Synthesis, antitumor and antibacterial activities of some novel tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.04.055

Two series of new tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines namely 2,3-disubstituted derivatives 3a-z and 2,4-disubstituted ones 6a-c were prepared and tested for their antitumor and antibacterial activities. The structures of the prepared compounds we

Full text of DOI:10.1016/j.ejmech.2013.04.055

Accepted Manuscript
Synthesis, antitumor and antibacterial activities of some novel
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives
Safinaz E. Abbas, Nagwa M. Abdel Gawad, Riham F. George, Yahya A. Akar
PII:
S0223-5234(13)00287-0
10.1016/j.ejmech.2013.04.055
EJMECH 6165
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 November 2012
Revised Date: 21 April 2013
Accepted Date: 26 April 2013
Please cite this article as: S.E. Abbas, N.M. Abdel Gawad, R.F. George, Y.A. Akar, Synthesis, antitumor
and antibacterial activities of some novel tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives,
European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.04.055.
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ACCEPTED MANUSCRIPT
Highlights
-
-
-
Two series of fused thieno[2,3-d]pyrimidines were synthesized.
They were tested for their antitumor and antibacterial activities.
3-Acridinyl derivatives had potent antitumor and antibacterial activities.

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Graphical abstract
R2
N
HN
O
R2
N
R1
R1
N
N
S
S
3a-z
6a-c
R1= 3,4,5-trimethoxyphenyl, styryl, R2= CSNH2, CONH2, CONHOH, heteroalicyclic, aryl, heteroaryl
rings
The target compounds 3a-z and 6a-c were prepared and tested for their antitumor and
antibacterial activities. Compounds 3z and 3r exerted the highest activity against MCF-7
and HEPG-2 cell lines, respectively.

ACCEPTED MANUSCRIPT
Synthesis, antitumor and antibacterial activities of some novel
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives
Safinaz E. Abbas, Nagwa M. Abdel Gawad, Riham F. George*, Yahya A. Akar.
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, El-Kasr El Aini Street,
11562, Cairo, Egypt
*Correspondence: Riham F. George, Pharmaceutical Chemistry Departement, Faculty of Pharmacy, Cairo
University, El-Kasr El-Eini Street, Cairo 11562, Egypt. Tel.: +002 02 26070436; Fax +002 02 23635140
E-mail: rihamfgeorge@yahoo.com
1

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Abstract
Two series of new tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines namely 2,3-
disubstituted derivatives 3a-z and 2,4-disubstituted ones 6a-c were prepared and tested
for their antitumor and antibacterial activities. The structures of the prepared compounds
were confirmed by spectral and elemental analyses. Compound 3z exhibited the highest
antitumor activity against breast MCF-7 with IC50= 0.19 ꢀM compared to Doxorubicin
(IC50= 5.46 ꢀM), while 3r was the most active one against liver HEPG-2 cancer cell line
with IC50= 1.29 ꢀM as regard to Doxorubicin (IC50= 7.36 ꢀM). Concerning the
antibacterial activity, compounds 3m and 3z exerted remarkable activity against the
tested bacterial species compared to Ampicillin, whereas compound 6c showed good
activity against only Gram positive species.
Keywords: thieno[2,3-d]pyrimidines, antitumor activity, antibacterial activity, MCF-7,
HEPG-2.
1. Introduction
Cancer is a major health problem worldwide. The relative mortality rate caused
by cancer is still very high in the developed countries, accounting for more than 20% of
all deaths. Among the various types of malignant tumors, breast cancer is the second
leading cause of death in women [1,2]. In the US and other western countries, alcohol-
related cirrhosis, and possibly nonalcoholic fatty liver disease associated with obesity,
account for the majority of liver cancer cases. Chronic infections with hepatitis B virus
(HBV) and hepatitis C virus (HCV) are associated with less than half of liver cancer
cases in the US, although they are the major risk factors for the disease worldwide [1].
Although chemotherapy is the mainstay of cancer therapy, the use of available
chemotherapeutics is often limited mainly due to undesirable side effects and a limited
choice of available anticancer drugs [3]. This clearly underlies the urgent need for
developing novel chemotherapeutic agents with more potent antitumor activities.
Many anticancer agents which act as tyrosine kinase inhibitors comprised the
pyrimidine group as a core moiety. This could be exemplified by different quinazoline
2

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derivatives such as gefitinib (IressaTM) [4] and tandutinib (MLN518) (phase II clinical
trials) [5] (Fig.1). In addition, the thieno[2,3-d]pyrimidine ring, which is considered as a
bioisostere of quinazoline, was used as a core for mechanism-based design and synthesis
of diversity of compounds of potential antitumor activity [6-19]. Furthermore, the
thienopyrimidine nucleus was adopted as a scaffold for many antimicrobial [20-26],
antiviral [20, 26-28], antiinflammatory [29] antidiabetic [30], antioxidant [31] and
anxiolytic agents [32]. In literature, it was documented that fusion of the thieno[2,3-
d]pyrimidine core with five, six or seven-membered cycloalkyl lipophilic moieties
conserved the antitumor activity [9, 14-17] as revealed by the 4-substituted-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine I which was described to exert high
anticancer activity [15]. Moreover, Wang et al. [7] reported that the presence of 3,4,5-
trimethoxyphenyl moiety at position 2 in compound 4a was critical for its cytotoxic
activity (Fig.1). Therefore, our research herein focused on the synthesis of two novel
series including 2,3- and 2,4-disubstituted tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines
hoping to obtain new derivatives with dual antitumor and antimicrobial activities. The
synthetic pathway was planned in such a way to accommodate a 3,4,5-trimethoxyphenyl
or styryl moiety at position 2 of the pyrimidine ring, in addition the 3- and 4- substituents
were selected to incorporate well documented pharmacophoric moieties of interest such
as amide, thioamide, N-hydroxyamide, aryl and heteroaryl moieties that proved to
contribute to antitumor and antibacterial activities such as benzoic acid [33],
benzenesulfonamide [34-36], indole [37,38], benzothiazole [39-41] and acridine [42-45].
The synthesized compounds were screened for their antitumor and antibacterial activities
to explore the effect of these structural modifications on the activity aiming to obtain new
candidates of remarkable activities and minimal side effects.
2- Results and discussion
2.1. Chemistry
The target compounds were prepared as outlined in Schemes 1 and 2. The starting
materials ethyl-2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate 1a and the 3-
carboxamide derivative 1b were prepared following the method of Gewald [46-48] via
the reaction of cyclohexanone and sulfur with either ethyl cyanoacetate or
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cyanoacetamide, respectively in the presence of diethylamine. Acylation procedure
described by Amr et al. [32] was applied to react 1a with either 3,4,5-trimethoxybenzoyl
chloride or cinnamoyl chloride in dry benzene to afford 2a,b, respectively (Scheme 1). 1H
NMR spectrum of 2a revealed the presence of singlet signal at 7.25 ppm corresponding
to the two aromatic protons of 3,4,5-trimethoxyphenyl in addition to the triplet quartet
pattern at 1.38 and 4.36 ppm of the ethyl group and two singlets at 3.91 and 3.96 ppm
attributed to the protons of the three methoxy groups. Similarly, 1H NMR spectrum of 2b
showed signals corresponding to the aromatic protons in addition to the olefinic ones
which were attributed to the styryl moiety at 6.75-7.81 ppm. Cyclization of 2a,b into 2,3-
disubstituted-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-ones 3a-z was
adopted using the reported reaction conditions [49]. This was achieved by refluxing 2a,b
with the appropriate amines in the presence of phosphorus oxychloride and phosphorus
pentoxide, the latter was added to accelerate the reaction for producing 3a-z (Scheme 1).
1H NMR spectra of 3a-z revealed the disappearance of the triplet quartet pattern of the
ethyl moiety and the presence of extra signals corresponding to the introduced groups (cf
experimental section). Reaction of the 3-carboxamide derivative 1b with 3,4,5-
trimethoxybenzaldehyde or cinnamaldehyde afforded 4a [7,8] or 4b [50,51], respectively
according to the reported procedures. Likewise, 1b was reacted with 2-nitro
cinnamaldehyde or α-methyl cinnamaldehyde in methanol and in the presence of catalytic
amount of hydrochloric acid to give compounds 4c or 4d, respectively (Scheme 2). IR
spectra of compounds 4c,d showed bands at 3363-3383 and 1639 cm-1 corresponding to
1
the NH and C=O groups, respectively. H NMR spectrum of 4d revealed two singlets at
3.49 and 8.26 ppm related to CH3 and olefinic protons, respectively in addition to the
other signals corresponding to the 2-styryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidine nucleus. The 4-chloro derivatives 5a,b were obtained via the reaction of
4a,b with phosphorus oxychloride [7,8,50]. Reaction of 5a,b with hydroxyurea or
acriflavine was carried out in the domestic microwave under solvent free condition to
yield the corresponding 4-substituted derivatives 6a-c (Scheme 2). Characterization of
the newly prepared derivatives 6a-c was verified by IR spectra that revealed the presence
of broad bands at 3406-3200 and 3314-3190 cm-1 corresponding to two NH and OH
1
groups in compounds 6a,b and NH and NH2 groups in compound 6c, respectively. H
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NMR spectra of compounds 6a,b showed two exchangeable signals at 6.50 and 7.80,
8.80 ppm correlated to OH and two NH protons, respectively, in addition to the usual
signals of the nucleus. Additionally, the presence of a singlet signal at 4.10 ppm and two
exchangeable signals at 5.40 and 9.80 ppm corresponding to CH3, NH2 and NH moieties,
respectively along with the extra aromatic signals of the acridinyl nucleus were observed.
2.2. Antitumor activity
All the prepared target compounds 3a-z , 4b-d and 6a-c were screened for their
antitumor activity against breast MCF-7 and liver HEPG-2 cancer cell lines at the
Pharmacology Unit of National Research Center according to Skehan et al method [52].
The obtained results (Table 1), revealed that the 2-styryl derivatives 3n-z exerted more
remarkable activity against both cell lines than the corresponding 3,4,5-trimethoxyphenyl
derivatives 3a-m.
Regarding the 2-(3,4,5-trimethoxy)phenyl derivatives 3a-m, the 3-thioamide
derivative 3a exerted weak activity against MCF-7 cancer cell line that increased in the
bioisostere carboxamide derivative 3b. Introduction of a polar hydroxyl group in
compound 3c led to a decrease in the antitumor activity against MCF-7 cell line. On the
other hand, compound 3c possessed higher activity against HEPG-2 cancer cell line than
the carboxamide 3b, while the thioamide analog 3a was inactive. In addition, it was
apparent that the presence of a sulfamoyl moiety in compound 3e produced a relatively
higher activity against MCF-7 compared to the carboxylic acid derivatives 3d, 3f and 3g,
whereby the activity was reversed as regard to the liver cell line. Furthermore,
substitution at position 3 with a five membered heterocyclic ring such as isoxazolidinone
as in 3h, showed a decrease in the antitumor activity against both cell lines. Ring
expansion of the substituent at position 3 to pyrimidinedione as in 3i led to a sharp
decrease in the antitumor activity. Moreover, the antitumor activity augmented,
especially against HEPG-2 cell line, by the incorporation of a benzothiazol-2-yl moiety at
position 3 as in 3j. Also, it was noticed that the presence of an electron withdrawing
group at position 5 of the benzothiazol-2-yl group as in 3l was more favorable for
enhancing the antitumor activity than the electron donating one as in 3k. Interestingly,
the
hybridization
of
the
2-(3,4,5-trimethoxy)phenyl-5,6,7,8-
5

