A convenient synthesis and molecular modeling study of novel pyrazolo[3,4-d]pyrimidine and pyrazole derivatives as anti-tumor agents

Article abstract of DOI:10.3109/14756366.2014.940936

An efficient method to obtain ethyl 5-amino-1-tosyl-1H-pyrazole-4-carboxylate (3) was outlined using condensation reactions of 4-methylbenzenesulfonylhydrazide with (E)-ethyl 2-cyano-3-ethoxyacrylate. The cyclocondensation reaction of this substrate and its hydrazide derivative with urea, thiourea, formamide, formic acid, d-glucose, o-phenylenediamine, 4-dimethylaminobenzaldehyde, anthracene-9-carbaldehyde, thioglycolic acid and carbon disulphide then with hydrazine hydrate analogues furnished a series of pyrazolo[3,4-d]pyrimidine, pyrazolo[3,4-d]oxazin-4-one, pyrazole-4-glucoside, 4-benzo[d]imidazole, 1,3-thiazolidinone, 1,3,4-oxadiazol-2(3H)-thione and 1,2,4-triazol-5(4H)-thione derivatives respectively. The structure of the compound 3 was supported by X-Ray crystallographic data. Orally administrated, one of each of the series of pyrazoles showed significant effects in mouse tumor model cancer cell lines (EAC) and two human cancer cell lines of Colon cancer (HCT-29) and Breast cancer (MCF-7) with docking studies.

Full text of DOI:10.3109/14756366.2014.940936

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.940936
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RESEARCH ARTICLE
A convenient synthesis and molecular modeling study of novel
pyrazolo[3,4-d]pyrimidine and pyrazole derivatives as anti-tumor agents
Ibrahim F. Nassar1, Saad R. Atta-Allah2, and Abdel-Sattar S. Hamad Elgazwy2
2
1Faculty of Specific Education, Ain Shams University (ASU), Abbassia, Cairo, Egypt and Department of Chemistry, Faculty of Science, Ain Shams
University, Abbassia, Cairo, Egypt
Abstract
Keywords
An efficient method to obtain ethyl 5-amino-1-tosyl-1H-pyrazole-4-carboxylate (3) was outlined
using condensation reactions of 4-methylbenzenesulfonylhydrazide with (E)-ethyl 2-cyano-3-
ethoxyacrylate. The cyclocondensation reaction of this substrate and its hydrazide derivative
with urea, thiourea, formamide, formic acid, D-glucose, o-phenylenediamine, 4-dimethylami-
nobenzaldehyde, anthracene-9-carbaldehyde, thioglycolic acid and carbon disulphide
then with hydrazine hydrate analogues furnished a series of pyrazolo[3,4-d]pyrimidine,
pyrazolo[3,4-d]oxazin-4-one, pyrazole-4-glucoside, 4-benzo[d]imidazole, 1,3-thiazolidinone,
1,3,4-oxadiazol-2(3H)-thione and 1,2,4-triazol-5(4H)-thione derivatives respectively. The struc-
ture of the compound 3 was supported by X-Ray crystallographic data. Orally administrated,
one of each of the series of pyrazoles showed significant effects in mouse tumor model cancer
cell lines (EAC) and two human cancer cell lines of Colon cancer (HCT-29) and Breast cancer
(MCF-7) with docking studies.
Anticancer, cycloadditions, cyclizations,
heterocycles, molecular modeling,
pyrazole, X-ray
History
Received 22 May 2014
Revised 15 June 2014
Accepted 23 June 2014
Published online 28 July 2014
Introduction
demonstrated significant antiviral activity35,36. There is no
much difference in the basic structures of pyrazolopyrimidines
and purines37. The above facts and our interest38 in the synthesis
of new biologically active isolated and fused substituted
heterocycles prompted us to synthesize new substituted pyrazoles
linked to five membered heterocycles and fused pyrazolopyr-
imidine and oxazine ring systems in order to increase its
biological activity and evaluate its activity as anticancer agents.
The chemistry of 1H-pyrazole-containing compounds is particu-
larly interesting because of their potential application in
medicinal chemistry as analgesic1,2, anti-inflammatory3,4, anti-
tumor5,6, antimicrobial7–11 and therapeutic agents12, as well as
based on their wide applications in agriculture as potent
insecticides13,14 and herbicides15,16, although scarcely found in
nature17. Due to many promising pharmacological, agrochemical,
and analytical applications, a number of substituted pyrazoles are
being used as inhibitors of heat-shock protein 90 (HSP90) and as
therapeutics of cancer and therefore they have been the focus of
many synthetic targets over the past decades18. Furthermore, it
has also been found that 1H-pyrazole based heterocyclic struc-
tures have attracted synthetic interest for being an essential
moieties in many chemotherapeutic agents with potential anti-
parasitic19,20, anti-malarial21 and antiviral activities22,23. As far as
the anticancer activity is concerned, literature citation revealed
that a wide range of pyrazole derivatives were reported to
contribute to a variety of anti-neoplastic potentials against a wide
range of cancer cell lines24–27. Moreover, many pyrazole deriva-
tives are associated with anti-fungal, anti-bacterial28 and anti-
pyretic29 properties. Pyrazolopyrimidines and related fused
heterocycles are of interest as potential bioactive molecules.
They are known to exhibit pharmacological activities such as
CNS-depressant30, anticancer31–33 and tuberculostatic34 activities.
Furthermore, some pyrazolo(3,4-d)pyrimidine derivatives
Experimental
Chemistry
General procedures
All melting points were measured using a Reichert Thermovar
apparatus (Depew, NY) and are uncorrected. Yields listed are of
isolated compounds. The IR spectra were recorded on a perkin-
Elmer model 1720 FTIR spectrometer (Waltham, MA) for KBr
disc. NMR spectra were recorded on a varian Gemini 300 BB NMR
1
Spectrometer (Buffalo, NY) at 300 MHz for H and 75 MHz for
13C. Chemical shifts were reported in d scale (ppm) relative toTMS
as a reference standard and the coupling constants J values are
given in Hz. Mass spectra were recorded on GC/MS Finnegan SSQ
7000 spectrophotometer and GC Ms-QP 1000 EX mass spectrom-
eter at 70 eV (Waltham, MA). The progress of the reactions was
monitored by TLC using aluminum silica gel plates 60 F245.
Elemental analyses were performed at the Microanalytical Centre
at Faculty of Science, Cairo University, Egypt.
