Synthesis of various fused heterocyclic rings from thiazolopyridine and their pharmacological and antimicrobial evaluations

Article abstract of DOI:10.1002/jhet.4011

Various fused-heterocyclic-derivatives containing thiazolopyridine moieties has been synthesized by allowing 5-aminothiazolo[3,2-a]pyridine derivative 1 to undergo annulations reactions with different reagents under different-reaction conditions. The biological assessment of compounds 2, 11, 14, 15, and 19 showed remarkable antimicrobial activities. In addition, selected derivatives of the products were screened for their anticancer activities against two tumor cell lines using MTT assay and the results showed that some of these compounds have potent cytotoxic effect, as concluded from their IC₅₀ values. Meanwhile, compounds 3a, 7 have exhibited very strong potency as anticancer candidates. Thiazolopyridine structures have been confirmed as a useful lead compounds for the development of new anticancer agents. Molecular docking showed that,-some of the synthesized compounds more suitable inhibitor against-ALR2 with farther alteration in future.

Full text of DOI:10.1002/jhet.4011

Received: 26 February 2020
DOI: 10.1002/jhet.4011
Revised: 12 April 2020
Accepted: 20 April 2020
A R T I C L E
Synthesis of various fused heterocyclic rings from
thiazolopyridine and their pharmacological and
antimicrobial evaluations
Naglaa F. H. Mahmoud
| Mina G. Balamon
Chemistry Department, Faculty of
Science, Ain Shams University, Cairo,
Egypt
Abstract
Various fused-heterocyclic-derivatives containing thiazolopyridine moieties
has been synthesized by allowing 5-aminothiazolo[3,2-a]pyridine derivative
1 to undergo annulations reactions with different reagents under different-
reaction conditions. The biological assessment of compounds 2, 11, 14, 15, and
19 showed remarkable antimicrobial activities. In addition, selected derivatives
of the products were screened for their anticancer activities against two tumor
cell lines using MTT assay and the results showed that some of these com-
pounds have potent cytotoxic effect, as concluded from their IC50 values.
Meanwhile, compounds 3a, 7 have exhibited very strong potency as anticancer
candidates. Thiazolopyridine structures have been confirmed as a useful lead
compounds for the development of new anticancer agents. Molecular docking
showed that,-some of the synthesized compounds more suitable inhibitor
against-ALR2 with farther alteration in future.
Correspondence
Naglaa F. H. Mahmoud, Chemistry
Department, Faculty of Science, Ain
Shams University, Cairo 11566, Egypt.
Email: naglaa.fawzy@yahoo.com
1 | INTRODUCTION
compounds were ascertained from the spectral and ele-
mental analyses. Some of the new compounds showed
Heterocyclic systems have attracted a great attention in the
last decade due to their importance in pharmaceutical
and medicinal chemistry. One of these heterocycles is the
thiazolidin-4-one scaffold which is considered as a back-
bone for the synthesis of different synthetic pharmaceuticals
targets having a wide spectrum of biological activities.[1–8]
On the other hand, literaturally nicotinonitrile moiety
also plays a significant role in drug design field.[9–17] There-
fore, the combination of new ring systems involving
thiazolo[3,2-a]-pyridine derivatives showed remarkable
antimicrobial,[18–23] antifungal,[24] anticancer,[25–32] and
antihypertensive activities.[33]
Due to all the aforementioned facts and in continua-
tion of our program regarding the synthesis of biologi-
cally active heterocyclic nuclei.[34–37] Herein, we used
5-aminothiazolo[3,2-a]pyridine derivative 1, as a key
starting material to construct different novel heterocyclic
compounds. The structures of the newly synthesized
remarkable biological activities.
2 | RESULTS AND DISCUSSION
The strategy for the construction of different heterocycles
bearing fused thiazolo[3,2-a]pyridine was emanated from
the utilization of 5-amino-2-(4-chlorobenzylidene)-7-(4-
chlorophenyl)-3-oxo-2,3-dihydro-7H-thiazolo[3,2-a]pyridine-
6,8-dicarbonitrile 1 as the building block.[38] In the pre-
sent work, the behavior of 1 towards malononitrile was
investigated. Therefore, compound 1 was allowed to react
with malononitrile in refluxing pyridine as shown in
(Scheme 1). Supposedly, two possible products 2 and 20
were anticipated to be yielded. When the reaction was
monitored with thin-layer chromatography (TLC), it rev-
ealed the presence of one spot. Unambiguously, both the
elemental analysis and spectral-data confirmed structure
J Heterocyclic Chem. 2020;1–15.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals LLC.
1

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MAHMOUD AND BALAMON
SCHEME 1 Reaction of 1 with
malononitrile
SCHEME 2 Reactions of
1 with different binucleophilic
reagents
2 and excluded structure 20. Compound 2 was obtained
by Michael addition on the α,β-unsaturated enone of
1 followed by intramolecular nucleophilic reaction of
the enol form on the cyano group as shown in Scheme 1.
As evidences, IR-spectrum revealed the absence of any
absorption band corresponding to C O group of the
thiazolinone ring while 1H-NMR showed singlet signal at
δ = 3.61 ppm due pyrane proton.
Treatment of an ethanolic solution of 1 with different
binucleophilic agents such as hydrazine hydrate and/or
phenyl hydrazine under reflux gave the corresponding
pyrazolo[3,4:4,5]thiazolo[3,2-a]pyridine derivatives 3a and

