Design, synthesis and anticancer activity of new polycyclic: Imidazole, thiazine, oxathiine, pyrrolo-quinoxaline and thienotriazolopyrimidine derivatives

Article abstract of DOI:10.3390/molecules26072031

In this article, we showed the synthesis of new polycyclic aromatic compounds, such as thienotriazolopyrimidinones, N-(thienotriazolopyrimidine) acetamide, 2-mercapto-thienotriazolopyrimidinones, 2-(((thienotriazolopyrimidine) methyl) thio) thieno-triazolopyrimidines, thieno-pyrimidotriazolo-thiazines, pyrrolo-triazolo-thienopyrimidines, thienopyrimido-triazolopyrrolo-quinoxalines, thienopyrimido-triazolo-pyrrolo-oxathiino-quinoxalinones, 1,4-oxathiino-pyrrolo-tri-azolothienopyrimidinones, imidazopyrrolotriazolothienopyrimidines and 1,2,4-triazoloimidazo-pyrrolotriazolothienopyrimidindiones, based on the starting material 2,3-diamino-6-benzoyl-5-methylthieno[2,3-d]pyrimidin-4(3H)-one (3). The chemical structures were confirmed using many spectroscopic ways (IR, ¹H, ¹³C, ?NMR and MS) and elemental analyses. A series of thiazine, imidazole, pyrrole, thienotriazolopyrimidine derivatives were synthesized and evaluated for their antiproliferative activity against four human cancer cell lines, i.e., CNE2 (nasopharyngeal), KB (oral), MCF-7 (breast) and MGC-803 (gastric) carcinoma cells. The compounds 20, 19, 17, 16 and 11 showed significant cytotoxicity against types of human cancer cell lines.

Full text of DOI:10.3390/molecules26072031

molecules
Article
Design, Synthesis and Anticancer Activity of New Polycyclic:
Imidazole, Thiazine, Oxathiine, Pyrrolo-Quinoxaline and
Thienotriazolopyrimidine Derivatives
Ameen Ali Abu-Hashem 1,2,* , Sami A. Al-Hussain 3 and Magdi E. A. Zaki 1,3
1
Heterocyclic Unit, National Research Centre, Photochemistry Department, Dokki, Giza 12622, Egypt;
mezaki@imamu.edu.sa or meazaki@gmail.com
Chemistry Department, Faculty of Science, Jazan University, Jazan 45142, Saudi Arabia
Department of Chemistry, Faculty of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU),
2
3
Riyadh 13318, Saudi Arabia; sahussain@imamu.edu.sa
*
Correspondence: aminaliabuhashem@yahoo.com or aaabuhashem@jazanu.edu.sa; Tel.: +20-012-2521-1700 or
966-591-363-915; Fax: +20-2-3337-0931
+
Abstract: In this article, we showed the synthesis of new polycyclic aromatic compounds, such as
thienotriazolopyrimidinones, N-(thienotriazolopyrimidine) acetamide, 2-mercapto-thienotriazolo-
pyrimidinones, 2-(((thieno-triazolopyrimidine) methyl) thio) thieno-triazolopyrimidines, thieno-
pyrimidotriazolo-thiazines, pyrrolo-triazolo-thienopyrimidines, thienopyrimido-triazolopyrrolo-
quinoxalines, thienopyrimido-triazolo-pyrrolo-oxathiino-quinoxalinones, 1,4-oxathiino-pyrrolo- tri-
azolothienopyrimidinones, imidazopyrrolotriazolothienopyrimidines and 1,2,4-triazoloimidazo-
pyrrolotriazolothienopyrimidindiones, based on the starting material 2,3-diamino-6-benzoyl-5-
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Abu-Hashem, A.A.;
Al-Hussain, S.A.; Zaki, M.E.A.
Design, Synthesis and Anticancer
Activity of New Polycyclic:
Imidazole, Thiazine, Oxathiine,
Pyrrolo-Quinoxaline and
methylthieno[2,3-d]pyrimidin-4(3H)-one (
3
). The chemical structures were confirmed using many
1
13
spectroscopic ways (IR, H, C,
−
NMR and MS) and elemental analyses. A series of thiazine,
imidazole, pyrrole, thienotriazolopyrimidine derivatives were synthesized and evaluated for their
antiproliferative activity against four human cancer cell lines, i.e., CNE2 (nasopharyngeal), KB (oral),
MCF-7 (breast) and MGC-803 (gastric) carcinoma cells. The compounds 20, 19, 17, 16 and 11 showed
Thienotriazolopyrimidine
significant cytotoxicity against types of human cancer cell lines.
Derivatives. Molecules 2021, 26, 2031.
https://doi.org/10.3390/
Keywords: thienopyrimidinone; thienotriazolopyrimidinone; 1,2,4-triazole; thiazine; pyrrole;
molecules26072031
oxathiinoquinoxaline; imidazole; imidazopyrrolotriazole; anticancer activity
Academic Editor: Brullo Chiara
Received: 6 March 2021
Accepted: 23 March 2021
Published: 2 April 2021
1
. Introduction
Cancer is one of the most recent serious diseases that afflict humans and ultimately
leads to their death. From here, researchers began to develop and discover much ef-
fective anticancer therapeutics, such as treatment of cancer using chemotherapy [ ] and
heterocyclic compounds, such as thienopyrimidine, triazolopyrimidine, quinoxaline and
imidazole derivatives containing anticancer drugs [ ], as follows: Azathioprine, Pimonida-
1
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
2
zole, Dacarbazine, Misonidazole, Fadrozole, Tipifarnib, Bendamustine, Indimitecan and
Nilotinib. Therefore, the chemical studies of each of thiophene, pyrimidine, triazole,
thiazine, pyrrole, quinoxaline and imidazole nucleus play a significant role in the syn-
thesis of a diversity of fused heterocyclic compounds having a wide range of biologi-
cal and pharmacological activities. Accordingly, previous scientific studies confirmed
that thienopyrimidine derivatives have various biological and pharmacological activi-
ties, such as antibiotics [
idant and antitumor agents [
anti-inflammatory and analgesic activities [11
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
3
], antimicrobial [
4
,5
], anticonvulsants [
] and mitotic arrest of breast cancer [10],
12], antiglaucoma agents [13], platelet ag-
6], antiviral [7], antiox-
8
], anticancer [
9
,
gregation inhibitors [14], anti-hyperlipidemia [15], antidepressant, anti-inflammatory and
antimicrobial activities [16]. In addition, the thiazolopyrimidine, 1,2,4-triazolopy- rim-
idine and thienotriazolopyrimidinone derivatives possess such biological activities as
4
.0/).
Molecules 2021, 26, 2031. https://doi.org/10.3390/molecules26072031
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2031
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anti-inflammatory and analgesic activity [17
,18], antimicrobial [19] and anticancer activity
through a potential of the enzyme (PARP-1) inhibition [20]. Lately, in connection to contin-
uing work in the synthesis and biological evaluation of new polycyclic fused thienopyrim-
idine, purine and 1,2,4-triazole systems, purine derivatives are of great importance and
wide applications in many biological activities, such as antitumor [21] and the potential
xanthine oxidase (XO)-inhibitory activities, as well as many biological activities when
fused with 1,2,4-triazole ring, shown in Figure 1, as the following: 7
,2,4-triazolopurines (A) [22], 1,2,4-triazolopurines (B) [23], thienotriazolopyrimidinones
C) and 2-sub-thienotriazolopyrimidinones (D) as a new class of the XO inhibitors [24].
β-D-ribofuranosyl-
1
(
Additionally, Azathioprine (E) is an anticancer drug that possesses considerable potential,
because of its ability to interfere with DNA prepared and then stop growth and division
cells and it is used for the treatment of metastatic malignant melanoma and cell carcinoma
of the pancreas [25].
Figure 1. Chemical structures of formerly synthesized drugs: triazolopurines and thienotriazolo- pyrimidines analogues.
The nitrogen atoms containing heterocycles, especially, display a different range of
biological activities, due to their similarities with numerous synthetic and natural molecules
with recognized biological activities [26]. The benzimidazole and imidazole rings have
been generally used as the substantial basic structure for the development of therapeutic
molecules of biological and pharmaceutical activities. An example of five-membered
heterocycles is imidazole, which is spread between the significant biological building blocks.
Thus, many drugs contain imidazole, such as the following: Sertaconazole is used as an
antifungal agent [27]. Omeprazole is antiulcer and controls the acid secretion in the stomach
and it is considered clinically superior to H -receptor antagonists [28,29]. Mizolastine is
2
an antihistaminic and potent antagonist at H receptor sites for the treatment of allergic
1
rhinoconjunctivitis and urticarial [30]. Candesartan is used as a receptor antagonist, because
it contains a bulky lipophilic group, carboxylic acid, the biphenyl group that is more
efficient than the tetrazole analogue [31]. Azanidazole is an antiparasitic drug and used
as an antibacterial and antiprotozoal drug [32]. Maribavir is an antiviral drug, which
is used in the treatment and prevention of human cytomegalovirus (HCMV) disease in
hematopoietic stem cell/bone marrow transplant patients [33], as shown in Figure 2.
This article is a supplement of our former work about the preparation of hetero-
cyclic compounds resulting from pyrimidine and thienopyrimidine derivatives, which
possess biological activities [
thienotriazolopyrimidinone, thienopyrimidotriazolopyrrolooxathiinoquinoxalines and
,2,4-triazoloimidazopyrrolotriazolothienopyrimidinedione derivatives were synthesized
4,5,8,11,12,19–21,34–48]. In this manuscript, these compounds,
1
from 2,3-diamino-6-benzoyl-5-methylthieno[2,3-d]pyrimidinone and evaluated as having
antiproliferative activities against types of human carcinoma cancer cell lines.

