Synthesis, characterization, and biological activities of some novel thienylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidines and related heterocycles

Article abstract of DOI:10.1002/jhet.4310

3-Cyano-5-ethoxycarbonyl-6-methyl-4-(2′-thienyl)-pyridine-2(1H)-thione (1) is synthesized and reacted with chloroacetamide or chloroacetonitrile to give 3-amino-5-ethoxycarbonyl-6-methyl-4(2′-thienyl)-thieno[2,3-b]pyridine-2-carboxamide 3a or its 2-carbon

Full text of DOI:10.1002/jhet.4310

Received: 6 April 2021
DOI: 10.1002/jhet.4310
Revised: 5 May 2021
Accepted: 27 May 2021
A R T I C L E
Synthesis, characterization, and biological activities of some
novel thienylpyrido[30,20:4,5]thieno[3,2-d]pyrimidines and
related heterocycles
Suzan Abuelhassan | Etify A.-G. Bakhite | Abdu E. Abdel-Rahman |
Ahmed F. M. El-Mahdy
Department of Chemistry, Faculty of
Abstract
Science, Assiut University, Assiut, Egypt
3-Cyano-5-ethoxycarbonyl-6-methyl-4-(20-thienyl)-pyridine-2(1H)-thione (1) is syn-
Correspondence
Etify A.-G. Bakhite, Department of
thesized and reacted with chloroacetamide or chloroacetonitrile to give 3-amino-
5-ethoxycarbonyl-6-methyl-4(20-thienyl)-thieno[2,3-b]pyridine-2-carboxamide 3a or
Chemistry, Faculty of Science, Assiut
University, Assiut 71516, Egypt.
its 2-carbonitrile analog 3b, respectively. Cyclocondensation of 3a with tri-
Email: etafy@aun.edu.eg
ethylorthoformate produced the corresponding pyridothienopyrimidineone 4,
which on heating with phosphorus oxychloride gave 4-chloropyrimidine derivative
5. Compound 5 was used as key intermediate for synthesizing compounds 6, 9, 10,
11, and 12 upon treatment with some nucleophilic reagents such as thiourea,
5-phenyl-s-triazole-3(1H)-thione, piperidine, morpholine, or hydrazine hydrate,
respectively. Reaction of pyridothienopyrimidinethione 6 with N-(4-tolyl)-
2-chloroacetamide or ethyl bromoacetate afforded the corresponding
S-substituted methylsulfanylpyrimidines 7 or 8. The condensation of 3b
with triethylorthoformate gave azomethine derivative 13, which was reacted with
hydrazine hydrate to give ethyl 3-amino-3,4-dihydro-4-imino-7-methyl-9-(20-thienyl)
pyrido[30,20:4,5]thieno[3,2-d]pyrimidine-8-carboxylate (14). Compounds 12 and 14
were used as precursors for synthesizing other new thienylpyridothienopyrimidines
as well as isomeric thienyl-s-triazolopyridothieno- pyrimidines. All synthesized com-
pounds were characterized by elemental and spectral analyses such as IR, 1H NMR,
and 13C NMR. In addition, majority of synthesized compounds were tested for their
antifungal activity against five strains of fungi. Moreover, compounds 3a, 5, 6, 8, and
22 were screened for their anticancer activity against HEPG-2 and MCF-7 cell lines.
1 | INTRODUCTION
cleavage [9], herbicidal [10], anti-tubercular [11], protein
kinase inhibition [12], and respiratory syndrome protease
inactivation [13].
Numerous pyridine-containing natural products and
synthetic organic compounds have diverse biophysio-
and pharmacological activity [14–18]. These scaffolds are
also of wide spread interest in supramolecular and coor-
dination chemistry, as well as for materials science [19].
Thieno[2,3-b]pyridines have received considerable
Sulfur-containing heterocycles opened the way for the
active research in the pharmaceutical chemistry. Nowa-
days thiophene and its derivatives are an essential class of
heterocyclic compounds that have a wide range of biologi-
cal applications such as anti-inflammatory [1], anticonvul-
sant [2], antibacterial [3], antitumor [4], antifungal [5],
anti-parasitic [6], antiviral [7], anti-nociceptive [8], DNA
J Heterocyclic Chem. 2021;1–18.
wileyonlinelibrary.com/journal/jhet
© 2021 Wiley Periodicals LLC.
1

2
ABUELHASSAN ET AL.
attention since they show a wide variety of bioactivity,
for example, antiviral [20,21], antidiabetic [22], antimi-
crobial [23–25], antitumor [26], antiparasitic [27], and
neurotropic [28]. Other thienopyridine modifies have
important pharmacologically activities including anti-
platelet drugs for the treatment of acute coronary
syndromes [29,30], antibacterial activity against a drug-
resistant S. epidermidis clinical strain [31], and cytotoxic
activity against human hepatocellular liver carcinoma
(HepG2) [32]. Actually, the key unit of thieno[2,3-b]pyri-
dines is found as the central fragment in many clinical
pharmaceuticals [33,34]. The pharmacological impor-
tance of thieno[2,3-b]pyridines has directed considerable
research activity toward the construction of the skeleton
of such heterocycles [35–37].
In view of the aforementioned findings, the present
project was designed to synthesize and characterize of
some new thiophene-tagged thieno[2,3-b]pyridines,
pyridothienopyrimidine, and s-triazolopyridothienopyrimidine
hoping to get novel condensed polycyclic compounds with
anticipated biological and medicinal importance. In addi-
tion, majority of synthesized compounds were tested for
their antifungal activity against five strains of fungi. More-
over, compounds 3a, 5, 6, 8, and 22 were screened for their
anticancer activity against HEPG-2 and MCF-7 cell lines.
thienyl)pyridine-2-(1H)-thione (1), which was prepared
from the reaction of 2-cyano-3-(20-thienyl)prop-
2-enethioamide with ethyl acetoacetate in the presence of
triethylamine according to the reported procedure
(Scheme 1) [38]. All characterization data of compound
1 are in agreement with those reported before [38].
Reaction of cyanopyridinethione 1 with chloroacetamide
by refluxing in ethanol containing anhydrous sodium car-
bonate for 30 min gave [3-cyano-5-ethoxycarbonyl-
6-methyl-4-(20-thienyl)pyridin-2-ylthio]acetamide (2). In
contrast, reaction of 1 with chloroacetonitrile under the
same conditions furnished 3-amino-5-ethoxycarbonyl-
6-methyl-4-(20-thienyl)thieno[2,3-b]pyridine-2-carbonitrile
(3b). Upon treatment of compound 2 with a catalytic
amount of sodium ethoxide in ethanol, it underwent intra-
molecular Thorpe-Ziegler cyclization affording 3-amino-
5-ethoxycarbonyl-6-methyl-4-(20-thienyl)thieno[2,3-b]
pyridine-2-carboxamide (3a) (Scheme 1).
IR spectrum of compound 2 showed characteristic
absorption bands in the regions 3413–3176 cmꢀ1 for
(NH2), 2223 cmꢀ1 for (C N), and 1681 cmꢀ1 for (C═O,
amide). While that of its isomer 3a revealed the disap-
1
pearance of ν C N band. H NMR spectrum of com-
pound 2 showed the presence of a singlet signal
corresponds to SCH2 group at δ 4.02, which disappeared
in the spectrum of 3a, which exhibits instead of a singlet
signal at δ 5.88 corresponds to NH2 group attached to
thiophene ring directly. IR spectrum of compound 3b
revealed the presence of three absorption bands in the
region 3480–3125 cmꢀ1 characteristic for NH2 group and
a band at 2198 cmꢀ1 for carbonitrile group.
2 | RESULTS AND DISCUSSION
2.1 | Synthesis and characterization
Our approach to the synthesis of the target compounds
started from 3-cyano-5-ethoxycarbonyl-6-methyl-4-(20-
Cyclocondensation reaction of compound 3a with
triethyl orthoformate afforded pyridothienopyrimidineone
SCHEME 1 Synthesis of
compounds 1, 2, and 3a,b

