Synthesis and antimicrobial activity of some new thiadiazoles, thioamides, 5-arylazothiazoles and pyrimido[4,5-d][1,2,4]triazolo[4,3-a]pyrimidines

Article abstract of DOI:10.3390/molecules21081072

A novel series of 1,3,4-thiadiazoles, 5-arylazothiazoles and hexahydropyrimido-[4,5-d] [1,2,4]triazolo[4,3-a]pyrimidines were synthesized via reaction of hydrazonoyl halides with each of alkyl carbothioates, carbothioamides and 7-thioxo-5,6,7,8-tetrahydropyrimido-[4,5-d]pyrimidine-2,4(1H,3H)-diones in the presence of triethylamine. The structures of the newly synthesized compounds were established based on their spectral data, elemental analyses and alternative synthetic routes whenever possible. Also, the newly synthesized compounds were screened for their antimicrobial activity against various microorganisms.

Full text of DOI:10.3390/molecules21081072

molecules
Article
Synthesis and Antimicrobial Activity of Some New
Thiadiazoles, Thioamides, 5-Arylazothiazoles and
Pyrimido[4,5-d][1,2,4]triazolo[4,3-a]pyrimidines
Abdou O. Abdelhamid 1,*, Ibrahim E. El Sayed 2, Mohamed Z. Hussein 3
and Mangoud M. Mangoud 3
1
Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt
Department of Chemistry, Faculty of Science, El Menoufia University, Shebin El Koom 32511, Egypt;
2
ibrahimtantawy@yahoo.co.uk
Environmental Research Department, National Center for Social and Criminological Research,
3
Ibn Khaldoun Square, Mohandesin, Zamalek, Giza 11561, Egypt; mohamedzaki1955@yahoo.com (M.Z.H.);
mangoud_online@yahoo.com (M.M.M.)
*
Correspondence: Abdelhamid45@gmail.com; Tel.: +20-0100-5205-750
Academic Editor: Richard A. Bunce
Received: 25 June 2016; Accepted: 10 August 2016; Published: 17 August 2016
Abstract: A novel series of 1,3,4-thiadiazoles, 5-arylazothiazoles and hexahydropyrimido-[4,5-d]
[1,2,4]triazolo[4,3-a]pyrimidines were synthesized via reaction of hydrazonoyl halides with each of
alkyl carbothioates, carbothioamides and 7-thioxo-5,6,7,8-tetrahydropyrimido-[4,5-d]pyrimidine-
2,4(1H,3H)-diones in the presence of triethylamine. The structures of the newly synthesized
compounds were established based on their spectral data, elemental analyses and alternative
synthetic routes whenever possible. Also, the newly synthesized compounds were screened for their
antimicrobial activity against various microorganisms.
Keywords: hydrazonoyl halides; thioamides; 1,3,4-thiadiazoles; thiazoles; pyrimido[4,5-d][1,2,4]
triazolo[4,3-a]pyrimidines; antimicrobials
1. Introduction
Hydrazonoyl halides have been widely used as reagents for the synthesis of heterocyclic
compounds, both through condensation reactions, and as precursors of nitrilimines, which can undergo
cycloaddition with dipolarophiles [
pharmacophores. They display a broad spectrum of biological activities, including antimicrobial [
anticancer [ ], antioxidant [ ], antidepressant [ ], anticonvulsant [ ], and antihypertensive
activity [10] as well as acetylcholinesterase inhibition for the treatment of Alzheimer disease [11 12].
1,2]. 1,3,4-Thiadiazoles are among the most common heterocyclic
3]
4
,5
6
7
8,9
,
In addition, thiazoles can found in drugs developed for the treatment of allergies [13], hypertension [14],
inflammation [15], schizophrenia [16], bacterial infections [17], HIV [18], sleep disorders [19] and
more recently, for the treatment of pain [20], as fibrinogen receptor antagonists with antithrombotic
activity [21], and as new inhibitors of bacterial DNA gyrase B [22]. 1,2,4-Triazolopyrimidines have
also attracted growing interest due to their important pharmacological activities, such as antitumor,
antimalarial, antimicrobial, anti-inflammatory, antifungal properties, and their potency in macrophage
activation [23–27].
2. Results and Discussion
Treatment of methyl 2-methylenehydrazinecarbodithioates 3a and [28] 4a with ethyl 2-chloro-2-
(2-phenylhydrazono)acetate (5a) in ethanol containing triethylamine gave ethyl 5-({[1H-pyrazol-4-
yl]methylene}hydrazono)-1,3,4-thiadiazole-2-caboxylates 9a and 10a, respectively (Scheme 1). The
Molecules 2016, 21, 1072; doi:10.3390/molecules21081072
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1072
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1
structure 9a was established by elemental analysis, H-NMR, IR spectroscopy, mass spectrometry
and alternative syntheses. Thus, (5E)-ethyl 5-hydrazono-4,5-dihydro-4-phenyl-1,3,4-thiadiazole-2-
carboxylate [29] (11) was reacted with 1a and 1b to give products identical in all aspects (m.p., mixed
m.p. and spectra) with 9a and 10a, respectively. In addition, benzyl 2-((1-phenyl-3-(p-tolyl)-1H-pyrazol-
4-yl)-methylene)-hydrazine-1-carbodithioate (3b) was reacted with 5a in ethanolic triethylamine to
give a product identical in all aspects (m.p., mixed m.p. and spectra) with 9a. In light of the foregoing
results, the mechanism outlined in Scheme 1 seems to be the most plausible pathway for the formation
of
, which undergoes intermolecular cyclization directly to yield intermediate
cycloaddition of nitrilimine (generated in situ from with triethylamine) to the C=S of
to yield intermediate . Formations of and are similar to the reaction of hydrazonoyl chloride
with 1-phenyl-1,4-dihydrotetrazole-5-thione [30] and 5-phenyl-1,3,4-thiadiazole-2(3H)-thione [31].
Intermediate was converted to by elimination of alkyl mercaptan. Analogously, 5b 5c were reacted
separately with 3a 3b 4a or 4b in ethanolic triethyamine to afford 2,3-dihydro-1,3,4-thiadiazoles 9b
and 10a–c, respectively (Scheme 1).
9
from the reaction of the
5
with 3a or 3b. The reaction involves initial formation of thiohydrazonate
or via 1,3-dipolar
or
7
8
6
5
3
4
8
7
8
8
9
,
,
,
–c
Scheme 1. Synthesis of 1,3,4-thiadiazole derivatives 9a–c and 10a–c.
Treatment of 3-(furan-2-yl)-1-(p-tolyl)prop-2-en-1-one (12a), 1-(furan-2-yl)-3-(3-(furan-2-yl)-
1-phenyl-1H-pyrazol-4-yl)prop-2-en-1-one (12b), 3-(3-(furan-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1-(p-
tolyl)prop-2-en-1-one (12c), 3-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1-(p-tolyl)prop-2-en-1-one (12d),