Molecules 2021, 26, 2031
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Figure 2. Chemical structures of formerly synthesized drugs: benzimidazoles and imidazoles.
2. Results and Discussion
2.1. Synthesis
The 2-(4-aminophenyl)-6-benzoyl-7-methylthieno[2,3-d][1,2,4]triazolo[1,5-a] pyrimidin-
(3H)-one ( ) was synthesized via the oxidative cyclization of 2,3-diamino- 6-benzoyl-5-
8
4
methylthieno[2,3-d]pyrimidin-4(3H)-one (
phoric acid at 250–280 C for 18–20 h. The IR spectra of compound
3
) [
4
,
18] and 4-aminobenzoic acid in polyphos-
◦
4
showed the presence
−
1
of a broad bands absorption at 3405 and 3355 cm of one NH and one NH group and two
2
−
1
bands absorption at 1680 and 1665 cm for two carbonyl groups. The proton nuclear mag-
1
netic resonance ( H–NMR) spectrum of compound
4
displayed two singlet broad signals
at
δ
6.10 and 10.70 ppm, agreeing to the three protons of the one NH and one NH group
2
(
D O-exchangeable). The reaction of the latter compound
4
with sodium ethoxide (NaOEt)
2
afforded the conforming sodium salt, which, upon treatment with acetic anhydride (Ac O),
2
provided the acetylated (5). In another way, the acetylated of compound 4 with acetic
anhydride, itself, for one hour formed the N-(4-(3-acetyl- 6-benzoyl-7-methyl-8-oxo-3,8-
dihydrothieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidin-2-yl) phenyl) acetamide (
5
) in 74% yield.
1
The H–NMR spectrum of compound
5
demonstrated one singlet broad signal at 11.90 ppm,
correspondent to the one proton of the one NH group with D O-exchangeable. Moreover,
2
refluxing 2,3-diamino-benzoyl-methylthieno- pyrimidinone (3) with a proper aromatic
aldehyde, namely benzaldehyde, 4-chloro- benzaldehyde or 4-methoxybenzaldehyde, re-
spectively, in dimethylformamide solution, having a catalyst amount of potassium iodide for
an extended time, gave the products 6-benzoyl-2-(4-substituted-phenyl)-7-methylthieno[2,3-
1
d][1,2,4]triazolo[1,5-a]pyrimidin- 8 (3H)-one (6a
of compound 6a established one singlet broad signal at
one proton of NH group with D O-exchangeable. The mass spectra of compounds 6a
showed molecular ion peaks at m/z 386 (M , 90%), 420 (M , 100%) and 416 (M , 95%),
respectively, as shown in Scheme 1.
–
c
) in good yields. The H–NMR spectrum
δ
10.55 ppm, corresponding to the
–c
2
+
+
+

Molecules 2021, 26, 2031
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Scheme 1. Synthesis of 2-(sub)-thieno[2,3-d][1,2,4]triazolopyrimidinone derivatives.
Heating of 2,3-diamino-benzoyl-methylthienopyrimidinone (3) with methylphenyl-
carbamodithioate and red mercury (II) oxide (HgO) catalyzed in dimethylformamide
to give 6-benzoyl-7-methyl-2-(phenylamino)thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidin-8
(
3H)-one (
7
) in good yield. IR spectrum of compound
7
revealed the presence of broad
−
1
−1
bands absorption at 3360 and 3320 cm of (2NH) groups and 1715 and 1676 cm of two
carbonyl groups, and H–NMR spectrum of compound
1
7 displayed two singlet broad
signals at
δ 9.60 and 10.80 ppm, conforming to the two protons of the (2NH) groups
with D O-exchangeable. Moreover, 6-benzoyl-7-methylthieno[2,3-d][1,2,4]triazolo[1,5-
2
a]pyri- midine-2,8(1H,3H)-dione (
methyl- thienopyrimidinone ( ) with ethyl-chloroformate and anhydrous potassium car-
bonate in dry acetone. IR spectra of compound showed the absence of some ab-
sorption bands for two amino groups, with the presence of bands absorption at 3352
8) was prepared by reacting of 2,3-diamino-benzoyl-
3
8
−
1
−1
and 3310 cm of (2NH) groups and 1722, 1685 1674 cm of three carbonyl groups.
1
The H–NMR spectrum of compound
8 designated two singlet broad signals at δ 10.20
and 11.10 ppm, corresponding to the two protons of the (2NH) groups. On the other
hand, heating and stirring of 2,3-diamino-benzoyl-methylthienopyrimidinone ( ) with
3
carbon disulfide in an methanolic potassium hydroxide solution led to the formation
of 6-benzoyl-2-mercapto-7-methylthieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidin-8(3H)-one
(
9
). IR spectra of compound
9
presented the presence of broad bands absorption at 3345
−
1
1
and 2372 cm of (NH) and (SH) groups, respectively, and H–NMR spectrum of com-
pound
9 revealed two singlet signals at 2.62 and 9.05 ppm, agreeing to two protons of
the (SH) and (NH) groups with D O-exchangeable, respectively. In addition, the re-
2
action of 2,3-diamino-benzoyl-methylthienopyrimidinone (3) with chloroacetic acid in
hydrochloric acid (4N, HCl) produced the 6-benzoyl-2-(chloromethyl)-7-methylthieno[2,3-
d][1,2,4]triazolo[1, 5-a] pyrimidin-8 (3H)-one (10). IR spectra of compound (10) obtained
−
1
−1
the presence of bands absorption at 3341 cm of (NH) and 1724 and 1680 cm of two
1
carbonyl groups, and H–NMR spectrum of compound 10 discovered one singlet signal at
9
.15 ppm, conforming to the one proton of the (NH) group (D O-exchangeable). Thus, the
2
heating under reflux of 2-mercapto-thienotriazolopyrimidinone (9) with 2-(chloromethyl)-

Molecules 2021, 26, 2031
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thienotriazolopyrimi- dinone (10) in an methanolic sodium metal solution produced
the 6-benzoyl-2-(((6-ben- zoyl-7-methyl-8-oxo-3,8-dihydrothieno[2,3-d][1,2,4]triazolo[1,5-
a]pyrimidin-2-yl)methyl)thio)-7-methylthieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidin-8(3H)-
1
one (11). The H–NMR spectra of compound 11 exhibited two broad singlet signals at
δ
9
.80 and 10.10 ppm, identical to the two protons of the (2NH) groups (D O-exchangeable).
2
+
Mass spectra of compounds (7- 11) showed molecular ion peaks at m/z 401 (M , 95%), 326
+
+
+
+
(M , 100%), 342 (M , 93%), 358 (M , 91%) and 664 (M , 90%), respectively as shown in
Scheme 2.
Scheme 2. Synthesis of methylthieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidinone derivatives.
In addition, 6-benzoyl-2-(mercaptomethyl)-7-methylthieno[2,3-d][1,2,4]triazolo[1,5-
a]pyrimidin-8 (1H)-one (12) was synthesized from 2,3-diamino-benzoyl-methylthieno-
pyrimidinone (
3) and mercaptoacetic acid, when refluxed in absolute ethanol. IR spec-
–
1
trum of compound (12) revealed absorption bands at 3370, 2355, 1730 and 1675 cm
13
of (NH), (SH) and two carbonyl groups, respectively. The C–NMR spectra of com-
pound (12) displayed signals at 20.3 and 30.1 ppm, agreeing to the two carbon atoms
of CH and CH groups, and also at 166.4 and 181.2 ppm, matching the two carbon
δ
3
2
atoms of the two carbonyl groups. Moreover, Alkylation of thienotriazolopyrimidinone
12), in dry dimethylormamide and potassium hydroxide solution with ethylbromoac-
(
0
0
0
0
etate, afforded 2-benzoyl-3-methylthieno[2”,3”:4 ,5 ]pyrimido[1 ,2 :2,3][1,2,4]triazolo[5,1-
c][1,4]thiazine-4,7(8H,10H)-dione (13). C–NMR spectra of compound (13) showed signals
at 20.5, 33.2 and 37.5 ppm, correspondent to three carbon atoms of one CH and two CH
13
δ
3
2
groups, respectively, and 165.9, 167.2 and 182.1 ppm, identical to three carbon atoms of the
three carbonyl groups. In addition, the synthesis of 6-benzoyl-7-methyl-8-oxo-3, 8-dihydro-
thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylic acid (14) was synthesized in 72%
yield from 2,3-diamino-benzoyl-methylthienopyrimidinone (
3), oxalic acid and anhydrous
potassium carbonate in dry dimethylformamide under refluxing conditions for 20–25 h. IR
–1
spectra of compound (14) showed absorption bands at 3375 and 3360 cm of OH and NH
–1
1
groups and 1745, 1735 and 1679 cm of three carbonyl groups. The H–NMR spectrum of

Molecules 2021, 26, 2031
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compound (14) displayed two singlet broad signals at
δ
10.20 and 12.45 ppm, correspond-
ing to the two protons of the NH and OH groups (with D O-exchangeable), respectively.
2
Furthermore, condensation and cyclization of thienotriazolopyrimidinone (12), oxalic acid
with the presence of anhydrous potassium carbonate in dry dimethyl- formamide gave
0
0
2
-benzoyl-8-hydroxy-9-mercapto-3-methylpyrrolo[1 ,2 :2,3][1,2,4] triazolo[1,5-a]thieno[2,3-
1
d]pyrimidin-4,7-dione (15). The H–NMR spectrum of compound (15) showed two singlet
signals at
δ 2.70 and 12.10 ppm, agreeing to the two protons of the SH and OH groups
(
D O-exchangeable). The mass spectrum of compounds 12
,
13
,
14 and 15 showed molec-
2
+
+
+
+
ular ion peaks at m/z 356 (M , 100%), 396 (M , 95%), 354 (M , 90%) and 410 (M , 98%),
respectively, as shown in Scheme 3.
Scheme 3. Synthesis of thiazine, pyrrolo-1,2,4-triazolo-thieno[2,3-d]pyrimidinones.
Additionally, the treatment of pyrrolotriazolothienopyrimidine-4,7-dione (15) with
o-phenylene-diamine in refluxing dimethylformamide and anhydrous potassium car-
0
00 000 00 00
bonate resulted in the formation of 2-benzoyl-3-methyl-8,13-dihydrothieno[2 ,3 :4 ,5 ]
0
0
00
0
0
0
0
pyrimido[1 ,2 :2 ,3 ][1,2,4]triazolo [1 ,5 :1,2]pyrrolo[3,4-b]quinoxaline-4,7-dione (16). The
1
H NMR spectra of compound 16 exhibited two singlet broad signals at
δ 11.50 and
1
1.57 ppm, conforming to the two protons of the two (2NH) groups (D O-exchangeable).
2
0
000 0000 000 000
000 000
”
Likewise, 2-Benzoyl-3-Methylthieno[2 ,3 :4 ,5 ]Pyrimido[1 ,2 :2
,3
1 , 5 :1 ,2 ]Pyrrolo[3 ,4 :5,6][1,4]Oxathiino[2,3-b]Quinoxaline-4,7-Dione (17) was synthe-
sized from the reaction of compound 15 with 2,3-dichloroquinoxaline in boiling absolute
”][1,2,4]Triazolo
0
0
00
0
0
0
0
[
13
ethanol with the presence of a catalytic amount of triethylamine. In the C–NMR spec-
trum of compound 17, it displayed signals at 166.5, 170.9 and 183.8 ppm, due to three
carbonyl groups. The pyrrolotriazolothienopyrimidine-4,7-dione (15) also reacted with
-halo-ketones as chloroacetyl-chloride in dimethylformamide with the presence of anhy-
δ
α