Address for correspondence: I. F. Nassar, Faculty of Specific Education,
Ain Shams University (ASU), Abbassia, Cairo, Egypt. E-mail:
ibrahimnassar72@yahoo.com
Reaction of sulfonylhydrazide
2. To a solution of 4-methylbenzenesulfonylhydrazide (1)
1
with cyanoacrylate

2
I. F. Nassar et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
(1.86 g, 0.01 mol) in absolute ethanol (20 ml) was added (E)-ethyl (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.6 (s, 1H, -NH exchangeable), 7.84
2-cyano-3-ethoxyacrylate (2) (1.69 g, 0.01 mol). The reaction (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.90 (s, 1H, N¼CH pyrazole), 8.70
mixture was heated at reflux temperature for 16 h, cooled to room (s, 1H, NH exchangeable); 13C NMR (DMSO-d6, 75 MHz):
temperature. The solvent was evaporated under reduced pressure. ꢁ ¼ 21.2 (CH3), 127.2–140.7 (Ar. C + C-3), 148.5 (C-7a), 150.6
The resulting solid was recrystallized from ethanol to afford ethyl (C-3a), 154.3, 155.5 (2C¼O); Anal. calcd. for C12H10N4O4S
5-amino-1-tosyl-1H-pyrazole-4-carboxylate (3) as a white solid, (306.30): C, 47.06; H, 3.29; N, 18.29; Found: C, 47.05; H, 3.25;
2.01 g, (64.97%) m.p. 135–137 ꢀC; dec. Diffraction-quality crystal N, 18.25.
was grown by slow diffusion of ethanol solution. IR (KBr) cmꢁ1
:
ꢀ (NH2) 3480, ꢀ (C¼O) 1693, ꢀ (C¼N) 1615; 1H NMR
(DMSO-d6, 300 MHz): ꢁ ¼ 1.29 (t, 3H, J ¼ 7.2, O–CH2CH3), 2.34
(s, 3H, CH3), 4.18 (q, 2H, J ¼ 2.7, O–CH2CH3), 4.22 (br.s, 2H,
NH2 exchangeable), 7.46 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.84 (d, 2H,
J ¼ 8.1 Hz, Ar–H), 7.90 (s, 1H, pyrazole N¼CH) ppm; 13C NMR
(DMSO-d6, 75 MHz): ꢁ ¼ 15.3 (CH3), 21.2 (CH3), 61.2 (CH2),
106.5 (C-4), 128–140 (Ar. C + C-3), 148.5 (C-5), 160.5 (C¼O)
ppm; GC/MS: m/z (%) 309 M+ (8.37), 245 (6.07), 199 (5.90), 155
(12.81), 91 (33.88), 63 (91.51), 44 (100). Anal. Calcd. for
C13H15N3O4S (309.34): C, 50.47; H, 4.89; N, 13.58; Found: C,
50.39; H, 4.90; N, 13.45%.
6-Thioxo-1-tosyl-6,7-dihydro-1H-pyrazolo3,4-dpyrimidin-4(5H)-
one (6b). 2.63 g (52.6%) brown powder, m.p. 190–191 ꢀC,
(ethanol); IR (KBr) cmꢁ1: ꢀ (NH) 3216, ꢀ (C¼O) 1670,
1
ꢀ (C¼S) 1623, ꢀ (C¼N) 1610; H NMR (DMSO-d6, 300 MHz)
ppm: ꢁ ¼ 2.34 (s, 3H, CH3), 5.90 (br. s, 1H, NH exchangeable),
7.46 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.84 (d, 2H, J ¼ 8.1 Hz, Ar–H),
7.9 (s, 1H, N¼CH pyrazole), 8.20 (br. s, 1H, NH exchangeable);
13C NMR (DMSO-d6, 75 MHz): ꢁ ¼ 21.2 (CH3), 127–140
(Ar. C + C-3), 148.6 (C-7a), 150.5 (C-3a), 155.23 (C¼O),
172 (C¼S); Anal. calcd. for C12H10N4O3S2 (322.36): C, 44.71;
H, 3.13; N, 17.38; Found: C, 44.65; H, 3.17; N, 17.46%.
Reaction of pyrazolocarboxylate 3 with formic acid. A suspen-
sion of 3 (3.09 g, 0.01 mol) in formic acid (85%; 20 ml) was
heated at reflux temperature for 10 h. A solid product was
obtained after cooling at room temperature was filtered off and
recrystallized from ethanol to give 1-tosylpyrazolo(3,4-
d1,3)oxazin-4(1H)-one (4) as a red powder 1.6 g (54.92%), m.p.
Formation
of
5-amino-1-tosyl-1H-pyrazol-4-carboxylic
acid (7). A mixture of the pyrazolocarboxylate 3 (9.27 g,
0.03 mol) and sodium hydroxide (4 g, 0.1 mol) in 20 ml water
was heated in an oil bath for 15 h. It was then cooled to 10 ꢀC and
acidified with HCl. The produced precipitate was filtered off,
washed with water, and recrystallized from ethyl acetate to afford
7 as a red solid, 5.06 g (59.96%) m.p. 230–231 ꢀC, IR (KBr)
cmꢁ1: ꢀ (OH) 3431, (NH) 3180, ꢀ (C¼O) 1640, 1H NMR
(DMSO-d6, 300 MHz) ppm: ꢁ ¼ 2.34 (s, 3H, CH3), 4.50 (br. s, 2H,
NH2 exchangeable), 7.49 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.84
(d, 2H, J ¼ 8.1 Hz, Ar–H), 7.9 (s, 1H, pyrazole N¼CH), 11.00
(br. s, 1H, OH exchangeable), 13C NMR (DMSO-d6,75 MHz):
ꢁ ¼ 21.2 (CH3), 106.2 (C-4), 128–140 (Ar. C + C-3), 146.6 (C-5),
165.0 (C¼O); Anal. Calcd. for C11H11N3O4S (281.29): C, 46.97,
H, 3.94, N, 14.94; Found: C, 46.84, H, 3.90, N, 14.84%.
1
135–136 ꢀC, IR (KBr) cmꢁ1: ꢀ (C¼O) 1644; H NMR (DMSO-
d6, 300 MHz) ppm: ꢁ ¼ 2.34 (s, 3H, CH3), 7.46 (d, 2H, J ¼ 8.1 Hz,
Ar–H), 7.84 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.9 (s, 1H, pyrazole
N¼CH), 8.14 (s, 1H, oxazine N¼CH); 13C NMR (DMSO-d6,
75 MHz): ꢁ ¼ 21.2 (CH3), 106.5 (C-3a), 128–140 (Ar. C + C-3),
147.0 (C-7a), 159.5 (C¼O), 162 (C-6); Anal. Calcd. for
C12H9N3O4S (291.28): C, 49.48, H, 3.11, N, 14.43; Found: C,
49.56, H, 3.0, N, 14.30.