ABUELHASSAN ET AL.
3
derivative 4. Heating compound 4 with an excess amount of
phosphorous oxychloride under neat conditions afforded
4-chloropyrimidine 5, which was considered a good syn-
thon for other thienylpyridothienopyrimidines. Thus, the
interaction of compound 5 with thiourea gave an adduct,
which on treatment with sodium hydroxide solution
followed by acidification with acetic acid furnished pyrimi-
dinethione 6 (Scheme 2).
spectrum revealed the disappearance of the singlet sig-
nal at δ 11.25 (NH) of compound 4. IR spectrum of com-
pound 6 showed the presence of a band at 3279 cmꢀ1
characteristic for NH group. 1H NMR spectrum of 6
displayed a singlet signal at δ 14.40 corresponds to NH
group.
On refluxing of the latter pyrimidinethione with
some α-halocarbonyl compounds such as N-(4-tolyl)-
2-chloroacetamide or ethyl bromoacetate in ethanol
containing sodium acetate for 2 h, the corresponding
S-substituted methylsulfanylpyridothienopyrimidines 7 and
8 were obtained in good yields (Scheme 2). In contrast,
compound 5 reacted with 5-phenyl-3-mercapto-2H-1,2,
4-triazole to give 3-[8-ethoxycarbonyl-7-methyl-9-(20-thi
enyl) pyrido[30,20:4,5]thieno[3,2-d]pyrimidin-4-ylsulfanyl]-5-
phenyl-2H-1,2,4-triazole (9) (Scheme 2).
IR spectrum of 4 showed the absence of the bands of
the two amino groups and presence of two bands at
3165 cmꢀ1 and 1662 cmꢀ1 characteristic for NH and
1
C═O groups of pyrimidineone 4, respectively. H NMR
spectrum of 4 displayed two singlet signals at δ 11.25
and δ 8.19 correspond to NH and pyrimidine-H, respec-
tively. IR spectrum of 5 revealed the absence of all afore-
mentioned bands of compound 4, and its 1H NMR
SCHEME 2 Synthesis of
compounds 4–9

4
ABUELHASSAN ET AL.
SCHEME 3 Synthesis of
compounds 10–14
IR spectrum of 7 exhibited two bands at 3248 cmꢀ1
TABLE 1 Heat of formation of compounds 15–18
characteristic for NH group and at 1687 cmꢀ1 for C═O of
anilide group. 1H NMR spectrum of 7 showed two singlet
signals at δ 9.37 and δ 4.09 corresponding to NH and
SCH2, respectively. IR spectrum of 8 showed the presence
of a band at 1724 cmꢀ1 characteristic for a non conju-
Compd.
No.
Compd.
No.
E (k.cal./Mol)
510.7438
E (k.cal./Mol)
493.6434
15
17
16
18
509.2006
486.8352
1
gated ester. H NMR spectrum of 8 showed the presence
of a singlet signal at δ 4.14 corresponding to SCH2 besides
other two signals equivalent to ethoxy group of the non
conjugated ester. IR spectrum of 9 revealed the presence
of a band at 3203 cmꢀ1 characteristic for NH function of
triazole ring. H NMR spectrum of 9 exhibited a broad
singlet signal at δ 15.20 referred to NH group.
ethyl 3-amino-3,4-dihydro-4-imino-7-methyl-9-(20-thienyl)
pyrido[30,20:4,5]thieno[3,2-d]pyrimidine-8-carboxylate (14)
(Scheme 3). The ester function of compounds 5 and 12–14
did not get affected by hydrazine hydrate under the
applied conditions.
1
Compound 5 underwent other nucleophilic displace-
ment reactions when treated with piperidine or morpholine
to furnish 4-substituted-8-ethoxycarbonyl-7-methyl-9-(20-
thienyl)pyrido[30,20:4,5]thieno[2,3-d]pyrimidines 10 and
11, respectively (Scheme 3). Compound 12 also produced
an excellent yield via condensation of chloropyrimidine
5 with hydrazine hydrate wherein only a nucleophilic
substitution of the labile chlorine atom by hydrazino
group occurred (Scheme 3).
IR spectrum of 13 proved the disappearance of the
amino group of 3b and presence of a band at 2212 cmꢀ1
characteristic for (C N). IR spectrum of 14 showed char-
acteristic absorption bands in the region 3313–3154 cmꢀ1
1
for (NH) and (NH2) groups. H NMR spectrum of 14 dis-
played two singlets at δ 10.20 and δ 5.74 due to NH and
NH2 groups, respectively.
Hydrazino compound 12 and its isomeric amino-
imino one 14 were used as precursors for synthesizing
other new thienylpyridothienopyrimidines as well as
thienyl-s-triazolopyridothienopyrimidines.
Thus, heating compound 12 with formic acid under
reflux for 4 h led to the formation of unexpected product
identified as s-triazolo compound 16 rather than the
expected isomer 15. On the same approach, the reaction
of 12 with glacial acetic acid furnished methyl-s-triazolo
compound 18 rather than the alternative one 17. From
thermodynamic point of view [39] the heat of formation
of compounds 16 and 18 are less than those of the
corresponding isomers 15 and 17 as shown in Table 1. So,
both compounds 16 and 18 seems to be more stable than
1H NMR spectrum of 11 showed a triplet signal at δ
3.93–3.95 equivalent to CH2OCH2 residue and a triplet
signal at δ 3.85–3.87 equivalent to CH2NCH2 residue of
morpholine ring. IR spectrum of 12 exhibited characteris-
tic absorption bands in the region 3341–3188 cmꢀ1 for
1
(NHNH2). H NMR spectrum of 12 displayed three sin-
glet signals at δ 9.14, δ 8.17, and δ 4.95 correspond to
NH, pyrimidine-H, and NH2 protons, respectively.
In contrast, condensation of o-aminocarbonitrile 3b
with triethyl orthoformate by refluxing in acetic anhy-
dride led to the formation of azomethine 13, which on
stirring with hydrazine hydrate in dioxane converted into

ABUELHASSAN ET AL.
5
the corresponding isomers 15 and 17. The probable
mechanism of this reaction starts with the usual forma-
tion of compounds 15 and 17, which underwent Dimroth
rearrangement to give the more stable ones 16 and 18,
respectively (Scheme 4). The mechanism of Dimroth
rearrangement is reported in our previous publication
[40]. The structure of compounds 16 and 18 was also con-
firmed by independent syntheses via heating compound
14 with triethylorthoformate and/ or acetic anhydride,
respectively (Scheme 4).
An attempt to synthesize s-triazolo derivative 15 in its
pure state via condensation of hydrazino compound 12
with triethyl orthoformate in the presence of acetic anhy-
dride has succeeded. In contrast, heating hydrazino com-
pound 12 with acetic anhydride did not give the expected
methyl-s-triazolo derivative 17 and instead of the tria-
cetylated product 19 was isolated. The synthesis of 17
may need triethyl orthoacetate, which was not pursued at
this time. However, heating compound 15 in formic acid
furnished its isomer 16 via Dimroth rearrangement
(Scheme 5).
compound 12, or amino and imino group of compound
14. Both 1H NMR spectrum of 15 and 16 showed a singlet
signal at δ 8.85 or δ 8.46 corresponds to triazole-H,
1
respectively. H NMR spectrum of 18 displated a singlet
signal at δ 2.65 corresponds to CH3 attached to triazole
moiety. IR spectrum of 19 showed a band at 1741 cmꢀ1
1
due to (C═O, acetyl). H NMR spectrum of 19 revealed
the presence of two singlets at δ 2.53 and δ 2.47 corre-
spond to two (COCH3) and one (COCH3), respectively.
The condensation of hydrazino compound 12 with
thiophene-2-carboxaldehyde gave the 4-(20-thienyl)methy-
lenehydrazinopyrimidine derivative 20. In addition, cyclo-
condensation of 12 with acetylacetone under neat conditions
gave 4-(3,5-dimethylpyrazol-1-yl)-8-ethox-ycarbonyl-7-methyl-
9-(20-thienyl)pyrido[30,20:4,5]thieno[3,2-d]pyrimidine (21)
(Scheme 5).
1H NMR spectrum of 20 showed the presence of two
singlets at δ 10.92 and δ 8.43 equivalent to one proton
(NH) and two protons (pyrimidine-H and N═CH),
1
respectively. H NMR of 21 displayed a singlet at δ 8.78
referred to pyrimidine-H and a singlet at δ 6.06 due to
pyrazole-H.
Condensation of compound 14 with thiophene-2-
carboxaldehyde by refluxing in ethanol did not give
IR spectrum of 15 revealed the disappearance of the
hydrazino group of compound 12. IR spectra of 16 and 18
revealed the disappearance of the hydrazino group of
SCHEME 4 Synthesis of
compounds 16 and 18

