.020 0.244
0.177 0.0003 0.019 0.008 0.177
12.5
25
50
0
4-(8-Cyano-7-(3,4-dimethoxyphenyl)-3-oxo-2,3-dihydro-
[1,2,4]triazolo[4,3-c] pyrimidin-5-ylamino)
benzenesulfonamide (17)
6
1
0.000 0.000 0.000 1.000
5
1.265 0.025 0.158 0.065 1.265
0.819 0.004 0.067 0.028 0.819
0.236 0.001 0.038 0.016 0.236
0.16 0.001 0.031 0.013 0.160
12.5
25
50
0
A mixture of triazolo derivative 16 (0.5 g, 1 mmol) and
sulfanilamide (0.26 g, 1 mmol) in DMF (5 mL) containing
few drops of TEA was refluxed for 15 h. Then, the reaction
mixture was cooled and poured gradually onto ice/water.
The obtained precipitate was filtered off, dried, and re-
crystallized from DMF/water. Yield: 66 %; mp (ꢁC): 289–
291. IR (KBr, cm-1): 3334, 3230, 3130 (2NH, NH2), 2208
(CN), 1657 (CO), 1337, 1144 (SO2NH2). 1H NMR
(DMSO-d6, d ppm): 3.75, 3.82 (2s, 6H, 2OCH3), 5.82 (s,
2H, NH2 exchangeable with D2O), 6.72–7.92 (m, 7H,
aromatic-H), and 8.00, 9.82 (2s, 2H, 2NH exchangeable
with D2O). MS m/z: M? 467 (30 %), 450 (12 %), 389
(20 %), 78 (100 %). Anal. calcd for C20H17N7O5S
(467.47): C, 51.38; H, 3.66; N, 20.97; S, 6.86. Found: C,
51.62; H, 3.49; N, 20.57; S, 6.63.
9
0
0.000 0.000 0.000 0.000
5
1.378 0.024 0.156 0.064 1.378
0.82 0.007 0.084 0.035 0.820
0.412 0.003 0.061 0.025 0.412
0.316 0.0005 0.023 0.010 0.316
12.5
25
50
0
11
13a
13b
17
0
0.000 0.000 1.000 0.000
5
1.513 0.023 0.154 0.062 1.513
0.897 0.047 0.218 0.089 0.897
0.349 0.013 0.117 0.049 0.349
0.195 0.0003 0.018 0.008 0.195
12.5
25
50
0
1
0.000 0.000 0.000 1.000
5
1.062 0.194 0.441 0.180 1.062
0.983 0.011 0.105 0.043 0.983
0.407 0.002 0.044 0.018 0.407
0.319 0.0003 0.018 0.007 0.319
12.5
25
50
0
Anti-tumor screening
1
0.000 0.000 0.000 1.000
Cell growth inhibition assay
5
1.438 0.013 0.116 0.048 1.438
0.943 0.004 0.064 0.026 0.943
0.422 0.002 0.051 0.021 0.422
0.238 0.0003 0.018 0.007 0.238
12.5
25
50
0
Cervical cancer cell lines (Hela cell lines) were obtained
from Cell Bank in National Cancer Institute, Cairo, Egypt.
The potential toxicity of the selected newly synthesized
derivatives was done by SRB using the method Skehan et al.
(1990) as follows: cells were plated in 96-multiwell plate
(104 cells/well) for 24 h before treatment with compounds to
allow attachment of cell to the wall of the plate. Different
concentrations of the compound under test (1, 2.5, 5 and
10 g/mL) were added to the cell monolayer triplicate wells
which were prepared for each individual dose. Monolayer
cells were incubated with the compounds for 48 h at 37 ꢁC
and in atmosphere of 5 % CO2. After 48 h, cells were fixed,
washed and stained with Sulfo-Rhodamine-B stain. Excess
stain was washed with acetic acid and attached stain was
recovered with Tris-EDTA buffer. Color intensity was
measured in an ELISA reader. Measurements were done six
times (n = 6) and averaged. The relation between surviving
fraction and drug concentration is plotted to get the survival
curve of each tumor cell line after the specified compound.
The variation correlation of the obtained data was presented
1
0.000 0.000 0.000 1.000
5
1.199 0.015 0.122 0.050 1.199
0.769 0.039 0.197 0.081 0.769
0.42 0.011 0.009 0.045 0.420
0.315 0.002 0.052 0.021 0.315
12.5
25
50
SEM standard error mean, SD standard deviation, V variance of
standard deviation
n = 6
in Table 3, using the following equation to obtain the stan-
dard deviation results:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
2
ðX ꢁ MÞ
n ꢁ 1
S ¼
while V (variance of standard deviation) = S2, R = sum
of, X = individual score, M = mean of all scores,
n = sample size (number of scores).
