Synthesis of potent anticancer thieno[2,3-d]pyrimidine derivatives

Article abstract of DOI:10.3184/174751912X13333849411283

As part of our program to identify novel cytotoxic agents, various series of hexahydrocycloocta[4,5]thieno[2,3-d] pyrimidines and pyrimidin-4-ones substituted by aryl at the C-2 position together with phenylethylamino, substituted amino, hydrazinyl or arylidenhydrazinyl substituents at the C-4 position were synthesised. These compounds were prepared as bioisosteres of gefitinib, an antitumour drug used for the treatment of gastrointestinal stromal tumours. All compounds exhibited antitumour activity against (HCT 116) cell line in vitro. Eight compounds (IC₅₀: 3.89, 4.65, 6.63, 6.94, 7.89, 9.53, 12.00 and 12.30 μg mL^-1, respectively) exhibited 4.3 to 1.3 fold more potent antitumour activity than imatinib (IC₅₀: 16.93 μg mL^-1). Also, a docking study of the newly synthesised compounds with the active site of CDK2 was described.

Full text of DOI:10.3184/174751912X13333849411283

266 RESEARCH PAPER
MAY, 266–275
JOURNAL OF CHEMICAL RESEARCH 2012
Synthesis of potent anticancer thieno[2,3-d]pyrimidine derivatives
M.M. Kandeel, Ashraf A. Mounir, Hanan M. Refaat* and Asmaa E. Kassab
Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
As part of our program to identify novel cytotoxic agents, various series of hexahydrocycloocta[4,5]thieno[2,3-d]
pyrimidines and pyrimidin-4-ones substituted by aryl at the C-2 position together with phenylethylamino, substituted
amino, hydrazinyl or arylidenhydrazinyl substituents at the C-4 position were synthesised. These compounds were
prepared as bioisosteres of gefitinib, an antitumour drug used for the treatment of gastrointestinal stromal tumours.
All compounds exhibited antitumour activity against (HCT 116) cell line in vitro. Eight compounds (IC50: 3.89, 4.65,
6.63, 6.94, 7.89, 9.53, 12.00 and 12.30 µg mL−1, respectively) exhibited 4.3 to 1.3 fold more potent antitumour activity
than imatinib (IC50: 16.93 µg mL−1). Also, a docking study of the newly synthesised compounds with the active site of
CDK2 was described.
Keywords: hexahydrocycloocta[4,5]thieno[2,3-d]pyrimidines, pyrimidin-4-ones, antitumour activity, docking study
The thieno[2,3-d]pyrimidine system is considered today a
relevant pharmacophore in a wide range of biological activi-
ties, and particularly it was found to be active against different
cancer types.1–6 Certain thieno[2,3-d] pyrimidines were
originally prepared as bioisosters of erlotinib (Tarceva®)7 and
gefitinib (Iressa®)8 (Fig. 1), a 4-substituted pyrimidine and a
4-amino quinazoline derivatives respectively, that had been
approved for the treatment of gastrointestinal stromal tumours
and lung cancer. Several studies indicated that the mechanism
by which the thieno[2,3-d]pyrimidines exert their cytotoxic
activities is strongly related to their inhibitory effect on
many enzymes and mediators, for example, receptor tyrosine
kinases (RTKs)9–11 and cyclin dependent kinases (CDKs).2,3,12,13
Recently, certain thieno[2,3-d]pyrimidines as ATPase inhibi-
tors of heat shock protein 90 (Hsp 90)14,15 were also identified.
Consequently, the thieno[2,3-d]pyrimidine ring system consti-
tutes an attractive target for the design of new anticancer
agents. In this context, a wide structure variation of thieno
[2,3-d]pyrimidines possessing an interesting biological profile
in terms of anticancer activity has been recently synthesised.
The structure variation includes substituted thieno1,10 or
cycloalkyl-fused thieno moiety,4,16,17 aromatic or aliphatic sub-
stitution on pyrimidine C-2,2,3 4-pyrimidone,2,3 4- substituted
amino9,11,17 or 4-hydrazinyl5,12,13 pyrimidine ring systems.
For example, the thieno[2,3-d]pyrimidine I (Fig. 2) showed
cytotoxic effect on leukaemia cell lines,1 whereas the lead
compounds II,2 III5 and IV11 (Fig. 2) exhibited inhibitory
activity against human colon tumour cell.
not previously screened as anticancer. We also introduced cer-
tain aryl substituents at the C-2 position and phenylethylamino,
substituted amino, hydrazine or arylidenhydrazinyl substitu-
ents at the C-4 position to substantiate the possible influence
of such substitution on the in vitro antitumour activities against
human colon carcinoma (HCT 116) cell line. Furthermore,
we also described a docking study of the newly synthesised
compounds with the active site of CDK2.
Results and discussion
The synthetic route to the target compounds is outlined in
Schemes 1 and 2. 2-Amino-4,5,6,7,8,9-hexahydrocycloocta[b]
thiophene-3-carboxamide (3) was selected as our primary
starting material for this series of reactions, and was prepared
via two steps procedure which involved the reaction of cyclooc-
tanone with cyanoacetamide 1 to give α-cyano-α-cyclooctyli-
deneacetamide 2 which was then reacted with sulfur and
diethylamine.18 One of the objectives of this study was the
preparation of compound 6, a structure analogue of compound
I (Fig. 2). Accordingly, compound 3 was cyclised with triethyl
orthoformate and acetic anhydride,18 followed by chlorination
with phosphorus oxychloride,18 to give compound 5, inevitable
for the preparation of the target compound 6. Preparation of
4-(2-phenylethylamino)-5,6,7,8,9,10-hexahydrocycloocta
[4,5]thieno[2,3-d] pyrimidine (6) was accomplished via the
reaction of 5 with 2-phenylethylamine. The 1H NMR spectrum
showed two triplet signals at δ 2.70–2.75 and 3.74–3.80
corresponding to CH2C6H5 and NHCH2 protons respectively.
In addition, NH proton appeared as an exchangeable singlet
signal at δ 6.49.
As part of our program to identify novel cytotoxic agents,
we decided to prepare and evaluate hexahydrocycloocta
[4,5]thieno[2,3-d]pyrimidine derivatives that could be consid-
ered as analogues of the previously reported potent anticancer
thieno[2,3-d]pyrimidines (Fig. 2). In order to explore the role
of the cycloalkyl moiety in the potency of the anticancer
activity, the hexahydrocycloocta ring was selected due to the
fact that hexahydrocycloocta[4,5]thieno[2,3-d]pyrimidine was
Condensation of the amino amide 3 with aromatic aldehydes
in ethanol in the presence of concentrated hydrochloric acid
afforded 7a,b, which were oxidised by heating in nitrobenzene
to yield the 2-substituted hexahydrocyclooctathieno[2,3-d]
pyrimidines 8a–c. Another pathway for the preparation of 8a–
c directly from the amino amide 3 involved the condensation
Fig. 1 Structures of clinically potent anticancer pyrimidines.
* Correspondent. E-mail: hanan-refaat@hotmail.com

JOURNAL OF CHEMICAL RESEARCH 2012 267
Fig. 2 Strategies for structural modifications of leads I, II, III and IV.
Scheme 1 Synthesis of compounds 1–9.