6
ABUELHASSAN ET AL.
SCHEME 5 Synthesis of
compounds 15, 16, and 19–21
SCHEME 6 Reaction of compound 14 with
Thiophene-2-carboxaldehyde; formation of
compounds 22 and 20
mainly the expected compound 22 [40] nor its isomer 22
[41] but the product was assigned as a mixture of 22 and
20 in a 2:1 ratio. The formation of 20 is attributed to
Dimroth rearrangement of 22 [41] (Scheme 6).
1H NMR spectra of all synthesized compounds as a quar-
tet signal at δ value ranged from 4.04–4.18 to 4.08–4.21
and a triplet signal at δ value ranged from 0.97–1.13 to
1.03–1.15. 1H NMR spectra of all synthesized compounds
exhibited a singlet signal at δ value ranged from 2.45 to
IR spectrum of 22 showed a band at 3245 cmꢀ1 for
1
1
(NH). H NMR spectrum of 22 showed a singlet at δ
2.81 due to methyl group attached to pyridine ring. H
10.09 correspond to (NH), a singlet at δ 9.11 for
pyrimidine-H, and a singlet at δ 8.50 for CH═N.
NMR spectra of compounds 5–11 showed the presence of
a singlet signal at δ value ranged from 8.28 to 8.93 corre-
sponds to pyrimidine-H. H NMR spectra of compounds
14–16, 18, and 19 showed the presence of a singlet signal
at δ value ranged from 7.89 to 9.16 corresponds to
pyrimidine-H. 13C NMR spectra of all synthesized com-
pounds exhibited collections of peaks, which are in agree-
ment with their proposed structures.
1
In general, the elemental analyses of all newly syn-
thesized compounds gave satisfactory results within 0.4
of the calculated values. IR spectra of these compounds
showed characteristic absorption band in the region from
1731 to 1704 cmꢀ1 for (C═O) of conjugated ester group
attached to pyridine ring. This ester group appears in the

ABUELHASSAN ET AL.
7
2.2 | Biological activity
against Aspergillus terreus with inhibition zone of
18 mm, which is near to that of Voriconazole reference
(20 mm). The product 13 that produced from the reac-
tion of compound 3b with triethyl orthoformate showed
moderate effect against Aspergillus flavus and Pencillium
purpurogenum with inhibition zones of 10 mm and
9 mm, respectively. Compounds 5 and 14 also have
moderate effect against Aspergillus flavus and Aspergil-
lus terreus. Compounds 7, 10, 19, 20, and 21 exhibited
very high activity toward Aspergillus niger with inhibi-
tion zones of 24 mm, 21 mm, 20 mm, 20 mm, and
24 mm, respectively. Hydrazino compound 12 show very
high activity against Pencillium purpurogenum with
inhibition zone of 27 mm compared with Voriconazole
reference (17 mm) and high activity against Candida
albicans with zone diameter 16 mm, which near to the
inhibition zone of the reference (18 mm). In addition,
compounds 7 and 18 exhibited very high activity against
Aspergillus terreus with inhibition zones of 20 mm and
25 mm compared with reference (20 mm) as well as
compounds 19, 20, and 21, which have great activity
against the same fungus with inhibition zones of
18 mm, 19 mm, and 19 mm, respectively. Compounds
20 and 21 also exhibited very high activity against Pen-
cillium purpurogenum and Candida albicans compared
2.2.1 | Antifungal activity
Resistance of various strains of the pathogenic bacteria
and fungi to the current antimicrobial therapy has
become one of the most threating problems worldwide.
Therefore, the main target of our study is synthesis of
new heterocyclic compounds having superior signifi-
cance in biological and medicinal chemistry. Accord-
ingly, majority of synthesized compounds were screened
in vitro for their antifungal activity against five strains of
fungi: Aspergillus flavus, Aspergillus niger, Aspergillus
terreus, Pencillium purpurogenum, and Candida albicans.
The antifungal activities of the majority of synthesized
compounds were examined as inhibition areas and were
summarized in Table 2 and illustrated in Figure 1. From the
data presented, it is clear that when compound 3a reacted
with triethyl orthoformate, the pyridothienopyrimidinone
4 that produced very high activity against Aspergillus niger,
Pencillium purpurogenum, and Candida albicans with inhi-
bition zones of 22 mm, 25 mm, and 25 mm, respectively,
compared with the reference fungus Voriconazole,
which posses inhibition zones of 20 mm, 17 mm, and
18 mm, respectively. Compound 4 showed high activity
TABLE 2 Comparison of inhibition zone diameters for the antifungal activity of compounds 3a, 4–15, and 18–22 with the Voriconazole
reference (mm)
Code of sample
A. flavus
A. niger
A. terreus
P. Purpurogenum
C. albicans
3b
4
–
–
–
–
–
–
+22
–
+18
+9
–
+25
+22
–
+25
+23
–
5
+15
–
6
–
7
–
+24
–
+20
–
–
+25
–
8
–
–
9
–
–
–
–
–
10
11
12
13
14
15
18
19
20
21
122
Ref.
–
+21
–
–
–
+15
–
–
–
–
–
–
–
+27
+9
–
+16
–
+10
+12
–
–
–
–
+11
–
–
–
–
–
–
–
+25
+18
+19
+19
–
–
+25
+12
+23
+24
–
–
+20
+20
+24
–
+15
+23
+25
–
–
–
–
24
20
20
17
18
Note: (+) susceptibility. (–) absence of susceptibility (Resistance). Reference of fungus Voriconazole 10 mg/ml.

8
ABUELHASSAN ET AL.
FIGURE 1 Comparison of
inhibition zone diameters for the
antifungal activity of the tested
compounds
with compound 20, which showed moderate activity
against Pencillium purpurogenum and Candida albicans.
Compound 7 showed very high activity against Candida
albicans with inhibition zone of 25 mm compared with
compound 10 which showed moderate activity with
inhibition zone of 15 mm.
chemicals were used as received (i.e., without further
purification). The reactions were followed with TLC
technique. 1H NMR and 13C NMR spectra were recorded
on a Varian A5 500 MHz spectrometer operating at
1
500 MHz for H and 125 MHz for 13C using CDCl3 or
DMSO-d6 as a solvent and tetramethylsilane as internal
standard. Chemical shifts are expressed in δ (ppm); cou-
1
pling constants (J values) are given in Hertz (Hz). H
2.2.2 | Anticancer activity
NMRs splitting patterns are designated as singlet (s),
doublet (d), triplet (t), quartet (q), or multiples (m). FT-
IR spectra were recorded on a Shimadzu 470 IR-
spectrophotometer (KBr; νmax in cmꢀ1). Microanalysis
was performed using a Perkin Elmer 2400 LS Series
CHN/O analyzer. Melting points were obtained using a
Gallan-Kamp apparatus and were reported uncorrected.
Rf values of all compounds were measured by using a
mixture of benzene: ethyl acetate with different ratios as
eluents.
Using the MTT assay compounds 3a, 5, 6, 8, and 22 were
studied for their cytotoxic activity involving MCF-7 and
HepG2 cell lines. As presented in Table 3 and Figures 2 and
3. It is clear from Table 3 that the thiophene moiety was
found to be crucial for the cytotoxic effect of the choice
cyclic products 3a, 5, 6, 8, and 22. The selected compounds
exhibited optimal cytotoxic effect against MCF-7 and
HepG2 cell lines. With IC50 in the μg/ml range. The cyto-
toxicity of the product of Thorpe-Ziegler cyclization of com-
pound 2 showed low cytotoxicity against MCF-7 and
HepG2 cell lines. By comparing the cytotoxicity of com-
pounds 5 and 6, it is obvious that the cytotoxicity of 5 was
higher than that of 6 against the two cell lines this may be
due to the presence of chlorine atom that is responsible for
its high potency. But when compound 6 reacted with ethyl
bromoacetate, product 8 showed high potency against
MCF-7 cell line compared with compound 6 this is due to
the presence of enrich electron group COOEt that is respon-
sible for its high potency. While the cytotoxicity of com-
pound 22 showed moderate effect against the two cell lines.
Results are given in concentrations that were able to cause
50% of cell growth inhibition (IC50).
3.1.1 | Synthesis of 3-cyano-5-ethoxcarbonyl-
6-methyl-4-(20-thienyl)pyridin-2-(1H)-thione (1)
To
a
mixture
of
2-cyano-3-(20-thienyl)prop-
2-enethioamide (5.82 g, 30 mmol) and ethyl acetoacetate
(3.90 ml, 30 mmol) in absolute ethanol (20 ml),
triethylamine (1 ml) was added. The resulting mixture
was heated under reflux for 5 h and then allowed to stand
overnight. The orange crystals that formed were collected
and recrystallized from ethanol to give compound 1;
yield: 78%; m.p.: 260ꢁC–262ꢁC, Rf: 0.76 (2:3). IR: 3168
(N─H), 2225 (C N), 1726 (C═O, ester). 1H NMR
(DMSO-d6): 14.37 (s, 1H, NH), 7.88–7.89 (d, 1H,
thiophene-H), 7.36–7.37 (d, 1H, thiophene-H), 7.21–7.22
(d, 1H, thiophene-H), 3.89–4.01 (q, 2H, OCH2), 2.44
(s, 3H, CH3), 0.90–0.92 (t, 3H, CH3, ester). 13C NMR
(DMSO-d6): 178.49, 164.49, 152.61, 147.48, 134.10, 130.27,
118.85, 115.94, 115.86, 114.11, 61.68, 17.95, 13.34. Anal
calcd. For C14H12N2O2S2: C, 55.24; H, 3.97; N, 9.20.
Found: C, 55.11; H, 3.82; N, 9.34.
3 | EXPERIMENTAL
3.1 | Chemistry
Analytical-grade reagents and solvents for synthesis
were obtained from Sigma Aldrich (USA). The