MAHMOUD AND BALAMON
3
SCHEME 3 Mechanistic
illustration for 3a and 3b formations
SCHEME 4 Reactions of
1 with different electrophilic agents
3b, respectively (Scheme 2). The IR-spectra of 3a and 3b
lack any carbonyl signals and 1H-NMR of compound
3a showed a broad singlet signal at δ = 10.81 ppm
corresponding to the NH group of the pyrazole moiety.
On the other hand, the mass spectrum of compound 3b
showed a peak at m/z = 539 (M+. + 1). A mechanistic
illustration for 3a and 3b formation commenced with
intramolecular-cyclocondensation with α,β-unsaturated car-
bonyl system of 1 followed by oxidation to give 3a and 3b
as depicted in Scheme 3. As alternative binucleophilic
agents, both of hydroxylamine hydrochloride and thiourea
were subjected to react with 1 to yield isoxazolo[50,40:4,5]
thiazolo[3,2-a]pyridine derivatives 4 and 5, respectively.
IR-spectra of compounds 4 and 5 lacked any carbonyl group
absorption band. H-NMR of compound 5 showed a broad
1
singlet signal at δ = 10.38 ppm due to NH pyrimidine.
In order to construct the four-ring fused system
pyrazolo[30,40:40,50]thiazolo [30,20:1,6]pyrido[2,3-d]pyrimi-
dine-6-carbonitrile 7, the thiazolopyridine derivative
1 was subjected to react with triethyl orthoformate
under reflux condition to give the corresponding deriva-
tive 6, which in turn was hydrazonolyzed with hydrazine
hydrate to get the target compound 7 (Scheme 4). The
1H-NMR spectrum compound 6 lacked any characteristic
band for the amino group and appearance of ethoxy sig-
1
nals at δ = 4.37 and 1.36 ppm. The H-NMR spectrum of

4
MAHMOUD AND BALAMON
SCHEME 5 Reactions of
1 with different active methylene
compounds
compound 7 showed an exchangeable broad singlet band
of NH2 protons at δ = 5.18 ppm besides two
exchangeable-broad singlet signals at δ = 12.79 and
11.15 ppm due to 2NH protons. Furthermore, 1 gave the
corresponding thiazolopyrido pyrimidine derivatives
8 and 9 by treatment with formamide and formic acid,
respectively (Scheme 4). The mass spectra of compounds
8 and 9 revealed the molecular ion peaks.
due to NH proton and multiplet signals due to cyclohexyl
protons at 2.16 to 1.62 ppm.
Pyrano[20,30:4,5]thiazolo[3,2-a]pyridine derivatives 12,
13, 14, 15, and 16 were obtained when compound
1 was reacted with different active methylene compounds
such as ethyl acetoacetate, diethyl malonate, benzyl cya-
nide, ethyl chloroacetate, cyanoacetic acid and/or ethyl
cyanoacetate, respectively (Scheme 5). Structural elucida-
tions of formed compounds were confirmed by exploiting
their spectral data. For instance, the IR-spectrum of
compound 12 exhibited a strong absorption band at
υ = 1725 cm−1 corresponding to (C O) group while
its 1H-NMR showed bands at δ = 4.10 and 1.25 ppm
corresponding to ethyl group protons. Finally, the mass
spectrum revealed the molecular ion peak at m/z 562.
For compound 13, the IR-spectrum of the compound
showed absorption bands at 1771 and 1710 cm−1 due to
On the other hand, when 1 was refluxed with acetic
anhydride, the thiazolopyrido pyrimidine derivative 10 was
yielded. The structure of 10 was confirmed by the spectral
data such as the IR-spectrum which exhibited strong
absorption bands at υ = 3428 cm−1 (NH), 2214 cm−1
1
(C N) and 1709, 1675 cm−1 (2C O). The H-NMR spec-
trum exhibited a singlet signal at δ = 1.91 ppm assigned to
the methyl protons.
Nucleophilic substitution of 1 with cyclohexyl bromide
yielded cyclohexylamino-thiazolo[3,2-a]pyridine derivative
11 (Scheme 4). The structure of compound 11 was con-
firmed by the IR-spectrum which exhibited an absorption
1
two C O groups. In addition, its H-NMR showed bands
at δ = 4.21 and 1.2 ppm corresponding to ethyl group
protons. The IR spectrum of compound 14 showed the
absence of any absorption band of C O (thiazolidinone).
1H-NMR of compound 14 showed-D2O-exchangeable
1
band at υ = 3204 cm−1 (NH). H-NMR of compound 11
showed D2O-exchangeable singlet signal at δ = 8.98 ppm

MAHMOUD AND BALAMON
5
in both cases the bis Schiff base 19 was yielded with differ-
ent yields (25% and 60% respectively). The mass spectrum
of 19 showed a peak at m/z 751 (M+. − 32).
2.1 | Antibacterial activity evaluation
The antimicrobial activities of the new compounds have
been screened using-agar diffusion method,[39,40] against
Staphylococcus aureus (G+ve), Pseudomonas aeruginosa
(G−ve), Candida-albicans (yeast), and Aspergillus niger
(fungus). As compiled in Table 1, it is obvious that most
of the tested compounds showed moderate to high anti-
microbial activities. In particular, compounds 2, 11, 14,
15, and 19 showed remarkable antimicrobial activities. It
is promising to consider those derivatives are having the
chance to be candidates as antimicrobial agents.
2.2 | Molecular docking studies
Molecular docking study of thiazolopyridine derivatives
was performed by Molecular Operating Environment
2014.09.[41–43] In order to understand the binding mode
of protein-ligand interactions, the crystal structure of the
human-ALR2 enzyme (1PWM) complexed with (ligand
Fidarestat) as an inhibitor was downloaded from a pro-
tein data bank. The molecular docking scoring function
was applied to-evaluate the binding affinities between
the (1PWM) and selected synthesized inhibitors (FID)
was re-docked into the active site of the enzyme, and
then replaced it with the tested derivatives in order to
compare the binding mode of ligand and the tested deriv-
atives (Figures 1-10, Table 2). We begin to carry out a
molecular docking study of the highest biologically active
newly synthesized derivatives. First, the enzyme prepared
for docking by adding lost hydrogen, reconnect the bonds
broken and fixing the-potential, also active sites has been
identified and we start to replace the known ligand (FID)
by our synthesized ligands.
The results of docking study were reported as -ALR2
(1PWM) binding free energy (δG). The negative values of
free energies refer to spontaneity of bindings (Table 2).
The docking results revealed that the highest binding
compound to ALR2 enzyme was 18 with binding energy
of −7.61 kcal/mol, this was attributed to the strong
hydrogen bond between the ligand and Lys 21, Ser
263, and Asn 272 from the enzyme and arenes interaction
of Leu228 and Arg 268. On the other hand, compound 14
also exhibit promising ALR2 enzyme inhibitory activity
(−7.16 kcal/mol). It showed hydrogen bond between the
ligand and Lys 21 and Asn 272. Finally, compound
2 showed hydrogen bond between the ligand and Lys
SCHEME 6 Reactions of 2 with benzoyl chloride, formamide,
and p-nitrobenzaldehyde
singlet signals at δ = 5.09 and 4.65 ppm due to four pro-
tons of 2NH2 groups. The mass spectra of compounds 15
and 16 revealed the molecular ion peaks at m/z 524 and
m/z 512, respectively.
For exploring the utilization of the pyrano[20,30:4,5]
thiazolo[3,2-a]pyridine derivative 2 as an useful key inter-
mediate in the synthesis of fused heterocyclic derivatives,
initially it was treated with benzoyl chloride and formam-
ide to give the pyrimidino[3000,4000:500,600]pyrano[200,300:40,50]
thiazolo[30,20:1,6]pyrido[2,3-d]pyrimidine derivatives 17
and 18, respectively (Scheme 6). The structure of 17 was
confirmed by the spectral data such as the IR which
lacked any band corresponding to the amino group. As an
1
additional proof, H-NMR spectrum showed the absence
of any broad singlet signals of the two NH2 protons and
showed D2O-exchangeable singlet signal at δ = 12.9 ppm
corresponding to the two NH protons. The mass spectrum
of compound 18 showed its molecular ion peak at
m/z 567.
Eventually, the reactivities of both amino groups in
2 has been proved to be comparable. This comparison was
carried out by allowing 2 to react with the electrophilic
agent 4-nitrobenzaldehyde. When 2 was treated with one
equivalent and/or two equivalents of 4-nitrobenzaldehyde,

