Synthesis, in-vitro cytotoxicity and antimicrobial evaluations of some novel thiazole based heterocycles

Article abstract of DOI:10.1248/cpb.c19-00681

Condensation of rhodanine (1) with pyrazol-3(2H)-one derivatives (2a-f) gave 5-substituted-2-thioxo-1,3- thiazolidin-4-one derivatives (3a-f). Reaction of compound (1) with 2-arylmethylidene-malononitrile (4a-d) yielded the unexpected derivatives (5a-d). The latter compounds were subjected to cyclization reactions with malononitrile under different basic conditions, hydroxylamine hydrochloride and/or thiourea to furnish the fused thiazole derivatives (6a-d) and (8-10a-d). Coupling of (1) with diazotized aromatic amines (11a-c) in pyridine afforded the arylhydrazones (12a-c). Fusion of latter compounds with malononitrile afforded the thiazolopyridazine derivatives (13a-c). The structures of the newly synthesized compounds were elucidated via spectral data and elemental analyses. The in-vitro cytotoxic activity of compounds (3a-f) against the cell line MCF-7 was evaluated. Also, the synthesized products were investigated for their antibacterial and antifungal activities against six standard organisms including the G- bacteria, Staphylococcus aureus and Bacillus subtilis, G+ bacteria, Escherichia coli and Proteus vulgaris in addition to fungi, Candida albicans and Aspergillus flavus.

Full text of DOI:10.1248/cpb.c19-00681

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Chem. Pharm. Bull. 67, 1314–1323 (2019)
Vol. 67, No. 12
Regular Article
Synthesis, in-Vitro Cytotoxicity and Antimicrobial Evaluations of Some
Novel Thiazole Based Heterocycles
Heba Kamal Abd El-Mawgoud
Department of Chemistry, Faculty of Women for Arts, Science and Education, Ain Shams University; Heliopolis,
Cairo 11767, Egypt.
Received August 11, 2019; accepted September 24, 2019
Condensation of rhodanine (1) with pyrazol-3(2H)-one derivatives (2a–f) gave 5-substituted-2-thioxo-1,3-
thiazolidin-4-one derivatives (3a–f). Reaction of compound (1) with 2-arylmethylidene-malononitrile (4a–d)
yielded the unexpected derivatives (5a–d). The latter compounds were subjected to cyclization reactions with
malononitrile under different basic conditions, hydroxylamine hydrochloride and/or thiourea to furnish the
fused thiazole derivatives (6a–d) and (8–10a–d). Coupling of (1) with diazotized aromatic amines (11a–c) in
pyridine afforded the arylhydrazones (12a–c). Fusion of latter compounds with malononitrile afforded the
thiazolopyridazine derivatives (13a–c). The structures of the newly synthesized compounds were elucidated
via spectral data and elemental analyses. The in-vitro cytotoxic activity of compounds (3a–f) against the
cell line MCF-7 was evaluated. Also, the synthesized products were investigated for their antibacterial and
antifungal activities against six standard organisms including the G+ bacteria, Staphylococcus aureus and
Bacillus subtilis, Gꢀ bacteria, Escherichia coli and Proteus vulgaris in addition to fungi, Candida albicans
and Aspergillus flavus.
Key words fused thiazole; cytotoxicity; antibacterial activity; antifungal activity
evaluate in-vitro for cytotoxic and antimicrobial activities.
Introduction
Rhodanines (2-thioxo-1,3-thiazolidin-4-ones) and rhoda-
nine-based molecules are accepted as advantaged heterocycles
in drugs discovery processes and in medicinal chemistry.1,2)
Results and Discussion
Chemistry Condensation of rhodanine (1) with the ap-
They have shown a varied range of pharmacological activi- propriate 4-(arylmethylideneamino)-1,5-dimethyl-2-phenyl-1H-
ties such as antimicrobial,3–5) anticonvulsant,6,7) antibacterial, pyrazol-3(2H)-one (2a–f) in boiling glacial acetic acid
antifungal and insecticidal activities.8–13) Furthermore, some containing a catalytic amount of anhydrous sodium acetate
of them were reported to possess antiviral,14) antidiabetic,15,16) afforded the corresponding 5-[4-(arylmethylideneamino)-1,5-
antiinflammatory17,18) and antimalarial19) activities. Moreover, dimethyl-2-phenyl-1H-pyrazol-3(2H)-ylidene]-2-thioxo-1,3-thi-
various substituted rhodanines were found to be anti-tubercu- azolidin-4-one (3a–f) in a high yield (Chart 1). The structures
lar,20,21) antiproliferative agent against human colon cancer,22) of the latter compounds were elucidated by their elemental
anti-human immunodeficiency virus (HIV),23–27) cyclooxy- analyses and spectral data (cf. Experimental).
genase (COX-2) inhibitors,28) potent PTP1B inhibitors,29) an-
Reaction of compound (1) with 2-arylmethylidenemalono-
tiglioma and cytotoxicity.30) Additionally, rhodanine-based nitrile (4a–d) in absolute ethanol in presence of piperidine
molecules have been popular as small molecule inhibitors of as a catalyst and stirring at room temperature furnished
numerous targets such as Hepatitis C virus NS3 protease,31) compounds (5a–d) in a good yield. The structures of these
β-lactamase,32) uridine diphospho-N-acetylmuramate/L-alanine compounds were identified to be the unexpected 5-arylme-
ligase,33) cathepsin D34) and c-Jun N-terminal kinase (JNK)- thylidene-2-thioxo-1,3-thiazolidin-4-one not the expected
stimulating phosphatase-1 (JSP-1),35) while some of rhodanine 5-amino-7-aryl-2-thioxo-3,7-dihydro-2H-pyrano[2,3-d]-1,3-
derivatives are used for the analysis of certain noble metal thiazole-6-carbonitrile (6a–d) on the basis of their IR, MS,
ions.36) Based on these considerations, the present work aimed 1H-NMR, 13C-NMR and elemental analyses. The IR spectra of
to synthesize some novel thiazole based heterocycles and to compounds (5a–d) revealed the presence of C=O group and
Chart 1. Synthesis of 5-[4-(Arylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-ylidene]-2-thioxo-1,3-thiazolidin-4-one Derivatives (3a–f)
e-mail: Heba.Kamal@women.asu.edu.eg
© 2019 The Pharmaceutical Society of Japan

Vol. 67, No. 12 (2019)
Chem. Pharm. Bull.
1315
there are no bands due to –NH2 or –C≡N groups. In addition rhodanine (1) and 2-arylmethylidenemalononitrile (4a–d) are
1
to the presence of signals due to=CH proton in H-NMR. reacted by Michael addition in presence of base to give the
Further verification for the structures of compounds (5a–d) intermediate (A), which upon enolization gave the intermedi-
was their synthesis directly by condensation of rhodanine (1) ate (B). This intermediate was expected to undergo the reverse
with aromatic aldehydes (7a–d) in presence of various cata- of a Michael reaction “β-elimination of malononitrile mol-
lysts as reported37–42) (identical mp, mixed mp and TLC). This ecule—pathway (a)” to yield compounds (5a–d). The presence
is agreeing with a previous result.20) However, the pyrano[2,3- of malononitrile in the reaction medium was detected using
d]thiazole derivatives (6a–d) were obtained by the reaction TLC. The intramolecular 6-exo-dig cyclization of the interme-
of the 5-arylmethylidene-thiazolidin-4-one derivatives (5a–d) diate (B)—pathway (b) to furnish the pyranothiazole deriva-
with malononitrile in boiling ethanol in presence of catalytic tives (6a–d) not happened under these conditions.
amount of triethylamine (Chart 2). The structures of com-
pounds (6a–d) were excluded on the basis of elemental analy- solute ethanol in presence of a catalytic amount of ammonium
ses and spectral data (cf. Experimental).
acetate furnished the thiazolo[4,5-b]pyridine-6-carbonitrile
Treatment of compounds (5a–d) with malononitrile in ab-
The proposed mechanism for the formation of compounds derivatives (8a–d). The structures of these compounds were
1
(5a–d) can be represented as shown in Chart 3. Initially, elucidated by their elemental analyses, IR, MS, H-NMR and
Chart 2. Reaction of Rhodanine (1) with 2-Arylidenemalononitrile (4a–d)
Chart 3. The Proposed Mechanism for the Formation of Compounds (5a–d) Not the Expected (6a–d)

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Chem. Pharm. Bull.