268 JOURNAL OF CHEMICAL RESEARCH 2012
Scheme 2 Synthesis of compounds 10–12.
of 3 with the selected aromatic aldehyde in dry dimethylfor-
mamide in the presence of concentrated hydrochloric acid. The
IR spectra of 7a,b showed two absorption bands at 3390, 3383
and 3188, 3182 cm−1 corresponding to two NH groups.Whereas
the C=O group appeared as an absorption band at 1635, 1641
cm−1, respectively. Further evidence was obtained from the 1H
NMR spectra of 7a,b that showed two exchangeable singlet
signals at δ 5.70, 9.08 and δ 5.72, 8.16 ppm corresponding to
two NH protons while C-2 proton appeared as a singlet signal
at δ 8.29 and δ 8.93 for compounds 7a and 7b, respectively.
ethanol in the presence of acetic acid gave the corresponding
4-arylidenehydrazinyl derivatives 12a–c. The 1H NMR spectra
of compounds 11a–c showed NH2 and NH peaks at around δ
4.63–4.78 and 7.86–8.09, respectively. Whereas the H NMR
spectra of 12a–c revealed the presence of singlet signals at δ
1
8.62–8.76 corresponding to N=CH protons.
Cytotoxic activity.
The in vitro growth inhibitory activity of all the prepared com-
pounds was evaluated using colon carcinoma cell line (HCT
116). For comparison purposes, the cytotoxicity of imatinib
(Gleevec®) (Fig. 1), a standard antitumour drug used for the
treatment of gasterointestinal tract tumours,19,20 was evaluated
under the same conditions. The IC50 (dose of the compound
which caused a 50% reduction of survival values) are shown
in Table 1. The results are represented graphically in Fig. 3.
From the analysis of Table 1, it was found that all compounds
showed significant antitumour activities. Interestingly, com-
pounds 11b, 7a, 12b, 8c, 9c, 8b, 11c and 9a (IC50: 3.89, 4.65,
6.63, 6.94, 7.89, 9.53, 12.00 and 12.30 µg mL−1, respectively)
exhibited 4.3 to 1.3 fold more potent antitumour activity than
imatinib (IC50: 16.93 µg mL−1) and were the most active among
their analogues. Further, compounds 6, 7b, 8a, 9b, 10a, b, c, e,
11a and 12c (IC50: 14.70, 16.80, 17.90, 17.50, 16.40, 19.00,
19.20, 19.30, 18.10 and 15.90 µg mL−1, respectively) showed
comparable cytotoxicity to imatinib. Moreover, compound
12a (IC50: 24.20 µg mL−1) was less active than imatinib and
compound 10d was the least active of all compounds.
1
Whereas the H NMR spectra of compounds 8a–c lacked the
signals corresponding to C-2 proton and showed an exchange-
able singlet signal at δ 12.10–12.84 corresponding to NH
proton which confirmed the structure.
On refluxing 2-aryl-5,6,7,8,9,10-hexahydrocycloocta[4,5]
thieno[2,3-d]pyrimidin-4(3H)-ones (8a–c), with phosphorus
oxychloride, the corresponding 4-chloro derivatives 9a–c was
produced. The IR spectra of compounds 9a–c proved as useful
in tracing the disappearance of the C=O and NH stretching
absorption bands of the parent compounds 8a–c.
The 4-substituted aminothieno[2,3-d]pyrimidine derivatives
10a–e were obtained through the reaction of 9a–c with the
appropriate primary amine in ethanol in the presence of
catalytic amount of triethylamine. The 1H NMR spectra of the
products 10a–e revealed the presence of NH proton exchange-
able signals resonating at the range of δ 3.45–6.78, in addition
to the expected signals corresponding to the different N-substi-
tuted groups which were indicative for the success of the
amination.
Structural changes at the C-2 phenyl group appear to have a
considerable effect on the anticancer activity. Compound 7a,
Hydrazinolysis of 9a–c afforded the 4-hydrazinyl deriva-
tives 11a–c, which upon reaction with aromatic aldeyhdes in

JOURNAL OF CHEMICAL RESEARCH 2012 269
Table 1 IC50 valuesa of compounds 6–12c and imatinib against
was also prepared keeping the optimum two carbon spacer.1
Compound 6, devoid of 2-aryl, was slightly more potent than
10a, 10d and 10e, with 2-aryl substituent.
colon carcinoma cell line (HCT 116)
Compound
a IC50 (µg mL-1)
6
7a
14.70
4.65
Docking study
CDK2 is a target enzyme for a wide range of antitumour drugs
due to its important role in the control of cell cycle by binding
with its associated cyclin that moves the cell from one phase
of the cell cycle to another one.21 Several thieno[2,3-d]
pyrimidines were reported as CDK2 and CDK4 inhibitors.12,13
Therefore we investigated the binding affinities of the newly
synthesised hexahydrocycloocta[4,5]thieno[2,3-d]pyrimidines
using Molecular Operating Environment (MOE 2008.10;
Chemical Computing Group, Canada) as the computational
software, into the target CDK2. The validation of the docking
accuracy was done by docking of the native ligand 4-
(3-hydroxyanilino)-6,7-dimethoxyquinazoline into its binding
site of CDK2. The docked results of the above mentioned
ligand were compared to the crystal structure of the ligand pro-
tein complex where both of them seemed exactly superim-
posed with binding free energy (∆Gb) of –14.07 kcal mol−1.
The ligand exhihited four hydrogen bonds between the pyrim-
idine N-1 and the OH of 4-(3-hydroxyanilino) along with
Asp145, Leu 83 and Lys33 (Fig. 4). These results indicated the
high accuracy of the MOE simulation in comparison with the
biological methods. The vicinity where 4-anilinoquinazoline
was situated was considered as the active site of CDK2.
In the present work, all the prepared new compounds were
docked using a rigid receptor / flexible ligand approach where
the binding affinity was evaluated by the binding free energy
(∆Gb, kcal mol−1) and hydrogen bond interactions. The com-
pounds which revealed the highest binding affinities (in other
words, lowest binding free energies), together with hydrogen
bond interactions within CDK2 are represented in Table 2.
Regarding the hydrogen bond interactions, the more linear
hydrogen bond is likely to be stronger, therefore the hydrogen
bond angle more than 120o is considered to be of a reasonable
strength. Also the formation of more and/or tighter hydrogen
bonds provides higher binding affinities.22,23
7b
16.80
17.90
9.53
8a
8b
8c
6.94
9a
12.30
17.50
7.89
9b
9c
10a
10b
10c
10d
10e
11a
11b
11c
12a
12b
12c
Imatinib
16.40
19.00
19.20
50.00
19.30
18.10
3.89
12.00
24.20
6.63
15.90
16.93
a IC50 (µg mL-1): dose of the compound which caused 50% reduc-
tion of survival.
Values were calculated from dose–response curves done in
triplicates for each compound.
The pyrimidone derivatives 7a,b and 8a–c were deeply
embedded into CDK2 binding site displayed ∆Gb between
–8.31 and –10.81 kcal/mol. Compounds 7a,b and 8c bound to
the active site of CDK2 through 2 hydrogen bonds with Leu
83, whereas compound 8b showed 2 hydrogen bonds with
Asp145 and Asn132. Compound 8a showed one hydrogen
bond with Asn132.
Fig. 3 Cytotoxicity of 6–12c and imatinib against (HCT 116)
cell line.
Docking study showed that the chloro derivatives 9a–c
displayed good ∆Gb between −10.08 and −12.77 kcal mol−1 but
did not bind to the active site of CDK2 through hydrogen
bonds.
The docking for compounds 11a–c exhibited high binding
affinities (∆Gb between –10.63 and −12.02 kcal mol−1) due to
more proper fitting into the target site by three hydrogen bonds
(with Leu 83 for 11a and with Asp145 and Lys33 for 11b)
and two hydrogen bonds (with Asp145 and Lys33 for 11c).