Molecules 2016, 21, 1072
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1,3-di(furan-2-yl)prop-2-en-1-one (12e) and 1-(furan-2-yl)-3-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-
prop-2-en-1-one (12f) with thiosemicarbazide in boiling ethanol containing potassium hydroxide
gave 5-(furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (13a), 3’,5-di(furan-2-yl)-
1’-phenyl-3,4-dihydro-1’H,2H-[3,4’-bipyrazole]-2-carbothioamide (13b), 3’-(furan-2-yl)-1’-phenyl-5-
(p-tolyl)-3,4-dihydro-1’H,2H-[3,4’- bipyrazole]-2-carbothioamide (13c), 1’-phenyl-3’,5-di-p-tolyl-3,4-
dihydro-1’H,2H-[3,4’-bipyrazole]-2-carbothioamide (13d), 3,5-di(furan-2-yl)-4,5-dihydro-1H-pyrazole-
1-carbothioamide (13e) and 5-(furan-2-yl)-1’-phenyl-3’-(p-tolyl)-3,4-dihydro-1’H,2H-[3,4’-bipyrazole]-
2-carbothioamide (13f), respectively (Scheme 2).
Scheme 2. Synthesis of thioamide derivatives 13a–f.
Thus, thioamide 13a was reacted with the hydrazonoyl halides 5b and 5c in boiling ethanol
containing triethylamine to give 2-(5-(furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-
5-(phenyldiazenyl)thiazole (14a) and 2-(5-(furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-
phenyl-5-(phenyldiazenyl)thiazole (15a), respectively (Scheme 3). Thus, benzenediazonium chloride
was reacted with 2-(5-(furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-phenyl-thiazole (16),
which was prepared via the reaction of 12a with 2-hydrazinyl-4-phenylthiazole [32] (17) or the reaction
of compound 13a with
ω
-bromoacetophenone in pyridine at 0–5 ◦C to give a product identical in all
aspects (mp., mixed mp. and spectra) with 15a (Scheme 3).
Scheme 3. 2-(5-(Furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-substituted-5-(phenyldiazenyl)thiazole
14a and 15a.

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Analogously, treatment of the appropriate 13b
–f
with the hydrazonoyl halides 5b and 5c in boiling
ethanol containing triethylamine afforded 2-substituted-4,5-dihydro-1H-pyrazol-1-yl)-4-subsituted-5-
(phenyldiazenyl)thiazoles 14b–f and 15b–f, respectively (Scheme 4).
S
Ph
N
R2 Cl (Br)
N
R3
NH2
R4
S
N
N
N
TEA
N
R3
+
N
N
R5
NH
R4
Ph
13b-f
5b, R2 = CH3CO
c, R2 = C6H5CO
14b-f, R5 = CH3
15b-f, R5 = C6H5
R3
R4
b
c
d
e
f
3-(furan-2-yl)-1-phenyl-1H-pyrazole-4-yl furan-2-yl
3-(furan-2-yl)-1-phenyl-1H-pyrazole-4-yl 4-CH3C6H4
1-phenyl-3-(p-tolyl)-1H-pyrazole-4-yl
furan-2-yl
4-CH3C6H4
furan-2-yl
furan-2-yl
1-phenyl-3-(p-tolyl)-1H-pyrazole-4-yl
Scheme 4. 4,5-Dihydro-1H-pyrazol-1-yl)-4-phenyl-5-(phenyldiazenyl)thiazole derivatives 14b–f and
15b–f.
On the other hand, treatment of ethyl 2-chloro-2-(2-phenylhydrazono)acetate (5a) with
5-(furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide
(13a), in boiling ethanol
containing triethylamine gave one isolable product according to TLC and proved to be 2-(5-(furan-2-
yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-5-(2-phenylhydrazono)thiazol-4(5H)-one (18a). Also,
benzenediazonium was reacted with 2-(5-(furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-
5(4H)-one (19), which was prepared via the reaction of ethyl chloroacetate with 13a, in ethanolic
◦
sodium acetate solution at 0–5 C to afford a product identical in all aspects (mp, mixed mp and
spectra) with 18a (Scheme 5)
Scheme 5. Synthesis of 2-(5-(furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-5-(2-phenyl-hydrazono)
thiazol-4(5H)-one 18a.
Similarly, carbothioamides 13b–d, and 13f were reacted with C-ethoxycarbonyl-N-phenylhydrazonyl
chloride in ethanolic triethylamine to give the corresponding thiazolone derivatives 18b–e, respectively
(Scheme 6).

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Scheme 6. Synthesis of 3,5-substituted 4,5-dihydro-1H-pyrazol-1-yl)-5-(2-phenylhydrazono)thiazol-4(5H)-one
18b–e.
In further experiments treatment of 5a with 5,6,7,8-tetrahydro-1,3-dimethyl-5-(1-phenyl-3-p-
tolyl-1H-pyrazol-4-yl)-7-thioxopyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione 20 in boiling chloroform
containing triethylamine produced ethyl 2,4-dioxo-1,2,3,4,5,7-hexahydro-pyrimido[5,4-e]-[1,2,4]-
triazolo[4,3-a]pyrimidine-9-carboxylate 26a in good yields (Scheme 7).
R2
R3
O
R3
O
R2 Cl (Br)
H3C
O
TEA
H3C
O
N
N
NH
SH
N
NH
+
N
N
NH
Ph
N
N
N
N
H
S
Ph
CH3
CH3
6a,c.d
5a,c.d
20
21
5,6a, R2 = COOC2H5
c, R2 = COC6H5
d, R2 = CONHC6H5
O
R3
O
R3
H3C
O
N
NH R2
S
H3C
N
H
N
NH
S
N
N
N
Ph
R2
O
N
N
H
Ph
CH3
N
22
N
CH3
23
O
N
R3
H3C
O
HS
R2
R3 =
N
N
N
N
CH3
- H2S
N
N
N
H
CH3
Ph
O
N
24
H3C
O
N
N
CH3
Ph
N
N
N
N
CH3
N
R2
O
R2
26
H3C
N
N
26a, R2 = COOC2H5
b, R2 = COC6H5
c, R2 = CONHC6H5
N
N
O
N
N
Ph
CH3
25
Scheme 7. Synthesis of 1,3-dimethyl-7-phenyl-5-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-9-substituted
5,7-dihydropyrimido[5,4-e][1,2,4]triazolo[4,3-a]pyrimidine-2,4(1H,3H)-dione 26a–c.