Vol. 67, No. 12 (2019)
Chart 4. Synthesis of 1,3-Thiazolo[4,5-b]pyridine (8a–d); 1,3-Thiazolo[4,5-c]isoxazole (9a–d) and 1,3-Thiazolo[4,5-d]pyrimidine (10a–d)
Chart 5. Synthesis of 1,3-Thiazolo[5,4-c]pyridazine Derivatives (13a–c)
13C-NMR. Compounds (8a–d) were also obtained by boiling
the pyrano[2,3-d]-1,3-thiazole derivatives (6a–d) in absolute
Table 1. Anticancer IC50 Values for Selected Compounds against MCF-7
ethanol containing a catalytic amount of ammonium acetate
Compound
MCF-7, IC50 values (µg/mL)
(identical mp, mixed mp, TLC and spectra). Furthermore, re-
fluxing compounds (5a–d) with hydroxylamine hydrochloride
in absolute ethanol containing anhydrous sodium acetate gave
the 1,3-thiazolo[4,5-c]isoxazole derivatives (9a–d). In addi-
tion, when the arylmethylidene derivatives (5a–d) were re-
acted with thiourea in boiling N,N-dimethylformamide (DMF)
containing few drops of triethylamine, 1,3-thiazolo[4,5-d]-
pyrimidine derivatives (10a–d) were obtained (Chart 4). The
spectral data and elemental analyses proved the structures of
compounds (9a–d) and (10a–d) (cf. Experimental).
3a
7.67 0.6
145 4.9
123 3.1
11.7 0.9
106 2.5
245 8.6
0.35 0.03
3b
3c
3d
3e
3f
Doxorubicin
inhibitory activity against MCF-7 human breast carcinoma
Ultimately, coupling of rhodanine (1) with diazotized aro- cell. The in-vitro cytotoxicity evaluation using viability assay
matic amines (11a–c) in pyridine at 0°C afforded the arylhy- was performed at the Regional Centre for Mycology and
drazone derivatives (12a–c) based on their spectral data as Biotechnology at Al-Azhar University, Cairo, Egypt, using
well as their synthesis using a reported method43) (identical Doxorubicin as reference standard drug. The inhibitory ac-
mp, mixed mp and TLC). The obtained arylhydrazones have tivity results are depicted in Table 1 (cf. Experimental). The
been utilized as starting materials for preparing fused pyrida- results of in-vitro inhibitory activities of the tested compounds
zine ring system. Fusion of the arylhydrazones (12a–c) with against MCF-7 cancer cell have the following descending
malononitrile in presence of ammonium acetate over melting order 3a>3d>3e>3c>3b>3f.
point for one hour gave the thiazolo[5,4-c]pyridazine deriva-
Antimicrobial Screening The new synthesised com-
tives (13a–c) (Chart 5). The structures of the latter compounds pounds were tested for their in-vitro antimicrobial activity
were confirmed based on elemental analyses and spectral data. against six standard organisms including the Gram-positive
Thus, the IR spectra of compounds (13a–c) indicated the pres- bacteria, Staphylococcus aureus [RCMB 010010] and Bacil-
ence of the absorption bands of the C≡N functional group at lus subtilis [RCMB 015 (1), NRRL B-543], Gram-negative
2202–2205cm−1. Also, the IR spectra revealed the absence of bacteria, Escherichia coli [RCMB 010052, ATCC 25955] and
absorption bands due to C=S functional group, as well as a Proteus vulgaris [RCMB 004 (1), ATCC 13315] in addition
strong smell of H2S gas was evolved during the reaction (cf. to fungi, Candida albicans [RCMB 005003 (1), ATCC 10231]
Experimental).
and Aspergillus flavus [RCMB 002002]. The antimicrobial
Cytotoxicity Evaluation The new synthesized com- screening was performed at the Regional Centre for Mycology
pounds (3a–f) were evaluated for human tumour cell growth and Biotechnology (RCMP) at Al-Azhar University, Cairo,

Vol. 67, No. 12 (2019)
Chem. Pharm. Bull.
1317
Table 2. Antimicrobial Screening for the All the Synthesized Compounds
Inhibition zone diameter (mm)
Gram-negative bacteria G−
Gram-positive bacteria G+
Staphylococcus Bacillus subtilis
Fungi
Candida albicans
[RCMB 005003 (1)]
[ATCC 10231]
Compound
Escherichia coli
Proteus vulgaris
[RCMB 004 (1)]
[ATCC 13315]
Aspergillus flavus
[RCMB 002002]
aureus
[RCMB 015 (1)]
[NRRL B-543]
[RCMB 010052]
[ATCC 25955]
[RCMB 010010]
3a
14
12
18
18
10
10
16
16
20
18
10
11
14
12
17
18
18
20
18
16
20
20
23
12
18
14
14
13
24
—
14
18
22
14
18
17
16
16
18
16
11
11
9
NA
NA
NA
NA
NA
NA
NA
18
19
12
21
13
11
19
9
NA
NA
NA
NA
NA
NA
NA
NA
18
NA
NA
NA
NA
NA
NA
13
16
17
13
9
3b
3c
3d
3e
3f
6a
6b
14
16
14
12
15
17
9
6c
19
6d
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
8
NA
NA
NA
NA
NA
18
8a
8b
12
18
21
17
22
16
15
21
8
8c
8d
7
9a
14
12
16
16
18
16
18
18
14
16
18
14
12
13
26
—
20
17
15
15
13
8
9b
13
9c
9d
12
14
10a
13
10b
18
10c
15
16
14
12
12
NA
NA
NA
25
—
20
17
16
20
NA
28
15
8
10d
17
12a
15
12b
NA
12
12
12c
24
13a
8
15
13b
NA
NA
30
14
13c
17
8
Gentamycin
Ketoconazole
—
—
20
—
16
* NA=No activity; Well diameter of the hole=6.0mm (100µL was tested), RCMB=Regional Center for Mycology and Biotechnology.
Egypt, using Gentamycin (minimum inhibitory concentra- 13c) showed very high antifungal activity against Candida
tion (MIC) 4µg/mL) and Ketoconazole (MIC 100µg/mL) as albicans, while eight compounds (9b, 9c, 9d, 10a, 12a, 12b,
reference standard drugs for bacteria and fungi, respectively. 13a and 13b) showed high antifungal activity against it. Fur-
The antimicrobial results are depicted in Table 2 (cf. Experi- thermore, four compounds (8d, 9b, 10a and 12c) showed very
mental).
high antifungal activity against Aspergillus flavus, as well as
The results indicated that five compounds (6c, 9d, 10c, 10d ten compounds (6b, 6c, 8c, 9a, 9c, 9d, 10c, 10d, 12a and 13a)
and 12a) showed high antimicrobial activity against Staphylo- showed high antifungal activity against Aspergillus flavus.
coccus aureus, and only one compound (3c) showed high anti- Ultimately, seven compounds (6a, 6d, 8a, 8b, 10b, 13b and
microbial activity against Bacillus subtilis. On the other hand, 13c) showed moderate antifungal activity against Aspergillus
all the synthesized compounds except (6c, 9d, 10c, 10d and flavus, as shown in Table 2.
12a) showed moderate antimicrobial activity against Staphy-
In conclusion, some compounds showed congruent results
lococcus aureus, as well as all of them except (3c, 8c and 8d) against the most tested microorganisms compared to the stan-
showed moderate antimicrobial activity against Bacillus subti- dard drugs Gentamycin and Ketoconazole.
lis, compared with the standard antibiotic Gentamycin.
