Single step synthesis of new fused pyrimidine derivatives and their evaluation as potent Aurora-A kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.05.033

A simple, facile, efficient and one pot three-component procedure for the synthesis of pyrazolo[1,5-a]pyrimidines, triazolo[1,5-a]pyrimidines and pyrimido[1,2-a]benzimidazoles ring systems incorporating phenylsulfonyl moiety was developed via the reaction

Full text of DOI:10.1016/j.ejmech.2011.05.033

European Journal of Medicinal Chemistry 46 (2011) 3690e3695
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Single step synthesis of new fused pyrimidine derivatives and their evaluation
as potent Aurora-A kinase inhibitors
,
b
,
c,
Mohamed R. Shaaban a , Tamer S. Saleh d, Abdelrahman S. Mayhoub e, Ahmad M. Farag b
*
a Department of Chemistry, Faculty of Applied Science, Umm Al-Qura University, Makkah 21955, Saudi Arabia
b Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt
c Department of Green Chemistry, National Research Center, Dokki, Giza 12622, Egypt
d Department of Chemistry, Faculty of Science, King AbdulAziz Univeristy, Jeddah 21589, Saudi Arabia
e Department of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple, facile, efficient and one pot three-component procedure for the synthesis of pyrazolo[1,5-a]
pyrimidines, triazolo[1,5-a]pyrimidines and pyrimido[1,2-a]benzimidazoles ring systems incorporating
phenylsulfonyl moiety was developed via the reaction of 1-aryl-2-(phenylsulfonyl)ethanone derivatives
1aed with the appropriate heterocyclic amine and triethyl orthoformate and evaluated as Aurora-A
kinase inhibitors. The cytotoxic activity of the newly synthesized compounds against HST116 colon
tumor cell line was investigated. 2,7-Diphenyl-6-(phenylsulfonyl)pyrazolo[1,5-a]pyrimidine (4b) and its
p-methoxy analogue 4c were found to be equipotent to Doxorubicin as a reference drug. Molecular
modeling study was carried out in order to rationalize the in vitro anti-tumor results.
Received 2 December 2010
Received in revised form
11 May 2011
Accepted 13 May 2011
Available online 20 May 2011
Keywords:
Aurora-A
Kinase inhibitors
Ó 2011 Elsevier Masson SAS. All rights reserved.
Colon tumor
Pyrazolo[1,5-a]pyrimidine
Triazolo[1,5-a]pyrimidine
Pyrimido[1,2-a]benzimidazole
Structureeactivity relationship (SAR)
Molecular modeling
1. Introduction
mitosis. It is associated with centrosome maturation and separation
and thereby regulates spindle assembly and stability [7]. Over-
Colon cancer is one of the most common tumors that occurs in the
western world and usually ranks high in incidence and mortality
among new malignant cases [1]; there isa strongcorrelation between
aurora kinase expressions and colon carcinoma. The expression of
aurora kinase protein was found to be related to grade of tumor, p16
expression, and telomerase activity. These findings are important to
select patients for clinical trials of Aurora kinase inhibitors [2e5].
Aurora-A is a member of a family of mitotic serine/threonine
kinases which includes also types B and C. It is implicated with
important processes during mitosis and meiosis whose proper
function is integral for healthy cell proliferation. Its activity peaks
have been observed during the G2 phase to M phase transition in
the cell cycle [6]. Aurora-A kinase plays an important role in
expression or deregulation of Aurora-A has been associated with
a high occurrence of cancer [8]. Deregulation of Aurora-A may lead
to cancer because it is required for the completion of cytokinesis.
If the cell begins mitosis, duplicates its DNA, but is then unable
to divide into two separate cells, it becomes an aneuploid (i.e. it
contains more chromosomes than normal). Aneuploidy is a trait of
many cancerous tumors [9]. Among compounds that can be used as
Aurora kinase inhibitors, pyrazolo[1,5-a]pyrimidine occupy an
important position [10].
In continuation of our recent work aiming at the synthesis of
a variety of heterocyclic systems with remarkable biological
importance [11e26], we report here on the utility of
b-keto-
sulfones as molecular scaffold for the synthesis of pyrazolo[1,5-a]
pyrimidine, 1,2,4-triazolo[1,5-a]pyrimidine and pyrimido[1,2-a]
benzimidazole derivatives. The synthesized compounds were
evaluated as Aurora-A kinase inhibitors and as anti-colon tumor
agents in order to explore new class of compounds that could be
optimized for potent anticancer agents. In addition, docking study
has been carried out to rationalize the biological activity.
