Synthesis of some novel thieno[3,2-d]pyrimidines as potential cytotoxic small molecules against breast cancer

Article abstract of DOI:10.1248/cpb.c13-00089

A variety of novel thieno[3,2-d]pyrimidines with different decorating functional groups were synthesized as a part of a study aiming to enrich the arsenal of chemotherapeutic agents for the treatment of cancer. The design of synthetic molecules based on DNA-interchelating properties by hydrogen bond formation. The reported compounds herein are: 4-aminothienopyrimidine derivatives 4a, b and their 4-substituted phenylamino analogues 8a, b; 4-thienopyrimidin-4-ones 5a, b; N-alkyl thienopyrimidin-4-ones 6a-g; 4-chlorothienopyrimidines 7a, b and thienopyrimidoquinazolinones 9a, b which are the structural mimics of 8a, b. The synthesized molecules were evaluated for their in vitro cytotoxic activity against human breast cancer cell line (MCF-7). Biological screening revealed varying cytotoxic potencies of the tested molecules compared with Doxorubicin as a reference drug. The cytotoxicity results from the study suggested that the synthesized molecules are potential antitumor agents and compound 4a was the most potent with an IC₅₀ 2.04 nM.

Full text of DOI:10.1248/cpb.c13-00089

June 2013
Regular Article
Chem. Pharm. Bull. 61(6) 637–647 (2013)
637
Synthesis of Some Novel Thieno[3,2-d]pyrimidines as Potential Cytotoxic
Small Molecules against Breast Cancer
,a
Manal Kandeel,a Mohammed Kamal Abdelhameid,* Kamal Eman,b and Madlen Berty Labibb
b
a Department of Organic Chemistry, Faculty of Pharmacy, Cairo University; Cairo 11562, Egypt: and Department
of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-suef University; Salah Salem St., Beni-suef 62511,
Egypt. Received January 31, 2013; accepted March 13, 2013; advance publication released online March 29, 2013
A variety of novel thieno[3,2-d]pyrimidines with different decorating functional groups were synthesized
as a part of a study aiming to enrich the arsenal of chemotherapeutic agents for the treatment of cancer.
The design of synthetic molecules based on DNA-interchelating properties by hydrogen bond formation.
The reported compounds herein are: 4-aminothienopyrimidine derivatives 4a,b and their 4-substituted
phenylamino analogues 8a,b; 4-thienopyrimidin-4-ones 5a,b; N-alkyl thienopyrimidin-4-ones 6a–g; 4-chloro-
thienopyrimidines 7a,b and thienopyrimidoquinazolinones 9a,b which are the structural mimics of 8a,b.
The synthesized molecules were evaluated for their in vitro cytotoxic activity against human breast cancer
cell line (MCF-7). Biological screening revealed varying cytotoxic potencies of the tested molecules compared
with Doxorubicin as a reference drug. The cytotoxicity results from the study suggested that the synthesized
molecules are potential antitumor agents and compound 4a was the most potent with an IC 2.04nm.
50
Key words heterocycle; thieno[3,2-d]pyrimidine; cytotoxic activity; breast cancer
The development of novel chemotherapeutic agents has greater interest than their constituting monocyclic fragments.
significantly progressed within the last 60 years, success in Thienopyrimidines occupies a special position among them
developing non-cytotoxic “targeted” drugs with fewer side as they serve as structural analogues for biogenic purines and
effects has occurred only in the last decade. Despite these potential nucleic acid antimetabolites.11) Literature screening
advances, the treatment of most types of solid tumors (e.g., revealed that many thienopyrimidine derivatives A, B and
breast and ovarian) is still a problem and survival rates re- C have been developed and showed pronounced cytotoxic
main significantly low.1) Doxorubicin (DOX) is an antitumour activity.12–20) In particular, they are currently an important
antibiotic and essential component of the chemotherapy of a group of compounds that have anticancer activity especially
large number of solid tumors, including breast cancer, soft against solid tumors (e.g., breast and ovarian).21–24) Several
tissue sarcomas, and aggressive lymphomas. Despite their mechanisms have been reported for the cytotoxic activity of
long-standing clinical utilization, the mechanism of action is thienopyrimidines including: inhibition of protein kinases
still unclear and subject to controversy. From a chemical point (PKs) either as competitive or noncompetitive inhibitors. It
of view, DOX is composed of a tetracyclic ring linked to the was reported that they exerted their action by inhibiting ty-
amino sugar; the mode of action of DOX is intercalation with rosine kinases (TKs) that represents a major advance in the
guanine base of DNA.2) DOX exerted its action by intercalat- treatment of solid tumors.25,26) Thienopyrimidine derivatives
ing DNA and consequently inhibiting the macromolecular D have been shown to prevent angiogenesis of tumor cells via
biosynthesis machinery and topoisomerase II (topo II).3) Topo- their potent inhibition of vascular endothelial growth factor
isomerases are crucial for the several DNA functions (e.g., receptor (VEGFR).27) Moreover, thienopyrimidine derivatives
replication and transcription). The main pharmacophoric fea- E exhibited inhibition of DNA replication during cell divi-
ture of DOX and other synthetic interclators is the hydrogen sion through the inhibition of cycle protein kinase (Cdc7) and
bonding formation with DNA.4) Mitoxantrone was a synthetic thienopyrimidine derivative F might be also apoptosis inducer
analog of DOX and is active in breast cancer with a similar in human breast cancer cells.28,29) Beside that, there are certain
mode of action. Isosteric substitution of one or more carbons groups of antitumor drugs working as topo II inhibitors that
of the benzene rings of Mitoxantrone with nitrogen atom they differ from topo II poisons (DNA intercalators) in inter-
has been employed as a strategy for the design of pixantrone fering with the steps of the catalytic cycle as they targeted the
(simplified analogue of mitoxantrone with an increased affin- N-terminal ATPase domain of topo II leading to inhibition of
ity to DNA due to the presence of hydrogen bonding sites).5–7) DNA synthesis. These groups have been synthesized to mimic
Guided with this bioisosteric modification, fused hetrocylic ATP, where they usually consist of a heterocyclic ring system
ring core could be used instead of the planner polycyclic ring that occupied the purine binding site and it served as a scaf-
system in the design of novel synthetic molecules as DNA fold for side chains that occupied the adjacent hydrophobic
intercalators.
regions I and II.30–36)
In addition, Pyrimidine is one of the most important
In the light of mentioned findings, and applying the concept
pharmacophoric ring exhibiting remarkable pharmacological of pharmacophoric hybridization, a number of small synthetic
activities as it is one of the essential building blocks of nu- molecules containing DNA-interchelating fragments and ki-
cleic acids, DNA and RNA.8) Moreover thiophene ring is also nase inhibition properties was designed as potential cytotoxic
known to possess anticancer activity.9,10) From biological point agents. The work reported the synthesis and cytotoxic activity
of view, fused hetero-aromatic ring systems are often of much of some novel small synthetic molecules in the aim of explor-
ing structure–activity relationship (SAR) of thienopyrimidine
ring for inhibition of DNA synthesis by comparison between
The authors declare no conflict of interest.