ABUELHASSAN ET AL.
9
TABLE 3 Toxicity of the most potent compounds against the MCF-7 and HepG2 cell lines
MCF-7
HepG2
Compd.
3a
%viability
99.4926
97.9904
96.4385
93.852
IC50
Toxicity
%viability
98.2062
95.902
IC50
Toxicity
>100
Non-toxic
> 100
Non-toxic
91.8477
90.8123
90.6227
95.0422
93.1786
92.5809
91.4557
10.7595
98.0032
91.944
92.8969
98.5421
97.7002
97.3511
96.7556
11.3244
99.5054
99.3817
97.9802
96.0223
43.075
5
41.5
82.8
>100
45.4
Very toxic
Harmful
33.6
Toxic
6
17.4
Harmful
Non-toxic
Non-toxic
85.0585
66.5825
15.0333
97.6961
96.1327
95.1728
91.8815
91.7992
98.5841
97.0796
95.9292
89.292
8
96.8825
96.5827
94.4045
93.0256
88.8489
99.9169
99.5015
95.9493
94.8899
18.0723
Non-toxic
Very toxic
>100
>100
22
67.8761
3.1.2 | Synthesis of 2-[3-cyano-
5-ethoxycarbonyl-6-methyl-4-(20-thienyl)
pyridin-2-ylthio] acetamide (2)
168.36, 165.88, 162.60, 158.43, 144.29, 132.85, 130.49,
127.95, 125.15, 114.61, 103.88, 61.82, 34.14, 23.01, 13.46.
Anal. calcd. For C16H15N3O3S2: C, 53.17; H, 4.18; N,
11.63. Found: C, 53.23; H, 4.22; N, 11.43.
To a suspension of compound 1 (3.04 g, 10 mmol) and
anhydrous sodium carbonate (2.33 g, 22 mmol) in etha-
nol (30 ml), chloroacetamide (0.94 g, 10 mmol) was
added. The mixture was heated under reflux for 30 min.
The product that formed on cooling was collected and
recrystallized from ethanol as white crystals of 2; Yield:
91%; m.p: 142ꢁC–144ꢁC, Rf: 0.74 (2:3). IR: 3413, 3300,
3176 (NH2), 2223 (C N), 1717 (C═O, ester), 1681 (C═O,
amide). 1H NMR (DMSO-d6): 7.0.89–7.90 (d, 1H,
thiophene-H), 7.65 (s, 1H, NH), 7.38 (s, 1H, thiophene-
H), 7.22–7.25 (t, 2H: NH and thiophene-H), 4.02 (s, 2H,
SCH2), 4.09–4.14 (q, 2H, OCH2), 2.54 (s, 3H, CH3), 1.00–
1.03 (t, 3H, CH3 of ester group). 13C NMR (DMSO-d6):
3.1.3 | Synthesis of 3-amino-
5-ethoxycarbonyl-6-methyl-4-(20-thienyl)thieno
[2,3-b]pyridine-2-carboxamide (3a)
Compound 2 (3.61 g, 10 mmol) was suspended in sodium
ethoxide solution (0.12 g sodium in 30 ml abs. ethanol)
and heated under reflux for 5 min. The solid that formed
was collected and recrystallized from ethanol to give
canary yellow crystals of 3a; yield: 95%; m. p.: 250ꢁC–
252ꢁC, Rf: 0.84 (2:3). IR (cmꢀ1): 3454, 3418, 3315, 3262,
1
3169 (2 NH2), 1715 (C═O, ester), 1655 (C═O, amide). H

10
ABUELHASSAN ET AL.
FIGURE 2 The relation between %viability and concentration of the compounds 3a, 5, 6, 8, and 22 against MCF-7 cell line
NMR (DMSO-d6): 7.877.88 (d, 1H, thiophene-H), 6.23–
7.28 (m, 2H, thiophene-H), 6.96 (s, 2H, CONH2), 5.88
(s, 2H, NH2 attached to thiophene ring), 4.04–4.08 (q, 2H,
OCH2), 2.57 (s, 3H, CH3), 0.97–1.00 (t, 3H, CH3 of ester
group). 13C NMR (DMSO-d6): 166.81, 166.65, 159.01,
154.12, 145.42, 136.62, 132.30, 130.03, 129.36, 127.77,
127.50, 120.76, 98.69, 61.45, 22.64, 13.59. Anal. calcd. For
C16H15N3O3S2: C, 53.17; H, 4.18; N, 11.63. Found: C,
53.34; H, 4.16; N, 11.50.
3.1.4 | Synthesis of 3-amino-
5-ethoxycarbonyl-6-methyl-4(20-thienyl)thieno
[2,3-b] pyridine-2-carbonitrile (3b)
To a suspension of compound 1 (3.04 g, 10 mmol) and
anhydrous sodium carbonate (2.33 g, 22 mmol) in etha-
nol (30 ml), chloroacetonitrile (0.76 ml, 10 mmol) was
added. The resulting mixture was refluxed for 30 min.
The precipitate that formed on cooling was collected,

ABUELHASSAN ET AL.
11
FIGURE 3 The relation between % viability and concentration of the compounds 3a, 5, 6, 8, and 22 against HepG2 cell line
washed several times with water, and recrystallized from
ethanol. Yield: 73%; m.p: 184ꢁC–186ꢁC, Rf: 0.80 (2:3). IR:
3480, 3383, 3125 (NH2), 2198 (C N), 1725 (C═O, ester).
1H NMR (CDCl3): 7.93–7.94 (d, 1H, thiophene-H),
7.28–7.31 (t, 2H, thiophene-H), 5.69 (s, 2H, NH2), 4.05–4.09
(q, 2H, OCH2), 2.57 (s, 3H, CH3), 0.97–1.00 (t, 3H, CH3 of
ester group). Anal. calcd. For C16H13N3O2S2: C, 55.96;
H, 3.82; N, 12.24. Found: C, 56.09; H, 3.61; N, 12.08.