6
MAHMOUD AND BALAMON
TABLE 1
Antimicrobial evaluations of the synthesized compounds
Clear zone (ϕ mm)
Sample number
Staphylococcus aureus
Pseudomonas aeruginosa
Candida albicans
Aspergillus niger
1
16
28
11
14
12
14
15
17
14
25
13
21
29
12
17
15
22
30
00
18
30
18
11
15
16
18
14
21
22
10
22
31
13
14
18
23
28
00
17
16
18
14
18
15
14
17
15
27
15
18
31
0
13
15
0
2
3a
3b
0
4
0
5
0
7
0
8
0
10
14
0
11
12
13
16
13
0
14
15
16
17
0
0
18
0
15
10
15
37
19
23
25
22
Streptomycin
Cyclohexamide
FIGURE 1 3D diagram of the interaction of compound 2 in
FIGURE 2 2D diagram of the interaction of compound 2 in
the active site of the ALR2 enzyme
the active site of the ALR2 enzyme

MAHMOUD AND BALAMON
7
FIGURE 3 3D diagram of the interaction of compound 3a in
the active site of the ALR2 enzyme
FIGURE 5 3D diagram of the interaction of compound 7 in
the active site of the ALR2 enzyme
FIGURE 4 2D diagram of the interaction of compound 3a in
the active site of the ALR2 enzyme
FIGURE 6 2D diagram of the interaction of compound 7 in
the active site of the ALR2 enzyme
21, Ser 263 and Asn 272 that makes ALR2 enzyme inhibi-
tory activity (−7.11 kcal/mol).
results on the antiproliferative activity of tested com-
pounds are summarized in (Table 3), the synthesized
compounds which exhibited very strong antiproliferative
activity are 3a and 7 with IC50: 6.10, 7.80 and 9.43, 8.24
μg against two carcinoma cell lines tested in this study
HePG-2 and MCF-7 cell lines, respectively, which are
comparable with the reference anticancer drug Doxorubi-
cin (DOX) with IC50: 4.50 and 4.17 μg. Compounds 2, 14,
and 18 also showed strong anticancer activity against two
carcinoma cell lines tested in this study with IC50: (19.04,
10.21), (13.91, 12. 65) and (16.28, 14.06) μg for HePG-2
and MCF-7 cell lines, respectively. While the other
2.3 | Antiproliferative activities
The results of the cytotoxic activity in vitro were
expressed as the IC50-concentration of the compound
(in μM) that inhibits proliferation of the cells by 50% as
compared to the untreated control cells. The synthesized
compounds are tested for anticancer activities against
two human tumor cells,[44,45] namely hepatocellular car-
cinoma (HePG-2) and mammary gland (MCF-7). The

8
MAHMOUD AND BALAMON
FIGURE 7 3D diagram of the interaction of compound 14 in
the active site of the ALR2 enzyme
FIGURE 9 3D diagram of the interaction of compound 18 in
the active site of the ALR2 enzyme
FIGURE 10 2D diagram of the interaction of compound 18 in
FIGURE 8 2D diagram of the interaction of compound 14 in
the active site of the ALR2 enzyme
the active site of the ALR2 enzyme
TABLE 2
The docking binding free energies of the synthesized compounds
Compound
number
Docking score
(kcal/mol)
No. of hydrogen bonding
2 (Lys 262 and Asn 272)
No. of (arenes) interaction
FID
2
−6.24
−7.11
−6.39
−6.91
−7.16
−7.61
—
3 (Lys 21, Ser 263 and Asn 272)
3 (Lys 262, Ser 263 and Val 264)
2 (Lys 21 and Asn 272)
—
3a
7
1 (Leu 228) (arene-H)
—
14
18
2 (Lys 21 and Asn 272)
—
3 (Lys 21, Ser 263 and Asn 272)
1 (Leu 228) (arene-H); 1 (Arg 268) (arene-+)

