Synthesis, cytotoxicity and toxicity of thieno[2,3-d]pyrimidine derivatives derived from 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene

Article abstract of DOI:10.1007/s00044-015-1433-6

The 4,5,6,7-tetrahydrobenzo[b]thiophene derivative 1 reacted with benzoylisothiocyanate to give N-benzoylthiourea derivative 3. The latter underwent ready cyclization to give the tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivative 4 which was used as t

Full text of DOI:10.1007/s00044-015-1433-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2015) 24:3932–3948
DOI 10.1007/s00044-015-1433-6
ORIGINAL RESEARCH
Synthesis, cytotoxicity and toxicity of thieno[2,3-d]pyrimidine
derivatives derived from 2-amino-3-cyano-4,5,6,7-
tetrahydrobenzo[b]thiophene
1
2
3
Mahmoud A. Abdelaziz
•
Hend M. El-Sehrawi
•
Rafat M. Mohareb
Received: 22 August 2014 / Accepted: 17 August 2015 / Published online: 29 August 2015
Ó Springer Science+Business Media New York 2015
Abstract The 4,5,6,7-tetrahydrobenzo[b]thiophene deriva-
tive 1 reacted with benzoylisothiocyanate to give N-ben-
zoylthiourea derivative 3. The latter underwent ready
cyclization to give the tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidine derivative 4 which was used as the key starting
compound for a series of heterocyclization reactions to pro-
duce thiophene, pyridine, pyrimidine and pyran derivatives.
The cytotoxicity of the newly synthesized products was eval-
uated using six cancer andone normalcell lines. The toxicity of
compounds with the optimal cytotoxicity was measured using
shrimp larvae.
et al. 2012; Ashalatha et al. 2007). The antioxidants that
scavenge reactive oxygen species may be of great value in
preventing the onset and propagation of oxidative diseases
such as autoimmune diseases, cardiovascular diseases,
neurovascular diseases (Kamat et al. 2013; Giordano et al.
2014) and neurodegenerative changes associated with
aging (Esposito et al. 2002). The homeostatic balance
between the reactive oxygen species (ROS) and endoge-
nous antioxidants is important in maintaining healthy tis-
sues. Excessive ROS states are important in diseases such
as acute respiratory distress syndrome and idiopathic pul-
monary fibrosis (Vianello et al. 2014). Most living organ-
isms possess enzymatic and nonenzymatic defense systems
against excessive production of the reactive oxygen spe-
cies. However, different external factors (smoke, diet,
alcohol and some drugs) and aging decrease the efficiency
of such protecting systems, resulting in disturbances of the
redox equilibrium established under healthy conditions
(Ogunro et al. 2013). In addition, the pyrimidine-based
derivatives such as thieno[2,3-d]pyrimidines have extreme
importance in medicinal chemistry, exhibiting pharmaco-
logical and therapeutic properties such as antidepressant
Keywords Tetrahydrobenzo[b]thiophene Á
Thieno[2,3-d]pyrimidine Á Cyanomethylene Á Cytotoxicity
Introduction
Several types of pyrimidines and thienopyrimidines have
attracted much interest due to their valuable pharmaco-
logical properties such as antiviral (Hafez et al. 2010),
antioxidant (Kotaiah et al. 2012) and antimalarial (Seerat
(Kotaiah et al. 2012), antibacterial (Aly et al. 2011; Dewal
et al. 2012; Abbas et al. 2013), antifungal (Leung et al.
2013), anti-inflammatory (Rizk et al. 2012; Ashour et al.
&
Rafat M. Mohareb
raafat_mohareb@yahoo.com
2013), antiplatelet (Bach et al. 2013), antihypertensive
Mahmoud A. Abdelaziz
m_mahmoud@ut.edu.sa
(Press et al. 1989), herbicidal (El-Sherbeny et al. 1995) and
plant growth regulatory (Kotaiah et al. 2012) properties.
Overview of the literature survey showed the impor-
tance of thieno[2,3-d]pyrimidines individually in the bio-
logical systems and led to assimilate that this moiety may
show synergistic effect. In the present work, we report the
details of the synthesis of novel thieno[2,3-d]pyrimidine
derivatives and in vitro antioxidant properties of them.
1
Department of Chemistry, Faculty of Science, University of
Tabuk, P. O. Box 741, Tabuk 71491,
Kingdom of Saudi Arabia
2
3
Pharmaceutical Chemistry Department, Faculty of Pharmacy
(Girls), Al-Azhar University, Nasr City, Cairo, Egypt
Department of Chemistry, Faculty of Science, Cairo
University, Giza, Egypt
1
23

Med Chem Res (2015) 24:3932–3948
3933
Results and discussion
Next, we studied the reactivity of compounds 7a, b
toward thiophene formation via the Gewald’s thiophene
synthesis (Scrowston et al. 1981; Mohareb et al., 2012;
Hala et al., 2011; Patel et al. 2003). Thus, the reaction of
either compounds 7a or 7b with elemental sulfur and either
malononitrile (9) or ethyl cyanoacetate (6a) gave the
thiophene derivatives 10a–d, respectively. On the other
hand, the reaction of either 7a or 7b with either ben-
zaldehyde (11a), 4-chlorobenzaldehyde (11b) or
4-methoxybenzaldehyde (11c) gave the benzylidene
derivatives 12a–f, respectively (Scheme 2). The analytical
and spectral data of the latter products are consistent with
their respective structures (see ‘‘Experimental’’ section).
Compounds 7a or 7b reacted with the aromatic diazo-
nium salts namely benzenediazonium chloride (13a),
4-chlorobenzenediazonium chloride (13b) or 4-methylben-
zenediazonium chloride (13c) to give the arylhydrazo
derivatives 14a-f, respectively. Reaction of compound 7b
with cyanomethylene reagents using different conditions
was studied. Thus, compound 7b reacted with either
malononitrile (9) or ethyl cyanoacetate (6b) in the presence
of ammonium acetate at 120 °C to give the condensation
products 15a and 15b, respectively. On the other hand,
carrying out the reaction of compound 7b with either
malononitrile (9) or ethyl cyanoacetate (6a) in sodium
ethoxide solution gave the pyridine-1-yl derivatives 16a and
16b, respectively (Scheme 3). The reaction took place at
first through condensation followed by the Michael addition
of the NH to the nitrile group. The analytical and spectral
data of 16a and 16b were in agreement with their respective
structures. Further confirmation for the structures of com-
pounds 16a and 16b was obtained through their synthesis
using another reaction route. Thus, either 15a or 15b when
heated in sodium ethoxide solution in a boiling water bath
gave the same two products 16a and 16b, respectively (same
m.p. and mixed m.p. and fingerprint IR spectra).
Herein, in order to extend our research on anticancer
heterocyclic derivatives with high inhibitory effects toward
some cancer cell lines, we report the synthesis of new
heterocyclic compounds derived from thieno[2,3-d]pyri-
dazine. Moreover, some of the newly synthesized products
were good candidates as anticancer drugs through their
screening toward cancer and normal cell lines. Thus,
4
,5,6,7-tetrahydrobenzo[b]thiophene reacted with ben-
zoylisothiocyanate to give the thiourea derivative 3. The
structure elucidation of compound 3 was based on analyt-
1
ical and spectral data. Thus, the H NMR spectrum showed
the presence of the four CH groups at d 1.80–1.84 and
2
2
.24–2.26 ppm, a multiplet at d 7.2–7.35 ppm equivalent
to the phenyl protons and two singlets at d 8.28 and 8.32
D O exchangeable) equivalent to the two NH groups. In
(
2
1
3
addition, the C NMR spectrum showed d 19.2, 22.1, 25.7,
2
8.6 (4 CH ), 119.2, 124.1, 124.9, 128.7, 130.4, 133.8,
2
1
38.0, 144.2 (thiophene, C H ), 165.3 (CO), 179.5 (C=S).
6
5
Compound 3 underwent cyclization when heated under
reflux in 1,4-dioxane, containing a catalytic amount of
triethylamine, to give the (4-amino-2-thioxo-5,6,7,-te-
trahydro[4,5]thieno[2,3-dpyrimidin-3(2H)-yl)(phe-
nyl)methanone (4). Compound
4
was previously
synthesized using another reaction routes (Amr et al. 2010;
Hacker et al. 2009; Leistner et al. 1989). Compound 4
underwent different heterocyclization reactions. Thus, it
reacted with hydrazine hydrate to give the tetrahy-
drobenzo[4,5]thieno[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine
derivative 5 (Scheme 1). The analytical and spectral data
of compound 5 are consistent with its proposed structure.
Compound 4 was reacted with either ethyl cyanoacetate
(
6a) or ethyl acetoacetate (6b) to give the amide derivatives
a or 7b (Scheme 1). The high yield of compounds 7a, b
7
encouraged us to use them for further work. In order to
expand the scope of the present work, compounds 7a, b
underwent ready cyclization when heated in sodium ethox-
ide solution using a boiling water bath to give the ben-
zo[4,5]thieno[3,2-e]pyrimido[1,2-c]pyrimidine derivatives
Compounds 16a, b reacted with phenyl isothiocyanate
(17) to give the pyrido[2,3-d]pyrimidine derivatives 18a and
18b, respectively. Structure of the latter products was con-
firmed on the basis of analytical and spectral data. Finally,
7a or 7b was reacted with either benzaldehyde (11a),
4-chlorobenzaldehyde (11b) or 4-methoxybenzaldehyde
(11c) and malononitrile to afford the pyran derivatives 19a–
f, respectively. The analytical and spectral data of 19a–f
were consistent with their respective structures (Scheme 4).
8a and 8b, respectively. The mass spectrum of compound 8a
showed molecular ion peak at m/e 390 equivalent to the
molecular formula C H N OS . The IR spectrum showed
2
0
14
4
2
-1
1
the presence of CN group stretching at t 2220 cm . The H
NMR spectrum revealed beside the expected signals, a
singlet at d 8.41 indicating the presence of the NH group.
1
3
The CNMR displayed d 19.0, 22.4, 25.5, 28.8 corre-
In vitro cytotoxic assay
sponding to the four CH groups of the cyclohexene ring, d
4
1
16.2 indicating the CN group, d 120.6, 123.6, 124.8, 129.3,
34.2, 136.8, 139.0, 143.7, 148.9, 151.6 (Bz, pyrimidine,
Chemicals
1
thiophene C), d 166.4 equivalent to the CO group of the
pyrimidine C-3, d 170.4 (C=N, C-7) and d 180.6 (C=S, C-6).
Fetal bovine serum (FBS) and L-glutamine were purchased
from Gibco Invitrogen Co. (Scotland, UK). RPMI-1640
123

3
934
Med Chem Res (2015) 24:3932–3948
Scheme 1 Synthesis of
compounds 3, 4, 5 and 7a, b
medium was purchased from Cambrex (New Jersey, USA).
Dimethyl sulfoxide (DMSO), doxorubicin, penicillin,
streptomycin and sulforhodamine B (SRB) were purchased
from Sigma Chemical Co. (Saint Louis, USA).
inactivated FBS, 2 mM glutamine and antibiotics (peni-
cillin 100 U/mL, streptomycin 100 lg/mL), at 37 °C in a
humidified atmosphere containing 5 % CO . Exponentially
2
5
growing cells were obtained by plating 1.5 9 10 cells/mL
for the seven human cancer cell lines including cells
4
derived from 0.75 9 10 cells/mL followed by 24 h of
Cell cultures
incubation. The effect of the vehicle solvent (DMSO) on
the growth of these cell lines was evaluated in all the
experiments by exposing untreated control cells to the
maximum concentration (0.5 %) of DMSO used in each
assay.
The cell cultures were obtained from the European Col-
lection of cell Cultures (ECACC, Salisbury, UK), and
human gastric cancer (NUGC and HR), human colon
cancer (DLD1), human liver cancer (HA22T and HEPG2),
human breast cancer (MCF), nasopharyngeal carcinoma
The heterocyclic compounds, prepared in this study,
were evaluated according to standard protocols for their
in vitro cytotoxicity (Combes et al. 2012; Roemer et al.
2008; Li et al. 2008) against six human cancer cell lines
including cells derived from human gastric cancer
(
HONE1) and normal fibroblast cells (WI38) were kindly
provided by the National Cancer Institute (NCI, Cairo,
Egypt). They grow as monolayer and routinely maintained
in RPMI-1640 medium supplemented with 5 % heat
1
23