Reaction of pyrazolocarboxylate 3 with formamide. A solution
of 3 (3.09 g, 0.01 mol) in formamide (10 ml) was heated on oil
bath at 180–190 ꢀC for 4 h. The solution was then cooled to room
temperature and diluted with 100 ml ice-cooled water. The
precipitate that obtained was filtered off and recrystallized from
ethanol to afford 1-tosyl-1H-pyrazolo(3,4-d)pyrimidin-4(5H)-one
(5) as a yellow powder, 1.6 g (55.11%) m.p. 125–126 ꢀC; IR (KBr)
Reaction of pyrazolocarboxylic acid 7 with Ac2O. The pyrazo-
locarboxilic acid 7 (11.25 g, 0.04 mol) was heated with 30 ml
acetic anhydride in an oil bath for 6 h. The mixture was cooled to
room temperature, poured onto ice-cooled water, and stirred for
30 min. The solid obtained was filtered, washed with water,
recrystallized from ethanol to afford 6-methyl-1-tosylpyra-
zolo(3,4-d1,3)oxazin-4(1H)-one (8) as a brown powder; 4.64 g
(37.99%) m.p. 280–281 ꢀC, IR (KBr) cmꢁ1: ꢀ (C¼O) 1696;
1H NMR (DMSO-d6) ppm: ꢁ ¼ 1.70 (s, 3H, CH3), 2.34 (s, 3H,
CH3), 7.46 (d, J ¼ 8.1, 2H, Ar–H), 7.70 (d, J ¼ 8.1, 2H, Ar–H),
7.90 (s, 1H, pyrazole CH); 13C NMR (DMSO-d6,75 MHz):
ꢁ ¼ 21.2 (CH3), 25.2 (CH3), 106.2 (C-9), 128–140
(Ar. C + C-3), 147 (C-7a), 154.3 (C¼O), 159.6 (C6); Anal.
Calcd. for C13H11N3O4S (305.31): C, 51.14, H, 3.63, N, 13.76;
Found: C, 51.0, H, 3.60, N, 13.80%.
1
cmꢁ1: ꢀ (NH) 3165, ꢀ (C¼O) 1700, ꢀ (C¼N) 1600; H NMR
(DMSO-d6, 300 MHz) ppm: ꢁ ¼ 2.34 (s, 3H, CH3), 7.46 (d, 2H,
J ¼ 8.1 Hz, Ar–H), 7.84 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.9
(s, 1H, pyrazole N¼CH), 8.0 (br.s, 1H, NH exchangeable), 9.0
(s, 1H, pyrimidine N¼CH); 13C NMR (DMSO-d6,75 MHz):
ꢁ ¼ 21.2 (CH3), 105.2 (C-3a), 128–140 (Ar. C + C-3), 145.5 (C-6),
147 (C-7a), 157.65 (C¼O); Anal. Calcd. for C12H10N4O3S
(290.30): C, 49.65; H, 3.47; N, 19.30; Found: C, 49.60; H,
3.35; N, 19.35%.
General procedure for preparing compounds (6a,b). Ethyl
5-amino-1-tosyl-1H-pyrazole-4-carboxylate (3) (5 g, 1.61 mol)
and urea (10 g, 16.66 mol) and/or thiourea (10 g, 13.15 mol)
were heated together in an oil path at 150 ꢀC for 30 min. The clear
solution went mushy and heating was continued for ten minutes at
170 ꢀC. The resulting solids were dissolved in dilute sodium
hydroxide solution then acidified with acetic acid to obtain the
crude products 6a or 6b.
Reaction
of
pyrazolocarboxylic
acid
7
with
o-phenylenediamine. A mixture of 7 (2.81 g, 0.01 mol) and
o-phenylenediamine (1.08 g, 0.01 mol) in DMF (30 ml)
was heated at reflux temperature for 6 h. The reaction mixture
was allowed to cool at room temperature and poured onto cold
water. The solid formed was filtered off, recrystallized from
ethanol to afford 4-(1H-benzodimidazol-2-yl)-1-tosyl-1H-pyrazol-
5-amine (9) as a brown solid; 2 g (56.59%) m.p. 170–172 ꢀC, IR
(KBr)cmꢁ1: ꢀ (NH2) 3307, ꢀ (NH) 3190, ꢀ (C¼N) 1602; 1H
1-Tosyl-1H-pyrazolo3,4-dpyrimidin-4,6(5H,7H)-dione (6a). 2.72 g NMR (DMSO-d6, 300 MHz): ꢁ ¼ 2.34 (s, 3H, CH3), 4.22 (br.s,
(54.88%) yellow powder, m.p. 290–291 ꢀC, (ethyl acetate); IR 2H, NH2 exchangeable), 7.19 (m, 2H, Ar–H), 7.46 (d, 2H,
1
(KBr) cmꢁ1: ꢀ (NH) 3210, ꢀ (C¼O) 1690, ꢀ (C¼N) 1610; H J ¼ 8.1 Hz, Ar–H), 7.6 (m, 2H, Ar–H), 7.70 (br. s,1H, NH
NMR (DMSO-d6, 300 MHz) ppm: ꢁ ¼ 2.34 (s, 3H, CH3), 7.46 exchangeable), 7.74 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.9 (s, 1H,

DOI: 10.3109/14756366.2014.940936
Molecular modeling of pyrazolo[3,4-d]pyrimidine and pyrazole derivatives
3
pyrazole N¼CH); 13C NMR (DMSO-d6,75 MHz): ꢁ ¼ 21.2 (CH3), 4-Amino-3-(5-amino-1-tosyl-1H-pyrazol-4-yl)-1H-1,2,4-triazole-
100.2 (C-4), 115.2–141.7 (Ar. C + C-3), 132.9 (C-5), 153.0 5(4H)-thione (13). A solution of compound 12 (3.37 g, 001 mol)
(C¼N); Anal. calcd. for: C17H15N5O2S (353.40): C, 57.78; H, and hydrazine hydrate (0.15 mol) in ethanol (30 ml) was refluxed
4.28; N, 19.82. Found: C, 57.67; H, 4.39; N, 19.70%.
for 6 h. The reaction mixture was cooled at room temperature and
the solvent was evaporated under reduced pressure. The solid that
obtained was crystallized from ethanol to afford 13 as a white
powder, 2.1 g (60%) m.p. 135–136 ꢀC, IR (KBr) cmꢁ1: ꢀ (NH2)
3460, ꢀ (NH) 3268, ꢀ (C¼S) 1696, ꢀ (C¼N) 1615; 1H NMR
(DMSO-d6, 300 MHz): ꢁ ¼ 2.0 (br. s, 2H, NH2 exchangeable),
2.34 (s, 3H, CH3), 4.22 (br. s, 2H, NH2 exchangeable), 7.0 (br. s,
1H, NH exchangeable),7.46 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.84 (d,
2H, J ¼ 8.1 Hz, Ar–H), 7.90 (s, 1H, pyrazole N¼CH); 13C NMR
(75 MHz, DMSO): ꢁ ¼ 21.2 (CH3), 98.2 (C-4), 128–142
(Ar. C + C-3), 148.0 (C-5), 149.8 (C¼N imine), 190.4 (C¼S);
Analysis Calcd. for C12H13N7O2S2 (351.41): C, 41.01, H, 3.73, N,
27.90; Found C, 41.10, H, 3.65, N, 27.80%.