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: mohorganic@hotmail.com

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Vol. 61, No. 6
Fig. 1. Structures of DOX, Mitoxantrone, Pixantrone and Some of the Literature Active Thienopyrimidines as Cytotoxic Molecules
different function groups on the cytotoxicity, where 4-amino- derivatives 3a–d were prepared either from the cyanoacet-
thienopyrimidines 4a,b and their 4-substituted phenylamino amides 1a,b adopting Gewald precautions or by reacting the
derivatives 8a,b were built to study the effect of substitution acyclic methylthio compounds 2a–d with sodium ethoxide in
on NH2 group by polar group (benzoic acid moiety) that can ethanol.39) The latter procedure was found to afford a better
form additional hydrogen bond on cytotoxicity, also planar yield than the former method. The IR spectra of 3a–d showed
tetracyclic compounds 9a,b which structurally related to the absorption bands at the range 3356–3200cm−1 due to two NH
synthetic intercalators were synthesized as structural mim- and NH2 groups. Compounds 3c,d showed the disappearance
ics of 8a,b to explore structural rigidity. In parallel, a series of C≡N group absorption band of the precursor 2c,d. The
of molecules was synthesized to compare the difference in 1H-NMR spectra of 3b–d lacked the presence of the singlet
activity between the NH2 group of derivatives 4a,b and other signal corresponding to CH2 protons of its precursor 2b–d but
function groups present in thienopyrimidin-4-ones 5a,b and showed an additional exchangeable singlet signal correspond-
4-chlorothienopyrimidines 7a,b. In this context, N-alkyl ing to NH2 protons at δ 6.41–6.68ppm. Compounds 3b,d
thienopyrimidin-4-ones 6a–g were synthesized as the struc- displayed a singlet signal at δ 2.25–2.26ppm indicating CH3
tural extension that could form extra hydrogen bond formation protons of p-tolyl moiety.
sites in hetrocyclic core. The synthetic molecules probably
4-Aminothienopyrimidines can be obtained from the cor-
displayed both DNA intercalators and topo inhibition struc- responding o-aminocyano derivatives by reaction a mixture of
tural features, consequently inhibition of DNA functions.
formamide and formic acid in DMF40) or formamide only.41)
The desired 4-aminothienopyrimidines 4a,b were obtained
by treating of the o-aminocyanothiophenes 3a,b with excess
Results and Discussion
Chemistry The synthetic strategies adopted for construct- formamide. 1H-NMR spectra revealed the presence of the
ing all the target molecules are illustrated in (Figs. 2–7). Ethyl signal due to NH2 protons in the aromatic region in addition
cyanooacetate reacts with the appropriate aromatic amine to to two exchangeable singlet signals appearing at δ 11.38–11.41
give cyanoacetamides 1a,b.37) It was reported that the reaction and 11.99–12.08ppm corresponding to two NH protons. Also,
of the acidic methylene compounds with phenyl isothiocyanate a singlet signal appeared at δ 8.43 corresponding to C2 proton
in a basic medium such as potassium hydroxide gave the suit- of thienopyrimidine ring. Moreover, the mass spectrum of 4b
able α-halo active methylene compound.38) The alkaline solu- displayed three molecular ion peaks at m/z 376, 375 and 374
tion of the 1a–b in N,N-dimethylformamide (DMF) was treat- corresponding to (M+1┐+ ·), (M┐+ ·) and (M-1┐+ ·), respectively.
ed with phenyl isothiocyanate followed by reaction with either
Synthesis of thienopyrimidinones from the corresponding
chloroacetonitrile to afford 2a,b or ethyl chloroacetate to give o-aminoesters via reaction with excess formamide was report-
1
2c,d. H-NMR spectra revealed the presence of a singlet peak ed in literature.42,43) Fusion of the parent o-aminoesters 3c,d
at δ 3.62–4.00ppm corresponding to CH2 protons, two ex- with excess formamide gave the thieno[3,2-d]pyrimidinones
changeable singlet signals at δ 8.80–9.58 and 10.16–11.42ppm 5a,b. The IR spectra of 5a,b indicated the presence of ab-
corresponding to the two NH protons. In addition, compounds sorption bands at 3438–3436, 3236–3182 and 1666–1639cm−1
2c,d showed a triplet signal at δ 1.15ppm and a quartet signal corresponding to OH, three NH and two C=O groups, re-
1
at δ 4.04ppm corresponding to CH3 and CH2 protons of the spectively. H-NMR spectra revealed the disappearance of the
ethoxide group, respectively. The key intermediate thiophene signals of ethoxy protons of the parent ester 3c,d whereas the

June 2013
639
Fig. 2. Synthesis of the Intermediates 2a–d and 3a–d
Fig. 4. Synthesis of the Thienopyrimidinones 5a,b
Fig. 3. Synthesis of 4-Aminothienopyrimidine Derivatives 4a,b
11.28–11.50ppm corresponding to three NH protons. In ad-
dition, OH proton of 6e and 6g appeared as an exchangeable
appearance of two exchangeable singlet signals at δ 11.43 and singlet signal at δ 11.45 and 11.49ppm, sequentially. The mass
11.47–11.53ppm and one singlet signal at δ 12.61–12.69 ppm spectra of 6a,b and 6g displayed molecular ion peaks at m/z
indicated NH protons and OH protons, respectively. Addi- 404, 495 and 525, respectively.
tionally, C2 proton of thienopyrimidine appeared as a singlet
Conversion of thienopyrimidinones to the correspond-
signal at δ 8.39 and 8.36ppm for compounds 5a and 5b. The ing chlorosubstituted thienopyrimidine derivatives could be
mass spectrum of 5a showed a molecular ion peak at m/z 362 achieved by using phosphorus oxychloride.45) Chlorination of
(M┐+) (46.73).
thienopyrimidinones 5a,b with excess phosphorus oxychloride
Moreover, conversion of thienopyrimidinone derivatives gave the 4-chlorothienopyrimidine derivatives 7a,b. Com-
into the corresponding N-alkyl derivatives could be achieved pounds 7a,b showed the disappearance of OH group absorp-
through reaction of the thienopyrimidinones with the respec- tion band at 3236–3182cm−1 of the precursor 5a,b.
tive alkyl halide in ethanolic solution of sodium ethoxide.44)
It has been reported that nucleophilic substitution of chlo-
Reacting thienopyrimidinones 5a or 5b with ethanolic potas- rosubstituted derivatives with different aromatic amines could
sium hydroxide solution followed by the addition of the appro- be operated in DMF.46) Reacting equimolar amounts of 7a,b
priate alkyl halide afforded the corresponding N-alkyl deriva- with anthranilic acid in DMF containing catalytic amounts of
tives 6a–g. IR spectra of compounds 6a–g showed an absorp- anhydrous potassium carbonate afforded the 4-anilino deriva-
1
1
tion band at 3510cm−1 indicating OH group. H-NMR spec- tives 8a,b. H-NMR spectra of 8a,b proved the structure as
trum of 7a showed a triplet signal at δ 1.30ppm and a quartet it showed an exchangeable singlet signal at δ 13.09ppm cor-
signal at δ 4.02ppm corresponding to CH3 and CH2 protons responding to acidic OH, whereas, the mass spectrum of 8a
1
of the ethoxide moiety. H-NMR spectra of 6b–g showed two displayed a molecular ion peak at m/z 481.
singlet signals at δ 4.86–4.93 and δ 8.61–8.65ppm indicating
Cyclization of the latter with excess thionyl chloride fur-
CH2 and C2 protons, respectively. Also, three exchangeable nished the thienopyrimidoquinazolinones 9a,b. IR spectra
singlet signals appeared at δ 9.21–10.46, 10.19–11.35 and showed two absorption bands at the range 3432–3224cm−1 due

640
Vol. 61, No. 6
1
to two NH groups. H-NMR spectrum lacked the presence of tion was plotted to obtain the survival curve of MCF7. The
the signals corresponding to NH, OH protons of 8a,b. Mass response parameter calculated was the IC50 value, which cor-
spectrum of 9b demonstrated three molecular ion peaks at m/z responds to the concentration required for 50% inhibition of
478 (M+1┐+ ·), 477 (M┐+ ·) and 476 (M-1┐+ ·).
cell viability. It was observed from the results obtained that
Biological Evaluation The antitumor screening of novel majority of the test compounds showed significant cytotoxic
synthesized compounds was carried in National Center Insti- activities against MCF7 with IC50 values ranging between
tute (NCI), Department of Cancer Biology, Cairo University, 2.04 and 35.36nm (Fig. 8). Eight compounds 4a,b, 5a,b, 8a,b
Cairo, Egypt.