123

Med Chem Res (2013) 22:659–673
Results and discussion
669
Chemistry
The key starting compound 6-(3,4-dimethoxyphenyl)-4-
oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1)
was synthesized by one-pot reaction of thiourea, ethyl-
cyanoacetate and 3,4-dimethoxybenzaldehyde in sodium
ethoxide solution with continuous stirring at room tem-
perature according to the reported method (Fathalla et al.,
2009). Chlorination of 1 was carried out by its refluxing
with POCl3/PCl5 for 8 h to obtain the chloro-derivative 2 in
75 % yield (Scheme 1). IR spectrum revealed the disap-
pearance of CO group at 1,730 cm-1. Mass spectrum
showed the two isotopic molecular ion peaks at 308, 310 in
a ratio (3:1). Compound 2 was considered as a precursor
for the synthesis of different heterocyclic rings fused with
2-thiopyrimidine ring. Thus, the treatment of 2 with
sodium azide in refluxing glacial acetic acid (Ali et al.,
2000) led to gain the desired tetrazolo derivative 3. Mass
spectrum of the derivative showed the molecular ion peak
at 314 (15 %). Upon reaction of 3 with an equimolar
amounts of sulfanilamide in refluxing DMF containing few
drops of TEA (Amin et al., 2009) afforded the benzene-
Molecular docking study
According to Table 1, molecular modeling simulation
study demonstrates that the designed derivatives could be
promising active hits as CDK2 inhibitors. Best affinities to
the active site of CDK2 were gained by the molecules
containing pyrimidine-benzenesulfonamide ring system
which are: 4, 6, 9, 11, 13a, 13b, 15, and 17. All of them
showed CDOCKER interaction energy less than that of the
lead compound. According to Table 2, showing the 3D
docking of the tested compounds in the binding site, the
presence of 3-ethoxy-5-pyrazolone (13a) exhibited the
highest affinity, while the replacement of the ethoxy group
of the pyrazolone ring with a methyl group (13b) or
exchange the pyrazolone moiety with tetrazole ring (4) led
to remarkable reduction in the affinity. Further affinity
decrease was observed by the molecules bearing imidazo-
pyrimidine and 5-amino-4-cyanopyrazole ring systems (6,
15). Least affinity was obtained by the molecules carrying
the triazole nucleus (9, 17).
Scheme 1 Synthetic protocols
of the starting
4-oxothiopyrimidine
1 and the intermediate
4-chlorothiopyrimidine
derivative 2
CHO
O
NH2
+
CN
COOC2H5
CN
alc.NaOH
HN
+
S
+
+
NH2
O
S
N
H
Ar
O
1
Halogenation
POCl3
Cl
CN
N
S
N
H
Ar
2
Ar =
O
O
123

670
Med Chem Res (2013) 22:659–673
sulfonamide derivative 4 in moderate yield. 1H NMR
spectrum of 4 demonstrated the presence of multiplet
signals at 7.01–7.99 ppm assigned for the seven aromatic
protons and a singlet signal at 5.20 ppm attributed to the
two protons of NH2, in addition to the presence of two
singlets at 3.69, 3.71 ppm due to 6H of 2OCH3 groups
and at 7.99 ppm corresponding to 1H of NH group.
Further treatment of the chloro-derivative 2 with the
amino acid glycine in refluxing DMF gave the imidazo-
pyrimidine analogue 5 in 66 % yield. IR spectrum of the
compound showed the appearance of a new absorption
band at 1,652 cm-1 assigned for the lactamic CO group.
Also, 1H NMR spectrum exhibited a singlet signal at
2.29 ppm indicating the two protons of methylene
group of imidazoline ring. Upon condensation of 5 with
equimolar amounts of sulfanilamide in refluxing DMF
afforded the benzenesulfonamide derivative 6 in moderate
1
yield. H NMR spectrum of 6 represented three singlets at
2.30, 3.75, 3.85 ppm referring to 2H of CH2 of imidaz-
oline ring and 6H of 2OCH3 groups. The seven aromatic
protons appeared multiplet signals at 6.80–7.70 ppm,
while NH2 and NH protons appeared as two singlet sig-
nals exchangeable with D2O at 5.62, 7.91 ppm. Synthesis
of hydrazinopyrimidine 7 as a key starting material for
many nitrogen bridge head compounds was performed by
the reaction of the chloro-derivative 2 with excess
hydrazine hydrate 99 % (Fathalla et al., 2009) in metha-
nol with continuous stirring at room temperature
1
(Scheme 2). H NMR spectrum exhibited the presence of
two singlet signals at 8.21 and 9.82 ppm representing the
two protons of 2NH and at 6.50 ppm due to the two
protons of NH2 group.