Med Chem Res (2015) 24:3932–3948
3935
(
(
NUGC), human colon cancer (DLD1), human liver cancer
HA22T and HEPG2), human breast cancer (MCF),
From Table 1, it is clear that for the benzylidene
derivatives 12a–f, compounds 12a and 12b with the cyano
moiety are less potent than compounds 12c–f, which might
nasopharyngeal carcinoma (HONE1) and
a normal
fibroblast cells (WI38). All of IC50 values are listed in
be due to the presence of either the OCH in 12c or the
3
Table 1. Twelve compounds 4, 7a, 8a, 8b, 10c, 10d, 12c,
COCH moiety as in 12d–f. It is of great value to note that
3
12d, 12e, 12f, 14e, 15b, 18b, 19b and 19e displayed sig-
nificant cytotoxicity against most of the cancer cell lines
among the compounds 12a–f, compound 12f with the
COCH and the Cl moieties is responsible for its maximum
3
tested (IC50 = 10–1000 nM). Normal fibroblasts cells
potency. Similarly concerning the arylhydrazo derivatives
14a–f, it is noticed that compound 14e with the COCH3
and Cl moieties showed the highest potency among the five
compounds. On the other hand, compound 14b with the CN
and Cl moieties showed high potency toward NUGC,
DLDI, HA22T, HEPG2 and HONE1 cell lines, but its
potency is less than that of 14e. Compounds 15a, b and
(
(
WI38) were affected to
a
much lesser extent
IC [ 10,000 nM). The reference compound used is the
5
0
CHS-828 which is a pyridyl cyanoguanidine antitumor
agent.
Structure activity relationship
16a, b showed low potency against the six cancer cell lines.
From Table 1, the newly synthesized compounds were
tested against the six cancer cell lines the human gastric
cancer (NUGC), human colon cancer (DLD1), human liver
cancer (HA22T and HEPG2), human breast cancer (MCF),
Concerning the 2,3-dihydropyrido[2,3-d]pyrimidin-7(8H)-
one derivatives 18a and 18b, it is clear that compound 18b
with the 4-hydroxy group showed more potency against the
six cancer cell lines than compound 18a with the 4-amino
moiety. Finally, regarding the pyran derivatives 19a–f, it is
obvious that compound 19b with electronegative groups
nasopharyngeal carcinoma (HONE1) and
fibroblast cells (WI38). The heterocyclic compounds 4, 7a,
a, 8b, 10c, 10d, 12c, 12d, 12e, 12f, 14e, 15b, 18b, 19b
a normal
8
the CN and Cl and compound 19e with COCH and the Cl
3
and 19e exhibited optimal cytotoxic effect against cancer
cell lines, with IC50 in the nM range. Comparing the
cytotoxicity of the tetrahydrobenzo[b]thiophene derivative
groups showed the highest cytotoxicity through such series
of compounds.
3
and the tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-
Toxicity
derivatives 4 exhibited clearly that cyclization of com-
pound 3 to the tricyclic product 4 increases the cytotoxicity
of the latter compound. On the other hand, the reaction of
compound 4 with hydrazine hydrate gave the 3-phenyl-
Bioactive compounds are often toxic to shrimp larvae.
Thus, in order to monitor these chemicals in vivo lethality
to shrimp larvae (Artemiasalina), Brine-Shrimp Lethality
Assay (Choudhary and Thomsen 2001) was used. Results
were analyzed with LC50 program to determine LC50 val-
ues and 95 % confidence intervals (Brayn et al. 1993).
Results are given in Table 2 for the compounds which
exhibited optimal cytotoxic effect against cancer cell lines,
respectively, the following twelve compounds: 4, 7a, 8a,
8b, 10c, 10d, 12c, 12d, 12e, 12f, 14e, 15b, 18b, 19b and
19e. The shrimp lethality assay is considered as a useful
tool for preliminary assessment of toxicity, and it has been
used for the detection of fungal toxins, plant extract toxi-
city, heavy metals, cyanobacteria toxins, pesticides, and
cytotoxicity testing of dental materials (Carballo et al.
2002), natural and synthetic organic compounds (Choud-
hary and Thomsen 2001). It has also been shown that A.
salina toxicity test results have a correlation with rodent
and human acute oral toxicity data. Generally, a good
correlation was obtained between A. salina toxicity test and
the rodent data. Likewise, the predictive screening poten-
tial of the aquatic invertebrate tests for acute oral toxicity
in man, including A. salina toxicity test, was slightly better
than the rat test for test compounds (Calleja and Persoone
1992).
6
,7,8,9-tetrahydrobenzo[4,5]thieno[2,3-d][1,2,4]triazolo
[
4,3-a]pyrimidin-5-amine5 through which the cytotoxicity
decreases against the tested cancer cell lines. Such decrease
in cytotoxicity is attributed to the replacement of the thione
moiety by the nitrogen of the hydrazine. On the other hand,
the reaction of compound 4 with either ethyl cyanoacetate
or ethyl acetoacetate produced compounds 7a and 7b,
respectively. The amide derivative 7a displayed remark-
able cytotoxicity which is more than that of compound 4
against the tested cancer cell lines. Concerning the hex-
ahydro-1H-benzo[4,5]thieno[3,2-e]pyrimido[1,2-c]pyrim-
idine derivatives 8a and 8b, it is clear that both of the two
compounds showed high cytotoxicity against NUGC,
DLDI, HA22T, HEPG2 and HONE1 cell lines.
Compound 8b with the COCH moiety is more potent
3
against the five cell lines than compound 8a with the CN
moiety. For the thiophene derivatives 10a–d, compounds
1
0c and 10d with the COOEt moiety are more potent than
0a and 10b. Moreover, compound 10a showed potency
1
against NUGC, DLDI, HA22T, HEPG2 and HONE1 cell
lines, but compound 10b showed potency against the six
cell lines.
123

3
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Med Chem Res (2015) 24:3932–3948
Scheme 2 Synthesis of
compounds 8a, b; 10a–d; and
1
2a–f
In order to prevent the toxicity results from possible false
Experimental
effects originated from solubility of compounds and
DMSO’s possible toxicity effect, solutions of the test com-
pounds were prepared in the suggested DMSO volume ran-
ges. It is clear from Table 2 that the N-(3-benzoyl-2-thioxo-
1
3
1
C NMR and H NMR spectra were recorded on Bruker
DPX400 instrument in DMSO with TMS as internal stan-
dard for protons and solvent signals as internal standard for
carbon spectra. Chemical shift values are mentioned in d
(ppm). Mass spectra were recorded on EIMS (Shimadzu)
and ESI-esquire 3000 Bruker Daltonics instrument. Ele-
mental analyses were carried out by the Microanalytical
Data Unit at Cairo University. The progress of all reactions
was monitored by TLC on 2 9 5 cm precoated silica gel
2
,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)-2-cyano-3-(4-methoxyphenyl)acrylamide (12c) and the
N -(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thi
eno[2,3-d]pyrimidin-4-yl)-3-oxo-2-(2-(4-chlorophenyl)hy-
drazono)-butanamide (14e) showed nontoxicity against the
tested organisms.
1
23

Med Chem Res (2015) 24:3932–3948
3937
Scheme 3 Synthesis of
compounds 14a–f; 15a, b; and
1
6a, b
6
0 F254 plates of thickness of 0.25 mm (Merck). Com-
0.01 mol) was added. The whole reaction mixture was
heated under reflux for 2 h and then poured onto ice/water,
and the formed solid product was collected by filtration.
Compound 3: Yellow crystals (EtOH), yield 80 %
pound 1 was synthesized through Gewald’s thiophene
synthesis as reported (Gewald et al. 1966). Compound 4
was synthesized earlier according to reported literature
-
1
(
Amr et al. 2010; Hacker et al. 2009; Leistner et al. 1989).
(2.73 g), mp 180–183 °C. IR (KBr) cm : 3460, 3325,
-1 1
057, 2220, 1688, 1662, 1625 cm . H NMR (DMSO-d ,
3
6
N-((3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
yl)carbamothioyl)benzamide (3)
400 MHz): d = 8.28, 8.32 (2s, 2H, D O exchangeable,
2
2NH), 7.28–7.35 (m, 5H, Bz), 2.24–2.26 (m, 4H, 2H-5, 2H-
1
), 1.80–1.84 (m, 4H, 2H-4, 2H-7). C NMR (DMSO-d ,
3
6
6
To s solution of benzoyl isothiocyanate [prepared by add-
ing benzoylchloride (0.01 mol) (1.40 g, 0.01 mol) to a
solution of ammonium thiocyanate (0.76 g, 0.01 mol) in
75 MHz): d = 179.5 (C=S, C-2), 165.3 (CO–Bz), 116.7
(CN), 144.2,138.0, 133.8, 130.4, 128.7, 124.9, 124.1,
0
0
119.2, (C-2, C-3, C-4 , C-5 , Bz), 28.6, 25.7, 22.1, 19.2
?
(C-4, C-5, C-6, C-7), EIMS m/z 341 [M] (20); Analysis
1
,4-dioxane and heating for 10 min] compound 1 (1.78 g,
123

3
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Med Chem Res (2015) 24:3932–3948
Ph
X
N
S
H C
3
N
1
,4-dioxane
16a,b
PhNCS
+
N
S
Et N
3
NH
S
17
O
N
1
8a, X = NH2
b, X = OH
NH2
O
NC
CHO
H C
2
CN
7a or 7b
CN
+
9
COPh
NH
EtOH/Et N
3
R
N
R
X
S
11a, R = H
b, R = Cl
c, R = OCH3
N
S
19
X
R
a
CN
H
b
c
CN
Cl
CN
OCH3
d
e
COCH3
H
Cl
OCH
COCH3
COCH3
f
3
Scheme 4 Synthesis of compounds 18a, b and 19a–f
Calcd for C H N OS (341.45): C, 59.80; H, 4.43; N,
2
product was triturated with ethanol and the formed solid
product was collected by filtration.
1
7
15
3
1
1
2.31; S, 18.78. Found: C, 59.66; H, 4.31; N, 12.46; S,
8.66.
3-Phenyl-6,7,8,9-tetrahydrobenzo[4,5]thieno[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidin-5-amine (5)
(
4-Amino-2-thioxo-5,6,7,8-
tetrahyobenzo[4,5]thieno[2,3-d]pyrimidin-3(2H)-
yl)(phenyl)methanone (4)
To a solution of compound 4 (3.41 g, 0.01 mol) in 1,4-
dioxane (40 mL) hydrazine hydrate (0.50 g, 0.01 mol) was
added. The reaction mixture was heated under reflux for
4 h and then poured onto ice/water containing few drops of
hydrochloric acid, and the formed solid product was col-
lected by filtration.
A solution of compound 3 (3.41 g, 0.01 mol) in 1,4-diox-
ane (40 mL) containing triethylamine (0.50 mL) was
heated under reflux for 6 h and then left to cool. The
solution was evaporated under vacuum and the remaining
1
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Med Chem Res (2015) 24:3932–3948
3939
b
Table 1 Cytotoxicity of the newly synthesized products against a variety of cancer cell lines [IC50 (nM)]
a
Compoundd
Cytotoxicity (IC50 in nM)
c
NUGC
DLDI
HA22T
HEPG2
HONE1
MCF
WI38
3
4
5
2213
380
2074
220
2187
59
1302
480
1802
1099
1180
1277
1028
1529
341
2172
629
1029
336
180
770
650
620
490
210
680
450
280
384
482
512
128
320
662
690
260
380
466
589
280
390
520
360
544
631
428
429
860
634
532
493
650
190
262
378
1288
48
2530
122
2135
3289
670
1729
480
7
7
8
8
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
1622
356
396
280
55
274
2169
2253
2540
1893
1655
1243
318
a
1652
270
490
b
36
528
0a
0b
0c
0d
2a
2b
2c
2d
2e
2f
2210
1128
40
2433
1892
68
1650
2377
312
1850
2138
328
1544
1290
824
86
42
203
253
210
2101
1135
38
1380
1240
46
2258
1278
120
2166
2359
337
2180
1266
441
2330
2555
180
48
33
28
260
348
38
222
234
30
210
65
252
226
122
59
270
1140
1584
326
1160
2270
1220
222
4a
4b
4c
4d
4e
4f
3138
232
1232
115
122
3268
60
2228
225
3328
370
2265
1180
35
2257
2560
61
322
2250
3330
66
2128
22
1180
623
1120
1270
2212
1355
2283
1480
148
1246
1290
3832
160
2458
1150
163
2255
447
3210
2366
66
1128
1355
1148
290
2334
2120
1220
221
2340
2440
2333
2229
2313
1260
480
2155
2457
2673
2332
2148
1140
860
5a
5b
6a
6b
8a
8b
9a
9b
9c
9d
9e
9f
1844
1140
63
2560
1328
232
1620
128
1760
264
2520
350
2088
470
1264
328
1145
3320
48
3218
2781
123
2276
3744
125
2672
1589
386
2711
1130
326
1253
25
2139
2315
2470
2067
1216
1245
2205
15
1286
18
CHS 828
a
NUGC, gastric cancer, DLDI, colon cancer, HA22T, liver cancer, HEPG2, liver cancer; HONEI, nasopharyngeal carcinoma; HR, gastric
cancer; MCF, breast cancer; WI38, normal fibroblast cells
b
The sample concentration produces a 50 % reduction in cell growth and CHS-828 is a pyridyl cyanoguanidine antitumor agent
4
IC50 against the normal fibroblast cells (WI38) are indicated as multiples of 10 nM
c
1
3
Compound 5: Yellow crystals (1,4-dioxane), yield 75 %
1
4H, 2H-7, 2H-8).
C NMR (DMSO-d , 75 MHz):
6
-
(
2.41 g), mp 177–179 °C. IR (KBr) cm : 3477, 3326,
d = 170.3, 172.8, 173.6 (3 C=N),145.0,141.1, 138.8,
132.6, 129.0, 124.6, 123.8, 120.3 (thiophene, pyrimidine,
Bz), 28.8, 25.7, 22.3,19.2(C-6, C-7, C-8, C-9), EIMS m/z
1
3
053, 1636.
H
NMR (DMSO-d6, 400 MHz):
d = 7.31–7.38 (m, 5H, Bz), 5.01 (s, 2H, D O exchange-
2
?
able, NH ), 2.24–2.29 (m, 4H, 2H-6, 2H-9), 1.79–1.84 (m,
2
321 [M] (18); Analysis Calcd for C H N S (321.41): C,
17 15 5
123

