Synthesis and anticancer activities of novel (tetrahydrobenzo [4,5] thieno [2,3-d] pyrimidine-4-yl)-pyrolidine-2-carboxylic acid derivatives

Article abstract of DOI:10.1007/s00044-016-1692-x

Cancer chemotherapy has been one of the major medical advances in the last few decades. The medications involved in treatment have a narrow therapeutic index, and often the responses produced are only just palliative as well as unpredictable. In contrast,

Full text of DOI:10.1007/s00044-016-1692-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1692-x
ORIGINAL RESEARCH
Synthesis and anticancer activities of novel (tetrahydrobenzo [4,5] thieno
[2,3-d] pyrimidine-4-yl)-pyrolidine-2-carboxylic acid derivatives
Laxmikant S. Pavase1 Dhananjay V. Mane1
●
Received: 10 February 2016 / Accepted: 14 July 2016
© Springer Science+Business Media New York 2016
Abstract Cancer chemotherapy has been one of the major
medical advances in the last few decades. The medications
involved in treatment have a narrow therapeutic index, and
often the responses produced are only just palliative as well
as unpredictable. In contrast, targeted therapy such as
Tyrosine kinases inhibitors, that has been introduced in
recent years is directed against cancer-specific molecules
and signaling pathways with some side effects Therefore,
we studied these class of molecules to obtain new candi-
dates of remarkable activities and minimal side effects. We
herein reported SAR driven synthesis of 16 novel molecules
and their anticancer profiles in specifically targeted human
Breast cell line MCF-7 and Human Colon Cancer Cell Line
HCT-15. Compounds 7e and 8f showed descent antitumor
activities when compared to Doxorubicine (GI50 o 0.1) for
human Breast cell line MCF-7. The synthesized analogs
were ensured by spectral and elemental analysis.
even in the developed countries. Surprisingly, among all
malignant cancer, breast cancer is leading in second posi-
tion. Alcohol-related cirrhosis, and nonalcoholic fatty liver
disease allied with obesity, is a major cause of liver cancer
cases in US and another western countries.
Clinically, cancer is known as a malignant neoplasm and
is a broad group of various diseases all involving unregu-
lated cell growth. Rapid and uncontrolled cell growth leads
to malignant tumors and occupy the nearby parts of the
body. The cancer may also spread to more distant parts of
the body through the lymphatic system or blood stream.
There are over 200 different known cancers that afflict
humans. Cancer can develop in almost any organ or tissue,
such as the lung, colon, breast, skin, bones, or nerve tissue.
Cancer can be detected in a number of ways including
presence of certain signs and symptoms, screening test, or
medical imaging. Once a possible cancer is detected, it is
diagnosed by microscopic examination of a tissue sample.
Cancer is usually treated with chemotherapy, radiation
therapy, and surgery.
●
●
●
Keywords Thieno pyrimidine Proline Gewald reaction
●
●
MCF-7 HCT-15 Antitumor activities
Tyrosine kinases are orally active, small molecules that
have a favorable safety profile and can be easily combined
with other forms of chemotherapy or radiation therapy.
Several tyrosine kinase inhibitors (TKIs) such as gefitinib
(Paez et al., 2004) (Fig. 1) erlotinib (Higgins et al., 2004)
(Fig. 1) and lapatinib (Johnston and Leary, 2006) (Fig. 1)
have gained market approval worldwide. TKIs are thus an
important new class of targeted therapy that interfere with
specific cell signaling pathways and thus allow target spe-
cific therapy for selected malignancies.
Introduction
Cancer disease has major health issue on mankind around
the globe. The relative rate of death by cancer is very high
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1692-x) contains supplementary material,
which is available to authorized users.
Pyrimidine and its fused ring system is present in cyto-
sine, adenine, guanine, and thiamine, which form a part of
ribonucleic acid, deoxyribonucleic acid, vitamins and co-
enzymes, and other purines. A pyrimidine nucleus fused
with another heterocycle act as a core moiety in the
* Laxmikant S. Pavase
laxmikantpavase@rediffmail.com
1
Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada
University, Aurangabad, Maharashtra 431004, India

Med Chem Res
Fig. 1 Representative examples
of anticancer quinazoline
compounds
H
N
O
F
N
N
O
Cl
HN
N
O
O
O
N
N
O
O
N
N
N
Gefitinib
Tandutinib
F
O
HN
N
Cl
HN
N
O
O
O
O
O
N
O
S
N
O
N
Erlotinib
Lapatinib
amide & hydrazide
discovery of bioactive molecules (Adepu et al., 2012).
Hydrazide and their heterocyclic products show evidence of
diverse biological activities including antibacterial, anti-
fungicidal, analgesic, antituberculosis, anticancer, anti-
inflammatory properties (Shiradkar et al., 2007; Gursoy and
Guzeldemirci-Ulusoy, 2007; Gemma et al., 2006). In
addition the thieno [2,3-d] pyrimidine core, which is eval-
uated as a bioisostere of the quinazoline core, was used in
the mechanism-based design and synthesis of new anti-
tumor agents (Habib et al., 2013).
C4 position
secondary
nitrogen
A number of thieno [2,3-d] pyrimidine derivatives with
different substituent at C-2 and C-4 positions were found to
exert potential antitumor activity (Pedeboscq et al., 2010;
Beckers et al., 2012). It is observed from the literature that
C-4 position must contain a hetero atom such as primary
nitrogen [Gefitinib (Iressa)] (Fig. 1) or secondary nitrogen
containing side chains on the quinazoline moiety [Tandu-
tinib (MLN518)] (Fig. 1) to have antitumor potency. From
the literature study it was observed that fusion of five, six,
or seven member cycloalkyl lipophilic moieties conserved
the antitumor activity (Amr et al., 2006; Pedeboscq et al.,
2010; Aponte et al., 2010; Golub et al., 2011; Lou et al.,
2011) as exhibited by the 4-sustituted 5,6,7,8-tetra-
hydrobenzo [4,5]thieno [2,3-d] pyrimidine 6.
In our current manuscript we have prepared a series of
new thieno [2,3-d] pyrimidines by introducing a five
member cycloalkyl ring containing secondary nitrogen
(L-proline) at C-4 position and synthesized pharmacophoric
moieties of interest such as amides and hydrazides
that proved to contribute to antitumor activities such as
7e and 8f.
C2 position
thiophene fused with
pyrimidine
lipophilic cyclohexyl
ring
Fig. 2 Essential elements of synthesized compounds
Tandutinib (Fig. 1) consist of C-4 substituted piperazine
attached to the quinazoline moiety and formed urea deri-
vative with the propoxy aniline.
We design the molecules after modifying the C-4 sub-
stitution by secondary nitrogen containing amino acid
L-proline and used the substituted anilines like gefitinib,
lapatinib and also substituted piperazine side chains to form
terminal amides replacing urea. (Fig. 2)
Hydrazide derivatives were also prepared to compare its
relationship with amide derivatives. We studied the struc-
tural modifications of thieno [2,3-d] pyrimidine by aiming
to obtain the new candidates of remarkable antitumor
activities.

Med Chem Res
Chemistry
corresponds to ethyl group of ester, and singlet of two
protons at δ 5.94 ppm corresponds to amino group. Simi-
1
The targeted compounds were prepared from acid inter-
mediate 6 as outlined in Scheme 1. The starting material
ethyl-2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-car-
boxylate 2 was prepared by following the method of
Gewald (Gewald, 1965; Sabnis et al., 1999) via the reaction
of cyclohexanone 1 and sulfur with ethyl cynoacetate in the
presence of morpholine. Cyclization of 2 to 3 is adopted
using the reported reaction condition by refluxing it in
formamide (Nirogi et al., 2011). The desired 4-chloro
derivative 4 was obtained via the reaction 3 with phos-
phorous oxychloride in reflux (Perspicace et al., 2009).