Molecules 2016, 21, 1072
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The 1H-NMR spectrum of 26a showed a triplet at
singlet at 2.33 of tolyl methyl group, a singlet at
a quartet at 4.13 due to the methylene of ethyl ester, a singlet at
pyrimidine H-4 and a multiplet at 6.90–7.83 that account for 14 aromatic protons and H-5 of pyrazole.
δ
1.18 due to methyl group of ethyl ester, a
δ
δ
3.33 from the two pyrimidinedione methyl groups,
5.62 that corresponding to the
δ
δ
δ
In the IR spectrum, an absorption peak at 1650 cm−1 corresponding to conjugated carbonyl groups
and an absorption near at 1615 cm−1 could account for the presence of the imino group.
The mechanism outlined in scheme 7 seems to be the most plausible pathway for the formation
of 26a from the reaction of
5 with 20: (1) 1,3-addition of the thiol tautomer 21 to the nitrilimine 6a
would give the thiohydrazonate ester 22 which could undergo nucleophilic cyclization to yield spiro
compounds 23. The latter ring could then open and cyclize to yield 26a with loss of hydrogen sulfide;
and (2) 1,3-cycloaddition of nitrilimine 6a to C=S of 20 would directly yield 22 (cf., Scheme 7). Attempts
to isolate the thiohydrazonate ester 22 or intermediates 23 and 24 did not succeed even under mild
conditions as they readily undergo in situ cyclization followed by elimination of hydrogen sulfide to
give the final product 26 in Scheme 7.
Analogously, reaction of 20 with each of 5b and 5d afforded 3-acetyl-7,9-dimethyl-1-phenyl-5-
(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-5,9-dihydropyrimido[4,5-d][1,2,4]triazolo[4,3-a]pyrimidine-
6,8(1H,7H)-dione (26b) and 7,9-dimethyl-6,8-dioxo-N,1-diphenyl-5-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-
yl)-1,5,6,7,8,9-hexahydropyrimido[4,5-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxamide (26c), respectively.
Based on the above results, structure 25 was ruled out. This structural assignment was also consistent
with literature reports which indicated that reaction of hydrazonoyl halides with 2-thioxopyrimidin-
4-one yielded regioselectively the corresponding 1,2,4-triazolo[4,3-a]pyrimidin-5-one derivatives [33].
Additionally, treatment of 5-(3-(furan-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,3-dimethyl-7-thioxo-5,6,7,8-
tetrahydropyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (27) with the hydrazonoyl chlorides 5a and
5d in boiling chloroform in the presence of triethylamine afforded ethyl 5-(3-(furan-2-yl)-1-phenyl-
1H-pyrazol-4-yl)-1,3-dimethyl-2,4-dioxo-7-phenyl-1,2,3,4,5,7-hexahydro-pyrimido[5,4-e][1,2,4]triazolo
[4,3-a]pyrimidine-9-carboxylate (28a) and 5-(3-(furan-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,3-dimethyl-2,4-
dioxo-N,7-diphenyl-1,2,3,4,5,7-hexahydro-pyrimido[5,4-e][1,2,4]-triazol[4,3-a]pyrimidine-9-carboxamide
(28b), respectively (Scheme 8).
Scheme 8. Synthesis of 5-(3-(furan-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,3-dimethyl-2,4-dioxo-7-phenyl-
9-substituted-1,2,3,4,5,7-hexahydropyrimido[5,4-e][1,2,4]triazolo-[4,3-a]pyrimidines 28a and 28b.
3. Antimicrobial Activity
Twenty seven of the newly synthesized target compounds were evaluated for their in vitro
antibacterial activity against Staphylococcus aureus and Bacillus subtilis as examples of Gram-positive
bacteria and Pseudomonas aeruginosa and Escherichia coli as examples of Gram-negative bacteria.
They were also evaluated for their in vitro antifungal activity against a representative panel of fungal
strains i.e., Aspergillus fumigatus, and Candida albicans. Ampicillin, Gentamicin and Amphotericin B are

Molecules 2016, 21, 1072
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used as reference drugs for in vitro antibacterial activity and for in vitro antifungal activity, respectively,
at The Regional Center for Mycology and Biotechnology at Al-Azhar University (Nasr City, Cairo,
Egypt). The results of testing for antimicrobial effects are summarized in Tables 1–3.
Table 1. Mean zone of inhibition beyond well diameter (6 mm) produced on a range of clinically
pathogenic microorganisms using (5 mg/mL) concentration of tested samples.
Streptococcus
pneumoniae
(Gram +ve
Bact.)
Pseudomonas
aeruginosa
(Gram −ve
Bact.)
Aspergillus
fumigatus
(Fungus)
Candida
albicans
(Fungus)
Bacillus subtilis
(Gram +ve
Bact.)
Escherichia coli
(Gram −ve
Bact.)
Compound
9a
0
0
0
0
0
0
0
9b
15.7
15.6
16.3
18.8
18.6
23.7
14.6
13.6
15.9
25.3
10.3
15.6
0
0
16.2
19.6
18.9
17.3
20.6
23.8
10.2
15.6
17.3
22.6
8.3
19.8
19.3
21.3
18.2
20
16.3
14.9
19.9
17.9
15.9
19.9
11.2
9.4
9c
0
0
10a
0
0
10b
0
0
10c
0
0
13b
25.4
16.9
11.7
15.1
17.6
11.3
12.5
0
32.4
9.8
17.3
0
13c
13f
15.3
13.3
33.7
10.9
13.3
11.2
13.4
30.8
14.9
13.9
20.3
14.2
12.9
14.9
20.6
23.3
15.6
20.8
12.7
-
0
14a
11.9
13.1
0
11.6
19.3
8.8
10.9
9.6
13.2
17.6
11.8
8.9
20.3
10.3
11.6
14.7
14.8
19.1
10.8
18.9
8.5
14b
14c
14e
17.3
12.6
17.3
19.5
19.4
19.6
18.2
13.9
10.9
12.2
17.3
21.8
15.6
17.5
13.3
-
10.3
0
15.1
12.3
13.6
9.7
0
11.7
11.3
11.8
12.5
15.3
11.7
0
14f
15a
16.9
22.3
20.8
15.4
21.3
17.1
15.4
14.7
13.9
14.9
16.2
16.3
18.6
23.7
-
18.3
16.5
18.9
12.9
17.2
11.1
14.8
10.9
15.6
10.4
14.9
13.4
15.4
25.4
-
15b
15c
15e
15f
18a
20
26a
26c
26d
27
28a
28b
0
-
-
Amphotericin B
Ampicillin
Gentamicin
-
-
23.8
-
32.4
-
-
-
17.3
19.9
Compound 9a had no activity against Aspergillus fumigates, Candida albicans, Streptococcus
pneumonia, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli.
•
•
•
•
•
•
•
Candida albicans and Pseudomonas aeruginosa were resistant to compounds 9a
10c, 10d, and 14f.
Aspergillus fumigatus was susceptible to compounds 13b, 14b, 15b, and 15c when compared to the
Amphotericin B standard.
Candida albicans was susceptible to compound 13b when compared to the Amphotericin
, 9b, 9c, 9d, 10a, 10b,
B standard.
Streptococcus pneumoniae was susceptible to compounds 13b, and 14b when compared to the
Ampicillin standard.
Bacillus subtilis was susceptible to compounds 13b
Ampicillin standard.
Pseudomonas aeruginosa was susceptible to compounds 13b
standard Gentamicin.
,
14b, and 15b when compared to the
15a, and 26d when compared to their
14b 15f, and 28a when compared to the
,
Escherichia coli was susceptible to compounds 10a
,
13b
,
,
Gentamicin standard.
The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that
will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory
concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to
an antimicrobial agent and also to determine the potency of new antimicrobial agents [34]. A MIC is