3
940
Med Chem Res (2015) 24:3932–3948
Table 2 Toxicity of compounds 4, 7a, 8a, 8b, 10c, 10d, 12c, 12d, 12e, 12f, 14e, 15b, 18b, 19b and 19e
a
Compound no.
Cons. (lg/ml)
Mortality
Toxicity
Harmful
LC50
Upper 95 % lim.
Lower 95 % lim
110.40
4
10
5
8
314.03
252.25
660.32
60.28
20.22
1002.2
–
1
00
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
000
10
0
7
8
8
1
1
1
1
1
1
1
1
1
1
1
a
a
10
Harmful
Harmful
Very toxic
Toxic
18.28
251.19
18.25
102.00
16.55
20.56
10.00
254.16
22.45
18.92
14.68
12.28
12.48
14.68
182.32
4.32
1
00
4
000
10
8
a
0
1
00
5
000
10
10
5
b
32.51
140.62
–
1
00
9
000
10
10
5
0c
0d
2c
2d
2e
2f
4e
5b
8b
9b
9e
1
00
8
000
10
10
0
Harmful
Nontoxic
Toxic
1
00
3
000
10
8
0
–
–
1
00
0
000
10
5
4
293.03
653.32
–
74.23
159.66
–
1
00
8
000
10
10
2
Very toxic
Harmful
Nontoxic
Harmful
Harmful
Harmful
Harmful
1
00
5
000
10
10
4
1
00
5
000
10
8
0
–
–
1
00
0
000
10
5
0
622.17
325.40
350.27
650.37
148.60
150.65
160.20
166.20
1
00
4
000
10
8
0
1
00
5
000
10
10
0
1
00
5
000
10
10
0
1
00
5
000
9
Ten organisms (A. salina) tested for each concentration
1
23

Med Chem Res (2015) 24:3932–3948
3941
6
3.53; H, 4.70; N, 21.79; S, 9.98. Found: C, 63.82; H, 4.85;
ethoxide solution [prepared by dissolving sodium metal
(0.46 g, 0.02 mol) in absolute ethanol (40 mL)] was heated in
a boiling water for 4 h. The reaction mixture was poured onto
ice/watercontainingfewdropsofhydrochloricacid(tillpH6),
and the formed solid product was collected by filtration.
Compound 8a: Pale yellow crystals (1,4-dioxane), yield
N, 21.84; S, 10.24.
N-(3-benzoyl-2-thioxo-2,,4,5,6,7,8-hexahy-
drobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-
cyanoacetamide(7a) and N-(3-benzoyl-2-thioxo-
-
1
2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-
d]pyrimidin-4-yl)-3-oxobutanamide (7b)
66 % (2.58 g), mp 193–196 °C. IR (KBr) cm : 3474,
1
3334, 3056, 2980, 2220, 1690, 1632. H NMR (DMSO-d ,
6
4
00 MHz): d = 8.41 (s, 1H, D O exchangeable NH),
2
General procedure: To a solution of compound 4 (3.41 g,
7.29–7.38 (m, 5H, Bz), 2.24–2.29 (m, 4H, 2H-9, 2H-12),
1
3
0
.01 mol) in dimethylformamide (40 mL), either ethyl
cyanoacetate (1.13 g, 0.01 mol) or ethyl acetoacetate
1.30 g, 0.01 mol) was added. The reaction mixture in each
1.81–1.87 (m, 4H, 2H-10, 2H-11), C NMR (DMSO-d6,
75 MHz): d = 180.6 (C=S, C-6), 170.4 (C=N, C-7), 166.4
(CO-pyrimidine C-3), 151.6, 148.9, 143.7, 139.0, 136.8,
134.2,129.3, 124.8, 123.6, 120.6(Bz, pyrimidine, thiophene
C), 116.2 (CN), 28.8, 25.5, 22.4, 19.0(C-9, C-10, C-11,
(
case was heated under reflux for 3 h and then poured onto
ice/water, and the formed solid product was collected by
filtration.
?
C-12), EIMS m/z 390 [M] (26); Analysis Calcd for C20
Compound 7a: Yellow crystals (1,4-dioxane), yield 80 %
H N OS (390.48): C, 61.52; H, 3.61; N, 14.35; S, 16.42.
4
1
4
2
-1
(
3.27 g), mp 233–235 °C. IR (KBr) cm : 3463, 3324,
Found: C, 61.38; H, 3.59; N, 14.49; S, 16.26.
1
058, 2988, 2220, 1687, 1679, 1633. H NMR (DMSO-d ,
3
4
7
2
Compound 8b: Yellow crystals (1,4-dioxane), yield
-
6
1
00 MHz): d = 8.39 (s, 1H, D O exchangeable NH),
76 % (3.10 g), mp 153–155 °C. IR (KBr) cm : 3456,
3341, 3053, 2986, 1715, 1684, 1632, 1210. H NMR
2
1
.32–7.39 (m, 5H, Bz), 5.20 (s, 2H, CH ), 2.26–2.30 (m, 4H,
2
1
H-4, 2H-7), 1.82–1.86 (m, 4H, 2H-5, 2H-6). C NMR
3
(DMSO-d , 400 MHz): d = 8.26 (s, 1H, D O exchange-
6
2
(
DMSO-d , 75 MHz): d = 180.4 (C=S, C-2), 170.2 (C=N,
able NH), 7.26–7.39 (m, 5H, Bz), 2.71 (s, 3H, CH3),
6
C-1), 163.6, 166.8 (CO–Bz, CO–NH), 144.8,140.9, 136.8,
2.24–2.36 (m, 4H, 2H-9, 2H-12), 1.80–1.85 (m, 4H, 2H-10,
1
3
1
43.2, 128.5, 124.8, 124.3, 120.6 (Bz, pyrimidine, thiophene
2H-11). C NMR (DMSO-d , 75 MHz): d = 180.6 (C=S,
6
C), 116.8 (CN), 28.6, 25.4, 22.6, 19.2(C-4, C-5, C-6, C-7),
?
EIMS m/z 408 [M] (31); Analysis Calcd for C H N O S
C-6), 170.6 (C=N, C-7),166.0,163.4 (CO-pyrimidine C-3,
COCH ), 152.3, 148.0, 142.7, 136.9, 133.6, 126.8, 124.9,
3
2
0
16
4
2
2
(
408.51): C, 58.80; H, 3.95; N, 13.72; S, 15.70. Found: C,
124.6, 123.8, 122.3 (Bz, pyrimidine, thiophene C), 28.9,
5
8.72; H, 4.21; N, 13.49; S, 15.63.
25.4, 22.5, 19.6, (C-9, C-10, C-11, C-12), 18.4 (CH ).
3
?
EIMS m/z 407 [M] (32); Analysis Calcd for C H N
Compound 7b: Yellow crystals (1,4-dioxane), yield
1
2
1
17
3
-
8
0 % (3.40 g), mp 170–173 °C. IR (KBr) cm1 : 3476,
O S (407.51): C, 61.90; H, 4.21; N, 10.31; S, 15.74.
2 2
3
364, 3055, 2983, 1710, 1681, 1633, 1200. H NMR
Found: C, 62.08; H, 4.13; N, 10.29; S, 15.52.
(
DMSO-d , 400 MHz): d = 8.22 (s, 1H, D O exchange-
6
2
able NH), 7.29–7.36 (m, 5H, Bz), 4.87 (s, 2H, CH ), 2.63
2
3,5-Diamino-N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-4-
cyanothiophene-2-carboxamide (10a), 5-amino-N-(3-
benzoyl-2-thioxo-2,3,5,6,7,8-
(
(
s, 3H, CH ), 2.27–2.32 (m, 4H, 2H-4, 2H-7), 1.81–1.86
3
1
m, 4H, 2H-5, 2H-6). C NMR (DMSO-d , 75 MHz):
3
6
d = 180.2 (C=S, C-2), 170.2 (C=N, C-1), 163.2, 164.8,
1
66.0 (CO–Bz, CO–NH, COCH ), 152.3, 146.7, 138.6,
3
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-4-
cyano-3-methylthiophene-2-carboxamide (10b),
ethyl 2,4-diamino-5-((3-benzoyl-2-thioxo-2,3,5,6,7,8-
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)carbamoyl)thiophene-3-carboxylate (10c)
and ethyl 2-amino-5-((3-benzoyl-2-thioxo-2,3,5,6,7,8-
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)carbamoyl)-4-methylthiophene-3-carboxylate
(10d)
1
33.2, 128.5, 124.8, 124.3, 121.9 (Bz, pyrimidine, thio-
phene C),116.3 (CN), 60.4 (s, 2H, CH ), 28.6, 25.4, 22.8,
2
?
1
9.4 (C-4, C-5, C-6, C-7), 18.3 (CH ). EIMS m/z 425 [M]
3
(
40); Analysis Calcd for C H N O S (425.52): C, 59.28;
21 19 3 3 2
H, 4.50; N, 9.88; S, 15.07. Found: C, 59.40; H, 4.43; N,
9
.75; S, 15.23.
2
1
-Oxo-4-phenyl-6-thioxo-2,6,9,10,11,12-hexahydro-
H-benzo[4,5]thieno[3,2-e]pyrimido[1,2-
c]pyrimidine-3-carbonitrile (8a) and 3-acetyl-4-
phenyl-6-thioxo-9,10,11,12-tetrahydrop-1H-
benzo[4,5]thieno[3,2-e]pyrimido-2(6H)-one (8b)
General procedure: To a solution of either compound 7a
(4.08 g, 0.01 mol) or 7b (4.25 g, 0.01 mol) in 1,4-dioxane
(40 mL) containing triethylamine (0.50 mL), both of ele-
mental sulfur (0.32 g, 0.01 mol) and of either malononitrile
(0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol)
were added. The whole reaction mixture, in each case, was
General procedure: A suspension of either compound 7a
(
4.08 g, 0.01 mol) or 7b (4.25 g, 0.01 mol) in sodium
123