L-Proline ethyl ester was introduced by replacing chloro
group of thieno [2,3-d] pyrimidine 4 in methanol to obtain
the ester intermediate 5. Tetrahydrobenzo [4,5]thieno [2,3-
d] pyrimidine-4yl-pyrrolidine-2-carboxylic acid 6 was
obtained after the alkaline hydrolysis of ester intermediate 5
by using lithium hydroxide.
larly, H NMR spectrum of 3 was identified by the broad
singlet at δ 12.31 and singlet at δ 8.00 ppm of the pyrimidin-
4(3H)-one. Infrared (IR) spectrum of 3 showed bands at
3363, 3383, and 1639 cm−1 corresponding to the NH and
C=O groups, respectively. Chloro intermediate 4 was
assigned by the shifted single signal to δ 8.72 ppm. Crude
ester intermediate 5 was used directly for hydrolysis reac-
tion, its formation was confirmed by the ESI-MS spectrum
showing ES-MS: m/z 332.3 (M+H)+. Acid intermediate 6
shows a characteristic triplet signal at δ 4.80 ppm corre-
sponds to chiral proton of L-proline and pyrimidine proton
at δ 8.32 ppm. Novel amides containing substituted anilines
7a–e and substituted piperazines 8a–f were prepared by
coupling selected amines with the acid core 6. Hydrazide
derivatives containing aromatic hydrazide 9a–e were also
prepared from 6. Secondary amines such as substituted
piperazines and primary amines such as substituted anilines
were selected to evaluate the structure activity relationship
among the novel analog. Hydrazide derivatives containing
1H NMR spectrum of 2 revealed the presence of triplet
signal at δ 1.33 ppm and quartet signal at δ 4.25 ppm
O
O
O
O
N
b
c
a
NH2
S
N
S
1
2
3
OH
Cl
OEt
N
N
N
N
N
d
e
O
O
N
N
S
4
S
S
N
5
6
Scheme 1 Synthetic route for 1-(5,6,7,8-tetrahydro-benzo[4,5]thieno
[2,3-d]pyrimidin-4-yl)-pyrrolidine-2-carboxylic acid (6). Reagents and
solvents: a ethyl cynoacetate, sulphur, morpholine, EtOH, Reflux;
b formamide; c POCl3 d L-proline ethyl ester hydrochloride, Et3N,
MeOH e. LiOH.H2O, THF: H2O
Scheme 2 Synthetic route for 1-
(5,6,7,8-tetrahydro-benzo[4,5]
OH
N
N
N
thieno[2,3-d]pyrimidin-4-yl)-
pyrrolidine-2-carboxylic acid
phenylamides (7a–e)
R
N
N
f
O
O
N
N
S
S
7a-e
f. R-Anilines, EDC.HCl/HOBt, DMF, RT
6
Compound 7a 7b
7c
7d 7e
R
H CH OCH Cl
F
3
3

Med Chem Res
Scheme 3 Synthetic route for
Piperazin-1-yl-[1-(5,6,7,8-
tetrahydro-benzo[4,5]thieno
[2,3-d]pyrimidin-4-yl)-
pyrrolidin-2-yl]-methanone
(8a–f)
R
N
OH
N
N
N
f
O
N
N
N
O
N
S
S
6
8a-f
f. R-Piperazines, EDC.HCl/HOBt, DMF, RT
Compound 8a 8b
8c
8d
8e
8f
F
N
N
N
R
CH Ph
3
OMe
Scheme 4 Synthetic route for 1-
(5,6,7,8-tetrahydro-benzo[4,5]
thieno[2,3-d]pyrimidin-4-yl)-
pyrrolidine -2-carboxylic acid
N’-aryl-hydrazide (9a–e)
O
H
N
OH
R
N
H
N
N
f
N
N
O
O
N
N
S
S
6
9a-e
f. R-Hydrazides, EDC.HCl/HOBt, DMF, RT
Compound
R
9a
9b
9c
9d
9e
O
S
Cl
OMe
CN
aromatic hydrazide were also studied for anticancer activ-
ities. (Schemes 2, 3, and 4)
detector instrument, Elemental analysis is reported from
Vario Micro Elementar instrument. Melting points were
taken on Veego melting point apparatus model VMP-D.
Material and methods
Experimental
Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carboxylate (2)
To a mixture of cyclohexanone (49 g, 0.5 mol), ethyl
2-cyanoacetate (56 g, 0.5 mol), and sulphur (16 g, 0.5 mol)
in 150 ml of ethanol was added morpholine (44 g, 0.5 mol).
The mixture was stirred for 8 h at room temperature. The
reaction mixture was diluted with water and the precipitate
was collected by filtration and recrystallized from ethanol.
Compound 2 as yellow solid (62 g, 55 %); m.p. = 115 °C;
1H NMR (CDCl3, 400 MHz) δ 5.94(s, 2H, Ar–NH2), 4.25
All the raw materials were obtained commercially and used
without further purification. IR spectra were recorded on
1
MIRacle10 shimadzu IR Affinity-1 instrument. H NMR
spectra were recorded using CDCl3 and DMSO-d6 as sol-
vent with tetramethylsilane as an internal standard on Var-
ian 400-MHz instruments. Elecronspray ionization-mass
spectra were recorded on LC-MS/MS Waters (Aquity) TQ

Med Chem Res
(q, 2H, J = 7 Hz, –O–CH2–), 2.69–2.60 (m, 2H, –CH2–
CH2–CH2–), 2.49–2.43(m, 2H, –CH2–CH2–CH2–), 1.76–
1.66 (m, 4H, –CH2–CH2–CH2–), 1.33(t, 3H, J = 7 Hz, –
OCH2–CH3); ES-MS: m/z 226.3 (M+H)+.
vacuum and to the remaining aqueous residue was added
1 N HCl solution in water to adjust the solution pH = 4, the
solid was precipitated out. The solid was filtered and dried
to obtain compound 6 as a yellowish solid (16 g, 70 %); IR
(KBr) νmax 3458, 3253, 2939, 1708, 1589, 1553, 1456,
1
5,6,7,8-Tetrahydrobenzo [4,5]thieno [2,3-d] pyrimidin-4
1338, 1219, 1060, 920, 769 cm−1; H NMR (DMSO-d6,
(3H)-one (3)
400 MHz) δ 8.23(s, 1H, pyrimidine–H), 4.80 (t, 1H, J = 8
Hz, –N–CH–CO), 3.82–3.89 (m, 2H, –N–CH2–CH2–),
2.80–2.86 (m, 4H, –CH2–CH2–CH2–), 1.75–1.98 (m, 6H, –
CH2–CH2–CH2–), 1.49–1.50 (m, 2H, CH2–CH2–CH2–);
ES-MS: m/z 302.2 (M+H)−.
The mixture of compound 2 (35 g, 0.16 mol) in 150 ml of
formamide was heated at 180 °C for 4 h and cooled down.
The mixture was poured into 200 ml water and filtered. The
solid was collected and recrystallized from ethanol. Com-
1
pound 3 was obtained as a yellow solid (25 g, 75 %); H
1-(5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidine-
NMR (CDCl3, 400 MHz) δ 12.31 (br s, 1H, –CO–NH–),
8.00 (s, 1H, =CH–N–), 2.85–2.88 (m, 2H, –CH2–CH2–
CH2–), 2.72–2.75 (m, 2H, –CH2–CH2–CH2–), 1.75–1.82
(m, 4H, –CH2–CH2–CH2–); ES-MS: m/z 207.2 (M+H)+.