Compounds 12a–c exhibited almost similar binding affinities
(∆Gb between –9.46 and –12.37 kcal mol−1) as compounds
11a–c. Compound 12a exhibited two hydrogen bonds with
Asp145 and Lys33, while compounds 12b,c showed one
hydrogen bond Asp86.
one of the most potent test compounds, included a 4-dimethyl-
aminophenyl group. This observation was in accordance
with the previously reported studies.12,13 Among the 2-aryl-
5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno[2,3-d]pyrimidin-
4(3H)-ones (8a–c) series, interestingly, 8c with C-2 thienyl
substituent was the most active. Our interest was then focused
on introducing substituents on C-4 to detect their effect on
activity. Compounds 9a–c, bearing chloro on C-4 maintained
relatively potent anticancer activity. Introduction of a hydra-
zinyl moiety at the 4-position, compounds 11a–c, improved
the anticancer activity over their 4-oxo analogues. Compound
11b, the most potent derivative of all the synthesised
compounds, had a 2-nitrophenyl substituent.
The 4-substituted amino compounds 6, 10a–e had good
docking scores between –10.39 and –14.33 kcal mol−1 and
bound to CDK2 active site through one hydrogen bond
(compound 6) or two hydrogen bonds (compound 10c) with
Lys33. On the other hand, compounds 10a,b,d,e displayed one
hydrogen bond with Asp86.
Unexpectedly, the 4-arylidenhydrazinyl derivatives 12a–c,
exhibited lower potency than their parent compounds 11a–c
(in contrast to reported studies12,13) however 12b, a 2-nitrophe-
nyl derivative was the most potent of its analogues. Further,
compounds 6 and 10a–e were designed taking in consideration
the previously reported works that signified the 4-phenethyl-
amino and 4-aminopropionic acid, with two carbon spacer,
as potent anticancer agents.1 Also, the 4-aminoethyl derivative
The comparative docking modes of the synthesised
compounds showed that compounds 6, 7a,b and 8a–c formed

270 JOURNAL OF CHEMICAL RESEARCH 2012
Fig. 4 2D and 3D structure of CDK2 complexed with 4-(3-hydroxyanilino)-6,7-dimethoxyquinazoline.
Table 2 The best docking results based on the binding free energy (∆Gb) of compounds docked into binding site of CDK2, the
distances and angles of hydrogen bonds between compounds and amino acids involved in CDK2
a
Hydrogen bonds between atoms of compounds and nucleotides
∆Gb
Compound
Ligand
(kcal mol−1)
Angle (o)
Distance (Ao)
Nucleotides
Atoms of compound
103.3
169.5
164.6
153.8
163.0
160.9
139.4
154.5
131.3
156.1
169.7
146.3
176.5
146.4
–
1.53
2.29
2.80
2.83
2.94
1.68
2.78
1.42
2.21
2.78
1.44
2.80
2.02
2.76
–
Asp145(C=O),
Asp145(C=O),
Leu83(NH),
Lys33(NH2)
Lys33(NH2)
Leu83(NH),
Leu83(C=O)
Leu83(NH),
Leu83(C=O)
Asn132(NH)
Asp145(C=O),
Asn132(NH2)
Leu83(NH),
Leu83(C=O)
–
OH(H-donor)
–14.07
OH(H-donor)
Pyrimidine N-1
OH(H-acceptor)
Pyrimidine N-1
–10.73
–10.34
6
7a
C=O
N-3H
C=O
–10.81
7b
N-3H
C=O
–8.31
–10.17
8a
8b
N-3H
C=O
C=O
–10.48
8c
N-3H
–
–10.08
–12.77
–10.62
–11.03
–11.10
–14.33
9a
9b
9c
10a
10b
10c
–
–
–
–
–
–
–
–
167.2
154.5
141.0
133.7
132.7
149.2
109.2
147.4
108.8
153.4
162.5
159.5
139.4
138.1
164.3
145.0
149.3
164.0
2.16
1.74
2.60
2.27
2.04
2.01
2.36
2.81
3.16
1.39
1.77
2.66
1.60
2.90
1.38
3.07
1.54
1.53
Asp86(C=O)
Asp86(C=O)
Lys33(NH2),
Lys33(C=O)
Asp86(C=O)
Asp86(C=O)
Leu83(C=O),
Leu83(NH),
Leu83(C=O)
Asp145(OH),
Asp145(C=O),
Lys33(NH2)
Asp145(C=O),
Lys33(NH2)
Asp145(C=O),
Lys33(NH2)
Asp86(C=O)
Asp86(C=O)
NH
NH
C=O
OH
NH
–11.65
–10.39
–10.63
10d
10e
11a
NH
NH
NH2(H-acceptor)
NH2(H-donor)
NH
–12.02
11b
NH2(H-donor)
NH2(H-acceptor)
NH2(H-donor)
NH2(H-acceptor)
OH(H-donor)
OH(H-donor)
NH
–10.93
–9.46
11c
12a
–12.37
–10.97
12b
12c
NH
a Binding free energy.

JOURNAL OF CHEMICAL RESEARCH 2012 271
under reflux for 15 h. The reaction mixture was then cooled; the
separated solid was filtered, dried and crystallised from ethanol.
M.p. 90–92 °C; yield 60%; IR (KBr) vmax: 3400 (NH), 1640 (C=N),
hydrogen bond via pyrimidine N-1 or N-3 (similar to the
ligand) in addition to other hydrogen bond via the electronega-
tive C-4 oxo group (compounds 7a,b and 8a–c). Compounds
10a–e, 11a–c and 8a–c displayed hydrogen bond between dif-
ferent nucleotides and the electronegative C-4 substituent only;
on the contrary, C-2 substituent exhibited no hydrogen bond
with different nucleotides. These different binding modes
intensify the role of the C-4 substituent to enhance the binding
affinities into CDK2 binding site and consequently improve
the anticancer activity of the thieno[2,3-d]pyrimidines. The
overall correlation between the growth inhibitory activities
(IC50, µg mL−1) of the synthesised thieno[2,3-d]pyrimidines
against colon carcinoma cell line, cited in Table 1 and the
binding affinities predicted by docking study was excellent
for most of the compounds. However, compound 10d showed
low antitumour activity in spite of the high binding affinity.
Figures 4–7 illustrate docking of ligand and compounds 7a,
11b and 12b respectively.
1
1560 (C=C) cm−1; H NMR (DMSO-d6) δ 1.05–1.20 (m, 2H, CH2),
1.35–1.46 (m, 4H, 2CH2), 1.50–1.60 (m, 2H, CH2), 2.70–2.75 (t, 2H,
J = 7.5 Hz, CH2C6H5), 2.82–2.89 (t, 2H, J = 5.7 Hz, CH2), 2.91–2.96
(t, 2H, J = 6.9 Hz, CH2), 3.74–3.80 (t, 2H, J = 7.5 Hz, CH2NH), 6.49
(s, 1H, NH, D2O exchangeable), 7.17–7.28 (m, 5H, ArH) and 8.31 (s,
1H, C2-H) ppm; MS [m/z, %]: 339 [M+2, 0.95], 338 [M+1, 1.74], 337
[M+, 4.58] and 57 [C4H9, 100]. Anal. Calcd for C20H23N3S (337.46):
C, 71.17; H, 6.86; N, 12.45. Found: C, 70.99; H, 6.50; N, 12.43%.