3
942
Med Chem Res (2015) 24:3932–3948
heated under reflux for 1 h and then poured onto ice/water
containing few drops of hydrochloric acid. The formed
solid product was collected by filtration.
2.23-2.38 (m, 4H, 2H-5, 2H-8), 1.83–1.89 (m, 4H, 2H-6, 2H-
1
7), 1.14 (t, 3H, J = 7.30 Hz, CH ). C NMR (DMSO-d ,
3
3
6
75 MHz): d = 180.0 (C=S, C-2), 170.3 (C=N, C-1), 166.8,
164.4, 163.2 (CO–Bz, CO–NH, CO–OEt), 154.2, 152.4,
146.2, 142.3, 136.9, 133.6, 126.3, 124.7, 124.6, 123.2, 122.8,
Compound 10a: Orange crystals (1,4-dioxane), yield
-
1
7
3 % (3.70 g), mp 231–233 °C. IR (KBr) cm : 3469, 3324,
1
059, 2987, 2220, 1688, 1684, 1630, 1210. H NMR
3
121.4 (Bz, pyrimidine, two thiophene C), 49.2 (CH ), 28.9,
2
(
DMSO-d , 400 MHz): d = 8.38 (s, 1H, D O exchangeable
25.8, 22.7, 19.7 (C-5, C-6, C-7, C-8), 18.5 (CH ), 16.3 (CH ,
3
6
2
3
?
NH), 7.33–7.42 (m, 5H, Bz), 4.21,4.48 (2s, 4H, D O
2
OCH CH ). EIMS m/z 552 [M] (32); Analysis Calcd for
2 3
exchangeable, 2 NH ), 2.25-2.28 (m, 4H, 2H-5, 2H-8),
2
C H N O S (552.68): C, 56.50; H, 4.38, N, 10.14; S,
26 24 4 4 3
1
.82–1.89 (m, 4H, 2H-6, 2H-7). C NMR (DMSO-d ,
3
1
7
1
1
17.41. Found: C, 56.69; H, 4.26; N, 10.09; S, 17.66.
6
5 MHz): d = 180.3 (C=S, C-2), 170.6 (C=N, C-1), 162.8,
66.8 (CO-Bz, CO–NH), 152.8, 149.0, 148.2, 146.5, 143.8,
39.0, 135.4, 134.2, 129.2, 124.9, 122.8, 120.8 (Bz, pyrim-
N-(3-Benzoyl-2-thioxo-2,3,5,6,7,8-
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-
cyano-3-phenylacrylamide (12a), N-(3-Benzoyl-2-
thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-
d]pyrimidin-4-yl)-2-cyano-3-(4-
idine, two thiophene C), 116.4 (CN), 28.8, 25.4, 22.7, 19.3,
?
C-5, C-6, C-7, C-8), EIMS m/z 506 [M] (18); Analysis
(
Calcd for C H N O S (506.62): C, 54.53; H, 3.58; N,
23 18 6 2 3
1
6.59; S, 18.99. Found: C, 54.80; H, 3.41; N, 16.33; S, 19.06.
chlorophenyl)acrylamide (12b), N-(3-Benzoyl-2-
thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-
d]pyrimidin-4-yl)-2-cyano-3-(4-
Compound 10b: Yellow crystals (1,4-dioxane), yield
-1
67 % (3.39 g), mp 210–212 °C. IR (KBr) cm : 3476, 3332,
3060, 2989, 2220, 1705, 1686, 1631, 1215. H NMR(DMSO-
1
methoxyphenyl)acrylamide (12c), N-(3-benzoyl-2-
thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-
d]pyrimidin-4-yl)-2-benzylidene-3-oxobutanamide
(12d), N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-
d , 400 MHz): d = 8.28 (s, 1H, D O exchangeable NH),
6
2
7
.28–7.37 (m, 5H, Bz),4.47 (s, 2H, D O exchangeable, NH ),
2 2
2
.69 (s, 3H, CH ), 2.22–2.35 (m, 4H, 2H-5, 2H-8), 1.82–1.86
3
1
m, 4H, 2H-6, 2H-7). C NMR (DMSO-d , 75 MHz):
3
(
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-
(4-chlorobenzylidene)-3-oxobutanamide (12e)
and N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-
6
d = 180.4 (C=S, C-2), 170.5 (C=N, C-1), 163.6, 166.3 (CO–
Bz, CO–NH), 154.0, 152.7, 146.2, 142.3, 136.9, 132.8, 126.6,
1
24.5, 124.3, 123.0,122.6, 121.8 (Bz, pyrimidine, two thio-
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2-
(4-methoxybenzylidene)-3-oxobutanamide (12f)
phene C), 117.0 (CN), 28.9, 25.5, 22.7, 19.4 (C-5, C-6, C-7,
?
C-8),18.7 (CH ). EIMS m/z 505 [M] (18); Analysis Calcd
3
for C H N O S (505.64): C, 57.01; H, 3.79N, 13.85; S,
24 19 5 2 3
General procedure: To a solution of either compound 7a
(4.08 g, 0.01 mol) or 7b (4.25 g, 0.01 mol) in 1,4-dioxane
(40 mL) containing piperidine (0.50 mL), either ben-
zaldehyde (1.06 g, 0.01 mol), 4-chlorobenzaldehyde
(1.40 g, 0.01 mol) or 4-methoxybenzaldehyde (1.36 g,
0.01 mol) was added. The whole reaction mixture was
heated under reflux for 4 h and then evaporated under
vacuum. The remaining product was triturated with diethyl
ether, and the formed solid product was collected by
filtration.
1
9.02. Found: C, 56.87; H, 4.09; N, 13.69; S, 18.89.
Compound 10c: Orange crystals (1,4-dioxane), yield
-
1
6
5 % (3.70 g), mp 180–184 °C. IR (KBr) cm : 3478,
1
320, 3062, 2984, 2887, 1690, 1686, 1630, 1212. H NMR
3
(
DMSO-d , 400 MHz): d = 8.33 (s, 1H, D O exchange-
6
2
able NH), 7.32–7.40 (m, 5H, Bz), 4.23,4.45 (2s, 4H, D O
2
exchangeable, 2 NH ), 4.21 (q, 2H, J = 7.02 Hz, CH ),
2
2
2
2
.22–2.29 (m, 4H, 2H-5, 2H-8), 1.83–1.89 (m, 4H, 2H-6,
1
H-7), 1.14 (t, 3H, J = 7.02 Hz, CH ). C NMR (DMSO-
3
3
d6, 75 MHz): d = 180.6 (C=S, C-2), 170.4 (C=N, C-1),
Compound 12a: Yellow crystals (1,4-dioxane), yield
-1
1
1
1
2
1
62.6, 163.8, 166.2 (CO–Bz, CO–NH, CO–OEt), 152.9,
50.3, 148.6, 146.5, 142.6, 139.0, 134.6, 132.9, 129.0,
24.9, 122.6, 120.3 (Bz, pyrimidine, two thiophene C),
70 % (3.48 g), mp 170–172 °C. IR (KBr) cm : 3455,
3331, 3056, 2984, 2222, 1689, 1680, 1632, 1210. H NMR
1
(DMSO-d , 400 MHz): d = 8.32 (s, 1H, D O exchangeable
6
2
8.7, 25.2, 22.9, 19.6, (C-5, C-6, C-7, C-8), 49.2 (CH ),
2
NH), 7.28–7.40 (m, 10H, 2Bz),6.01 (s, 1H, CH=C),
?
6.0 (CH ), EIMS m/z 553 [M] (28); Analysis Calcd for
2.26–2.29 (m, 4H, 2H-5, 2H-8), 1.80–1.88 (m, 4H, 2H-6,
1
3
3
C H N O S (553.67): C, 54.23; H, 4.19; N, 12.65; S,
3
2H-7). C NMR (DMSO-d , 75 MHz): d = 180.2 (C=S,
2
5
23
5
4
6
1
7.37. Found: C, 54.59; H, 4.52; N, 12.49; S, 17.22.
C-2), 170.4 (C=N, C-1),163.2, 166.4 (CO–Bz, CO–NH),
152.6, 149.2, 148.2, 146.5, 143.8, 139.0, 135.4, 128.6, 124.2,
123.9, 120.4, 119.6 (2Bz, pyrimidine, thiophene C), 116.7
(CN), 109.6, 114.3 (CH=C), 28.8, 25.6, 22.4, 19.1 (C-5, C-6,
Compound 10d: Yellow crystals (1,4-dioxane), yield
-1
7
1 % (3.92 g), mp 166–168 °C. IR (KBr) cm : 3483,
1
328, 3063, 2984, 2887, 1702, 1689, 1633, 1213. H NMR
3
?
(
DMSO-d , 400 MHz): d = 8.31 (s, 1H, D O exchangeable
C-7, C-8). EIMS m/z 496 [M] (21); Analysis Calcd for
6
2
NH), 7.29–7.36 (m, 5H, Bz), 4.48 (s, 2H, D O exchangeable,
2
C H N O S (496.60): C, 65.30; H, 4.06; N, 11.28; S,
27 20 4 2 2
NH ), 4.21 (q, 2H, J = 7.30 Hz, CH ), 2.65 (s, 3H, CH ),
2
12.91. Found: C, 65.29; H, 3.81; N, 11.08; S, 12.76.
2
3
1
23