Also, the results revealed that four compounds (3a, 3c, 3f
and 9a) showed high antimicrobial activity against Proteus
Experimental
Chemistry Melting points of the reaction products were
vulgaris. On the other hand, twenty compound (3b, 3d, 3e, determined in open capillary tubes on an electro-thermal
6a, 6b, 6c, 6d, 8a, 8b, 8c, 8d, 9b, 9c, 9d, 10a, 10c, 10d, 12a, melting point apparatus (MEL-TEMP II) and were uncor-
12b and 12c) showed moderate antimicrobial activity against rected. TLC was used in the monitoring of the progress of
Proteus vulgaris, with respect to Gentamycin.
all reactions and in the checking of the homogeneity of the
In comparison with the well-known antifungal Ketocon- synthesized compounds. It was run on TLC aluminium silica
azole, seven compounds (6c, 9a, 10b, 10c, 10d, 12c and gel sheets 60F254 (Merck) using UV light (254 and 365nm) for

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Chem. Pharm. Bull.
Vol. 67, No. 12 (2019)
detection. The FTIR were recorded on a Bruker (Model Alpha
5-[4-(4-Chlorobenzylideneamino)-1,5-dimethyl-2-
II) Infrared spectrometer at the Central Lab, Faculty of Sci- phenyl-1H-pyrazol-3(2H)-ylidene]-2-thioxo-1,3-thiazolidin-4-
1
ence, Ain Shams University, Cairo, Egypt. The H-NMR and one (3c)
13C-NMR spectra were measured on a Bruker Avance (III)
Yellow crystals; yield 86.4%; mp 222–224°C. 1H-NMR
1
400MHz spectrometer (400MHz for H-NMR and 100MHz (DMSO-d6) δ: 2.45 (3H, –CH3, s), 3.19 (3H, –NCH3, s),
for 13C-NMR) spectrometer in dimethyl sulfoxide (DMSO)-d6 7.36–7.83 (9H, Ar–H, m), 9.57 (1H, =CH, s), 13.48 (1H, –NH,
as solvent, using tetramethylsilane (TMS) as internal reference exchangeable with D2O, s). 13C-NMR (DMSO-d6) δ: 19.51,
and chemical shifts (δ) are expressed in ppm at the Center 34.78, 112.4, 126.55, 127.60, 128.70, 128.85, 129.08, 132.15,
for Discovery Research and Development at Faculty of Phar- 131.50, 135.20, 135.39, 140.85, 144.05, 163.25, 166.65, 198.15.
macy, Ain Shams University, Cairo, Egypt. Mass spectra were IR (KBr) υ: 3148 (N–H), 3032 (stretching C–Harom.), 2919,
recorded at (70ev) and carried out on direct probe controller 2843 (C–Haliph.), 1690 (C=O), 1652 (C=N), 1235 (C=S), 822
inlet part to single quadrupole mass analyzer in Thermo Sci- (bending C–Harom. p-disubstituted ring), 726, 674 (bending C–
entific GCMS Model (ISQ LT) using Thermo X-Caliber Soft- Harom. monosubstituted ring) cm−1. MS (70ev) m/z (%): 442.5
ware at the Regional Center for Mycology and Biotechnology (M+ +2, 5.34), 440.5 (M+, 24.80), 384.5 (31.16), 56 (100).
at Al-Azhar University, Naser City, Cairo, Egypt. Elemental Anal. Calcd for C21H17ClN4OS2 (440.97): C, 57.20; H, 3.89; N,
analyses were performed at the Central Lab, Faculty of Sci- 12.71. Found: C, 57.33; H, 3.92; N, 12.65.
ence, Ain Shams University, Cairo, Egypt.
General Procedure for Synthesis of Compounds pyrazol-3(2H)-ylidene]-2-thioxo-1,3-thiazolidin-4-one (3d)
(3a–f) A mixture of rhodanine (1) (0.01mol, 1.33g) and
Orange crystals; yield 88.7%; mp 244–246°C. 1H-NMR
5-[4-(4-Nitrobenzylideneamino)-1,5-dimethyl-2-phenyl-1H-
4-(arylmethylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol- (DMSO-d6) δ: 2.51 (3H, –CH3, s), 3.26 (3H, –NCH3, s),
3(2H)-one (2a–f) (0.01mol) in glacial acetic acid (20mL) in 7.37–8.30 (9H, Ar–H, m), 9.66 (1H, =CH, s), 13.61 (1H,
presence of anhydrous sodium acetate (0.03mol, 2.5g) was –NH, exchangeable with D2O, s). 13C-NMR (DMSO-d6) δ:
heated under reflux for 2h. After cooling, the solid product 31.35, 108.65, 125.65, 126.02, 126.25, 127.10, 128.15, 128.85,
obtained was filtered off, dried and recrystallized from etha- 132.35, 135.05, 137.20, 140.58, 149.35, 162.09, 168.19, 199.01.
nol to give (3a–f).
5-[4-(Benzylideneamino)-1,5-dimethyl-2-phenyl-1H- 2847 (C–Haliph.), 1699 (C=O), 1668 (C=N), 1523 (NO2, asym.),
pyrazol-3(2H)-ylidene]-2-thioxo-1,3-thiazolidin-4-one (3a)
IR (KBr) υ: 3169 (N–H), 3040 (stretching C–Harom.), 2917,
1340 (NO2, sym.), 1231 (C=S), 844 (bending C–Harom. p-di-
Yellow crystals; yield 89.3%; mp 200–202°C. 1H-NMR substituted ring), 763, 680 (bending C–Harom. monosubstituted
(DMSO-d6) δ: 2.51 (3H, –CH3, s), 3.37 (3H, –NCH3, s), ring) cm−1. MS (70ev) m/z (%): 451 (M+, 12.64), 405 (14.07),
7.48–7.60 (10H, Ar–H, m), 7.65 (1H, =CH, s), 13.82 (1H, 374 (17.29), 360 (9.96), 329 (12.64), 122 (20.63), 91 (25.66), 77
–NH, exchangeable with D2O, s). 13C-NMR (DMSO-d6) δ: (100). Anal. Calcd for C21H17N5O3S2 (451.52): C, 55.86; H, 3.79;
15.40, 39.59, 108.65, 119.75, 125.65, 126.02, 128.65, 128.85, N, 15.51. Found: C, 55.97; H, 3.84; N, 15.59.
129.91, 130.93, 131.20, 132.09, 133.44, 163.25, 169.86, 196.18.
5-[1,5-Dimethyl-4-(naphthalen-2-ylmethyleneamino)-2-
IR (KBr) υ: 3139 (N–H), 3036 (stretching C–Harom.), 2916, phenyl-1H-pyrazol-3(2H)-ylidene)-2-thioxo-1,3-thiazolidin-4-
2848 (C–Haliph.), 1696 (C=O), 1668 (C=N), 1225 (C=S), 759, one (3e)
672 (bending C–Harom. monosubstituted ring) cm−1. MS (70ev)
Yellow crystals; yield 89%; mp 258–260°C. 1H-NMR
m/z (%): 406 (M+, 3.80), 347 (4.19), 329 (6.55), 319 (12.49), 316 (DMSO-d6) δ: 2.53 (3H, –CH3, s), 3.40 (3H, –NCH3, s),
(3.69), 303 (4.10), 272 (5.82), 134 (100), 103 (5.14), 90 (35.80), 7.58–7.78 (5H, phenyl protons, m), 7.96–8.07 (7H, naphthyl
87 (30.67), 77 (26.66), 59 (60.42). Anal. Calcd for C21H18N4OS2 protons, m), 8.19 (1H, =CH, s), 13.91 (1H, –NH, exchangeable
(406.52): C, 62.04; H, 4.46; N, 13.78. Found: C, 62.19; H, 4.42; with D2O, s). 13C-NMR (DMSO-d6) δ: 19.05, 34.89, 108.65,
N, 13.87.