MAHMOUD AND BALAMON
9
compounds that showed moderate antiproliferative activ-
ity are 3b, 4, 5, 8, 11, 13, and16; compounds 1, 10, 12, 15,
and 19 show weak cytotoxicity activity against all cell
lines (Table 3). In conclusion, thiazolopyridine structures
have been confirmed as a useful lead compounds for
the development of new anticancer agents. Our initial
goal to prepare synthetic derivatives with higher antican-
cer activity could be achieved.
Graphical correlation between tested compounds and
their IC50 against HePG-2 and MCF-7 is depicted in
Figure 11.
TABLE 3
Antiproliferative activities of some compounds
against human tumor cells
2.4 | Structure activity relationship
In vitro cytotoxicity IC50 (μM)a
Compound number
HePG-2
MCF-7
Upon investigations of the results revealed from the
assessment of the tested compounds as inhibitors against
two different tumor cell lines, it is obvious that activities
are dependent on existence of certain structural features
of the tested compounds. The most cytotoxic derivatives
were found in 2, 3a, 14, and 18 since it has potent which
may attribute to the presence of the amino and imino
groups. Therefore, as a conclusion, derivatives with more
than one amino and imino group exhibited remarkable
activities as anti-tumor candidates when compared with
those having one only (Figures 12 and 13).
Doxorubicin
4.50 0.3
87.18 4.5
19.04 1.7
6.10 0.5
48.17 2.9
35.49 2.4
23.84 1.9
9.43 0.8
35.81 2.3
82.76 4.0
28.22 2.1
54.39 3.2
13.91 1.1
64.54 3.4
42.72 2.7
>100
4.17 0.2
85.32 4.3
10.21 1.0
7.80 0.7
1
2
3a
3b
4
42.17 2.6
26.67 2.1
17.23 1.7
8.24 0.8
5
7
8
29.01 2.2
65.49 3.4
21.15 1.9
49.79 2.8
12.65 1.2
53.58 3.1
33.88 2.4
91.14 5.1
14.06 1.4
78.26 3.8
10
11
12
14
15
16
17
18
19
3 | CONCLUSIONS
The present work aimed to the development of novel mole-
cules containing thiazolopyridine moieties. The biological
assessment of compounds 2, 11, 14, 15, and 19 showed
remarkable antimicrobial activities. Meanwhile, selected
derivatives of the products were screened for their antican-
cer activities against two tumor cell lines using MTT assay
and the results showed that some of these compounds
3a, 7 have exhibited very strong potency as anticancer can-
didates. Thiazolopyridine structures have been confirmed
16.28 1.3
72.35 3.6
aIC50 (μM): 1-10 (very-strong), 11-20 (strong), 21-50 (moderate),
51-100 (weak), and above 100 (non-cytotoxic).
In vitro Cytotoxicity IC (µM)
50
HePG2 MCF-7
120
100
80
60
40
20
0
FIGURE 11 Graphical correlation
DOX
1
2
3a 3b
4
5
7
8
10 11 12 14 15 16 17 18 19
between tested compounds and their
IC50 against HePG-2 and MCF-7
Compounds

10
MAHMOUD AND BALAMON
FIGURE 12 High anticancer
activities
FIGURE 13 Moderate
anticancer activities
as a useful lead compounds for the development of new
anticancer agents. Molecular docking showed that some of
the synthesized compounds are more suitable inhibitor
against ALR2 with farther alteration in future.
Analytical Center of Cairo University, Egypt, the Micro
Analytical-Center of Ain-Shams University, or the Main
Defense Chemical Laboratory. Elemental analyses were
realized at the Faculty of Science, Ain Shams University.
The antimicrobial activities were determined at Al-Azhar
University, Faculty of Science. The anticancer evaluation
was performed at the Pharmacology Department, Faculty
of Pharmacy, Mansoura University, Egypt. The chemical
reactions were monitored by TLC.
4 | EXPERIMENTAL
Melting points were measured on a Griffin melting point
apparatus. The IR spectra were measured with a Pye Uni-
cam SP 2000 infrared spectrophotometer using the KBr
wafer technique. The EI-MS spectra were determined
4.1 | 5-Amino-2-(4-chlorobenzylidene)-
7-(4-chlorophenyl)-3-oxo-2,3-dihydro-7H-
thiazolo[3,2-a]pyridine-6,8-
1
using an AE1 MS 902 mass spectrometer. The H-NMR
(300 MHz) and 13C-NMR (75 MHz) were measured on
Varian Gemini instrument with chemical shifts (δ)
expressed in ppm downfield from TMS as internal stan-
dard in deuterated dimethylsulfoxide (DMSO-d6). All the
spectral measurements were performed at the Micro
dicarbonitrile (1)
A mixture of 4-chlorobenzaldehyde (0.02 mol, 2.81 g),
malononitrile (0.02 mol, 1.32 g) and thioglycolic acid