Synthesis of 2-aryl-1,2,3,5,6,7,8,9,10-octahydrocycloocta[4,5]thieno
[2,3-d]pyrimidin-4(1H)-ones (7a,b); general procedure
The appropriate aldehyde (0.007 mol) was added to a solution of ami-
noamide 3 (1.12 g, 0.005 mol) in absolute ethanol (5 mL) containing
one drop of concentrated hydrochloric acid. The mixture was then
refluxed for 36 h and set aside to cool at room temperature. The sepa-
rated solid was filtered, washed with ethanol, dried and crystallised
from ethanol.
2-(4-Dimethylaminophenyl)-1,2,3,5,6,7,8,9,10-octahydrocycloocta
[4,5]-thieno[2,3-d]pyrimidin-4(1H)-one (7a): M.p. >300 °C; yield
Experimental
1
76%; IR (KBr) vmax: 3390, 3182 (2NH), 1635 (C=O) cm−1; H NMR
Melting points were obtained on a Griffin apparatus and are uncor-
rected. Microanalyses for C, H and N were carried out at the micro-
analytical center, Cairo University. IR spectra were recorded on a
Shimadzu 435 spectrometer, using KBr discs. 1H NMR spectra were
performed on a joel NMR FXQ-200 MHz spectrometer, using TMS as
the internal standard. Mass spectra were recorded on a GCMP-QP1000
EX Mass spectrometer. Progress of the reactions were monitored by
TLC using precoated aluminum sheet silica gel MERCK 60F 254 and
was visualised by UV lamp.
(CDCl3) δ 1.25–1.35 (m, 2H, CH2), 1.40–1.55 (m, 2H, CH2), 1.60–
1.80 (m, 4H, 2CH2), 2.75–2.85 (m, 2H, CH2), 3.05–3.15 (m, 2H, CH2),
3.09 (s, 6H, N(CH3)2), 5.70 (s, 1H, NH, D2O exchangeable), 6.72
(d, 2H, J = 8.0 Hz, ArH), 7.66 (d, 2H, J = 8.1 Hz, ArH), 8.29 (s, 1H,
C2-H) and 9.08 (s, 1H, NH, D2O exchangeable) ppm; MS [m/z, %]:
358 [M+3, 0.97], 357 [M+2, 2.08], 355 [M+, 0.43] and 79 [C6H7, 100].
Anal. Calcd for C20H25N3OS (355.48): C, 67.56; H, 7.08; N, 11.82.
Found: C, 67.94; H, 6.99; N, 11.44%.
2-(2-Nitrophenyl)-1,2,3,5,6,7,8,9,10-octahydrocycloocta[4,5]thi-
eno[2,3-d]pyrimidin-4(1H)-one (7b): M.p. 168–170 °C; yield 63%;
IR (KBr) vmax: 3383, 3188 (2NH), 1641 (C=O), 1517, 1336 (NO2)
cm−1; 1H NMR (CDCl3) δ 1.24–1.40 (m, 2H, CH2), 1.45–1.55 (m, 2H,
4-(2-Phenylethylamino)-5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno
[2,3-d]pyrimidine (6): A mixture of the chloro derivative 5 (0.25 g,
0.001 mol) and 2-phenylethylamine (0.12 g, 0.001 mol) and triethyl-
amine (0.36 mL, 0.003 mol) in absolute ethanol (12 mL) was heated
Fig. 5 The proposed binding mode of compound 7a in the binding site of CDK2, created with Molsoft ICM-Pro software.

272 JOURNAL OF CHEMICAL RESEARCH 2012
Fig. 6 The proposed binding mode of compound 11b in the binding site of CDK2, created with Molsoft ICM-Pro software.
CH2), 2.95–3.10 (m, 2H, CH2), 3.00 (s, 6H, N(CH3)2), 6.74 (d, 2H,
J = 7.2 Hz, ArH), 8.00 (d, 2H, J = 7.2 Hz, ArH) and 12.10 (s, 1H, NH,
D2O exchangeable) ppm. Anal. Calcd for C20H23N3OS (353.46): C,
67.95; H, 6.55; N, 11.88. Found: C, 68.19; H, 6.37; N, 11.68%.
2-(2-Nitrophenyl)-5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno[2,3-d]-
pyrimidin-4(3H)-one (8b): M.p. 170–172 °C (ethanol); yield (method
A) 70%, (method B) 70%; IR (KBr) vmax: 3120 (NH), 1651(C=O),
CH2), 1.60–1.74 (m, 4H, 2CH2), 2.81–2.86 (t, 2H, J = 6.3 Hz, CH2),
3.02–3.08 (t, 2H, J = 6.3 Hz, CH2), 5.72 (s, 1H, NH, D2O exchange-
able), 7.59–7.77 (m, 3H, ArH), 8.09 (d, 1H, J = 7.2 Hz, ArH); 8.16 (s,
1H, NH, D2O exchangeable) and 8.93 (s, 1H, C2-H) ppm; MS [m/z,
%]: 359 [M+2, 1.55], 358 [M+1, 4.53], 357 [M+, 13.54] and 222
[C11H14N2OS, 100]. Anal. Calcd for C18H19N3O3S (357.41): C, 60.48;
H, 5.35; N, 11.75. Found: C, 60.80; H, 5.28; N, 11.55%.
1
1533, 1346 (NO2) cm−1; H NMR (DMSO-d6) δ 1.25–1.35 (m, 2H,
Synthesis of 2-aryl-5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno[2,3-
d]pyrimidin-4(3H)-ones (8a–c); general procedure
Method A: A mixture of aminoamide 3 (2.25 g, 0.01 mol) and the
appropriate aromatic aldehyde (0.03 mol) in dry dimethylformamide
(25 mL) containing concentrated hydrochloric acid (0.2 mL) was
refluxed for 24 h. The mixture was cooled, filtered and the precipitate
was crystallised from the appropriate solvent.
Method B: A mixture of either 7a or 7b (0.001 mol) and nitroben-
zene (5 mL) was refluxed for 5 h. The reaction mixture was subjected
to steam distillation to remove nitrobenzene and the solid obtained
was collected, washed with petroleum ether, dried and crystallised
from the appropriate solvent.
CH2), 1.37–1.45 (m, 2H, CH2), 1.60–1.70 (m, 4H, 2CH2), 2.86–2.90
(t, 2H, J = 6.0 Hz, CH2), 3.07–3.10 (t, 2H, J = 6.0 Hz, CH2), 7.80–
7.89 (m, 3H, ArH), 8.18 (d, 1H, J = 8.1 Hz, ArH) and 12.84 (s, 1H,
NH, D2O exchangeable) ppm; MS [m/z, %]: 356 [M+1, 11.24], 355
[M+, 21.89] and 55 [C4H7, 100]. Anal. Calcd for C18H17N3O3S (355.40):
C, 60.82; H, 4.82; N, 11.82. Found: C, 61.00; H, 5.00; N, 11.59%.
2-(2-Thienyl)-5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno[2,3-d]-
pyrimidin-4(3H)-one (8c): M.p. >300 °C (n-butanol); yield (method
A) 45%; IR (KBr) vmax: 3167 (NH), 1651 (C=O) cm−1; 1H NMR
(DMSO-d6) δ 1.25–1.35 (m, 2H, CH2), 1.38–1.50 (m, 2H, CH2), 1.55–
1.70 (m, 4H, 2CH2), 2.80–2.90 (m, 2H, CH2), 3.00–3.10 (m, 2H,
CH2), 7.19, 7.24 (dd, 1H, J = 5.2, 3.6 Hz, thiophene H), 7.84 (d, 1 H,
J = 5.2 Hz, thiophene H), 8.19 (d, 1H, J = 3.6 Hz, thiophene H) and
12.64 (s, 1H, NH, D2O exchangeable) ppm. Anal. Calcd for
C16H16N2OS2 (316.43): C, 60.72; H, 5.09; N, 8.85. Found: C, 61.05; H,
5.06; N, 8.98%.