Molecules 2016, 21, 1072
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generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent
against an organism [35]. MIC was determined by the broth micro dilution method using 96-well
micro-plates [36,37]. Pseudomonas aeruginosa showed an the same as the MIC value of 12.5 mg/mL of
the tested compound 13b suggesting high inhibitory activity compared to that of Gentamicin. Also,
compound 10a showed an MIC value of 3.9 against Escherichia coli which was the same MIC value of
Gentamicin against Escherichia coli (Table 2).
Table 2. Antimicrobial activity as MICS, (mg/mL) of tested samples against tested microorganisms.
Streptococcus
pneumoniae
(Gram +ve
Bact.)
Pseudomonas
aeruginosa
(Gram −ve
Bact.)
Aspergillus
fumigatus
(Fungus)
Candida
albicans
(Fungus)
Bacillus subtilis
(Gram +ve
Bact.)
Escherichia coli
(Gram −ve
Bact.)
Compound
9d
7.81
31.25
7.81
3.9
12.5
25
12.5
25
0.49
-
-
0
0
3.9
3.9
31.25
3.9
3.9
15.63
3.9
62.5
-
0.49
-
1.95
3.9
7.81
3.9
3.9
7.95
1.95
62.5
-
0
15.63
3.9
10a
0
10b
10c
0
0
0
12.5
50
25
0
15.63
31.25
15.63
31.25
15.63
25
-
-
3.9
0
13b
62.5
12.5
62.5
50
0.49
-
14b
15f
28a
Amphotericin B
Ampicillin
Gentamicin
-
-
0.24
-
-
15.63
The half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a substance
in inhibiting a specific biological or biochemical function. This quantitative measure indicates how
much of a particular substance is needed to inhibit a given biological process by half. And here the
biological function is the growth of microorganisms Aspergillus fumigatus, Syncephalastrum racemosum,
Geotrichum candidum, Candida albicans, Streptococcus pneumoniae, Bacillus subtilis, Pseudomonas aeruginosa,
and Escherichia coli (Table 3).
Table 3. Antimicrobial activity as half maximal inhibitory concentration (IC50) (mg/mL) of tested
samples against tested microorganisms.
Streptococcus
pneumoniae
(Gram +ve
Bact.)
Bacillus
subtillis
(Gram +ve
Bact.)
Pseudomonas
aeruginosa
(Gram −ve
Bact.)
Aspergillus
fumigatus
(Fungus)
Syncephalastrum Geotrichum
Candida
albicans
(Fungus)
Escherichia
coli (Gram
−ve Bact.)
Compound
racemosum
candidum
(Fungus)
(Fungus)
9d
10a
43.21
64.31
42.63
35.24
11.24
-
36.28
34.28
33.42
27.58
16.84
-
18.24
31.17
19.63
24.63
9.32
-
0
17.52
31.25
76.34
25.12
-
10.58
-
15.63
18.24
37.25
22.41
-
5.29
-
0
27.34
31.56
28.37
34.25
-
0
0
10b
0
0
0
0
-
-
10c
Amphotericin B
Ampicillin
Gentamicin
12.68
-
-
-
-
-
-
17.96
16.24
4. Experimental Section
4.1. General Information
All melting points were determined on an Electrothermal apparatus (Bibby Sci. Lim. Stone,
Staffordshire, UK) and are uncorrected. IR spectra were recorded (KBr discs) on a FT-IR 8201 PC
1
spectrophotometer (Shimadzu, Tokyo, Japan). H-NMR spectra were recorded in CDCl3 and DMSO-d6
solutions on a Gemini 300 MHz spectrometer (Varian, Mercury VX-300 NMR spectrometer, Bruker
BioSpin GmbH, Rheinstetten, Germany) and chemical shifts are expressed in
δ ppm units using TMS
as an internal reference. Mass spectra were recorded on a Shimadzu GC-MS QP1000 EX instrument.
(Tokyo, Japan) Elemental analyses were carried out at the Microanalytical Center of Cairo university.
Hydrazonoyl halides 5a–d were prepared as previously reported [38–41]. Antimicrobial screening was
performed at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.