and 9a,b exhibited enhanced antitumor activities compared
Preliminary in Vitro Antitumor Screening Most of the with that of DOX. Compounds 7a (IC50=8.21 nm) was found
newly synthesized compounds were evaluated for their in to be nearly as active as DOX. Compounds 6a, 6e and 7b
vitro cytotoxic activity against human breast cancer cell line showed a moderate activity, while compounds 6b–d and 6f,g
MCF7. DOX was one of the most effective anticancer agents were less active than the reference drug.
and therefore was used as a reference drug in this study. The
SAR Structurally, the biologically screened molecules be-
relationship between surviving fraction and drug concentra- long to a structure category of compounds with thienopyrimi-
dine heterocyclic core that have hydrogen bond acceptor donor
pairs at 6 and 7 positions but with different functional groups
decoration at 3 and 4 positions These molecules are classified
to four types of thienopyrimidine derivatives: first, 4-amino-
thienopyrimidine derivatives, 4a,b and their 4-phenylamino
analogues 8a,b; second, rigid derivatives 9a,b which are the
structural mimics of 8a,b; third, 4-chlorothienopyrimidines
7a,b, thienopyrimidine-4-ones 5a,b and N-alkyl thienopyrim-
idine-4-ones 6a–g. Examining the activity of the molecules,
we can deduce that 4-aminothienopyrimidine derivatives 4a,b
represent the most potent cytotoxic molecules, especially 4a
with an IC50 of 2.04nm. Substitution of amino group with
Fig. 6. Synthesis of 4-Chlorothienopyrimidines 7a,b
Fig. 5. Synthesis of N-Alkyl Thienopyrimidine-4-ones 6a–g
Fig. 7. Synthesis of 4-Substituted Phenylaminothienopyrimidines 8a,b and Thienopyrimidoquinazolinones 9a,b

June 2013
641
Fig. 8. Results of the in Vitro Cytotoxic Activity of the Synthesized Molecules and DOX against MCF7 Cells
Table 1. Results of the in Vitro Cytotoxic Activity of 4a,b, 8a,b and Table 2. Results of the in Vitro Cytotoxic Activity of 9a,b against
7a,b against MCF7 Cells MCF7 Cells
a)
Cpd. No.
R
R1
IC50 (nm )
a)
Cpd. No.
R
IC50 (nm )
4a
4b
7a
7b
8a
8b
H–
CH3–
H–
CH3–
H–
–NH2
–NH2
–Cl
2.04 0.10
4.28 0.18
8.21 011
10.35 0.15
3.34 0.16
4.76 0.15
9a
9b
H–
CH3–
4.96 0.14
5.91 0.11
–Cl
a) The values given are means of three experiments.
–NHC6H4COOH
–NHC6H4COOH
CH3–
ing suggested explanations, the compounds may be targeted
both the mentioned modes of actions for the reported leads
molecules as intercalators (5a–g and 9a,b) or kinase inhibitors
a) The values given are means of three experiments.
aromatic carboxylic acid moiety slightly diminished its activ- (4a,b, 7a,b and 8a,b). Further studies are still needed to de-
ity (4a,b compared with 8a,b). Moreover, replacement of the termine the exact mechanism of the antitumor action as well
amino group with chloride radical 7a,b decreased the activity as to explore the SAR of other positions of the nucleus.
(Table 1).
Cyclization of 4-substituted aminothienopyrimidines 8a,b
afforded mimic structures 9a,b and increased the activity,
probably due geometrical similarity with DOX (Table 2).
Conclusion
In summary, a series of thieno[3,2-d]pyrimidine derivatives
were designed and synthesized as small synthetic molecules
Thienopyrimidinones 5a,b displayed a potent activity, al- with antitumor activity. The results of cytotoxic screening
though extending the structure of 5a,b by a polar chain was were in good agreement with our design rationale except com-
hypothesized to increase the activity by increasing the number pounds 6a–g as the additional hydrogen bond formation moi-
of hydrogen bonding sites in the molecules, our results were ety introduced in 3 position of the fused ring decreased their
to the contrary of this hypothesis, where the substitution of activity. All the target molecules were evaluated for their in
5a or 5b with ethyl or alkyl aryl amide radicals to produce vitro cytotoxicity against MCF7. The cytotoxicity results con-
3-alkyl thienopyrimidinones 6a–g decreased the cytotoxic ac- cluded that there are three levels of activity compared with the
tivity. Order of activity for alkyl substitution 4-pyrimidinones reference, compounds 4a,b, 5a,b, 8a,b and 9a,b, exhibited
was H>C2H5>CH2CONHPh (Table 3).
significant cytotoxic activity which was even higher than that
Substituting by methyl group on p-position of phenyl amide of DOX. Compound 4a was the most potent with IC50 2.04nm.
moiety at 7 position decreased activity. The cytotoxic activity Compound 7a was equipotent as DOX. Compounds 6a, 6e and
of the synthesized molecules may be attributed to the follow- 7b showed moderate activity. Compounds 6b–d and 6f,g were

642
Vol. 61, No. 6
Table 3. Results of the in Vitro Cytotoxic Activity of 5a,b and 6a–g
against MCF7 Cells
dried, crystallized from ethanol to give 2a–d.
2-Cyano-3-cyanomethylthio-N-phenyl-3-(phenylamino)-
acrylamide (2a): Yield, 0.07g (84%); mp 186–187°C; Anal.
Calcd for C18H14N4OS (334.39): C, 64.65; H, 4.22; N, 16.75.
Found: C, 64.90; H, 4.33; N, 16.94. IR (KBr, cm−1): 3404–3296
(2NH), 2181 (C≡N), 1636 (C=O); 1H-NMR (DMSO-d6) δ:
4.00 (s, 2H, CH2), 6.97–7.28 (m, 3H, ArH), 7.30–7.44 (m, 3H,
ArH), 7.55–7.66 (m, 2H, ArH), 7.69 (d, J=2.7Hz, 2H, ArH),
8.90 (s, 1H, NH, D2O exchangeable), 10.18 (s, 1H, NH–CO,
D2O exchangeable).
a)
Cpd. No.
R
R1
IC50 (nm )
5a
5b
6a
6b
6c
H–
CH3–
CH3–
H–
H–
H–
CH3CH2–
4.08 0.08
4.62 0.12
10.41 0.11
15.38 0.15
20.14 0.14
2-Cyano-3-cyanomethylthio-3-(phenylamino)-N-p-tolyl
Acrylamide (2b): Yield, 0.08g (92%); mp 180–182°C; Anal.
Calcd for C19H16N4OS (348.42): C, 65.50; H, 4.63; N, 16.08.
Found: C, 65.26; H, 4.36; N, 15.67. IR (KBr, cm−1): 3411–3255
(2NH), 2183 (C≡N), 1645 (C=O); 1H-NMR (DMSO-d6)
δ: 2.24 (s, 3H, CH3 of p-tolyl moiety), 3.99 (s, 2H, CH2),
7.05–7.10 (m, 2H, ArH), 7.37–7.44 (m, 3H, ArH), 7.52–7.54 (m,
2H, ArH), 7.64 (d, J=2.7Hz, 2H, ArH), 8.80 (s, 1H, NH, D2O
C6H5–NHCOCH2–
4-CH3–C6H4–
NHCOCH2–
C6H5–NHCOCH2–
4-OH–C6H4–
NHCOCH2–
4-CH3–C6H4–
NHCOCH2–
4-OH–C6H4–
NHCOCH2–
H–
6d
6e
CH3–
H–
22.75 0.15
10.13 0.13
exchangeable), 10.16 (s, 1H, NH–CO, D2O exchangeable); MS
6f
CH3–
CH3–
35.36 0.16
13.43 0.13
┐+ ·
m/z (%): 349 [M+1┐+ ·, 2.54], 348 [M┐+ ·, 8.23], 77 [C6H5
100].