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tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine with acridinyl nucleus resulted in
compound 3m that exhibited a prominent increase in the antitumor activity against both
cell lines (IC50= 0.86, 5.50 ꢀM compared to Doxorubicin IC50= 5.46, 7.36 ꢀM,
respectively) (Table 1).
Concerning the antitumor activity of the 2-styryl derivatives 3n-z, it was found
that the 3-thioamide 3n and its carboxamide analog 3o were nearly equipotent showing
moderate antitumor activity against both cell lines. On the other hand, the activity
significantly decreased in the N-hydroxyl derivative 3p. Furthermore, the sulfonamido
derivative 3r exhibited higher activity than the carboxylic acid candidates 3q, 3s and 3t
against MCF-7 and HEPG-2 as revealed from its IC50 values (0.43 and 1.29 ꢀM,
respectively) which were in the same time more potent than those expressed by
Doxorubicin (IC50=5.46 and 7.36 ꢀM, respectively). The incorporation of
isoxazolidinone ring as in 3u revealed mild activity against MCF-7 cell line and a
remarkably higher activity against HEPG-2 (IC50= 6.86 ꢀM) even higher than
Doxorubicin. Derivative 3v, carrying the larger pyrimidinedione ring, demonstrated a
slight increase in the activity against the MCF-7 cell line. Additionally, it was observed
that the 3-benzothiazol-2-yl derivative 3w and its 5-nitro analog 3y were more potent
than Doxorubicin against MCF-7 as expressed by their IC50 values (4.30 and 1.23 ꢀM,
respectively). Interestingly, it was noticed that the 3-acridin-3-yl derivative 3z revealed
the highest activity against breast cancer cell line (IC50=0.19 ꢀM) which was considered
to be the most active among all the tested compounds. Prominent activity of 3z against
the liver cell line (IC50=5.82 ꢀM) was also remarked. Considering the 3-unsubstituted-2-
styryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-ones 4b-d, they revealed
moderate to weak activity against both cell lines. It was noteworthy that the incorporation
of hydroxy urea moiety at position 4 of the 2-(3,4,5-trimethoxyphenyl) and 2-styryl
derivatives as in 6a and 6b, respectively resulted in improved activity against both cell
lines, especially HEPG-2 (IC50= 2.32 and 3.27 ꢀM, respectively). Finally, incorporation
of the acridin-3-yl amino moiety at position 4 of the pyrimidine ring as in compound 6c
contributed to a distinctive increase in the antitumor activity against MCF-7 and HEPG-2
(IC50= 0.58, 1.36 ꢀM, respectively).
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2.3. Antibacterial activity
All the target compounds 3a-z, 4b-d and 6a-c were evaluated in vitro for their
antibacterial activities at the Microanalytical Center, Faculty of Science, Cairo University
according to the reported procedure [53]. They were tested against Bacillus subtilis
(ATCC 6051) and Staphylococcus aureus (ATCC 12600) as Gram positive strains and
Escherichia coli (ATCC 11775) and Pseudomonas aeruginosa (ATCC 10145) as Gram
negative strains. The obtained results were represented in Table 2, where it could be
noticed that the tested compounds showed mild activity in comparison to the reference
drug Ampicillin. Only compounds 3m and 3z containing the 3-acridin-3-yl moiety
revealed broad spectrum activity comparable to that of Ampicillin. Furthermore, their
MIC values against Staphylococcus aureus and Pseudomonas aeruginosa obtained from
the broth microdilution method [54] were illustrated in Table 3. On the other hand, the 2-
styryl derivatives 3q, 3u and 3v were equipotent to Ampicillin against Staphylococcus
aureus. Additionally, 3-carboxamide-2-(3,4,5-trimethoxyphenyl) derivative 3b was
selective against Pseudomonas aeruginosa. Moreover, the presence of acridinylamino
moiety at position-4 of the 2-styryl derivative 6c shifted the activity against only Gram
positive bacteria.
3. Conclusion
A series of novel 2,3-disubstituted-4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidines 3a-z were prepared via reaction of ethyl 2-amino-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-carboxylate 1a with the appropriate acid chloride to
obtain 2a,b. The latter were further reacted with different amines in the presence of
phosphorus oxychloride and phosphorus pentoxide to give 3a-z. Additionally, reaction of
the 3-carboxamide derivative 1b with the appropriate aldehyde produced compounds 4a-
d. Chlorination of 4a,b yielded 5a,b which reacted with hydroxyurea or acriflavine to
afford 6a-c.
The target compounds 3a-z, 4b-d and 6a-c were screened for their antitumor
activity against MCF-7 breast and HEPG-2 liver cancer cell lines and for their
antibacterial activity against G (+) and G (-) bacteria. It was concluded that the presence
of the styryl moiety at position 2 was more favorable for the antitumor activity than the
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3,4,5-trimethoxyphenyl group. In addition, substitution at position 3 with p-
sulfamoylphenyl 3r, benzothiazol-2-yl 3w, 5-nitrobenzothiazol-2-yl 3y or 6-amino-10-
methyl-acridin-3-yl 3m, 3z was essential for increased activity. Compound 3z exhibited
the highest antitumor activity against MCF-7 with IC50= 0.19 ꢀM, in addition to
compounds 3m, 3r, 3w, 3y and 6c that showed higher activity than that expressed by
Doxorubicin (IC50= 5.46 ꢀM). Compound 3r was the most active one against liver
HEPG-2 cancer cell line (IC50= 1.29 ꢀM), in addition to compounds 3j, 3m, 3u, 3z and
6a-c that demonstrated distinct activity compared with Doxorubicin (IC50= 7.36 ꢀM).
Furthermore, the 3-acridin-3-yl derivatives 3m and 3z exerted remarkable activity against
the tested bacterial species indicating dual antitumor and antibacterial activities, while the
4-acridinyl amino derivative 6c showed good activity against only the G (+) species.
Finally, it could be concluded that some of the newly prepared 2,3- and 2,4-disubstituted
derivatives exerted remarkable antitumor and antibacterial activities compared to the 3-
unsubstituted ones.
4. Experimental
4.1. Chemistry
Melting points were recorded on Gallenkamp electric melting point apparatus. IR
spectra cm-1 (KBr) were recorded on Shimadzu FT-IR 8400S infrared spectrophotometer.
13
NMR spectra were recorded on JEOL-ECA500 (1H: 500, C: 125 MHz) using TMS as
an internal reference. Mass spectra (EI) were recorded on 70 eV with Kratos MS
equipment and a Varian MAT311A Spectrometer. Elemental analyses were performed at
the Regional center for mycology and biotechnology, Al-Azhar University, Egypt.
Reactions were monitored on Merck silicagel 60 F254 aluminum sheets. TLC spots were
visualized by inspection of plates under UV light (254 and 365 nm). All commercial
reagents were obtained either from Aldrich, or Oxford and used without further
purification.
Compounds 1a,b [46-48], 4a [7, 8], 4b [50, 51], 5a [7, 8], 5b [50] were prepared
according to the reported procedures
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4.1.1. General procedure for the preparation of (2a,b)
A mixture of equimolar amounts of 1a and the appropriate acid chloride (4 mmol)
in dry benzene (10 ml) was refluxed for 3 h. The solid separated on cooling was filtered,
washed and crystallized from ethanol.
4.1.1.1.
Ethyl-2-(3,4,5-trimethoxybenzamido)-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-carboxylate (2a)
Yellow crystals, mp 170-172oC , 93 % yield; IR (cm-1): ν 3400 (NH), 3050 (CH
aromatic), 2943-2843 (CH aliphatic), 1786 (C=O ester), 1713 (C=O amide), 1686 (NH
1
bending), 1589-1466 (C=C); H NMR (CDCl3): δ 1.38 (t, J= 7.20 Hz, 3H, CH2CH3),
1.80-1.81 (m, 4H, 2CH2), 2.68 (br.s, 2H, CH2), 2.80 (br.s, 2H, CH2), 3.91 (s, 3H, OCH3),
3.96 (s, 6H, 2 OCH3), 4.36 (q, J= 7.20 Hz, 2H, CH2CH3), 7.25 (s, 2H, aromatic protons),
12.32 (s, 1H, NH); MS (m/z, %): 419 (M+, 5.00), 420 (1.74), 195 (28.47), 63 (100);
Anal. Calcd. for C21H25NO6S (419.49): C, 60.13; H, 6.01; N, 3.34; found: C, 60.18; H,
5.98; N, 3.42.
4.1.1.2.
Ethyl-2-cinnamamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carboxylate (2b)
Yellow crystals, mp 157-159oC , 90 % yield; IR (cm-1): ν 3237 (NH), 3050-3020
(CH aromatic), 2974-2839 (CH aliphatic), 1665 (C=O ester), 1651 (C=O amide), 1626
(NH bending), 1599-1497 (C=C); 1H NMR (CDCl3): δ 1.53 (t, J= 7.65 Hz, 3H, CH2CH3),
1.86-1.87 (m, 4H, 2CH2), 2.78-2.79 (m, 2H, CH2), 2.94-2.95 (m, 2H, CH2), 4.54 (q, J=
7.65 Hz, 2H, CH2CH3), 6.75 (d, J=16.05 Hz, 1H, CH=CH-ph), 7.41-7.58 (m, 5H, 5
aromatic protons), 7.81 (d, J= 16.05 Hz, 1H, CH=CH-ph), 12.13 (s, 1H, NH); MS (m/z,
%): 355 (M+, 2.82), 148 (100); Anal. Calcd. for C20H21NO3S (355.45): C, 67.58; H, 5.95;
N, 3.94; found: C, 67.65; H, 5.98; N, 4.11.
4.1.2. General procedure for preparation of (3a-z)
To a solution of 2a,b (5 mmol) in phosphorus oxychloride (10 ml) was added a
solution of the appropriate amine (5 mmol) and phosphorus pentoxide (5 mmol) in
phosphorus oxychloride (5 ml), and the mixture was refluxed for 5h. After cooling, the
9