2-(4-Dimethylaminophenyl)-5,6,7,8,9,10-hexahydrocycloocta[4,5]-
thieno[2,3-d]pyrimidin-4(3H)-one (8a): M.p. >300 °C (n-butanol);
yield (method A) 51%, (method B) 70%; IR (KBr) vmax: 3160 (NH),
1
1651 (C=O) cm−1; H NMR (DMSO-d6) δ 1.25–1.35 (m, 2H, CH2),
1.36–1.45 (m, 2H, CH2), 1.55–1.70 (m, 4H, 2CH2), 2.80–2.90 (m, 2H,

JOURNAL OF CHEMICAL RESEARCH 2012 273
Fig. 7 The proposed binding mode of compound 12b in the binding site of CDK2, created with Molsoft ICM-Pro software.
Synthesis of 2-aryl-4-chloro-5,6,7,8,9,10-hexahydrocycloocta[4,5]
thieno[2,3-d]pyrimidines (9a–c); general procedure
CH2), 7.74, 7.79 (dd, 1H, J = 7.8 Hz, ArH), 7.80, 7.82 (dd, 1H, J =
7.8 Hz, ArH), 8.00 (d, 1H, J = 7.8 Hz, ArH) and 8.10 (d, 1H, J =
7.8 Hz, ArH) ppm; MS [m/z, %]: 376 [M+3, 11.20], 375 [M+2, 43.00],
374 [M+1, 36.70], 373 [M+, 95.50] and 134 [C7H4NO2, 100]. Anal.
Calcd for C18H16ClN3O2S (373.84): C, 57.82; H, 4.31; N, 11.23.
Found: C, 57.61; H, 4.39; N, 10.88%.
4-Chloro-2-(2-thienyl)-5,6,7,8,9,10-hexahydrocycloocta[4,5]thi-
eno-[2,3-d]pyrimidine (9c): M.p. 148–150 °C; yield 30%; IR (KBr)
vmax: 1556 (C=C) cm−1; 1H NMR (CDCl3) δ 1.20–1.35 (m, 4H, 2CH2),
1.50–1.60 (m, 2H, CH2), 1.70–1.85 (m, 2H, CH2), 2.93–2.96 (t, 2H,
J = 6.2 Hz, CH2), 3.14–3.20 (t, 2H, J = 6.2 Hz, CH2), 7.12, 7.16 (dd,
1H, J = 5.2, 3.6 Hz, thiophene H), 7.48 (d, 1H, J = 5.2 Hz, thiophene
H) and 8.03 (d, 1H, J = 3.6 Hz, thiophene H) ppm. Anal. Calcd for
C16H15ClN2S2 (334.87): C, 57.38; H, 4.51; N, 8.36. Found: C, 57.60;
H, 4.51; N, 8.20%.
Phosphorus oxychloride (15 mL) was added to the corresponding
thienopyrimidone 8a–c (0.003 mol) and the mixture was refluxed for
1 h. The reaction mixture was concentrated under reduced pressure
then poured into ice cold water (100 mL). The precipitated product
was filtered, washed with water (2×10 mL), dried and crystallised
from ethanol.
4-Chloro-2-(4-dimethylaminophenyl)-5,6,7,8,9,10-hexahydrocy-
cloocta-[4,5]thieno[2,3-d]pyrimidine (9a): M.p. 190–192 °C; yield
60%; IR (KBr) vmax: 1606 (C=N), 1556 (C=C) cm−1; 1H NMR (CDCl3)
δ 1.25–1.40 (m, 2H, CH2), 1.45–1.55 (m, 2H, CH2), 1.70–1.90 (m, 4H,
2CH2), 2.92–2.98 (t, 2H, J = 5.6 Hz, CH2), 3.06 (s, 6H, N(CH3)2),
3.14–3.20 (t, 2H, J = 6.0 Hz, CH2), 6.83 (d, 2H, J = 9.0 Hz, ArH) and
8.37 (d, 2H, J = 9.0 Hz, ArH) ppm. Anal. Calcd for C20H22ClN3S
(371.91): C, 64.58; H, 5.96; N, 11.29. Found: C, 64.45; H, 5.68; N,
11.00%.
Synthesis of 2-aryl-4-substituted amino-5,6,7,8,9,10-hexahydrocyclo-
octa[4,5]thieno[2,3-d] pyrimidines (10a–e); general procedure
A mixture of 9a–c (0.001 mol), the selected primary amine (0.001 mol)
and triethylamine (0.36 mL, 0.003 mol) in absolute ethanol (12 mL)
was heated under reflux for 15 h. The separated solid after cooling
was filtered, dried and crystallised from ethanol.
4-Chloro-2-(2-nitrophenyl)-5,6,7,8,9,10-hexahydrocycloocta[4,5]
thieno-[2,3-d]pyrimidine (9b): M.p. 138–140 °C; yield 62%; IR (KBr)
vmax: 1530, 1358 (NO2) cm−1; 1H NMR (DMSO-d6) δ 1.25–1.33
(m, 2H, CH2), 1.45–1.53 (m, 2H, CH2), 1.66–1.80 (m, 4H, 2CH2),
3.03–3.06 (t, 2H, J = 6.0 Hz, CH2), 3.14–3.18 (t, 2H, J = 6.0 Hz,

274 JOURNAL OF CHEMICAL RESEARCH 2012
2-(4-Dimethylaminophenyl)-4-(2-phenylethylamino)-5,6,7,8,9,10-
4-Hydrazinyl-2-(2-nitrophenyl)-5,6,7,8,9,10-hexahydrocycloocta[4,5]-
thieno[2,3-d]pyrimidine (11b): M.p. 236–238 °C; yield 60%; IR
(KBr) vmax: 3304, 3280 (NH / NH2), 1627 (C=N) cm−1; 1H NMR
(DMSO-d6) δ 1.20–1.30 (m, 2H, CH2), 1.45–1.55 (m, 2H, CH2), 1.60–
1.75 (m, 4H, 2CH2), 2.90–2.95 (t, 2H, CH2), 3.00–3.10 (t, 2H, CH2),
4.63 (s, 2H, NH2, D2O exchangeable), 7.69–7.82 (m, 3H, 2ArH and
NH), 7.89 (d, 1H, J = 7.2 Hz, ArH) and 8.25 (d, 1H, J = 7.2 Hz, ArH)
ppm. Anal. Calcd for C18H19N5O2S (369.43): C, 58.51; H, 5.18; N,
18.95. Found: C, 58.79; H, 4.99; N, 18.79%.
hexahydrocycloocta[4,5]thieno[2,3-d]pyrimidine (10a): M.p. 160–
1
162 °C; yield 70%; IR (KBr) vmax: 3456 (NH), 1607(C=N) cm−1; H
NMR (DMSO-d6) δ 1.10–1.20 (m, 2H, CH2), 1.40–1.70 (m, 6H,
3CH2), 2.80–2.90 (m, 2H, CH2), 2.95–3.10 (m, 4H, CH2 and CH2C6H5),
3.00 (s, 6H, N(CH3)2), 3.60–3.80 (m, 2H, NHCH2), 6.51 (s, 1H, NH,
D2O exchangeable), 6.82 (d, 2H, J = 8.2 Hz, ArH), 7.15–7.40 (m, 5H,
ArH) and 8.23 (d, 2H, J = 8.2 Hz, ArH) ppm. Anal. Calcd for
C28H32N4S (456.63): C, 73.64; H, 7.06; N, 12.27. Found: C, 73.64; H,
7.00; N, 12.21%.