4-yl)-pyrolidine-2-carboxylic acid phenyl-amide (7a)
To a solution of 1-(5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-
d] pyrimidine-4-yl)-pyrolidine-2-carboxylic acid 6 (1 g,
0.0033 mol) in DMF (5 ml) was added EDC.HCl (0.95 g,
0.0050 mol) and aniline (0.306 g, 0.0033 mol) followed by
the HOBt (0.443 g, 0.0033 mol). The reaction mixture was
stirred for 6 h and quenched with water (50 ml); the brown
solid that came out was filtered, dried, and washed with
diethyl ether to give compound 7a as an off-white solid
(0.82 g, yield = 66 %); m.p. = 127–129 °C; IR (KBr) νmax
3264, 2931, 1674, 1538, 1522, 1450, 1217, 1149, 1037,
851 cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 10.0 (s, 1H, –
CO–NH–), 8.21 (s, 1H, pyrimidine–H), 7.59 (d, J = 8 Hz,
2H, Ar–H), 7.27 (t, 2H, J = 8 Hz, Ar–H), 7.0–7.02 (t, 1H, J
= 7.6 Hz, Ar–H), 4.96 (t, 1H, J = 7.6 Hz, N–CH–CO),
3.92–3.96 (m, 1H, –Ar–CH2–CH2–), 3.69 (t, 1H, J = 8 Hz,
–N–CH2–CH2–), 2.85–3.01 (m, 4H, –CH2–CH2–CH2–),
2.33–2.38 (m, 1H, –CH2–CH2–CH2–), 1.72–1.98 (m, 6H, –
CH2–CH2–CH2–), 1.49–1.51 (m, 1H, –CH2–CH2–CH2–);
13C NMR (DMSO-d6, 100 MHz) δ 22.3, 22.9, 25.3 , 25.4,
28.6, 29.8, 53.8, 62.6, 117.2, 119.3, 119.3, 123.1, 127.8,
128.6, 128.6, 131.8, 139.1, 150.4, 157.5, 167.1, 170.7; ES-
MS: m/z 379.3 (M+H)+; anal. calcd. for C21H22N4OS; C,
66.64; H, 5.86; N, 14.80; found: C, 66.61; H, 5.89; N,
14.82.
4-Chloro-5,6,7,8-tetrahydrobenzo [4,5]thieno[2,3-d]
pyrimidin-4(3H)-one (4)
A suspension of compound 3 (25 g, 0.12 mol) in 150 ml of
POCl3 was heated at reflux for 2 h. POCl3 was removed at
reduced pressure and the residue was poured onto ice and
filtered. The solid was washed with water and dried.
Compound 4 was obtained as a brown solid (23 g, 85 %);
1H NMR (CDCl3, 400 MHz) δ 8.72 (s, 1H), 3.10–3.12 (m,
2H, –CH2–CH2–CH2–), 2.88–2.90 (m, 2H, –CH2–CH2–
CH2–), 1.75–1.92–1.95 (m, 4H, –CH2–CH2–CH2–); ES-
MS: m/z 225.3 (M+H)+.
1-(5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidine-
4-yl)-pyrolidine-2-carboxylic acid ethyl ester (5)
To a clear solution of compound 4 (20 g, 0.089 mol) in
methanol 200 ml was added L-proline ethyl ester hydro-
chloride (16 g, 0.089 mol) and triethylamine (27 ml, 0.267
mol) , and the reaction mixture was stirred for 3 h. Methanol
was removed under reduced pressure and residue was taken
in EtOAc, washed with water, 1 N HCl solution in water
and saturated NaHCO3 solution in water. Collected organic
layer was dried over Na2SO4 and removed under reduced
pressure to yield ester intermediate as a yellowish gel (25 g,
84 %) 5. This was used without further purification. ES-MS:
m/z 332.3 (M+H)+.
1-(5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidine-
4-yl)-pyrolidine-2-carboxylic acid p-tolylamide (7b)
Brownish solid (0.85 g, yield = 66 %); m.p. = 136–138 °C;
IR (KBr) νmax 3234, 2918, 1664, 1530, 1508, 1436, 1210,
1
1146, 1037, 843 cm−1 ; H NMR (DMSO-d6, 400 MHz) δ
1-(5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidine-
4-yl)-pyrolidine-2-carboxylic acid (6)
9.9 (s, 1H, –CO–NH–), 8.12 (s, 1H, pyrimidine–H), 7.46 (d,
J = 9.6 Hz, 2H, Ar–H), 7.08 (d, 2H, J = 8.4 Hz, Ar–H), 4.95
(t, 1H, J = 7.2 Hz, –N–CH–CO), 3.89–3.94 (m, 1H, Ar–
CH2–CH2–), 3.68 (t, J = 7.6 Hz, 1H, –N–CH2–CH2–),
2.85–2.97 (m, 4H, –CH2–CH2–CH2–), 2.32–2.35 (m, 1H, –
CH2–CH2–CH2–), 2.0 (s, 3H, –CH3), 1.75–1.98 (m, 6H, –
CH2–CH2–CH2–), 1.47–1.51 (m, 1H, –CH2–CH2–CH2–);
A suspension of ester compound 5 (25 g, 0.075 mol) in 225
ml THF and 25 ml water was added to lithium hydroxide
monohydrate (4.2 g, 0.11 mol) at 0 °C, and the reaction
mixture was stirred for 12 h. THF was distilled out under

Med Chem Res
13C NMR (DMSO-d6, 100 MHz) δ 20.4, 22.3, 22.9 , 25.4,
25.8, 28.6, 29.5, 53.8, 62.5, 117.2, 119.3, 119.3, 127.8,
128.9, 128.9, 131.8, 131.9, 136.6, 150.4, 157.5, 167.1,
170.5; ES-MS: m/z 393.3 (M+H)+; anal. calcd. for
C22H24N4O2S; C, 64.68; H, 5.92; N; 13.71; found: C,
64.66; H, 5.91; N, 13.74.
1143, 1058, 833 cm−1; H NMR (DMSO-d6, 400 MHz) δ
10.05(s, 1H, –CO–NH–), 8.21 (s, 1H, pyrimidine–H), 7.59
(d, J = 4.8 Hz, 2H, Ar–H), 7.11 (t, J = 8.4 Hz, 2H, Ar–H),
4.94 (t, 1H, –N–CH–CO, J = 8.8 Hz), 3.91–3.94 (m, 1H,
Ar–CH2–CH2), 3.71 (t, 1H, J = 7.6 Hz, –N–CH2–CH2–),
2.85–2.97 (m, 4H, –CH2–CH2–CH2–), 2.32–2.36 (m, 1H, –
CH2–CH2–CH2–), 1.74–1.98 (m, 6H, –CH2–CH2–CH2–),
1.50–1.51 (m, 1H, –CH2–CH2–CH2–); 13C NMR (DMSO-
d6, 100 MHz) δ 22.3, 22.8, 25.3, 25.8, 28.5, 29.8, 53.8,
62.5, 115.0, 115.3, 117.3, 120.9, 121.0, 127.8, 131.8,
135.5, 150.4, 157.5, 159.0, 167.1, 170.6 ; ES-MS: m/z
397.3 (M+H)+; anal. calcd. for C21H21FN4OS; C, 63.62; H,
5.34; N, 14.13; found: C, 63.66; H, 5.37; N, 14.11.
1
1-(5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidine-
4-yl)-pyrolidine-2-carboxylic acid (4-methoxy-phenyl)-
amide (7c)
Yellowish solid (0.9 g, yield = 67 %); m.p. = 135–137 °C;
IR (KBr) νmax 3264, 2948, 1674, 1532, 1516, 1442, 1216,
1
1152, 1057, 841 cm−1; H NMR (DMSO-d6, 400 MHz) δ
9.82(s, 1H, –CO–NH–), 8.20 (s, 1H, pyrimidine–H), 7.45–
7.47 (m, 2H, Ar–H), 6.82–6.84 (m, 2H, Ar–H), 4.92 (t, 1H,
–N–CH–CO), 3.84–3.87 (m, 1H), 3.66–3.67 (s, 1H), 3.68
(s,3H, –OCH3), 2.83–2.89 (m, 4H, –CH2–CH2–CH2–),
2.53–2.56 (m, 1H, –CH2–CH2–CH2–), 1.72–1.89 (m, 6H, –
CH2–CH2–CH2–), 1.48–1.49 (m, 1H, –CH2–CH2–CH2–);
13C NMR (DMSO-d6, 100 MHz) δ 22.2, 22.8, 25.3 , 25.8,
28.5, 29.5, 53.8, 55.1, 62.4, 113.7, 113.7, 117.3, 120.8,
120.8, 127.8, 131.7, 132.2, 150.3, 155.1, 157.5, 167.1,
170.2; ES-MS: m/z 408.3 (M+H)+; anal. calcd. for
C22H24N4O2S; C, 64.68; H, 5.92; N; 13.71; found: C,
64.66; H, 5.91; N, 13.74.