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reaction mixture was poured onto ice/water and neutralized with 5% sodium hydroxide
solution; the formed precipitate was filtered, washed with water and crystallized from the
ethanol.
4.1.2.1.
4-Oxo-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidine-3 (4H)-carbothioamide (3a)
Brown crystals, mp 155-156oC , 80 % yield; IR (cm-1): ν 3441-3390 (NH2),
3036-3001 (CH aromatic), 2940-2835 (CH aliphatic), 1748 (C=O), 1593 (NH bending),
1
1558-1454 (C=C), 1223 (C=S); H NMR (CDCl3): δ 1.42 (s, 2H, NH2), 1.87-1.91 (m,
4H, 2CH2), 2.79 (br.s., 2H, CH2), 2.94 (br.s., 2H, CH2), 3.92 (s, 3H, OCH3), 3.96 (s, 6H,
2 OCH3), 7.49 (s, 2H, aromatic protons); 13C NMR (CDCl3): δ 22.7, 23.0, 24.5, 26.6 (4C
of cyclohexene), 56.3 ( 2OCH3), 61.0 (OCH3), 105.2 (2C o-OCH3), 113.1, 127.6, 127.8,
141.7, 141.9, 147.6, 153.4 (aromatic carbons + thiophene carbons), 163.2 (C2 of
pyrimidine), 169.1 (C=O), 180.0 (C=S); Anal. Calcd. for C20H21N3O4S2 (431.53): C,
55.67; H, 4.91; N, 9.74; found: C, 55.65; H, 4.98; N, 9.91.
4.1.2.2.
4-Oxo-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidine-3 (4H)-carboxamide (3b)
Yellow crystals, mp 230-231oC, 90 % yield; IR (cm-1): ν 3280 (NH2), 3050 (CH
aromatic), 2940-2839 (CH aliphatic), 1760, 1680 (2C=O), 1632 (NH bending), 1589-
1
1458 (C=C); H NMR (CDCl3): δ 1.66 (s, 2H, NH2), 1.86-1.90 (m, 4H, 2CH2), 2.79
(br.s., 2H, CH2), 2.95 (br.s., 2H, CH2), 3.93 (s, 3H, OCH3), 3.97 (s, 6H, 2 OCH3), 7.50 (s,
2H, aromatic protons); Anal. Calcd. for C20H21N3O5S (415.46): C, 57.82; H, 5.09; N,
10.11; found: C, 57.78; H, 5.14; N, 10.23.
4.1.2.3.
N-Hydroxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5] thieno[2,3-d]pyrimidine-3 (4H)-carboxamide (3c)
Grey crystals, mp 167-168oC , 77 % yield; IR (cm-1): ν 3300, 3280 (NH, OH),
3050 (CH aromatic), 2940-2839 (CH aliphatic), 1750, 1680 (2C=O), 1632 (NH bending),
1
1589-1458 (C=C); H NMR (CDCl3): δ 1.84-1.89 (m, 4H, 2CH2), 2.79 (br.s., 2H, CH2),
2.94 (br.s., 2H, CH2), 3.93 (s, 3H, OCH3), 3.96 (s, 6H, 2 OCH3), 6.30 (s, 1H, OH), 7.50
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(s, 2H, aromatic protons), 12.23 (s, 1H, NH); Anal. Calcd. for C20H21N3O6S (431.46): C,
55.67; H, 4.91; N, 9.74; found: C, 55.73; H, 4.96; N, 9.89.
4.1.2.4.
4-[4-Oxo-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidin-3 (4H)-yl] benzoic acid (3d)
Light brown Crystals, mp 220-222oC , 86 % yield; IR (cm-1): ν 3221 (OH
carboxylic), 3050 (CH aromatic), 2997-2851 (CH aliphatic), 1748 (2C=O), 1597-1462
1
(C=C); H NMR (CDCl3): δ 1.88 (s, 4H, 2CH2), 2.78-2.80 (m, 2H, CH2), 2.95-2.98 (m,
2H, CH2), 3.80 (s, 3H, OCH3), 3.92 (s, 6H, 2 OCH3), 7.20-7.80 (m, 6H, aromatic
protons), 11.30 (s, 1H, OH); MS (m/z, %): 491 (M+-1, 2.62), 373 (49.13), 195 (100);
Anal. Calcd. for C26H24N2O6S (492.54): C, 63.40; H, 4.91; N, 5.69; found: C, 63.42; H,
4.97; N, 5.88.
4.1.2.5.
4-(4-Oxo-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3 (4H)-yl) benzenesulfonamide (3e)
Greyish white Crystals, mp 170-172oC , 80 % yield; IR (cm-1): ν 3476-3391
(NH2), 3036 (CH aromatic), 2997-2835 (CH aliphatic), 1748 (C=O), 1593 (NH bending),
1558-1454 (C=C), 1323, 1180 (S=O); 1H NMR (CDCl3): δ 1.86 (s, 4H, 2CH2), 2.76-2.78
(m, 2H, CH2), 2.98-3.02 (m, 2H, CH2), 3.88 (s, 3H, OCH3), 3.95 (s, 6H, 2 OCH3), 7.20-
8.10 (m, 6H, aromatic protons), 8.50 (s, 2H, NH2); 13C NMR (CDCl3): δ 22.0, 22.9, 25.2,
25.3 (4C of cyclohexene), 56.5 (2OCH3), 61.1 (OCH3), 105.3 (2C o-OCH3), 116.2, 124.9,
132.3, 134.8, 142.0, 153.3, 155.3 (aromatic carbons + thiophene carbons), 158.3 (C=O),
162.4 (C2 of pyrimidine); Anal. Calcd. for C25H25N3O6S2 (527.61): C, 56.91; H, 4.78; N,
7.96; found: C, 56.88; H, 4.82; N, 8.10.
4.1.2.6.
2-(4-Oxo-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidin -3 (4H)-yl)-3-phenylpropanoic acid
(3f)
Yellowish white Crystals, mp 177-178oC , 69 % yield; IR (cm-1): ν 3275 (OH
carboxyic), 3036 (CH aromatic), 2997-2835 (CH aliphatic), 1748 (C=O), 1632 (C=O),
1
1593-1454 (C=C); H NMR (CDCl3): δ 1.87-1.90 (m, 4H, 2CH2), 2.74-2.76 (m, 2H,
11