Scheme 2 Synthetic protocols
of the compounds tetrazolo[1,5-
Cl
N
NHNH2
CN
CN
O
CN
c]pyrimidine 3, imidazo[1,2-
c]pyrimidine 5 and their
respective benzenesulfonamide
derivatives 4, 6 and synthesis of
4-hydrazinylthiopyrimidine
derivative 7
N
N
v
iii
N
S
N
H
Ar
S
N
H
Ar
S
N
H
Ar
2
5
7
iv
i
N
N
N
CN
O
N
N
CN
N
Ar1
N
N
Ar
H
S
N
H
Ar
6
3
ii
N
N
N
CN
N
Ar1
N
N
Ar
H
4
Ar =
Ar1 =
O
O
S
O
O
NH2
i) sodium azide, glacial acetic acid, reflux 8h.
ii) & iv) sulfanilamide, DMF/ dps TEA, reflux 15h.
iii) glycine, DMF, reflux 9h.
v) hydrazine hydrate, methanol, r. t, stirr 8h.
123

Med Chem Res (2013) 22:659–673
671
Upon refluxing 7 with acetic anhydride for 4 h the
desired triazolo[4,3-c]pyrimidine derivative 8 was obtained
in 70 % yield. 1H NMR spectrum showed the presence of a
singlet signal at 2.31 ppm corresponding to the three pro-
tons of CH3 of the triazole ring. The other protons of the
molecule appeared at their expected regions. The deriva-
tive 8 was in turn condensed with equimolar amounts of
sulfanilamide in refluxing DMF containing few drops of
TEA to give the benzenesulfonamide derivative 9 in
moderate yield. 1H NMR spectrum of the compound 9
exhibited a singlet signal at 2.13 ppm due to CH3, two
singlet signals at 3.70, 3.88 ppm due to 2OCH3, multiplet
signals at 6.70–7.80 ppm due to 7H aromatic protons, in
addition to two singlets at 5.92 and 9.80 ppm due to NH2
and NH protons. Cyclocondensation of the hydrazinyl
derivative 7 with bielectrophilic species such as acetyl
NHNH2
HN
N
N
N
CN
CN
CN
N
N
O
vi
N
xv
S
N
H
Ar
S
N
H
Ar
S
N
H
16
Ar
x
8
i
i
i
i
7
i
i
v
x
xi
R1
NC
N
vii
N
N
N
O
H2N
xvi
N
N
CN
Ar
CN
Ar
CN
N
N
N
S
N
H
S
N
H
Ar
N
H
S
10
14
N
R1=12a= OC2H5
12b=CH3
N
HN
N
CN
Ar
CN
Ar
N
O
N
xii
Ar1
N
H
N
9
ix
xiv
Ar1
R1
N
N
H
17
N
O
N
NC
CN
N
N
Ar1
N
HN
N
Ar
N
H2N
N
CN
Ar
CN
N
R1=13a= OC2H5
13b= CH3
N
Ar1
Ar1
N
H
N
N
N
Ar
H
11
15
Ar =
Ar1
=
O
O
S
O
O
NH2
vi) acetic anhydride, reflux 4h.
viii) acetylacetone, DMF, reflux 4h.
x) diethyl malonate, DMF, reflux 4h.
xi) ethyl acetoacetate, DMF, reflux 4h.
xiii) 2-(ethoxymethylene) malononitrile, DMF, reflux 6h.
xv) ethyl chloroformate, pyridine, reflux 6h.
vii), ix), xii), xiv) &xvi) sulfanilamide, DMF, reflux 15h.