Med Chem Res (2015) 24:3932–3948
3943
Compound 12b: Yellow crystals (1,4-dioxane), yield
-
146.2, 144.2, 136.9, 133.6, 126.3, 124.9, 124.2, 123.9,
123.0, 121.7, (2Bz, pyrimidine, thiophene C), 28.5, 25.8,
1
7
0 % (3.72 g), mp 188-190 °C. IR (KBr) cm : 3483,
1
312, 3058, 2984, 2220, 1710, 1688, 1630, 1211. H NMR
3
22.9, 19.6 (C-5, C-6, C-7, C-8),19.3 (CH ).EIMS m/z 548
3
?
[M] (21); Analysis Calcd for C H ClN O S (548.07):
(
DMSO-d , 400 MHz): d = 8.03 (s, 1H, D O exchange-
6
2
28 22
3 3 2
able NH), 7.26–7.39 (m, 9H, C H , C H ), 5.99 (s, 1H,
6
C, 61.36; H, 4.05, N, 7.67; S, 11.70. Found: C, 61.49; H,
4.25; N, 6.83; S, 11.92.
5
6 4
CH=C), 2.24-2.38 (m, 4H, 2H-5, 2H-8), 1.80–1.87 (m, 4H,
1
3
2
H-6, 2H-7). C NMR (DMSO-d , 75 MHz): d = 180.6
Compound 12f: Pale yellow crystals (1,4-dioxane), yield
1
6
-
(
C=S, C-1), 170.3 (C=N, C-2), 166.1, 164.8 (CO–Bz, CO–
NH), 154.2, 153.4, 146.2, 142.6, 136.9, 133.4, 129.6,
26.6, 124.5, 123.8, 122.8, 120.9 (2Bz, thiophene, pyrim-
67 % (3.64 g), mp 277–279 °C. IR (KBr) cm : 3484,
3318, 3058, 2988, 2887, 1720–1684, 1636, 1202. H NMR
1
1
(DMSO-d , 400 MHz): d = 8.34 (s, 1H, D O exchange-
6
2
idine C), 116.7 (CN), 28.7, 25.5, 22.4, 19.6 (C-5, C-6, C-7,
?
C-8), EIMS m/z 531 [M] (22); Analysis Calcd for C27
able NH), 7.30–7.46 (m, 9H, C H , C H ), 6.06 (s, 1H,
6 5 6 4
CH=C), 2.78, 3.01 (2s, 6H, 2CH ), 2.22–2.34 (m, 4H, 2H-
3
1
5, 2H-8), 1.88–1.93 (m, 4H, 2H-6, 2H-7). C NMR
3
H ClN O S (531.05): C, 61.07; H, 3.61; N, 10.55; S,
1
9
4 2 2
1
2.08. Found: C, 60.91; H, 3.80; N, 10.76; S, 11.89.
(DMSO-d , 75 MHz): d = 180.5 (C=S, C-2), 170.2 (C=N,
6
Compound 12c: Orange crystals (1,4-dioxane), yield
-
C-1), 163.2, 164.5, 166.7 (CO–Bz, CO–NH,
1
5
8 % (3.05 g), mp 220–222 °C. IR (KBr) cm : 3445,
COCH ),153.6, 151.9, 146.2, 143.6, 136.9, 133.8, 126.3,
3
1
328, 3055, 2989, 2884, 2222, 1695, 1688, 1632, 1218. H
3
125.3, 124.6, 123.9, 122.8, 121.3(2Bz, pyrimidine, thio-
NMR (DMSO-d , 400 MHz): d = 8.09 (s, 1H, D O
phene C), 24.24 (OCH ), 28.6, 25.8, 22.9, 19.4 (C-5, C-6,
3
6
2
?
C-7, C-8), 19.8 (CH ). EIMS m/z 543 [M] (12); Analysis
exchangeable NH), 7.31–7.38 (m, 9H, C H , C H ),6.11 (s,
6
5
6
4
3
1
8
7
H, CH=C), 2.88 (s, 3H, CH ),2.23-2.26 (m, 4H, 2H-5, 2H-
3
Calcd for C H N O S (543.65): C, 64.07; H, 4.64, N,
29 25 3 4 2
1
), 1.81–1.87 (m, 4H, 2H-6, 2H-7), C NMR (DMSO-d6,
3
7.73; S, 11.80. Found: C, 63.86; H, 4.43; N, 7.49; S, 11.66.
5 MHz): d = 180.3 (C=S, C-2), 170.9 (C=N,
C-1),164.2,162.9 (CO-Bz, CO–NH),153.3, 150.3, 148.6,
2-((3-Benzoyl-2-thioxo-2,3,5,6,7,8-
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
1
1
2
5
45.9, 142.8, 138.2, 133.6, 136.3, 129.0, 124.9, 122.8,
20.1(2Bz, pyrimidine, thiophene C), 116.8 (CN), 28.9,
0
yl)amino)-2-oxo-N phenylacetohydrazonoyl cyanide
(14a),2-((3-Benzoyl-2-thioxo-2,3,5,6,7,8-
5.5, 22.9, 19.8 (C-5, C-6, C-7, C-8),18.3 (CH ). EIMS m/z
3
?
26 [M] (20); Analysis Calcd for C H N O S
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
0
2
8
22
4
3
2
(
526.62): C, 63.86; H, 4.21; N, 10.64; S, 12.18. Found: C,
yl)amino)-2-oxo-N (4-chlorophenyl)-
acetohydrazonoyl cyanide (14b), 2-((3-Benzoyl-2-
6
3.93; H, 4.30; N, 10.44; S, 12.09.
Compound 12d: Yellow crystals (1,4-dioxane), yield
-
thioxo-2,3,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-
0
1
6
8 % (3.49 g), mp 213–215 °C. IR (KBr) cm : 3456,
d]pyrimidin-4-yl)amino)-2-oxo-N (4-
methylphenyl)aceto-hydrazonoyl cyanide (14c),
N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-
1
330, 3060, 2986, 2880, 1710–1687, 1638, 1218. H NMR
3
(
DMSO-d , 400 MHz): d = 8.28 (s, 1H, D O exchange-
6
2
able NH), 7.31–7.42 (m, 10H, 2Bz),6.02 (s, 1H,
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-3-
oxo-2-(2-phenylhydrazono)butanamide (14d), N-(3-
benzoyl-2-thioxo-2,3,5,6,7,8-
CH=C),2.68 (s, 3H, CH ), 2.24–2.38 (m, 4H, 2H-5, 2H-8),
3
1
.85–1.89 (m, 4H, 2H-6, 2H-7), C NMR (DMSO-d6,
3
1
7
1
1
1
2
5 MHz): d = 180.3 (C=S, C-2), 170.6 (C=N, C-1), 166.4,
64.2, 163.6 (CO–Bz, CO–NH, COCH ), 154.3, 152.4,
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-3-
oxo-2-(2-(4-chlorophenyl)hydrazono)butanamide
(14e) and N-(3-benzoyl-2-thioxo-2,3,5,6,7,8-
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-3-
oxo-2-(2-(4-methylphenyl)hydrazono)-butanamide
(14f)
3
46.2, 144.2, 136.9, 133.6, 126.3, 124.7, 124.6, 123.9,
22.8, 121.6 (2Bz, pyrimidine, thiophene C), 28.5, 25.8,
2.9, 19.4 (C-5, C-6, C-7, C-8), 18.9 (CH ), EIMS m/z 513
3
?
M] (21); Analysis Calcd for C H N O S (513.63): C,
[
2
8 23 3 3 2
6
5.48; H, 4.51, N, 8.18; S, 12.49. Found: C, 65.72; H, 4.49;
N, 8.09; S, 12.66.
General procedure: To a solution of either compound 7a
(4.08 g, 0.01 mol) or 7b (4.25 g, 0.01 mol) in ethanol
(40 mL) containing sodium hydroxide (5 mL, 10 %), a
cold solution of either benzenediazoniumchloride
(0.01 mol), 4-chlorobenzenediazonium chloride (0.01 mol)
or 4-methylbenzenediazonium chloride (0.01 mol) [pre-
pared by the addition of sodium nitrite solution (0.70 g,
0.01 mol) to a cold solution (0–5 °C) of the aniline (0.94 g,
0.01 mol), 4-chloroaniline (1.27 g, 0.01 mol) or 4-methy-
laniline (1.08 g, 0.01 mol) in concentrated hydrochloric
Compound 12e: Pale yellow crystals (1,4-dioxane),
1
-
yield 87 % (4.77 g), mp [ 300 °C. IR (KBr) cm : 3476,
1
326, 3060, 2988, 2883, 1708–1688, 1634, 1209. H NMR
3
(
DMSO-d , 400 MHz): d = 8.28 (s, 1H, D O exchange-
6
2
able NH), 7.31–7.42 (m, 9H, C H , C H ), 6.04 (s, 1H,
6 5
6
4
CH=C), 2.73 (s, 3H, CH ), 2.26–2.36 (m, 4H, 2H-5, 2H-8),
3
1
.86–1.90 (m, 4H, 2H-6, 2H-7). C NMR (DMSO-d6,
3
1
7
1
5 MHz): d = 180.1 (C=S, C-2), 170.7 (C=N, C-1),163.4,
64.8, 166.2 (CO–Bz, CO–NH, COCH ), 154.8, 152.4,
3
123

3
944
Med Chem Res (2015) 24:3932–3948
acid (10 mL, 18 M) was added with continuous stirring]
was added with continuous stirring. The whole reaction
mixture was kept with stirring for 2 h, and the formed solid
product was collected by filtration.
(C=S, C-1), 172.4, 170.1 (2C=N), 166.3, 164.2, 162.4
(CO–Bz, CO–NH, COCH ), 154.3, 151.9, 146.8, 144.2,
3
136.9, 133.8, 126.6, 124.7, 124.6, 123.9, 122.8, 120.8 (2Bz,
pyrimidine, thiophene C), 19.0 (CH ), 28.7, 26.0, 23.6,
3
?
19.6 (C-5, C-6, C-7, C-8). EIMS m/z 529 [M] (18);
Compound 14a: Yellow crystals (1,4-dioxane), yield
-
1
8
6 % (4.41 g), mp 144–146 °C. IR (KBr) cm : 3478,
Analysis Calcd for C H N O S (529.63): C, 61.23; H,
27 23 5 3 2
1
321, 3053, 2986, 2220, 1688, 1683, 1634, 1212. H NMR
3
4.38, N, 13.22; S, 12.11. Found: C, 61.63; H, 4.61; N,
13.47; S, 12.36.
(
DMSO-d , 400 MHz): d = 8.26, 3.11 (2s, 2H, D O
6
2
exchangeable, 2NH), 7.32–7.39 (m, 10H, 2Bz), 2.21–2.28
1
Compound 14e: Yellow crystals (1,4-dioxane), yield
-1
3
(
m, 4H, 2H-5, 2H-8), 1.82–1.84 (m, 4H, 2H-6, 2H-7).
C
79 % (4.46 g), mp 294–297 °C. IR (KBr) cm : 3477, 3340,
3060, 2989, 2885, 1705–1686, 1632, 1210. H NMR
1
NMR (DMSO-d , 75 MHz): d = 180.4 (C=S, C-2), 170.3,
6
1
1
1
72.3 (2C=N), 166.6, 163.8 (CO–Bz, CONH)), 153.8,
49.2, 148.7, 146.5, 143.8, 139.0, 136.3, 134.2, 129.3,
24.2, 121.5, 120.8 (2Bz, pyrimidine, thiophene C), 116.9
(DMSO-d , 400 MHz): d = 8.24, 8.32 (2s, 2H, D O
6
2
exchangeable, 2NH), 7.28–7.40 (m, 9H, C H , C H ), 2.68
6
4
6 5
(s, 3H, CH ), 2.23–2.38 (m, 4H, 2H-5, 2H-8),1.84–1.88 (m,
3
1
3
(
CN), 29.0, 25.3, 22.6, 19.4, (C-5, C-6, C-7, C-8), EIMS m/z
?
4H, 2H-6, 2H-8). C NMR (DMSO-d , 75 MHz): d = 180.3
6
5
12 [M] (28); Analysis Calcd for C H N O S
6 20 6 2 2
(C=S, C-2), 172.3,170.4 (2C=N), 163.2, 164.6, 166.4 (CO–
2
(
512.61): C, 60.92; H, 3.93; N, 16.39; S, 12.51. Found: C,
Bz, CO–NH, COCH ), 153.6, 152.4, 120.9, 123.3, 124.1,
3
6
1.28; H, 3.68; N, 16.59; S, 12.66.
124.3, 124.9, 126.8, 132.4, 135.2, 144.2, 146.2, (2Bz,
Compound 14b: Yellow crystals (1,4-dioxane), yield
-
pyrimidine, thiophene C), 19.6 (CH ), 28.7, 25.9, 22.6, 19.9
3
1
?
(C-5, C-6, C-7, C-8), EIMS m/z 564 [M] (16); Analysis
5
6 % (3.06 g), mp 193–195 °C. IR (KBr) cm : 3480,
1
310, 3055, 2986, 2221, 1690, 1687, 1632, 1214. H NMR
3
Calcd for C H ClN O S (564.07): C, 57.49; H, 3.93, N,
2
7
22
5 3 2
(
DMSO-d , 400 MHz): d = 8.32, 8.12 (2s, 2H, D O
12.42; S, 11.37. Found: C, 57.74; H, 4.02; N, 12.42; S, 11.88.
6
2
exchangeable, 2NH), 7.41–7.28 (m, 9H, C H , C H ),
6
Compound 14f: Yellow crystals (1,4-dioxane), yield
-
5
6 4
1
2
2
.26–2.37 (m, 4H, 2H-5, 2H-8), 1.82–1.87 (m, 4H, 2H-6,
1
77 % (4.19 g), mp [ 300 °C. IR (KBr) cm : 3474, 3339,
3056, 2980, 2881, 1706–1686, 1633, 1210. H NMR
3
1
H-7). C NMR (DMSO-d , 75 MHz): d = 180.6 (C=S,
6
C-2),170.8, 172.2 (2C=N), 164.6, 166.4 (CO–Bz, CO–NH),
(DMSO-d , 400 MHz): d = 8.20,8.34 (2s, 2H, D O
6
2
1
1
1
54.0, 153.6, 146.2, 142.6, 136.6, 133.7, 129.6, 125.3,
24.8, 122.9, 122.5, 120.4 (2Bz, thiophene, pyrimidine C),
exchangeable, 2NH), 7.28–7.43 (m, 9H, C H , C H ), 2.64,
6 5 6 4
3.11 (2s, 6H, 2CH ), 2.25-2.38 (m, 4H, 2H-5, 2H-8),
3
1
1.84–1.91 (m, 4H, 2H-6, 2H-7). C NMR (DMSO-d ,
3
16.9 (CN), 28.9, 25.3, 22.6, 19.5 (C-5, C-6, C-7, C-8),
?
6
EIMS m/z 547 [M] (18); Analysis Calcd for C H
2
75 MHz): d = 180.6 (C=S, C-2), 172.8,170.11 (C=N),
163.0, 164.6, 166.9 (CO–Bz, CO–NH, COCH ), 153.8,
6
19
ClN O S (547.05): C, 57.09; H, 3.50; N, 15.36; S, 11.72.
6
2
2
3
Found: C, 56.93; H, 3.70; N, 15.49; S, 11.93.
151.6, 146.4, 143.9, 136.9, 133.8, 126.8, 125.0, 123.9,
122.7, 121.6, 120.2, 28.9, 25.7, 22.8, 19.8 (C-5, C-6, C-7,
Compound 14c: Orange crystals (1,4-dioxane), yield
-
1
6
2 % (3.27 g), mp 132-135 °C. IR (KBr) cm : 3458, 3319,
C-8), (2Bz, pyrimidine, thiophene C), 19.6, 19.8 (2CH ),
3
1
053, 2988, 2882, 2220, 1705, 1686, 1630, 1220. H NMR
?
EIMS m/z 543 [M] (8); Analysis Calcd for C H N O S
3
2
8
25
5
3
2
(
DMSO-d , 400 MHz): d = 8.12, 8.34 (2s, 2H, D O
(543.66): C, 61.86; H, 4.64, N, 12.88; S, 11.80. Found: C,
61.96; H, 4.84; N, 12.70; S, 11.73.
6
2
exchangeable, 2NH), 7.30–7.36 (m, 9H, C H , C H ), 2.82
6 4
6
5
(
s, 3H, CH ), 2.24–2.28 (m, 4H, 2H-5, 2H-8), 1.83-1.89 (m,
3
1
3
4
H, 2H-6, 2H-7). C NMR (DMSO-d , 75 MHz): d = 180.1
N-(3-Benzoyl-2-thioxo-2,3,5,6,7,8-
6
(
C=S, C-2), 172.3,170.6 (C=N), 164.2, 162.9 (CO–Bz, CO–
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-4,4-
dicyano-3-methylbut-3-enamide (15a) and ethyl 5-((3-
benzoyl-2-thioxo-2,3,5,6,7,8-
NH), 153.8, 150.3, 148.6, 144.2, 143.2, 138.2, 135.8, 133.6,
1
1
29.0, 124.7, 123.6, 120.3 (2Bz pyrimidine, thiophene C),
16.6 (CN), 19.5 (CH ), 28.6, 25.5, 23.4, 19.4 (C-5, C-6, C-7,
3
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)amino)-2-cyano-3-methyl-5-oxopent-2-enoate (15b)
?
C-8), EIMS m/z526 [M] (18); Analysis Calcd for C H
2
7
22
N O S (526.63): C, 61.58; H, 4.21; N, 15.96; S, 12.18.
6 2 2
Found: C, 61.79; H, 4.44; N, 16.27; S, 12.34.
General procedure: To a dry solid of 7b (4.25 g, 0.01 mol)
containing ammonium acetate (0.50 g), either malononitrile
(0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol)
was added. The whole reaction mixture was heated in an oil
bath at 120 °C for 1 h and the formed solid product upon
cooling was triturated with ethanol and collected by filtration.
Compound 15a: Pale orange crystals (acetic acid), yield
Compound 14d: Yellow crystals (1,4-dioxane), yield
-
1
8
1 % (4.29 g), mp 120–123 °C. IR (KBr) cm : 3449,
1
323, 3055, 2988, 2880, 1703–1689, 1638, 1220. H NMR
3
(
DMSO-d , 400 MHz): d = 8.24, 8.32 (2s, 2H, D O
6
2
exchangeable, 2NH), 7.26–7.40 (m, 10H, 2Bz), 2.66 (s, 3H,
CH ), 2.22–2.37 (m, 4H, 2H-5, 2H-8), 1.82–1.87 (m, 4H,
3
1
3
-1
2
H-6, 2H-7). C NMR (DMSO-d , 75 MHz): d = 180.1
74 % (3.50 g), mp 210–212 °C. IR (KBr) cm : 3483, 3324,
6
1
23