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4.1.1. General Procedure for the Synthesis of Thiadiazoles 9a–c and 10a–c
Mixtures of alkyl carbodithioate (3a, 3b, 4a or 4b) (5 mmol), hydrazonoyl halides (5a, 5b, or 5c)
(5 mmol) and triethylamine (0.75 mL, 5 mmol) in ethanol (20 mL) were stirred at room temperature for
3 h. The resulting solid was collected and recrystallized from acetic acid (dioxane) to give 9a
–c and
10a–c, respectively, in good yields.
Ethyl (Z)-5-(((Z)-(1,1’-diphenyl-3’-(p-tolyl)-1H,1’H-[3,4’-bipyrazol]-4-yl)methylene)hydrazono)-4-phenyl-
4,5-dihydro-1,3,4-thiadiazole-2-carboxylate (9a). Yellow solid from glacial acetic acid, yield (1.9 g, 75%),
◦
mp: 176–177 C; IR (KBr, cm−1): 3078 (=C–H), 2993–2862 (–C–H), 1708 (–C=O), 1608 (–C=N); 1H-NMR:
δ
1.45 (t, 3H, –CH2CH3), 2.43 (s, 3H, 4-CH3C6H4), 4.44 (q, 2H, –OCH2CH3), 7.26–7.97 (m, 18H, Ar–H),
8.95 (s, 1H pyrazole-H-5); MS (m/z): 510 (M + 2, 9), 509 (M + 1, 32), 508 (M+, 72), 259 (52), 246 (41),
91 (57), 77 (100); Anal. Calcd. for C28H24N6O2S (508.59): C, 66.12; H, 4.76; N, 16.52; S, 6.30; found: C,
66.10; H, 4.75; N, 16.53; S, 6.33.
1-((Z)-5-(((Z)-(1,1’-Diphenyl-3’-(p-tolyl)-1H,1’H-[3,4’-bipyrazol]-4-yl)methylene)hydrazono)-4-phenyl-4,5-
dihydro-1,3,4-thiadiazol-2-yl)ethan-1-one (9b). Yellow solid from glacial acetic acid, yield (2 g, 84%), mp:
165–166 ◦C; IR (KBr, cm−1): 3040 (=C–H), 2869 (–C–H), 1674 (–C=O), 1604 (–C=N); 1H-NMR:
δ 2.43 (s,
3H, 4-CH3C6H4), 2.68 (s, 3H, –CO–CH3), 7.27–8.02 (m, 14H, Ar–H), 8.50 (s, 1H, –C=H), 8.85 (s, 1H,
pyrazole-H-5); MS (m/z): 479 (M + 1, 25), 478 (M+, 32), 310 (35), 307 (34), 150 (33), 138 (38), 126 (30), 122
(39), 91 (37), 77 (26); Anal. Calcd. for C27H22N6OS (478.57): C, 67.75; H, 4.61; N, 17.58; S, 6.70; found: C,
67.76; H, 4.63; N, 17.56; S, 6.71.
((Z)-5-(((Z)-(1,1’-Diphenyl-3’-(p-tolyl)-1H,1’H-[3,4’-bipyrazol]-4-yl)methylene)hydrazono)-4-phenyl-4,5-
dihydro-1,3,4-t◦hiadiazol-2-yl)(phenyl)methanone (9c). Orange solid from dioxane, yield (2.27 g, 84%),
1
mp: 264–265 C; IR (KBr, cm−1): 2863 (C–H), 1700 (–C=O), 1608 (–C=N); H-NMR:
δ
2.44 (s, 3H,
4-CH3C6H4), 7.27–8.49 (m, 19H, Ar–H), 8.54 (s, 1H, –C=H), 8.96 (s, 1H, pyrazole-H-5); MS (m/z): 542
(M + 2, 57), 541 (M + 1, 59), 540 (M+, 68), 525 (55), 510 (53), 494 (46), 479 (54), 468 (70), 458 (51), 439 (54),
421 (60), 412 (62), 371 (100), 355 (53), 282 (78), 135 (64), 105 (75); Anal. Calcd. for C32H24N6OS (540.64):
C, 71.09; H, 4.47; N, 15.54; S, 5.93; found: C, 71.07; H, 4.48; N, 15.54; S, 5.95.
Ethyl (Z)-5-(((Z)-(3’-(furan-2-yl)-1,1’-diphenyl-1H,1’H-[3,4’-bipyrazol]-4-yl)methylene)hydrazono)-4-phenyl-
4,5-dihydro-1,3,4-thiadiazole-2-carboxylate (10a). Yellow solid from ethanol, yield (2.2 g, 91%), mp:
171–173 C; IR (KBr, cm−1): 3108 (=C-H), 2980 (–C–H), 1738 (–C=O), 1603 (–C=N), 1545 (C=C);
◦
1H-NMR:
δ 1.44 (t, 3H, –CH2CH3), 4.45 (q, 2H, –OCH2CH3), 6.55 (d, 1H, furyl-H), 6.94 (q, 1H, furyl-H),
7.27–8.03 (m, 11H, Ar–H + 1furyl-H), 8.47 (s, 1H, CH=N), 8.89 (s, 1H, pyrazole-H-5); MS (m/z): 485 (M
+ 1, 9), 484 (M+, 22), 221 (100), 135 (30), 120 (27), 92 (46), 78 (60); Anal. Calcd. for C25H20N6O3S (484.53):
C, 61.97; H, 4.16; N, 17.34; S, 6.62; found: C, 61.99; H, 4.15; N, 17.35; S, 6.60.
1-((Z)-5-(((Z)-(3’-(Furan-2-yl)-1,1’-diphenyl-1H,1’H-[3,4’-bipyrazol]-4-yl)methylene)hydrazono)-4-phenyl-
4,5-dihydro-1,3,4-thiadiazol-2-yl)ethan-1-one (10b). Yellow solid from glacial acetic acid, yield (1.3 g, 65%),
◦
mp: 200–202 C; IR (KBr, cm−1): 3021 (=C–H), 2918 (–C–H), 1683 (C=O), 1604 (C=N), 1548 (C=C);
1H-NMR:
δ 2.63 (s, 3H, CO–CH3), 6.55 (q, 1H, furyl), 6.89 (d, 1H, furyl), 7.27–7.96 (m, 11H, Ar–H +
1furyl-H), 8.28 (s, 1H, N=CH), 8.91 (s, 1H, pyrazole-H-5); MS (m/z): 454 (M+, 33), 426 (100), 221 (34),
193 (16), 119 (14), 92 (12), 78 (23), 65 (15); Anal. Calcd. for C24H18N6O2S (404.50): C, 63.40; H, 3.98; N,
18.50; S, 7.05; found: C, 63.42; H, 3.99; N, 18.49; S, 7.07.
5-(-((3-(Furan-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazono)-4-phenyl-4,5-dihydro-1,3,4-thiadiazol-
2-yl)(phenyl)methanone (10c). Red solid from glacial acetic acid, yield (2.1 g, 83%), mp: 228–230 ◦C
;
1
IR (KBr, cm−1): 3061 (=C–H), 1725 (–C=O), 1601 (–C=N), 1547 (C=C); H-NMR:
δ 6.56 (q, 1H,
furyl-H), 6.85 (d, 1H, furyl-H), 7.27–8.35 (m, 16H, Ar–H + 1furyl-H), 8.35 (s, 1H, CH=N), 8.90 (s, 1H,
pyrazole-H-5); MS (m/z): 518 (M + 2, 1), 517 (M + 1, 5), 516 (M+, 31), 502 (12), 487 (97), 167 (10), 135 (25),
131 (10), 235 (17), 221 (49), 193 (24), 129 (17), 119 (11), 106 (100), 78 (45), 65 (30), 51 (45); Anal. Calcd. for
C29H20N6O2S (516.57): C, 67.43; H, 3.90; N, 16.27; S, 6.21; found: C, 67.40; H, 3.89; N, 16.27; S, 6.23.