Scheme 3 Synthetic protocols of the compounds triazolo[4,3-c] pyrimidine 8, pyrazolopyrimidines 10, 12a, b, 14, triazolo[4,3-c]pyrimidine 16
their respective benzenesulfonamide derivatives 9, 11, 13a, b, 15, 17
123

672
Med Chem Res (2013) 22:659–673
Table 4 The effect of some
newly synthesized compounds
against human cervix carcinoma
cell line (Hela)
acetone, diethylmalonate, and ethylacetoacetate in reflux-
ing DMF furnished the pyrazolo derivatives 10, 12a, b,
respectively. 1H NMR spectrum of 10 represented two
singlet signals at 2.15 and 2.16 ppm accomplished for the
Compounds
IC50 (lM)
4
0.039
0.042
0.048
0.041
0.042
0.046
0.047
6
1
9
six protons of the two CH3 groups. H NMR spectrum of
11
13a
13b
17
12a represented the characteristic triplet-quartet pattern of
the ethyl group at 1.13 and 4.03 ppm and CH2 protons
of pyrazoline ring appeared as a singlet signal at 2.40 ppm,
while 1H NMR spectrum of 12b showed a singlet signal at
2.21 ppm referring to the 3H of CH3 group and the
methylene protons of the pyrazoline ring appeared as a
singlet signal at 2.46 ppm. Further treatment of 10, 12a, b
with equimolar amounts of sulfanilamide was carried out to
get the benzenesulfonamide analogues 11, 13a, b, respec-
tively. Mass spectra of the new derivatives exhibited their
molecular ion peaks. Nucleophilic reaction of the hydra-
zinyl derivative 7 with 2-(ethoxymethylene) malononitrile
in refluxing DMF or with ethylchloroformate in refluxing
pyridine afforded the desired 4-cyano-5-amino pyrazolo
and/or triazolopyrimidine derivatives 14, 16 respectively.
IR spectrum of the compound 14 exhibited a strong
stretching band at 2,210 cm-1 assigned for 2CN groups,
Doxorubicin (Dox) 0.016
4 (IC50 0.039 lM). The obtained data insure that the
modifications which were carried out for the lead thiazolyl
pyrimidine compound furnished novel derivatives of higher
potency as anti-cancer agents but still less active than the
reference Doxorubicin. This means that further modifica-
tions are required for getting new anti-cancer agents of
higher efficacy and selectivity.
1
Conclusion
while its H NMR spectrum revealed a singlet signal at
5.23 ppm due to the two protons of NH2 group. The other
protons of the molecule were present at their expected
regions; 6H of 2OCH3 appeared at 3.75, 3.80 ppm as two
singlets, 4H aromatic protons appeared at 6.90–7.53 ppm
as a multiplet signal and NH proton appeared as an
exchangeable signal at 8.00 ppm. IR spectrum of the
derivative 16 exhibited the lactamic CO group at
1,657 cm-1. The formation of the respective benzenesul-
fonamide analogues 15, 17 was carried out by the reaction
of equimolar amounts of 14 and/or 16 with sulfanilamide
(Scheme 3). 1H NMR spectra of the two derivatives
showed the seven aromatic protons as multiplets at 6.72–
7.92 ppm in addition to the other expected protons at their
correct regions.
In this study design, molecular modeling simulation study
and synthesis were carried out for a new series of pyr-
imidine-benzenesulfonamide derivatives as CDK2 inhibi-
tors taking the derivative 4-(2-amino-4-methylthiazol-5-
yl)-N-(3-nitrophenyl) pyrimidin-2-amine as a lead com-
pound. Docking results indicated that the novel agents
exhibited better affinities to the active site of CDK2 than
the lead compound. In vitro anti-proliferative activity
evaluation against Hela cell lines indicated that although
the compounds (4, 6, 9, 11, 13a, 13b, 17) are more potent
cytotoxic agents than the lead derivative but they are still
less active than the reference drug Doxorubicin and further
optimization is required to get more active anti-cancer
compounds.
Anti-tumor activity
Acknowledgments This study was supported by the National
Research Centre, Dokki, Cairo, Egypt.
In order to correlate between the obtained molecular
modeling simulation results of the tested compounds and
their anti-proliferative activity, the analogues that showed
the best molecular docking results (4, 6, 9, 11, 13a, 13b,
17) were selected as representative examples to evaluate
their in vitro inhibitory effects against cellular proliferation
in human cultured Hela cell lines using Doxorubicin as a
reference drug. Depending upon the data of Table 4, there
is a good correlation between both studies since all the
evaluated derivatives appeared to be approximately equi-
potent cytotoxic agents with IC50 values (0.039–0.047 lM)
lower than that of the lead derivative (III) (IC50 0.5 lM).
Best result was obtained by tetrazolopyrimidine derivative
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