12
ABUELHASSAN ET AL.
3.1.5 | Synthesis of 8-ethoxycarbonyl-
7-methyl-9-(20-thienyl)pyrido[30,20:4,5]thieno
[3,2-d] pyrimidine-4-(3H)-one (4)
yellow precipitate formed. It was collected and crystal-
lized from ethanol as yellow fine needles; yield: 80%; m.
p.: 298ꢁC–300ꢁC, Rf: 0.82 (1:4). IR: 3279 (N─H), 1704
1
(C═O, ester). H NMR (DMSO-d6): 14.40 (s, 1H, NH);
A mixture of compound 3a (3.61 g, 10 mmol) and triethyl
orthoformate (2 ml) in acetic anhydride (20 ml) was
refluxed for 4 h. The solid that formed was collected and
crystallized from ethanol as greenish white needles; yield:
93%; m.p.: >360ꢁC, Rf: 0.92 (3:2). IR: 3165 (N─H), 1724
(C═O, ester), 1662 (C═O, pyrimidineone). 1H NMR
(CDCl3): 11.25 (s, 1H, NH); 8.19 (s, 1H, pyrimidine-H),
7.62 (s, 1H, thiophene-H), 7.17–7.22 (m, 2H, thiophene-
H), 4.18–4.22 (q, J = 5 Hz, 2H, OCH2), 2.82 (s, 3H, CH3),
1.11–1.13 (t, J = 5 Hz, 3H, CH3 of ester group). 13C NMR
(CDCl3): 167.64, 161.78, 160.82, 157.71, 152.63, 145.80,
145.15, 142.60, 133.45, 131.08, 29.59, 127.81, 125.15,
124.03, 63.36, 22.68, 14.34. Anal.calcd. For C17H13N3O3S2:
C, 54.97; H, 3.53; N, 11.31. Found: C, 54.73; H, 3.50;
N, 11.42.
8.28 (s, 1H, pyrimidine-H), 7.78–7.80 (t, J = 5 Hz, 1H,
thiophene-H), 7.15–7.17 (t, J = 5 Hz, 1H, thiophene-H).
4.08–4.12 (q, J = 7 Hz, 2H, OCH2), 2.63 (s, 3H, CH3),
1.00–1.03 (t, J = 7 Hz, 3H, CH3 of ester group). 13C NMR
(DMSO-d6): 185.77, 176.27, 172.43, 165.58, 155.85, 155.67,
148.97, 146.05, 142.56, 139.18, 138.49, 138.11, 136.26,
132.88, 71.03, 32.34, 23.04. Anal. calcd. For
C17H13N3O2S3: C, 52.69; H, 3.38; N, 10.84. Found: C, 52.86;
H, 3.22; N, 10.57.
3.1.8 | Synthesis of 4-substituted
8-ethoxycarbonyl-7-methyl-9-(20-thienyl)pyrido
[30,20: 4,5]thieno[3,2-d]pyrimidines 7,8; general
procedure
To a suspension of compound 6 (3.87 g, 10 mmol) and
3.1.6 | Synthesis of 4-chloro-
sodium acetate anhydrous (3.0 g, 22 mmol) in ethanol
8-ethoxycarbonyl-7-methyl-9-(20-thienyl)pyrido
[30,20:4,5] thieno[3,2-d]pyrimidine (5)
(40 ml),
N-(4-tolyl)-2-chloroacetamide
or
ethyl
bromoacetate (10 mmol) was added. The resulting mix-
ture was heated under reflux for 2 h. The precipitate that
formed was collected and recrystallized from ethanol to
give compounds 7 or 8, respectively.
Compound
4
(3.71 g, 10 mmol) in phosphorus
oxychloride (20 ml) was heated under refluxed on a water
bath for 4 h. The cooled reaction mixture was poured
with vigorous stirring into ice-water (50 ml). The sepa-
rated solid was collected and crystallized from ethanol as
pale green needles; yield: 98%; m.p.: 184ꢁC–186ꢁC, Rf:
Synthesis of 8-ethoxycarbonyl-7-methyl-9-(20-thienyl)-
4-[N-(4-tolyl)carbamoyl-methylsulfanyl]pyrido[30,20:4,5]
thieno[3,2-d]pyrimidine (7)
1
0.77 (3:2). IR: 1731 (C═O, ester). H NMR (CDCl3): 8.86
It is obtained as a pale green needle by using N-(4-tolyl)-
2-chloroacetamide; yield: 70%; m.p.: 198ꢁC–200ꢁC, Rf:
0.83 (2:3). IR: 3248 (N─H), 1720 (C═O, ester), 1687
(C═O, anilide). 1H NMR (DMSO-d6): 9.37 (1H, NH), 8.93
(1H, pyrimidine-H), 7.05–7.59 (m, 7H: Ar─H and
thiophene-H), 4.16–4.20 (q, J = 7 Hz, 2H, OCH2), 4.09
(s, 2H, SCH2), 2.78 (s, 3H, CH3), 2.27 (s, 3H, CH3 of
4-tolyl group), 1.10–1.13 (t, J = 7 Hz, 3H, CH3 of ester
group). 13C NMR (DMSO-d6): 168.28, 166.92, 163.55,
162.90, 158.95, 154.79, 154.00, 140.71, 135.97, 134.75,
130.44, 130.02, 129.77, 128.87, 128.65, 127.65, 127.42,
123.24, 118.51, 62.66, 34.85, 24.15, 21.56, 14.47. Anal.
calcd. For C26H22N4O3S3: C, 58.41; H, 4.15; N, 10.48.
Found: C, 58.31; H, 4.19; N, 10.60.
(s, 1H, pyrimidine-H), 7.57–7.59 (d, 1H, thiophene-H),
7.18 (s, 1H, thiophene-H), 7.16 (s, 1H, thiophene-
H), 4.15–4.19 (q, J = 7.5 Hz, 2H, OCH2), 2.78 (s, 3H,
CH3), 1.09–1.12 (t, J = 7.5 Hz, 3H, CH3 of ester group),
13C NMR (CDCl3): 168.01, 163.89, 159.41, 157.67, 155.20,
154.98, 141.00, 131.15, 130.45, 130.06, 128.90, 127.53,
123.53, 62.36, 24.14, 14.46. Anal. calcd. For
C17H12ClN3O2S2: C, 52.37; H, 3.10; N, 10.78. Found: C,
52.25; H, 3.14; N, 10.91.
3.1.7 | Synthesis of 8-ethoxycarbonyl-
7-methyl-9-(20-thienyl)pyrido[30,20:4,5]thieno
[3,2-d] pyrimidin-4-(3H)-thione (6)
Synthesis of ethyl [8-ethoxycarbonyl-7-methyl-9-(20-
thienyl)pyrido[30,20:4,5] thieno[3,2-d]pyrimidin-
4-ylsulfanyl]acetate (8)
Compound 5 (3.89 g, 10 mmol) and thiourea (0.76 g,
10 mmol) in (20 ml) ethanol for 3 h were heated under
reflux for 3 h. The precipitate that formed on cooling was
collected and then dissolved in 5% sodium hydroxide
solution. The resulting solution was filtered and the clear
filtrate was acidified with diluted acetic acid whereby a
It is obtained as a white needle by using ethyl
bromoacetate; yield: 80%; m.p.: 130ꢁC, Rf: 0.68 (1:4). IR
1
(cmꢀ1): 1724 (C═O, ester), 1712 (C═O, ester). H NMR
(DMSO-d6): 8.78 (s, 1H, pyrimidine-H), 7.54–7.57 (t, 1H,