Med Chem Res (2015) 24:3932–3948
3945
1
058, 2986, 2223, 2220, 1690, 1688, 1630. H NMR (DMSO-
3
Compound 16a: Yellow crystals (1,4-dioxane), yield
-
1
d , 400 MHz): d = 8.38 (s, 1H, D O exchangeable NH),
77 % (3.65 g), mp 190–192 °C. IR (KBr) cm : 3473,
3331, 3056, 2983, 2873, 2220, 1688, 1684, 1630. H NMR
6
2
1
7
.31–7.39 (m, 5H, Bz), 5.25 (s, 2H, CH ), 3.01 (s, 3H, CH ),
3
2
2
.24–2.29(m, 4H, 2H-5, 2H-8), 1.80–1.87(m, 4H, 2H-6, 2H-7),
(DMSO-d , 400 MHz): d = 7.34-7.36 (m, 5H, C H ), 6.03
(s, 1H, pyridine H-5),4.30 (s, 2H, D O exchangeable,
2
6
6 5
1
3
C NMR (DMSO-d , 75 MHz): d = 19.9 (CH ), 20.3, 22.4,
6
3
2
5.2, 28.9 (4 CH ), 117.2,116.4 (2CN), 152.8, 151.8, 148.9,
2
NH ), 3.11 (s, 3H, CH ), 2.24–2.30 (m, 4H, 2H-5, 2H-
2
3
1
3
143.7, 139.0, 135.9, 134.8, 129.1, 125.3, 123.9, 120.8 (C H
6
8),1.82–1.88 (m, 4H, 2H-6, 2H-7), C NMR (DMSO-d6,
75 MHz): d = 180.1 (C=S, C-2), 170.8 (C=N, C-1), 164.1,
162.3 (CO–Bz, CO, pyridine C-6), 154.2, 152.4, 148.9,
143.7, 140.0, 135.3, 134.5, 129.0, 124.3, 123.9, 122.4,
120.3 (Bz, pyridine, pyrimidine, thiophene C), 116.6 (CN),
5
pyrimidine, thiophene C), 166.4,164.2 (CO–Bz, CO–NH),
?
70.2 (C=N, C-1), 180.3 (C=S, C-2). EIMS m/z 473 [M] (28);
1
Analysis Calcd for C H N O S (473.57): C, 60.88; H, 4.04;
24 19 5 2 2
N, 14.79; S, 13.54. Found: C, 60.72; H, 3.89; N, 14.53; S, 13.66.
Compound 15b: Yellow crystals (acetic acid), yield 90 %
28.9, 25.0, 22.8, 20.1 (C-5, C-6, C-7, C-8), 19.8 (CH ),
3
-1
?
EIMS m/z 473 [M] (20); Analysis Calcd for C H N
(
4.69 g), mp 254–258 °C. IR (KBr) cm : 3456, 3341,
2
4
19
5
1
055, 2986, 2220, 1688, 1683, 1630, 1212. H NMR
3
O S (473.57): C, 60.87; H, 4.04; N, 14.79; S, 13.54.
2 2
(
DMSO-d , 400 MHz): d = 8.28 (s, 1H, D O exchangeable
Found: C, 60.68; H, 3.99; N, 14.61; S, 13.72..
2-Amino-1-(3-benzoyl-2-thioxo-2,3,5,6,7,8-hexahydro
benzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-4-methyl-6-oxo-1,
6-dihydropyridine-3-carbonitrile
6
2
NH), 7.28–7.39 (m, 5H, Bz), 5.21 (s, 2H, CH ), 4.23 (q, 2H,
2
J = 7.22 Hz, CH , OCH CH ), 2.89 (s, 3H, CH ), 2.22-2.38
2
2
3
3
(
m, 4H, 2H-5, 2H-8), 1.80–1.87 (m, 4H, 2H-6, 2H-7), 1.14
1
3
t, 3H, J = 7.22 Hz, CH . OCH CH ), C NMR (DMSO-
(
Compound 16b: Yellow crystals (1,4-dioxane), yield
3
2
3
-1
d6, 75 MHz): d = 180.3 (C=S, C-2), 170.8 (C=N, C-1),
1
70 % (3.64 g), mp 176–178 °C. IR (KBr) cm : 3444, 3337,
1
3054, 2986, 1688, 1683, 1630, 1212. H NMR (DMSO-d ,
64.8, 163.4, 162.6 (CO–Bz, CO–NH, COCH ),154.1,
3
6
1
48.0, 142.7, 136.9, 126.8, 124.9, 123.8, 122.2 (Bz, pyrim-
400 MHz): d = 7.25–7.37 (m, 5H, Bz), 6.22 (s, 1H, pyridine
H-5),4.63 (s, 2H, D O exchangeable, NH ), 4.23 (q, 2H,
idine, thiophene C), 116.6 (CN), 36.8 (OCH CH ), 28.9,
2
3
2
2
2
5.4, 22.6, 19.8 (C-5, C-6, C-7, C-8), 18.8 (CH ), 16.2
3
J = 9.93 Hz, OCH CH ), 2.76 (s, 3H, CH ), 2.20–2.39 (m,
2 3 3
?
OCH CH ), EIMS m/z 520 [M] (18); Analysis Calcd for
(
4H, 2H-5, 2H-8), 1.82–1.87 (m, 4H, 2H-6, 2H-7), 1.16 (t, 3H,
1
2
3
3
C H N O S (520.61): C, 59.98; H, 4.65; N, 10.76; S,
2
J = 6.93 Hz, OCH CH ). C NMR (DMSO-d , 75 MHz):
2 3 6
2
6
24
4
4
1
2.32. Found: C, 60.09; H, 4.44; N, 10.63; S, 12.52.
d = 180.1 (C=S, C-2), 170.5 (C=N, C-1), 162.8, 163.2,
64.8 (CO-Bz, CO-OEt, CO-pyridine C-6), 154.1, 150.4,
1
2
-Amino-1-(3-benzoyl-2-thioxo-2,3,5,6,7,8-
147.2, 142.9, 136.9, 133.6, 126.2, 124.9, 123.3, 122.1, 121.5,
120.3 (Bz, pyridine, pyrimidine, thiophene C), 36.5 (OCH2
CH ), 28.3, 25.8, 22.4, 19.9(C-5, C-6, C-7, C-8), 18.6 (CH ),
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-4-
methyl-6-oxo-1,6-dihydropyridine-3-carbonitrile
3
3
?
(
16a) and ethyl 2-amino-1-(3-benzoyl-2-thioxo-
,3,5,6,7,-hexahydrobenzo[4,5]thieno[2,3-
16.3 (OCH CH ). EIMS m/z 520 [M] (22); Analysis Calcd
2 3
2
for C H N O S (520.61): C, 59.98; H, 4.65; N, 10.76; S,
26 24 4 4 2
d]pyrimidin-4-yl)-4-methyl-6-oxo-1,65-
dihydropyridine-3-carboxylate (16b)
12.32. Found: C, 60.21; H, 4.59; N, 10.49; S, 12.48.
4-Amino-5-methyl-3-phenyl-2-thioxo—(2-thioxo-
Method (A): General procedure: To a suspension of 7b
(
2,3,5,6,7,8-hexahydrobenzo[4,5]-thieno[2,3-
d]pyrimidin-4-yl)-2,3-dihydropyrido[2,3-
4.25 g, 0.01 mol) in sodium ethoxide [prepared by dis-
solving sodium metal (0.46 g, 0.02 mol) in absolute etha-
nol (40 ml)], either malononitrile (0.66 g, 0.01 mol) or
ethyl cyanoacetate (1.13 g, 0.01 mol) was added. The
whole reaction mixture was heated in a boiling water bath
for 6 h and then poured onto ice/water containing few
drops of hydrochloric acid (till pH 6), and the formed solid
product was collected by filtration.
d]pyrimidin-7(8H)-one (18a) and 4-hydroxy-5-
methyl-3-phenyl-2-thioxo—(2-thioxo-2,3,5,6,7,8-
hexahydrobenzo[4,5]-thieno[2,3-d]pyrimidin-4-yl)-
2,3-dihydropyrido[2,3-d]pyrimidin-7(8H)-one (18b)
General procedure: To a solution of either compound 16a
(4.73 g, 0.01 mol) or 16b (5.20 g, 0.01 mol) in 1,4-dioxane
(40 mL) containing triethylamine (0.50 mL) phenylisoth-
iocyanate (1.30 g, 0.01 mol), the whole reaction mixture
was heated under reflux for 4 h and then poured onto ice/
water containing few drops of hydrochloric acid, and the
formed solid product was collected by filtration.
Method (B): To a suspension of either compound 15a
(
4.73 g, 0.01 mol) or 15b (5.20 g, 0.01 mol) in sodium
ethoxide [prepared by dissolving sodium metal (0.46 g,
.02 mol) in absolute ethanol (40 mL)] was heated under
0
reflux in a boiling water bath for 4 h. The solid product
formed upon pouring onto ice/water containing few drops
of hydrochloric acid (till pH 6) and the formed solid pro-
duct was collected by filtration.
Compound 18a: Yellow crystals (1,4-dioxane), yield 80 %
-
1
(4.04 g), mp [300 °C. IR (KBr) cm : 3481, 3348, 3054,
1
2987, 2878, 1689, 1638. H NMR (DMSO-d , 400 MHz):
6
123