* Corresponding author. Department of Chemistry, Faculty of Applied Science,
Umm Al-Qura University, Makkah 21955, Saudi Arabia. Tel.: þ966 545917568;
fax: þ966 255635580.
E-mail address: rabiemohamed@hotmail.com (M.R. Shaaban).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.033

M.R. Shaaban et al. / European Journal of Medicinal Chemistry 46 (2011) 3690e3695
3691
2. Results and discussion
2.1. Chemistry
Treatment of 1-aryl-2-(phenylsulfonyl)ethanones 1aec with the
appropriate heterocyclic amine and triethyl orthoformate in one
pot, afforded the target fused heterocyclic ring systems. Thus, when
compounds 1aec were treated with 5-aminopyrazole derivatives
2aeg and triethyl orthoformate, in the presence of a catalytic
amount of piperidine, they afforded the corresponding 1H-pyrazolo
[1,5-a]pyrimidine derivatives 4ael (Scheme 1). The other possible
structure 5 for the reaction products was ruled out on the basis of
the 1H NMR spectra of the isolated products. For example, the 1H
NMR spectrum of compound 4a, taken as a typical example of the
prepared series, revealed a singlet signal at
d 9.11 assigned for the
pyrimidine CH-2 in structure 4 and not CH-4 in structure 5 (Scheme
1). A further evidence for the structures of the products 4ael stems
from their independent synthesis via condensation of 1aec with
triethyl orthoformate in acetic anhydride and subsequent
condensation of so formed 1-aryl-3-ethoxy-2-(phenylsulfonyl)
Fig. 1. X-ray crystal structure of compound 4b.
or carbonyl functions. Moreover, the 1H NMR spectrum of
compound 8a, taken as a typical example of the prepared series,
prop-2-ene-1-ones
3 with 5-aminopyrazole derivatives 2aeg
which afforded products identical in all respects (m.p., mixed m.p.
and IR) with those obtained from a three-component one step
reaction of the sulfone 1 with the aminopyrazole derivatives 2aeg
and triethyl orthoformate (Scheme 1). Moreover, the proposed
structure of products 4ael was further unequivocally confirmed by
X-ray crystallography of compound 4b, taken as a typical example
of prepared series (Fig. 1).
Similarly, when the sulfone derivatives 1a,b,d were treated with
2-aminobenzimidazole (6), under the same reaction conditions,
they afforded, in each case, only one isolable product. The isolated
products were identified as 2-aryl-3-(phenylsulfonyl)pyrimido[1,2-
a]benzimidazole derivatives 8aec (Scheme 2). The IR spectrum of
the reaction products revealed, in each case, no bands due to amino
revealed two singlet signals at
pyrimidine protons, respectively, in addition to an aromatic
multiplet in region 7.15e7.95. The products 8aec are assumed to
d 2.42 and 9.24 due to methyl and
d
be formed via the addition of the exocyclic amino group in 2-
aminobenzimidazole to the activated double bond in the ethoxy-
methylene derivative 3 to give the non-isolable acyclic intermedi-
ates 7aec, which undergo intramolecular cyclization and
subsequent aromatization via the loss of ethanol and water mole-
cules, under the reaction conditions, to afford the final products
8aec as depicted in Scheme 2.
The sulfone derivatives 1a,b reacted also with 3-amino-1,2,4-
triazole (9) and afforded high yields of 7-aryl-6-(phenylsulfonyl)
[1,2,4]triazolo[1,5-a]pyrimidine derivatives 10a,b (Scheme 3). The
structures of the reaction products were assigned based on their
elemental analyses and spectral data (cf. Experimental part).
O
O
O
O
S
O
S
2.2. Pharmacology
Ph
Ph
Ac2O
R
CH(OEt)3
OEt
O
R
2.2.1. Anticancer activity
1a-c
3
All newly synthesized compounds have been subjected to
biochemical assays; i.e. Aurora-A inhibition activity, and cell-based
assay using HCT116 colon tumor cell line in four deferent concen-
trations. Doxorubicin (AdriablastinaÒ) was used as a reference drug
in both assays. Aurora-A inhibition assay was summarized in
Table 1, and in vitro cytotoxic was summarized in Table 2.
The parameter used in this study is half inhibition concentration
(IC50), was calculated with regard to a saline control group and the
potency was calculated with regard to the percentage of the change
of the Doxorubicin and the tested compounds, as depicted in
Table 1. In the present study, compounds that showed IC50 values
NH2
NH
R1
R2
N
2a-g
Ph
O
N
N
R1
R2
S
O
N
NH2
R1
R2
CH(OEt)3
4a-l
1a-c
R
NH
N
more than 25
mg/ml were considered inactive.