Molecules 2021, 26, 2031
7 of 20
drous potassium carbonate or in acetic acid with the presence of zinc dust to afford the
0
0
0
0
corresponding 8-benzoyl-9-methyl-[1,4]oxathiino[3”,2”:3 ,4 ]pyrrolo[1 ,2 :2,3][1,2,4]triazo-
1
lo[1,5-a]thieno[2,3-d]pyrimidine-3,10,13 (2H)-trione (18). The H–NMR spectrum of com-
pound 18 showed one singlet signal at 5.10 ppm, conforming to the two protons of the
CH , and multiple singlet signals at
δ
7.61–7.90 ppm, agreeing to the five protons of the
2
phenyl group. Moreover, the reaction of pyrrolotriazolothienopyrimidine-4,7-dione (15
)
with thiourea in a mixture of dimethylformamide or dioxane in the presence of catalytic
amount of piperidine under control (TLC) afforded the 7-benzoyl-2-mercapto-8- methyl-
0
0
0
0
1
H-imidazo[4”,5”:3 ,4 ]pyrrolo[1 ,2 :2,3][1,2,4]triazolo[1,5-a]thieno[2,3-d]pyrimidi- ne-9,
1
2-dione (19). The IR spectrum of compound 19 displayed the presence of broad band
−
1
−1
absorption at
ν
3355 cm of one (NH) group, 2357 cm of one (SH) group and 1729, 1681
−
1
1
and 1670 cm of the three carbonyl groups. The H–NMR spectrum of compound 19
demonstrated two singlet broad signals at
δ 10.83 and 11.90 ppm, corresponding to the
two protons of the (SH) and (NH) groups, respectively (D O-exchangeable). Furthermore,
2
refluxed 19 with benzo-hydrazide in dry dimethylformamide and anhydrous potassium
carbonate formed a new heterocyclic compound, 10- benzoyl-9-methyl-3-phenyl- 1H-
0
00 000
0
0
0
0
[
[
1,2,4]triazolo[4 ,3 :1”,2”]imidazo[4”,5”:3 ,4 ]pyrrolo[1 ,2 :2,3][1,2,4]triazolo[1,5-a]thie- no
1
2,3-d]pyrimidine-5,8-dione (20). The H–NMR spectrum of compound 20 revealed one
singlet signal at δ 12.01 ppm for one proton of NH (D O-exchangeable). The mass spectra of
2
+
+
compounds 16
4
–
20 exhibited the molecular ion peaks at m/z 466 (M , 100%), 536 (M , 94%),
50 (M , 90%), 434 (M , 85%) and 518 (M , 100%), respectively, as shown in Scheme 4.
+
+
+
Scheme 4. Synthesis of polycyclic: thienopyrimidotriazolopyrrolo; -quinoxaline, 1,4-oxathiino and
imidazole derivatives.

Molecules 2021, 26, 2031
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2.2. Biological Activities
2.2.1. Anticancer Screening (In Vitro Cytotoxicity)
The new compounds that were synthesized, such as thieno[2,3-d]pyrimidinones, thienotri-
azolopyrimidinones, thienopyrimido-triazolo-thiazines, pyrrolo-triazolothieno- pyrimidines,
thieno-pyrimido-triazolopyrrolo-quinoxalines, thieno-pyrimidotriazolo-pyrrolo-oxathiino-quin-
oxalines, 1,4-oxathiino-pyrrolotriazolo-thienopyrimidin-triones, imidazopyrrolotriazolothienop-
yrimidin-diones and 1,2,4-triazoloimidazopyrrolotriazol- othienopyrimidin-diones, were tested
for their in vitro cytotoxicity using the standard method to test cell growth rate and toxicity
of the culture (MTT) method [34,38,40,48] against the human nasopharyngeal carcinoma
cells (CNE2), oral carcinoma cells (KB), breast adenocarcinoma cells (MCF-7) and gastric
carcinoma cells (MGC-803). The MTT method is based on the reduction of soluble 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, as shown in Table 1. Through
the final results of the effect of compounds against the types of human carcinoma cell lines,
we found many compounds, such as 20
activity. Some of the compounds, 20 19, exhibited the highest effect of cytotoxicity and
outperformed the 5-Fluorouracil-positive control. Many compounds had moderate results
in activity against cancer cells, such as 15 13 and 12 and the rest of the compounds in
the manuscript, had a weak effect against cancer cells. Consequently, the compounds 20
17 16 11 and 18 possess the best activities against all carcinoma cell lines as follows:
the CNE2 (IC : Comp. 20 19 17 16 11 and 18 gives 11.6–11.7, 11.8–11.9, 12.1 and 12.4
M); the KB (IC : Comp. 20 19 17 16
M); the MCF-7 (IC : Comp. 20 19
–19, 17–16, 11 and 18 gave the highest anticancer
–
, 5, 7,
,
19,
,
,
–
,
– ,
50
µ
µ
–
,
–
,
,
11 and 18 gives 10.5, 10.7, 10.8–10.9, 11.1 and 11.3
16 11 and 18 gives 11.3–11.4, 11.5–11.6, 11.8 and
19 17 16 11 and 18 gives 10.9, 11.1, 11.3, 11.5,
M), respectively. All results of the compounds as anticancer activities are
50
–
17
– ,
–
5
0
1
1
1.9
µ
M); the MGC-803 (IC : Comp. 20
,
– ,
5
0
1.7 and 11.9
µ
mentioned in Table 1.
Table 1. Cytotoxic activity of the new compounds against different human cancer cell lines
In Vitro Cytotoxicity IC50 (µM)
Compounds
CNE2 a
KB a
MCF-7 a
MGC-803 a
1
2
3
4
5
>50
>50
>50
>50
>50
>50
>50
>50
33.5 ± 1.8
21.3 ± 1.1
13.1 ± 1.1
26.3 ± 1.4
24.5 ± 1.5
25.1 ± 1.2
14.2 ± 1.7
29.1 ± 1.2
18.3 ± 1.7
19.1 ± 1.6
12.1 ± 1.2
16.1 ± 1.4
15.4 ± 1.3
20.5 ± 1.8
12.7 ± 1.2
11.9 ± 1.5
11.8 ± 1.4
12.4 ± 1.5
11.7 ± 1.3
11.6 ± 1.1
11.8 ± 1.4
32.2 ± 1.2
20.5 ± 1.3
12.3 ± 1.4
25.8 ± 1.7
23.1 ± 1.2
24.2 ± 1.3
13.5 ± 1.5
28.7 ± 1.1
17.2 ± 1.5
18.4 ± 1.2
11.1 ± 1.3
15.3 ± 1.3
14.1 ± 1.2
19.8 ± 1.5
11.7 ± 1.6
10.9 ± 1.2
10.8 ± 1.1
11.3 ± 1.4
10.7 ± 1.5
10.5 ± 1.5
10.7 ± 1.2
34.1 ± 1.3
20.9 ± 1.2
12.5 ± 1.3
26.9 ± 1.9
24.2 ± 1.4
25.5 ± 1.5
13.7 ± 1.1
29.5 ± 1.8
17.8 ± 1.4
18.7 ± 1.3
11.8 ± 1.5
15.9 ± 1.2
14.8 ± 1.1
19.8 ± 1.4
12.1 ± 1.2
11.6 ± 1.1
11.5 ± 1.4
11.9 ± 1.3
11.4 ± 1.1
11.3 ± 1.2
11.5 ± 1.3
35.7 ± 1.5
21.5 ± 1.4
12.8 ± 1.2
27.5 ± 1.3
25.2 ± 1.1
26.8 ± 1.8
13.9 ± 1.4
30.2 ± 1.6
18.5 ± 1.3
19.2 ± 1.5
11.7 ± 1.4
16.5 ± 1.1
15.7 ± 1.2
20.4 ± 1.6
12.2 ± 1.4
11.5 ± 1.3
11.3 ± 1.5
11.9 ± 1.1
11.1 ± 1.2
10.9 ± 1.4
11.1 ± 1.1
6
a
6
6
b
c
7
8
9
1
0
1
2
3
4
5
6
7
8
9
0
1
1
1
1
1
1
1
1
1
2
5
-Fluorouracil
a
CNE2 cells are drug-sensitive human nasopharyngeal carcinoma cells; KB cells are drug-sensitive human
oral carcinoma cells; MCF-7 cells are drug-sensitive human breast adenocarcinoma cells and MGC-803 cells are
drug-sensitive human gastric carcinoma cells.