,
6g
(2-Cyano-1-phenylamino-2-phenylcarbamoyl-vinylsulfanyl)-
acetic Acid Ethyl Ester (2c): Yield, 0.08g (84%); mp
202–204°C; Anal. Calcd for C20H19N3O3S (381.45): C, 62.97;
a) The values given are means of three experiments.
less active DOX. The study suggested that the newly syn- H, 5.02; N, 11.02; Found: C, 63.26; H, 5.08; N, 11.33. IR
thesized molecules are potential antitumor agents but further (KBr, cm−1): 3442–3345 (2NH), 2200 (C≡N), 1728–1656
1
studies are needed to investigate the mechanism of action of (2C=O). H-NMR (DMSO-d6) δ: 1.13–1.18 (t, J=6.9Hz, 3H,
these cytotoxic molecules.
–OCH2CH3), 3.64 (s, 2H, CH2), 4.00–4.08 (q, J=6.9Hz, 2H,
–OCH2CH3), 7.04–7.09 (m, 2H, ArH), 7.18–7.23 (m, 1H, ArH),
7.26–7.46 (m, 3H, ArH), 7.51 (d, J=7.8Hz, 2H, ArH), 7.55
Experimental
Chemistry Melting points (°C, uncorrected) were deter- (d, J=7.8Hz, 2H, ArH), 9.58 (s, 1H, NH, D2O exchangeable),
mined in open capillaries on a Graffin melting point appara- 11.34 (s, 1H, NH, D2O exchangeable); MS m/z (%): 382 [M+
tus. IR spectra were determined as KBr discs on Shimadzu IR
1
┐+ ·, 13.90], 381 [M┐+ ·, 28.60], 77 [100].
(2-Cyano-1-phenylamino-2-p-tolyl carbamoyl-vinylsulfanyl)-
435 Spectrophotometer and values were represented in cm−1.
1H-NMR spectra (in DMSO-d6) were carried out on Varian acetic Acid Ethyl Ester (2d): Yield, 0.07g (71%); mp 200–
Gemini 300 MHZ Spectrophotometer, at nuclear magnetic 202°C; Anal. Calcd for C21H21N3O3S (395.47): C, 63.78; H,
resonance, Cairo University, Egypt, using TMS as internal 5.35; N, 10.63; Found: C, 63.91; H, 5.49; N, 10.85; IR (KBr,
standard and chemical shifts were recorded in ppm on δ cm−1): 3412–3340 (2NH), 2192 (C≡N), 1745–1654 (2C=
scale.13C-NMR were carried out on Bruker AC 200 (200MHz) O); 1H-NMR (DMSO-d6) δ: 1.13–1.17 (t, J=7.2Hz, 3H,
spectrometer, Institute for synthetic chemistry, Vienna. Uni- –OCH2CH3) 2.24 (s, 3H, CH3 of p-tolyl moiety), 3.62 (s, 2H,
versity of Technology, Vienna, Austria Electron impact Mass CH2), 3.99–4.07 (q, J=7.2Hz, 2H, –OCH2CH3), 7.05–7.19 (m,
Spectra were recorded on Hewlett Packard 5988 Spectrom- 3H, ArH), 7.28–7.41 (m, 4H, ArH), 7.49–7.52 (t, J=8.4Hz,
eter. Element analysis was carried out at the Micro analytical 2H, ArH), 9.50 (s, 1H, NH, D2O exchangeable), 11.42 (s, 1H,
Center, Cairo University, Giza, Egypt. All compounds were NH, D2O exchangeable); MS m/z (%): 396 [M+1┐+ ·, 8.40], 395
within 0.5% of the theoretical values. Pre-coated silica gel [M┐+ ·, 19.50], 215 [100].
plates (silica gel 0.25mm, MERCK 60F 254, Germany) were
4-Amino-N-aryl-5-substituted-2-phenylaminothiophene-
used for thin layer chromatography, benzene–ethyl acetate 3-carboxamides (3a–d) General Procedure. Method A: To
(8.5:1.5mL) mixture was used as a developing solvent system a well stirred sodium ethoxide solution (prepared from sodium
and the spots were visualized by UV lamp. Compounds 1a,b (0.46g, 0.02mol) in 20mL absolute ethanol), N-aryl-2-cyano-
and 3a are synthesized according to reported methods.37,47)
3-phenylamino-3-(substituted methylthio)-acrylamides 2a–d
N-Aryl-2-cyano-3-phenylamino-3-(substituted methylthio)- (0.005mol) was added. The reaction mixture was heated under
acrylamides (2a–d) General Procedure: To a cold suspen- reflux for half an hour and set aside to cool at room tempera-
sion of finely grinded KOH (1.4g, 0.025mol) in DMF (20mL), ture. The separated solid was filtered, washed with ethanol
N-aryl-2-cyanoacetamides 1a or 1b (0.025mol) was added and and crystallized from ethanol to give 3a–d.
the mixture was stirred at room temperature for 3h. Phenyl
Method B: An ethanolic solution of sodium ethoxide (pre-
isothiocyanate (3.4g, 0.025mol) was added and the mixture pared from sodium (2.3g, 0.1mol) in 40mL absolute ethanol)
was stirred at room temperature for other 3h. The active was added dropwise to a well stirred solution of N-aryl-2-cy-
methylene chloro derivative (0.025mol) was then added and anoacetamides 1a or 1b (0.1mol) in absolute ethanol (20mL).
the mixture was left on stirring for additional 24h at room After complete addition, the reaction mixture was stirred
temperature. The mixture was triturated with cold water (ca. at room temperature for 2h. Phenyl isothiocyanate (13.5g,
50mL). The resultant solid product was collected by filtration, 0.1mol) was added and the mixture was heated on a water-

June 2013
643
bath at 70°C for half an hour then cooled to 25°C. Chloroace- 128.9 (s), 129.1 (s), 134.4 (s), 137.3 (s), 140.2 (s), 145.8 (s), 154.2
tonitrile (7.55g, 0.1mol) was then added, and the temperature (s), 160.2 (s), 163.2 (s), 166.8 (s).
was raised to 60°C for half an hour more, followed by addition
4-Amino-6-phenylamino-N-p-tolyl Thieno[3,2-d]pyrimidine-
of an ethanolic solution of sodium ethoxide (prepared from 7-carboxamide (4b): Yield, 22.50g (60%); mp 280–282°C;
sodium (1.15g, 0.05mol) in 10mL absolute ethanol) and heat- Anal, Calcd for C20H17N5OS (375.45): C, 63.98; H, 4.56; N,
ing was continued for additional 3h. After cooling, water was 18.65; Found: C, 63.90; H, 4.40; N, 18.41. IR (KBr, cm−1):
1
added and the separated product was filtered and crystallized 3422–3220 (2NH, NH2), 1644 (C=O); H-NMR (DMSO-d6)
from ethanol to afford 3a,b.
δ: 2.29 (s, 3H, CH3 of p-tolyl moiety), 7.17–7.20 (m, 3H, ArH),
4-Amino-5-cyano-N-p-tolyl-2-phenylaminothiophene-3-car- 7.26–7.33 (m, 4H, ArH+NH2, D2O exchangeable), 7.49–7.51
boxamide (3b): Yield, 26.20g (75%); mp 280–282°C; Anal. (m, 2H, ArH), 7.61 (d, J=8.4Hz, 2H, ArH), 8.45 (s, 1H,
Calcd for C19H16N4OS (348.42): C, 65.50; H, 4.63; N, 16.08; C2-H), 11.40 (s, 1H, NH, D2O exchangeable), 11.95 (s, 1H,
Found: C, 65.61; H, 4.42; N, 16.46. IR (KBr, cm−1): 3356–3224 NHCO, D2O exchangeable); MS m/z (%): 376 [M+1┐+ ·, 9.89],
1
(2NH, NH2), 2186 (C≡N), 1644 (C=O); H-NMR (DMSO-d6) 375 [M┐+ ·, 21.00], 107 [100].