O
O
2a-g
S
N
Ph
N
R2
4a-l
R
R1 R2
R
R
N
a
b
c
d
e
f
H
H
H
H
H
H
Cl
Cl
CH3
H
H
H
H
R1
Ph
O
O
C6H5
R
S
C6H4-OCH3-p
C6H4-Cl-p
5a-l
Ph
H
N
-EtOH
-H2O
CH(OEt)3
N
N
Br CH3
O
S
1a-c
NH2
O
N
N
C6H5
Br
H
O
H
N
H
g
h
i
C6H5
EtO
H
C6H4-OCH3-p
C6H5
HN
CH3
CH3
CH3
CH3
H
N
6
8
j
C6H5
Br
H
7
a, R= H
b, R= Cl
c, R= Br
k
l
C6H4-Cl-p
C6H4-CH3-p
H
Scheme 1. Synthesis of compounds 4ael.
Scheme 2. Synthesis of compounds 8aec.

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M.R. Shaaban et al. / European Journal of Medicinal Chemistry 46 (2011) 3690e3695
Table 2
R
In vitro cytotoxic activity of the newly synthesized compounds and doxorubicin
against HCT116 colon tumor cell line.
Compound
Fraction surviving
Concentration ( g/ml)
IC50 (mg/ml)
Ph
S
H
N
m
N
CH(OEt)3
O
N
N
1a,b
NH2
N
1
2.5
5
10
O
N
Doxorubicin
0.523
0.774
0.683
0.516
0.560
0.529
0.629
0.987
0.824
1.00
1.00
1.00
1.00
0.984
0.950
1.00
0.689
0.786
0.439
0.698
0.455
0.436
0.508
0.373
0.420
0.953
0.794
0.745
0.701
0.665
0.764
0.885
0.905
0.748
0.302
0.377
0.420
0.671
0.430
0.288
0.420
0.253
0.271
0.894
0.778
0.701
0.530
0.382
0.408
0.845
0.889
0.624
0.294
0.316
0.291
0.597
0.377
0.217
0.420
0.183
0.240
0.797
0.744
0.253
0.309
0.333
0.246
0.820
0.790
0.209
0.256
0.243
1.30
>25
N
1
0
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
8a
8b
8c
10a
10b
1.27
1.28
1.64
2.21
1.34
>25
>25
18.00
14.00
9.30
10.50
>25
>25
16.3
1.97
2.15
9
a, R= H
b
, R= Cl
Scheme 3. Synthesis of compounds 10a and 10b.
2.3. Molecular modeling study
Molecular modeling study has been performed in order to
rationalize the obtained biological results and get insight the
ligandeprotein interactions. A molecular docking study was per-
formed on all compounds, which were built with Sybyl 7.1 and
minimized to 0.01 kcal/mol by the Powell method, using Gastei-
gereHueckel charges and the Tripos force fields. The energy-opti-
mized compounds were docked into the active site of Aurora-A
kinase, using GOLD software.
kinase assay came in accordance with the conducted computa-
tional model since it showed that compound 4d has one fifth
inhibition activity of its methoxy analogue 4c (Table 1). On the
other hand, it was assumed that the relatively similar cytotoxic
activity of compounds 4c and 4d (Table 2) and the great difference
in enzymatic inhibition assay (Table 1) could be due to other
factors such as solubility and partition coefficient. This hypothesis
is supported by the biological results of compound 4a which
showed no significant cytotoxic activity in comparison with that of
compounds 4b and 4c (Table 2). However, it revealed only 1e2
fold(s) IC50 values more than 4b and 4c, respectively, in the
enzymatic inhibition assay (Table 1). This led us to the conclusion
that certain degree of lipophilicity is required for the activity, and
it was assumed that increasing the cLog P of compounds might
lead to an increase in their cytotoxic properties. Therefore, Scheme
2 was adopted in which the phenyl group was fused with
heterocyclic ring system and compounds 8aec were synthesized.
Unfortunately, the enzymatic inhibition assay (Table 1) and cell
cytotoxic assay (Table 2) of compounds 8aec showed no activity.
The docking study suggests that the low activity of compounds
8aec may be due to the interference of the fused pyrimido[1,2-a]
benzimidazole moiety with the methyl group of Thr217. The
All active compounds showed relatively similar binding poses;
therefore compound 4b was taken as a representative example of
the series (Fig. 2A). These compounds suggested, by docking study,
to be bound in an allosteric binding site 3e4 A away from ADP
binding site (Fig. 2A). In more details, the two phenyl rings at
position 7 and that attached to sulfone moiety showed a strong
ꢀ
edge-to-face
pep stacking with the phenyl ring of Thr219. This
strong hydrophobic interaction is further supported by two
hydrogen-bonds between sulfone moiety and Gln223 (Fig. 2B).