Formation of 5-amino-1-tosyl-1H-pyrazole-4-carbohydrazide
(10).
A solution of the pyrazolocarboxylate 3 (3.09 g,
0.01 mol) and hydrazine hydrate (0.15 mol) in ethanol (50 ml)
was stirred at room temperature for 2 h. The solid formed after
filtration was recrystallized from ethanol to yield 10 as a white
solid; 1.33 g (45.03%) m.p. 140–142 ꢀC, IR (KBr) cmꢁ1: ꢀ (NH2)
3457, ꢀ (NH) 3219, ꢀ (C¼O) 1630; 1H NMR (DMSO-d6,
300 MHz) ppm: ꢁ ¼ 2.34 (s,3H, CH3), 4.22 (br. s, 2H, NH2
exchangeable), 5.8 (br. s, 2H, NH2 exchangeable), 7.46 (d, 2H,
J ¼ 8.1 Hz, Ar–H), 7.87 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.9 (s, 1H,
pyrazole N¼CH), 8.0 (br. s, 1H, NH exchangeable); 13C NMR
(DMSO-d6,75 MHz): ꢁ ¼ 21.2 (CH3), 128–140 (Ar. C + C-3),
148.0 (C-5), 150.2 (C-4), 165.0 (C¼O) ppm; Anal. Calcd. for
C11H13N5O3S (295.32): C, 44.74; H, 4.44; N, 23.71; Found: C,
44.65; H, 4.36; N, 23.74%.
Generals procedure for preparation of the shiff’s bases
(15, 18). A solution of 10 (2.95 g, 0.01 mol) and the appropriate
aldehydes, (4-(dimethylamino)-benzaldehyde 14 or anthracene-
9-carbaldehyde 17 (0.01 mol) in ethanol (30 ml) and a few drops
of acetic acid was heated at reflux temperature for 6 h. The
reaction mixture was cooled at room temperature and the solvent
was evaporated under reduced pressure. The resulting solid was
crystallized from ethanol to obtain the shiff’s bases 15 and/or 18.
Reaction of carbohydrazide 10 with D-glucose. To a well stirred
solution of the D-glucose (0.9 g, 5 mmol) in water (1 ml), and
glacial acetic acid (1 ml); was added the carbohydrazide 10
(1.47 g, 5 mmol) in ethanol (15 ml). The mixture was heated under
reflux for 3 h. and the resulting solution was concentrated and left
to cool. The precipitate formed was filtered off, washed with
water, then dried and recrystallized from ethanol to afford
5-amino-N0-(d-glucopyranosyl)-1-tosyl-1H-pyrazole-4-carbohy-
drazide (11) as a white powder, 1.37 g (59.9%) m.p. 143–144 ꢀC;
IR (KBr) cmꢁ1: ꢀ (OH) 3460–3449, ꢀ (NH) 3265,ꢀ 1695 (C¼O),
5-Amino-N0-(4-(dimethylamino)benzylidene)-1-tosyl-1H-pyra-
zole-4-carbohydrazide (15). This compound was obtained as a
brown solid (benzene), 2.34 g (54.86%) m.p. 100–101 ꢀC, IR (KBr)
cmꢁ1: ꢀ (NH2) 3455, ꢀ (NH) 3266, ꢀ (C¼O) 1666, ꢀ (C¼N) 1600;
1H NMR (DMSO-d6, 300 MHz) ppm: ꢁ ¼ 2.34 (s, 3H, CH3), 3.0
(s, 6H, 2CH3), 4.22 (br. s, 2H, NH2 exchangeable), 6.77 (d, 2H,
J ¼ 8.4 Hz, Ar–H), 7.0 (br. s, 1H, NH exchangeable), 7.50 (d, 2H,
J ¼ 8.2 Hz, Ar–H), 7.70 (d, 2H, J ¼ 8.4 Hz, Ar–H), 7.86 (d, J ¼ 8.2,
1
ꢀ (C¼N) 1616; H NMR (DMSO-d6, 300 MHz): ꢁ ¼ 2.34 (s, 3H,
CH3), 3.30–3.35 (m, 2H, H-60,600), 3.70 (m, 1H, H-50), 4.12 (m,
1H, H-40), 4.27 (t, J ¼ 7.4 Hz, 1H, H-30), 4.22 (br. s, 2H, NH2
exchangeable), 4.34 (dd, J ¼ 7.4 Hz, J ¼ 7.8 Hz, 1H, H-20), 4.44
(m, 1H, OH), 4.45 (d, J ¼ 6.4 Hz, 1H, OH), 5.20 (m, 1H, OH),
5.60 (t, J ¼ 4.6 Hz, 1H, OH), 5.79 (t, J ¼ 4.6 Hz, 1H, OH), 7.0 (br.
s, 1H, NH exchangeable), 7.47 (d, 2H, J ¼ 8.1 Hz, Ph-H), 7.6
(s, 1H, N¼CH), 7.87(d, 2H, J ¼ 8.4 Hz, Ph-H), 7.89 (s, 1H,
pyrazole N¼CH); 13C NMR (DMSO-d6, 75 MHz): ꢁ ¼ 21.2
(CH3), 62.1 (C-60), 63.0 (C-50), 69.2 (C-40), 74.3 (C-30),
75.7 (C-20), 128–142 (Ar. C + C-3), 148.0 (C-5), 150.5 (C-4),
151.5 (C-10), 164.2 (C¼O) ppm, Anal. calcd. for: C17H23N5O8S
(457.46): C, 44.63; H, 5.07; N, 15.31. Found: C, 44.56; H, 5.15;
N, 15.37%.
2H, Ar–H), 7.90 (s,1H, pyrazole N¼CH), 9.67 (s,1H, N¼CH); 13
C
NMR (DMSO-d6, 75 MHz): ꢁ ¼ 21.2 (CH3), 41.0 (2CH3), 128–142
(Ar. C + C-3), 148.0 (C-5), 149.3 (N¼CH), 151 (C-4), 164.2
(C¼O) ppm, Anal. Calcd. for C20H22N6O3S (426.49): C, 56.32, H,
5.20, N, 19.70; Found C, 56.36, H, 5. 30, N, 19.73%.