(4-Methyl-piperazine-1-yl)-[1-(5,6,7,8-tetrahydro-benzo
[4,5]thieno[2,3-d] pyrimidine-4-yl)-pyrolidine-2-yl]-
methanone (8a)
To a solution of 1-(5,6,7,8-tetrahydro-benzo [4,5]thieno
[2,3-d] pyrimidine-4-yl)-pyrolidine-2-carboxylic acid
6
(1 g, 0.0033 mol) in DMF (5 ml) was added EDC.HCl
(0.950 g, 0.0050 mol) and 1-methyl piperazine (0.331 g,
0.0033 mol) followed by the HOBt (0.443 mg, 0.0033 mol).
The reaction mixture was stirred for 6 h and was quenched
with water (50 ml), solid that comes out was filtered, dried,
and washed with diethyl ether to give compound 8a as a
yellowish solid, (0.80 g, yield = 62 %); m.p. = 80–82 °C, IR
(KBr) νmax 3418, 2905, 1635, 1548, 1548, 1431, 1361,
1-(5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidine-
4-yl)-pyrolidine-2-carboxylic acid (4-chloro-phenyl)-amide
(7d)
1
1230, 948, 742 cm−1; H NMR (DMSO-d6, 400 MHz) δ
8.20 (s, 1H, pyrimidine–H), 5.38 (t, 1H, N–CH–CO), 3.51–
3.56 (m, 4H, –CH2–CH2–CH2–), 3.28–3.31 (m, 2H, –CH2–
CH2–CH2–), 2.72–2.83 (m, 4H, –CH2–CH2–CH2–), 2.62–
2.68 (m, 4H, –CH2–CH2–CH2–), 2.31 (m, 3H, –CH3),
1.94–1.99 (m, 5H, –CH2–CH2–CH2–), 1.72–1.82 (m, 3H, –
CH2–CH2–CH2–), 1.45–1.48 (m, 1H, –CH2–CH2–CH2–);
13C NMR (DMSO-d6, 100 MHz) δ 22.3, 23.5, 24.2, 25.8,
28.6, 28.6, 43.6, 47.3, 47.6, 49.2, 48.8, 54.6, 54.6, 58.2,
127.6, 131.5, 145.7, 155.9, 167.1, 170.4; ES-MS: m/z 386.1
(M+H)+; anal. calcd. for C20H27N5OS; C, 62.31; H, 7.06;
N, 18.17; found: C, 62.33; H, 7.03; N, 18.21.
0.92 g, yield = 67 %; m.p. = 139–141 °C; IR (KBr) νmax
3265, 2945, 1668, 1530, 1512, 1446, 1210, 1145, 1060,
835 cm−1 ; 1H NMR (DMSO-d6, 400 MHz) δ 10.01(s, 1H, –
CO–NH–), 8.21 (s, 1H, pyrimidine–H), 7.62 (d, 2H, J = 7.2
Hz, Ar–H), 7.33 (d, 2H, J = 9.2 Hz Ar–H), 4.94 (t, 1H, J =
8 Hz, –N–CH–CO), 3.92–3.94 (m, 1H, Ar–CH2–CH2–),
3.70 (t, J = 7.2 Hz, 1H, –N–CH2–CH2–), 2.74–2.96 (m, 5H,
–CH2–CH2–CH2–), 2.0–2.35 (m, 1H, –CH2–CH2–CH2–),
1.87–2.0 (m, 5H, –CH2–CH2–CH2–), 1.47–1.50 (m, 1H, , –
CH2–CH2–CH2–); 13C NMR (DMSO-d6, 100 MHz) δ 21.4,
22.3, 22.9 , 25.4, 25.7, 28.6, 29.6, 53.8, 62.5, 115.4, 117.8,
119.3, 127.4, 127.9, 132.8, 136.6, 150.2, 157.5, 159.4,
167.1, 170.5; ES-MS: m/z 413.4 (M+H)+; anal. calcd. for
C21H21ClN4OS; C, 61.08; H, 5.13; N; 13.57; found: C,
61.09; H, 5.17; N, 13.54.
(4-Phenyl-piperazine-1-yl)-[1-(5,6,7,8-tetrahydro-benzo
[4,5]thieno[2,3-d] pyrimidine-4-yl)-pyrolidine-2-yl]-
methanone (8b)
Yellowish solid (0.80g, yield = 62 %); m.p. = 86–88 °C; IR
(KBr) νmax 3428, 2915, 2843, 1665, 1578, 1542, 1428,
1359, 1228, 948, 768 cm−1; 1H NMR (DMSO-d6, 400
MHz) δ 8.16 (s, 1H, pyrimidine–H), 7.23 (t, 2H, J = 8.4Hz,
Ar–H), 6.96 (s, 1H, Ar–H), 6.98 (d, 1H, J = 8.4Hz, Ar–H),
6.82 (t, 1H, J = 8.4Hz, Ar–H), 5.38 (t, 1H, J = 8Hz, N–CH–
CO), 3.81–3.86 (m, 3H, –CH2–CH2–CH2–), 3.67–3.71 (m,
1H, –CH2–CH2–CH2–), 3.60 (br s, 2H, –CH2–CH2–CH2–),
1-(5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidine-
4-yl)-pyrolidine-2-carboxylic acid (4-fluoro-phenyl)-amide
(7e)
Brownish solid (0.85 g, yield = 65 %); m.p. = 131–133 °C;
IR (KBr) νmax 3261, 2943, 1668, 1527, 1510, 1448, 1215,

Med Chem Res
3.28–3.31 (m, 2H, –CH2–CH2–CH2–). 3.14–3.17 (m, 2H, –
CH2–CH2–CH2–), 3.09 (br s, 1H, –CH2–CH2–CH2–), 2.83–
2.88 (m, 3H, –CH2–CH2–CH2–). 2.31–2.32 (m, 1H, –CH2–
CH2–CH2–), 1.94–1.99 (m, 3H, –CH2–CH2–CH2–), 1.72–
1.99 (m, 3H, –CH2–CH2–CH2–), 1.45–1.48 (m, 1H, –CH2–
CH2–CH2–); 13C NMR (DMSO-d6, 100 MHz) δ 22.3, 22.8,
25.3 , 25.8, 28.6, 28.6, 41.5, 44.9, 48.5, 48.8, 58.2, 58.2,
115.8, 116.8, 119.3, 127.6, 128.9, 128.9, 131.5, 131.6,
150.4, 150.8, 156.9, 167.1, 170.2; ES-MS: m/z 448.4 (M
+H)+; anal. calcd. for C25H29N5OS; C, 67.09; H, 6.53; N,
15.65; found: C, 67.07; H, 6.57; N, 15.67.
C26H31N5O2S; C, 65.38; H, 6.54; N, 14.66; found: C,
65.37; H, 6.58; N, 14.69.
(4-Pyridine-2-yl-piperazine-1-yl)-[1-(5,6,7,8-tetrahydro-
benzo[4,5]thieno[2,3-d] pyrimidine-4-yl)-pyrolidine-2-yl]-
methanone (8e)
Yellowish solid (850 mg, yield = 57 %); m.p. = 90–92 °C;
IR (KBr) νmax 3425, 2927, 2856, 1653, 1587, 1527, 1439,
1348, 1228, 985, 799 cm−1; 1H NMR (DMSO-d6, 400
MHz) δ 8.39 (d, 2H, J = 8.4Hz, Ar–H), 8.18 (s, 1H, Ar–H),
7.44(s, 1H, Ar–H), 6.66–6.68 (m, 2H, Ar–H), 5.37 (t, 1H, J
= 8.4Hz, –N–CH–CO–), 3.73–3.89 (m, 7H, –CH2–CH2–
CH2–), 3.54 (br s, 2H, –CH2–CH2–CH2–), 2.83–2.89 (m,
4H, –CH2–CH2–CH2–), 2.35–2.38 (m, 1H, –CH2–CH2–
CH2–). 1.91–2.01 (m, 3H, –CH2–CH2–CH2–), 1.78–1.82
(m, 3H, –CH2–CH2–CH2–), 1.45–1.49 (m, 1H, –CH2–
CH2–CH2–); 13C NMR (DMSO-d6, 100 MHz) δ 22.3, 22.8,
25.3 , 25.9, 28.6, 28.6,28.6, 42.3, 41.7, 43.2, 44.7, 53.4,
58.3, 110.4, 116.9, 126.4, 132.5, 151.6, 156.9, 157.9,
157.9, 163.2, 167.1, 170.5; ES-MS: m/z 448.3 (M+H)+;
anal. calcd. for C24H28N6OS; C, 64.26; H, 6.29; N, 18.73;
found: C, 64.23; H, 6.23; N, 18.75.