These two interactions orient the rest of the molecule towards the
magnesium ions deeply in the active pocket allowing the nitrogen
atom at position 4 to create one more hydrogen-bond with
hydroxyl group of Thr213 (Fig. 2B). In this particular example,
compound 4b, there is no definite known role for phenyl ring at
position-2, however, the observed higher activity of the corre-
sponding 4-methoxy derivative 4c (Table 2) could be explained by
the formation of an extra bond with magnesium ion (Fig. 2C). So
far, the conducted computational model suggested that the
methoxy-containing derivative 4c docked deeper than other cor-
responding compounds that lack the methoxy moiety. To test our
computational model, the methoxy group was replaced with
chlorine atom; therefore, compound 4d was synthesized. It was
expected that the chlorine-containing analogue will reveal less
activity than the corresponding methoxy group, since it has less
ability for hydrogen-bond formation than oxygen. The aurora-A
consequences of interference include docking of the compounds
ꢀ
3 A away from original pose and inability for forming
pep
stacking with Thr219. Next, as the fused phenyl group showed no
biological activity, we decided to remove the phenyl group at
position-2 to decrease the cLog P. So that, Scheme 3 was adopted
and compounds 10a,b were synthesized. As expected, decreasing
the lipophilicity led to a decrease in the cytotoxic properties of the
prepared compounds (Table 2).
Table 1
Aurora-A inhibition activity.
The docking study suggests that substitution of phenyl ring at
position-8 (compounds 4gel) could interfere with the C-backbone
of Pro259, Gln260 and Asn261 amino acid residues. However, there
is no definite explanation for the moderate activity of the chlori-
nated compound 10b in comparison with 4gel. It was assumed
that, the lacking of substitution at position-8 may provide more
flexibility in the active pocket. The moderate cytotoxic effect of
compound 10b may be due to the interference with other proteins
rather than the Aurora-A kinase itself. This explanation is sup-
ported by the high IC50 value of compound 10b in Aurora-A kinase
inhibition assay (Table 1).
Compound
IC50
(
m
M)
Compound
IC50 (mM)
Doxorubicin
0.040
0.101
0.051
0.025
0.120
NA
0.039
NA
NA
4i
4j
NA
NA
NA
NA
NA
NA
NA
4a
4b
4c
4d
4e
4f
4k
4l
8a
8b
8c
4g
4h
10a
10b
0.104
9.238
NA: not active.

M.R. Shaaban et al. / European Journal of Medicinal Chemistry 46 (2011) 3690e3695
3693
3. Experimental
3.1. Chemistry
3.1.1. General
All melting points were measured on a Gallenkamp melting
point apparatus (Weiss-Gallenkamp, London, UK). The infrared
spectra were recorded in potassium bromide disks on a pye Unicam
SP 3300 and Shimadzu FT IR 8101 PC infrared spectrophotometers
(Pye Unicam Ltd. Cambridge, England and Shimadzu, Tokyo, Japan,
respectively). The NMR spectra were recorded on a Varian Mercury
VX-300 NMR spectrometer (Varian, Palo Alto, CA, USA). 1H spectra
were run at 300 MHz and 13C spectra were run at 75.46 MHz in
deuterated chloroform (CDCl3) or dimethyl sulphoxide (DMSO-d6).
Chemical shifts were related to that of the solvent. Mass spectra
were recorded on a Shimadzu GCMS-QP 1000 EX mass spectrom-
eter (Shimadzu) at 70 eV. Elemental analyses were carried out at
the Micro-analytical Center of Cairo University, Giza, Egypt.
X-ray crystallography was carried out on Kappa CCD Enraf
Nonius FR 590 diffractometer, National Research Center, Dokki,
Cairo, Egypt.
The sulfone derivatives 1aed [27], 1-aryl-3-ethoxy-2-(phenyl-
sulfonyl)prop-2-ene-1-one 3 [28], pyrazolo[1,5-a]pyrimidines 4iel
[19], and pyrimido[1,2-a]benzimidazoles 8c [19], were prepared
according to the reported literature.