5-Amino-N0-(anthracen-9-ylmethylene)-1-tosyl-1H-pyrazole-4-
carbohydrazide (18). This compound was obtained as a brown
solid (benzene), 2.56 g (52.98%) m.p. 97–98 ꢀC, IR (KBr) cmꢁ1: ꢀ
1
(NH2) 3458, ꢀ (NH) 3265, ꢀ (C¼O) 1666, ꢀ (C¼N) 1610; H
NMR (DMSO-d6, 300 MHz) ppm: ꢁ ¼ 2.38 (s, 3H, CH3), 4.22 (br.
s, 2H, NH2 exchangeable), 7.0 (br. s, 1H, NH exchangeable), 7.46
(d, J ¼ 8.4, 2H, Ar–H), 7.6 (m, 2H, Ar–H), 7.70 (m, 2H, Ar–H),
7.84 (d, J ¼ 8.2, 2H, Ar–H), 7.90 (s, 1H, N¼CH pyrazole), 8.20
(d, J ¼ 8.4, 2H, Ar–H), 9.0 (d, J ¼ 9.3, 2H, Ar–H), 9.05 (s, 1H,
Ar–H), 11.48 (s, 1H, -N¼CH); 13C NMR (DMSO-d6,75 MHz):
ꢁ ¼ 21.2 (CH3), 125.5–142.8 (Ar. C + C-3), 149.3 (CH¼N), 148.0
(C-5), 151.6 (C-4), 164.2 (C¼O), Anal. Calcd. for C26H21N5O3S
(483.14): C, 64.58, H, 4.38, N, 14.48; Found C, 64.47, H, 4.25, N,
14.55%.
5-(5-Amino-1-tosyl-1H-pyrazol-4-yl)-1,3,4-oxadiazole-2(3H)-thione
(12). To a solution of 10 (3.37 g, 0.01 mol) in ethanol (20 ml)
was added a potassium hydroxide solution (0.56 g, 0.01 mol) in
water (2 ml) and carbon disulphide (4 ml). The mixture was
heated under reflux for 10 h. The solvent was evaporated and
the residue was dissolved in water, filtered, and the filtrate
was acidified with dilute hydrochloric acid with continuous
stirring. The precipitate that formed was filtered off, washed
with water and recrystallized from ethanol to yield 2.7 g (80%) 12
as a yellow solid; m.p. 140–141 ꢀC; IR (KBr) cmꢁ1: ꢀ (NH2)
3252, ꢀ (NH) 3141, ꢀ (C¼S) 1670, ꢀ (C¼N) 1600; 1H NMR
(DMSO-d6, 300 MHz): ꢁ ¼ 2.34 (s,3H, CH3), 4.20 (br. s, 2H, NH2
exchangeable), 7.1 (br. s, 1H, NH exchangeable); 7.46 (d, 2H,
J ¼ 8.1 Hz, Ar–H), 7.84 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.90 (s, 1H,
pyrazole N¼CH),13C NMR (DMSO-d6, 75 MHz): ꢁ ¼ 21.2 (CH3),
General
procedure
for
preparing
compounds
(16, 19). A mixture of compounds 15 or 18 (0.01 mol) and
thioglycolic acid (0.01 mol) in benzene (30 ml) was stirred at
room temperature for 30 min, then refluxed for 10 h, cooled to
room temperature. The solvent was removed under reduced
pressure and the solid that obtained was recrystallized from the
proper solvent to yield 16 and/or 19.
98.2 (C-4), 128–142 (Ar. C + C-3), 148.0 (C-5), 156.5 (C¼N 5-Amino-N-(2-(4-(dimethylamino)phenyl)-4-oxothiazolidin-3-yl)-
imine), 190 (C¼S) ppm; Anal. calcd. for: C12H11N5O3S2 1-tosyl-1H-pyrazole-4-carboxamide (16). This compound was
(337.38): C, 42.72; H, 3.29; N, 20.76; Found: C, 42.65; H, obtained as a yellow powder (ethyl acetate) 1.9 g (37.95%)
3.35; N, 20.80%.
m.p. 209–210 ꢀC, IR (KBr) cmꢁ1: ꢀ (NH2) 3458, ꢀ (NH) 3266,

4
I. F. Nassar et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
1
ꢀ (C¼O) 1690, ꢀ (C¼N) 1610; H NMR (DMSO-d6, 300 MHz) violet and 50% methanol then made up to volume with DD water
ppm: ꢁ ¼ 2.39 (s, 3H, CH3), 3.0 (s, 6H, 2CH3), 3.6 (s, 2H, CH2), and filtered through Whatmann filter paper. The Colon cancer
4.22 (br. s, 2H, NH2 exchangeable), 5.20 (s, 1H, thiazolidine CH), cells (HCT) and Breast cancer cells (MCF-7) were propagated in
6.90 (d, 2H, J ¼ 8.4 Hz, Ar–H), 7.0 (br. s, 1H, NH exchangeable), Dulbecco’s modified Eagle’s medium (DMEM) supplemented
7.2 (d, 2H, J ¼ 8.2 Hz, Ar–H), 7.47 (d, 2H, J ¼ 8.4 Hz, Ar–H), 7.84 with 10% heat-inactivated fatal bovine serum, 1% glutamine,
(d, J ¼ 8.2, 2H, Ar–H), 7.9 (s,1H, pyrazole N¼CH); 13C NMR HEPES buffer and 50 mg/ml gentamycine. All cells were main-
(DMSO-d6,75 MHz): ꢁ ¼ 21.2 (CH3), 39.4 (CH2), 41.0 (2CH3), tained at 37 ꢀC in a humidified atmosphere with 5% CO2 and were
64.2 (CH), 128.0–145.0 (Ar. C + C-3), 148.0 (C-5), 151.5 (C-4), subtracted two times a week.
165.0 (C¼O), 169 (C¼O); Anal. Calcd. for C22H24N6O4S2
(500.59): C, 52.78, H, 4.83, N, 16.79; Found C, 52.69, H, 4.79, X-ray crystallography
N, 16.85%.
A single crystal of compound 3 was obtained by slow evaporation
from a solution of ethanol. The crystal structure was solved and
refined using data collection. The chemical structure and ring-
5-Amino-N-(2-(4-(anthracen-9-yl)-4-oxothiazolidin-3-yl)-1-tosyl-
1H-pyrazole-4-carboxamide (19). This compound was obtained
as a yellow powder (ethanol) 2.5 g (44.83%) m.p. 200–201 ꢀC, IR
labeling system is shown in Figure 1. Crystallographic data
(KBr) cmꢁ1: ꢀ (NH2) 3456, ꢀ (NH) 3266, ꢀ (C¼O) 1670, (C¼N)
(excluding structure factors) for the structure in this paper have
been published40.