4-Hydrazinyl-2-(2-thienyl)-5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno-
[2,3-d]pyrimidine (11c): M.p. 218–220 °C; yield 50%; IR (KBr) vmax
:
4-(Ethylamino)-2-(2-nitrophenyl)-5,6,7,8,9,10-hexahydrocyclo-
3363, 3292 (NH / NH2), 1630 (C=N) cm−1; H NMR (DMSO-d6)
δ 1.10–1.25 (m, 2H, CH2), 1.40–1.50 (m, 2H, CH2), 1.55–1.70 (m, 4H,
2CH2), 2.85–2.90 (m, 2H, CH2), 2.95–3.10 (m, 2H, CH2), 4.78 (s, 2H,
NH2, D2O exchangeable), 7.15 (dd, 1H, thiophene H), 7.65 (d, 1H,
thiophene H), 7.96 (d, 1H, thiophene H) and 8.09 (s, 1H, NH, D2O
exchangeable) ppm. Anal. Calcd for C16H18N4S2 (330.45): C, 58.14;
H, 5.48; N, 16.95. Found: C, 58.34; H, 5.30; N, 16.90%.
1
octa-[4,5]thieno[2,3-d]pyrimidine (10b): M.p. 110–112 °C; yield
1
97%; IR (KBr) vmax: 3385 (NH), 1531,1348 (NO2) cm−1; H NMR
(DMSO-d6) δ 1.13–1.17 (t, 3H, J = 6.9 Hz, CH2CH3), 1.20–1.25
(m, 2H, CH2), 1.45–1.50 (m, 2H, CH2), 1.55–1.70 (m, 4H, 2CH2),
2.87–2.90 (t, 2H, J = 6.0 Hz, CH2), 3.01–3.05 (t, 2H, J = 6.1 Hz,
CH2), 3.47–3.51(m, 2H, J = 6.9 Hz, CH2CH3), 6.78 (t, 1H, J = 6.0 Hz,
NH, D2O exchangeable), 7.64, 7.70 (dd, 1H, J = 7.8 Hz, ArH), 7.73,
7.76 (dd, 1H, J = 7.8 Hz, ArH), 7.83 (d, 1H, J = 7.8 Hz, ArH) and 8.13
(d, 1H, J = 7.8 Hz, ArH) ppm; MS [m/z, %]: 384 [M+2, 21.79], 383
[M+1, 82.60], 382 [M+, 100] and 365 [M-OH, 35.25]. Anal. Calcd for
C20H22N4O2S (382.47): C, 62.80; H, 5.79; N, 14.64. Found: C, 62.90;
H, 5.80; N, 14.52%.
Synthesis of 2-aryl-4-arylidenhydrazinyl-5,6,7,8,9,10-hexahydrocyclo
octa[4,5]thieno[2,3-d]pyrimidines (12a–c); general procedure
A mixture of hydrazino derivative 11a–c (0.003 mol), the selected
aromatic aldehyde (0.003 mol) and few drops of glacial acetic acid in
absolute ethanol (17 mL) was heated under reflux for 6 h. The reaction
mixture was cooled, the separated solid was filtered, dried and crystal-
lised from the appropriate solvent.
4-(2-Hydroxybenzylidenhydrazinyl)-2-(4-dimethylaminophenyl)-
5,6,7,8, 9,10-hexahydrocycloocta[4,5]thieno[2,3-d]pyrimidine (12a):
M.p. 234–236 °C (n-butanol); yield 90%; IR (KBr) vmax: 3420 (OH),
3340 (NH), 1608 (C=N) cm−1; 1H NMR (DMSO-d6) δ 1.20–1.30
(m, 2H, CH2), 1.50–1.60 (m, 2H, CH2), 1.65–1.85 (m, 4H, 2CH2),
2.90–3.10 (m, 2H, CH2), 3.05 (s, 6H, N(CH3)2), 3.20–3.30 (m, 2H,
CH2), 6.83 (d, 2H, J = 7.4 Hz, ArH), 6.99–7.09 (m, 2H, J = 8.6 Hz,
ArH), 7.35 (d, 1H, ArH), 7.50 (d, 1H, ArH), 8.36 (d, 2H, J = 7.4 Hz,
ArH), 8.76 (s, 1H, N=CH), 10.37 (s, 1H, NH, D2O exchangeable)
and 12.41 (s, 1H, OH, D2O exchangeable) ppm. Anal. Calcd for
C27H29N5OS (471.57): C, 68.76; H, 6.19; N, 14.85. Found: C, 68.92;
H, 5.94; N, 14.67%.
3-[(2-Nitrophenyl-5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno[2,3-d]-
pyrimidin)-4-yl]aminopropionic acid (10c): M.p. 140–142 °C; yield
50%; IR (KBr) vmax: 3200–2501 (NH and OH), 1650 (C=O), 1531,
1
1361 (NO2) cm−1; H NMR (DMSO-d6) δ 1.25–1.33 (m, 2H, CH2),
1.45–1.55 (m, 2H, CH2), 1.65–1.80 (m, 4H, 2CH2), 2.22 (t, 2H,
J = 6.3 Hz, CH2COOH), 2.86 (t, 2H, J = 6.3 Hz, NHCH2), 3.04–3.08
(t, 2H, J = 6.0 Hz, CH2), 3.16–3.20 (t, 2H, J = 6.0 Hz, CH2), 3.45
(br s, 2H, OH and NH, D2O exchangeable), 7.75, 7.79 (dd, 1H,
J = 7.8 Hz, ArH), 7.83, 7.88 (dd, 1H, J = 7.8 Hz, ArH) 8.00 (d, 1H,
J = 7.8 Hz, ArH) and 8.11 (d, 1H, J = 7.8 Hz, ArH) ppm; MS [m/z,
%]: 426 [M+, 5.17] and 89 [C3H7NO2,100]. Anal. Calcd for
C21H22N4O4S (426.48): C, 59.13; H, 5.20; N, 13.13. Found: C, 59.10;
H, 5.17; N, 12.85%.
2-(2-Nitrophenyl)-4-(2-phenylethylamino)-5,6,7,8,9,10-hexahydro-
cycloocta[4,5]thieno[2,3-d]pyrimidine (10d): M.p. 92–94 °C; yield
60%; IR (KBr) vmax: 3457 (NH), 1533, 1367 (NO2), 1566 (C=N) cm−1;
1H NMR (CDCl3) δ 1.20–1.40 (m, 4H, 2CH2), 1.42–1.75 (m, 4H,
2CH2), 2.60–2.66 (t, 2H, J = 6.3 Hz, CH2), 2.82–2.88 (t, 2H, J =
6.3 Hz, CH2), 2.96–3.02 (t, 2H, J = 6.4 Hz, CH2C6H5), 3.80–3.89
(q, 2H, J = 6.4 Hz, NHCH2), 5.28(t, 1H, J = 6.2 Hz, NH, D2O
exchangeable), 7.23–7.32 (m, 5H, ArH), 7.50–7.67 (m, 3H, ArH) and
8.19 (d, 1H, J = 7.6 Hz, ArH) ppm. Anal. Calcd for C26H26N4O2S
(458.56): C, 68.09; H, 5.71; N, 12.21. Found: C, 67.99; H, 5.45; N,
12.50%.