3.1.2. Pyrazolo[1,5-a]pyrimidines 4ael, pyrimido[1,2-a]
benzimidazoles 8aec and triazolo[1,5-a]pyrimidine derivatives
10a,b
3.1.2.1. General procedure
3.1.2.1.1. Method A. A solution of 1-aryl-2-(phenylsulfonyl)
ethanone (1a) (10 mmol) and an equivalent molar ratio of the
appropriate heterocyclic amine 2, 6 or 9 in triethyl orthoformate
(20 mL), in the presence of piperidine (0.3 mL), was heated under
reflux for 4 h. The excess solvent was removed by distillation under
reduced pressure and the residue was left to cool. The precipitated
solid product was collected by filtration, washed with ethanol,
dried and finally recrystallized from DMF/EtOH to afford the cor-
responding pyrazolo[1,5-a]pyrimidines 4ael, pyrimido[1,2-a]
benzimidazole 8aec and triazolo[1,5-a]pyrimidine derivatives
10a,b, respectively.
3.1.2.1.2. Method B. To a mixture of 1-aryl-3-ethoxy-2-(phe-
nylsulfonyl)prop-2-en-1-one 3 [28] (10 mmol) and appropriate
aminopyrazole derivative 2 (10 mmol) in absolute EtOH (25 mL)
were added few drops of piperidine and the reaction mixture was
refluxed for 1 h, then left to cool. The formed solid product was
filtered off, washed with ethanol and recrystallized from EtOH/DMF
to afford products identical in all respects (mp, mixed mp and
spectra) with those obtained by Method A above.
The physical and spectral data of the newly synthesized
compounds 4aeh, 8a,b and 10a,b are listed below.
3.1.2.2. 2-Methyl-7-phenyl-6-(phenylsulfonyl)pyrazolo[1,5-a]pyrimi-
dine (4a). Yield (82%); m.p. > 300 ꢀC; IR (KBr) ymax/cmꢁ1: 1590 (C]
N), 1147, 1318 (SO2); 1H NMR (DMSO-d6):
d
2.31 (s, 3H, CH3), 6.76 (s,
1H, pyrazole-3-CH), 7.13e7.54 (m, 10H, ArH0s), 9.12 (s, 1H, pyrimi-
dine-5-CH); 13C NMR (DMSO-d6):
14.48, 97.64, 120.78, 127.11,
d
127.37, 127.72, 129.02, 129.36, 130.21, 133.56, 140.48, 146.95, 148.33,
149.60, 158.18; MS (m/z): 349 (Mþ); Anal. Calcd for C19H15N3O2S
(349.41): C, 65.31; H, 4.33; N, 12.03; S, 9.18%. Found: C, 65.40; H,
4.36; N, 12.00; S, 9.11%.
Fig. 2. (A and B) Binding mode of proposed 1st ranked pose for compound 4b into the
catalytic site of Aurora-A kinase enzyme; (C) Binding mode of proposed 1st ranked
pose for compound 4c into the catalytic site of Aurora-A kinase enzyme. The important
amino acids were represented as green sticks. Mg ions were represented as gray
spheres.
3.1.2.3. 2,7-Diphenyl-6-(phenylsulfonyl)pyrazolo[1,5-a]pyrimidine
(4b). Yield (75%); m.p. 252 ꢀC; IR (KBr) ymax/cmꢁ1: 1582 (C]N),
1154, 1308 (SO2); 1H NMR (DMSO-d6):
d 7.31 (s, 1H, pyrazole-3-CH),

3694
M.R. Shaaban et al. / European Journal of Medicinal Chemistry 46 (2011) 3690e3695
7.22e7.77 (m, 15H, ArH0s), 9.17 (s, 1H, pyrimidine-5-CH); 13C NMR
(DMSO-d6): 94.98, 120.51, 121.56, 126.29, 127.04, 127.10, 127.56,
128.62, 128.91, 129.47, 130.27, 131.36, 133.46, 140.39, 147.32, 148.57,
150.20, 158.03; MS (m/z): 411 (Mþ); Anal. Calcd for C24H17N3O2S
(411.48): C, 70.05; H, 4.16; N,10.21; S, 7.79%. Found: C, 70.16; H, 4.10;
N, 10.18; S, 7.78%.
3.1.2.10. 4-Phenyl-3-(phenylsulfonyl)pyrimido[1,2-a]benzimidazole
d
(8a). Yield (81%); mp > 300 ꢀC; IR (KBr) ymax/cmꢁ1: 1608 (C]N),
1159, 1311 (SO2); 1H NMR (DMSO-d6): 7.11e8.68 (m, 14H, ArH0s),
d
10.35 (s, 1H, pyrimidine-4-CH); 13C NMR (DMSO-d6):
d 114.05,
119.48, 120.62, 122.917, 127.25,127.30,127.57, 128.71, 128.72, 128.90,
129.27, 133.47, 136.58, 139.83, 141.27, 144.79, 161.48; MS (m/z): 385
(Mþ). Anal. Calcd for C22H15N3O2S (385.44): C, 68.55; H, 3.92; N,
10.90; S, 8.32%. Found: C, 68.65; H, 3.96; N, 10.83; S, 8.26%.