[4-(2-Fluoro-phenyl)-piperazine-1-yl)-[1-(5,6,7,8-
tetrahydro-benzo[4,5]thieno[2,3-d] pyrimidine-4-yl)-
pyrolidine-2-yl]-methanone (8c)
Yellow solid (1.2 g, yield = 62 %), m.p. = 83–85 °C; IR
(KBr) νmax 3435, 2938, 2863, 1660, 1598, 1546, 1438,
1357, 1224, 968, 788 cm−1; 1H NMR (DMSO-d6, 400
MHz) δ 8.19 (s, 1H, pyrimidine–H), 7.00–7.18 (m, 4H, Ar–
H), 5.37 (t, 1H, J = 8Hz, N–CH–CO), 3.82–3.90 (m, 3H),
3.72 (t, 3H, J = 6.8Hz, –N–CH2–CH2–), 3.03–3.14 (m, 3H,
–CH2–CH2–CH2–), 2.83–2.94 (m, 5H, –CH2–CH2–CH2–).
2.32 (br s, 1H, –CH2–CH2–CH2–), 1.94–1.98 (m, 3H, –
CH2–CH2–CH2–), 1.76–1.81 (m, 3H, –CH2–CH2–CH2–),
1.47–1.49 (m, 1H, –CH2–CH2–CH2–); 13C NMR (DMSO-
d6, 100 MHz) δ 22.3, 22.8, 25.3 , 25.9, 28.6, 40.2, 41.7,
45.2, 50.2, 50.7, 58.3, 58.3, 115.9, 116.1, 119.6, 119.6,
122.7, 122.8, 124.9, 127.7, 131.8, 150.4, 156.9, 167.1,
170.2; ES-MS: m/z 466.4 (M+H)+; anal. calcd. for
C25H28FN5OS; C, 64.49; H, 6.06; N, 15.04; found: C,
64.51; H, 6.08; N, 15.07.
(4-Pyrimidine-2-yl-piperazine-1-yl)-[1-(5,6,7,8-tetrahydro-
benzo[4,5]thieno[2,3-d] pyrimidine-4-yl)-pyrolidine-2-yl]-
methanone (8f)
Yellowish solid (1.3 g, yield = 87 %); m.p. = 88–90 °C ; IR
(KBr) νmax 3421, 2931, 2854, 1651, 1585, 1529, 1442,
1355, 1226, 981, 796 cm−1; 1H NMR (DMSO-d6, 400
MHz) δ 8.39 (d, 2H, J = 2 Hz, Ar–H), 8.18 (s, 1H, Ar–H),
6.67 (d, 1H, J = 4.8 Hz, Ar–H), 5.37 (t, 1H, J = 8.4 Hz, –N–
CH–CO–), 3.71–3.91 (m, 8H, –CH2–CH2–CH2–), 3.54 (br
s, 2H, –CH2–CH2–CH2–), 2.83–2.89 (m, 4H, –CH2–CH2–
CH2–), 2.31–2.35 (m, 1H, –CH2–CH2–CH2–), 1.91–2.01
(m, 3H, –CH2–CH2–CH2–), 1.70–1.82 (m, 3H, –CH2–
CH2–CH2–), 1.45–1.49 (m, 1H, –CH2–CH2–CH2–); 13C
NMR (DMSO-d6, 100 MHz) δ 22.3, 22.8, 25.3 , 25.9, 28.6,
28.6, 40.2, 41.5, 43.2, 44.7, 53.4, 58.3, 110.4, 116.9, 127.7,
131.5, 150.4, 156.9, 157.9, 157.9, 161.1, 167.1, 170.5; ES-
MS: m/z 450.3 (M+H)+; anal. calcd. for C23H27N7OS; C,
61.45; H, 6.05; N, 21.81; found: C, 61.47; H, 6.08; N,
21.83.
[4-(3-Methoxy-phenyl)-piperazine-1-yl)-[1-(5,6,7,8-
tetrahydro-benzo[4,5]thieno[2,3-d] pyrimidine-4-yl)-
pyrolidine-2-yl]-methanone (8d)
Brownish solid (900 mg, yield = 57 %); m.p. = 89–91 °C;
IR (KBr) νmax 3415, 2918, 2859, 1657, 1589, 1526, 1438,
1351, 1214, 978, 789 cm−1; 1H NMR (DMSO-d6, 400
MHz) δ 8.22 (s, 1H, pyrimidine–H), 6.98–7.18 (m, 2H, Ar–
H), 6.38–6.42 (m, 2H, Ar–H), 5.37 (t, 1H, J = 8.4 Hz, –N–
CH–CO–), 3.85–3.91 (m, 3H, –CH2–CH2–CH2–), 3.62–
3.72 (m, 3H, –CH2–CH2–CH2–), 3.83 (s, 3H, –OCH3)
3.01–3.12 (m, 3H, –CH2–CH2–CH2–), 2.83–2.94 (m, 5H, –
CH2–CH2–CH2–). 2.38 (br s, 1H, –CH2–CH2–CH2–), 1.94–
1.98 (m, 3H, –CH2–CH2–CH–), 1.76–1.80 (m, 3H, –CH2–
CH2–CH2–), 1.47–1.49 (m, 1H); 13C NMR (DMSO-d6,
100 MHz) δ 22.3, 23.5, 23.5, 24.2, 25.3 , 25.9, 28.6, 40.2,
42.7, 45.2, 50.2, 50.7, 55.8, 58.3, 115.9, 118.1, 119.6,
122.7, 124.9, 127.7, 131.8, 150.4, 156.9, 161.6, 167.1,
170.2; ES-MS: m/z 478.2 (M+H)+; anal. calcd. for
3-Chloro-benzoic acid N′-[1-(5,6,7,8,-tetrahydro-benzo
[4,5]thieno[2,3-d] pyrimidine-4-yl)-pyrrolidine-2-
carbonyl]-hydrazide (9a)
To a solution of 1-(5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-
d] pyrimidine-4-yl)-pyrolidine-2-carboxylic acid 6 (1 g,
0.0033 mmol) in DMF (5 ml) was added EDC.HCl
(0.950 g, 0.0050 mol) and 3-chloro-benzoic acid hydrazide

Med Chem Res
(0.561 g, 0.0033 mol) followed by the HOBt (0.443 g,
0.0033 mol). The reaction mixture was stirred for 6 h and
quenched with water (50 ml); the off-white color solid that
came out was filtered, dried, and washed with diethyl ether
to give compound 9a (1.3 g, yield = 87 %); m.p. = 115–117
°C; IR (KBr) νmax 3228, 3064, 2935, 1716, 1647, 1531,
1446, 1323, 1236, 1138, 964, 742 cm−1; 1H NMR (DMSO-
d6, 400 MHz) δ 10.45 (s, 1H, –NH–NH–), 10.05 (s, 1H, –
NH–NH–), 8.26 (s, 1H, pyrimidine–H), 7.88 (d, 1H, J =
1.6 Hz, Ar–H), 7.81 (d, 1H, J = 8 Hz, Ar–H), 7.62–7.64 (m,
1H, Ar–H), 7.50–7.54 (m, 1H, Ar–H), 4.95 (t, 1H, J = 8 Hz,
–N–CH–CO–), 3.90–3.94 (m, 1H, –CH2–CH2–CH2–),
3.65–3.69 (m,1H, –CH2–CH2–CH2–), 3.11–3.15 (m, 2H,
–CH2–CH2–CH2–), 2.33–2.40 (m, 2H, –CH2–CH2–CH2–),
1.95–2.02 (m, 4H, –CH2–CH2–CH2–), 1.74–1.79 (m, 4H, –
CH2–CH2–CH2–); 13C NMR (DMSO-d6, 100 MHz) δ 23.3,
23.8, 25.3, 25.8, 28.6, 28.6, 52.4, 62.9, 115.9, 116.1, 122.8,
124.9, 127.7, 131.8, 132.5, 134.3, 135.2 151.4, 158.7,
164.3, 167.1, 170.2; ES-MS: m/z 455.3 (M+H)+; anal.
calcd. for C22H22ClN5O2S: C, 57.95; H, 4.86; N, 15.36;
found: C, 57.93; H, 4.84; N, 15.33.