2-(2-Nitrophenyl)-4-(2-thienylidenhydrazinyl)-5,6,7,8,9,10-hexahydro-
cycloocta[4,5]thieno[2,3-d]pyrimidine (12b): M.p. 128–130 °C (ethanol);
1
yield 50%; IR (KBr) vmax: 3380 (NH), 1618 (C=N) cm−1; H NMR
(DMSO-d6) δ 1.10–1.20 (m, 2H, CH2), 1.35–1.50 (m, 2H, CH2), 1.55–
1.70 (m, 4H, 2CH2), 2.87–2.91 (t, 2H, J = 6.0 Hz, CH2), 3.01–3.05
(t, 2H, J = 6.0 Hz, CH2), 7.12, 7.15 (dd, 1H, J = 5.0, 3.6 Hz, thiophene
H), 7.44 (d, 1H, ArH), 7.63–7.91 (m, 3H, ArH), 8.10 (d, 1H, J =
7.8 Hz, ArH), 8.23 (d, 1H, J = 7.5 Hz, ArH), 8.66 (s, 1H, N=CH) and
10.25 (s, 1H, NH, D2O exchangeable) ppm. Anal. Calcd for
C23H21N5O2S2 (463.57): C, 59.58; H, 4.56; N, 15.10. Found: C, 59.89;
H, 4.68; N, 15.06%.
4-(2-Phenylethylamino)-2-(2-thienyl)-5,6,7,8,9,10-hexahydrocycloocta-
[4,5]thieno[2,3-d]pyrimidine (10e): M.p. 194–196 oC; yield 80%;
2-(2-Thienyl)-4-(2-thienylidenhydrazinyl)-5,6,7,8,9,10-hexahydro-
1
IR (KBr) vmax: 3464 (NH), 1568(C=N) cm−1; H NMR (DMSO-d6)
cycloocta[4,5]thieno[2,3-d]pyrimidine (12c): M.p. 128–130 °C (ethanol);
1
yield 90%; IR (KBr) vmax: 3342 (NH), 1608 (C=N) cm−1; H NMR
δ 1.15–1.25 (m, 2H, CH2), 1.45–1.60 (m, 4H, 2CH2), 1.65–1.70 (m,
2H, CH2), 2.70–2.85 (m, 2H, CH2), 2.86–2.95 (m, 2H, CH2), 2.96–
3.10 (m, 2H, CH2C6H5), 4.15–4.50 (m, 2H, NHCH2), 6.71 (s, 1H, NH,
D2O exchangeable), 7.20–7.40 (m, 6H, ArH), 7.67 (d, 1H, ArH) and
7.90 (d, 1H, ArH) ppm. Anal. Calcd for C24H25N3S2 (419.58): C, 68.69;
H, 6.00; N, 10.01. Found: C, 68.66; H, 5.71; N, 9.71%.
(CDCl3) δ 1.10–1.25 (m, 2H, CH2), 1.30–1.45 (m, 2H, CH2), 1.50–
1.75 (m, 4H, 2CH2), 2.70–2.80 (m, 2H, CH2), 3.05–3.15 (m, 2H, CH2),
6.90–7.10 (m, 2H, thiophene H), 7.26–7.31(m, 2H, thiophene H),
7.99–8.04 (m, 2H, thiophene H) and 8.62 (s, 1H, N=CH) ppm. Anal.
Calcd for C21H20N4S3 (424.59): C, 59.40; H, 4.74; N, 13.19. Found: C,
59.18; H, 4.70; N, 12.94%.
Cytotoxic activity studies Anticancer activity studies were done
at Cairo University, National Cancer Institute, Cancer Biology
Department, Pharmacology Unit.
Compounds 6–12c were tested at concentrations between 1 and
10 µg mL−1 using SulfoRhodamine-B (SRB) assay for cytotoxic
activity against human colon tumour cell line (HCT116). Imatinib
which is 2-substituted aminopyrimidine derivative was chosen as a
reference standard anticancer drug because it showed potency against
gasterointestinal tract tumours. 19,20
Synthesis of 2-aryl-4-hydrazinyl-5,6,7,8,9,10-hexahydrocycloocta[4,
5]thieno[2,3-d]pyrimidines (11a–c); general procedure
A mixture of chloro derivative 9a–c (0.002 mol) and hydrazine hydrate
(99%, 0.62 g, 0.012 mol) in absolute ethanol (20 mL) was refluxed
for 6 h. The reaction mixture was then cooled and the precipitate was
filtered, dried and crystallised from n-butanol.
4-Hydrazinyl-2-(4-dimethylaminophenyl)-5,6,7,8,9,10-hexahydro-
cycloocta[4,5]thieno[2,3-d]pyrimidine (11a): M.p. 218–220 °C; yield
1
50%; IR (KBr) vmax: 3327, 3300 (NH / NH2), 1608 (C=N) cm−1; H
NMR (DMSO-d6) δ 1.16–1.20 (m, 2H, CH2), 1.40–1.50 (m, 2H, CH2),
1.55–1.66 (m, 4H, 2CH2), 2.85–2.90 (m, 2H, CH2), 2.95–3.05 (m, 2H,
CH2), 2.99 (s, 6H, N(CH3)2), 4.73 (s, 2H, NH2, D2O exchangeable),
6.76 (d, 2H, J = 8.8 Hz, ArH), 7.86 (s, 1H, NH, D2O exchangeable)
and 8.28 (d, 2H, J = 8.8 Hz, ArH) ppm; MS [m/z, %]: 370 [M+3,
2.19], 369 [M+2, 7.41], 368 [M+1, 23.73], 367 [M+, 100], 352
[M-NH, 22.89], 351[M-NH2, 56.36], 323[M-CH3N=CH2-H, 14.36],
and 77 [C6H5, 4.14]. Anal. Calcd for C20H25N5S (367.50): C, 65.35; H,
6.85; N, 19.05. Found: C, 65.55; H, 6.55; N, 18.86%.
Measurement of potential cytotoxicity by SRB assay Potential cyto-
toxicity of the compounds was tested using the method of Skehan
et al24 as follows. Cells were plated in 96 multiwell plate (104 cells/
well) for 24 h before treatment with the compound(s) to allow attach-
ment to the wall of the plate. Different concentrations of the com-
pounds (0, 1, 2.5, 5 and 10 µg mL−1) were added to the cell monolayer
triplicate wells were prepared for each individual dose. Monolayer
cells were incubated with the compound(s) for 48 h at 37 °C in atmo-
sphere of 5% CO2. After 48 h, cells were fixed, washed and stained

JOURNAL OF CHEMICAL RESEARCH 2012 275
4
5
A.E. Amr, A.M. Mohamed, S.F. Mohamed, N.A. Abdel-Hafez and
A.G. Hammam, Bioorg. Med. Chem., 2006, 14, 5481.
with SulfoRhodamine-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. The relation between sur-
viving fraction and drug concentration is plotted to get the survival
curve of each tumour cell line after the specified compound.
J.C. Aponte, A.J. Vaisberg, D. Castillo, G. Gonzalez,Y. Estevez, J. Arevalo,
M. Quiliano, M. Zimic, M. Verástegui, E. Málaga, R.H. Gilman,
J.M. Bustamante, R.L. Tarleton, Y. Wang, S.G. Franzblau, G.F. Pauli,
M. Sauvain and G.B. Hammonda, Bioorg. Med. Chem., 2010, 18, 2880.
A. Gangjee, Y. Qiu and R.L. Kisliuk, J. Heterocyclic Chem., 2004, 41,
941.