MAHMOUD AND BALAMON
11
(0.01 mol, 0.92 mL) in absolute ethanol containing drops
of triethyl amine (5 drops) was heated under reflux for
2 hours. The formed precipitate was filtered off, washed
with ethanol and recrystallized from dioxane to give 1.
Yield (75%), mp 268ꢀC-270ꢀC, (dioxane). Anal.
Calcd. For C22H12Cl2N4OS (451): C, 58.55; H, 2.68; N,
12.41. Found: C, 58.52; H, 2.64; N, 12.46. FT-IR (KBr)
4.4 | 8-Amino-3,6-bis(4-chlorophenyl)-
1-phenyl-1H,6H-pyrazolo[30,40:4,5]thiazolo
[3,2-a]pyridine-5,7-dicarbonitrile (3b)
To a solution of compound 1 (0.01 mol, 4.51 g) in abso-
lute ethanol (20 mL) phenyl hydrazine (0.02 mol, 2 mL)
was added in the presence of drops of piperidine (5 drops).
The reaction mixture was heated under reflux for
20 hours. The reaction mixture was poured into ice/HCl.
The formed precipitate was filtered off, dried and rec-
rystallized from dioxane to give 3b.
Yield (64%), mp >300ꢀC (dioxane). Anal. Calcd.
For C28H16Cl2N6S (538): C, 62.34; H, 2.99; N, 15.58.
Found: C, 62.31; H, 3.03; N, 15.61. FT-IR (KBr) (cm−1):
3476-3388 (NH2), 2216 (CN), 1580 (C N). 1H-NMR
(DMSO-d6) δ: 7.82-7.27 (m, 13H, H-Ar), 6.28 (br.s, 2H,
NH2, D2O exchangeable) and 4.65 (s, 1H, H-pyridine).
13C-NMR (DMSO-d6) δ: 161.5, 156.4, 151.7, 142.1, 140.0,
139.4, 134.6, 131.8, 131.6, 130.3 (2), 129.3 (4), 128.9 (2),
128.5 (2), 126.0, 123.3 (2), 118.3, 117.4, 93.0, 61.4, 57.6,
37.1. MS, m/z (%): 539 (M+. + 1, 1.5%).
1
(cm−1): 3379, 3284 (NH2), 2208 (CN), 1719 (C O). H-
NMR (DMSO-d6): δ 7.84 (br.s, 2H, NH2, D2O exchange-
able), 7.66 (s, 1H, CH-arylidine), 7.54-7.47 (m, 8H, H-Ar),
and 4.65 (s, 1H, H pyridine). MS, m/z (%): 451
(M.+, 8.3%).
4.2 | 2,9-Diamino-4,7-bis
(4-chlorophenyl)-4H,7H-pyrano[20,30:4,5]
thiazolo[3,2-a]pyridine-
3,6,8-tricarbonitrile (2)
A mixture of compound 1 (0.01 mol, 4.51 g) and
malononitrile (0.01 mol, 0.66 g) in dry pyridine (20 mL)
was heated under reflux for 10 hours. The reaction mix-
ture was poured into ice/HCl. The formed precipitate
was filtered off, dried and recrystallized from ethanol to
give 2.
Yield (44%), mp >300ꢀC (ethanol). Anal. Calcd. For
C25H14Cl2N6OS (517): C, 58.04; H, 2.73; N, 16.24.
Found: C, 58.06; H, 2.70; N, 16.26. FT-IR (KBr) (cm−1):
3406-3156 (2NH2), 2211 (CN). 1H-NMR (DMSO-d6)
δ: 7.90-7.31 (m, 8H, H-Ar), 6.61 (br.s, 2H, NH2, exchange-
able), 6.49 (br.s, 2H, NH2, D2O exchangeable), 4.10
(s, 1H, H-pyridine), 3.61 (s, 1H, H-pyrane). MS, m/z (%):
517 (M+., 37.6%).
4.5 | Amino-3,6-bis(4-chlorophenyl)-6H-
isoxazolo[50,40:4,5]thiazolo[3,2-a]pyridine-
5,7-dicarbonitrile (4)
A mixture of compound 1 (0.01 mol, 4.51 g) and hydrox-
ylamine hydrochloride (0.01 mol, 0.68 g) in alcoholic
NaOH (20%) (30 mL) was heated under reflux for
20 hours. The reaction mixture was poured into ice/HCl.
The formed residue was filtered off, dried and rec-
rystallized from benzene/ethanol to give 4.
Yield (54%), mp 96ꢀC-98ꢀC (benzene/ethanol). Anal.
Calcd. For C22H11Cl2N5OS (464): C, 56.91; H, 2.39; N,
15.08. Found: C, 56.89; H, 2.37; N, 15.11. FT-IR (KBr)
(cm−1): broad band centered at 3442 (NH2), 2207 (CN),
1645 (C C), 1594 (C C). 1H-NMR (DMSO-d6) δ:
7.82-7.50 (m, 8H, H-Ar), 5.57 (br.s, 2H, NH2, D2O
exchangeable), and 4.15 (s, 1H, H- pyridine). MS, m/z
(%): 464 (M+., 16%).
4.3 | 8-Amino-3,6-bis(4-chlorophenyl)-
1H,6H-pyrazolo[30,40:4,5]thiazolo[3,2-a]
pyridine-5,7-dicarbonitrile (3a)
A mixture of compound 1 (0.01 mol, 4.51 g), hydrazine
hydrate (0.02 mol, 1 mL) in absolute ethanol (30 mL) was
heated under reflux for 20 hours. The reaction mixture
was poured into ice/HCl. The formed precipitate was fil-
tered off, dried and recrystallized from mixed benzene/
ethanol to give 3a.
4.6 | Amino-4,7-bis(4-chlorophenyl)-
2-thioxo-1,2-dihydro-7H-pyrido[20,10:2,3]
thiazolo [4,5-d]pyrimidine-
Yield (70%), mp 112ꢀC-114ꢀC (benzene/ethanol).
Anal. Calcd. For C22H12Cl2N6S (463): C, 57.03; H,
2.61; N, 18.14; Found: C, 57.01; H, 2.63; N, 18.13. FT-IR
(KBr) (cm−1): 3329-3216 (NH2, NH), 2211 (CN), 1599
6,8-dicarbonitrile (5)
A solution of compound 1 (0.01 mol, 4.51 g), thiourea
(0.01 mol, 0.76 g) in ethyl alcohol (20 mL) and alc.
sodium hydroxide (40%) (30 mL) was heated under reflux
for 10 hours. The reaction mixture was poured into
ice/HCl. The formed solid was filtered off, washed by
1
(C N). H-NMR (DMSO-d6) δ: 10.81 (br.s, 1H, NH, D2O
exchangeable), 8.04-7.17 (m, 8H, H-Ar), 4.81 (br.s, 2H,
NH2, D2O exchangeable), and 4.73 (s, 1H, H-pyridine).
MS, m/z (%): 422 (M+. − 41, 4.5%).