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2.2.2. Structural Activity Relationship (SAR)
The relationship of the chemical structures (SARs) of the prepared compounds to
the results mentioned in this study, in terms of the ability of these compounds to apply
cytotoxicity activity to human carcinoma cells, is due to several possibilities, including
the following: (
,4-thiazine, pyrrolo-1,2,4-triazole, pyrroloquinoxaline, oxathiinoquinoxaline, 1,4-oxa-
thiinopyrrolotriazole, imidazopyrrolotriazole and 1,2,4-triazoloimidazopyrrolo-triazole
moieties may be requested for a broad spectrum of the cytotoxicity activity. ( ) 1,2,4-
A) The presence of the thienopyrimidinones, 1,2,4-triazolopyrimidinone,
1
B
triazoloimidazopyrrolotriazolothienopyrimidindione (20), imidazopyrrolotriazolo- thienopy-
rimidindione (19), thienopyrimidotriazolopyrrolooxathiinoquinoxalindione (17), thieno-
pyrimidotriazolopyrroloquinoxalindione (16), 2-(((6-benzoyl-thienotriazolo- pyrimidin-2-
yl)methyl) thio)-thienotriazolopyrimidinene (11) and 1,4-oxathiinopyrrolo- triazolothienop-
yrimidin-trione (18) demonstrated great anticancer activity in vitro, and this corresponds to
previous scientific studies, because numerous heterocyclic compounds have pharmacologi-
cal activities, such as imidazoles, benzimidazoles, thieno-[2,3-d]pyrimidines, 1,2,4-triazoles,
thiazolopyrimidines, pyrrolothiazolo-pyrimidines, pyrrolotriazines, pyrrolotriazepines,
triazolopyrrolothiazolopyrimidines, quinolines, quinoxalines, thiazolidinones, thiazines,
thienotriazoles, thiophenes, phenyl, benzoyl and methyl, which have displayed potent
anticancer activity [2,5,8,10,20,25,34,38,40,48–50]. (C) Thence, the compounds 20, 19, 17,
16,
11 and 18 offered high cytotoxicity against all human carcinoma cell lines, such as
nasopharyngeal (CNE2), oral (KB), breast (MCF-7) and gastric (MGC-803), during the
comparison with 5-Fluorouracil as a standard drug, shown in Table 2. From studying the
chemical structures of compounds 20
are preferred for the following reasons: (
–
16 and 11, it becomes clear that these compounds
) they contain thienotriazolopyrimidinene as
I
the basis in these compounds. (II) Some compounds containing pyrrole, imidazole and
triazole rings are all linked with the thienotriazolopyrimidinene basis, such as compound
20. (III) Some compounds that contain pyrrole and imidazole rings are all linked with
the thienotriazolopyrimidinene basis, such as compound 19. (IV) Some compounds that
contain pyrrole, oxathiine and quinoxaline rings are all linked with the thienotriazolopy-
rimidinene basis, such as compound 17. (V) Some compounds that contain pyrrole and
quinoxaline rings are all linked with the thienotriazolopyrimidinene basis, such as com-
pound 16. (VI) Some compounds that contain pyrrole and oxathiine rings are all linked
with the thienotriazolopyrimidinene basis, such as compound 18. (VII) Some compounds
linked with two molecules of thienotriazolopyrimidinene, such as compound 11. (VIII
)
These are in addition to many functional groups, such as methyl and phenyl, and many
heteroatoms, such as nitrogen, oxygen and sulfur.
Table 2. The best new compounds to cytotoxic activity against different human cancer cell lines.
In Vitro Cytotoxicity IC50 (µM)
Compounds/Chemical Structures
CNE2 a
KB a
MCF-7 a
MGC-803 a
1
1
2.1 ± 1.2
11.1 ± 1.3
11.8 ± 1.5
11.7 ± 1.4
1.9 ± 1.5
10.9 ± 1.2
11.6 ± 1.1
11.5 ± 1.3

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Table 2. Cont.
In Vitro Cytotoxicity IC50 (µM)
Compounds/Chemical Structures
CNE2 a
KB a
MCF-7 a
MGC-803 a
1
1
1.8 ± 1.4
2.4 ± 1.5
10.8 ± 1.1
11.5 ± 1.4
11.3 ± 1.5
11.9 ± 1.1
11.3 ± 1.4
10.7 ± 1.5
11.9 ± 1.3
11.4 ± 1.1
1
1
1
1.7 ± 1.3
1.6 ± 1.1
1.8 ± 1.4
11.1 ± 1.2
10.9 ± 1.4
11.1 ± 1.1
10.5 ± 1.5
10.7 ± 1.2
11.3 ± 1.2
11.5 ± 1.3
a
CNE2 cells are drug-sensitive human nasopharyngeal carcinoma cells; KB cells are drug-sensitive human oral carcinoma cells; MCF-7
cells are drug-sensitive human breast adenocarcinoma cells and MGC-803 cells are drug-sensitive human gastric carcinoma cells.
3. Experimental Section
3.1. General Information
All melting points were taken on an Electrothermal IA 9100 series digital melting point
apparatus (Shimadzu, Tokyo, Japan). Elemental analyses were performed on Vario EL
Elementar, Langenselbold, Germany). Microanalytical data were processed in the microan-
(
alytical center, Faculty of Science, Cairo University and National Research Centre. The IR
spectra (KBr disc) were recorded using a Perkin–Elmer 1650 spectrometer (Waltham, MA,
USA). NMR spectra were determined using JEOL 270 MHz and JEOL JMS-AX 500 MHz
(JEOL, Tokyo, Japan) spectrometers with Me4Si as an internal standard. Mass spectra were
recorded on an EI Ms-QP 1000 EX instrument (Shimadzu, Tokyo, Japan) at 70 eV. Biological
evaluations were done by the anticancer unit, Mansoura University, Faculty of Pharmacy
(Department of Pharmacognosy), 35516, Egypt. All starting materials and solvents were
purchased from Sigma–Aldrich (Saint Louis, MO, USA).
3.2. Synthesis of 2,3-Diamino-6-Benzoyl-5-Methylthieno[2,3-d]Pyrimidin-4(3H)-One (3)
Compound (3.42 g, 0.01 mol) in 15 mL of hydrazine hydrate was heated under reflux
2
for 7 h. The reaction mixture was cooled and suspended in 50 mL of water. The solid was
collected by suction filtration, washed with water, and recrystallized from ethanol to afford
◦
compound
(
3
(75%). M. p.: 270–272 C. IR (KBr): νmax = 3400, 3385 (2NH ), 1650, 1622
2
−
1 1
2CO), 1585 (C=N) cm ; H–NMR (500 MHz, DMSO-d ):
δ
= 2.20 (s, 3H, CH ), 6.41 (br. s,
6
3

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2
H, CNH ), 7.43–7.94 (m, 5H, Ar-H), 11.49 (br. s, 2H, NNH ) ppm; 13C–NMR (125.7 MHz,
2
2
DMSO-d6):
δ
= 19.2, 121.3, 128.6, 129.3, 131.5, 132.3, 135.3, 142.2, 146.4, 149.5, 158.1, 168.9
+
ppm; MS (70 eV): m/z = 300 (M , 100%); Anal. Calc. (Found) for C H N O S (300.34): C,
14
12
4
2
55.99 (55.90); H, 4.03 (4.10); N, 18.66 (18.60).
3
.3. Synthesis of
2
-(4-Aminophenyl)-6-Benzoyl-7-Methylthieno[2,3-d][1,2,4]Triazolo[1,5-a]Pyrimidin-8(3H)-One (4)
Equimolar quantities of 2,3-diamino-benzoyl-methylthienopyrimidinone (3) (3 g,
0
.01 mol) and 4-aminobenzoic acid (1.37 g, 0.01 mol) were dissolved in polyphospho-
◦
ric acid (30 mL) and refluxed for 18–20 h at 250–280 C under control (TLC), with constant
stirring. The reaction mixtures were cooled and slowly poured into crushed ice with
constant stirring. The pH of the reaction mixture was detected to alkaline by adding 4N of
anhydrous sodium carbonate. The final precipitates were filtered under pressure, washed
with distilled water and recrystallized from ethanol as yellow crystals in 70% yield, m.
◦
p. > 350 C. IR (KBr): νmax = 3405, 3355 (NH NH), 3045 (CH, aryl), 2940 (CH, alkyl),
2
,
−
1 1
1
680, 1665 (2C=O), 1620 (C=N), 1585 (C=C) cm ; H–NMR (DMSO-d ):
δ
= 2.10 (s, 3H,
6
CH ), 6.10 (br. s, 2H, NH ), 7.50–7.90 (m, 9H, Ar-H), 10.70 (br,1H, NH, D O-exchangeable);
3
2
2
13
C–NMR (DMSO-d6) δ = 20.4 (1C, CH ), 115.1, 119.4, 121.3, 127.1, 128.5, 129.9, 131.5, 135.7,
3
1
40.9, 143.1, 147.2, 148.5, 151.8, 162.9 (18C, Ar-C), 165.1, 181.4 (2C, 2C=O); MS (70 eV):
+
m/z = 401 (M , 100%); Anal. Calc. (Found) for C H N O S (401.44): C, 62.83 (62.75); H,
21
15
5
2
3.77 (3.70); N, 17.45 (17.64).
3
.4. Synthesis of N-(4-(3-Acetyl-6-Benzoyl-7-Methyl-8-oxo-3,8-Dihydrothieno[2,3-d][1,2,4]
Triazolo[1,5-a]Pyrimidin-2-yl)Phenyl) Acetamide (5)
◦
Method A: To a warm (60–80 C) solution of sodium ethoxide (prepared by dissolving
0
.23 g of sodium metal in 35 mL ethanol), compound
4
(4.01 g, 10 mmol) was added, and
◦
heating (60–80 C) was continued for 40 min. The mixture was allowed to cool to room
temperature, and acetic anhydride (10 mmol) was added. The mixture was stirred under
reflux for 8–10 h, allowed to cool to room temperature and finally poured into 100 mL cold
water. The solid product precipitate was filtered off and washed with 100 mL water. The
solid obtained was filtered off, dried and crystallized from the proper solvent to produce
compound
5. Method B: A mix of compound 4 (4.01 g, 0.01 mol) and acetic anhydride
(
30 mL) was refluxed with heat for one hour, checked via (TLC). After cooling, the solution
was concentrated and poured into crushed ice. The separated solid was filtered off, dried
◦
and recrystallized from benzene as yellowish crystals in 74% yield, m. p. > 350 C. IR
(
1
7
KBr): νmax = 3325 (NH), 3040 (CH, aryl), 2935 (CH, alkyl), 1705, 1685, 1680, 1675 (4C=O),
−
622 (C=N), 1580 (C=C) cm ; H–NMR (DMSO-d ): δ = 1.90, 2.05, 2.15 (3s, 9H, 3CH ),
6 3
1 1
13
.52–7.93 (m, 9H, Ar-H), 11.90 (br, s.1H, NH, D O-exchangeable); C–NMR (DMSO-d6)
2
mboxemphδ = 20.7, 22.5, 26.3 (3C, 3CH ), 118.8, 122.1, 123.4, 126.7, 128.6, 129.5, 132.2, 136.8,
3
1
39.5, 141.4, 143.7, 148.5, 152.2, 163.5 (18C, Ar-C), 164.8, 169.2, 175.1, 182.7 (4C, 4C=O); MS
+
(
(
70 eV): m/z = 485 (M , 100%); Anal. Calc. (Found) for C H N O S (485.52): C, 61.85
61.77); H, 3.94 (3.85); N, 14.42 (14.51).
25 19 5 4
3
.5. General Procedure for Synthesis of 6-Benzoyl-2-(4-Substituted-Phenyl)-7-Methylthieno[2,3-
d][1,2,4]Triazolo[1,5-a]Pyrimidin-8(3H)-One (6a–c)
A mixture of 2,3-diamino-benzoyl-methylthienopyrimidinone (3) (3 g, 0.01 mol), the
appropriate aromatic aldehyde (0.01 mol) and the catalytic amount of potassium iodide
(
1
0.3 g, 0.002 mol) in dimethyl- formamide (40 mL) was heated and refluxed for 20–22 h at
80–200 C; the progress of reaction was monitored by TLC, and, at the end of reaction, the
◦
solvent was allowed to cool to room temperature and poured into water (100 mL). Then
the obtained solid products were filtered off, dried and crystallized from the appropriate
solvent to give compounds 6a–c in good yields, respectively.