J.D. Moyer, E.G. Barbacci, K.K. Iwata, L. Arnold, B. Boman,
A. Cunningham, C. Diorio, J. Doty, M.J. Morin, M.P. Moyer, M. Neveu,
V.A. Pollack, L.R. Pustilink, M.M. Reynolds, D. Salon, A. Theleman and
P. Miller, Cancer Res., 1997, 57, 4838.
A.E. Wakeling, S.P. Guy, J.R. Woodburn, S.E. Ashton, B.J. Curry,
A.J. Barker and K.H. Gibson, Cancer Res., 2002, 62, 5749.
T.R. Rheault, T.R. Caferro, S.H. Dickerson, K.H. Donaldson, M.D. Gaul,
A.S. Goetz, R.J. Mullin, O.B. McDonald, K.G. Petrov, D.W. Rusnak,
L.M. Shewchuk, G.M. Spehar, A.T. Truesdale, D.E. Vanderwall,
E.R. Wood and D.E. Uehling, Bioorg. Med. Chem. Lett., 2009, 19, 817.
Docking steps All the docking studies were carried out on an Intel
Pentium 1.6 GHz processor, 512 MB memory with Windows XP
operating system using Molecular Operating Environment (MOE
2008.10; Chemical Computing Group, Canada) as the computational
software. All the minimisations were performed with MOE until a
6
7
−1
RMSD gradient of 0.05 kcal mol−1 A° with MMFF94x force-field
8
9
and the partial charges were automatically calculated.
In order to perform docking, some preliminary steps were done.
25Enzyme structures were checked for missing atoms, bonds and con-
tacts. Water of crystallisation was manually deleted. Hydrogens and
partial charges were added to the system using Protonate 3D applica-
tion. The active site was generated using the residues close to the
4-anilino- quinazoline atoms. The interactions of the ligand with the
amino acids of the active site were studied (Fig. 7).
Docking of compounds The compounds were constructed using the
builder module and were energy minimised using the MMFF94x
forcefield. Hydrogens and partial charges were added to the system
using Protonate 3D application. All antagonist structures were docked
into the active site by using the MOE Dock tool.
10 Y. Dai, Y. Guo, R.R. Frey, Z. Ji, M.L. Curtin, A.A. Ahmed, D.H. Albert,
L.Arnold, S.S.Arries, T. Barlozzari, J.L. Bauch, J.J. Bouska, P.F. Bousquet,
G.A. Cunha, K.B. Glaser, J. Guo, J. Li, P.A. Marcotte, K.C. Marsh,
M.D. Moskey, L.J. Pease, K.D. Stewart, V.S. Stoll, P. Tapang, N. Wishart,
S.K. Davidsen and M.R. Michaelides, J. Med. Chem., 2005, 48, 6066.
11 S. Pédeboscq, D. Gravier, F. Casadebaig, G. Hou, A. Gissot, F. De Giorgi,
F. Ichas, J. Cambar and J. Pometan, Eur. J. Med. Chem. 2010, 45, 2473.
12 T. Horiuchi, J. Chiba, K. Uoto and T. Soga, Bioorg. Med. Chem. Lett.,
2009, 19, 305.
Conformational analysis of compounds The algorithm generated
conformations from a single 3D conformation by conducting a sys-
tematic search. In this way, all combinations of angles were created
for each compound. A collection of poses was generated from the
pool of ligand conformations using Triangle Matcher placement
method. Poses were generated by superposition of ligand atom triplets
and triplets of points in the receptor binding site in a systematic way.
Poses generated by the placement methodology were scored using an
available method implemented in MOE, the London dG scoring func-
tion which estimates the free energy of binding of the ligand from a
given pose. The top 30 poses for each ligand were output in a MOE
database. Each resulting ligand pose was then subjected to MMFF94x
energy minimisation. The minimised docking conformations were
then rescored using London dG scoring method.25
13 T. Horiuchi, M. Nagata, M. Kitagawa, K. Akahane and K. Uoto, Bioorg.
Med. Chem., 2009, 17, 7850.
14 Y.L. Janin, Drug Discov. Today, 2010, 15, 342.
15 J.R. Porter, C.C. Fritz and K.M. Depew, Curr. Opin. Chem. Biol., 2010, 14,
412.
16 A.B. Pinkerton, T.T. Lee, T.Z. Hoffman, Y. Wang, M. Kahraman,
T.G. Cook, D. Severance, T.C. Gahman, S.A. Noble, A.K. Shiaub and
R.L. Davis, Bioorg. Med. Chem. Lett., 2007, 17, 3562.
17 C. Wu, M. Coumar, C. Chu, W. Lin, Y. Chen, C. Chen, H. Shiao, S. Rafi,
S.Wang, H. Hsu, C. Chen, C. Chang, T. Chang, T. Lien, M. Fang, K. Yeh,
C. Chen, T. Yeh, S. Hsieh, J. Hsu, C. Liao, Y. Chao and H. Hsieh, J. Med.
Chem. 2010, 53, 7316.
18 Y.P. Arya, Indian J. Chem., 1972, 1141.
19 G.D. Demetri, Eur. J. Cancer, 2002, 38, S52.
20 A.T. Van Oosterom, I. Judson, J. Verweij, S. Stroobants, E.D. Di Paola,
S. Dimitrijevic, M. Martens, A. Webb, R. Sciot, M. Van Glabbeke,
S. Silberman and O.S. Nielsen, The Lancet, 2001, 358, 1421.
21 G.L. Patrick, In An Introduction to Medicinal Chemistry, 4th edn,
Pergamon, Oxford, 2008, pp. 496.
Received 21 December 2011; accepted 5 March 2012
Paper 1101054 doi: 10.3184/174751912X13333849411283
Published online: 10 May 2012
22 E. Akaho, C. Fujikawa, H.I. Runion, C.R. Hill, H. Nakano, J. Chem.
Software, 1999, 5, 147.
23 H.I. Ali, K. Tomita, E. Akaho, H. Kambara, S. Miura, H. Hayakawa, N.
Ashida, Y. Kawashima, T. Yamagishi, H. Ikeya, F. Yoneda, T. Nagamatsu,
Bioorg. Med. Chem., 2007, 15, 242.
24 P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney and M.R. Boyd, J. Natl. Cancer Inst.,
1990, 82, 1107.
25 F. Payton-Stwart, R.S. Khupse, C. Boue, E.V. Skripnikova, H. Ashe, S.L.
Tilghman, B.S. Beckman, T.E. Cleveland, J.A. Melachlan, D. Bhatnager,
T.E. Wiese, P. Erhardt, M.E. Burow, Steroids, 2010, 75, 870.
References
1
2
3
J. Katada, K. Iijima, M. Muramatsu, M. Takami, E. Yasuda, M. Hayashi,
M. Hattori and Y. Hayashi, Bioorg. Med. Chem. Lett., 1999, 9, 797.
Y.D. Wang, S. Johnson, D. Powell, J.P. McGinnis, M. Miranda and
S.K. Rabindran, Bioorg. Med. Chem. Lett., 2005, 15, 3763.
L.D. Jennings, S.L. Kincaid, Y.D. Wang, G. Krishnamurthy, C.F. Beyer,
J.P. McGinnis, M. Miranda, C.M. Discafani and S.K. Rabindran, Bioorg.
Med. Chem. Lett., 2005, 15, 4731.

Copyright of Journal of Chemical Research is the property of Science Reviews 2000 Ltd. and its content may
not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written
permission. However, users may print, download, or email articles for individual use.