Molecules 2016, 21, 1072
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4.1.2. General Procedure for the Synthesis of Chalcones 12a–f
10% NaOH solution (0.2 g, 10 mL, 5 mmol) was added dropwise to a mixture of the appropriate
2-acetylfuran (0.54 g, 5 mmol) or 4-methylacetophenone (0.67 mL, 5 mmol) and the appropriate
of 1-phenyl-3-(p-tolyl)-1H-pyrazole-4-carbaldehyde (1a) (1.3 g, 5 mmol), 3-(furan-2-yl)-1-phenyl-1H-
pyrazole-4-carbaldehyde (1.2 g, 5 mmol) (1b) or furfuraldehyde (1c) (0.48 g, 5 mmol) in ethanol (30 mL),
at 0–5 ◦C while stirring. The precipitate that formed was filtered, washed with ethanol (10 mL), and
recrystallized from ethanol to give 12a–f, respectively.
(E)-3-(Furan-2-yl)-1-(p-tolyl)prop-2-en-1-one (12a). Mp: 64–65 ◦C (lit. mp: 62–64 ◦C) [42].
(E)-1-(Furan-2-yl)-3-(3-(furan-2-yl)-1-phenyl-1H-pyrazol-4-yl)prop-2-en-1-one (12b). Yellow solid from
◦
ethanol, yield (1.5 g, 90%), mp: 155–156 C; IR (KBr, cm−1): 3103 (=C–H), 1652 (C=O); 1H-NMR:
δ 6.54
(q, 1H, furyl-H), 6.59 (q, 1H, furyl-H), 6.90 (q, 1H, furyl-H), 7.27–8.33 (m, 11H, ArH’s + 2CH=CH +
3furyl-H + pyrazole-H-5); MS (m/z): 332 (M + 2, 2), 331 (M + 1, 16), 330 (M+, 100), 314 (1), 300 (22), 242
(4), 270 (25), 214 (5), 91 (7); Anal. Calcd. for C20H14N2O3 (330.34): C, 72.72; H, 4.27; N, 8.48; found: C,
72.69; H, 4.28; N, 8.49.
(E)-3-(3-(Furan-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1-(p-tolyl)prop-2-en-1-one (12c). Yellow solid, yield (1.63 g,
92%), mp: 146–147 ◦C; IR (KBr, cm−1): 3122 (=C-H aromatic), 3069 (=C–H), 2919 (–C–H), 1651 (C=O);
1H-NMR:
δ 2.44 (s, 3H, 4-CH3C6H4), 6.54 (q, 1H, furyl-H), 6.89 (d, 1H, furyl-H), 7.26–8.19 (m, 12H,
Ar–H’s + 2H + 1furyl-H), 8.25 (s, 1H, pyrazole-H-5); MS (m/z): 355 (M + 1, 2), 354 (M+, 7), 308 (20), 281
(25), 234 (43), 209 (27), 208 (42), 207 (28), 181 (13), 180 (43), 179 (20), 178 (15), 168 (16), 167 (36), 166 (100),
154 (13), 153 (44), 152 (32), 140 (38), 139 (10), 127 (13), 126 (10), 115 (16), 114 (10), 113 (13), 63 (10), 29
(30), 27 (13); Anal. Calcd. for C23H18N2O2 (354.40): C, 77.95; H, 5.12; N, 7.90; found: C, 77.97; H, 5.10;
N, 7.91.
◦
(E)-3-(1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1-(p-tolyl)prop-2-en-1-one (12d). mp: 135–136 C (lit. mp.:
101–103 ◦C) [43].
(E)-1,3-di(Furan-2-yl)prop-2-en-1-one (12e). Mp: 89–90 ◦C (lit. mp: 88–90 ◦C) [39].
1-(Furan-2-yl)-3-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)prop-2-en-1-one (12f) Yellow solid, yield (1.5 g, 86%),
◦
mp: 154–155 C; IR (KBr, cm−1): 3138 (=C–H aromatic), 3105 (=C–H), 2922 (C–H), 1649 (C=O); 1H-NMR:
δ
2.44 (s, 3H, 4-CH3C6H4), 6.56 (q, 1H, furyl-H), 7.23–7.97 (m, 13H, ArH’s + 2CH=CH + 2furyl-H), 8.35
(s, 1H, pyrazole-H-5); MS (m/z): 356 (M + 2, 3), 355 (M + 1, 20), 354 (100), 339 (11), 273 (17), 188 (10),
186 (20), 172 (20), 171 (24), 170 (17), 157 (10), 156 (17), 143 (11), 142 (11), 130 (24), 129 (14), 128 (15); Anal.
Calcd. for C23H18N2O2 (354.40): C, 77.95; H, 5.12; N, 7.90; found: C, 77.97; H, 5.14; N, 7.88.
4.1.3. Synthesis of Carbothioamide Derivatives 13a–f
Mixtures of chalcones 12a–f (5 mmol) and thiosemicarbazide (0.46 g, 5 mmol) in ethanol (20 mL)
were refluxed for 3 h. The resulting solid was collected and recrystallized from acetic acid to give
13a–f, respectively.
◦
5-(Furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (13a). Mp: 190–191 C. (lit. mp:
280–282 ◦C) [44].
3’,5-di(Furan-2-yl)-1’-phenyl-3,4-dihydro-1’H,2H-[3,4
'
-bipyrazole]-2-carbothioamide (13b). Yellow solid,
yield (1.7 g, 85%), mp: 285–287 ◦C; IR (KBr, cm−1): 3334 (N–H), 3120 (=C–H); 1H-NMR:
δ
3.15 (dd,
1H, pyrazoline-H), 3.90 (q, 1H, pyrazoline-H), 6.20 (dd, 1H, pyrazoline-H), 6.65 (m, 2H, furyl-H),
6.67 (q, 1H, furyl-H), 7.05 (d, 1H, furyl-H), 7.44–7.91 (m, 9H, Ar–H + 1furyl-H + 2N–H), 8.95 (s, 1H,
pyrazole-H-5); MS (m/z): 404 (M + 1, 2), 403 (M+, 9), 300 (6), 256 (1), 228 (10), 209 (36), 196 (100), 181
(58), 164 (50), 136 (25), 121 (6), 93 (9), 77 (7); Anal. Calcd. for C21H17N5O2S (403.46): C, 62.52; H, 4.25;
N, 17.36; S, 7.95; found: C, 62.55; H, 4.26; N, 17.34; S, 7.94.