3.11–3.14 (m, 1H, –CH2–CH2–CH2–), 2.85 (br s, 3H,
–CH2–CH2–CH2–), 2.67–2.74 (m, 1H, –CH2–CH2–CH2–),
2.32–2.40 (m, 1H, –CH2–CH2–CH2–); 13C NMR
(DMSO-d6, 100 MHz) δ 23.3, 24.2, 25.6, 25.9, 29.5, 28.6,
52.3, 62.9, 115.8, 116.1, 123.1, 124.7, 126.8, 127.7, 128.2,
132.8, 133.3, 152.3, 157.8, 162.2, 164.3, 167.1, 170.2; ES-
MS: m/z 446.2 (M+H)+; anal. calcd. for C23H22N6O2S:
C, 61.87; H, 4.97; N, 18.82; found: C, 61.83; H, 4.94;
N, 18.81.
Furan-2-carboxylic acid N′-[1-(5,6,7,8,-tetrahydro-benzo
[4,5]thieno[2,3-d] pyrimidine-4-yl)-pyrrolidine-2-
carbonyl]-hydrazide (9d)
Brownish solid (1.1 g, yield = 84 %); m.p. = 118–120 °C;
IR (KBr) νmax 3238, 3080, 2938, 1741, 1674, 1553, 1469,
1
1345, 1248, 1141, 976, 758 cm−1; H NMR (DMSO-d6,
400 MHz) δ 10.22 (s, 1H, –NH–NH–); 9.89 (s, 1H, –NH–
NH), 8.22 (s, 1H, Ar–H), 7.86 (s, 1H, Ar–H), 7.23–7.225
(m, 1H, Ar–H), 6.68 (br s, 1H, Ar–H), 4.84 (t, 1H, J = 8Hz,
–N–CH–CO–), 3.91–3.93 (m, 1H, –CH2–CH2–CH2–),
3.73–3.76 (m, 1H, –CH2–CH2–CH2–), 3.13–3.17 (m,
2H, –CH2–CH2–CH2–), 2.95–2.98 (m, 2H, –CH2–CH2–
CH2–), 2.58–2.62 (m, 2H, –CH2–CH2–CH2–), 2.25–2.45
(m, 3H, –CH2–CH2–CH2–), 1.85–2.05 (m, 3H, –CH2–
CH2–CH2–); 13C NMR (DMSO-d6, 100 MHz) δ 23.3,
23.5, 25.4, 25.7, 28.2, 28.5, 52.3, 55.9, 62.9, 111.7, 113.5,
126.8, 132.8, 146.1, 148.3, 157.3, 158.7, , 164.3, 167.1,
170.2; ES-MS: m/z 412.2 (M+H)+; anal. calcd. for
C20H21N5O3S: C, 58.38; H, 5.14; N, 17.02; found: C,
57.34; H, 5.12; N, 17.01.
4-Methoxy-benzoic acid N′-[1-(5,6,7,8,-tetrahydro-benzo
[4,5]thieno[2,3-d] pyrimidine-4-yl)-pyrrolidine-2-
carbonyl]-hydrazide (9b)
1.1 g, yield = 74 %; m.p. = 89–91 °C; IR (KBr) νmax 3225,
3066, 2938, 1718, 1650, 1533, 1448, 1323, 1236, 1137,
1
962, 740 cm−1; H NMR (DMSO-d6, 400 MHz) δ 10.20
(s,1H, –NH–NH–), 9.95 (s, 1H, –NH–NH), 8.14 (s, 1H, Ar–
H), 7.85 (d, 2H, J = 8 Hz, Ar–H), 7.01 (d, 2H, J = 8.4 Hz,
Ar–H), 4.93–4.97 (t, 1H, J = 8 Hz –N–CH–CO–), 3.95 (br
s, 1H, –CH2–CH2–CH2–), 3.80 (s, 3H), 3.77–3.78 (m, 1H,
–CH2–CH2–CH2–), 3.06–3.10 (m, 2H), 2.87 (br s, 3H,
–CH2–CH2–CH2–), 2.40 (br s, 1H, –CH2–CH2–CH2–),
1.77–2.03 (m, 6H, –CH2–CH2–CH2–); 13C NMR (DMSO-
d6, 100 MHz) δ 23.2, 23.8, 25.6, 25.9, 28.3, 28.6, 52.3,
55.9, 62.9, 114.5, 116.1, 123.1, 124.7, 126.8, 127.7, 132.8,
133.5, 151.4, 158.7, 162.2, 164.3, 167.1, 170.2; ES-MS:
m/z 451.3 (M+H)+; anal. calcd. for C23H25N5O3S: C, 61.18;
H, 5.58; N, 15.51; found: C, 61.15; H, 5.55; N, 15.49.
Thiophene-2-carboxylic acid N′-[1-(5,6,7,8,-tetrahydro-
benzo[4,5]thieno[2,3-d] pyrimidine-4-yl)-pyrrolidine-2-
carbonyl]-hydrazide (9e)
Yellowish solid (1.2 g, yield = 85 %); m.p. = 122–124 °C;
IR (KBr) νmax 3239, 3075, 2931, 1751, 1678, 1557, 1471,
1
1346, 1249, 1140, 978, 753 cm−1; H NMR (DMSO-d6,
400 MHz) δ 10.18 (s, 1H, –NH–NH–), 9.85 (s, 1H, –NH–
NH), 8.22 (s, 1H, Ar–H), 7.66 (s, 1H, Ar–H), 7.27–7.29 (m,
1H, Ar–H), 6.70 (br s, 1H, Ar–H), 4.81–4.83 (m, 1H, –N–
CH–CO–), 3.91–3.93 (m,1H, –CH2–CH2–CH2–), 3.73–
3.76 (m, 1H, –CH2–CH2–CH2–), 3.15–3.18 (m, 2H, –CH2–
CH2–CH2–), 2.95–2.98 (m, 2H, –CH2–CH2–CH2–), 2.58–
2.62 (m, 2H, –CH2–CH2–CH2–), 2.25–2.46 (m, 3H, –CH2–
CH2–CH2–), 1.89–2.03 (m, 3H, –CH2–CH2–CH2); 13C
NMR (DMSO-d6, 100 MHz) δ 23.3, 23.5, 25.4, 25.7, 28.2,
28.5, 52.3, 55.9, 62.9, 123.1, 124.7, 126.8, 127.7, 132.8,
133.5, 158.7, 162.2, 164.3, 167.1, 170.2; ES-MS: m/z 428.2
(M+H)+; anal. calcd. for C20H21N5O2S2: C, 56.19; H, 4.95;
N, 16.38; found: C, 56.16; H, 4.94; N, 16.35.
4-Cyno-benzoic acid N′-[1-(5,6,7,8,-tetrahydro-benzo[4,5]
thieno[2,3-d] pyrimidine-4-yl)-pyrrolidine-2-carbonyl]-
hydrazide (9c)
Yellowish solid (0.9 g, yield = 61 %); m.p. = 117–119 °C;
IR (KBr) νmax 3228, 3069, 2978, 1738, 1670, 1553, 1468,
1
1343, 1246, 1139, 972, 748 cm−1; H NMR (DMSO-d6,
400 MHz) δ 10.59 (s, 1H, –NH–NH–), 10.02 (s, 1H, NH–
NH–), 8.26 (s, 1H, Ar–H), 7.96–8.08 (m, 4H, Ar–H), 4.95
(t, 1H, J = 8 Hz, –N–CH–CO–), 3.90–3.96 (m, 1H, –CH2–
CH2–CH2–), 3.67 (t, 1H, J = 8 Hz, –CH2–CH2–CH2–),

Med Chem Res
Biology
(TGI) was calculated from Ti = Tz. The LC50 (concentration
of drug resulting in a 50 % reduction in the measured protein
at the end of the drug treatment as compared to that at the
beginning) indicating a net loss of cells following treatment is
calculated from [(Ti−Tz)/Tz] × 100 = −50.