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3
6
.6. Synthesis of
-Benzoyl-7-Methyl-2-Phenylthieno[2,3-d][1,2,4]Triazolo[1,5-a]Pyrimidin-8(3H)-One (6a)
The compound was obtained from the reaction of compound and benzaldehyde
3
(
1.06 g, 0.01 mol) with DMF/KI, as white crystals, crystallized from dioxane in 70% yield, m.
◦
p. > 350 C. IR (KBr): νmax = 3350 (NH), 3060 (CH, aryl), 2937 (CH, alkyl), 1710, 1672 (2C=O),
1
1
−
1 1
628 (C=N), 1580 (C=C) cm ; H–NMR (DMSO-d ):
δ
3
= 2.17 (s, 3H, CH3), 7.40–7.84 (m,
6
1
0H, Ar-H), 10.55 (br,1H, NH, D O-exchangeable); C–NMR (DMSO-d6) δ = 20.2 (1C,
2
CH ), 121.2, 128.1, 128.6, 128.7, 128.9, 129.5, 130.4, 132.1, 138.7, 142.4, 143.6, 148.5, 151.9,
3
+
1
62.2 (18C, Ar-C), 164.9, 182.7 (2C, 2C=O); MS (70 eV): m/z = 386 (M , 90%); Anal. Calc.
(
Found) for C H N O S (386.43): C, 65.27 (65.20); H, 3.65 (3.74); N, 14.50 (14.40).
21 14 4 2
3
.7. Synthesis of
-Benzoyl-2-(4-Chlorophenyl)-7-Methylthieno[2,3-d][1,2,4]Triazolo[1,5-a]Pyrimidin-8(3H)-One (6b
6
)
The compound was obtained from the reaction of compound and 4-chlorobenzaldehyde
3
(
1.40 g, 0.01 mol) with DMF/KI, as white crystals, crystallized from methanol in 80% yield, m.
◦
p. > 350 C. IR (KBr): νmax = 3355 (NH), 3065 (CH, aryl), 2940 (CH, alkyl), 1715, 1670 (2C=O),
1
−1 1
630 (C=N), 1582 (C=C) cm ; H–NMR (DMSO-d ):
δ
= 2.20 (s, 3H, CH ), 7.35–7.41 (dd,
6
3
2
H, J = 7.50, 7.53 Hz, 4-chlorophenyl), 7.46–7.52 (dd, 2H, J = 7.54, 7.58 Hz, 4-chlorophenyl),
13
7.62–7.90 (m, 5H, Ar-H), 10.66 (br,1H, NH, D O-exchangeable); C–NMR (DMSO-d6) δ = 20.5
2
(
1
1C, CH ), 121.7, 126.8, 128.5, 128.7, 129.4, 129.7, 132.3, 135.6, 137.1, 142.3, 143.2, 148.7, 152.3,
63.1 (18C, Ar-C), 165.5, 183.1 (2C, 2C=O); MS (70 eV): m/z = 420 (M , 100%); Anal. Calc.
3
+
(
Found) for C H ClN O S (420.87): C, 59.93 (59.85); H, 3.11 (3.19); N, 13.31 (13.39).
21 13 4 2
3
.8. Synthesis of 6-Benzoyl-2-(4-Methoxyphenyl)-7-Methylthieno[2,3-d][1,2,4]Triazolo[1,5-
a]Pyrimidin-8(3H)-One (6c)
The compound was obtained from the reaction of compound 3 and 4-methoxy- ben-
zaldehyde (1.36 g, 0.01 mol) with DMF/KI, as yellowish crystals, crystallized from benzene
◦
in 72% yield, m. p. > 350 C. IR (KBr): νmax = 3358 (NH), 3067 (CH, aryl), 2945 (CH, alkyl),
−
1 1
1
718, 1674 (2C=O), 1633 (C=N), 1585 (C=C) cm ; H–NMR (DMSO-d ):
δ
= 2.25 (s, 3H,
6
CH ), 3.75 (s, 3H, OCH ), 7.05–7.12 (dd, 2H, J = 7.51, 7.54 Hz, 4- methoxyphenyl), 7.32–7.39
3
3
(
dd, 2H, J = 7.56, 7.59 Hz, 4- methoxyphenyl), 7.65–7.95 (m, 5H, Ar-H), 10.72 (br,1H, NH,
13
D O-exchangeable); C–NMR (DMSO-d6)
δ = 21.1, 58.4 (2C, 2CH ), 121.9, 122.2, 124.5,
2
3
1
28.4, 128.9, 129.8, 132.5, 136.7, 142.3, 143.6, 148.2, 152.5, 161.3, 163.4, (18C, Ar-C), 166.1,
+
1
83.8 (2C, 2C=O); MS (70 eV): m/z = 416 (M , 95%); Anal. Calc. (Found) for C H N O S
22
16
4
3
(
416.45): C, 63.45 (63.52); H, 3.87 (3.79); N, 13.45 (13.37).
3
.9. Synthesis of
-Benzoyl-7-Methyl-2-(Phenylamino)Thieno[2,3-d][1,2,4]Triazolo[1,5-a]Pyrimidin-8(3H)-One (
6
7)
To a suspension of 2,3-diamino-benzoyl-methylthienopyrimidinone (
and red mercury (II) oxide (2.16 g, 0.01 mol) in 40 mL of dimethylformamide was added
.83 g, 0.01 mol, of the corresponding methylphenylcarbamodithioate in 5 mL of DMF
3) (3 g, 0.01 mol)
1
at room temperature. The mixture was refluxed for 15–18 h under control (TLC). After
cooling, the mixture was filtrated, and to the filtrate were added 50 mL of water. The
precipitate thus obtained was filtrated off, washed with water, dried and recrystallized
◦
from dioxane in 76% yield as yellow crystals, m. p. > 350 C. IR (KBr): νmax = 3360, 3320
−
1
(
2NH), 3065 (CH, aryl), 2940 (CH, alkyl), 1715, 1676 (2C=O), 1630(C=N), 1585 (C=C) cm
;
1
H–NMR (DMSO-d6):
δ = 2.20 (s, 3H, CH ), 7.15–7.80 (m, 10H, Ar-H), 9.60, 10.80 (br, 2H,
3
13
2
NH, D O-exchangeable); C–NMR (DMSO-d6)
δ = 20.7 (1C, CH ), 120.8, 122.1, 123.4,
3
2
1
1
28.7, 129.2, 129.8, 131.6, 137.5, 137.9, 142.4, 143.1, 143.9, 152.3, 161.9 (18C, Ar-C), 163.8,
+
81.4 (2C, 2C=O); MS (70 eV): m/z = 401 (M , 95%); Anal. Calc. (Found) for C H N O S
21
15
5
2
(
401.44): C, 62.83 (62.75); H, 3.77 (3.70); N, 17.45 (17.52).