12
MAHMOUD AND BALAMON
water several times, dried and recrystallized from ben-
zene to give 5.
(br.s, 2H, NH2, D2O exchangeable), 4.27 (s, 1H, H-pyri-
dine). MS, m/z (%): 505 (M+., 14%).
Yield (66%), mp 184ꢀC-186ꢀC (benzene). Anal. Calcd.
For C23H12Cl2N6S2 (507): C, 54.44; H, 2.38; N, 16.56
Found: C, 54.43; H, 2.36; N, 16.62. FT-IR (KBr) (cm−1):
4.9 | 4-Amino-8-(4-chlorobenzylidene)-
5-(4-chlorophenyl)-9-oxo-8,9-dihydro-5H-
thiazolo[30,20:1,6]pyrido[2,3-d]pyrimidine-
6-carbonitrile (8)
1
3342-3192 (NH2, NH), 2193 (CN), 1651 (C N). H-NMR
(DMSO-d6) δ: 10.38 (br.s, 1H, NH, D2O exchangeable),
7.95 (br.s, 2H, NH2, D2O exchangeable), 7.68-7.08 (m, 8H,
H-Ar), and 4.41 (s, 1H, H-pyridine). 13C-NMR (DMSO-d6)
δ, 180.4, 164.6, 161.5, 151.7, 140.3, 138.4, 136.6, 131.8,
131.2, 130.6 (2), 130.4, 128.9 (2), 128.5 (2), 126.0, 118.3,
117.1, 80.0, 69.9, 57.6., 37.1. MS, m/z (%): 444 (M+.
− 62, 7%).
A mixture of compound 1 (0.01 mol, 4.51 g) and formam-
ide (20 mL) was heated under reflux for 12 hours. The
reaction mixture was poured into ice/water and then the
formed residue was filtered off, dried and recrystallized
from ethanol to give 8.
Yield (40%), mp >300ꢀC (ethanol). Anal. Calcd. For
C23H13Cl2N5OS (478): C, 57.75; H, 2.74; N, 14.64.
Found: C, 57.71; H, 2.77; N, 14.61. FT-IR (KBr) (cm−1):
3332, 3203 (NH2), 2207 (CN), 1714 (C O), 1628 (C N).
MS, m/z (%): 480 (M+. + 2, 20%).
4.7 | Ethyl-N-(2-(-4-chlorobenzylidene)-
7-(4-chlorophenyl)-6,8-dicyano-3-oxo-
2,3-dihydro-7H-thiazolo[3,2-a]pyridin-5-yl)
formimidate (6)
A solution of compound 1 (0.01 mol, 4.51 g) in triethyl
orthoformate (20 mL) was heated under reflux for
6 hours. The reaction mixture was concentrated and then
the formed precipitate was collected and recrystallized
from ethanol to give 6.
4.10 | 8-(4-Chlorobenzylidene)-
5-(4-chlorophenyl)-4,9-dioxo-
3,5,8,9-tetrahydro-4H-thiazolo[30,20:1,6]
pyrido[2,3-d]pyrimidine-6-carbonitrile (9)
Yield (44%), mp 253ꢀC-255ꢀC (ethanol). Anal. Calcd.
For C25H16Cl2N4O2S (507): C, 59.18; H, 3.18; N, 11.04.
Found: C, 58.99; H, 3.12; N, 11.07. FT-IR (KBr) (cm−1):
A solution of compound 1 (0.01 mol, 4.51 g) in formic
acid (20 mL) was heated under reflux for 10 hours. The
reaction mixture was cooled, and the precipitated solid
was collected by filtration and recrystallized from ben-
zene to give 9.
Yield (20%), mp >300ꢀC (benzene). Anal. Calcd. For
C23H12Cl2N4O2S (479): C, 57.63; H, 2.52; N, 11.69.
Found: C, 57.65; H, 2.56; N, 11.68. FT-IR (KBr) (cm−1):
3187 (NH), 2205 (CN), 1706, 1658 (2C O), 1604 (C N).
MS, m/z (%): 479 (M+., 41%).
1
2209 (CN), 1739 (C O), 1640 (C N). H-NMR (DMSO-
d6) δ: 8.22 (s, 1H, N CH), 7.82-7.53 (m, 8H, H-Ar), 6.79
(s, 1H, CH-arylidene), 4.82 (s, 1H, H-pyridine), 4.37
(q, 2H, CH2) 1.36 (t, 3H, CH3). MS, m/z (%):
506 (M+. − 1, 25%).
4.8 | 3-Amino-5,8-bis(4-chlorophenyl)-
4-imino-3,5-dihydro-4H,10H-pyrazolo
[30,40:40,50]thiazolo[30,20:1,6]pyrido[2,3-d]
pyrimidine-6-carbonitrile (7)
4.11 | 8-(4-Chlorobenzylidene)-
5-(4-chlorophenyl)-2-methyl-4,9-dioxo-
3,5,8,9-tetrahydro-4H-thiazolo[30,20:1,6]
pyrido[2,3-d]pyrimidine-6-carbonitrile (10)
Compound 6 (0.01 mol, 5.07 g) was fused with hydra-
zine hydrate (0.02 mol, 1 mL) for 8 hours. The reaction
mixture was poured into ice/HCl and the formed pre-
cipitate was filtered off, dried and recrystallized from
dioxane to yield 7.
Yield (21%), mp >300ꢀC (dioxane). Anal. Calcd. For
C23H14Cl2N8S (505): C, 54.66; H, 2.79; N, 22.17.
Found: C, 54.69; H, 2.80; N, 22.22. FT-IR (KBr) (cm−1):
3331-3211 (NH2, 2NH), 2210 (CN), 1602 (C N). 1H-
NMR (DMSO-d6) δ: 12.79 (br.s, 1H, NH, D2O exchange-
able), 11.15 (br.s, 1H, NH, D2O exchangeable), 8.36
(s, 1H, H-pyrimidine), 7.65-7.26 (m, 8H, H-Ar), 5.18
A solution of compound 1 (0.01 mol, 4.51 g) in acetic anhy-
dride (15 mL) and pyridine (5 mL) was heated under reflux
for 24 hours. The reaction mixture was cooled and poured
into ice/water with stirring and refrigerated overnight. The
separated solid was collected by filtration, washed by
water, dried and recrystallized from toluene to give 10.
Yield (65%), mp >300ꢀC (toluene). Anal. Calcd. For
C24H14Cl2N4O2S (493): C, 58.43; H, 2.86; N, 11.36.
Found: C, 58.40; H, 2.89; N, 11.40. FT-IR (KBr) (cm−1):