δ: 2.25 (s, 3H, CH3 of p-tolyl moiety), 6.41 (s, 2H, NH2, D2O
N-Aryl-4-oxo-6-phenylamino-3,4-dihydrothieno[3,2-d]-
exchangeable), 7.06–7.11 (m, 3H, ArH), 7.26–7.37 (m, 4H, pyrimidine-7-carboxamides (5a,b) General Procedure: A
ArH), 7.47 (d, J=8.1Hz, 2H, ArH), 9.66 (s, 1H, NH, D2O ex- suspension of ethyl 3-amino-4-arylcarbamoyl-5-phenylamino-
changeable), 9.72 (s, 1H, NH–CO, D2O exchangeable); MS m/z thiophene-2-carboxylates 3c or 3d (0.01mol) and formamide
(%): 349 [M+1┐+ ·, 20.50], 348 [M┐+ ·, 82.60], 107 [C7H9N┐+ ·
100].
,
(30mL) was heated under reflux for 8h in an oil bath at
160–180°C. After cooling to room temperature, the reaction
3-Amino-5-phenylamino-4-phenyl Carbamoyl Thiophene- mixture was poured onto ice-cold water (ca. 80mL) and the
2-carboxylic Acid Ethyl Ester (3c): Yield, 24.22g (63%); mp formed precipitate was collected by filtration, washed with
245–247°C; Anal. Calcd for C20H19N3O3S (381.45): C, 62.97; water, dried and crystallized from chloroform to afford 5a,b.
H, 5.02; N, 11.02; Found: C, 62.87; H, 5.39; N, 11.24. IR (KBr,
4-Oxo-N-phenyl-6-phenylamino-3,4-dihydrothieno[3,2-d]-
1
cm−1): 3356–3316 (2NH, NH2), 1663–1638 (2C=O); H-NMR pyrimidine-7-carboxamide (5a): Yield, 2.16g (60%); mp
(DMSO-d6) δ: 1.19–1.23 (t, J=6.9Hz, 3H, –OCH2CH3), 257–259°C; Anal. Calcd for C19H14N4O2S (362.41): C, 62.97; H,
4.11–4.18 (q, J=6.9Hz, 2H, –OCH2CH3), 6.68 (s, 2H, NH2, 3.89; N, 15.46; Found: C, 63.02; H, 4.19; N, 15.26. IR (KBr,
D2O exchangeable), 7.03–7.10 (m, 3H, ArH), 7.28–7.39 (m, 5H, cm−1): 3438–3232 (2NH), 1642 (2C=O); H-NMR (DMSO-d6)
1
ArH), 7.62 (d, J=7.5Hz, 2H, ArH), 9.69 (s, 1H, NH, D2O ex- δ: 7.08–7.13 (m, 2H, ArH), 7.21–7.26 (m, 1H, ArH), 7.35–7.39
changeable), 9.72 (s, 1H, NH–CO, D2O exchangeable); MS m/z (m, 2H, ArH), 7.49–7.51 (m, 2H, ArH), 7.71 (d, J=8.7Hz, 2H,
(%): 382 [M+1┐+ ·, 18.60], 381 [M┐+ ·, 67.70], 242 [100].
ArH), 8.39 (s, 1H, C2-H), 11.43 (s, 1H, NH, D2O exchange-
3-Amino-5-phenylamino-4-p-tolyl Carbamoyl Thiophene- able), 11.53 (s, 1H, NH–CO, D2O exchangeable), 12.69 (s,
2-carboxylic Acid Ethyl Ester (3d): Yield, 27.42g (61%); mp 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ: 117.3
258–260°C; Anal. Calcd for C21H21N3O3S (395.47): C, 63.78; (s), 120.9 (s), 123.2 (s), 126.8 (s), 128.0 (s), 129.1 (s), 130.5 (s),
H, 5.35; N, 10.63; Found: C, 63.90; H, 5.00; N, 10.51. IR (KBr, 137.3 (s), 140.2 (s), 144.5 (s), 147.1 (s), 155.1 (s), 160.2 (s), 161.8
1
cm−1): 3340–3262 (2NH, NH2), 1667–1625 (2C=O); H-NMR (s), 165.4 (s); MS m/z (%): 363 [M+1┐+ ·, 10.82], 362 [M┐+ ·
,
(DMSO-d6) δ: 1.19–1.23 (t, J=6.9Hz, 3H, –OCH2CH3), 2.26 46.73], 269 [100].
(s, 3H, CH3 of p-tolyl moiety), 4.11–4.18 (q, J=7.2Hz, 2H,
4-Oxo-6-phenylamino-N-p-tolyl-3,4-dihydrothieno[3,2-d]-
–OCH2CH3), 6.68 (s, 2H, NH2, D2O exchangeable), 7.09–7.12 pyrimidine-7-carboxamide (5b): Yield, 2.45g (65%); mp
(m, 3H, ArH), 7.30–7.39 (m, 4H, ArH), 7.50 (d, J=8.4Hz, 2H, 262–264°C; Anal. Calcd for C20H16N4O2S (376.43): C, 63.81;
H2,6), 9.63 (s, 1H, NH, D2O exchangeable), 9.69 (s, 1H, NH– H, 4.28; N, 14.88; Found: C, 64.09; H, 4.31; N, 14.61. IR (KBr,
1
CO, D2O exchangeable); MS m/z (%): 397 [M+2┐+ ·, 11.50], cm−1): 3436–3236 (2NH), 1639 (2C=O); H-NMR (DMSO-d6)
396 [M+1┐+ ·, 16.20], 395 [M┐+ ·, 82.00], 242 [100].
δ: 2.30 (s, 3H, CH3 of p-tolyl moiety), 7.18–7.24 (m, 3H, ArH),
4-Amino-N-aryl-6-phenylaminothieno[3,2-d]pyrimidine- 7.48–7.50 (m, 4H, ArH), 7.58 (d, J=8.7Hz, 2H, ArH), 8.36
7-carboxamides (4a,b) General Procedure: A mixture of (s, 1H, C2-H), 11.43 (s, 1H, NH, D2O exchangeable), 11.47
4-amino-N-aryl-5-cyano-2-phenylamino-thiophene-3-carbox- (s, 1H, NH–CO, D2O exchangeable), 12.61 (s, 1H, NH, D2O
amides 3a or 3b (0.1mol) and formamide (30mL) was heated exchangeable) 13C-NMR (DMSO-d6) δ: 14.2 (q, CH3), 116.3
under reflux for 2h in an oil bath at 160°C. After cooling (s), 119.5 (s), 123.2 (s), 126.1 (s), 127.9 (s), 128.3 (s), 130.9 (s),
to room temperature, the reaction mixture was poured onto 138.4 (s), 140.9 (s), 144.1 (s), 147.6 (s), 156.6 (s), 161.0 (s), 162.1
ice-cold water (ca. 80mL) and the formed precipitate was col- (s), 167.9 (s).
lected by filtration, washed with water, dried and crystallized
from the proper solvent to give 4a,b.
N-Aryl-4-oxo-3-substituted-6-phenylaminothieno[3,2-d]-
pyrimidine-7-carboxamides (6a–g) General Procedure:
4-Amino-N-phenyl-6-phenylaminothieno[3,2-d]pyrimidine- To a solution of potassium hydroxide (1.12g, 0.02mol) in
7-carboxamide (4a): Yield, 23.50g (65%); mp 297–299°C; absolute ethanol (20mL), N-aryl-4-oxo-6-phenylamino-3,4-
Anal. Calcd for C19H15N5OS (361.42): C, 63.14; H, 4.18; N, dihydrothieno[3,2-d]pyrimidine-7-carboxamides 5a or 5b
19.38; Found: C, 63.00; H, 4.00; N, 19.01. IR (KBr, cm−1): (0.001mol) was added and the mixture was left on stirring
1
3450–3250 (2NH, NH2), 1687 (C=O); H-NMR (DMSO-d6) at room temperature for 2h. The appropriate alkyl halide
δ: 7.08–7.12 (m, 3H, ArH), 7.21–7.35 (m, 5H, ArH+NH2, D2O (0.02mol) was then added and the reaction mixture was heat-
exchangeable), 7.37–7.51 (m, 2H, ArH), 7.72 (d, J=8.4Hz, 2H, ed under reflux for 20h. After cooling to room temperature,
ArH), 8.43 (s, 1H, C2-H), 11.38 (s, 1H, NH, D2O exchange- the reaction mixture was poured onto crushed ice (20g). The
able), 12.08 (s, 1H, NHCO, D2O exchangeable); 13C-NMR precipitated solid was filtered, washed with water, dried and
(DMSO-d6) δ: 116.3 (s), 119.2 (s), 122.5 (s), 123.5 (s), 125.6 (s), crystallized from the proper solvent to yield 6a–g.