Values were calculated for each of these three parameters
if the level of activity was reached; however, if the effect
was not reached or was exceeded, the values for that
parameter were expressed as greater or less than the max-
imum or minimum concentration tested.
Antitumor activity
All the prepared target compounds 7a–7e, 8a–8f, and 9a–9e
were screened for their antitumor activities against breast
MCF-7 cell line and colon HCT-15 cell lines at Anti-Cancer
Drug screening facility, Tata memorial centre, Navi Mumbai.
Experimental procedure for Sulforhodamine B (SRB) assay
(Vichai and Kirtikara, 2006) The cell lines were grown in
RPMI 1640 medium containing 10 % fetal bovine serum
and 2 mM L-glutamine. For present screening experiment,
cells were inoculated into 96-well microtiter plates in 90 µL
at 5000 cells per 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 experi-
mental drugs. Experimental drugs were solubilized in
appropriate solvent to prepare stock of 10−2 concentration.
At the time of experiment four 10-fold serial dilutions were
made using complete medium. Aliquots of 10 µl of these
different drug dilutions were added to the appropriate
microtiter wells already containing 90 µl of medium,
resulting in the required final drug concentrations.
GI50 Growth inhibition of 50 % (GI50) calculated from [(Ti−Tz)/(C
−Tz)] × 100 = 50, drug concentration resulting in a 50 %
reduction in the net protein increase
TGI Drug concentration resulting in TGI will calculated from Ti =
Tz
LC50 Concentration of drug resulting in a 50 % reduction in the
measured protein at the end of the drug treatment as compared
to that at the beginning) indicating a net loss of 50 % cells
following treatment is calculated from [(Ti−Tz)/Tz] × 100 =
−50.
All tested compounds showing TGI 4 100 and LC50
4
100 for both the breast MCF-7 and colon HCT-15 cell lines.
After compound addition, plates were incubated at
standard conditions for 48 h and assay was terminated by
the addition of cold TCA. Cells were fixed in situ by the
gentle addition of 50 µl of cold 30 % (w/v) TCA (final
concentration, 10 % TCA) and incubated for 60 min at 4 °C.
The supernatant was discarded; the plates were washed five
times with tap water and air dried. SRB solution (50 µl) at
0.4 % (w/v) in 1 % acetic acid was added to each of the
wells, and plates were incubated for 20 min at room tem-
perature. After staining, unbound dye was recovered and the
residual dye was removed by washing five times with 1 %
acetic acid. The plates were air dried. Bound stain was
subsequently eluted with 10 mM trizma base, and the
absorbance was read on an Elisa plate reader at a wave-
length of 540 nm with 690 nm reference wavelength.
Percent growth was calculated on a plate-by-plate basis for
test wells relative to control wells. Percent growth was
expressed as the ratio of average absorbance of the test well to
the average absorbance of the control wells * 100. Using the
six absorbance measurements [time zero (Tz), control growth
(C), and test growth in the presence of drug at the four con-
centration levels (Ti)], the percentage growth was calculated
at each of the drug concentration levels. The dose response
parameters were calculated for each test article. Growth
inhibition of 50 % (GI50) was calculated from [(Ti−Tz)/(C
−Tz)] × 100 = 50, which is the drug concentration resulting in
a 50 % reduction in the net protein increase (as measured
by SRB staining) in control cells during the drug incubation.
The drug concentration resulting in total growth inhibition
Results and discussion
A substituted aniline derivatives often improves the activity
of different pharmacophores and hence plays an important
role for various SAR studies. It was also found that these
substituted aniline derivatives could be potential anticancer
agents [Gefitinib (Iressa)] Fig. 1. With the above facts,
here we have designed and prepared five novel compounds,
(7a–e) (Table 1).
The variation was brought across acid core 6 with five
different anilines to get the desired derivatives (7a–e). In the
above series, 4-Fluoro aniline compound 7e has shown
remarkable inhibitory activity in MCF-7 cell line (GI50
o
0.1 µmol/L) and good inhibitory activity in HCT-15 cell line
(GI50 = 56.9 µmol/L). All the other aniline derivatives
showed very poor inhibitory activity in HCT-15 cell line. 4-
Chloro aniline 7d revealed inferior activity in MCF-7 cell
line than the 4-Fluoro aniline compound 7e. 4-Methoxy
aniline 7c found to show better activity than 4-Methyl
aniline 7b in MCF-7 cell line, for HCT-15 cell line both are
equally inferior. Here, electron withdrawing group found to
improving the potential of inhibiting the anticancer activ-
ities in MCF-7 cell line; therefore it was observed that
compound 7e and compound 7c are superior to compound
7d and compound 7b, respectively.
Only compound 7d and compound 7e were showed
notable contribution among the compounds 7a–e in Human
Colon Cancer Cell Line HCT-15. In conclusion, between

Med Chem Res
Table 1 GI50 of the tested compounds against human tumor cell line MCF-7 and HCT-15
R
O
N
H
N
H
N
R
N
N
H
R
N
N
O
O
N
O
N
N
N
S
N
S
N
S
N
7a-e
8a-f
9a-e
Compounds
R
GI50 (µmol/L)
Breast MCF-7
Colon HCT-15
7a
H
>100
78.5
61.4
32.2
<0.1
>100
24.0
20.8
68.7
20.3
<0.1
>100
88.7
>100
71.8
>100
<0.1
>100
>100
>100
71.8
7b
CH3
7c
OCH3
7d
Cl
7e
F
56.9
8a
CH3
>100
67.3
8b
Ph
8c
2-Fluoro phenyl
3-Methoxy phenyl
Pyridine
55.2
8d
>100
71.8
8e
8f
Pyrimidine
3-Chloro phenyl
4-Methoxy phenyl
4-Cyno phenyl
2-Furan
63.5
9a
>100
>100
>100
>100
>100
<0.1
9b
9c
9d
9e
2-Thiophene
Doxorubicine
Reference
the two studied cell lines, compound 7e was found to be
potent for MCF-7 cell line.
could not make to contribute towards the inhibitory activity
as compare to aryl substituted piperazine derivatives 8b, 8c,
and 8d. Between the substituted phenyl piperazine deriva-
tives 4-(2-fluoro phenyl) piperazine compound 8c and 4-(3-
methoxy phenyl) piperazine compound 8d, compound 8c is
more superior to compound 8d. Electron withdrawing
group increases inhibitory activity of 8c and it is also
comparable to compound 8d in MCF-7 cell line.
For Human Colon Cancer Cell Line HCT-15 activities it
was found that compound 8c (GI50 = 55.7 µmol/L) is the
good inhibitor among the 8a–f. Compound 8b (GI50 = 67.3
µmol/L) is inferior to 8c, because the electron withdrawing
fluoro group at second position of the phenyl ring was
present in 8c. Compound 8e and compound 8f followed the
activity trend as they were followed for the MCF-7 cell line.
Compound 8f was superior to compound 8e in HCT-15 cell
line (Fig. 3).
Secondary nitrogen side chains on the quinazoline moi-
ety [Tandutinib (MLN518)] Fig 1 exhibits antitumor
potency when piperazine was coupled with aniline, to get
the terminal urea. In order to enhance the activity, here we
have modified our synthetic approach and accordingly
prepared piperazine 4-substituted amide derivatives 8a–f
from same acid core 6.
In our SAR study, 4-pyridinyl piperazine compound 8e,
showed intermittent activities in both the cell lines corre-
spond to 4-pyrimidinyl piperazine compound 8f and
4-phenyl piperazine compound 8b. Insertion of the nitrogen
atom in the aromatic ring attached to the piperazine ring
increases the activity of compound 8b from (GI50 = 24.0
µmol/L) to compound 8e (GI50 = 20.3 µmol/L) for MCF-7
cell line and the trend continues still increase in inhibitory
activity reaches to remarkable (GI50 o 0.1 µmol/L) value in
compound 8f. Alkyl substituted piperazine derivative 8a
Hydrazide side chains attached to the acid 6 was
observed very poorly in showing anticancer activities in

Med Chem Res
Conclusion
N
N
N
A series of novel tetrahydrobenzo [4,5]thieno [2,3-d] pyr-
imidine-4yl-pyrrolidine-2-carboxamides were synthesized
by a facile six step procedure. Their structures were char-
acterized by spectral and elemental analysis. The pre-
liminary bioassay results imply that some of the compounds
exhibit first-rate tumor cell inhibitor activity against human
Breast MCF-7 and Colon HCT-15 cell lines. Compound 7e
and compound 8f were the new finding from this research
work and it will be studied further in near future.