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3
6
.10. Synthesis of
-Benzoyl-7-Methylthieno[2,3-d][1,2,4]Triazolo[1,5-a]Pyrimidine-2,8(1H,3H)-Dione (8)
To a solution of 2,3-diamino-benzoyl-methylthienopyrimidinone (3) (3 g, 0.01 mol)
and anhydrous potassium carbonate (1.4 g, 0.01 mol) in dry acetone (50 mL), ethyl- chlo-
roformate (1 mL, 0.01 mol) was added to the solution dropwise; after the completion of
the addition, the mixture was refluxed for 14–17 h, allowed to cool at room temperature,
and then poured into the cool water. The final product was obtained by being filtrated off,
washed with ethanol and recrystallized from methanol in 84% yield as yellowish crystals,
◦
m. p. > 350 C. IR (KBr): νmax =3352, 3310 (2NH), 3050 (CH, aryl), 2945 (CH, alkyl), 1722,
−
1 1
1
685, 1674 (3C=O), 1632 (C=N), 1588 (C=C) cm ; H–NMR (DMSO-d ):
CH ), 7.55–7.77 (m, 5H, Ar-H), 10.20, 11.10 (br, 2H, 2NH, D O-exchangeable); C–NMR
δ
= 2.23 (s, 3H,
6
1
3
3
2
(
(
(
DMSO-d6)
δ
= 21.1 (1C, CH ), 121.1, 128.4, 129.2, 131.8, 138.1, 142.6, 143.7, 152.2, 162.5
3
+
11C, Ar-C), 163.5, 166.2, 182.7 (3C, 3C=O); MS (70 eV): m/z = 326 (M , 100%); Anal. Calc.
Found) for C H N O S (326.33): C, 55.21 (55.15); H, 3.09 (3.17); N, 17.17 (17.26).
15
10
4
3
3
6
.11. Synthesis of
-Benzoyl-2-Mercapto-7-Methylthieno[2,3-d][1,2,4]Triazolo[1,5-a]Pyrimidin-8(3H)-One (9)
KOH (0.56g, 0.01mol) was dissolved through stirring in 20 mL of anhydrous methanol
in a 250 mL flask. Carbon disulfide (0.89 g, 0.01 mol) was dissolved in anhydrous methanol
5 mL) and added dropwise to the stirring solution, followed by reflux. The solution of
(
2
,3-diamino-benzoyl-methyl- thienopyrimidinone (
3
) (3 g, 0.01 mol) in methanol (30 mL)
◦
was added to the above reaction mixture and stirring under reflux (75–80 C) for 8–10 h.
Initially, the solution was yellow, which then slowly turned to brown, and, as the reaction
progressed, the evolution of hydrogen sulfide was observed. The reaction mixture was
monitored via TLC (Ethyl-acetate: petroleum ether, 1:3). After completion of the reaction,
the mixture was poured into a beaker containing ice water and acidified with 4N, HCl to
maintain pH 4–5. The final precipitate was separated, then filtered and recrystallized from
◦
the benzene in 70% yield as yellow crystals, m. p. > 350 C. IR (KBr): νmax = 3345 (NH),
3
040 (CH, aryl), 2937 (CH, alkyl), 2372 (SH), 1718, 1678 (2C=O), 1627(C=N), 1584(C=C)
−
1 1
cm ; H–NMR (DMSO-d ):
δ = 2.17 (s, 3H, CH ), 2.62 (s, 1H, SH), 7.61–7.88 (m, 5H, Ar-H),
6
3
13
9.05 (br, 1H, NH, D O-exchangeable); C–NMR (DMSO-d6) δ = 19.8 (1C, CH ), 120.7, 128.7,
2
3
1
29.5, 132.1, 138.6, 142.3, 143.5, 148.9, 151.8, 161.7 (12C, Ar-C), 164.5, 181.4 (2C, 2C=O); MS
+
(
(
70 eV): m/z = 342 (M , 93%); Anal. Calc. (Found) for C H N O S (342.39): C, 52.62
52.55); H, 2.94 (2.86); N, 16.36 (16.45).
15
10
4
2 2
3
.12. Synthesis of
-Benzoyl-2-(Chloromethyl)-7-Methylthieno[2,3-d][1,2,4]Triazolo[1,5-a]Pyrimidin-8(3H)-One (10
6
)
Chloroacetic acid (0.95 g, 0.01 mol) was dissolved in 35 mL of 4N, HCl and stirred into
the mixture for nearly 30 min. 2,3-diamino-benzoyl-methylthienopyrimidinone ( ) (3 g,
.01 mol) was added to the above solution with constant stirring; we continued stirring
3
0
◦
with reflux (100 C) for nearly 7–9 h. The solid product was established by TLC (Ethyl
acetate: petroleum ether, 1:1). The above hot solution was poured into ice cold water with
stirring, followed by dropwise addition of con NH ; then the yellowish precipitate was
3
filtered. The precipitate was then recrystallized from the dioxane in 79% yield as yellowish
◦
crystals, m. p. > 350 C. IR (KBr): νmax = 3341 (NH), 3035 (CH, aryl), 2933 (CH, alkyl),
−
1 1
1
724, 1680 (2C=O), 1625 (C=N), 1581(C=C) cm ; H–NMR (DMSO-d ):
δ
= 2.23 (s, 3H,
6
CH ), 4.45 (s, 2H, CH ), 7.60–7.86 (m, 5H, Ar-H), 9.15 (br, 1H, NH, D O-exchangeable);
3
2
δ
2
13
C–NMR (DMSO-d6)
= 20.5 (1C, CH ), 50.2 (1C, CH ), 120.4, 128.4, 129.2, 132.3, 138.1,
3
2
1
42.5, 143.7, 152.2, 155.9, 162.5 (12C, Ar-C), 165.3, 181.8 (2C, 2C=O); MS (70 eV): m/z = 358
+
(
M , 91%); Anal. Calc. (Found) for C H ClN O S (358.80): C, 53.56 (53.50); H, 3.09 (3.15);
16
11
4
2
N, 15.62 (15.71).

Molecules 2021, 26, 2031
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3
.13. Synthesis of
6
-Benzoyl-2-(((6-Benzoyl-7-Methyl-8-oxo-3,8-Dihydrothieno[2,3-d][1,2,4]Triazolo[1,5-a]Pyrimidin-
2-yl)Methyl)thio)-7-Methylthieno[2,3-d][1,2,4]Triazolo[1,5-a]Pyrimidin-8(3H)-One (11)
The 0.22 g, 0.01 mol, of sodium metal was added to a solution of 2-mercapto-methyl-
thieno-1,2,4-triazolopyrimidine (9) (3.42 g, 0.01 mol) in 40 mL anhydrous methanol and
stirred vigorously for 35 min. The 2-chloromethyl-methylthieno- 1,2,4-triazolopyrimi-
dine (10) (3.58g, 0.01 mol) was added to the above reaction mixture in portions and
◦
continually stirred for (90–100 C) nearly 5–8 h. A yellow compound so obtained was
filtered, washed with the methanol and dried. The product was established via TLC (Ethyl
acetate: petroleum ether, 1:1). The solid precipitate was then filtered and recrystallized
◦
from the DMF in 68% yield as yellow crystals, m. p. > 350 C. IR (KBr): νmax = 3355 (2NH),
3
050 (CH, aryl), 2940 (CH, alkyl), 1730, 1728, 1683, 1675 (4C=O), 1628 (C=N), 1585 (C=C)
−
1 1
cm ; H–NMR (DMSO-d ):
δ = 2.16, 2.28 (s, 6H, 2CH ), 4.05 (s, 2H, CH ), 7.35–7.95 (m,
6
3 2
13
10H, Ar-H), 9.80, 10.10 (br, 2H, 2NH, D O-exchangeable); C–NMR (DMSO-d6)
δ
= 20.3,
2
2
0.8 (2C, 2CH ), 33.7 (1C, CH ), 120.6, 120.9, 128.2, 128.5, 129.4, 129.8, 132.5, 132.7, 138.3,
3
2
1
1
38.6, 142.1, 142.5, 143.2, 143.6, 144.3, 152.4, 152.8, 156.1, 163.1, 163.4 (24C, Ar-C), 166.1,
66.5, 182.2, 182.8 (4C, 4C=O); MS (70 eV): m/z = 664 (M , 90%); Anal. Calc. (Found) for
+
C H N O S (664.73): C, 56.01 (56.12); H, 3.03 (3.10); N, 16.86 (16.73).
31
20
8
4 3
3
.14. Synthesis of 6-Benzoyl-2-(Mercaptomethyl)-7-Methylthieno[2,3-d][1,2,4]Triazolo[1,5-
a]Pyrimidin-8(1H)-One (12)
A mixture of 2,3-diamino-benzoyl-methylthienopyrimidinone (3) (3 g, 0.01 mol) and
thioglycolic acid (0.92 g, 0.01 mol) in absolute ethanol (25 mL). The reaction solution was
heated under reflux for 8–10 h by under control (TLC), then left to cool to room temperature.
The separated crystalline product was filtered, washed with ethanol and crystallized from
◦
methanol in 73% yield as yellow crystals, m. p. > 350 C. IR (KBr): νmax = 3370 (NH), 3040
−
1
(
CH, aryl), 2950 (CH, alkyl), 2355 (SH), 1730, 1675 (2C=O), 1629 (C=N), 1580 (C=C) cm
;
1
H–NMR (DMSO-d6):
δ = 2.25 (s, 3H, CH ), 2.60 (s, 1H, SH), 4.25 (s, 2H, CH ), 7.55–7.82
3
2
13
(
m, 5H, Ar-H), 10.10 (br, 1H, NH, D O-exchangeable); C–NMR (DMSO-d6)
δ
= 20.3 (1C,
2
CH ), 30.1 (1C, CH ), 120.1, 128.6, 129.5, 131.8, 137.6, 142.4, 143.5, 157.1, 158.8, 160.2 (12C,
3
2
+
Ar-C), 166.4, 181.2 (2C, 2C=O); MS (70 eV): m/z = 356 (M , 100%); Anal. Calc. (Found) for
C H N O S (356.42): C, 53.92 (53.84); H, 3.39 (3.44); N, 15.72 (15.78).
16
12
4
2 2
0 0 0 0
.15. Synthesis of 2-Benzoyl-3-Methylthieno[2”,3”:4 ,5 ]Pyrimido[1 ,2 :2,3][1,2,4]Triazolo
3
[
5,1-c][1,4] Thiazine-4,7(8H,10H)-Dione (13)
A well-stirred and ice-cooled suspension of finely powdered potassium hydroxide
(0.56 g, 0.01 mol) and thienotriazolopyrimidinone (12) (3.56 g, 0.01 mol) in dry dimethyl-
formamide (30 mL). After complete addition, heating and stirring was continued at room
◦
temperature for 2–4 h. The reaction mixture was cooled to 0 C, treated with the appropri-
ate halogenated compound as ethylbromoacetate (1.10 mL, 0.01 mol), heated and stirred
at room temperature for 18–22 h under control (TLC), then poured onto ice water. The
resulting product was filtered off, dried and recrystallized from dimethylformamide in
◦
8
(
0% yield as yellowish crystals, m. p. > 350 C. IR (KBr): νmax = 3050 (CH, aryl), 2955
−
1 1
CH, alkyl), 1733, 1682, 1677 (3C=O), 1631 (C=N), 1582 (C=C) cm ; H–NMR (DMSO-d ):
6
δ = 2.28 (s, 3H, CH ), 3.25 (s, 2H, CH , thiazine), 3.70 (s, 2H, CH , thiazine), 7.60–7.88 (m,
3
2
2
5
H, Ar-H); 13C–NMR (DMSO-d6)
δ
= 20.5 (1C, CH ), 33.2, 37.5 (2C, 2CH ), 120.4, 128.7,
3
2
1
29.6, 131.9, 137.8, 142.7, 143.6, 158.5, 159.6, 161.3 (12C, Ar-C), 165.9, 167.2, 182.1 (3C, 3C=O);
+
MS (70 eV): m/z = 396 (M , 95%); Anal. Calc. (Found) for C H N O S (396.44): C, 54.53
18
12
4
3 2
(
54.62); H, 3.05 (3.13); N, 14.13 (14.05).
3
.16. Synthesis of 6-Benzoyl-7-Methyl-8-oxo-3,8-Dihydrothieno[2,3-d][1,2,4]Triazolo[1,5-
a]Pyrimidine-2-Carbox-ylic Acid (14)
Method A: A mixture of 2,3-diamino-benzoyl-methylthienopyrimidinone (3) (3 g,
0.01 mol), oxalic acid (0.9 g, 0.01 mol) and anhydrous potassium carbonate (1.4 g, 0.01 mol)