MAHMOUD AND BALAMON
13
3428 (NH), 2214 (CN), 1709 (C O), 1675 (C O), 1635
(C N). H-NMR (DMSO-d6) δ: 12.33 (br.s, 1H, NH, D2O
exchangeable), 7.95 (s, 1H, CH-arylidene), 7.93-6.98 (m,
8H, H-Ar), 4.63 (s, 1H, H-pyridine), and 1.91 (s, 3H,
CH3). MS, m/z (%): 493 (M+., 25%).
1H-NMR (DMSO-d6) δ: 7.95-7.01 (m, 8H, H-Ar), 6.14 (br.
s, 2H, NH2, D2O exchangeable), 5.45 (s, 1H, H-pyridine),
5.10 (s, 1H, H-pyrane), 4.20 (q, 2H, CH2), 2.01 (s, 3H,
CH3), 1.20 (t, 3H, CH3). MS, m/z (%): 563 (M+., 42%).
1
4.15 | Ethyl 9-amino-4,7-bis
(4-chlorophenyl)-6,8-dicyano-2-oxo-2H,7H-
pyrano[20,30:4,5]thiazolo[3,2-a]pyridine-
3-carboxylate (13)
4.12 | 2-(4-Chlorobenzylidene)-
7-(4-chlorophenyl)-5-(cyclohexylamino)-
3-oxo-2,3-dihydro-7H-thiazolo[3,2-a]
pyridine-6,8-dicarbonitrile (11)
Yield (38%), mp 183ꢀC-184ꢀC (ethanol). Anal. Calcd. For
C27H16Cl2N4O4S (563): C, 57.56; H, 2.86; N, 9.94.
Found: C, 57.58; H, 2.84; N, 9.91. FT-IR (KBr) (cm−1):
3332-3204 (NH2), 2206 (CN), 1771 (C O), 1710 (C O),
A mixture of compound 1 (0.01 mol, 4.51 g) and bromo-
cyclohexane (0.01 mol, 1.63 mL) in alcoholic sodium
hydroxide (40%) (30 mL) was heated under reflux for
8 hours. The reaction mixture was poured into ice/HCl.
The formed precipitate was filtered off, dried and rec-
rystallized by benzene to give 11.
Yield (33%), mp 295ꢀC-296ꢀC (benzene). Anal. Calcd.
For C28H22Cl2N4OS (533): C, 63.04; H, 4.16; N, 10.50.
Found: C, 63.02; H, 4.19; N, 10.53. FT-IR (KBr) (cm−1):
3204 (NH), 2188 (CN), 1719 (C O), 1641 (C C). 1H-
NMR (DMSO-d6) δ: 8.98 (br.s, 1H, NH, D2O exchange-
able), 8.13 (s, 1H, CH-arylidene), 7.95-6.92 (m, 8H, H-Ar),
4.48 (s, 1H, H-pyridine), 2.16-1.62 (m, 11H, H-cyclohex-
ane). 13C-NMR (DMSO-d6) δ, 161.5, 157.3, 152.0, 140.3,
138.4, 133.6, 133.3, 133.2, 131.3, 130.4 (2), 129.0 (2), 128.9
(3), 127.5, 118.3, 117.1, 70.5, 58.2, 57.6, 37.8, 32.9 (2), 25.7,
25.1 (2). MS, m/z (%): 530 (M+. − 2, 18%).
1
1609 (C C). H-NMR (DMSO-d6) δ: 7.65-7.09 (m, 8H, H-
Ar), 5.75 (br.s, 2H, NH2, D2O exchangeable), 5.10 (s, 1H,
H-pyridine), 4.20 (q, 2H, CH2), and 1.23 (t, 3H, CH3). MS,
m/z (%): 537 (M+. − 26, 0.3%).
4.16 | 2,9-Diamino-4,7-bis
(4-chlorophenyl)-3-phenyl-4H,7H-pyrano
[20,30:4,5]thiazolo[3,2-a]pyridine-
6,8-dicarbonitrile (14)
A solution of benzyl cyanide (0.02 mol, 2.34 g) in the least
amount of dry benzene (10 mL) was stirred at room temper-
ature for 10 minutes. Then solid sodium methoxide (0.23 g
of sodium metal in 10 mL of absolute methanol then evapo-
ration till dryness) was added and a solution of 1 (0.01 mol,
4.51 g) in dry benzene (10 mL) was added dropwise within
30 minutes. With stirring to benzyl cyanide solution, the
reaction mixture was heated using water bath for 10 minutes,
cooled, collected the formed precipitate and recrystallized
from mixed benzene/ethanol to give 14.
Yield (40%), mp 244ꢀC-245ꢀC (benzene/ethanol).
Anal. Calcd. For C30H19Cl2N5OS (568): C, 63.39; H,
3.37; N, 12.32. Found: C, 63.42; H, 3.40; N, 12.29. FT-IR
(KBr) (cm−1): 3344-3213 (2NH2), 2214 (CN), 1637 (C C).
1H-NMR (DMSO-d6) δ: 7.95-7.01 (m, 13H, H-Ar), 5.09
(br.s, 2H, NH2, D2O exchangeable), 4.65 (br.s, 2H, NH2,
D2O exchangeable), 4.43 (s, 1H, H-pyridine), and 3.90 (s,
1H, H-pyrane). MS, m/z (%): 570 (M+. + 2, 10%).
4.13 | The general method for preparing
12, 13, 15, 16
A solution of compound 1 (0.01 mol, 4.51 g) in sodium
ethoxide (0.5 g of sodium in 25 mL of ethanol) and ethyl
acetoacetate and/or diethyl malonate and/or benzyl cyanide
and/or ethyl chloroacetate and/or ethyl cyanoacetate and/or
cyano acetic acid (0.01 mol) was heated under reflux for
8 hours. The reaction mixture was poured into ice/HCl. The
formed precipitate was filtered off, dried and recrystallized
from proper solvent to yield 12, 13, 15, and 16, respectively.
4.14 | Ethyl 9-amino-4,7-bis
(4-chlorophenyl)-6,8-dicyano-2-methyl-
4H,7H-pyrano[20,30:4,5]thiazolo[3,2-a]
pyridine-3-carboxylate (12)
4.17 | 9-Amino-3-chloro-4,7-bis
(4-chlorophenyl)-2-oxo-2H,7H-pyrano
[20,30:4,5]thiazolo[3,2-a]pyridine-
6,8-dicarbonitrile (15)
Yield (30%), mp 285ꢀC-287ꢀC (ethanol). Anal. Calcd. For
C28H20Cl2N4O3S (563): C, 59.69; H, 3.58; N, 9.94.
Found: C, 59.67; H, 3.61; N, 9.97. FT-IR (KBr) (cm−1):
3383-3217 (NH2), 2214 (CN), 1725 (C O), 1625 (C C).
Yield (25%), mp >300ꢀC (dioxane). Anal. Calcd. For
C24H11Cl3N4O2S (526): C, 54.83; H, 2.11; N, 10.66.