ABUELHASSAN ET AL.
13
thiophene-H), 7.15–7.18 (t, 2H, thiophene-H), 4.15–4.24
(m, 4H, two OCH2), 4.14 (s, 2H, SCH2), 2.76 (s, 3H, CH3),
1.25–1.28 (t, J = 7 Hz, 3H, CH3 of ester group), 1.08–1.11
(t, J = 7 Hz, 3H, CH3 of ester group). Anal. calcd. For
C21H19N3O4S3: C, 53.26; H, 4.04; N, 8.87. Found: C, 53.51;
H, 4.20; N, 8.56.
C22H22N4O2S2: C, 60.25; H, 5.06; N, 12.78. Found: C, 60.00;
H, 5.24; N, 12.53.
4-Morpholino-8-ethoxycarbonyl-7-methyl-9-(20-thienyl)
pyrido[30,20:4,5]thieno [3,2-d]pyrimidine (11)
It was obtained as white needles; yield: 91%; m.p.:
1
146ꢁC–147ꢁC, Rf: 0.89 (1:4). IR: 1720 (C═O, ester). H
NMR (CDCl3): 8.49 (s, 1H, pyrimidine-H), 7.54 (s, 1H,
thiophene-H), 7.14–7.16 (t, J = 5 Hz, 2H, thiophene-H),
4.13–4.17 (q, J = 7 Hz, 2H, OCH2), 3.93–3.95 (t, J = 5 Hz,
4H, two OCH2), 3.85–3.87 (t, J = 5 Hz, 4H, two NCH2),
2.74 (s, 3H, CH3), 1.08–1.11 (t, J = 7 Hz, 3H, CH3 of ester
group). 13C NMR (CDCl3): 168.56, 162.39, 159.11, 157.60,
156.73, 154.83, 139.87, 135.57, 130.09, 129.43, 128.25,
127.22, 123.74, 114.85, 67.40, 62.49, 47.34, 23.93, 14.47.
Anal. calcd. For C21H20N4O3S2: C, 57.26; H, 4.58;
N, 12.72. Found: C, 57.00; H, 4.61; N, 12.48.
3.1.9 | Synthesis of 3-[8-ethoxycarbonyl-
7-methyl-9-(20-thienyl)pyrido[30,20:4,5]thieno
[3,2-d]pyrimidin-4-ylsulfanyl]-5-phenyl-2H-
1,2,4-triazole (9)
To a mixture of chloropyrimidine 5 (g, 10 mmol) and
using 5-phenyl-3-mercapto-2H-1,2,4-triazole in ethanol
(30 ml); sodium acetate anhydrous (3.0 g, 22 mmol), was
added. The resulting mixture was heated under reflux for
4 h. The precipitate that formed on cooling was collected
and recrystallized from ethanol to give a pale green
needles of compound 9; yield: 90%. m.p.: 198ꢁC–200ꢁC,
Rf: 0.78 (2:3). IR: 3203 (N─H), 1726 (C═O, ester). 1H
NMR (DMSO-d6): 15.20 (s, 1H, NH), 8.84 (s, 1H,
pyrimidine-H), 6.92–8.03 (m, 8H: Ar-H and thiophene-
H), 4.08–4.12 (q, J = 7 Hz, 2H, OCH2), 2.64 (s, 3H, CH3),
0.99–1.02 (t, J = 7 Hz, 3H, CH3 of ester group). 13C NMR
(DMSO-d6): 176.12, 171.60, 167.24, 164.94, 163.88, 163.44,
148.95, 142.53, 141.35, 139.35, 139.06, 138.58, 138.26,
136.74, 136.38, 135.59, 131.24, 129.04, 125.71, 121.97,
70.98, 65.40, 32.39, 28.01, 23.02. Anal. calcd. For
C25H18N6O2S3: C, 56.59; H, 3.42; N, 15.84. Found: C,
56.33; H, 3.37; N, 15.69.
3.1.11 | Synthesis of 4-hydrazino-
8-ethoxycarbonyl-7-methyl-9-(20-thienyl)pyrido
[30,20: 4,5]thieno[3,2-d]pyrimidine (12)
A mixture of compound 5 (3.89 g, 10 mmol) and hydra-
zine hydrate (3 ml) in ethanol (20 ml) was heated under
reflux for 2 h. The product that formed was collected and
recrystallized from ethanol to give white needles of com-
pound 12; yield: 94%; m.p.:280ꢁC–282ꢁC, Rf: 0.87 (2:3).
IR: 3341, 3292, 3227, 3188 (NHNH2), 1729 (C═O, ester),
1
1644 (C═N). H NMR (DMSO-d6): 9.14 (s, 1H, NH), 8.17
(s, 1H, pyrimidine-H), 7.75–7.76 (d, J = 5 Hz, 1H,
thiophene-H), 7.10–7.15 (m, 2H, thiophene-H), 4.95 (s,
2H, NH2), 4.06–4.10 (q, J = 7 Hz, 2H, OCH2), 2.63 (s, 3H,
3.1.10 | Reaction of compound 5 with
piperidine or morpholine; synthesis of
compound 10 and 11; general procedure
CH3), 1.00–1.03 (t, J = 7 Hz, 3H, CH3 of ester group). 13
C
NMR (DMSO-d6): 176.80, 173.96, 170.26, 164.81, 163.51,
147.34, 143.91, 138.41, 137.60, 137.23, 135.97, 131.44,
120.33, 70.83, 32.24, 23.05. Anal. calcd. For
C17H15N5O2S2: C, 52.97; H, 3.92; N, 18.17. Found: C, 52.80;
H, 3.87; N, 18.35.
A mixture of chlorocompound 5 (1.95 g, 5 mmol) and
piperidine or morpholine (4 ml) was gently heated under
reflux for 3 h. The reaction mixture was triturated with
ethanol (10 ml) and then left to cool. The precipitate that
formed was collected and recrystallized from ethanol to
give compound 10 or 11, respectively.
3.1.12 | Synthesis of 2-cyano-
3-ethoxymethyleneamino-5-ethoxycarbonyl-
6-methyl-4-(20-thienyl)thieno[2,3-b]
pyridine (13)
4-Piperidino-8-ethoxycarbonyl-7-methyl-9-(20-thienyl)
pyrido[30,20:4,5]thieno [3,2-d]pyrimidine (10)
It was obtained as pale yellow white needles; yield: 85%;
m.p.: 156ꢁC, Rf: 0.89 (1:4). IR: 1725 (C═O, ester). 1H
NMR (CDCl3): δ 8.48 (s, 1H, pyrimidine-H), 7.54–7.55
(s, 1H, thiophene-H), 7.14–7.15 (d, 2H, thiophene-H),
4.11–4.16 (q, 2H, OCH2), 3.89–3.92 (t, 4H, CH2NCH2),
2.73 (s, 3H, CH3), 1.73–1.76 (s, 6H, CH2CH2CH2), 1.07–
1.10 (t, 3H, CH3 of ester group). Anal. calcd. For
A mixture of compound 3b (3.43 g, 10 mmol), triethyl
orthoformate (5 ml) and acetic anhydride (20 ml) was
heated under reflux for 4 h. The precipitate that formed
after cooling was collected and recrystallized from etha-
nol to give pale yellow crystals of 13; yield: 92%, m.p.:
152ꢁC–153ꢁC, Rf: 0.77 (2:3). IR: 2212 (C N), 1730 (C═O
ester), 1633 (C═N). 1H NMR (CDCl3): 7.63 (s, 1H,