ACCEPTED MANUSCRIPT
CH2), 2.90-2.93 (m, 2H, CH2), 3.40-3.55 (m, 2H, CH2), 3.70-3.72 (m, 1H, CH), 3.85 (s,
3H, OCH3), 3.95 (s, 6H, 2 OCH3), 7.20-7.50 (m, 7H, aromatic protons), 11.30 (s, 1H,
OH); MS (m/z, %): 520.10 (M+, 8.18), 521.55 (M++1, 1.38), 373 (62.38), 195 (90.89),
135 (100); Anal. Calcd. for C28H28N2O6S (520.60): C, 64.60; H, 5.42; N, 5.38; found: C,
64.57; H, 5.46; N, 5.54.
4.1.2.7.
3-(1H-indol-2-yl)-4-(4-Oxo-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-
tetrahydro benzo[4,5]thieno[2,3-d]pyrimidin -3 (4H)-yl) propanoic acid (3g)
Brown Crystals (from charcoal/ethanol), mp > 300oC , 88 % yield; IR (cm-1): ν
3329 (NH), 3167 (OH carboxylic), 3059 (CH aromatic), 2920-2847 (CH aliphatic), 1670
1
(2C=O), 1601 (NH bending), 1535-1458 (C=C); H NMR (CDCl3): δ 1.87-1.88 (m, 4H,
2CH2), 2.76-2.78 (m, 2H, CH2), 2.92-2.95 (m, 2H, CH2), 3.42-3.53 (m, 2H, CH2), 3.69-
3.71 (m, 1H, CH), 3.89 (s, 3H, OCH3), 3.95 (s, 6H, 2 OCH3), 6.70-7.40 (m, 7H, aromatic
13
protons), 11.10 (s, 1H, OH), 12.07 (s, 1H, NH); C NMR (DMSO): δ 22.8, 23.1, 24.3,
25.3 (4C of cyclohexene), 26.3 (CH2CH), 56.5 (CH2CH), 56.6 ( 2OCH3), 60.8 (OCH3),
105.4 (2C o-OCH3), 104.9, 112.9, 116.2, 120.0, 123.0, 124.9, 126.0, 127.8, 131.7, 132.0,
135.0, 139.0, 141.3, 147.1, 153.7 (aromatic carbons + thiophene carbons + indol
carbons), 158.3 (C=O), 167.1 (C2 of pyrimidine), 168.4 (COOH); MS (m/z, %): 559.20
(M+, 23.54), 202.20 (29.30), 90.50 (100); Anal. Calcd. for C30H29N3O6S (559.63): C,
64.39; H, 5.22; N, 7.51; found: C, 64.41; H, 5.25; N, 7.67.
4.1.2.8.
3-(3-Oxoisoxazolidin-4-yl)-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-
tetrahydrobenzo [4,5]thieno [2,3-d]pyrimidin -4 (3H)-one (3h)
Yellow Crystals, mp 187-188oC , 90% yield; IR (cm-1): ν 3310 (NH), 3040 (CH
aromatic), 2936-2839 (CH aliphatic), 1748 (C=O), 1660 (C=O), 1632 (NH bending),
1
1593-1454 (C=C); H NMR (CDCl3): δ 1.87 (s, 4H, 2CH2), 2.68-2.72 (m, 2H, CH2),
2.96-2.98 (m, 2H, CH2), 3.48-3.60 (m, 2H, CH2), 3.80 (s, 3H, OCH3), 3.90 (s, 6H, 2
13
OCH3), 4.05-4.10 (m, 1H, CH), 7.50 (s, 2H, aromatic protons), 12.23 (s, 1H, NH); C
NMR (CDCl3): δ 22.9, 23.1, 24.5, 26.5 (4C of cyclohexene), 56.4 ( 3OCH3), 60.6, 61.0
(2C of isoxazolidine), 104.9 (2C o-OCH3), 112.0, 127.1, 127.9, 141.8, 148.1, 153.4
(aromatic carbons + thiophene carbons), 163.1 (C=O), 167.0 (C=O of isoxazolidinone);
12