644
Vol. 61, No. 6
3-Ethyl-4-oxo-6-phenylamino-N-p-tolyl-3,4-dihydro- ArH), 8.62 (s, 1H, C2-H), 9.21 (s, 1H, NH, D2O exchangeable),
thieno[3,2-d]pyrimidine-7-carboxamide (6a): Yield, 0.23g 10.18 (s, 1H, NH–CO, D2O exchangeable), 11.34 (s, 1H, NH–
(57%); mp 270–272°C; Anal. Calcd for C22H20N4O2S (404.48): CO, D2O exchangeable), 11.45 (s, 1H, OH, D2O exchangeable);
C, 65.33; H, 4.98; N, 13.85; Found: C, 65.29; H, 4.90; N, 14.10. 13C-NMR (DMSO-d6) δ: 50.6 (t, CH2), 114.3 (s), 117.5 (s),
IR (KBr, cm−1): 3395–3264 (2NH), 1648 (C=O); 1H-NMR 120.3 (s), 123.1 (s), 125.1 (s), 126.9 (s), 127.3 (s), 130.1 (s), 131.1
(DMSO-d6) δ: 1.28–1.32 (t, J=6.3Hz, 3H, –OCH2CH3), (s), 131.9 (s), 138.3 (s), 140.6 (s), 143.2 (s), 147.1 (s), 155.8 (s),
2.28 (s, 3H, CH3 of p-tolyl moiety), 4.01–4.03 (q, J=6.3Hz, 156.2 (s), 160.9 (s), 161.7 (s), 165.2 (s), 167.1 (s).
2H, –OCH2CH3), 7.16–7.23 (m, 5H, ArH), 7.46–7.51 (m, 2H,
4-Oxo-6-phenylamino-N-p-tolyl-3-(p-tolylcarbamoyl-
ArH), 7.55 (d, J=6.9Hz, 2H, ArH), 8.65 (s, 1H, C2-H), 11.23 methyl)-3,4-dihydrothieno[3,2-d]pyrimidine-7-carboxamide
(s, 1H, NH, D2O exchangeable), 11.44 (s, 1H, NH–CO, D2O (6f): Yield, 0.38g (73%); mp >300°C; Anal. Calcd for
exchangeable); 13C-NMR (DMSO-d6) δ: 13.4 (q, CH3), 20.3 C29H25N5O3S (523.61): C, 66.52; H, 4.81; N, 13.38; Found: C,
(q, CH3), 36.3 (t, CH2), 115.9 (s), 119.3 (s), 122.9 (s), 126.5 (s), 66.29; H, 5.06; N, 13.14. IR (KBr, cm−1): 3453–3200 (3NH),
1
126.9 (s), 129.3 (s), 131.2 (s), 138.2 (s), 140.6 (s), 143.2 (s), 147.4 1665–1644 (2C=O); H-NMR (DMSO-d6) δ: 2.24 (s, 3H, CH3
(s), 156.5 (s), 160.9 (s), 161.7 (s), 166.4 (s); MS m/z (%): 405 of p-tolyl moiety), 2.28 (s, 3H, CH3 of p-tolyl moiety), 4.89 (s,
[M+1┐+ ·, 5.73], 404 [M┐+ ·, 19.90], 107 [100].
2H, CH2), 7.10–7.26 (m, 5H, ArH), 7.45–7.48 (m, 4H, ArH),
4-Oxo-N-phenyl-6-phenylamino-3-phenylcarbamoyl- 7.59 (d, J=8.1Hz, 4H, ArH), 8.61 (s, 1H, C2-H), 10.36 (s, 1H,
methyl-3,4-dihydro-thieno[3,2-d]pyrimidine-7-carboxamide NH), 11.24 (s, 1H, NH), 11.50 (s, 1H, NH).
(6b): Yield, 0.38g (78%); mp 354–356°C; Anal. Calcd for
3-(4 -Hyd roxy phenylcarbamoylmethyl)- 4 -oxo- 6 -
C27H21N5O3S (495.55): C, 65.44; H, 4.27; N, 14.13; Found: C, phenylamino-N-p-tolyl-3,4-dihydrothieno[3,2-d]pyrimidine-7-
65.22; H, 4.42; N, 13.77. IR (KBr, cm−1): 3421–3276 (3NH), carboxamide (6g): Yield, 0.39g (74%); mp 298–300°C; Anal.
1670–1647 (2C=O); 1H-NMR (DMSO-d6) δ: 4.93 (s, 2H, Calcd for C28H23N5O4S (525.58): C, 63.99; H, 4.41; N, 13.33;
CH2), 7.05–7.16 (m, 3H, ArH), 7.24–7.42 (m, 6H, ArH), 7.50 Found: C, 64.37; H, 4.48; N, 13.33; IR (KBr, cm−1): 3515 (OH),
(d, J=4.2Hz, 2H, ArH), 7.59 (d, J=8.1Hz, 2H, ArH), 7.73 (d, 3401–3270 (3NH), 1665–1640 (2C=O); 1H-NMR (DMSO-
J=7.8Hz, 2H, ArH), 8.65 (s, 1H, C2-H), 10.46 (s, 1H, NH, d6) δ: 2.30 (s, 3H, CH3 of p-tolyl moiety), 4.87 (s, 2H, CH2),
D2O exchangeable), 11.35 (s, 1H, NH–CO, D2O exchangeable), 6.70 (d, J=8.7Hz, 2H, ArH), 7.19–7.28 (m, 5H, ArH), 7.36
11.46 (s, 1H, NH–CO, D2O exchangeable); MS m/z (%): 496 (d, J=8.7Hz, 2H, ArH), 7.47–7.51 (m, 2H, ArH), 7.62 (d,
[M+1┐+ ·, 14.66], 495 [M┐+ ·, 46.17], 269 [100].
J=8.7Hz, 2H, ArH), 8.63 (s, 1H, C2-H), 9.22 (s, 1H, NH,
4-Oxo-N-phenyl-6-phenylamino-3-(p-tolylcarbamoyl- D2O exchangeable), 10.19 (s, 1H, NH–CO, D2O exchangeable),
methyl)-3,4-dihydrothieno[3,2-d]pyrimidine-7-carboxamide 11.28 (s, 1H, NH-CO, D2O exchangeable), 11.49 (s, 1H, OH,
(6c): Yield, 0.32g (63%); mp >300°C; Anal. Calcd for D2O exchangeable); MS m/z (%): 525 [M┐+ ·, 16.97] and 283
C28H23N5O3S (509.58): C, 66.00; H, 4.55; N, 13.74; Found: C, [100].