119.70, 125.65, 126.17, 126.55, 127.20, 127.69, 128.00, 132.30,
5-[4-(4-Methoxybenzylideneamino)-1,5-dimethyl-2- 133.54, 134.85, 135.05, 152.25, 166.85, 191.75. IR (KBr) υ:
phenyl-1H-pyrazol-3(2H)-ylidene]-2-thioxo-1,3-thiazolidin-4- 3138 (N–H), 3048 (stretching C–Harom.), 2916, 2835 (C–Haliph.),
one (3b)
1699 (C=O), 1670 (C=N), 1224 (C=S), 760, 699 (bending
Yellow crystals; yield 87.5%; mp 238–240°C. 1H-NMR C-Harom. monosubstituted ring) cm−1. MS (70ev) m/z (%):
(DMSO-d6) δ: 2.49 (3H, –CH3, s), 3.00 (3H, –NCH3, s), 3.81 456 (M+, 100), 379 (1.48), 365 (2.91), 338 (1.87), 329 (1.26),
(3H, –OCH3, s), 7.07–7.55 (9H, Ar–H, m), 9.15 (1H, =CH, 316 (1.44), 302 (4.13), 154 (9.85), 140 (23.92), 127 (21.76), 118
s), 13.51 (1H, –NH, exchangeable with D2O, s). 13C-NMR (3.03), 91 (45.61), 77 (31.48). Anal. Calcd for C25H20N4OS2
(DMSO-d6) δ: 19.78, 31.35, 56.01, 108.65, 115.49, 123.21, (456.58): C, 65.76; H, 4.42; N, 12.27. Found: C, 65.90; H, 4.45;
125.65, 126.04, 127.95, 128.85, 129.35, 130.38, 131.89, 133.05, N, 12.33.
161.69, 170.57, 196.26. IR (KBr) υ: 3127 (N–H), 3055 (stretch-
5-[4-(Benzo[d][1,3]dioxol-5-ylmethyleneamino)-1,5-
ing C–Harom.), 2917, 2847 (C–Haliph.), 1682 (C=O), 1655 (C=N), dimethyl-2-phenyl-1H-pyrazol-3(2H)-ylidene]-2-thioxo-1,3-
1236 (C=S), 820 (bending C–Harom. p-disubstituted ring), 776, thiazolidin-4-one (3f)
734 (bending C–Harom. monosubstituted ring) cm−1. MS (70ev)
Yellow crystals; yield 88.5%; mp 278–280°C. 1H-NMR
m/z (%): 436 (M+, 100), 421 (3.76), 405 (8.69), 380 (7.78), (DMSO-d6) δ: 2.49 (3H, –CH3, s), 3.31 (3H, –NCH3, s), 6.13
360 (2.69), 345 (13.52), 329 (12.94), 316 (1.30), 302 (1.66), (2H, –CH2, s), 7.07–7.16 (8H, Ar–H, m), 7.54 (1H, =CH,
289 (1.34), 147 (21.31), 134 (3.10), 120 (52.40), 107 (11.30), 91 s), 13.73 (1H, –NH, exchangeable with D2O, s). 13C-NMR
(18.28), 76 (73.69), 56 (8.86). Anal. Calcd for C22H20N4O2S2 (DMSO-d6) δ: 18.79, 34.49, 104.95, 108.67, 118.53, 120.01,
(436.55): C, 60.53; H, 4.62; N, 12.83. Found: C, 60.71; H, 4.65; 125.50, 125.65, 126.02, 126.55, 126.78, 127.75, 129.55, 132.35,
N, 12.75.
139.80, 140.58, 152.25, 155.48, 166.65, 198.15. IR (KBr) υ:
3141 (N–H), 3042 (stretching C–Harom.), 2983, 2892 (C–Haliph.),

Vol. 67, No. 12 (2019)
Chem. Pharm. Bull.
1319
1687 (C=O), 1627 (C=N), 1217 (C=S), 745, 701 (bending C- 100), 321.5 (M+, 3.41), 305.5 (6.67), 279.5 (6.72), 210 (1.36),
Harom. monosubstituted ring) cm−1. MS (70ev) m/z (%): 450 255.5 (1.29), 230.5 (9.85), 111.5 (9.51), 91 (1.40). Anal. Calcd
(M+, 13.80), 435 (11.04), 391 (16.70), 373 (16.70), 359 (13.83), for C13H8ClN3OS2 (321.81): C, 48.52; H, 2.51; N, 13.06. Found:
330 (10.68), 120 (21.16), 91 (18.64), 77 (8.52), 59 (100). Anal. C, 48.40; H, 2.55; N, 13.15.
Calcd for C22H18N4O3S2 (450.53): C, 58.65; H, 4.03; N, 12.44.
Found: C, 58.78; H, 4.08; N, 12.53.
General Procedure for Synthesis of Compounds (5a–d)
5-Amino-7-(4-nitrophenyl)-2-thioxo-3,7-dihydro-2H-
pyrano[2,3-d]-1,3-thiazole-6-carbonitrile (6d)
Dark brown crystals; yield 77.2%; mp 180–182°C. H-NMR
1
Method A: A solution of compound (1) (0.01mol, 1.33g) and (DMSO-d6) δ: 4.49 (1H, pyran H-4, s), 7.22 (2H, –NH2,
2-arylmethylidenemalononitrile (4a–d) in absolute ethanol in exchangeable with D2O, s), 7.32–7.86 (4H, Ar–H, m), 8.01
presence of piperidine as a catalyst was stirred at room tem- (1H, –NH, exchangeable with D2O, s). 13C-NMR (DMSO-d6)
perature for 3h. The solid product obtained was filtered off, δ: 56.28, 40.41, 84.05, 117.85, 126.59, 128.37, 142.65, 143.08,
dried and recrystallized from ethanol to afford (5a–d).
146.65, 151.75, 189.20. IR (KBr) υ: 3351, 3256, 3106 (–NH2,
N–H), 3081 (stretching C–Harom.), 2918, 2850 (C–Haliph.), 2212
Method B: According to literature.37–42)
General Procedure for Synthesis of Compounds (6a–d) (C≡N), 1646 (C=C), 1514 (NO2 asym.), 1345 (NO2 sym.),
A mixture of 5-arylmethylidene-derivatives (5a–d) (0.01mol) 1230 (C=S), 839 (bending C–Harom. p-disubstituted ring) cm−1.
and malononitrile (0.01mol, 0.66g) was heated for 12h in MS (70ev) m/z (%): 332 (M+, 1.67), 316 (1.23), 290 (2.04), 266
(30mL) of absolute ethanol in presence of few drops of trieth- (3.72), 241 (2.70), 210 (2.96), 122 (26.74), 91 (2.07), 66 (2.34),
ylamine under reflux. After cooling at room temperature, the 46 (100). Anal. Calcd for C13H8N4O3S2 (332.36): C, 46.98; H,
resulted precipitate was filtered off, dried and recrystallized 2.43; N, 16.86. Found: C, 47.01; H, 2.48; N, 16.94.
from ethanol to give (6a–d).
5-Amino-7-phenyl-2-thioxo-3,7-dihydro-2H-pyrano[2,3- Method A: A solution of compounds (5a–d) (0.01mol), malo-
d]-1,3-thiazole-6-carbonitrile (6a)
nonitrile (0.01mol, 0.66g) and ammonium acetate (1g) in ab-
General Procedure for Synthesis of Compounds (8a–d)
Reddish brown crystals; yield 84%; mp 248–250°C. solute ethanol (30mL) was heated under reflux for 16h and al-
1H-NMR (DMSO-d6) δ: 4.41 (1H, pyran H-4, s), 6.91 (2H, lowed to cool at room temperature. The solid product obtained
–NH2, exchangeable with D2O, s), 7.09–7.44 (5H, Ar–H, m), was filtered off, washed with water, dried and recrystallized
7.65 (1H, –NH, exchangeable with D2O, s). 13C-NMR (DMSO- from ethanol to afford the thiazolopyridine derivatives (8a–d).
d6) δ: 39.79, 103.89, 116.95, 128.50, 129.28, 137.62, 160.90,
Method B: A solution of equimolar amounts of the py-
165.37, 189.2. IR (KBr) υ: 3307, 3211, 3150 (–NH2, N–H), ranothiazole derivatives (6a–d) (0.01mol) and ammonium
3082 (stretching C–Harom.), 2925, 2855 (C–Haliph.), 2216 (C≡N), acetate (0.01mol) in absolute ethanol (30mL) was heated for
1643 (C=C), 1236 (C=S), 762, 703 (bending C–Harom. mono- 3h under reflux. The solid product obtained was filtered off,
substituted ring) cm−1. MS (70ev) m/z (%): 287 (M+, 26.72), washed with water, dried and recrystallized from ethanol to
271 (5.35), 245 (6.78), 221 (21.36), 210 (49.78), 205 (8.51), 196 give (8a–d).