14
MAHMOUD AND BALAMON
Found: C, 54.88; H, 2.12; N, 10.70. FT-IR (KBr) (cm−1):
broad band at 3417 (NH2), 2207 (CN), 1716 (C O), 1633
(C C). H-NMR (DMSO-d6) δ: 8.55 (br.s, 2H, NH2, D2O
exchangeable), 7.64-7.33 (m, 8H, H-Ar), and 4.12 (s, 1H,
H-pyridine). MS, m/z (%): 526 (M+., 10%).
Yield (36%), mp >300ꢀC (ethanol). Anal. Calcd. For
C27H16Cl2N8OS (571): C, 56.75; H, 2.82; N, 19.61.
Found: C, 56.71; H, 2.85; N, 19.60. FT-IR (KBr) (cm−1):
3418-3219 (2NH2), 2206 (CN), 1630 (C N). 1H-NMR
(DMSO-d6) δ: 8.15, 7.92 (s, 2H, H-pyrimidine), 7.76-7.51
(m, 8H, H-Ar), 6.12 (br.s, 4H, 2NH2, D2O exchangeable),
4.25 (s, 2H, H-pyridine, H-pyrane). MS, m/z (%): 567 (M+.
− 4, 13%).
1
4.18 | 9-Amino-4,7-bis(4-chlorophenyl)-
2-oxo-2H,7H-pyrano[20,30:4,5]thiazolo[3,2-a]
pyridine-3,6,8-tricarbonitrile (16)
4.21 | 4,7-Bis(4-chlorophenyl)-2-(([E]-
4-nitrobenzylidene)amino)-9-([4-nitro
benzylidene]amino)-4H,7H-pyrano[20,30:4,5]
thiazolo[3,2-a]pyridine-3,6,8-tri
carbonitrile (19)
Yield (42%), mp 254ꢀC-256ꢀC (ethanol). Anal. Calcd. For
C25H11Cl2N5O2S (516): C, 58.15; H, 2.15; N, 13.56.
Found: C, 58.18; H, 2.13; N, 13.60. FT-IR (KBr) (cm−1):
3330-3204 (NH2), 2202 (CN), 1721 (C O), 1641 (C C).
1H-NMR (DMSO-d6) δ: 7.86-7.51 (m, 8H, H-Ar), 5.88 (br.
s, 2H, NH2, D2O exchangeable), and 4.25 (s, 1H, H-pyri-
dine). MS, m/z (%): 512 (M+. − 4, 0.13%).
A mixture of compound 2 (0.01 mol, 5.17 g) and p-nitro
benzaldehyde (0.01 mol and/or 0.02 mol) in glacial acetic
acid (30 mL) was heated under reflux for 8 hours. The
formed precipitate was filtered off, dried and rec-
rystallized from ethanol to give 19.
4.19 | 5,8-Bis(4-chlorophenyl)-4,9-dioxo-
2,11-diphenyl-3,5,9,12-tetrahydro-4H,8H-
pyrimidino[3000,2000:500,600]pyrano[200,300:40,50]
thiazolo[30,20:1,6]pyrido[2,3-d]pyrimidine-
6-carbonitrile (17)
Yield (25%) and/or (60%), mp >300ꢀC (ethanol). Anal.
Calcd. For C39H20Cl2N8O5S (783): C, 59.78; H, 2.57; N,
14.30. Found: C, 59.81; H, 2.56; N, 14.35. FT-IR (KBr)
1
(cm−1): 2210 (CN), 1624 (C N). H-NMR (DMSO-d6) δ:
10.16 (s, 2H, H-benzylidenimin), 8.43-7.57 (m, 16H, H-
Ar), 5.62 (s, 1H, H-pyridine), and 5.15 (s, 1H, H-pyrane).
13C-NMR (DMSO-d6) δ: 170.2, 163.5 (2), 155.3, 152.0,
150.3 (2), 143.3, 140.1 (2), 139.6 (2), 131.4 (2), 130.4 (4),
128.9 (4), 127.5 (4), 124.0 (4), 118.3, 117.1 (2), 84.1, 76.3,
76.1, 70.5, 41.2, 38.2. MS, m/z (%): 784 (M+. + 1, 24%).
A mixture of compound 2 (0.01 mol, 5.17 g) and benzoyl
chloride (20 mL) was heated under reflux for 24 hours.
After evaporation of the solvent, the formed product was
recrystallized with petroleum ether (80ꢀC-100ꢀC) to
give 17.
Yield (54%), mp 114ꢀC-116ꢀC, petroleum ether (80ꢀC-
100ꢀC). Anal. Calcd. For C39H22Cl2N6O3S (725): C,
64.56; H, 3.06; N, 11.58. Found: C, 64.55; H, 3.09; N,
11.61. FT-IR (KBr) (cm−1): 3305 (2NH), 2220 (CN), 1667
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
1
(2C O), 1602 (C C). H-NMR (DMSO-d6) δ: 12.91 (br.s,
ORCID
2H, 2NH, D2O exchangeable), 7.92-7.46 (m, 18H, H-Ar),
5.41 (s, 1H, H-pyridine), and 4.19 (s, 1H, H-pyrane). MS,
m/z (%): 551 (M+. − 173, 3%).
Naglaa F. H. Mahmoud https://orcid.org/0000-0002-
6655-5741
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