3
946
Med Chem Res (2015) 24:3932–3948
d = 8.29 (s, 1H, NH), 7.26–7.38 (m, 5H, Bz),6.06 (s, 1H,
pyridine H-5), 4.30 (s, 2H, D O exchangeable, NH ), 2.69 (s,
(0.66 g, 0.01 mol), either benzaldehyde (1.08 g, 0.01 mol),
4-chlorobenzaldehyde (1.40 g, 0.01 mol) or 4-methoxy-
benzaldehyde (1.36 g, 0.01 mol) was added. The whole
reaction mixture, in each case, was heated under reflux for
6 h and then poured onto ice/water containing few drops of
hydrochloric acid, and the formed solid product was col-
lected by filtration.
2
2
3
2
1
H, CH ), 2.22–2.33 (m, 4H, 2H-5, 2H-8), 1.80–1.87 (m, 4H,
3
13
H-6, 2H-7), C NMR (DMSO-d , 75 MHz): d = 183.2,
6
0
0
80.0 (2C=S, C-2, C-2 ), 172.4, 170.3 (2C=N, C-1, C-1 ), 163.8
(CO, pyridine C-6), 28.7, 25.3, 22.4, 20.4 (C-5, C-6, C-7, C-8),
154.0, 153.6, 150.3, 148.9, 143.9, 140.0, 135.63, 128.4, 123.7,
1
22.6, 121.4, 119.4 (Bz, pyridine, two pyrimidine, thiophene
Compound 19a: Orange crystals (ethanol), yield 93 %
-1
?
C), 19.6 (CH ). EIMS m/z 504 [M] (20); Analysis Calcd for
(5.23 g), mp 183–185 °C. IR (KBr) cm : 3493-3332,
3056, 2989, 2228, 2226, 2222, 1686, 1630, 1218. H NMR
3
1
C H N OS (504.66): C, 57.12; H, 3.99; N, 16.65; S, 19.06.
20
24
6
3
Found: C, 57.04; H, 3.68; N, 16.82; S, 18.79.
(DMSO-d , 400 MHz): d = 8.28 (s, 1H, D O exchange-
6
2
Compound 18b: Yellow crystals (1,4-dioxane), yield
-
able, NH), 7.25–7.40 (m, 10H, 2Bz), 6.13(s, 1H, pyran
1
6
3
4
8
5 % (3.29 g), mp 190–192 °C. IR (KBr) cm : 3489,
H-4),4.26 (s, 2H, D O, NH ), 2.21–2.34 (m, 4H, 2H-5, 2H-
2
2
1
321, 3056, 2988, 1680, 1633, 1200. H NMR (DMSO-d ,
13
8), 1.81–1.88 (m, 4H, 2H-6, 2H-7), C NMR (DMSO-d6,
75 MHz): d = 180.2 (C=S, C-2), 170.8 (C=N, C-1), 164.4
(CO-Bz), 154.0, 149.2, 148.7, 146.8, 144.3, 142.5, 140.2,
139.0, 136.3, 134.2, 129.3, 125.4, 124.8, 124.0, 120.6,
120.3 (2Bz, pyran, pyrimidine, thiophene C), 117.0, 116.8
(2CN), 42.3 (pyran C-4), 29.3, 25.5, 22.8, 19.8 (C-5, C-6,
6
00 MHz): d = 10.20 (s, 1H, D O exchangeable, OH),
2
.22 (s, 1H, D O exchangeable, NH), 7.23–7.38 (m, 5H,
2
Bz), 6.21 (s, 1H, pyridine H-5), 2.22–2.37 (m, 4H, 2H-5,
2
H-8), 2.77 (s, 3H, CH ), 1.80–1.87 (m, 4H, 2H-6, 2H-
3
1
3
7
). C NMR (DMSO-d , 75 MHz): d = 180.2, 182.1
6
0
0
?
(
2C=S, C-2, C-2 ), 170.3, 171.3 (2C=N, C-1, C-1 ), 163.8
C-7, C-8), EIMS m/z 562 [M] (21); Analysis Calcd for
(
CO, pyridine C-6), 19.8, 22.2, 25.9, 28.6 (C-5, C-6, C-7,
C H N O S (562.67): C, 64.04; H, 3.94; N, 14.94; S,
30 22 6 2 2
C-8), 154.3, 150.6, 147.6, 142.9, 140.3, 138.6, 136.9,
33.6, 126.2, 125.9, 124.9, 123.8, 122.1, 121.8, 120.1 (Bz,
11.40. Found: C, 64.29; H, 3.88; N, 14.73; S, 11.62.
1
Compound 19b: Orange crystals (ethanol), yield 63 %
1
-
pyridine, two pyrimidine, thiophene C), 19.4 (CH ), EIMS
3
(3.76 g), mp 166–168 °C. IR (KBr) cm : 3483, 3318,
3054, 2986, 227, 2223, 1690, 1688, 1210. H NMR
?
m/z 505 [M] (28); Analysis Calcd for C H N O S
1
2
4
19
5
2
3
(
505.64): C, 57.01; H, 3.79; N, 13.85; S, 19.02. Found: C,
(DMSO-d , 400 MHz): d = 8.18 (s, 1H, D O exchange-
6
2
5
6.88; H, 3.84; N, 14.06; S, 19.29
able, NH), 7.26–7.40 (m, 9H, C H , C H ),6.37 (s, 1H,
6 5 6 4
pyran H-4), 4.40 (s, 2H, D O exchangeable, NH ),
2
2
2
-Amino-6-((3-benzoyl-2-thioxo-2,3,5,6,7,8-
2.22–2.38 (m, 4H, 2H-5, 2H-8), 1.84–1.89 (m, 4H, 2H-6,
1
3
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)amino)-4-phenyl-4H-pyran-3,5-dicarbonitrile
2H-7), C NMR (DMSO-d , 75 MHz): d = 180.4 (C=S,
6
C-2), 170.2 (C=N, C-1), 164.8 (CO–Bz), 154.2, 153.2,
150.1, 148.3, 146.2, 142.6, 136.3, 134.9, 128.4, 125.8,
124.3, 123.3, 122.9, 121.9, 120.3, 119.8, (2Bz, pyran,
thiophene, pyrimidine C), 117.0, 116.6 (2CN), 42.6 (pyran
C-4), 28.9, 25.6, 22.7, 19.4 (C-5, C-6, C-7, C-8), EIMS m/z
(
19a), 2-amino-6-((3-benzoyl-2-thioxo-2,3,5,6,7,8-
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)amino)-4-(4-chlorophenyl)-4H-pyran-3,5-
dicarbonitrile (19b), 2-amino-6-((3-benzoyl-2-
thioxo-2,3,5,6,7,8-hexahydrobenzo-[4,5]thieno[2,3-
d]pyrimidin-4-yl)amino)-4-(4-methoxyphenyl)-4H-
pyran-3,5-dicarbonitrile (19c), 5-acetyl-2-amino-6-
?
597 [M] (21); Analysis Calcd for C H ClN O
3
0
21
6
2
S2(597.11): C, 60.35; H,.55; N, 14.07; S, 10.74. Found: C,
60.59; H, 3.62; N, 13.4; S, 10.88.
(
(3-benzoyl-2-thioxo-2,3,5,6,7,8-
Compound 19c: Orange crystals (1,4-dioxane), yield
-
1
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)amino)-4-phenyl-4H-pyran-3-carbonitrile (19d),
79 % (4.68 g), mp 184–186 °C. IR (KBr) cm : 3442,
3337, 3055, 2984, 2867, 2227, 2224, 1690, 1633, 1222. H
1
5
-acetyl-2-amino-6-((3-benzoyl-2-thioxo-2,3,5,6,7,8-
NMR (DMSO-d , 400 MHz): d = 8.28 (s, 1H, D O
6
2
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)amino)-4-(4-chlorophenyl)-4H-pyran-3-
carbonitrile (19e) and 5-acetyl-2-amino-6-((3-
benzoyl-2-thioxo-2,3,5,6,7,8-
exchangeable, NH), 7.32–7.38 (m, 9H, C H , C H ), 6.20
6 5 6 4
(s, 1H, pyran H-4), 4.38(s, 2H, D O exchangeable, NH ),
2
2
2.90 (s, 3H, CH ), 2.23–2.29 (m, 4H, 2H-5, 2H-8),
3
1
1.81–1.88 (m, 4H, 2H-6, 2H-7). C NMR (DMSO-d ,
3
6
hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)amino)-4-(4-methoxyphenyl)-4H-pyran-3-
carbonitrile (19f)
75 MHz): d = 180.3 (C=S, C-2), 170.9, (C=N, C-1), 163.4
(CO–Bz), 154.2, 153.2, 150.3, 148.6, 144.5, 143.8, 140.5,
138.8, 135.5, 133.9, 129.3, 128.6, 124.7, 123.8, 122.4,
120.1 (2Bz, pyran, pyrimidine, thiophene C),116.8, 117.2
General procedure: To a solution of either compound 7a
(2CN), 42.4 (pyran C-4), 19.8 (CH ), 28.4, 25.8, 23.7, 19.3
3
?
(C-5, C-6, C-7, C-8), EIMS m/z 592 [M] (22); Analysis
(
(
4.08 g, 0.01 mol) or 7b (4.25 g, 0.01 mol) in ethanol
40 mL) containing triethylamine (0.50 mL) malononitrile
Calcd for C H N O S (592.69): C, 62.82; H, 4.08; N,
31 24 6 3 2
1
23