1600; 1H NMR (DMSO-d6, 300 MHz) ppm: ꢁ ¼ 2.34 (s, 3H, CH3),
3.6 (s, 2H, CH2), 4.22 (br. s, 2H, NH2 exchangeable), 5.13 (s,1H,
CH), 7.0 (br. s, 1H, NH exchangeable), 7.13 (d, J ¼ 7.8, 2H,
Results and discussion
Ar–H), 7.33 (d, J ¼ 7.8, 2H, Ar–H), 7.46 (d, 2H, J ¼ 8.4 Hz, Ar–H),
Chemistry
7.9 (s,1H, pyrazole N¼CH), 8.20 (m, 2H, Ar–H), 8.40 (d, J ¼ 8.1,
2H, Ar–H), 8.60 (s, 1H, Ar–H), 8.74 (d, J ¼ 8.2, 2H, Ar–H); 13C
Reaction of p-toluenesulfonylhydrazide (1) with (E)-ethyl
2-cyano-3-ethoxyacrylate (2) in absolute ethanol under reflux
NMR (DMSO-d6,75 MHz): ꢁ ¼ 21.2 (CH3), 36.4 (CH2), 64.2 (CH),
afforded the pyrazole derivative 3.
The structure of compound 3 was evidenced from its spectral
125.0–142.0 (Ar. C + C-3), 148.0 (C-5), 151.0 (C-4), 164.2 (C¼O),
168.0 (C¼O); Anal. Calcd. for C28H23N5O4S2 (557.64): C, 60.31,
and analytical data. Its IR spectrum showed bands at 3483, 1693
H, 4.16, N, 12.56; Found C, 60.30, H, 4.10, N, 12.55.
and 1615 cmꢁ1 corresponding to NH2, C¼O and C¼N groups
1
Biology
respectively, its H NMR spectrum showed signals at ꢁ 1.29 ppm
as triplet (3H) and a quartet at ꢁ 4.18 ppm (2H) indicating the
presence of the ethyl ester group and also showed a singlet at
ꢁ 2.34 ppm (3H) for the tosylmethyl group, in addition to an
exchangeable singlet for NH2 at 4.22 ppm. The CH of the pyrazole
at position 3 was appeared as a singlet at ꢁ 7.90 ppm and the
phenyl ring protons were appeared as two doublets at 7.46 and
7.84 ppm with J coupling 8.1 Hz. 13C NMR of compound 3
supported its structure. The mass spectrum of 3 revealed the
presence of the molecular ion peak at m/z 309 (M+, 8.37%).
Moreover, the X-ray diffraction40 of 3 (Figure 1) added a clear
evidence for its proposed structure following the cyclization
pathway of the intermediate outlined in Scheme 1.
Materials used in the study
EAC cell lines were supplied from National Cancer Institute,
Cairo, Egypt. Colon cancer (HCT-29) and Breast cancer (MCF-7),
cell lines were obtained from Centre of Mycology and
Biotechnology, Al-Azhar University, DMSO, crystal violet and
trypan blue dye were purchased from Sigma (St. Louis, MO).
DMEM, RPMI-1640, Fatal bovine serum (FBS), HEPES buffer
solution, L-glutamine, gentamycin and 0.25% trypsin-EDTA were
purchased from (BioWhittaker@ Lonza, Belgium). Anticancer
activity was performed at Centre of Mycology and Biotechnology,
Al-Azhar University, Nasr city, Cairo, Egypt.
When compound 3 was refluxed with formic acid, the
pyrazolo(3,4-d1,3)oxazinone derivative 4 was obtained. IR spec-
trum of 4 showed the band of C¼O of the oxazinone ring at
1644 cmꢁ1. Its 1H NMR spectrum displayed a singlet at 8.13 ppm
of the CH of the oxazinone ring and 13C NMR spectrum of
4 showed a signal at ꢁ 154.0 ppm of the C¼O group. However,
refluxing compound 3 with formamide afforded the pyrazolo(3,4-
d)pyrimidin-4(5H)-one derivative 5. Its IR spectrum revealed
bands at 3164, 1700 and 1590 cmꢁ1 characteristic for NH, C¼O
and C¼N groups respectively, while its 1H NMR spectrum
showed an exchangeable singlet signal of the NH proton at
Evaluation of cytotoxic effects of the chemical compounds
Method of trypan blue exclusion cytotoxicity assay. The in vitro
cytotoxicity of the newly synthesized pyrazole derivatives were
subjected to a preliminary screening for their anti-tumor activity
in breast mouse tumor (EAC) using trypan blue exclusion
method39. Stock solution of each of the tested samples was
prepared of 100 mg of the tested material dissolved in 100 mL
DMSO and 900 mL saline. From the stock solution, various
dilutions of the tested materials were prepared. EAC cells were
obtained by needle aspiration of Ascite under aseptic conditions.
10% of the tumor cell suspension (2.5 ꢂ 106) was prepared in
saline (0.1 ml Ascite + 0.9 ml saline). From the stock solution,
five concentrations of the tested compound (200, 100, 50, 30 and
10) mg/ml were prepared in five sterile test tubes, then 100 mL of
10% Ascite suspension was added, the volume was then
completed to 1 ml by adding saline solution. The test tubes
were incubated at 37 ꢀC for 1 h. The supernatant was discarded
from each tube, and one drop of trypan blue solution was added to
the pellet. The number of viable cells were counted for trypan
blue dye exclusion in a hemocytometer and examined by light
microscopy. The viability assay for each compound was per-
formed in triplicate.
Cell viability assay for human cell lines using crystal
violet. Crystal violet stain (1%) is composed of 5% (w/v) crystal
Figure 1. ORTEP Diagram of compound 3.

DOI: 10.3109/14756366.2014.940936
Molecular modeling of pyrazolo[3,4-d]pyrimidine and pyrazole derivatives
5
ꢁ 8.0 ppm in addition to a singlet at 9.0 ppm attributed to the CH derivative 6b respectively. The structures of compounds 6a, b
of the pyrimidine ring. 13C NMR spectrum of 5 showed a signal were confirmed by studying their IR, 1H NMR, 13C NMR spectra
of the C¼O group at 157.6 ppm in addition to the other signals for and elemental analyses which are in accord with the assigned
carbons of the fused rings.
structures cf. experimental. While refluxing 3 with NaOH (10%
On the other hand, fusion of 3 with urea and/or thiourea at solution) followed by acidification with HCl afforded the acid
150 ꢀC yielded the pyrazolo(3,4-d)pyrimidine-4,6-dione deriva- derivative 7, which upon heating with Ac2O gave the pyr-
tive 6a and/or 6-thioxo-pyrazolo(3,4-d)pyrimidine-4(5H)-one azolo(3,4-d1,3)oxazin-4(1H)-one derivative 8. Furthermore,
O
S
Scheme 1. A proposed mechanism for the
reaction of 4-methylbenzenesulfonylhydra-
zide (1) with (E)-ethyl 2-cyano-3-ethoxya-
crylate (2) at refluxing temperature.