(18.63), 91 (10.87), 82 (12.32), 77 (46.17), 66 (100). Anal. Calcd
5-Amino-7-phenyl-2-thioxo-2,3,4,7-tetrahydro-1,3-thiazolo-
for C13H9N3OS2 (287.36): C, 54.34; H, 3.16; N, 14.62. Found: [4,5-b]pyridine-6-carbonitrile (8a)
C, 54.49; H, 3.21; N, 14.72.
Reddish brown crystals; yield 81.3%; mp 183–185°C.
5-Amino-7-(4-methoxyphenyl)-2-thioxo-3,7-dihydro-2H- 1H-NMR (DMSO-d6) δ: 4.18 (1H, pyridine H-4, s), 6.95–7.73
pyrano[2,3-d]-1,3-thiazole-6-carbonitrile (6b)
(8H, Ar–H, –NH2, –NH pyridine, m), 8.18 (1H, –NH thiazole,
Brown crystals; yield 82.8%; mp 230–232°C. 1H-NMR exchangeable with D2O, s). 13C-NMR (DMSO-d6) δ: 39.49,
(DMSO-d6) δ: 3.83 (3H, –OCH3, s), 4.90 (1H, pyran H-4, s), 60.92, 103.58, 116.96, 127.01, 128.09, 128.55, 137.60, 140.35,
7.05 (2H, –NH2, exchangeable with D2O, s), 7.13–7.62 (4H, 152.98, 189.87. IR (KBr) υ: 3445, 3317, 3204, 3162 (–NH2, two
Ar–H, m), 7.78 (1H, NH, exchangeable with D2O, s). 13C-NMR N-H), 3082 (stretching C–Harom.), 2920, 2850 (C–Haliph.), 2213
(DMSO-d6) δ: 39.68, 52.40, 103.91, 116.95, 128.50, 129.28, (C≡N), 1627 (C=C), 1233 (C=S), 762, 700 (bending C–Harom.
137.62, 150.53, 160.48, 165.37, 189.34. IR (KBr) υ: 3307, 3210, monosubstituted ring) cm−1. MS (70ev) m/z (%): 286 (M+,
3150 (–NH2, N–H), 3081 (stretching C–Harom.), 2934, 2841 1.66), 270 (5.33), 244 (18.26), 220 (100), 209 (18.97), 2014
(C–Haliph.), 2216 (C≡N), 1644 (C=C), 1230 (C=S), 824 (bend- (1.51), 195 (1.68), 91 (4.20), 82 (1.86), 77 (1.77), 66 (1.21). Anal.
ing C–Harom. p-disubstituted ring) cm−1. MS (70ev) m/z (%): Calcd for C13H10N4S2 (286.38): C, 54.52; H, 3.52; N, 19.56.
317 (M+, 100), 301 (1.28), 286 (1.42), 275 (5.98), 251 (4.13), Found: C, 54.65; H, 3.57; N, 19.66.
226 (4.95), 210 (1.48), 107 (12.26), 91 (3.96), 66 (12.07). Anal.
5-Amino-7-(4-methoxyphenyl)-2-thioxo-2,3,4,7-tetrahydro-
Calcd for C14H11N3O2S2 (317.39): C, 52.98; H, 3.49; N, 13.24. 1,3-thiazolo[4,5-b]pyridine-6-carbonitrile (8b)
Found: C, 53.11; H, 3.51; N, 13.31.
Reddish brown crystals; yield 79.4%; mp 214–216°C.
5-Amino-7-(4-chlorophenyl)-2-thioxo-3,7-dihydro-2H- 1H-NMR (DMSO-d6) δ: 3.74 (3H, –OCH3, s), 4.39 (1H, pyri-
pyrano[2,3-d]-1,3-thiazole-6-carbonitrile (6c)
dine H-4, s), 7.21–7.82 (7H, Ar–H, –NH2, –NH pyridine, m),
Buff crystals; yield 79.8%; mp 156–158°C. 1H-NMR 8.24 (1H, –NH thiazole, exchangeable with D2O, s). 13C-NMR
(DMSO-d6) δ: 4.15 (1H, pyran H-4, s), 7.20 (2H, –NH2, ex- (DMSO-d6) δ: 49.73, 51.08, 57.60, 103.85, 117.58, 121.35,
changeable with D2O, s), 7.33–7.63 (4H, Ar–H, m), 7.65 (1H, 129.52, 143.05, 145.65, 159.90, 162.00, 192.35. IR (KBr) υ:
–NH, exchangeable with D2O, s). 13C-NMR (DMSO-d6) δ: 3459, 3313, 3207, 3135 (–NH2, two N–H), 3094 (stretching
39.37, 103.91, 116.95, 129.28, 129.84, 130.86, 131.90, 137.62, C–Harom.), 2917, 2843 (C–Haliph.), 2211 (C≡N), 1645 (C=C),
160.48, 165.37, 188.74. IR (KBr) υ: 3307, 3211, 3150 (–NH2, 1241 (C=S), 821 (bending C–Harom. p-disubstituted ring) cm−1.
N–H), 3080 (stretching C–Harom.), 2919, 2850 (C–Haliph.), 2214 MS (70ev) m/z (%): 316 (M+, 2.09), 300 (67.35), 285 (20.32),
(C≡N), 1645 (C=C), 1238 (C=S), 825 (bending C–Harom. p- 274 (100), 250 (8.95), 225 (8.16), 209 (28.84), 107 (39.84), 91
disubstituted ring) cm−1. MS (70ev) m/z (%): 323.5 (M+ +2, (47.94), 66 (4.04). Anal. Calcd for C14H12N4OS2 (316.40): C,

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Chem. Pharm. Bull.
Vol. 67, No. 12 (2019)
53.14; H, 3.82; N, 17.71. Found: C, 53.20; H, 3.87; N, 17.81.
(KBr) υ: 3230, 3139 (two N–H), 3033 (stretching C–Harom.),
5-Amino-7-(4-chlorophenyl)-2-thioxo-2,3,4,7-tetrahydro-1,3- 2930, 2848 (C–Haliph.), 1663 (C=C), 1240 (C=S), 824 (bend-
thiazolo[4,5-b]pyridine-6-carbonitrile (8c)
ing C–Harom. p-disubstituted ring) cm−1. MS (70ev) m/z (%):
Buff crystals; yield 78.1%; mp 128–130°C. 1H-NMR 266 (M+, 100), 235 (57.95), 190 (2.85), 159 (8.53), 107 (65.23),
(DMSO-d6) δ: 4.47 (1H, pyridine H-4, s), 7.46–7.88 (7H, 76 (1.92). Anal. Calcd for C11H10N2O2S2 (266.34): C, 49.61; H,
Ar–H, –NH2, –NH pyridine, m), 8.37 (1H, –NH thiazole, 3.78; N, 10.52. Found: C, 49.79; H, 3.85; N, 10.61.
exchangeable with D2O, s). 13C-NMR (DMSO-d6) δ: 40.93,
3-(4-Chlorophenyl)-3,6-dihydro-1,3-thiazolo[4,5-c]-
89.63, 118.37, 123.30, 128.51, 129.80, 136.40, 141.07, 142.49, isoxazole-5(1H)-thione (9c)
195.66. IR (KBr) υ: 3432, 3332, 3204, 3148 (–NH2, two N-H),
Buff crystals; yield 75.3%; mp 296–298°C. 1H-NMR
3081 (stretching C–Harom.), 2919, 2850 (C–Haliph.), 2212 (C≡N), (DMSO-d6) δ: 5.69 (1H, isoxazole H-3, s), 7.55–7.59 (4H,
1648 (C=C), 1236 (C=S), 820 (bending C–Harom. p-disubsti- Ar–H, m), 10.95 (1H, –NH isoxazole, exchangeable with
tuted ring) cm−1. MS (70ev) m/z (%): 320.5 (M+, 24.18), 322.5 D2O, s), 12.05 (1H, –NH thiazole, exchangeable with D2O, s).