ACCEPTED MANUSCRIPT
Anal. Calcd. for C22H23N3O6S (457.50): C, 57.76; H, 5.07; N, 9.18 ; found: C, 57.72; H,
5.13; N, 9.26.
4.1.2.9.
6-(4-Oxo-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidin -3 (4H)-yl)pyrimidine-2,4(1H, 3H)-
dione (3i)
Yellow Crystals, mp 173-175oC , 90 % yield; IR (cm-1): ν 3356, 3271 (2NH),
3078 (CH aromatic), 2936-2835 (CH aliphatic), 1748 (C=O), 1728, 1655 (2C=O of
1
pyrimidine), 1636 (NH bending), 1589-1462 (C=C); H NMR (CDCl3): δ 1.87-1.90 (m,
4H, 2CH2), 2.79 (s, 2H, CH2), 2.96 (s, 2H, CH2), 3.89 (s, 3H, OCH3), 3.95 (s, 6H, 2
OCH3), 6.50 (s, 1H, =CH of pyrimidinedione), 7.50 (s, 2H, aromatic protons), 10.90 (s,
1H, NH), 11.1 (s, 1H, NH); Anal. Calcd. for C23H22N4O6S (482.51): C, 57.25; H, 4.60;
N, 11.61; found: C, 57.31; H, 4.57; N, 11.83.
4.1.2.10.
3-(Benzo[d]thiazol-2-yl)-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-tetrahydro
benzo[4,5] thieno[2,3-d]pyrimidin -4 (3H)-one (3j)
Brown Crystals, mp 190-191oC , 85 % yield; IR (cm-1): ν 3050 (CH aromatic),
2940-2835 (CH aliphatic), 1748 (C=O), 1593-1454 (C=C); 1H NMR (DMSO-d6): δ 1.66-
1.72 (m, 4H, 2CH2), 2.63-2.68 (s, 4H, 2CH2), 3.71 (s, 3H, OCH3), 3.80 (s, 6H, 2 OCH3),
7.20 (br.s., 2H, benzothiazole protons), 7.34 (s, 2H, trimethoxyphenyl protons), 8.00
(br.s., 1H, benzothiazole proton), 8.38 (s, 1H, benzothiazole protons); MS (m/z, %):
505.55 (M+, 4.75), 373.15 (20.78), 195 (69.32), 149 (100); Anal. Calcd. for
C26H23N3O4S2 (505.61): C, 61.76; H, 4.59; N, 8.31; found: C, 61.80; H, 4.61; N, 8.49.
4.1.2.11.
3-(5-Methylbenzo[d]thiazol-2-yl)-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5] thieno[2,3-d]pyrimidin -4 (3H)-one (3k)
Yellow crystals, mp 163-165oC , 65 % yield; IR (cm-1): ν 3036 (CH aromatic),
1
2997-2839 (CH aliphatic), 1748 (C=O), 1593-1454 (C=C); H NMR (CDCl3): δ 1.60 (s,
3H, CH3), 1.86-1.88 (m, 4H, 2CH2), 2.78-2.82 (m, 2H, CH2), 2.98-3.10 (m, 2H, CH2),
3.88 (s, 3H, OCH3), 3.95 (s, 6H, 2 OCH3), 7.20-7.90 (m, 5H, aromatic protons); Anal.
13

ACCEPTED MANUSCRIPT
Calcd. for C27H25N3O4S2 (519.64): C, 62.41; H, 4.85; N, 8.09; found: C, 62.47; H, 4.93;
N, 8.18.
4.1.2.12.
3-(5-Nitrobenzo[d]thiazol-2-yl)-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5] thieno[2,3-d]pyrimidin -4 (3H)-one (3l)
Greyish white crystals, mp 180-182oC , 91 % yield; IR (cm-1): ν 3036 (CH
1
aromatic), 2997-2835 (CH aliphatic), 1748 (C=O), 1632 (C=N), 1593-1454 (C=C); H
NMR (CDCl3): δ 1.86-1.88 (m, 4H, 2CH2), 2.75-2.77 (m, 2H, CH2), 2.96-2.99 (m, 2H,
CH2), 3.82 (s, 3H, OCH3), 3.96 (s, 6H, 2 OCH3), 7.49-8.40 (m, 5H, aromatic protons);
13C NMR (CDCl3): δ 22.1, 22.8, 25.3, 29.8 (4C of cyclohexene), 56.5 ( 2OCH3), 61.1
(OCH3), 105.5 (2C o-OCH3), 106.5, 110.7, 116.2, 125.1, 127.4, 132.3, 134.8, 142.0,
153.1, 154.7 (aromatic carbons + thiophene carbons + benzothiazole carbons), 158.4
(C=O), 162.5 (C2 of pyrimidine); MS (m/z, %): 551 (M++1, 5.34), 373.65 (10.11),
195.35 (78.66), 80.45 (100); Anal. Calcd. for C26H22N4O6S2 (550.61): C, 56.72; H, 4.03;
N, 10.18; found: C, 56.73; H, 4.11; N, 10.26.
4.1.2.13.
3-(6-Amino-10-methylacridin-3-yl)-2-(3,4,5-trimethoxyphenyl)-5,6,7,8-
tetrahydrobenzo[4,5] thieno[2,3-d]pyrimidin -4 (3H)-one (3m)
Brown crystals, mp 213-215oC , 93 % yield; IR (cm-1): ν 3364, 3217 (NH2), 3040
(CH aromatic), 2940-2835 (CH aliphatic), 1748 (C=O), 1639 (NH2 bending), 1601
1
(C=N), 1562-1497 (C=C); H NMR (CDCl3): δ 1.86-1.92 (m, 4H, 2CH2), 2.66-2.70 (m,
4H, 2CH2), 3.93 (s, 3H, OCH3), 3.95 (s, 6H, 2 OCH3), 4.00 (s, 3H, CH3), 5.50 (s, 1H,
NH), 7.11-7.58 (m, 9H, aromatic protons); Anal. Calcd. for C33H31N4O4S+ (579.69): C,
68.37; H, 5.39; N, 9.66; found: C, 68.44; H, 5.45; N, 9.78.
4.1.2.14.
(E)-4-Oxo-2-styryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-
3(4H)-carbothioamide (3n)
Brown crystals, mp 117-119oC , 69 % yield; IR (cm-1): ν 3383, 3348 (NH2), 3032
(CH aromatic), 2927-2851 (CH aliphatic), 1680 (C=O), 1643 (NH2 bending), 1535-1447
1
(C=C); H NMR (CDCl3): δ 1.40 (s, 2H, NH2), 1.84-1.86 (m, 4H, 2CH2), 2.68-2.70 (m,
2H, CH2), 2.98-3.08 (m, 2H, CH2), 6.90 (d, J= 16 Hz, 1H, CH=CH-ph), 7.26-7.50 (m,
5H, aromatic protons), 7.95 (d, J=16 Hz, 1H CH=CH-ph); MS (m/z, %): 367 (M+, 1.51),
14

ACCEPTED MANUSCRIPT
204 (1.34), 104 (100); Anal. Calcd. for C19H17N3OS2 (367.49): C, 62.10; H, 4.66; N,
11.43; found: C, 62.14; H, 4.69; N, 11.51.
4.1.2.15.
(E)-4-Oxo-2-styryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-
3(4H)-carboxamide (3o)
Green crystals, mp 130-131oC , 88 % yield; IR (cm-1): ν 3426, 3356 (NH2), 3040
(CH aromatic), 2870-2735 (CH aliphatic), 1651 (2C=O), 1581 (NH2 bending), 1492-
1
1480 (C=C); H NMR (CDCl3): δ 1.70 (s, 2H, NH2), 1.84 (s, 4H, 2CH2), 2.77 (s, 2H,
CH2), 2.90 (s, 2H, CH2), 6.90 (d, J= 15 Hz, 1H, CH=CH-ph), 7.29-7.39 (m, 5H, aromatic
protons), 7.90 (d, J=15 Hz, 1H, CH=CH-ph); 13C NMR (CDCl3): δ 22.1, 22.9, 25.3, 29.8
(4C of cyclohexene), 116.6 (CH=CH-ph), 118.4, 127.5, 128.1, 128.8, 130.0, 132.6,
134.8, 141.9, 158.6 (aromatic carbons + thiophene carbons + CH=CH-ph), 155.2 (C2 of
pyrimidine), 162.3 (CONH2), 169.8 (C=O); MS (m/z, %): 352 (M++1, 8.67), 204
(24.70), 105 (97.97), 77 (100); Anal. Calcd. for C19H17N3O2S (351.42): C, 64.94; H, 4.88;
N, 11.96; found: C, 64.99; H, 4.86; N, 12.04.
4.1.2.16.
(E)-N-Hydroxy-4-Oxo-2-styryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidine-3(4H)-carboxamide (3p)
Grey crystals, mp 123-125oC , 92 % yield; IR (cm-1): ν 3410 (OH), 3287 (NH),
3032 (CH aromatic), 2936-2855 (CH aliphatic), 1690 (2C=O), 1631 (NH bending), 1539
(C=C); 1H NMR (CDCl3): δ 1.85-1.87 (m, 4H, 2CH2), 2.67-2.78 (m, 2H, CH2), 3.00-3.10
(m, 2H, CH2), 6.35 (s, 1H, OH), 6.92 (d, J= 16 Hz, 1H, CH=CH-ph), 7.30-7.50 (m, 5H,
aromatic protons), 7.90 (d, J=16 Hz, 1H, CH=CH-ph), 12.10 (s, 1H, NH); Anal. Calcd.
for C19H17N3O3S (367.42): C, 62.11; H, 4.66; N, 11.44; found: C, 62.14; H, 4.69; N,
11.46.
4.1.2.17.
(E)-4-(4-Oxo-2-styryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-3 (4H)-yl) benzoic acid (3q)
Yellowish brown crystals, mp 143-145oC , 90 % yield; IR (cm-1): ν 3200 (OH
carboxylic), 3055-3028 (CH aromatic), 2924-2851 (CH aliphatic), 1659 (2C=O), 1604
1
(C=N), 1560-1450 (C=C); H NMR (CDCl3): δ 1.83-1.88 (m, 4H, 2CH2), 2.70-2.78 (m,
15