14
ABUELHASSAN ET AL.
N═CH), 7.44–745 (d, 1H, thiophene-H), 7.04–7.05 (d,
J = 5 Hz, 1H, thiophene-H), 6.97–6.98 (d, J = 5 Hz, 1H,
thiophene-H), 4.08–4.12 (q, J = 7 Hz, 2H, OCH2 of ester
119.88, 62.43, 23.79, 14.46. Anal. calcd. For
C18H13N5O2S2: C, 54.67; H, 3.31; N, 17.71. Found: C,
54.45; H, 3.38; N, 17.61.
group), 3.73–3.77 (q,
J = 7 Hz, 2H, OCH2 of
methanimidate group), 2.68 (s, 3H, CH3), 1.17–1.20
(t, J = 7 Hz, 3H, CH3 of methanimidate group), 1.04–1.07
(t, J = 7 Hz, 3H, CH3 of ester group). 13C NMR (CDCl3):
167.85, 161.16, 157.44, 156.84, 151.62, 138.67, 134.68,
130.26, 130.02, 127.87, 127.09, 123.66, 118.52, 114.50,
92.99, 63.72, 62.47, 23.77, 14.40. Anal. calcd. For
C19H17N3O3S2: C, 57.13; H, 4.29; N, 10.52. Found: C,
57.16; H, 4.24; N, 10.37.
3.1.15 | Synthesis of 8-ethoxycarbonyl-
9-methyl-7-(20-thienyl)-[1,2,4]triazolo[200,300-c]
pyrido[30,20:4,5]thieno[2,3-e]pyrimidine (16)
Method A
A mixture of hydrazino compound 12 (0.38 g, 1 mmol)
and formic acid (5 ml) was heated under reflux for 5 h.
The solid product was collected and crystallized from eth-
anol as white crystals of compound 16; yield: 75%; m.p.:
1
3.1.13 | Synthesis of 3-Amino-3,4-dihydro-
4-imino-8-ethoxycarbonyl-7-methyl-9-(20-
thienyl) pyrido[30,20:4,5]thieno[3,2-d]
pyrimidine (14)
194ꢁC–196ꢁC, Rf: 0.78 (2:3). IR: 1720 (C═O, ester). H
NMR (CDCl3): 9.16 (s, 1H, pyrimidine-H), 8.46 (s, 1H,
triazole-H), 7.57–7.58 (m, 3H, thiophene-H), 7.18–7.19 (d,
2H, thiophene-H), 4.15–4.19 (q, J = 7 Hz, 2H, OCH2),
2.77 (s, 3H, CH3), 1.09–1.12 (t, J = 7 Hz, 3H, CH3 of ester
group). 13C NMR (CDCl3): 167.74, 162.62, 156.67, 155.92,
148.87, 144.65, 139.13, 137.60, 134.91, 130.35, 129.58,
128.07, 127.29, 124.07, 121.22, 62.60, 23.89, 14.35. Anal.
calcd. For C18H13N5O2S2: C, 54.67; H, 3.31; N, 17.71.
Found: C, 54.82; H, 3.24; N, 17.50.
To a suspension of compound 13 (3.99 g, 10 mmol) in
Dioxane (10 ml), hydrazine hydrate 99% (2 ml) was
added. The reaction mixture was stirred at room tempera-
ture for 2 h. The solid that formed was collected and rec-
rystallized from ethanol to give compound 14; yield: 96%,
m.p.: 218ꢁC–220ꢁC, Rf: 0.52 (1:4). IR: 3313, 3282, 3154
(NH2, NH), 1720 (C═O, ester). 1H NMR (DMSO-d6):
10.20 (1H, NH), 7.89 (1H, pyrimidine-H), 7.10–7.76 (m,
3H, thiophene-H), 5.74 (s, 2H, NH2), 4.07–4.11 (q, 2H,
OCH2), 2.61 (s, 3H, CH3), 1.00–1.03 (t, 3H, CH3 of ester).
13C NMR (DMSO-d6): 167.10, 161.58, 153.89, 149.89,
149.70, 144.50, 133.63, 129.39, 128.64, 128.49, 126.52,
123.70, 107.09, 61.40, 55.75, 22.62, 18.52, 13.55. Anal.
calcd. For C17H15N5O2S2: C, 52.97; H, 3.92; N, 18.17.
Found: C, 53.14; H, 3.77; N, 18.00.
Method B
A mixture of compound 14 (0.38 g, 1 mmol) and triethyl
orthoformate (5 ml) was heated under reflux for 5 h. The
solid product was collected and recrystallized from etha-
nol as white crystals of compound 16; yield: 70%; m.p.:
194ꢁC–196ꢁC (m.m.p.: 194ꢁC–196ꢁC). The data of FT-IR,
1H-NMR, and 13C-NMR of this product are identical to
those reported in method A.
Method C
Compound 15 (0.39 g, 1 mmol) in formic acid (5 ml) was
heated under reflux for 5 h. The solid product was col-
lected and crystallized from ethanol as white crystals of
compound 16; yield: 96%; m.p.: 194ꢁC–196ꢁC (m.m.p.:
194ꢁC–196ꢁC). The data of FT-IR, 1H-NMR, and 13C-
NMR of this product are identical to those reported in
both methods A and B.
3.1.14 | Synthesis of 8-ethoxycarbonyl-
9-methyl-7-(20-thienyl)-[1,2,4]triazolo[400,300-c]
pyrido[30,20:4,5]thieno[2,3-e]pyrimidine (15)
To a mixture of hydrazino compound 12 (0.39 g, 1 mmol)
and triethyl orthoformate (5 ml), few drops of acetic
anhydride were added. The reaction mixture was heated
under reflux for 5 h. The solid product was collected and
recrystallized from ethanol as yellowish crystals of com-
pound 15; yield: 77%; m.p.: 258ꢁC–260ꢁC, Rf: 0.53 (2:3).
3.1.16 | Synthesis of 2-methyl-
8-ethoxycarbonyl-9-methyl-7-(20-thienyl)-[1,2,4]
triazolo [200,300-c]pyrido[30,20:4,5]thieno[2,3-e]
pyrimidine (18)
1
IR: 1728 (C═O, ester). H NMR (CDCl3): 9.00 (s, 1H,
pyrimidine-H), 8.85 (s, 1H, triazole-H), 7.54–7.55 (d, 1H, -
thiophene-H), 7.14–7.17 (t, 2H, thiophene-H), 4.14–4.18
(q, J = 7 Hz, 2H, OCH2), 2.74 (s, 3H, CH3), 1.09–1.11
(t, J = 7 Hz, 3H, CH3 of ester group). 13C NMR (CDCl3):
168.28, 162.10, 156.36, 145.88, 141.71, 138.55, 135.39,
134.74, 134.17, 130.22, 129.79, 128.21, 127.27, 123.90,
Method A
Hydrazino compound 12 (0.38 g, 1 mmol) in glacial
acetic acid (5 ml) was heated under reflux for 5 h. the
precipitate that formed on cooling was collected and