(M+ + 2, 16.64), 304.5 (46.35), 278.5 (15.72), 209 (13.32), 13C-NMR (DMSO-d6) δ: 40.60, 124.08, 126.67, 129.68, 133.03,
254.5 (23.22), 229.5 (20.50), 111.5 (40.18), 91 (100). Anal. 134.42, 146.18, 166.78. IR (KBr) υ: 3189, 3137 (two N–H),
Calcd for C13H9ClN4S2 (320.82): C, 48.67; H, 2.83; N, 17.46. 3026 (stretching C–Harom.), 2920, 2849 (C–Haliph.), 1662 (C=C),
Found: C, 48.78; H, 2.86; N, 17.52.
1239 (C=S), 820 (bending C–Harom. p-disubstituted ring) cm−1.
5-Amino-7-(4-nitrophenyl)-2-thioxo-2,3,4,7-tetrahydro-1,3- MS (70ev) m/z (%): 272.5 (M+ + 2, 8.17), 270.5 (M+, 100),
thiazolo[4,5-b]pyridine-6-carbonitrile (8d)
194.5 (3.55), 179.5 (8.17), 159 (40.92), 111.5 (16.31), 91 (9.38),
Brown crystals; yield 76.4%; mp 135–137°C. 1H-NMR 76 (4.15). Anal. Calcd for C10H7ClN2OS2 (270.76): C, 44.36; H,
(DMSO-d6) δ: 4.53 (1H, pyridine H-4, s), 7.58–7.90 (7H, 2.61; N, 10.35. Found: C, 44.53; H, 2.59; N, 10.29.
Ar–H, –NH2, –NH pyridine, m), 8.43 (1H, –NH thiazole, ex-
3-(4-Nitrophenyl)-3,6-dihydro-1,3-thiazolo[4,5-c]isoxazole-
changeable with D2O, s). 13C-NMR (DMSO-d6) δ: 41.85, 90.13, 5(1H)-thione (9d)
119.08, 123.90, 126.84, 128.79, 130.20, 142.19, 144.90, 195.80.
Buff crystals; yield 73.9%; mp 270–272°C. 1H-NMR
R (KBr) υ: 3432, 3343, 3204, 3108 (–NH2, two N–H), 3081 (DMSO-d6) δ: 5.72 (1H, isoxazole H-3, s), 7.57–7.62 (4H,
(stretching C–Harom.), 2922, 2852 (C–Haliph.), 2215 (C≡N), 1630 Ar–H, m), 10.98 (1H, –NH isoxazole, exchangeable with
(C=C), 1515 (NO2 asym.), 1344 (NO2 sym.), 1231 (C=S), 844 D2O, s), 12.09 (1H, –NH thiazole, exchangeable with D2O,
(bending C–Harom. p-disubstituted ring) cm−1. MS (70ev) m/z s).13C-NMR (DMSO-d6) δ: 40.88, 124.72, 126.96, 129.87,
(%): 331 (M+, 100), 315 (97.87), 289 (47.05), 265 (10.71), 240 133.41, 134.69, 146.37, 167.03. IR (KBr) υ: 3202, 3108 (two
(44.50), 109 (9.51), 122 (42.24), 91 (33.30), 66 (45.07). Anal. N–H), 3015 (stretching C–Harom.), 2920, 2849 (C–Haliph.), 1657
Calcd for C13H9N5O2S2 (331.37): C, 47.12; H, 2.74; N, 21.13. (C=C), 1512 (NO2 asym.), 1341 (NO2 sym.), 1241 (C=S), 843
Found: C, 47.28; H, 2.78; N, 21.21.
(bending C–Harom. p-disubstituted ring) cm−1. MS (70ev) m/z
General Procedure for Synthesis of Compounds (9a–d) (%): 281 (M+, 28.72), 235 (22.56), 205 (5.21), 159 (14.95), 122
A mixture of 5-arylidene-derivatives (5a–d) (0.01mol) and (58.76), 76 (100). Anal. Calcd for C10H7N3O3S2 (281.31): C,
hydroxylamine hydrochloride (0.01mol, 0.69g) in absolute 42.70; H, 2.51; N, 14.94. Found: C, 42.78; H, 2.54; N, 15.01.
ethanol (30mL) and anhydrous sodium acetate (0.01mol,
General Procedure for Synthesis of Compounds (10a–d)
0.82g) was heated under reflux for 15h. After cooling at room A mixture of compounds (5a–d) (0.01mol) and thiourea
temperature, the reaction mixture was poured on ice with con- (0.01mol, 0.76g) in dimethylformamide (10mL) in presence
tinuous stirring. The formed precipitate was filtered off, dried of few drops of triethylamine was heated under reflux for 9h.
and recrystallized from ethanol to give (9a–d).
After cooling at room temperature, the reaction mixture was
3-Phenyl-3,6-dihydro-1,3-thiazolo[4,5-c]isoxazole-5(1H)- poured on ice. The formed precipitate was filtered off, dried
thione (9a)
and recrystallized from the ethanol solvent to give (10a–d).
7-Phenyl-1,3-thiazolo[4,5-d]pyrimidine-2,5(3H,4H)-dithione
Buff crystals; yield 72.1%; mp 256–258°C. 1H-NMR
(DMSO-d6) δ: 5.61 (1H, isoxazole H-3, s), 7.32–7.48 (5H, (10a)
Ar–H, m), 10.89 (1H, –NH isoxazole, exchangeable with
Brown crystals; yield 70.4%; mp 160–162°C. 1H-NMR
D2O, s), 11.96 (1H, –NH thiazole, exchangeable with D2O, s). (DMSO-d6) δ: 6.98–7.58 (6H, Ar–H, NH pyrimidine, m),
13C-NMR (DMSO-d6) δ: 39.56, 123.44, 125.71, 128.50, 131.27, 7.67 (1H, –NH thiazole, exchangeable with D2O, s). 13C-NMR
133.82, 145.09, 165.88. IR (KBr) υ: 3149, 3112 (two N–H), (DMSO-d6) δ: 128.50, 129.32, 130.17, 130.55, 133.00, 134.49,
3027 (stretching C–Harom.), 2925, 2852 (C–Haliph.), 1650 (C=C), 174.63, 178.04. IR (KBr) υ: 3349, 3212 (two N–H), 3058
1247 (C=S), 759, 684 (bending C–Harom. monosubstituted (stretching C–Harom.), 1599 (C=N), 1237 (C=S), 763, 678
ring) cm−1. MS (70ev) m/z (%): 236 (M+, 1.58), 205 (19.39), (bending C–Harom. monosubstituted ring) cm−1. MS (70ev)
160 (31.99), 159 (26.40), 145 (27.56), 91 (90.41), 77 (62.80), 76 m/z (%): 277 (M+, 2.16), 201 (1.75), 200 (1.69), 186 (1.93), 174
(100). Anal. Calcd for C10H8N2OS2 (236.31): C, 50.83; H, 3.41; (3.24), 147 (12.48), 130 (25.43), 103 (1.21), 91 (100), 77 (1.96),
N, 11.85. Found: C, 50.72; H, 3.38; N, 11.78.