Molecules 2021, 26, 2031
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in dry dimethyl- formamide (40 mL); the mixture was refluxed for 20–25 h under control
(
TLC). The final product was filtered off, washed with cold water, dried and crystallized
from the proper solvent. Method B: A mixture of 2,3-diamino-benzoyl- methylthienopy-
rimidinone (
3
) (3 g, 0.01 mol) and oxalic acid (0.9 g, 0.01 mol) in a solution of phosphorus
◦
pentoxide and methanesulfonic acid (PPMA) at 110–125 C for 22–27 h under control (TLC).
(
(
PPMA was prepared by adding phosphorus pentoxide (30 g) to methane sulfonic acid
210 mL) in portions and stirring the mixture at 80 C until the phosphorus pentoxide had
◦
completely dissolved.) Then, the mixture was allowed to cool at room temperature and
then poured into the cool water. The solid product was obtained by being filtrated off,
washed with ethanol and recrystallized from dioxane in 72% yield as yellow crystals, m.
◦
p. > 350 C. IR (KBr): νmax = 3375 (br, OH), 3360 (NH), 3058 (CH, aryl), 2960 (CH, alkyl),
−
1 1
1
745, 1735, 1679 (3C=O), 1630 (C=N), 1585 (C=C) cm ; H–NMR (DMSO-d ):
δ
= 2.30 (s,
6
3
H, CH ), 7.63–7.90 (m, 5H, Ar-H), 10.20 (br, 1H, NH, D O-exchangeable), 12.45 (br, 1H,
3
2
13
OH, D O-exchangeable); C–NMR (DMSO-d6) δ = 20.8 (1C, CH ), 120.6, 128.9, 129.5, 132.1,
2
3
1
37.9, 142.4, 142.8, 143.8, 151.7, 160.9 (12C, Ar-C), 164.8, 169.1, 183.5 (3C, 3C=O); MS (70 eV):
+
m/z = 354 (M , 90%); Anal. Calc. (Found) for C H N O S (354.34): C, 54.23 (54.31); H,
16
10
4
4
2.84 (2.75); N, 15.81 (15.90).
0 0
.17. Synthesis of 2-Benzoyl-8-Hydroxy-9-Mercapto-3-Methylpyrrolo[1 ,2 :2,3][1,2,4]Triazolo[1,5-
3
a]Thieno[2,3-d]Pyrimidine-4,7-Dione (15)
A solution of thienotriazolopyrimidinone (12) (3.56 g, 0.01 mol), oxalic acid (0.9 g,
0.01 mol) and anhydrous potassium carbonate (1.4 g, 0.01 mol) in dry dimethylformamide
(
45 mL). The reaction mixture was refluxed for 17–20 h under control (TLC). The formed
precipitate was filtered off, washed with cold water, dried and crystallized from benzene
◦
in 70% yield as yellowish crystals, m. p. > 350 C. IR (KBr): νmax = 3380 (br, OH), 3070 (CH,
−
1
aryl), 2955 (CH, alkyl), 2360 (SH), 1728, 1680, 1673 (3C=O), 1633 (C=N), 1591 (C=C) cm
;
1
H–NMR (DMSO-d6):
δ = 2.32 (s, 3H, CH ), 2.70 (s, 1H, SH), 7.56–7.87 (m, 5H, Ar-H), 12.10
3
13
(
br, 1H, OH, D O-exchangeable); C–NMR (DMSO-d6) δ = 21.1 (1C, CH ), 98.5 (1C, C-SH,
2
3
pyrrole), 120.4, 128.5, 129.4, 132.3, 138.6, 142.6, 143.5, 152.2, 159.7, 160.4 (12C, Ar-C), 178.7
+
(
1C, C-OH, pyrrole) 165.1, 169.5, 183.9 (3C, 3C=O); MS (70 eV): m/z = 410 (M , 98%); Anal.
Calc. (Found) for C H N O S (410.42): C, 52.68 (52.75); H, 2.46 (2.40); N, 13.65 (13.71).
18
10
4
4 2
3
.18. Synthesis of 2-Benzoyl-3-Methyl-8,13-
0
00 000
0
0
0
0
Dihydrothieno[2 ,3 :4”,5”]Pyrimido[1”,2”:2 ,3 ][1,2,4]triazolo[1 ,5 :1,2]Pyrrolo[3,4-
b]Quinoxaline-4,7-Dione (16)
A mixture of compound 15 (4.10 g, 0.01 mol) and anhydrous potassium carbonate (1.4
g, 0.01 mol) in dry dimethylformamide (40 mL) was refluxed for 7–10 h under control (TLC).
The product formed was filtered off, washed with methanol, dried and recrystallized from
◦
dioxane in 64% yield as yellow crystals, m. p. > 350 C. IR (KBr): νmax = 3385–3370 (2NH),
−
1
3
075 (CH, aryl), 2960 (CH, alkyl), 1735, 1685, 1675 (3C=O), 1635 (C=N), 1595 (C=C) cm
;
1
H–NMR (DMSO-d6):
δ = 2.35 (s, 3H, CH ), 7.01–7.92 (m, 9H, Ar-H), 11.50, 11.57 (2s, 2H,
3
13
2
NH, D O-exchangeable); C–NMR (DMSO-d6)
δ = 21.7 (1C, CH ), 114.2, 115.5, 119.1,
3
2
1
1
19.6, 120.4, 120.8, 128.9, 129.8, 132.5, 138.1, 142.5, 143.2, 152.8, 160.1, 162.4 (20 C, Ar-C),
66.7, 170.1, 183.5 (3C, 3C=O); MS (70 eV): m/z = 466 (M , 100%); Anal. Calc. (Found) for
+
C H N O S (466.48): C, 61.80 (61.73); H, 3.03 (3.10); N, 18.02 (18.08).
24
14
6
3
3
.19. Synthesis of 2-Benzoyl-3-
0
000 0000 000 000
000 000
0
0
0
0
Methylthieno[2 ,3 :4 ,5 ]Pyrimido[1 ,2 :2”,3”][1,2,4]Triazolo[1”,5”:1 ,2 ]Pyrrolo[3 ,4 :5,6]
[
1,4]Oxathiino[2,3-b]Quinoxaline-4,7-Dione (17)
A mixture of compound 15 (4.10 g, 0.01 mol) and 2,3- dichloroquinoxaline (1.99 g,
0
.01 mol) in absolute ethanol (40 mL) containing 0.3 mL of TEA as a catalyst was refluxed
for 24–28 h under control (TLC). The reaction solution was cooled, and the deposited solid
was filtered off, dried and recrystallized from DMF in 66% yield as yellow crystals, m.
◦
p. > 350 C. IR (KBr): νmax = 3080 (CH, aryl), 2960 (CH, alkyl), 1730, 1680, 1675 (3C=O),