ACCEPTED MANUSCRIPT
2H, CH2), 2.80-2.87 (m, 2H, CH2), 6.75 (d, J= 16.05 Hz, 1H, CH=CH-ph), 7.40-7.59 (m,
5H, aromatic protons), 7.80 (d, J=16.05 Hz, 1H, CH=CH-ph), 7.99 (d, J= 7.65 Hz, 2H,
aromatic protons), 8.25 (d, J= 7.65 Hz, 2H, aromatic protons), 11.28 (s, 1H, OH); 13C
NMR (CDCl3): δ 22.1, 22.9, 23.0, 25.3 (4C of cyclohexene), 111.1 (CH=CH-ph), 116.5,
118.4, 127.4, 127.9, 128.1, 128.8, 130.4, 132.4, 134.8, 135.1, 141.9, 142.4, 155.3
(aromatic carbons + thiophene carbons + CH=CH-ph), 158.9 (C=O), 163.6 (C2 of
pyrimidine), 170.4 (COOH); Anal. Calcd. for C25H20N2O3S (428.50): C, 70.07; H, 4.70;
N, 6.54; found: C, 70.11; H, 4.76; N, 6.58.
4.1.2.18.
(E)-4-(4-Oxo-2-styryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-3 (4H)-yl) benzenesulfonamide (3r)
Black crystals, mp 150-151oC , 70 % yield; IR (cm-1): ν 3356-3156 (NH2), 3059-
3028 (CH aromatic), 2928-2855 (CH aliphatic), 1680 (C=O), 1634 (NH2 bending), 1597-
1535 (C=C), 1312, 1161 (S=O); 1H NMR (CDCl3): δ 1.83-1.86 (m, 4H, 2CH2), 2.72-2.78
(m, 2H, CH2), 2.95-3.00 (m, 2H, CH2), 6.80 (d, J= 16 Hz, 1H, CH=CH-ph), 7.35-7.86
(m, 9H, aromatic protons), 7.93 (d, J=16 Hz, 1H, CH=CH-ph), 8.50 (s, 2H, NH2); MS
(m/z, %): 463 (M+, 32.05), 282 (30.67), 63.75 (100); Anal. Calcd. for C24H21N3O3S2
(463.57): C, 62.18; H, 4.57; N, 9.06; found: C, 62.22; H, 4.61; N, 9.14.
4.1.2.19.
(E)-4-(4-Oxo-2-styryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-3 (4H)-yl) phenylpropanoic acid (3s)
Brown crystals, mp 119-121oC , 93 % yield; IR (cm-1): ν 3271-3159 (OH
carboxylic), 3059-3028 (CH aromatic), 2928-2851 (CH aliphatic), 1680 (C=O), 1650
1
(C=O), 1632 (C=N), 1562-1454 (C=C); H NMR (CDCl3): δ 1.85 (s, 4H, 2CH2), 2.68-
2.78 (m, 2H, CH2), 2.85-2.94 (m, 2H, CH2), 3.79-3.80 (m, 2H, CH2CH), 3.98-4.10 (m,
1H, CH2CH), 6.74 (d, J= 16.05 Hz, 1H, CH=CH-ph), 7.39-7.56 (m, 7H, aromatic
protons), 7.81 (d, J=16.05 Hz, 1H, CH=CH-ph), 7.96-8.25 (m, 3H, aromatic protons),
11.25 (s, 1H, OH carboxylic); 13C NMR (CDCl3): δ 22.0, 22.9, 24.5, 25.2 (4C of
cyclohexene), 26.5 (CH2CH), 55.5 (CH2CH), 111.5 (CH=CH-ph), 118.4, 127.5, 128.1,
128.3, 128.7, 128.9, 130.4, 132.6, 134.5, 141.9, 155.3 (aromatic carbons + thiophene
carbons + CH=CH-ph), 158.9 (C=O), 163.6 (C2 of pyrimidine), 169.8 (COOH); Anal.
16

ACCEPTED MANUSCRIPT
Calcd. for C27H24N2O3S (456.56): C, 71.03; H, 5.30; N, 6.14; found: C, 71.02; H, 5.37;
N, 6.08.
4.1.2.20.
(E)-3-(1H-indol-2-yl)-4-(4-Oxo-2-styryl-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin -3 (4H)-yl) propanoic acid (3t)
Brown crystals (from charcoal/ethanol), mp > 300oC , 83 % yield; IR (cm-1): ν
3375 (NH), 3175 (OH carboxylic), 3059-3032 (CH aromatic), 2924-2851 (CH aliphatic),
1
1748 (2C=O), 1624 (NH bending), 1539-1497 (C=C); H NMR (CDCl3): δ 1.82 (s, 4H,
2CH2), 2.68-2.78 (m, 2H, CH2), 2.85-2.95 (m, 2H, CH2), 3.73-3.75 (m, 2H, CH2), 4.20-
4.25 (m, 1H, CH), 6.63 (d, J= 15.30 Hz, 1H, CH=CH-ph), 7.39-7.58 (m, 8H, aromatic
protons), 7.81 (d, J=15.30 Hz, 1H, CH=CH-ph), 7.97-7.98 (m, 2H, aromatic protons),
11.26 (s, 1H, OH carboxylic), 12.06 (s, 1H, NH); Anal.Calcd.for C29H25N3O3S (495.59):
C, 70.28; H, 5.08; N, 8.48; found: C, 70.31; H, 5.14; N, 8.62.
4.1.2.21.
(E)-3-(3-Oxoisoxazolidin-4-yl)-2-styryl-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin -4 (3H)-one (3u)
Grey crystals, mp 140-142oC , 90 % yield; IR (cm-1): ν 3406 (NH), 3063-3032
(CH aromatic), 2924-2851 (CH aliphatic), 1670 (2C=O), 1636 (NH bending), 1558-1458
1
(C=C); H NMR (CDCl3): δ 1.84 (s, 4H, 2CH2), 2.77 (s, 2H, CH2), 3.05 (m, 2H, CH2),
3.72-3.74 (m, 1H, 1H of CH2), 3.89-3.90 (m, 1H, 1H of CH2), 4.23-4.36 (m, 1H, CH),
6.28 (s, 1H, NH), 6.91 (d, J= 16.80 Hz, 1H, CH=CH-ph), 7.23-7.59 (m, 5H, aromatic
protons), 7.90 (d, J=16.85 Hz, 1H, CH=CH-ph); Anal. Calcd. for C21H19N3O3S (393.46):
C, 64.10; H, 4.87; N, 10.68; found: C, 64.08; H, 4.89; N, 10.77.
4.1.2.22.
(E)-6-(4-Oxo-2-styryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-3(4H)-yl)pyrimidine-2,4(1H, 3H)-dione (3v)
Brown crystals, mp 110-112oC , 66 % yield; IR (cm-1): ν 3375-3348 (2NH), 3063
(CH aromatic), 2928-2855 (CH aliphatic), 1659 (C=O), 1640 (2C=O), 1580 (NH
bending), 1535-1450 (C=C); 1H NMR (CDCl3): δ 1.86 (s, 4H, 2CH2), 2.79 (s, 2H, CH2),
2.98 (m, 2H, CH2), 6.20 (s, 1H, =CH of pyrimidinedione), 6.90 (d, J= 16 Hz, 1H,
CH=CH-ph), 7.30-7.50 (m, 5H, aromatic protons), 7.90 (d, J=16 Hz, 1H, CH=CH-ph),
13
10.8 (s, 1H, NH), 11.20 (s, 1H, NH); C NMR (CDCl3): δ 22.0, 22.9, 24.2, 25.3 (4C of
17