3-(4-Methoxyphenyl)-3,6-dihydro-1,3-thiazolo[4,5-c]- 2.54; N, 15.15. Found: C, 47.81; H, 2.49; N, 15.22.
isoxazole-5(1H)-thione (9b) 7-(4-Methoxyphenyl)-1,3-thiazolo[4,5-d]pyrimidine-
Yellow crystals; yield 69.8%; mp 210–212°C. 1H-NMR 2,5(3H,4H)-dithione (10b)
76 (1.29). Anal. Calcd for C11H7N3S3 (277.39): C, 47.63; H,
(DMSO-d6) δ: 3.74 (3H, –OCH3, s), 5.63 (1H, isoxazole H-3,
Brown crystals; yield 73.1%; mp 134–136°C. 1H-NMR
s), 7.48–7.55 (4H, Ar–H, m), 10.89 (1H, –NH isoxazole, ex- (DMSO-d6) δ: 3.86 (3H, –OCH3, s), 7.12–7.60 (5H, Ar–H,
changeable with D2O, s), 12.01 (1H, –NH thiazole, exchange- –NH pyrimidine, m), 7.84 (1H, –NH thiazole, exchangeable
able with D2O, s). 13C-NMR (DMSO-d6) δ: 39.80, 55.09, with D2O, s). 13C-NMR (DMSO-d6) δ: 55.60, 115.05, 128.13,
123.98, 126.33, 129.02, 131.76, 133.92, 145.70, 166.45. IR 129.45, 130.34, 130.89, 133.01, 134.69, 174.78, 178.20. IR

Vol. 67, No. 12 (2019)
Chem. Pharm. Bull.
1321
(KBr) υ: 3330, 3239 (two N-H), 3074 (stretching C–Harom.),
5-[2-(4-Chlorophenylhydrazono)-2-thioxo-1,3-thiazolidin-4-
2933, 2834 (C–Haliph.), 1600 (C=N), 1241 (C=S), 824 (bending one (12b)
C–Harom. p-disubstituted ring) cm−1. MS (70ev) m/z (%): 307
Orange crystals; yield 94%; mp 248–250°C. 1H-NMR
(M+, 1.91), 276 (1.31), 231 (100), 200 (4.29), 177 (36.55), 174 (DMSO-d6) δ: 7.29–7.46 (4H, Ar–H, m), 11.07 (1H, –NH, ex-
(5.47), 133 (1.40), 130 (11.71), 107 (2.01), 76 (2.85). Anal. Calcd changeable with D2O, s), 13.82 (1H, –NH thiazole, exchange-
for C12H9N3OS3 (307.41): C, 46.88; H, 2.95; N, 13.67. Found: able with D2O, s). 13C-NMR (DMSO-d6) δ: 193.13, 165.24,
C, 46.69; H, 2.89; N, 13.58.
7-(4-Chlorophenyl)-1,3-thiazolo[4,5-d]pyrimidine- (two N–H), 3081 (stretching C–Harom.), 1698 (C=O), 1602
2,5(3H,4H)-dithione (10c)
142.50, 129.75, 126.77, 126.64, 116.30. IR (KBr) υ: 3241, 3189
(C=N), 1216 (C=S), 824 (bending C–Harom. p-disubstituted
Buff crystals; yield 69.5%; mp 210–212°C. 1H-NMR ring) cm−1. MS (70ev) m/z (%): 273.5 (M+ + 2, 10.31), 271.5
(DMSO-d6) δ: 7.42–7.79 (5H, Ar–H, NH pyrimidine, m), 8.06 (M+, 6.00), 195.5 (8.00), 180.5 (2.81), 160 (9.44), 145 (7.63),
(1H, –NH thiazole, exchangeable with D2O, s). 13C-NMR 140.5 (19.02), 131 (9.10), 126.5 (7.50), 111.5 (4.85), 76 (100).
(DMSO-d6) δ: 128.78, 129.71, 130.58, 131.58, 133.28, 134.69, Anal. Calcd for C9H6ClN3OS2 (271.75): C, 39.78; H, 2.23; N,
175.09, 179.27. IR (KBr) υ: 3329, 3247 (two N–H), 3054 15.46. Found: C, 39.62; H, 2.28; N, 15.57.
(stretching C–Harom.), 1618 (C=N), 1220 (C=S), 822 (bending
5-[2-(4-Nitrophenylhydrazono)-2-thioxo-1,3-thiazolidin-4-
C–Harom. p-disubstituted ring) cm−1. MS (70ev) m/z (%): 313.5 one (12c)
(M+ + 2, 2.81), 311.5 (M+, 8.69), 200 (37.38), 196.5 (2.73),
Brown crystals; yield 91.8%; mp 186–188°C. 1H-NMR
181.5 (2.95), 174 (5.58), 137.5 (1.76), 130 (2.19), 115 (100), (DMSO-d6) δ: 7.40–7.88 (4H, Ar–H, m), 11.14 (1H, –NH, ex-
111.5 (10.16). Anal. Calcd for C11H6ClN3S3 (311.83): C, 42.37; changeable with D2O, s), 13.87 (1H, –NH thiazole, exchange-
H, 1.94; N, 13.48. Found: C, 42.59; H, 1.98; N, 13.59.
7-(4-Nitrophenyl)-1,3-thiazolo[4,5-d]pyrimidine- 144.03, 129.50, 126.89, 126.82, 116.90. IR (KBr) υ: 3246, 3191
2,5(3H,4H)-dithione (10d) (two N–H), 3097 (stretching C–Harom), 1691 (C=O), 1595
able with D2O, s). 13C-NMR (DMSO-d6) δ: 193.42, 167.38,
Reddish brown crystals; yield 67%; mp 178–180°C. (C=N), 1551 (NO2 asym.), 1330 (NO2 sym.), 1225 (C=S),
1H-NMR (DMSO-d6) δ: 7.51–7.88 (5H, Ar–H, NH pyrimi- 840 (bending C–Harom p-disubstituted ring) cm−1. MS (70ev)
dine, m), 8.32 (1H, –NH thiazole, exchangeable with D2O, s). m/z (%): 282 (M+, 10.88), 236 (12.32), 206 (100), 191 (23.02),
13C-NMR (DMSO-d6) δ: 128.91, 129.84, 130.65, 131.90, 133.77, 160 (10.73), 151 (1.27), 145 (27.97), 137 (6.94), 131 (12.60),
134.87, 176.23, 180.04. IR (KBr) υ: 3344, 3222 (two N–H), 122 (6.31), 91 (11.43), 76 (4.24), 46 (4.96). Anal. Calcd for
3076 (stretching C–Harom.), 1592 (C=N), 1515 (NO2 asym.), C9H6N4O3S2 (282.30): C, 38.29; H, 2.14; N, 19.85. Found: C,
1342 (NO2 sym.), 1234 (C=S), 831 (bending C–Harom. p- 38.47; H, 2.21; N, 19.97.
disubstituted ring) cm−1. MS (70ev) m/z (%): 322 (M+, 2.64),
General Procedure for Synthesis of Compounds (13a–c)
276 (12.48), 246 (100), 231 (1.81), 207 (1.20), 200 (2.18), 192 An equimolar mixture of compounds (12a–c) (0.01mol) and
(1.96), 174 (1.28), 148 (1.45), 130 (9.01), 122 (7.84), 115 (2.88), malononitrile (0.01mol, 0.66g) in ammonium acetate (2g) was
91 (2.15), 76 (20.70). Anal. Calcd for C11H6N4O2S3 (322.39): C, fused for 1h. The reaction mixture was left to stand at room
40.98; H, 1.88; N, 17.38. Found: C, 40.83; H, 1.92; N, 17.47.
temperature and triturated with ethanol. The solid product was
General Procedure for Synthesis of Compounds (12a–c) collected by filtration, washed with water, dried and recrystal-
Method A: A solution of (1) (0.01mol, 1.33g) in pyridine lized from ethanol to give (13a–c).