O
O
S
NH
O
O
S
NH
HN
NH
O
H N
1
2
HN
CO Et
2
CO Et
2
O
2
O
C
C
N
O
NH
CO Et
C
NH
2
I
II
O
S
O
O
O
H
S
N N
N N
O
NH
2
NH
2
CO Et
2
CO Et
2
3
III
O
O
N
N
HCO H
N
2
S
O
N
O
O
4
NH
N
HCONH
2
N
S
5
O
O
O
CO Et
2
NH
X
N
N
N
N
N
H
6
X
H N
NH
NH
2
2
2
N
S
N
H
O
X = O, S
S O
O
N
N
O
DMF
NH
O
9
2
CO H
2
S
O
NH
NH
2
3
O
N
NH
2
i) NaOH
ii) HCl
2
N
O
N
N
S O
O
N
Me
7
S
O
8
Ac O
O
2
O
CONHNH
2
HN
HC
N
NH -NH . H O
N
2
2
2
D-glucose
EtOH, AcOH
H
HO
H
OH
H
NH
N
2
N
N
H N
2
S O
O
OH
OH
O
O
S
10
H
CH OH
2
11
Scheme 2. Preparation pathways for compounds 4–11.

6
I. F. Nassar et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
reaction of 7 with o-phenylenediamine in DMF at reflux ethanol gave the pyrazolothione derivative 13 in a good yield. The
temperature afforded the benzo(d)imidazolopyrazole derivative IR spectra of 12 and 13 revealed bands attributable to NH2, NH
9. The structures of compounds 7, 8 and 9 were confirmed by IR, and C¼N groups, respectively. Further support for their assigned
1H NMR, 13C NMR spectra and elemental analyses cf. structures is gained from their 1H NMR that revealed the presence
experimental.
of signals of NH2, NH groups in 12 as well as one additional
Reaction of 3 with hydrazine hydrate in absolute ethanol at singlet signal for NH2 group at 2.0 ppm for compound 13.
reflux temperature yielded the acid hydrazide derivative 10. Its IR Their 13C NMR spectra correspond very well with their
spectrum showed absorption bands of NH2, NH and C¼O groups assigned structures. Heating of the hydrazide derivative
respectively at 3457, 3219 and 1630 cmꢁ1. The 1H NMR 10 with 4-dimethylaminobenzaldehyde 14 and/or anthracene-
spectrum of 10 showed a singlet at ꢁ 8.0 ppm corresponding to 9-carbaldehyde 17 in ethanol and in the presence of a catalytic
the NH group and a singlet at ꢁ 5.8 ppm of the –NH2 group of the amount of acetic acid, the Shiff’s bases 15, 18 were afforded in
hydrazide. The 13C NMR spectrum of 10 showed a signals of the good yields, which upon reaction with thioglycolic acid in
C¼O group at 165.0 ppm.
benzene at reflux temperature afforded the thiazolidinone
The hydrazide 10 is used as a key starting material for the derivatives 16, 19 in good yields (Scheme 3).
preparation of novel interesting heterocyles and sugar hydrazide.
The structures of compounds 15–19 were evidenced from their
Thus, when compound 10 was allowed to react with D-glucose in IR spectra that revealed bands characteristic for NH2, NH and CO
an aqueous solution of ethanol and a catalytic amount of glacial groups. The 1H NMR and 13C NMR spectra correspond very well
acetic acid, the corresponding hydrazone 11 was obtained. The IR with their assigned structures.
spectrum of 11 showed the bands of the OH groups of the glucose
at 3460–3449 cmꢁ1. Its 1H NMR spectrum displayed the presence Anticancer activity
of the CH2, CH and OH groups of the sugar at their specific
The anticancer activity of the newly synthesized compounds were
regions and so the 13C NMR spectrum showed the presence of the
assessed in mouse tumor model cancer cell line (EAC) and two
sugar hydrazone carbons as outlined in the experimental section
human cancer cell lines, Colon cancer (HCT-29), and Breast
(Scheme 2).
cancer (MCF-7) cell lines, using trypan blue exclusion assay and
On the other hand, refluxing the acid hydrazide 10 with carbon
crystal violet for cytotoxicity assay. These assays are based on the
disulfide in alcoholic KOH afforded 1,3,4-oxadiazole-2(3H)-
alteration in the membrane integrity as determined by the uptake
thione derivative 12. Reaction of 12 with hydrazine hydrate in
of the dye by dead cells, thereby giving a direct indication on cell
H
N
S
N
N
NH
2
N
NH
2
N
S O
O
H
N
S
N
13
O
NH -NH . H O
2
2
2
CS
N
2
NH
2
N
KOH
N
S O
O
S
EtOH
N
O
H
12
HN
O
C NHN
O
O
NHNH
N
2
NH
O
N
2
N
NH
2
N
N
N
S O
O
N
NH
S O
O
N
2
H
S O
O
15
HSCH COOH
2
14
16
10
O
C
H
NHN
S
N
CHO
N
N
NH
2
HN
O
S
O
O
O
HSCH COOH
17
18
N
2
NH
2
N
S O
O
19
Scheme 3. Preparation pathways for compounds 12–19.

DOI: 10.3109/14756366.2014.940936
Molecular modeling of pyrazolo[3,4-d]pyrimidine and pyrazole derivatives
7
viability. Cytotoxicity was considered as anticancer activity. force field with the Steepest Descent followed by truncated
Treatment of cell lines by chemical materials caused inhibition of Newton Conjugate gradient protocol. Partial atomic charges were
the growth of cells.
computed using the OPLS-2005force field. The LigPrep is a
utility in Schrodinger software suite that combines tools for
generating3D structures from 1D (Smiles) and 2D (SDF)
representation, searching for tautomers and steric isomers and
geometry minimization of ligand. Finally, different poses had
been prepared with different tautomeric and steric features for
docking studies.
Cytotoxicity evaluation
The cells were seeded in 96-well plate at a cell concentration of
1 ꢂ 104 cells per well in 100 mL of growth medium. Fresh medium
containing different concentrations of the tested samples was
added after 24 h of seeding. Serial two-folds dilutions of the tested
chemical compounds were added to confluent cell monolayer
dispensed into 96-well, flat-bottomed microtiter plates were
incubated at 37 ꢀC in a humidified incubator with 5% CO2 for
48 h. Three wells were used for each concentration of the test
sample. Control cells were incubated without test sample and/or
without DMSO. (NB: The low DMSO percent used in the assay
(maximal 0.1%) was found to be not effective). After incubation
of cells for 24 h at 37 ꢀC, various concentrations of samples (1.56,
3.125, 6.25, 12.5, 25, and 50) mg were added. Then the incubation
continued for 48 h and the viable cells yield were determined by
using trypan blue exclusion assay. All experiments were carried
out in triplicate. The cytotoxicity of each compound was
calculated, and the percentage of viable cells was plotted against
the concentration. The preliminary investigation of the anticancer
activity of the newly synthesized pyrazole derivatives was
screened against EAC. The IC50 values of these compounds
were determined compared to Doxorubicin.