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3’-(Furan-2-yl)-1’-phenyl-5-(p-tolyl)-3,4-dihydro-1’H,2H-[3,4’-bipyrazole]-2-carbothioamide (13c). Yellow
solid, yield (1.7 g, 80%), mp: 263–265 ◦C; IR (KBr, cm−1): 3265 (N–H), 3136 (=C–H aromatic), 3048
(=C–H), 2917 (–C–H); 1H-NMR:
δ 2.34 (s, 3H, 4-CH3C6H4), 3.18 (dd, 1H, pyrazoline-H), 3.93 (q, 1H,
pyrazoline-H), 6.16 (dd, 1H, pyrazoline-H), 6.65–7.84 (m, 14H, Ar–H + 3furyl-H + 2N–H), 8.08 (s, 1H,
pyrazole-H-5); MS (m/z): 427 (M+, 5), 408 (11), 386 (18), 344 (25), 302 (48), 260 (100), 231 (6), 203 (3), 153
(1); Anal. Calcd. for C24H21N5OS (427.52): C, 67.43; H, 4.95; N, 16.38; S, 7.50; found: C, 67.39; H, 4.95;
N, 16.38; S, 7.52.
1’-Phenyl-3’,5-di-p-tolyl-3,4-dihydro-1’H,2H-[3,4’-bipyrazole]-2-carbothioamide (13d). White solid from
dioxane, yield (1.7 g, 75%), mp: 276–277 ◦C; IR (KBr, cm−1): 3430; 3115 (N–H), 3046 (=C–H aromatic),
2918 (–C–H); 1H-NMR:
δ 2.34 (s, 3H, 4-CH3C6H4), 2.35 (s, 3H, 4-CH3C6H4), 3.18 (dd, 1H, pyrazoline-H),
3.93 (q, 1H, pyrazoline-H), 6.16 (dd, 1H, pyrazoline-H), 6.66–7.82 (m, 15H, Ar–H + 2N–H), 8.07 (s, 1H,
pyrazole-H-5); MS (m/z): 452 (M + 1, 6), 253 (17), 250 (13), 225 (16), 221 (10), 206 (19), 193 (20), 174 (14),
151 (9), 142 (29), 116 (10), 110 (16), 108 (17), 107 (16), 91 (100), 84 (43), 82 (26), 79 (20), 82 (26), 77 (12);
Anal. Calcd. for C27H25N5S (451.59): C, 71.81; H, 5.58; N, 15.51; S, 7.10; found: C, 71.79; H, 5.57; N,
15.52; S, 7.12.
◦
3,5-di(Furan-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (13e). Mp: 164–166 C. (lit. mp: 162–163 ◦C) [45].
5-(Furan-2-yl)-1 -phenyl-3’-(p-tolyl)-3,4-dihydro-1’H,2H-[3,4’-bipyrazole]-2-carbothioamide (13f). White
solid, yield (1.6 g, 76%), mp: 247–248 C;IR (KBr, cm−1): 3255 (N–H), 3145 (=C–H), 2919 (–C–H);
1H-NMR:
2.37 (s, 3H, 4-CH3C6H4), 3.05 (dd, 1H, pyrazoline-H), 3.85 (q, 1H, pyrazoline-H), 6.06 (dd,
'
◦
δ
1H, pyrazoline-H), 6.65–7.90 (m, 14H, Ar–H + 3furyl-H + 2N–H), 8.01 (s, 1H, pyrazole-H-5); MS (m/z):
427 (M+, 7), 384 (8), 354 (10), 325 (9), 296 (6), 270 (5), 254 (11), 240 (8), 213 (9), 103 (100), 75 (32); Anal.
Calcd. for C24H21N5OS (427.52): C, 67.43; H, 4.95; N, 16.38; S, 7.50; found: C, 67.41; H, 4.95; N, 16.37;
S, 7.51.
4.1.4. 5-Arylazothiazole Derivatives 14a–f and 15a–f
Method A
Mixtures of the appropriate thioamides 13a–f (5 mmol), the appropriate 5b and 5c (5 mmol) and
triethylamine (0.75 mL, 5 mmol) in ethanol (20 mL) were refluxed for 3 h. The resulting solid was
collected and recrystallized from acetic acid (or dioxane) to give 14a–f and 15a–f, respectively.
Method B
Benzenediazonium chloride (5 mmol), which was prepared from aniline (0.45 mL, 5 mmol),
hydrochloric acid (6 N, 6 mL), and sodium nitrite (0.35 g, 5 mmol), was added dropwise with stirring
to a cold solution of 2-(5-(furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-phenylthiazole (16
)
(1.92 g, 5 mmol) and sodium acetate trihydrate (1.3 g, 10 mmol) in ethanol (50 mL). The reaction
◦
mixture was stirred in an ice bath at 0–5 C for 3 h. The result solid was collected and recrystallized to
give a product identical in all aspects (mp, mixed mp. and spectra) with 15a.
(E)-2-(3-(Furan-2-yl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-5-(phenyldiazenyl)-thiazole (14a).
◦
1
Red solid, yield (1.5 g, 70%), mp: 179–180 C; IR (KBr, cm−1): 2920 (–C–H); H-NMR:
δ 2.37 (s,
3H, 4-CH3C6H4), 2.50 (s, 3H, –CH3), 3.56 (dd, 1H, pyrazoline-H), 3.90 (dd, 1H, pyrazoline-H), 5.90 (dd,
1H, pyrazoline-H), 6.43 (dd, 1H, furyl-H), 6.52 (dd, 1H, furyl-H), 7.31–7.76 (m, 10H, Ar–H + 1furyl-H);
MS (m/z): 427 (M+, 1), 326 (12), 236 (16), 232 (14), 206 (39), 204 (11), 194 (31), 147 (51), 146 (14), 134
(23), 133 (41), 129 (11), 121 (32), 118 (12), 117 (100), 115 (10), 107 (24), 91 (17), 73 (24); Anal. Calcd. for
C24H21N5OS (427.52): C, 67.43; H, 4.95; N, 16.38; S, 7.50; found: C, 67.41; H, 4.94; N, 16.37; S, 7.51.
(E)-2-(3’,5-Di(furan-2-yl)-1’-phenyl-3,4-dihydro-1’H,2H-[3,◦4'-bipyrazol]-2-yl)-4-methyl-5-(phenyldiazen-yl)-
thiazole (14b). Red solid, yield (1.5 g, 55%), mp: 275–277 C; IR (KBr, cm−1): 3146 (=C–H), 2922 (–C–H);
1H-NMR:
δ 2.49 (s, 3H, –CH3), 3.13 (dd, 1H, pyrazoline-H), 3.97 (dd, 1H, pyrazoline-H), 6.17 (dd, 1H,