ACCEPTED MANUSCRIPT
cyclohexene), 74.5, 112.8, 117.8, 126.6, 126.9, 127.7, 128.9, 129.3, 131.6, 146.4, 146.9
(aromatic carbons + thiophene carbons + pyrimidinedione carbons + olefinic carbons),
162.9, 167.3 (2C=O of pyrimidinedione), 168.1 (C=O); Anal. Calcd. for C22H18N4O3S
(418.47): C, 63.14; H, 4.34; N, 13.39; found: C, 63.22; H, 4.37; N, 13.52.
4.1.2.23.
(E)-3-(Benzo[d]thiazol-2-yl)-2-styryl-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d] pyrimidin -4 (3H)-one (3w)
Brown crystals, mp 100-103oC , 60 % yield; IR (cm-1): ν 3059-3028 (CH
1
aromatic), 2920-2851 (CH aliphatic), 1748 (C=O), 1663 (C=N), 1628-1493 (C=C); H
NMR (CDCl3): δ 1.83-1.85 (m, 4H, 2CH2), 2.65-2.75 (m, 2H, CH2), 2.80-2.90 (m, 2H,
CH2), 6.90 (d, J= 15 Hz, 1H, CH=CH-ph), 7.28-7.90 (m, 10H, aromatic protons +
13
CH=CH-ph); C NMR (DMSO-d6): δ 22.7, 23.0, 25.8, 26.9 (4C of cyclohexene), 111.0
(CH=CH-ph), 127.5, 127.6, 127.8, 129.1, 130.1, 132.0, 135.4, 137.9, 145.1, 152.8, 157.9
(aromatic carbons + benzothiazole carbons + CH=CH-ph), 166.3 (C=O); Anal. Calcd. for
C25H19N3OS2 (441.57): C, 68.00; H, 4.34; N, 9.52; found: C, 68.03; H, 4.34; N, 9.63.
4.1.2.24.
(E)-3-(5-Methylbenzo[d]thiazol-2-yl)-2-styryl-5,6,7,8-tetrahydrobenzo
[4,5] thieno[2,3-d]pyrimidin -4 (3H)-one (3x)
Grey crystals, mp 155-156oC , 90 % yield; IR (cm-1): ν 3063 (CH aromatic),
1
2936-2859 (CH aliphatic), 1643 (C=O), 1535-1458 (C=C); H NMR (CDCl3): δ 1.83-
1.85 (m, 4H, 2CH2), 2.10 (s, 3H, CH3), 2.78-2.79 (m, 2H, CH2), 2.93-2.96 (m, 2H, CH2),
6.80 (d, J= 15 Hz, 1H, CH=CH-ph), 7.29-7.90 (m, 9H, aromatic protons + CH=CH-ph);
Anal. Calcd. for C26H21N3OS2 (455.59): C, 68.54; H, 4.65; N, 9.22; found: C, 68.60; H,
4.72; N, 9.31.
4.1.2.25.
(E)-3-(5-Nitrobenzo[d]thiazol-2-yl)-2-styryl-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin -4 (3H)-one (3y)
Green crystals, mp 133-135oC , 90 % yield; IR (cm-1): ν 3063-3032 (CH
1
aromatic), 2932-2855 (CH aliphatic), 1651 (C=O), 1631 (C=N), 1580-1454 (C=C); H
NMR (CDCl3): δ 1.87-1.89 (m, 4H, 2CH2), 2.78-2.80 (m, 2H, CH2), 2.95-3.02 (m, 2H,
CH2), 6.90 (d, J= 16 Hz, 1H, CH=CH-ph), 7.35-8.60 (m, 9H, aromatic protons +
18

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CH=CH-ph); Anal. Calcd. for C25H18N4O3S2 (486.57): C, 61.71; H, 3.73; N, 11.51;
found: C, 61.69; H, 3.76; N, 11.73.
4.1.2.26.
(E)-3-(6-Amino-10-methylacridin-3-yl)-2-styryl-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin -4 (3H)-one (3z)
Brown crystals, mp 260-261oC , 90 % yield; IR (cm-1): ν 3360, 3217 (NH2), 3050
(CH aromatic), 2920-2851 (CH aliphatic), 1640 (C=O), 1636 (NH2 bending), 1605
1
(C=N), 1493 (C=C); H NMR (CDCl3): δ 1.87-1.90 (m, 4H, 2CH2), 2.77-2.79 (m, 2H,
CH2), 2.92-2.94 (m, 2H, CH2), 3.49 (s, 3H, CH3), 3.78 (s, 2H, NH2), 6.75 (d, J= 16.05
Hz, 1H, CH=CH-ph), 7.40-7.57 (m, 12H, aromatic protons), 7.83 (d, J=16.05 Hz, 1H,
13
CH=CH-ph); C NMR (CDCl3): δ 23.0, 24.5, 25.3, 26.3 (4C of cyclohexene), 111.3
(CH=CH-ph), 114.1, 116.6, 118.4, 119.4, 119.8, 127.3, 127.5, 128.1, 128.3, 128.8, 129.1,
134.4, 134.8, 135.1, 142.0, 142.4, 144.1, 149.7, 155.3 (aromatic carbons + thiophene
carbons + CH=CH-ph), 162.7 (C=O), 163.6 (C2 of pyrimidine); Anal. Calcd. for
C32H27N4OS+ (515.65): C, 74.54; H, 5.28; N, 10.87; found: C, 74.61; H, 5.33; N, 10.96.
4.1.3. General procedure for the preparation of (4a-d)
To a mixture of equimolar amounts of 2b and the appropriate aldehyde (5 mmol)
in methanol (10 ml), hydrochloric acid (5 drops) was added and the solution was stirred
at room temperature for 5 h. The obtained precipitate was filtered, washed and
crystallized from ethanol.
4.1.3.1.
2-(3,4,5-Trimethoxyphenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidin-4(3H) one (4a)[7, 8]
1H NMR (CDCl3): δ 1.87-1.89 (m, 4H, 2CH2), 2.79 (s, 2H, CH2), 2.94 (s, 2H,
CH2), 3.96 (s, 3H, OCH3), 3.99 (s, 6H, 2OCH3), 7.49 (s, 2H, aromatic protons), 12.23 (s,
1H, NH).
4.1.3.2.
(E)-2-Styryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-
4(3H)one (4b)[50, 51]
IR (cm-1): ν 3363(NH), 3039 (CH olifenic and aromatic), 2927 (CH aliphatic),
1647 (C=O), 1616 (C=N); 1H NMR (DMSO-d6): δ 1.73-1.76 (m, 4H, 2CH2), 2.70 (s, 2H,
19