(20mL) was cooled to 0°C, stirred and treated gradually with
6-Amino-3-imino-2-phenyl-2,3-dihydro-1,3-thiazolo[5,4-c]-
cooled solution of aryl diazonium chloride (11a–c) [prepared pyridazine-4-carbonitrile (13a)
from (0.02mol, 1.4g) NaNO2 dissolved in (5mL) water, added
Dark brown crystals; yield 69%; mp >300°C. 1H-NMR
with keeping temperature between 0–5°C to (0.01mol) of (DMSO-d6) δ: 6.81–7.41 (8H, Ar–H, =NH, –NH2, m).
amines namely aniline, p-chloroaniline and/or p-nitroaniline 13C-NMR (DMSO-d6) δ: 102.01, 116.75, 125.45, 126.05, 129.00,
dissolved in (10mL) of conc. HCl]. After complete addition, 142.95, 151.70, 158.60, 158.75, 162.90. IR (KBr) υ: 3323, 3183
the reaction mixture was stirred for 1h at 0°C. The solid (–NH2, =NH), 3054 (stretching C–Harom.), 2205 (C≡N), 1616,
product formed was collected, washed with water, dried and 1596 (C=N), 756, 690 (bending C–Harom. monosubstituted
recrystallized from ethanol to give (12a–c).
ring) cm−1. MS (70ev) m/z (%): 268 (M+, 1.76), 252 (11.24),
191 (39.08), 163 (1.20), 150 (10.24), 118 (6.95), 105 (9.07), 77
(100). Anal. Calcd for C12H8N6S (268.30): C, 53.72; H, 3.01; N,
Method B: According to literature.43)
5-(2-Phenylhydrazono)-2-thioxo-1,3-thiazolidin-4-one (12a)
Orange crystals; yield 93.5%; mp 216–218°C. 1H-NMR 31.32. Found: C, 53.55; H, 3.07; N, 31.41.
(DMSO-d6) δ: 7.18–7.38 (5H, Ar–H, m), 10.99 (1H, –NH, ex-
6-Amino-2-(4-chlorophenyl)-3-imino-2,3-dihydro-1,3-
changeable with D2O, s), 13.67 (1H, –NH thiazole, exchange- thiazolo[5,4-c]pyridazine-4-carbonitrile (13b)
1
able with D2O, s). 13C-NMR (DMSO-d6) δ: 193.05, 165.11,
Reddish brown crystals; yield 82%; mp >300°C. H-NMR
141.97, 129.48, 126.28, 125.93, 116.08. IR (KBr) υ: 3236, (DMSO-d6) δ: 6.94–7.81 (7H, Ar–H, =NH, –NH2, m).
3160 (two N–H), 3054 (stretching C–Harom.), 1695 (C=O), 13C-NMR (DMSO-d6) δ: 102.18, 116.97, 125.85, 126.63, 129.17,
1597 (C=N), 1212 (C=S), 743, 686 (bending C–Harom. mono- 143.13, 151.76, 158.56, 158.90, 163.05. IR (KBr) υ: 3321, 3173
substituted ring) cm−1. MS (70ev) m/z (%): 237 (M+, 7.40), (–NH2, =NH), 3081 (stretching C–Harom.), 2202 (C≡N), 1613,
145 (13.17), 160 (16.52), 146 (8.36), 131 (5.83), 106 (16.70), 1594 (C=N), 824 (bending C–Harom. p-disubstituted ring)
92 (22.47), 91 (3.77), 77 (100). Anal. Calcd for C9H7N3OS2 cm−1. MS (70ev) m/z (%): 304.5 (M+ + 2, 100), 302.5 (M+,
(237.30): C, 45.55; H, 2.97; N, 17.71. Found: C, 45.76; H, 2.93; 59.00), 286.5 (4.40), 267 (3.09), 237.5 (6.41), 228.5 (5.85),
N, 17.84.
191 (3.53), 111.5 (5.97), 74 (4.85), 65 (1.51). Anal. Calcd for
C12H7ClN6S (302.74): C, 47.61; H, 2.33; N, 27.76. Found: C,
47.75; H, 2.28; N, 27.85.

1322
Chem. Pharm. Bull.
Vol. 67, No. 12 (2019)
6-Amino-3-imino-2-(4-nitrophenyl)-2,3-dihydro-1,3- depicted in Table 1.
thiazolo[5,4-c]pyridazine-4-carbonitrile (13c) Antimicrobial Screening The susceptibility tests were
1
Reddish brown crystals; yield 73.7%; mp >300°C. H-NMR performed according to recommendations of National Com-
(DMSO-d6) δ: 7.14–8.33 (7H, Ar–H, =NH, –NH2, m). mittee for Clinical Laboratory Standards, 1993. Screening
13C-NMR (DMSO-d6) δ: 102.76, 117.28, 125.93, 127.00, 129.57, tests regarding the inhibition zone were carried out by the
143.00, 151.95, 158.84, 159.11, 163.23. IR (KBr) υ: 336, 3200, well diffusion method.46) The inoculum suspension was pre-
3191 (–NH2, =NH), 3097 (stretching C–Harom), 2202 (C≡N), pared from colonies grown overnight on an agar plate and
1616, 1590 (C=N), 1514 (NO2 asym.), 1334 (NO2 sym.), 842 inoculated into Mueller–Hinton broth (fungi using malt broth).
(bending C–Harom p-disubstituted ring) cm−1. MS (70ev) m/z A sterile swab was immersed in the suspension and used to
(%): 313 (M+, 100), 297 (10.80), 287 (11.05), 267 (2.46), 239 inoculate Mueller-Hinton agar plates (fungi using malt agar
(36.89), 191 (23.09), 122 (9.14), 74 (11.80), 46 (6.40). Anal. plates). The compounds were dissolved in DMSO at con-
Calcd for C12H7N7O2S (313.29): C, 46.00; H, 2.25; N, 31.30. centration of 10mg/mL. The inhibition zone was measured
Found: C, 46.16; H, 2.31; N, 31.20.
around each well after 24h at 37°C. Controls using DMSO
Cytotoxicity Evaluation The new synthesized com- were adequately done. The antimicrobial screening was per-
pounds (3a–f) were evaluated for human tumour cell growth formed using Gentamycin (MIC 4µg/mL) and Ketoconazole
inhibitory activity against MCF-7 human breast carcinoma (MIC 100µg/mL) as reference standard drugs for bacteria and
cell, which was obtained from VACSERA Tissue Culture fungi, respectively. The antimicrobial results are depicted in
Unit. The measurements of cell growth and the viabilities Table 2.
were determined as described in the literature.44,45) The in-
vitro cytotoxicity evaluation using viability assay was per-
formed using the standard drug Doxorubicin as reference.
For cytotoxicity assay, the cells were seeded in 96-well
plate at a cell concentration of 1×104 cells per well in 100µL
of growth medium. Fresh medium containing different con-
centrations of the test sample was added after 24h of seeding.
Serial two-fold dilutions of the tested chemical compound
were added to confluent cell monolayers dispensed into
96-well, flat-bottomed microtiter plates (Falcon, NJ, U.S.A.)
using a multichannel pipette. The microtiter plates were in-
cubated at 37°C in a humidified incubator with 5% CO2 for a
period of 24h. Three wells were used for each concentration
of the test sample. Control cells were incubated without test
sample and with or without DMSO. The little percentage of
DMSO present in the wells (maximal 0.1%) was found not
to affect the experiment. After incubation of the cells for at
37°C, for 24h, the viable cells yield was determined by a colo-
rimetric method.
In brief, after the end of the incubation period, media were
aspirated and the crystal violet solution (1%) was added to
each well for at least 30min. The stain was removed, and
the plates were rinsed using tap water until all excess stain
is removed. Glacial acetic acid (30%) was then added to all
wells and mixed thoroughly, and then the absorbance of the
plates was measured after gently shaken on Microplate reader
(TECAN, Inc., U.S.A.), using a test wavelength of 490nm. All
results were corrected for background absorbance detected in
wells without added stain. Treated samples were compared
with the cell control in the absence of the tested compounds.
All experiments were carried out in triplicate. The cell cyto-
toxic effect of each tested compound was calculated. The opti-
cal density was measured with the microplate reader (Sunrise,
TECAN, Inc.) to determine the number of viable cells and the
percentage of viability was calculated as [(ODt/ODc)]×100%
where ODt is the mean optical density of wells treated with
the tested sample and ODc is the mean optical density of un-
treated cells. The relation between surviving cells and drug
concentration is plotted to get the survival curve of each
tumor cell line after treatment with the specified compound.
The IC50 was estimated from graphic plots of the dose re-
sponse curve for each conc. using GraphPad Prism Software
(San Diego, CA, U.S.A.). The inhibitory activity results are
Conflict of Interest The author declares no conflict of
interest.
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