66.03; H, 4.26; N, 13.67. IR (KBr, cm−1): 3408–3281 (3NH),
N-Aryl-4-chloro-6-phenylaminothieno[3,2-d]pyrimidine-
1665–1648 (2C=O); 1H-NMR (DMSO-d6) δ: 2.24 (s, 3H, 7-carboxamides (7a,b) General Procedure:
A
mix-
CH3 of p-tolyl moiety), 4.90 (s, 2H, CH2), 7.10–7.15 (m, 3H, ture of phosphorus oxychloride (15mL) and N-aryl-4-
ArH), 7.23–7.26 (m, 1H, ArH), 7.36–7.49 (m, 8H, ArH), 7.72 oxo-6-phenylamino-3,4-dihydrothieno[3,2-d]pyrimidine-7-
(d, J=7.8Hz, 2H, ArH), 8.63 (s, 1H, C2-H), 10.36 (s, 1H, NH, carboxamides 5a or 5b (0.001mol) and was heated under
D2O exchangeable), 11.34 (s, 1H, NH–CO, D2O exchangeable), reflux in an oil bath at 120°C for 8h. Excess phosphorus
11.46 (s, 1H, NH–CO); 13C-NMR (DMSO-d6) δ: 20.9 (q, CH3), oxychloride was distilled off under vacuum and the residual
52.3 (t, CH2), 115.9 (s), 118.1 (s), 119.4 (s), 121.9 (s), 125.4 (s), syrup was poured while stirring over crushed ice. The formed
126.6 (s), 127.0 (s), 129.6 (s), 131.4 (s), 132.2 (s), 139.1 (s), 141.3 precipitate was collected by filtration, washed with water,
(s), 143.5 (s), 147.8 (s), 155.4 (s), 156.9 (s), 161.2 (s), 162.3 (s), dried and crystallized from benzene to afford 7a,b.
166.1 (s), 167.8 (s); MS m/z (%): 509 [M┐+ ·, 42.20], 310 [100].
4-Chloro-N-phenyl-6-phenylaminothieno[3,2-d]pyrimidine-
4-Oxo-6-phenylamino-3-phenylcarbamoylmethyl-N-p- 7-carboxamide (7a): Yield, 0.26g (68%); mp 182–184°C; Anal.
tolyl-3,4-dihydrothieno[3,2-d]pyrimidine-7-carboxamide (6d): Calcd for C19H13ClN4OS (380.85): C, 59.92; H, 3.44; N, 14.71;
Yield, 0.37g (73%); mp 298–300°C; Anal. Calcd for Found: C, 60.02; H, 3.70; N, 15.00. IR (KBr, cm−1): 3413–3249
C28H23N5O3S (509.58): C, 66.00; H, 4.55; N, 13.74; Found: C, (2NH), 1661 (C=O); 1H-NMR (DMSO-d6) δ: 7.23–7.34 (m,
65.98; H, 4.65; N, 13.51. IR (KBr, cm−1): 3436–3279 (3NH), 2H, ArH), 7.36–7.47 (m, 2H, ArH), 7.62 (d, J=8.4Hz, 2H,
1
1670–1645 (2C=O); H-NMR (DMSO-d6) δ: 2.30 (s, 3H, CH3 ArH), 7.74–7.79 (m, 2H, ArH), 7.95 (d, J=8.4Hz, 2H, ArH),
of p-tolyl moiety), 4.93 (s, 2H, CH2), 7.07–7.10 (t, J=7.8Hz, 9.11 (s, 1H, C2-H), 10.42 (s, 2H, 2NH, D2O exchangeable);
2H, ArH), 7.19–7.35 (m, 6H, ArH), 7.49–7.51 (m, 2H, ArH), 13C-NMR (DMSO-d6) δ: 117.3 (s), 120.9 (s), 122.5 (s), 123.2
7.57–7.63 (m, 4H, ArH), 8.65 (s, 1H, C2-H), 10.45 (s, 1H, NH, (s), 126.5 (s), 127.9 (s), 129.1 (s), 134.4 (s), 137.3 (s), 140. 2(s),
D2O exchangeable), 11.28 (s, 1H, NH–CO, D2O exchangeable), 145.8 (s), 154.2 (s), 160.2 (s), 163.2 (s), 166.2 (s); MS m/z (%):
11.50 (s, 1H, NH–CO, D2O exchangeable).
380 [M┐+ ·, 9.52], 362 [100].
3-(4-Hydroxyphenylcarbamoylmethyl)-4-oxo-N-phenyl-
4-Chloro-6-phenylamino-N-p-tolylthieno[3,2-d]pyrimidine-
6-phenylamino-3,4-dihydrothieno[3,2-d]pyrimidine-7- 7-carboxamide (7b): Yield, 0.23g (59%); mp 160–162°C; Anal.
carboxamide (6e): Yield, 0.36g (70%); mp 337–9°C; Anal. Calcd for C20H15ClN4OS (394.88): C, 60.83; H, 3.83; N, 14.19;
Calcd for C27H21N5O4S (511.55): C, 63.39; H, 4.14; N, 13.69; Found: C, 60.74; H, 4.09; N, 14.07. IR (KBr, cm−1): 3420–3245
Found: C, 63.51; H, 4.16; N, 13.38. IR (KBr, cm−1): 3510 (OH), (2NH), 1680 (C=O); 1H-NMR (DMSO-d6) δ: 2.36 (s, 3H,
1
3399–3279 (3NH), 1660–1643 (2C=O); H-NMR (DMSO-d6) CH3 of p-tolyl moiety), 7.22–7.27 (m, 3H, ArH), 7.60 (m, 2H,
δ: 4.86 (s, 2H, CH2), 6.69 (d, J=7.2Hz, 2H, ArH), 7.10–7.25 ArH), 7.75 (m, 2H, ArH), 7.94 (d, J=8.4Hz, 2H, ArH), 9.10 (s,
(m, 4H, ArH), 7.34–7.48 (m, 6H, ArH), 7.71 (d, J=7.2Hz, 2H, 1H, C2-H), 10.43 (s, 2H, 2NH, D2O exchangeable); 13C-NMR

June 2013
645
Table 4. The Surviving Fraction of MCF7 Cells for 4 Concentrations of Each Compound (µg/mL) and Their IC50 (µg/mL)
The surviving fraction at each concentration (µg/mL)
Cpd. No.
IC50 (µg/mL)
1
2.5
5
10
DOX
4a
4b
5a
5b
6a
6b
6c
6d
6e
6f
6g
7a
7b
8a
8b
9a
9b
0.360 0.014
0.210 0.012
0.638 0.042
0.523 0.012
0.910 0.015
0.472 0.020
0.572 0.009
0.672 0.031
0.735 0.012
0.523 0.013
0.734 0.015
0.521 0.022
0.309 0.021
0.455 0.017
0.694 0.018
0.531 0.012
0.815 0.013
0.307 0.027
0.317 0.026
0.155 0.011
0.465 0.021
0.423 0.031
0.685 0.045
0.419 0.019
0.373 0.012
0.470 0.032
0.360 0.020
0.387 0.011
0.626 0.019
0.360 0.010
0.229 0.014
0.467 0.020
0.326 0.015
0.350 0.030
0.405 0.033
0.226 0.017
0.343 0.018
0.095 0.018
0.243 0.011
0.187 0.023
0.504 0.030
0.361 0.022
0.410 0.014
0.438 0.030
0.314 0.013
0.313 0.020
0.307 0.023
0.346 0.030
0.250 0.016
0.193 0.012
0.233 0.025
0.346 0.017
0.224 0.011
0.240 0.020
0.370 0.033
0.142 0.022
0.165 0.016
0.143 0.019
0.356 0.031
0.380 0.023
0.259 0.021
0.471 0.032
0.295 0.021
0.324 0.015
0.272 0.024
0.453 0.031
0.339 0.033
0.273 0.042
0.268 0.031
0.453 0.013
0.123 0.030
0.339 0.018
4.40 0.09
0.74 0.10
1.81 0.07
1.47 0.04
3.91 0.11
4.21 0.13
7.62 0.12
10.26 0.21
11.59 0.22
5.18 0.06
18.51 0.22
7.05 0.10
3.58 0.08
4.08 0.10
1.60 0.07
2.35 0.08
2.29 0.10
2.82 0.12
(DMSO-d6) δ: 20.4 (q, CH3), 115.9 (s), 120.3 (s), 123.1 (s), pyrimido[3,4-b]quinazolin-7-ones (9a,b) General Proce-
124.2 (s), 126.2 (s), 128.1 (s), 129.3 (s), 134.3 (s), 136.3 (s), dure: mixture of N-aryl-4-(2-carboxy-phenylamino)-6-
141.1 (s), 146.8 (s), 155.1 (s), 161.4 (s), 163.2 (s), 166.4 (s). phenylamino-thieno[3,2-d]pyrimidine-7-carboxamides 8a or
A
N-Aryl-4-(2-carboxy-phenylamino)-6-phenylaminothieno- 8b (0.01mol) and excess thionyl chloride (10mL) was heated
[3,2-d]pyrimidine-7-carboxamides (8a,b) General Pro- under reflux for 4h. Distillation of excess thionyl chloride
cedure:
A mixture of the respective N-aryl-4-chloro-6- under vacuum afforded the target compounds 9a,b which
phenylamino-thieno[3,2-d]pyrimidine-7-carboxamides 7a or were crystallized from the appropriate solvent.