ABUELHASSAN ET AL.
15
recrystallized from ethanol as white crystals of compound
with ethanol for 3 h. The solid product was collected and
recrystallized from ethanol as yellow crystals; yield: 95%;
m.p.: 254ꢁC–256ꢁC, Rf: 0.83 (1:4). IR: 3196 (NH), 1726
18; yield: 81%; m.p.: 230ꢁC–232ꢁC, Rf: 0.85 (1:4).. IR: 1718
1
(C═O, ester). H NMR (CDCl3): 9.03 (s, 1H, pyrimidine-
1
H), 7.55–7.56 (d, 1H, thiophene-H), 7.16–7.17 (d, 2H,
thiophene-H), 4.14–4.18 (q, 2H, OCH2), 2.75 (s, 3H, CH3
attached to pyridine), 2.65 (s, 3H, CH3 attached to tri-
azole), 1.08–1.11 (t, 3H, CH3 of ester group). 13C NMR
(CDCl3): 168.29, 166.54, 162.60, 156.61, 149.05, 144.68,
139.13, 137.41, 134.96, 130.23, 129.66, 128.28, 127.35,
124.12, 120.11, 62.55, 23.85, 15.47, 14.46. Anal. calcd. For
C19H15N5O2S2 (409.48): C, 55.73; H, 3.69; N, 17.10.
Found: C, 55.45; H, 3.52; N, 17.01.
(C═O, ester). H NMR (CDCl3): 10.92 (s, 1H, NH), 8.43
(s, 2H, pyrimidine-H, N═CH), 7.13–7.67 (m, 6H,
thiophene-H), 4.17–4.22 (q, J = 8 Hz, 2H, OCH2), 2.79 (s,
3H, CH3), 1.11–1.14 (t, J = 8 Hz, 3H, CH3 of ester group).
Anal. calcd. For C22H17N5O2S3: C, 55.10; H, 3.57; N,
14.60. Found: C, 55.23; H, 3.40; N, 14.41.
3.1.19 | Synthesis of 4-(3,5-Dimethylpyrazol-
1-yl)-8-ethoxycarbonyl-7-methyl-9-(20-thienyl)
pyrido[30,20:4,5]thieno[3,2-d]pyrimidine (21)
Method B
A mixture of compound 14 (0.39 g, 1 mmol) and acetic
anhydride (5 ml) was heated under reflux for 5 h. The
crystalline precipitate that formed on cooling was col-
lected by filtration and recrystallized from ethanol as
white crystals of compound 18; yield: 94%; m.p.: 230ꢁC–
232ꢁC (m.m.p.: 230ꢁC–132ꢁC). The data of FT-IR, 1H-
NMR, and 13C-NMR of this product are identical to those
reported in Method A.
A mixture of hydrazino compound 12 (0.38 g, 1 mmol)
and acetylacetone (10 ml) was gently refluxed for 3 h. the
mixture was then mashed with ethanol (15 ml) and left
to cool. The solid product was collected and recrystallized
from ethanol as buff crystals; yield: 95%; m.p.: 200ꢁC–
1
202ꢁC, Rf: 0.55 (1:4). IR: 1731 (C═O, ester). H NMR
(CDCl3): 8.78 (s, 1H, pyrimidine-H), 7.57–7.58 (d,
J = 5 Hz, 1H, thiophene-H), 7.17–7.21 (m, 2H,
thiophene-H), 6.06 (s, 1H, pyrazole-H), 4.14–4.18 (q,
J = 7 Hz, 2H, OCH2), 2.77 (s, 3H, CH3), 2.76 (s, 3H,
CH3), 2.36 (s, 3H, CH3 attached to pyrazole ring), 1.09–
1.12 (t, J = 7 Hz, 3H, CH3 of ester group). 13C NMR
(CDCl3): 168.61, 167.05, 158.07, 158.09, 154.46, 153.55,
151.95, 144.14, 139.71, 135.59, 129.59, 129.43, 128.19,
127.27, 122.88, 119.83, 111.61, 62.52, 24.07, 16.13, 14.46,
14.39. Anal. calcd. For C22H19N5O2S2: C, 58.78; H,
4.26; N, 15.58. Found: C, 58.90; H, 4.15; N, 15.37.
3.1.17 | Synthesis of
4-(1,2,2-triacetylhydrazino)-8-ethoxycarbonyl-
7-methyl-9-(20-thienyl)pyrido[30,20:4,5]thieno
[3,2-d]pyrimidine (19)
A mixture of hydrazino compound 12 (0.38 g, 1 mmol)
and acetic anhydride (5 ml) was refluxed for 5 h. The pre-
cipitate that formed was collected and recrystallized from
ethanol as white crystals of compound 20; yield: 89%; m.
p.: 174ꢁC–176ꢁC, Rf: 0.88 (2:3). IR: 1741 (C═O, acetyl),
1714 (C═O, ester). 1H NMR (CDCl3): 8.81 (s, 1H,
pyrimidine-H), 7.56–7.58 (d, 1H, thiophene-H), 7.16–7.17
(d, 2H, thiophene-H), 4.14–4.18 (q, J = 7 Hz, 2H, OCH2),
2.76 (s, 3H, CH3), 2.53 (s, 6H, two COCH3), 2.47 (s, 3H,
COCH3), 1.08–1.11 (t, J = 7 Hz, 3H, CH3 of ester group).
13C NMR (DMSO-d6):): 171.24, 170.28, 167.37, 163.37,
158.62, 153.74, 153.29, 139.77, 134.22, 129.52, 128.83,
127.77, 126.72, 121.79, 120.71, 118.05, 104.93, 61.90,
24.90, 23.89, 23.41, 13.75. Anal. calcd. For
C23H21N5O5S2: C, 54.00; H, 4.14; N, 13.69. Found: C,
53.71; H, 4.10; N, 13.43.
3.1.20 | Reaction of compound 14 with
thiophene-2-carboxaldehyde; formation of
compounds 20 and 22
A mixture of compound 14 (0.38 g, 1 mmol) and
thiophene-2-carboxaldehyde (2 ml) ethanol (30 ml) was
heated under reflux for 3 h. The precipitate that formed
on hot was collected and recrystallized from dioxane
and identified as 4-(20-thienyl)methylenehydrazino-
8-ethoxycarbonyl-7-methyl-9-(20-thienyl)pyrido[30,20:4,5]
thieno[3,2-d]pyrimidine (20) which is identical to that
described above in all aspects; yield: 24%. The filtrate
from the above crude compound was allowed to cool
whereby a crystalline compound precipitated. It was col-
lected and recrystallized from ethanol to give pale yellow
needles of 3-(20-[thienyl]methylene)amino-3,4-dihydro-
4-imino-8-ethoxycarbonyl-7-methyl-9(20-thienyl) pyrido
[30,20:4,5]thieno[3,2-d]pyrimidine (22; yield: 50%; m.p.:
232ꢁC–234ꢁC, Rf: 0.78 (1:4). IR: 3245 (NH), 1726 (C═O,
3.1.18 | Synthesis of 4-(20-thienyl)
methylenehydrazino-8-ethoxycarbonyl-
7-methyl-9-(20-thienyl)pyrido[30,20:4,5]thieno
[3,2-d]pyrimidine (20)
A mixture of hydrazino compound 12 (0.38 g, 1 mmol)
and thiophene-2-carboxaldehyde (2 ml) was refluxed
1
ester). H NMR (CDCl3): 10.09 (s, 1H, NH), 9.11 (s, 1H,

16
ABUELHASSAN ET AL.
pyrimidine-H), 8.50 (s, 1H, CH═N), 7.09–8.03 (m, 6H,
thiophene-H), 4.17–4.21 (q, J = 5 Hz, 2H, OCH2), 2.81 (s,
3H, CH3), 1.13–1.15 (t, J = 5 Hz 3H, CH3 ester). Anal.
calcd. For C22H17N5O2S3: C, 55.10; H, 3.57; N, 14.60.
Found: C, 55.00; H, 3.31; N, 14.77.
removed, and the cells were washed five times with dis-
tilled water. Aliquots of 70 μl SRB solution (0.4% w/v)
were added and incubated in a dark place at room tem-
perature for 10 min. Plates were washed three times with
1% acetic acid and allowed to air-dry overnight. Then,
150 μl of TRIS (10 μM) was added to dissolve protein-
bound SRB stain; the absorbance was measured at
540 nm using a BMG L ABTECH® -FLUO star Omega
microplate reader (Ortenberg, Germany). The effect of
the vehicle solvent (DMSO) on the growth of these cell
lines was evaluated in all the experiments by exposing un
reacted control cells to the maximum concentration
(0.5%) of DMSO used in each assay.
3.2 | Biological activity
3.2.1 | Antifungal activity
This study was carried out by using the disk diffusion
method, which was reported by Kwon-Chung and Ben-
nett [42], because of the inhibition zone diameters were
clear with good definition. The inhibition zones (mm) of
the screened compounds were compared with those of
Voriconazole, which was used as a reference for the anti-
fungal tests.
4 | CONCLUSION
In this paper, we have reported a facile approach for
synthesis of new thienylthieno[2.3-b]pyridines and
thienylpyrido[30,20:4,5]thieno[3,2-d]pyrimidines started
3.2.2 | Anticancer activity
from
3-cyano-5-ethoxcarbonyl-6-methyl-4-(20-thienyl)
pyridine-2-(1H)-thione (1). In addition, 4-hydrazino-
8-ethoxycarbonyl-7-methyl-9-(20-thienyl)pyrido[30,20:4,5]
thieno[3,2-d]pyrimidine (12) and 3-Amino-3,4-dihydro-
4-imino-8-ethoxycarbonyl-7-methyl-9-(20-thienyl)pyrido
[30,20:4,5]thieno[3,2-d]pyrimidine (14) were used as pre-
cursors for synthesizing novel thienylpyrido[30,20:4,5]
thieno[3,2-d]pyrimidines as well as their condensed s-
triazolo derivatives. All synthesized compounds were
characterized, elemental and spectral analyses such as
The SRB method of monitoring in vitro cytotoxicity was
used with multi well plates. The stock concentration of
the entire synthesized compounds in DMSO was
10 μg/ml, and this was used to prepare the working dilu-
tion. The final DMSO concentration used in the experi-
ments was ≤0.5% as the working concentration. The
compounds in serial dilutions (0.01, 0.1, 1.0, 10, and
100 μg/ml) were added after 24 h of culture and the cells
were cultured for another 24 h at 37ꢁC. The cell viability
was determined in each experiment using MTT colori-
metric assay [43,44]. Data were calculated as percent of
cell viability.
1
IR, H NMR, and 13C NMR. In addition, majority of
synthesized compounds were screened in vitro for their
antifungal activity against five strains of fungi. More-
over, some of the synthesized compounds were
screened in vitro for their anticancer activity against
HEPG-2 and MCF-7 cell lines. The results obtained,
encourage us to publish this research in your esteemed
journal, JHC.
Cell cultures
MCF7: Breast Adenocarcinoma and HepG2: Hepatocellu-
lar carcinoma cell lines were obtained from Nawah Sci-
entific Inc., (Mokatam, Cairo, Egypt). Cells were
maintained in DMEM media supplemented with
100 mg/ml of streptomycin, 100 units/ml of penicillin,
10% of heat-inactivated fetal bovine serum in humidified,
and 5% (v/v) CO2 atmosphere at 37ꢁC.
DATA AVAILABILITY STATEMENT
Data available in supplementary material of the article.
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How to cite this article: S. Abuelhassan, E. A.-
G. Bakhite, A. E. Abdel-Rahman, A. F. M. El-
Mahdy, J Heterocyclic Chem 2021, 1. https://doi.
org/10.1002/jhet.4310
SUPPORTING INFORMATION
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article.