Med Chem Res (2015) 24:3932–3948
3947
1
1
4.18; S, 10.82. Found: C, 62.68; H, 4.19; N, 14.35; S,
0.44.
Conclusions
Compound 19d: Pale orange crystals (1,4-dioxane),
-
In summary, we have shown herein that our strategy is
compatible with the synthesis of a wide range of tetrahy-
drobenzo[b]thiophene derivatives and particularly when
being incorporated into heterocyclic and fused derivatives.
The cytotoxicity of the newly synthesized products was
evaluated against human gastric cancer (NUGC and HR),
human colon cancer (DLD1), human liver cancer (HA22T
and HEPG2), human breast cancer (MCF), nasopharyngeal
carcinoma (HONE1) and normal fibroblast cells (WI38).
The results showed that compounds 4, 7a, 8a, 8b, 10c, 10d,
12c, 12d, 12e, 12f, 14e, 15b, 18b, 19b and 19e exhibited
optimal cytotoxic effect against cancer cell lines with IC50
in the nM range. In addition, compounds 12c and 14e
showed no toxicity when tested in vivo lethality to shrimp
larvae (Artemiasalina).
1
yield 74 % (4.29 g), mp 222-225 °C. IR (KBr) cm
:
1
438, 3303, 3058, 2980, 2893, 2220, 1688, 1634, 1210. H
3
NMR (DMSO-d , 400 MHz): d = 8.26 (s, 1H, D O
6
2
exchangeable, NH), 7.27–7.43 (m, 10H, 2Bz), 6.38 (s, 1H,
pyran H-4), 4.40 (s, 2H, D O exchangeable, NH ), 2.72 (s,
2
2
3
4
H, CH ), 2.24-2.39 (m, 4H, 2H-7, 2H-8), 1.83–1.88 (m,
3
1
3
H, 2H-6, 2H-7). C NMR (DMSO-d6, 75 MHz):
d = 180.5 (C=S, C-2)170.3 (C=N, C-1), 164.2, 166.3 (CO–
Bz, COCH ), 154.3, 151.9, 146.8, 144.2, 143.2, 136.9,
3
1
1
33.8, 129.7, 126.8, 125.2, 124.8, 123.6, 122.5, 121.3,
20.5, 119.3 (2Bz, pyran, pyrimidine, thiophene C), 116.8
(
CN), 42.5 (pyran C-4), 19.4 (CH ), 28.9, 26.6, 23.8,
3
?
9.4(C-5, C-6, C-7, C-8), EIMS m/z 579 [M] (36);
1
Analysis Calcd for C H N O S (579.69): C, 64.23; H,
31 25 5 3 2
4
1
.35, N, 12.08; S, 11.06. Found: C, 64.07; H, 4.44; N,
1.95; S, 11.30.
Acknowledgments The authors would like to acknowledge finan-
cial support for this work from the Deanship of Scientific Research
Compound 19e: Yellow crystals (1,4-dioxane), yield
-
(
DSR), University of Tabuk, Saudi Arabia, under grant no. S-0083-
436.
1
5
5 % (3.38 g), mp 190–193 °C. IR (KBr) cm : 3488,
1
1
319, 3058, 2980, 2880, 2220, 1720-1688, 1631, 1212. H
3
NMR (DMSO-d , 400 MHz): d = 8.30 (s, 1H, D O
6
2
References
exchangeable, NH), 7.27–7.41 (m, 9H, C H , C H ), 6.12
6
4
6 5
(
s, 1H, pyran H-4), 4.30 (s, 2H, D O exchangeable NH ),
2 2
Abbas SE, Gawad NMA, George RF, Akar YA (2013) Synthesis,
antitumor and antibacterial activities of some novel tetrahy-
drobenzo[4,5]thieno[2,3-d]pyrimidine derivatives. Eur J Med
Chem 65:195–204
Aly HM, Saleh NM, Elhady HA (2011) Design and synthesis of some
new thiophene, thienopyrimidine and thienothiadiazine deriva-
tives of antipyrine as potential antimicrobial agents. Eur J Med
Chem 46:4566–4572
3
1
7
1
1
1
.13 (s, 3H, CH ), 2.23-2.36 (m, 4H, 2H-5, 2H-8),
3
1
3
.82–1.89 (m, 4H, 2H-6, 2H-7), C NMR (DMSO-d6,
5 MHz): d = 180.4 (C=S, C-2), 170.9 (C=N, C-1),164.3,
65.4 (CO–Bz, COCH ), 120.5, 121.8, 122.7, 123.9, 125.3,
3
26.0, 127.3, 129.0, 132.5, 134.0, 137.4, 140.1, 143.9,
46.4, 152.3, 153.4 (2Bz, pyran, pyrimidine, thiophene
C),116.6 (CN), 42.4 (pyran C-4),19.9, 22.4, 25.6, 28.7 (4
?
CH ), 19.7 (CH ). EIMS m/z 614 [M] (18); Analysis
Amr AEGE, Sherif MH, Assy MG, Al-Omar MA, Ragab I (2010)
Antiarrhythmic, serotonin antagonist and antianxiety activities of
novel substituted thiophene derivatives synthesized from
2-amino-4,5,6,7-tetrahydro-N-phenylbenzo[b]thiophene-3-car-
boxamide. Eur J Med Chem 45:5935–5942
Ashalatha BV, Narayana B, Raj KK, Kumari NS (2007) Synthesis of
some new bioactive 3-amino-2-mercapto-5,6,7,8-tetrahy-
dro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one derivatives. Eur J
Med Chem 42:719–728
2
3
Calcd for C H ClN O S (614.13): C, 60.63; H, 3.94, N,
3
1
24
5 3 2
1
1
1.40; S, 10.44. Found: C, 60.82; H, 3.69; N, 11.28; S,
0.68.
Compound 19f: Orange crystals (1,4-dioxane), yield
-
1
7
3 % (4.45 g), mp 180–183 °C. IR (KBr) cm : 3480,
1
320, 3056, 2987, 2882, 2218, 1686, 1630, 1210. H NMR
3
Ashour HM, Shaaban OG, Rizk OH, El-Ashmawy IM (2013)
0
0
(
DMSO-d , 400 MHz): d = 8.30 (s, 1H, D O exchange-
Synthesis and biological evaluation of thieno [2 ,3 :4,5]pyrim-
ido[1,2-b][1,2,4]triazines and thieno[2,3-d][1,2,4]triazolo[1,5-
a]pyrimidines as anti-inflammatory and analgesic agents. Eur J
Med Chem 62:341–351
6
2
able, NH), 7.29–7.38 (m, 9H, C H , C H ), 6.12 (s, 1H,
6
5
6 4
pyran H-4), 4.37 (s, 2H, D O exchangeable, NH), 2.78,
2
2
1
7
1
1
1
.89 (2s, 6H, 2CH ), 2.21–2.34 (m, 4H, 2H-5, 2H-8),
3
Bach P, Bostr o¨ m J, Brickmann K, Giezen JJJ, Groneberg RD, Harvey
DM, Sullivan MO, Zetterberg F (2013) Synthesis, structure–
property relationships and pharmacokinetic evaluation of ethyl
6-aminonicotinate sulfonylureas as antagonists of the P2Y12
receptor. Eur J Med Chem 65:360–375
Brayn B, Timothy M, Tore S (1993) General and applied toxicology,
vol I, 2nd edn. Macmillan, London, p 52
1
.81–1.86 (m, 4H, 2H-6, 2H-7). C NMR (DMSO-d ,
3
6
5 MHz): d = 180.2 (C=S, C-2), 170.6 (C=N, C-1), 164.8,
65.2 (CO–Bz, COCH ), 154.6, 153.2, 152.4, 147.7, 146.2,
3
44.2, 135.2, 133.6, 132.4, 130.4, 126.9, 125.0, 124.9,
24.4, 123.9, 123.3, 120.2, 119.8 (2Bz, pyran, pyrimidine,
Calleja MC, Persoone G (1992) The potential of ecotoxicological
tests for the prediction of acute toxicity in man as evaluated on
the 1st 10 chemicals on the meic program. Atla 20:396–405
Carballo JL, Inda ZLH, P e´ rez P, Gr a´ valos MDG (2002) A comparison
between two brine shrimp assays to detect in vitro cytotoxicity in
marine natural products. BMC Biotechnol 2:17
thiophene C),116.3 (CN), 42.6 (pyran C-4), 19.8, 19.9
OCH , COCH ), 28.9, 25.8, 22.3, 19.8 (C-5, C-6, C-7,
(
3
3
?
C-8), EIMS m/z 609 [M] (28); Analysis Calcd for C32
H N O S (609.71): C, 63.04; H, 4.46, N, 11.49; S, 10.52.
27 5 4 2
Found: C, 63.32; H, 4.69; N, 11.82; S, 11.63.
123

3
948
Med Chem Res (2015) 24:3932–3948
Choudhary MI, Thomsen WJ (2001) Bioassay techniques for drug
development. Harwood Academic Publishers, Amsterdam,
pp 9–10
Dewal MB, Wani AS, Vidaillac C, Oupick y´ D, Rybak MJ, Firestine
SM (2012) Thieno[2,3-d]pyrimidinedione derivatives as antibac-
terial agents. Eur J Med Chem 51:145–153
El-Sherbeny MA, El-Ashmawy MB, El-Subbagh HI, El-Emam AA,
Badria FA (1995) Synthesis, antimicrobial and antiviral evalu-
ation of certain thienopyrimidine derivatives. Eur J Med Chem
Leung CY, Langille AM, Mancuso J, Tsantrizos YS (2013) Discovery
of thienopyrimidine-based inhibitors of the human farnesyl
pyrophosphate synthase—parallel synthesis of analogs via a
trimethylsilylylidene intermediate. Biorg Med Chem
21:2229–2240
Mohareb RM, Al-Omran F (2012) Reaction of pregnenolone with
cyanoacetylhydrazine: novel synthesis of hydrazide–hydrazone,
pyrazole, pyridine, thiazole, thiophene derivatives and their
cytotoxicity evaluations. Steroids 77:1551–1559
3
0:445–449
Esposito E, Rotilio D, Matteo VD, Giulio CD, Cacchio M, Algeri S
2002) A review of specific dietary antioxidants and the effects
Ogunro PS, Eegunranti BA, Atiba AS, Oke EO, Akande JO (2013)
Status of antioxidant defense and lipid peroxidation in
schizophrenics with positive, negative and cognitive symptoms.
Free Radicals Antioxid 3:20–24
Patel MV, Rohde JJ, Gracias V, Kolasa T (2003) An efficient one-pot
synthesis of substituted 2-arylbenzo[b]thiophene derivatives.
Tetrahedron Lett 44:6665–6667
Press JB, McNally JJ, Keiser JA, Offord SJ, Katz LB, Giardino E,
Falotico R, Tobia AJ (1989) Furo[3,4-d]pyrimidine-2,4-dione
derivatives with antihypertensive activity. Analogues of
thienopyrimidine-2,4-diones. Eur J Med Chem 24:627–630
Rizk OH, Shaaban OG, El-Ashmawy IM (2012) Design, synthesis
and biological evaluation of some novel thienopyrimidines and
fused thienopyrimidines as anti-inflammatory agents. Eur J Med
Chem 55:85–93
(
on biochemical mechanisms related to neurodegenerative pro-
cesses. Neurobiol Aging 23:719–735
Gewald K, Schinke E, Bottcher H (1966) Heterocyclization using
acidic CH nitriles, VIII. 2-aminothiophene from activated
cyanomethylenes and sulfur. Chem Berichte 99:94–100
Giordano S, Usmar VD, Zhang J (2014) Autophagy as an essential
cellular antioxidant pathway in neurodegenerative disease.
Redox Biol 2:82–90
H a¨ cker HG, Elsinghorst PW, Michels S, Daniels J, Schnakenburg G,
G u¨ tschow M (2009) (Benzoylimino)thiazolidin-4-ones: forma-
tion by an Alternative Ring Closure and Analysis of Rotational
Barriers. Synthesis 7:1195–1203
Hafez HN, Husseina HA, El-Gazzar AB (2010) Synthesis of
substituted thieno[2,3-d]pyrimidine-2,4-dithiones and their
S-glycoside analogues as potential antiviral and antibacterial
agents. Eur J Med Chem 45:4026–4034
Kamat PK, Kalani A, Givvimani S, Sathnur PB, Tyagi SC, Tyagi N
(2013) Hydrogen sulfide attenuates neurodegeneration and
Scrowston RM (1981) Recent advances in the chemistry of benzo
[b] thiophenes. Adv Hetrocycl Chem 29:171–249
Seerat F, Anindra S, Reshu S, Rajkamal T, Sanjeev KS, Pandey SK,
Renu T, Rama PT (2012) One pot efficient diversity oriented
synthesis of polyfunctionalstyrylthiazolopyrimidines and their
bio-evaluation as antimalarial and anti-HIV agents. Eur J Med
Chem 55:195–204
Vianello A, Arcaro G, Battistella L, Pipitone E, Vio S, Concas A,
Paladini L, Gallan F, Marchi MR, Tona F, Iliceto S (2014)
Noninvasive ventilation in the event of acute respiratory failure
in patients with idiopathic pulmonary fibrosis. J Crit Care
29:562–567
neurovascular dysfunction induced by intracerebral-administered
homocysteine in mice. Neuroscience 252:302–319
Kotaiah Y, Harikrishna N, Nagaraju K, Rao CV (2012) Synthesis and
antioxidant activity of 1,3,4-oxadiazole tagged thieno[2,3-
d]pyrimidine derivatives. Eur J Med Chem 58:340–345
Leistner S, G u¨ tschow M, Wagner G (1989) MehrcyclischeAzine-
mitHeteroatomen in 1- und 3-Stellung, 22. Mitt. EineeinfacheSyn-
these von 2-Alkylthio-4-amino-thieno[2,3-d]pyrimidine. Arch
Pharm 322:227–230
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