7b (0.01mol), anthranilic acid (1.4g, 0.01mol) and anhydrous
2-Phenylamino-3-N-phenylcarbamoylthieno[3′,2′-4,5]-
potassium carbonate (1.4g, 0.01mol) in DMF (15mL) was pyrimido[3,4-b]quinazolin-7-one (9a): Yield, 2.50g (54%);
heated under reflux for 24h. After cooling at room tempera- mp >300°C; Anal. Calcd for C26H17N5O2S (463.51): C, 67.37;
ture, the reaction mixture was poured onto crushed ice (20g). H, 3.70, N, 15.11; Found: C, 67.20; H, 3.80; N, 14.86; IR
1
The precipitated solid was filtered, washed with water, dried (KBr, cm−1): 3413–3300 (2NH), 1650–1636 (2C=O); H-NMR
and crystallized from the proper solvent to yield 8a,b.
(DMSO-d6) δ: 7.14–7.21 (m, 4H, ArH), 7.32–7.37 (m, 2H,
4-(2-Carboxy-phenylamino)-N-phenyl-6-phenylaminothieno- ArH), 7.60–7.68 (m, 5H, ArH), 7.91–7.94 (m, 3H, ArH), 8.26
[3,2-d]pyrimidine-7-carboxamide (8a): Yield, 2.91g (61%); mp (s, 1H, C5-H), 10.74 (s, 2H, 2NH, D2O exchangeable).
>300°C; Anal. Calcd for C26H19N5O3S (481.53): C, 64.85; H,
2-Phenylamino-3-N-p-tolylcarbamoylthieno[3′,2′-4,5]-
3.98; N, 14.54; Found: C, 64.59; H, 3.63; N, 14.37. IR (KBr, pyrimido[3,4-b]quinazolin-7-one (9b): Yield, 2.32g (49%);
1
cm−1): 3545–3260 (OH, 3NH), 1679–1629 (2C=O); H-NMR mp >300°C; Anal. Calcd for C27H19N5O2S (477.54): C, 67.91;
(DMSO-d6) δ: 7.16–7.42 (m, 4H, ArH), 7.54–7.74 (m, 6H, H, 4.01; N, 14.67; Found: C, 68.20; H, 4.28; N, 14.73; IR
ArH), 7.93–7.98 (m, 4H, ArH), 8.45 (s, 2H, C2-H, NH, D2O (KBr, cm−1): 3432–3224 (2NH), 1660–1640 (2C=O); H-NMR
1
exchangeable), 10.23 (s, 2H, 2NH, D2O exchangeable), 13.09 (DMSO-d6) δ: 2.30 (s, 3H, CH3 of p-tolyl moiety), 7.04–7.20
(s, 1H, OH, D2O exchangeable); MS m/z: 481 [M┐+ ·, 10.97], (m, 5H, ArH), 7.59–7.65 (m, 4H, ArH), 7.89 (d, J=8.7Hz, 4H,
344 [100].
ArH), 8.22 (s, 1H, C5-H), 10.79 (s, 2H, 2NH, D2O exchange-
4-(2-Carboxy-phenylamino)-6-phenylamino-N-p-tolylthieno- able); MS m/z: 478 [M+1┐+ ·, 1.71], 477 [M┐+ ·, 5.20], 442 [100].
[3,2-d]pyrimidine-7-carboxamide (8b): Yield, 2.57g (52%); mp
280–282°C; Anal. Calcd for C27H21N5O3S (495.55): C, 65.44;
Biological Evaluation
Materials and Methods The human breast adenocar-
H, 4.27; N, 14.13; Found: C, 65.18; H, 4.15; N, 13.97; IR (KBr, cinoma cell line (MCF7) was obtained as a gift from NCI,
cm−1): 35453260 (OH, 3NH), 16801634 (2C=O); 1H-NMR MD, U.S.A. All chemicals and solvents were purchased
(DMSO-d6) δ: 2.30 (s, 3H, CH3 of p-tolyl moiety), 7.05 (d, from Sigma-Aldrich. The cytotoxicity calculated using trans-
J=8.4Hz, 2H, ArH), 7.147.20 (m, 3H, ArH), 7.557.65 (m, formed curves (Graph pad prism software, version 5.01, 2007,
6H, ArH), 7.89 (d, J=8.1Hz, 2H, ArH), 8.20 (s, 2H, C2-H+ GPW5-739310-RAG-3437).
NH, D2O exchangeable), 8.53 (s, 1H, NH, D2O exchangeable),
Measurement of Potential Cytotoxicity The cytotoxic
10.80 (s, 1H, NH, D2O exchangeable), 13.09 (s, 1H, OH, D2O activity of the newly synthesized compounds was measured
exchangeable); 13C-NMR (DMSO-d6) δ: 23.4 (q, CH3), 117.3 in vitro on MCF7 using Sulforhodamine-B stain (SRB) assay
(s), 119.1 (s), 120.2 (s), 120.9 (s), 122.5 (s), 123.2 (s), 126.5 (s), applying the method of Skehan et al.48) Cells were plated in
127.9 (s), 129.1 (s), 132.4 (s), 135.3 (s), 137.3 (s), 134.4 (s), 140.2 96-multiwell plate (104 cells/well) for 24h before treatment
(s), 145.3 (s), 145.8 (s), 154.2 (s), 160.2 (s), 163.2 (s), 167.2 (s).
with the test compounds to allow attachment of the cells
2-Phenylamino-3-N-arylcarbamoylthieno[3′,2′-4,5]- to the wall of the plate. Test compounds were dissolved in

646
Vol. 61, No. 6
Fig. 9. Survival Curve of MCF7 Cells at 4 Concentrations (µg/mL) for
4-Aminothienopyrimidine 4a
Fig. 11. Survival Curve of MCF7 Cells at 4 Concentrations (µg/mL) for
4-Substituted Phenylaminothienopyrimidine 8a
Fig. 12. Survival Curve of MCF7 Cells at 4 Concentrations (µg/mL)
for Thienopyrimidoquinazolinone 9a
Fig. 10. Survival Curve of MCF7 Cells at 4 Concentrations (µg/mL) for
Thienopyrimidine-4-ones 5a
dimethyl sulfoxide (DMSO) and diluted with saline to the
appropriate volume. Different concentrations of the test com-
pound (0, 2.5, 5, 10µg/mL) were added to the cell monolayer.
Triplicate wells were prepared for each individual dose. Mono-
layer cells were incubated with the test compound for 48h at
37°C in atmosphere of 5% CO2. After 48h, cells were fixed
with trichloroacetic acid, washed with water and stained for
30min with 0.4% (w/v) Sulforhodamine-B stain dissolved with
1% acetic acid. Excess stain was removed by four washes with
1% acetic acid and attached stain was recovered with Tris
ethylenediamine tetraacetic acid (EDTA) buffer colour inten-
sity which was measured in enzyme-linked immunosorbent
assay (ELISA) reader. The percentage of cell survival was
calculated as follows: survival fraction=optical density (OD)
(treated cells)/OD (control cells) and the results are showed in
(Table 4).
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