3.1.2.4. 2-(4-Methoxyphenyl)-7-phenyl-6-(phenylsulfonyl)pyrazolo
[1,5-a]pyrimidine (4c). Yield (86%); m.p. 204 ꢀC; IR (KBr) ymax/cmꢁ1
:
1593 (C]N), 1152, 1308 (SO2); 1H NMR (DMSO-d6):
CH3), 7.30 (s, 1H, pyrazole-3-CH), 6.94e7.77 (m, 14H, ArH0s), 9.12 (s,
1H, pyrimidine-5-CH); 13C NMR (DMSO-d6):
55.11, 94.49, 112.40,
d
3.70 (s, 3H,
3.1.2.11. 4-(4-Chlorophenyl)-3-(phenylsulfonyl)pyrimido[1,2-a]benz-
imidazole (8b). Yield (80%); mp > 300 ꢀC; IR (KBr) ymax/cmꢁ1: 1604
d
(C]N), 1159, 1316 (SO2); 1H NMR (DMSO-d6):
d
7.15e8.67 (m, 13H,
ArH0s), 10.33 (s, 1H, pyrimidine-4-CH); 13C NMR (DMSO-d6):
114.06, 119.61, 120.66, 120.96, 122.917, 127.33, 127.43, 127.92,
121.26, 123.82, 125.30, 126.07, 126.26, 127.18, 129.74, 131.58, 133.71,
135.40, 140.51, 145.45, 147.23, 150.30, 158.19; 160.49; MS (m/z): 441
(Mþ); Anal. Calcd for C25H19N3O3S (441.50): C, 68.01; H, 4.34; N,
9.52; S, 7.26%. Found: C, 68.10; H, 4.30; N, 9.49; S, 7.24%.
d
128.92, 130.64, 133.65, 134.45, 135.48, 139.98, 141.35, 144.94,
160.26; MS (m/z): 419 (Mþ); Anal. Calcd for C22H14ClN3O2S
(419.88): C, 62.93; H, 3.36; N, 10.01; S, 7.64%. Found: C, 62.99; H,
3.34; N, 9.95; S, 7.63%.
3.1.2.5. 2-(4-Chlorophenyl)-7-phenyl-6-(phenylsulfonyl)pyrazolo
[1,5-a]pyrimidine (4d). Yield (80%); m.p. 240 ꢀC; IR (KBr) ymax
/
cmꢁ1: 1590 (C]N), 1152, 1308 (SO2); 1H NMR (DMSO-d6):
1H, pyrazole-3-CH), 6.92e7.78 (m, 14H, ArH0s), 9.12 (s, 1H, pyrim-
idine-5-CH); 13C NMR (DMSO-d6):
94.58, 114.29, 121.41, 122.25,
d
7.33 (s,
3.1.2.12. 7-Phenyl-6-(phenylsulfonyl)-[1,2,4]triazolo[1,5-a]pyrimi-
dine (10a). Yield (83%); mp > 300 ꢀC; IR (KBr) ymax/cmꢁ1: 1615 (C]
d
N), 1151, 1312 (SO2); 1H NMR (DMSO-d6):
d
7.36e7.73 (m, 10H,
ArH0s), 8.62(s, 1H, triazole-2-CH), 9.16 (s, 1H, pyrimidine-5-CH); 13C
NMR (DMSO-d6): 124.28, 126.37, 126.77, 127.47, 127.77, 129.01,
123.96, 126.32, 127.01, 127.22, 127.99, 128.21, 131.64, 133.70, 135.52,
140.68, 147.26, 150.39, 158.32, 160.61; MS (m/z): 445 (Mþ); Anal.
Calcd for C24H16ClN3O2S (445.92): C, 64.64; H, 3.62; N, 9.42; S,
7.19%. Found: C, 64.71; H, 3.59; N, 9.40; S, 7.17%.
d
130.81, 133.91, 139.92, 150.52, 153.37, 155.76, 157.74; MS (m/z): 336
(Mþ); Anal. Calcd for C17H12N4O2S (336.37): C, 60.70; H, 3.60; N,
16.66; S, 9.53%. Found: C, 60.79; H, 3.55; N, 16.62; S, 9.53%.