Molecules 2021, 26, 2031
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−
1 1
1
9
635 (C=N), 1595 (C=C) cm ; H–NMR (DMSO-d ):
δ
= 2.37 (s, 3H, CH ), 7.25–7.92 (m,
6
3
H, Ar-H); 13C–NMR (DMSO-d6)
δ
= 21.9 (1C, CH ), 119.5, 120.8, 127.1, 127.4, 128.5, 129.2,
3
1
29.8, 130.6, 132.4, 135.7, 137.9, 139.3, 140.5, 142.7, 143.5, 143.9, 149.6, 152.4, 160.8, 163.5
+
(
(
22C, Ar-C), 166.5, 170.9, 183.8 (3C, 3C=O); MS (70 eV): m/z = 536 (M , 94%); Anal. Calc.
Found) for C H N O S (536.54): C, 58.20 (58.28); H, 2.25 (2.16); N, 15.66 (15.74).
26
12
6
4 2
3
8
.20. Synthesis of
0
0
0
0
-Benzoyl-9-Methyl-[1,4]Oxathiino[3”,2”:3 ,4 ]Pyrrolo[1 ,2 :2,3][1,2,4]Triazolo[1,5-
a]Thieno[2,3-d]Pyrimidine-3,10,13(2H)-Trione (18)
Method A: A mix of compound 15 (4.10 g, 0.01 mol) and chloro-acetyl-chloride (1.13 g,
0
.01 mol) was stirred under reflux in dimethylformamide (45 mL) in the presence of
anhydrous potassium carbonate (0.015 mol) for 22–26 h under control (TLC). The reaction
solution was allowed to cool to room temperature, poured into cooled water (100 mL)
and neutralized. The final solid formed was filtered off, dried and crystallized from the
appropriate solvent. Method B: To the stirring solution of compound 15 (4.10 g, 0.01 mol)
and chloro-acetyl-chloride (1.13g, 0.01 mol) in glacial acetic acid (40 mL), activated zinc
dust (1.3g, 0.02 mol) was added portion-wise at room temperature over a period of one
hour. Stirring was continued for an additional 4–6 h. The reaction solution was heated on a
◦
water bath (90–95 C) for 12–17 h under control (TLC). After allowing the reaction mixture
to cool to room temperature, it was poured into cold water (100 mL). The solid precipitate
was filtered, washed with water, dried and crystallized from DMF in 68% yield as yellowish
◦
crystals, m. p. > 350 C. IR (KBr): νmax = 3077 (CH, aryl), 2958 (CH, alkyl), 1737, 1727,
−
1 1
1
679, 1672 (4C=O), 1631 (C=N), 1593 (C=C) cm ; H–NMR (DMSO-d ): δ = 2.34 (s, 3H,
6
δ
13
CH ), 5.10 (s, 2H, CH ), 7.61–7.90 (m, 5H, Ar-H); C–NMR (DMSO-d6)
= 21.3 (1C, CH3),
3
2
8
0.6 (1C, CH , oxathiine), 90.2 (1C, CH, oxathiine), 120.4, 128.6, 129.5, 132.1, 138.5, 142.4,
2
1
43.1, 152.3, 155.8, 161.2, 162.7 (13C, Ar-C), 165.9, 171.6, 183.2, 192.5 (4C, 4C=O); MS (70 eV):
+
m/z = 450 (M , 90%); Anal. Calc. (Found) for C H N O S (450.44): C, 53.33 (53.42); H,
20
10
4
5 2
2.24 (2.17); N, 12.44 (12.36).
3
.21. Synthesis of 7-Benzoyl-2-Mercapto-8-Methyl-1H-
0
0
0
0
Imidazo[4”,5”:3 ,4 ]Pyrrolo[1 ,2 :2,3][1,2,4]Triazolo[1,5-a]Thieno[2,3-d]Pyrimidine-9,12-Dione (19
)
Method A: A mixture of compound 15 (4.10 g, 0.01 mol) and thiourea (0.76 g, 0.01 mol)
in 45 mL of DMF was refluxed for 14–18 h under control (TLC). The final product formed
was filtered off, washed with methanol, dried and recrystallized from a suitable solvent.
Method B: A mix of compound 15 (4.10 g, 0.01 mol) and thiourea (0.76 g, 0.01 mol) was
stirred under reflux in dioxane (45 mL) in the existence of the catalytic amount of piperidine
(1 mL) for 18–22 h. The reaction solution was allowed to cooling at room temperature and
poured into cooled water, and the final precipitate was filtered off, washed with methanol,
◦
dried and crystallized from DMF in 70% yield as brownish crystals, m. p. > 350 C. IR (KBr):
νmax = 3355 (NH), 3050 (CH, aryl), 2945 (CH, alkyl), 2357(SH), 1729, 1681, 1670 (3C=O),
−
626 (C=N), 1586 (C=C) cm ; H–NMR (DMSO-d ): δ = 2.28 (s, 3H, CH ), 7.55–7.82 (m,
6 3
1 1
1
13
5
H, Ar-H), 10.83 (s, 1H, SH), 11.90 (s, 1H, NH, D O-exchangeable); C–NMR (DMSO-d6)
2
δ
= 21.1 (1C, CH ), 120.3, 128.4, 129.3, 130.1, 132.5, 137.8, 140.3, 142.7, 143.2, 157.4, 161.6,
3
1
63.2 (14C, Ar-C), 164.7 (1C,1C=O), 168.9 (1C, C-SH, imidazole), 170.2, 183.4 (2C, 2C=O);
+
MS (70 eV): m/z = 434 (M , 85%); Anal. Calc. (Found) for C H N O S (434.45): C, 52.53
19
10
6
3 2
(
52.62); H, 2.32 (2.40); N, 19.34 (19.27).
3
.22. Synthesis of 10-Benzoyl-9-Methyl-3-Phenyl-1H-
000 000 0 0 0 0
1,2,4]Triazolo[4 ,3 :1”,2”]Imidazo[4”,5”:3 ,4 ]Pyrrolo[1 ,2 :2,3][1,2,4]Triazolo[1,5-
[
a]Thieno[2,3-d]Pyrimidine-5,8-Dione (20)
Method A: A mixture of compound 19 (4.34 g, 0.01 mol) and benzohydrazide (1.36 g,
0.01 mol) and anhydrous potassium carbonate (1.4 g, 0.01 mol) in dry dimethylformamide
(40 mL). The reaction solution was heated under reflux for 13–16 h under control (TLC),

Molecules 2021, 26, 2031
17 of 20
and then allowed to cool and poured into ice water. The formed solid product was collected
by filtration and crystallized from the proper solvent. Method B: A solution of compound
19 (4.34 g, 0.01 mol) and benzohydrazide (1.36 g, 0.01 mol) in ethanol (40 mL) containing
sodium ethoxide [prepared by dissolving sodium metal (0.23 g, 0.01 mol) in ethanol]
was heated under reflux for 11–15 h, the reaction mixture was cooled and the deposited
precipitate was filtered off, washed with ice water/ethanol and acidified with 10% HCl.
The formed precipitate was filtered, dried and crystallized from dioxane in 73% yield
◦
as yellowish crystals, m. p. > 350 C. IR (KBr): νmax = 3372 (NH), 3058 (CH, aryl), 2951
−
1 1
(
CH, alkyl), 1733, 1684, 1676 (3C=O), 1631 (C=N), 1579 (C=C) cm ; H–NMR (DMSO-d ):
6
δ
= 2.32 (s, 3H, CH ), 7.15–7.98 (m, 10H, Ar-H), 12.01 (s, 1H, NH, D O-exchangeable);
3
2
1
3
C–NMR (DMSO-d6)
δ
= 21.6 (1C, CH ), 119.2, 120.7, 127.8, 128.2, 129.4, 129.8, 130.5, 131.8,
3
1
1
32.9, 136.5, 138.7, 142.5, 143.7, 150.8, 151.2, 155.4, 160.9, 162.7 (22C, Ar-C), 165.1, 168.8,
+
83.4 (3C, 3C=O); MS (70 eV): m/z = 518 (M , 100%); Anal. Calc. (Found) for C H N O S
26
14
8
3
(
518.51): C, 60.23 (60.33); H, 2.72 (2.65); N, 21.61 (21.68).
3.23. Pharmacological Screening
3.23.1. Ethics Approval and Consent to Participate
No humans or animals were used in this study, nevertheless: all the procedures are
carried out under the Medical Research Ethics Committee of Mansoura University, Faculty
of Pharmacy, Department of Pharmacognosy, 35516, Egypt.
3.23.2. Human and Animal Rights
No humans or animals were used in the study. The research was conducted according
to ethical standards in vitro.
3.23.3. Chemicals and Drugs
Types of human carcinoma cancer cell line (CNE2, KB, MCF-7 and MGC-803) are de-
rived from the National Cancer institute, Cairo University, Cairo, Egypt, and 5-Fluorouracil
and DMSO were purchased from Sigma–Aldrich (Saint Louis, MO, USA).
3.23.4. Materials and Methods (In Vitro Cytotoxicity)
The in vitro cytotoxicity of the synthesized compounds against different cancer cell
lines was performed with the MTT assay, according to the method found in [34,38,40,48].
The MTT assay is based on the reduction of the soluble 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazoliumbromide (MTT) into a blue–purple formazan product, mainly by
mitochondrial reductase activity inside living cells. The cells used in the cytotoxicity assay
were cultured in the suitable cell culture medium (RPMI 1640) medium supplemented
with 10% fetal calf serum. Cells suspended in the medium (2Y’ 104/mL) were plated in
◦
9
6-well culture plates and incubated at 37 C in a 5% CO incubator. After 12 h, the test
2
◦
sample (2 mL) was added to the cells (2Y’ 104) in 96-well plates and cultured at 37 C for
3
3
days. The cultured cells were mixed with 20 mL of MTT solution and incubated for 4 h at
7 C. The supernatant was carefully removed from each well, and 100 mL of DMSO was
◦
added to each well to dissolve the formazan crystals, which were formed by the cellular
reduction of MTT. After mixing with a mechanical plate mixer, the absorbance of each well
was measured by a microplate reader using a test wavelength of 570 nm. The results were
expressed as the IC , which is the concentration of the drugs inducing a 50% inhibition of
50
cell growth of treated cells, when compared to the growth of control cells. Each experiment
was performed at least 3 times. There was a good reproducibility between replicate wells
with standard errors below 10%.
4
. Conclusions
In the current work, we successfully prepared and developed 2,3-diamino-6-benzoyl-
-methylthieno[2,3-d]pyrimidin-4(3H)-one ( ) as starting material to synthesize various
5
3
new heterocyclic, such as thieno[2,3-d][1,2,4]triazolo[1,5-a] pyrimidinone-thieno[2,3-d]

Molecules 2021, 26, 2031
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[
1,2,4]triazolo [1,5-a]pyrimidinones, thienopyrimidotriazolothiazine-diones, pyrrolotria-
zolothienopyrimidines, thienopyrimidotriazolopyrroloquinoxalindiones, thienopyrimi-
dotriazolopyrrolo-oxathiinoquinoxalines, 1,4-oxathiinopyrrolo-triazolothienopyrimidin-
triones, imidazopyrrolotriazolothienopyrimidindiones and 1,2,4-triazoloimidazopyrrolo-
triazolothienopyrimidine-diones, which were prepared and described using different meth-
ods and high techniques that produced good yields. In addition, the anticancer properties
of the new compounds were studied in vitro; we in screened all compounds for cytotoxic
and antiproliferative effects against the types of human carcinoma cancer cell line (CNE2
,
KB MCF-7 and MGC-803). The compounds 20 19 17 16 11 and 18 showed potent cyto-
,
,
,
,
,
toxic and anticancer efficacy, because these compounds contain many functional groups
and many heterocyclic, such as the thienopyrimidine, 1,2,4-triazole, 1,4-thiazine, pyrrole,
quinoxaline, 1,4-oxathiine and imidazole moieties, according to previous scientific studies.
1,2,4-triazolo-imidazopyrrolotriazolo-thienopyrimidin-dione (20) is a promising anticancer
molecule with multiple modes of actions, such as antiproliferative and angiopreventive
effects, together with its remarkable apoptosis-inducing action [48–50].
Author Contributions: A.A.A.-H., S.A.A.-H. and M.E.A.Z. participated in the synthesis, purification
and characterization of the new compounds; the Department of Pharmacognosy, Faculty of Pharmacy,
Mansoura University, Egypt, participated in the evaluation of the newly synthesized compounds
for anticancer activities. S.A.A.-H. participated in the work of physical and chemical experiments
and some of the measurements for the prepared compounds, with the interpretation of many results.
M.E.A.Z. participated in the interpretation of spectroscopy of new compounds and the review of
the manuscript. A.A.A.-H. participated in the interpretation of the results, writing, revision and
correspondence to the journal of molecules until the manuscript was accepted. All authors have read
and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: “Not applicable” this study not involving humans or animals.
Informed Consent Statement: “Not applicable” this study not involving humans.
Data Availability Statement: All data are contained within the article and the experimental section.
Acknowledgments: The authors are extremely grateful to prof. Farid A. Badria and the Department
of Pharmacognosy, Faculty of Pharmacy, Mansoura University, 35516 Egypt, for helping us to screen
the newly synthesized compounds for anticancer activity.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the synthesized compounds are available from the authors.
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