H
N
N
F
N
O
N
N
O
N
N
S
N
S
7e
8f
Fig. 3 Representative examples of primary evolutionary tumor cell
inhibitors with GI50 o 0.1 in MCF-7 cell line
Acknowledgments Authors are thankful to Dr. Aarti Juwekar and
Dr. Jyoti Kode of Anti-Cancer Drug screening facility (ACDSF), Tata
memorial centre, Navi Mumbai, Maharashtra, India for screening of
compounds for anticancer activities.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
MCF-7 Positive control
MCF-7 with 8f
References
Adepu R, Rambhau D, Prasad B, Meda C, Kandale A, Krishna G, Reddy
C, Chennuru L, Parsa K, Pal M (2012) Novel thieno [2, 3-d] pyr-
imidine: their design, synthesis, crystal structure analysis and
pharmacological evaluation. Org Biomol Chem 10:5554–5569
Amr A, Mohamed A, Mohamed S, Abdel-Hafez N, Hammam A
(2006) Synthesis and anticancer activities of new pyridine, pyrane
and pyrimidine derivatives fused with nitrobenzosubetrone moi-
ety. Bioorg Med Chem 14:5481–5488
MCF-7 with 7e
MCF-7 control
Aponte J, Vaisberg A, Castillo D, Gonzalez G, Estevez Y, Arevalo J,
Quiliano M, Zimic M, Verastegui M, Málaga E, Gilman R,
Bustamante J, Tarleton R, Wang Y, Franzblau S, Pauli G, Sau-
vain M, Hammonda G (2010) Trypanoside, anti-tuberculosis,
Fig. 4 Images of anticancer activities of 7e and 8f
leishmanicidal,
and
cytotoxic
activities
of
tetra-
Human Colon Cancer Cell Line HCT-15. All the synthe-
sized compounds 9a–e was reportedly showed (GI50 4 100
µmol/L). Compound 9b (GI50 = 88.7 µmol/L) and com-
pound 9d (GI50 = 71.8 µmol/L) exhibiting the anticancer
activities for the human Breast Cancer MCF-7 cell line.
Compound 9b contains 4-methoxy benzoic acid hydrazide
side chain and compound 9d was with 2-Furoic acid
hydrazide side chain. Compounds 9b and 9d have an
oxygen atom present in hydrazide analog and it might be
resemblance to phenolic and ethereal type of oxygen con-
taining Erlotinib (Fig. 1) to show some activities (Fig. 4).
Between the three series of compounds containing sub-
stituted anilines 7a–7e, substituted piperazine 8a–8f and
hydrazide side chains 9a–9e, it was found that secondary
substituted piperazine derivatives are exhibiting good inhi-
bitory activities in both the MCF-7 and HCT-15 cell lines.
Primary substituted anilines resulted in improved activities
in MCF-7 cell line than HCT-15 cell line and hydrazide
compounds were very poor in both the MCF-7 and HCT-15
cell lines.
hydrobenzothienopyrimidines. Bioorg Med Chem 18:2880–2886
Beckers T, Sellmer A, Eichhorn E, Pongratz H, Schächtele C, Totzke
F, Kelter G, Krumbach R, Fiebig H, Böhmer F, Mahboobi S
(2012) Novel inhibitors of epidermal growth factor receptor;(4-
(arylamino)-7H-pyrrolo[2,3-d]pyrimidine-6-yl)(1H-indol-2-yl)
methanones and(1-H-indol-2-yl)(4-(phenylamino)thieno[2,3-d]
pyrimidine-6-yl)methanones. Bioorg Med Chem 20:125–136
Gemma S, Kukreja G, Fattorusso C, Persico M, Romano M, Altarelli
M, Savini L, Campiani G, Fattorusso E, Basilico N, Tarmelli D,
Yardley V, Butini S (2006) Synthesis of N1-arylidene-N2-qui-
nolyl- and N2-acrydinylhydrazones as potent antimalarial agents
active against CQ-resistant P. falciparum strains. Bioorg Med
Chem Lett 16:5384–5388
Gewald K (1965) Heterocyclen aus CH-aciden Nitrilen, VII. 2-Amino-
thiophene aus α-Oxo-mercaptanen und methylenaktiven Nitrilen.
Chem Ber 98:3571–3577
Golub A, Bdzohla V, Briukhovetska N, Balanda A, Kukharenko O,
Koyey I, Ostrynska O, Yarmoluk S (2011) Synthesis and biolo-
gical evaluation of substituted (thieno[2,3-d] pyrimidin-4-ylthio)
carboxylic acids as inhibitors of human protein kinase CK2. Eur J
Med Chem 46:870–876
Gursoy E, Guzeldemirci-Ulusoy N (2007) Synthesis and primary
cytotoxicity evaluation of new imidazo [2, 1-b] thiazole deriva-
tives. Eur J Med Chem. 42:320–326

Med Chem Res
Habib N, Soliman R, EI-Tomboys A, EI-Hawash, Soad A, Shaaban,
Omaima G (2013) Synthesis and biological evolution of novel
series of thieno[2,3-d]pyrimidine derivatives as anticancer and
antimicrobial agent. Med Chem Res 22:3289–3308
Higgins B, Kolinsky K, Smith M, Rashed M, Adames V, Linn M,
Wheeldon E, Gand L, Birnboeck H, Hoffmann G (2004) Anti-
tumor activity of erlotinib (OSI-774, Tarceva) alone or in com-
bination in human non-small cell lung cancer tumor xenograft
models. Anticancer Drugs 15:503–512
Eck M, Sellers W, Johnson B, Meyerson M (2004) EGFR
mutations in lung cancer: correlation with clinical response to
gefitinib therapy. Science 304:1497–1500
Pedeboscq S, Gravier D, Casadebaig F, Hou G, Gissot A, Giorgi F,
Ichas F, Cambar J, Pometan J (2010) Synthesis of and study
of antiproliferative activity of novel thienopyrimidines on
glioblastoma cells. Eur J Med Chem 45:2473–2479
Perspicace E, Hesse S, Kirsch G, Yemloul M, Lecomte C (2009)
Unexpected C=O bond formation in Suzuki coupling of
Johnston S, Leary A (2006) Lapatinib: a novel EGFR/ HER2 tyrosine
kinase inhibitor for cancer. Drugs Today 42:441–453
4-chlorothieno[2,3-d]pyrimidines.
459–464
J
Heterocycl Chem 46:
Lou J, Liu Z, Li Y, Zhou M, Zhang Z, Zeng S, Wang R, Li J (2011)
Synthesis and anti-tumor activities of N′-benzylidene-2-(4-oxo-
Sabnis R, Rangnekar D, Sonawane N (1999) 2-Aminothiophenes by
the Gewald reaction. J Heterocycl Chem 36(2):333–345
Shiradkar M, Murahari K, Gangadasu H, Suresh T, Kalyan C,
Panchal D, Kaur R, Burange P, Ghogare J, Mokale V, Raut M
(2007) Synthesis of new S-derivatives of clubbed triazolyl thia-
zole as anti-My-cobacterium tuberculosis agents. Bioorg Med
Chem 15:3997–4008
thieno[2,3-d]pyrimidin-3(4H)-yl)acetohydrazone
derivatives.
Bioorg Med Chem Lett 21:6662–6666
Nirogi R, Kambhampati R, Kothmirkar P, Arepalli S, Pamuleti N,
Reddy G, Shinde A, Dubey P (2011) Convenient and efficient
synthesis of some novel fused thieno pyrimidines using Gewald’s
reaction. Synth Commun 41(19):2835–2851
Paez J, Janne P, Lee J, Tracy S, Greulich H, Gabriel S, Herman P,
Kaye F, Linderman N, Boggon T, Naoki K, Sasaki H, Fujji Y,
Vichai V, Kirtikara K (2006) Sulforhodamine B Colorimetric assay for
cytotoxicity screening. Nat Protoc 1:1112–1116