3.1.2.6. 3-Bromo-2-methyl-7-phenyl-6-(phenylsulfonyl)pyrazolo[1,5-
a]pyrimidine (4e). Yield (73%); m.p. 260 ꢀC; IR (KBr) ymax/cmꢁ1
:
3.1.2.13. 7-(4-chlorophenyl)-6-(phenylsulfonyl)-[1,2,4]triazolo[1,5-a]
1596 (C]N),1150,1301 (SO2); 1H NMR (DMSO-d6):
7.16e7.80 (m, 10H, ArH0s), 9.19 (s, 1H, pyrimidine-5-CH); 13C NMR
(DMSO-d6): 22.99, 95.14, 120.51, 120.56, 121.90, 126.42, 127.66,
d
2.31 (s, 3H, CH3)
pyrimidine (10b). Yield (80%); mp > 300 ꢀC; IR (KBr) ymax/cmꢁ1
:
1618 (C]N), 1155, 1313 (SO2); 1H NMR (DMSO-d6):
9H, ArH0s), 8.69 (s, 1H, triazole-2-CH), 9.52 (s, 1H, pyrimidine-5-
CH); 13C NMR (DMSO-d6):
124.36, 125.77, 127.49, 127.97, 129.20,
d 7.32e7.66 (m,
d
128.88, 130.32, 133.51, 140.23, 146.00, 148.17, 149.02, 155.85; MS (m/
z): 428 (Mþ); Anal. Calcd for C19H14BrN3O2S (428.30): C, 53.28; H,
3.29; N, 9.81; S, 7.49%. Found: C, 53.35; H, 3.25; N, 9.78; S, 7.49%.
d
131.44, 134.08, 135.94, 139.94, 149.35, 153.37, 155.70, 157.74; MS (m/
z): 370 (Mþ); Anal. Calcd for C17H11ClN4O2S (370.81): C, 55.06; H,
2.99; N, 15.11; S, 8.65%. Found: C, 55.00; H, 2.97; N, 15.10; S, 8.74%.
3.1.2.7. 3-Bromo-2,7-diphenyl-6-(phenylsulfonyl)pyrazolo[1,5-a]
pyrimidine (4f). Yield (75%); m.p. 240 ꢀC; IR (KBr) ymax/cmꢁ1: 1593
3.2. Pharmacology
(C]N); 1149, 1301 (SO2); 1H NMR (DMSO-d6):
d
7.16e7.70 (m, 15H,
ArH0s), 9.27 (s, 1H, pyrimidine-5-CH); 13C NMR (DMSO-d6):
d
94.88,
3.2.1. In vitro anti-tumor screening
121.51, 122.66, 126.89, 127.14, 127.40, 127.56, 128.72, 128.92, 129.57,
130.57, 131.56, 133.36, 140.49, 147.82, 148.97, 151.20, 159.03; MS (m/
z): 490 (Mþ); Anal. Calcd for C24H16BrN3O2S (490.37): C, 58.78; H,
3.29; N, 8.57; S, 6.54%. Found: C, 58.88; H, 3.25; N, 8.51; S, 5.54%.
All newly synthesized compounds were subjected to the
in vitro disease-oriented primary anti-tumor screening against
human HCT116 colon tumor cell line. The human tumor cell line of
the cancer screening panel was grown in RPMI 1640 medium
containing 5% fetal bovine serum and 2 mM L-glutamine. For
3.1.2.8. 7-(4-Chlorophenyl)-2-phenyl-6-(phenylsulfonyl)pyrazolo
a typical screening experiment, cells were inoculated into 96-well
microtiter plates in 100 mL at plating densities 10,000 cells/well.
After cell inoculation, the microtiter plates were incubated at
37 ꢀC, 5% CO2, 95% air, and 100% relative humidity for 24 h prior to
addition of experimental drugs. After 24 h, two plates were fixed
in situ with TCA, to represent a measurement of the cell pop-
ulation at the time of compound addition. Experimental
compounds as well as Doxorubicin were solubilized in DMSO at
400-fold the desired final maximum test concentration and stored
frozen prior to use. At the time of drug addition, an aliquot of
frozen concentrate was thawed and diluted to twice the desired
final maximum test concentration with complete medium con-
taining 50 mg/mL gentamicin. Additional four 10-fold or 1/2 log
serial dilutions were made to provide a total of four drug
[1,5-a]pyrimidine (4g). Yield (78%); m.p. 220 ꢀC; IR (KBr) ymax
/
cmꢁ1: 1590 (C]N), 1159, 1309 (SO2); 1H NMR (DMSO-d6):
1H, pyrazole-3-CH), 7.32e7.86 (m, 14H, ArH0s), 9.14 (s, 1H, pyrimi-
dine-5-CH); 13C NMR (DMSO-d6):
95.03, 120.39, 120.41, 120.49,
d 7.35 (s,
d
121.58, 126.28, 127.75, 128.64, 128.99, 129.51, 131.29, 131.45, 133.60,
135.27, 140.39, 147.26, 150.14, 158.03; MS (m/z): 445 (Mþ), Anal.