Protein preparation. The X-ray crystal structure of Cyclin
Dependent Kinase2 (CDK2) was obtained from the RCSB protein
data bank41 (pdb: 3pj8). After evaluating numbers of entries, the
best protein was selected by analyzing the protein with
Table 1. Docking score, binding energy and IC50 of the new compounds
against mouse tumor model (EAC), HCT (colon) and MCF-7 (breast
cancer cell lines).
IC50
(mg/ml)
(EAC)
IC50
(mg/ml)
(HCT)
IC50
(mg/ml)
(MCF-7)
Tested
compounds
Docking
score
Binding
energy
4
5
6a
7
9
10
12
15
18
19
200
200
85
200
191
144
186
180
33
ꢁ6.6
ꢁ1.5
ꢁ8.3
ꢁ6.5
ꢁ5.0
ꢁ5.48
ꢁ5.6
ꢁ4.6
ꢁ5.9
ꢁ4.6
ꢁ51.125
ꢁ54.981
ꢁ56.097
ꢁ55.761
ꢁ46.954
ꢁ57.263
ꢁ44.426
ꢁ63.754
ꢁ87.567
ꢁ56.904
29.58
10.8
24.34
10.24
Results and discussion for biology
The data expressed in (Table 1 and Figure 2) represents the
cytotoxicity effect of the two compounds 6a and 18 in colon
cancer cells (HCT), the data showed that compound 18 exhibit the
most potent antitumor activity (IC50 ¼ 10.24 m/ml) as compared to
compound 6a (IC50 ¼ 29.58 m/ml). The anticancer activity of the
two compounds were also screened against breast cancer cell lines
(MCF-7), in which both compounds exhibited anticancer activity
with IC50 values (18 ¼ 10.24 m/ml and 6a ¼ 24.34 m/ml) respect-
ively (Table 1, Figure 3).
From the data we concluded that the higher anticancer
activity of compound 18 is attributed to the presence of the
tosyl, C¼N, and C¼O groups in addition to NH2 group that
facilitates the H-Bonding with the active sites which increase its
reactivity.
200
120
100
80
60
40
20
0
6a
18
Molecular modeling
Ligand preparation. The structures of the newly synthesized
compounds were drawn by using ChemDraw and converted to 3D
structure with the help of 3D optimization tool. By using the
LigPrep, the drawn ligand was geometry optimized by using the
Optimized Potentials for Liquid Simulations-2005(OPLS-2005)
0
1.56
3.125
6.25
12.5
25
50
Concentration (µg/ml)
Figure 2. In vitro cytotoxicity of the compounds 6a and 18 on human
cancer cell line (HCT).
120
100
80
60
40
20
0
Figure 3. In vitro cytotoxicity of the
compounds 6a and 18 on breast cancer cell
line (MCF-7).
6a
18
conc.
0
1.56
3.125
6.25
12.5
25
50
Concentration (µg/ml)

8
I. F. Nassar et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure 4. Electrostatic and hydrogen bond interactions of compound 18 with active site of CDK2.
Figure 5. Schematic interaction of compound
18 with amino acids in the active pocket of
CDK2.
Ramachandran plot and ProCheck using SAVS server based on Docking studies. The docking studies on compounds prepared
ligand and number of disallowed regions42,43. After selection, through LigPrep were carried out in the active site of the protein.
protein preparation wizard of Schrodinger suite44 has been used to Receptor Vander Waals scaling for the non-polar atoms was
prepare protein. The protein was preprocessed separately by set to 0.9 which makes the protein site ‘‘roomier’’ by moving
deleting the substrate cofactor as well as the crystallographic ally back the surface of non-Polar Regions of the protein and
observed water molecules (water without H bonds), correcting the ligand. This kind of adjustments emulate to some extent the
mistakes in PDB file, optimizing hydrogen bonds. After assigning effect of breathing motion to the protein site, it is a kind of
charge and protonation state finally energy minimization with root giving breathing to the receptor, this approach softens the active
mean square deviation (RMSD) value of 0.30 A was done using site region of the receptor making it flexible45. The prepared
˚
OPLS2005 force field.
protein and the ligand were employed to build energy grids

DOI: 10.3109/14756366.2014.940936
Molecular modeling of pyrazolo[3,4-d]pyrimidine and pyrazole derivatives
9
Figure 6. Hydrogen bond interactions of compound 18 with active site of CDK2.
˚
using the default value of protein atom scaling (1.0 A) within a Acknowledgements
cubic box, centered on the centroid of the X-ray ligand pose.
After Grid generation, the ligand was docked with the protein
by using Glide 5.5 module in extra precision mode (XP) which
The authors thank Prof. Dr. Peter G. Jones Institut fur Anorganische und
Analytische Chemie, Technische Universitat, Braunschweig, Postfach
3329, 38023 Braunschweig, Germany for solving the crystal structure for
uses MCSA (Monte Carlo Based Simulated Algorithm) based Ethyl 5-amino-1-(4-methylbenzenesulfonyl)- pyrazole-4-carboxylate (3).
minimization. The best docked pose (with lowest Glide Score
value) obtained from Glide was analyzed46,47. The binding
energy was calculated by Prim module. All the data are shown
in Table (1). Figures 4–6 represents the Electrostatic and
responsible for the content and writing of this article.
hydrogen bond interactions of compound 18 with active site of
Declaration of interest
The authors report no conflicts of interest. The authors alone are
CDK2 and the interaction with amino acids in the active pocket of
CDK2.
References
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2. Gursoy SA, Demirayak S, Capan G, Vural K. Synthesis and
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preliminary evaluation of new 5-pyrazolinone derivatives as anal-
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crystallographic data. Reactions of this substrate with urea,
gesic agents. Eur J Med Chem 2000;35:359–64.
3. Tsuji K, Nakamurana K, Konishi N, et al. Studies on anti-
thiourea, formamide and formic acid furnished the bicyclic
pyrazolooxazine and pyrazolo-pyrimidine ring systems. The
hydrazide derivative was used as a key compound for synthesizing
1,3,4-oxadiazole, 1,2,4-triazole, thiazole and glucoside deriva-
tives. Among the compounds screened, compound 18 showed the
most potent anticancer activity and compound 6a exhibited
moderate anticancer activity towards (EAC) cancer cell line,
while the rest of the other tested compounds exerted week
anticancer activity. The two compounds 6a and 18 were sub-
jected to further evaluation of their antitumor activity in the two
human cell lines (HCT-29 and MCF-7) using Crystal violet
cytotoxicity assay and showed good to high activities. The results
from docking study also represent that compound 18 had the
more Electrostatic and hydrogen bond interactions with active site
of CDK2.
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