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CH2), 2.84 (m, 2H, CH2), 6.94 (d, J= 16.05 Hz, 1H, CH=CH-ph), 7.20-7.59 (m, 5H,
aromatic protons), 7.84 (d, J=16.05 Hz, 1H, CH=CH-ph), 12.30 (s, 1H, NH); Anal. calcd.
for C18H16N2OS (308.40): C, 70.10; H, 5.23; N, 9.08; found: C, 70.23; H, 5.16; N, 9.20.
4.1.3.3.
(E)-2-(2-Nitrostyryl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-
4(3H) one (4c)
Orange crystals, mp 208-210oC, 90% yield; IR (cm-1): ν 3363(NH), 3066 (CH
1
olefinic and aromatic), 2931 (CH aliphatic), 1639 (C=O), 1616 (C=N); H NMR
(CDCl3): δ 1.86-1.87 (m, 4H, 2CH2), 2.78-2.79 (s, 2H, CH2), 2.94-2.95 (m, 2H, CH2),
6.75 (d, J= 16.05 Hz, 1H, CH=CH-ph), 7.39-7.50 (m, 4H, aromatic protons), 7.81 (d,
J=16.05 Hz, 1H, CH=CH-ph), 12.10 (s, 1H, NH); Anal. calcd. for C18H15N3O3S (353.39):
C, 61.18; H, 4.28; N, 11.89; found: C, 61.22; H, 4.17; N, 11.54.
4.1.3.4.
(E)-2-(1-Phenylprop-1-en-2-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d] pyrimidin-4(3H) one (4d)
Yellow crystals, mp 240-243oC, 90% yield; IR (cm-1): ν 3383(NH), 3070 (CH
1
olefinic and aromatic), 2931 (CH aliphatic), 1639 (C=O), 1616 (C=N); H NMR
(CDCl3): δ 1.86-1.88 (m, 4H, 2CH2), 2.69-2.78 (s, 2H, CH2), 2.95 (s, 2H, CH2), 3.49 (s,
3H, CH3), 7.40-8.04 (m, 5H, aromatic protons), 8.26 (s, 1H, =CH-ph), 12.15 (s, 1H, NH);
Anal. calcd. for C19H18N2OS (322.42): C, 70.78; H, 5.63; N, 8.69; found: C, 70.69; H,
5.60; N, 8.71.
4.1.4. General procedure for the preparation of (6a-c)
A mixture of equimolar amounts of 5a,b and the appropriate amine (5 mmol) was
heated in domestic microwave at 1000 W under solvent free condition for 45 seconds.
The obtained product was crystallized from ethanol.
4.1.4.1.
(E)-1-Hydroxy-3-[2-(3,4,5-trimethoxyphenyl-5,6,7,8-
tetrahydrobenzo[4,5]thieno [2,3-d]pyrimidin-4-yl]urea (6a)
Yellow crystals, mp >300oC, 93% yield; IR (cm-1): ν 3406-3237 (2NH, OH),
3050 (CH aromatic), 2936-2839 (CH aliphatic), 1655 (C=O), 1630 (NH bending), 1589-
20

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1
1485 (C=C); H NMR (CDCl3): δ 1.86-1.88 (m, 4H, 2CH2), 2.77-2.79 (m, 2H, CH2),
2.96-2.99 (m, 2H, CH2), 3.90 (s, 3H, OCH3), 4.00 (s, 6H, 2OCH3), 6.5 (s, 1H, OH), 7.70
13
(s, 2H, aromatic protons), 7.80 (s, 2H, 2NH); C NMR (DMSO-d6): δ 22.6, 23.1, 24.5,
26.3 (4C of cyclohexene), 56.5 (2OCH3), 60.7 (OCH3), 106.7, 130.0, 131.1, 131.6, 136.3,
141.5, 153.7 (aromatic carbons + thiophene carbons), 150.7 (C=O), 157.2 (C4 of the
pyrimidine), 165.5 (C2 of the pyrimidine); Anal. Calcd. for C20H22N4O5S (430.48): C,
55.80; H, 5.15; N, 13.02; found: C, 55.78; H, 5.14; N, 13.15.
4.1.4.2.
(E)-1-Hydroxy-3-(2-styryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidin-4-yl)urea (6b)
Brown crystals, mp 225-227 C, 92% yield; IR (cm-1): ν 3387-3200 (2NH, OH),
o
3030 (CH aromatic), 2940 (CH aliphatic), 1659(C=O), 1630 (NH bending), 1589-1400
(C=C); 1H NMR (CDCl3): δ 1.87-1.89 (m, 4H, 2CH2), 2.75-2.77 (m, 2H, CH2), 2.93-2.95
(m, 2H, CH2), 6.50 (s, 1H, OH), 6.80 (d, J= 16.05 Hz, 1H, CH=CH-ph), 7.20-8.20 (m,
6H, aromatic protons + CH=CH-ph), 8.80 (s, 2H, 2NH); 13C NMR (CDCl3): δ 22.3, 23.0,
25.3, 25.9 (4C of cyclohexene), 111.7 (CH=CH-ph), 115.5, 126.4, 128.1, 128.6, 138.4,
138.7, 145.9 (aromatic carbons + thiophene carbons+ CH=CH-ph), 151.0 (C=O), 160.5
(C4 of the pyrimidine), 160.9 (C2 of pyrimidine); MS (m/z, %): 366 (M+, 3.72), 105
(100); Anal. Calcd. for C19H18N4O2S (366.44): C, 62.28; H, 4.95; N, 15.29; found: C,
62.32; H, 4.93; N, 15.48.
4.1.4.3.
(E)-3-(6-Amino-10-methylacridin-3-ylamino)-2-styryl-5,6,7,8-
tetrahydrobenzo [4,5]thieno [2,3-d]pyrimidine (6c)
Brown crystals, mp >300 oC, 95% yield; IR (cm-1): ν 3314-3190 (NH, NH2), 3030
1
(CH aromatic), 2936 (CH aliphatic), 1639 (NH bending), 1601-1443(C=C); H NMR
(CDCl3): δ 1.86-1.88 (m, 4H, 2CH2), 2.75-2.77 (m, 2H, CH2), 2.95-2.97 (m, 2H, CH2),
4.10 (s, 3H, CH3), 5.40 (s, 2H, NH2), 6.80 (d, J=16.05 Hz, 1H, CH=CH-ph), 7.28-8.30
(m, 13H, aromatic protons + CH=CH-ph), 9.80 (s, 1H, NH); Anal. Calcd. for C32H28N5S+
(514.66): C, 74.68; H, 5.48; N, 13.61; found: C, 74.66; H, 5.51; N, 13.84.
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4.2. Antitumor activity
The potential cytotoxicity of the tested compounds and Doxorubicin as a
reference standard was evaluated using the method of Skehan et al. [52]. Cells were
plated in 96-multiwell plate (104 cells/well) for 24 h before treatment with the prepared
compounds to allow the attachment of cells to the wall of the plate. The tested
compounds were dissolved in dimethylsulfoxide (DMSO) and diluted 1000-fold in the
assay. Concentrations 0, 5, 12.5, 25, and 50 ꢀg/ml of the tested compounds were added
to the cell monolayer. The monolayer cells were incubated with the compounds for 48 h
at 37 C, in atmosphere of 5% CO2. After 48 h, the cells were fixed, washed and stained
with Sulfo-Rhodamine-B stain (SRB). Excess stain was washed with acetic acid. The
attached stain was recovered with Tris EDTA buffer. Cell survival and drug activity
were determined by measuring color intensity using an ELISA reader. Data are
representative of the individual experiment, performed in three replicates for each
individual dose and measured by SRB assay. Control values did not exhibit significant
changes compared to the DMSO vehicle. The IC50 was determined using a program
Graph-Pad PRISM version 5. Mean and standard error were determined by SPSS 11
software. The obtained IC50 values were illustrated in Table 1.
4.3. Antibacterial activity
Antibacterial activity of the tested compounds was determined using a modified
Kirby-Bauer disc diffusion method [53]. Of the many media available, National
Committee for Clinical Laboratory Standards NCCLS recommend Mueller-Hinton agar
due to its good batch-to-batch reproducibility. Plates were incubated with Staphylococcus
aureus (ATCC 12600) and Bacillus subtilis (ATCC 6051) (as Gram positive bacteria)
while Pseudomonas aeruginosa (ATCC 10145) and Escherichia coli (ATCC 11775) (as
Gram negative bacteria) at 35-37 C for 24-48 h, then the diameters of the inhibition
zones were measured in millimeters. Standard discs of Ampicillin served as positive
control for antibacterial activity and filter discs impregnated with DMSO solvent served
as a negative control. Blank paper discs (Schleicher & Schuell, Spain) with a diameter of
8.0 mm were impregnated with the tested concentration of the stock solutions. The zone
22

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diameters were measured with slipping calipers of the National Committee for Clinical
Laboratory Standards (NCCLS, 1993). The results expressing the inhibition zone in mm
were represented in Table 2. The MIC values obtained from the broth microdilution
method [54] for the most active compounds 3m and 3z were represented in Table 3.
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ACCEPTED MANUSCRIPT
Figures captions:
Figure 1. Representative anticancer quinazoline and thieno[2,3-d]pyrimidines and our
proposed derivatives.
Tables captions:
Table 1. IC50 of the tested compounds against human tumor cell lines
Table 2. Inhibition zone (mm) of the tested compounds
Table 3. MIC values (ꢀg/ml) of tested samples against tested microorganisms
27