Calcd for C24H16ClN3O2S (445.92): C, 64.64; H, 3.62; N, 9.42; S,
7.19%. Found: C, 64.70; H, 3.60; N, 9.40; S, 7.17%.
3.1.2.9. 7-(4-Chlorophenyl)-2-(4-methoxyphenyl)-6-(phenylsulfonyl)
pyrazolo[1,5-a]pyrimidine (4h). Yield (88%); m.p. 232 ꢀC; IR (KBr)
ymax/cmꢁ1: 1596 (C]N), 1151, 1301 (SO2); 1H NMR (DMSO-d6):
d
3.75 (s, 3H, CH3), 7.31 (s, 1H, pyrazole-3-CH), 6.97e7.79 (m, 14H,
ArH0s), 9.13 (s, 1H, pyrimidine-5-CH); 13C NMR (DMSO-d6):
d
55.12,
concentrations (1, 2.5, 5 and 10 mg/mL) plus control. Aliquots of
94.60, 112.32, 122.26, 123.85, 125.31, 126.27, 126.36, 127.18, 129.74,
132.58, 133.81, 135.44, 141.51, 145.55, 147.33, 151.30, 158.29; 161.49;
MS (m/z): 475 (Mþ); Anal. Calcd for C25H18ClN3O3S (475.95): C,
63.09; H, 3.81; N, 8.83; S, 6.74%. Found: C, 63.18; H, 3.79; N, 8.78; S,
6.72%.
100 mL of these different drug dilutions were added to the
appropriate microtiter wells already containing 100 mL of
medium, resulting in the required final drug concentrations.
Following drug addition, the plates were incubated for an addi-
tional 48 h at 37 ꢀC, 5% CO2, 95% air, and 100% relative humidity.

M.R. Shaaban et al. / European Journal of Medicinal Chemistry 46 (2011) 3690e3695
3695
For adherent cells, the assay was terminated by the addition of
cold TCA. Cells were fixed in situ by the gentle addition of 50 mL of
cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated
for 60 min at 4 ꢀC. The supernatant was discarded, and the plates
were washed five times with tap water and air-dried. Sulforhod-
amine B (SRB) solution (100 mL) at 0.4% (w/v) in 1% acetic acid
was added to each well, and plates were incubated for 10 min at
room temperature. After staining, unbound dye was removed by
washing five times with 1% acetic acid and the plates were air-
dried. Bound stain was recovered with TriseEDTA buffer. Color
intensity was measured in ELISA reader.
original point (0, 0, and 0) of the X, Y, Z axes that were used as the
referenced point of the GOLD docking program.
4. X-ray crystallography
A single crystal of compound 4b was obtained by slow evapo-
ration from a mixture of ethanol: DMF (2:1). The crystal structure
was solved and refined using maxus (nonius, Deflt and MacScience,
ꢀ
Japan) [33] Mo-K
a
radiation (
l
¼ 0.71073 A) and a graphite mono-
chromator were used for data collection.
The chemical formula and ring labeling system are shown in
Fig. 1.
The relation between surviving fraction and drug concentra-
tions is plotted to get the survival curve of HCT116 tumor cell line
after addition of specified compound. The parameter used here is
IC50, was calculated for each trial [29e31].
Crystallographic data (excluding structure factors) for the
structure in this paper have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication number
CCDC 687842. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union, Road, Cambridge CB2 1EZ, UK [fax:
144 (0)1223 336033 or e-mail: deposit@ccdc.cam.ac.uk].
3.2.2. Aurora assays
As prescribed by Fancelli et al. [32], the Aurora proteins were
produced in insect cells as a GST-fusion protein and purified
using GST affinity chromatography and gel filtration. The
biochemical activity of compounds was determined by incuba-
tion with Aurora kinase and substrate, followed by quantitation
of the phosphorylated product. Compounds were 3-fold serially
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at room temperature in the presence of ATP/P [33]
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m
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g
-ATP mix
m
m
g
m
m
m
3
m
m
m
m
m
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ꢀ
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GOLD suggested. The center of the binding domain was defined as