Molecules 2016, 21, 1072
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pyrazoline-H), 6.65–7.91 (m, 16H, Ar–H + 3-furyl-H), 8.95 (s, 1H, pyrazole-H-5); MS (m/z): 545 (4), 520
(14), 505 (35), 504 (10), 492 (13), 491 (17), 478 (10), 460 (10), 270 (48), 252 (17), 241 (18), 223 (10), 176 (19),
121 (40), 106 (11), 83 (10), 71 (11); Anal. Calcd. for C30H23N7O2S (545.61): C, 66.04; H, 4.25; N, 17.97; S,
5.88; found: C, 66.02; H, 4.24; N, 17.98; S, 5.89.
(E)-2-(3’-(Furan-2-yl)-1’-phenyl-5-(p-tolyl)-3,4-dihydro-1’H,2H-[3,4’-bipyrazol]-2-yl)-4-methyl-5-(phenyl-
diazenyl)-thiazole (14c). Red solid, yield (1.7 g, 60%), mp: 250–252 ◦C; IR (KBr, cm−1): 3139 (=C–H),
2917 (–C–H); 1H-NMR:
δ 2.30 (s, 3H, 4-CH3C6H4), 2.50 (s, 3H, –CH3), 3.18 (dd, 1H, pyrazoline-H), 3.97
(dd, 1H, pyrazoline-H), 6.19 (dd, 1H, pyrazoline-H), 6.65–7.88 (m, 17H, Ar–H + 3furyl-H), 8.08 (s, 1H,
pyrazole-H-5); MS (m/z): 569 (M+, 4), 484 (16), 454 (14), 358 (42), 317 (60), 289 (34), 230 (34), 130 (16),
103 (26), 77 (14); Anal. Calcd. for C33H27N7OS (569.68): C, 69.57; H, 4.78; N, 17.21; S, 5.63; found: C,
69.55; H, 4.78; N, 17.20; S, 5.64.
(E)-4-Methyl-2-(1’-phenyl-3’,5-di-p-tolyl-3,4-dihydro-1’H,2H-[3,4
'-bipyrazol]-2-yl)-5-(phenyldiazenyl)-thiazole
(
14d). Orange solid, yield (2.1 g, 70%), mp: 210–211 ◦C; IR (KBr, cm−1): 3136 (=C–H aromatic), 3051
1
(=C–H), 2950 (–C–H); H-NMR:
δ 2.40 (s, 6H, 4-CH3C6H4), 2.63 (s, 3H, –CH3), 3.65; 3.9 (m, 2H,
pyrazoline-H), 5.1 (q, 1H, pyrazoline-H), 6.99–8.95 (m, 19H, Ar–H + pyrazole-H-5); MS (m/z): 593 (M+,
2), 581 (12), 578 (16), 574 (35), 300 (33), 299 (100), 298 (11), 288 (11), 287 (19), 286 (77), 285 (15), 241 (10),
227 (24), 211 (18); Anal. Calcd. for C36H31N7S (593.74): C, 72.82; H, 5.26; N, 16.51; S, 5.40; found: C,
72.85; H, 5.27; N, 16.49; S, 5.39.
(E)-2-(3,5-Di(furan-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-5-(phenyldiazenyl)-thiazole (14e). Red solid,
yield (1.3 g, 66%), mp: 201–203 ◦C; IR (KBr, cm−1): 3122 (=C–H), 2950 (–C–H); 1H-NMR:
δ 2.54 (s, 3H,
–CH3), 3.50 (dd, 1H, pyrazoline-H), 3.89 (dd, 1H, pyrazoline-H), 5.90 (dd, 1H, pyrazoline-H), 6.43–6.73
(m, 3H, furyl-H), 7.14 (t, 1H, furyl-H), 7.37–7.97 (m, 7H, Ar–H +2 furyl-H); MS (m/z): 405 (M+2, 3), 404
(M + 1, 21), 403 (M+, 75), 388 (41), 361 (64), 275 (12), 146 (7), 130 (14); Anal. Calcd. for C21H17N5O2S
(403.46): C, 62.52; H, 4.25; N, 17.36; S, 7.95; found: C, 62.49; H, 4.25; N, 17.37; S, 7.96.
(E)-2-(5-(Furan-2-yl)-1’-phenyl-3’-(p-tolyl)-3,4-dihydro-1’H,2H-[3,4’-bipyrazol]-2-yl)-4-methyl-5-(phenyldiazenyl)
-thiazole (14f). Orange solid, yield (2.1 g, 75%), mp: 216–219 ◦C; IR (KBr, cm−1): 3120 (=C–H), 2914
(–C–H); 1H-NMR:
δ 2.39 (s, 3H, 4-CH3C6H4), 2.47 (s, 3H, –CH3), 4.49 (q, 2H, pyrazoline-H), 5.95 (q, 1H,
pyrazoline-H), 6.70 (dd, 1H, furyl-H), 7.05 (d, 1H, furyl-H),7.28–7.61 (m, 15H, Ar–H + 1furyl-H), 8.52
(s, 1H, pyrazole-H-5); MS (m/z): 569 (M+, 7), 553 (10), 398 (12), 370 (15), 248 (51), 235 (100), 91 (27);
Anal. Calcd. for C33H27N7OS (569.68): C, 69.57; H, 4.78; N, 17.21; S, 5.63; found: C, 69.58; H, 4.79; N,
17.22; S, 5.62.
(E)-2-(5-(Furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-phenyl-5-(phenyldiazenyl)-thiazole (15a).
◦
Orange solid, yield (1.9 g, 76%), mp: 233–234 C; IR (KBr, cm−1): 3147 (=C–H), 2930 (–C–H); 1H-NMR:
δ
2.38 (s, 3H, 4-CH3C6H4), 3.60 (dd, 1H, pyrazoline-H), 4.00 (dd, 1H, pyrazoline-H), 6.00 (dd, 1H,
pyrazoline-H), 6.46 (q, 1H, furyl-H), 6.65 (d, 1H, furyl-H), 7.33–8.05 (m, 15H, Ar–H + 1furyl-H); MS
(m/z): 489 (M+, 2), 428 (10), 335 (18), 321 (10), 293 (18), 163 (11), 165 (9), 155 (100), 92 (10), 91 (68), 43 (9);
Anal. Calcd. for C29H23N5OS (489.59): C, 71.14; H, 4.74; N, 14.30; S, 6.55; found: C, 71.15; H, 4.73; N,
14.31; S, 6.52.
(E)-2-(3’,5-di(Furan-2-yl)-1’-phenyl-3,4-dihydro-1’H,2H-[3,4’-◦bipyrazol]-2-yl)-4-phenyl-5-(phenyldiazenyl)-
thiazole (15b). Orange solid, yield (1.8 g, 60%), mp: 270–272 C; IR (KBr, cm−1): 3150 (=C–H); 1H-NMR:
δ
3.08 (q, 1H, pyrazoline-H), 3.96 (q, 1H, furyl-H), 6.18 (q, 1H, pyrazoline-H), 6.66–8.10 (m, 22H, Ar–H
+ pyrazole-H-5 + 6furyl-H); MS (m/z): 607 (M+, 2), 331 (14), 284 (100), 169 (94), 127 (17), 109 (57), 43
(86); Anal. Calcd. for C35H25N7O2S (607.68): C, 69.18; H, 4.15; N, 16.13; S, 5.28; found: C, 69.19; H, 4.16;
N, 16.15; S, 5.23.
(E)-2-(3’-(Furan-2-yl)-1’-phenyl-5-(p-tolyl)-3,4-dihydro-1’H,2H-[3,4
-thiazole (15c). Orange solid, yield (2.3 g, 74%), mp: 240–242 ◦C; IR (KBr, cm−1): 3139 (=C–H), 2920
(–C–H); 1H-NMR:
2.34 (s, 3H, 4-CH3C6H4), 3.18 (dd, 1H, pyrazoline-H), 3.39 (dd, 1H, pyrazoline-H),
'-bipyrazol]-2-yl)-4-phenyl-5-(phenyldiazenyl)
δ

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6.11 (dd, 1H, pyrazoline-H), 6.64–8.08 (m, 23H, Ar–H + pyrazole-H-5 + 3furyl-H); MS (m/z): 631 (M+,
4), 477 (9), 409 (65), 297 (6), 271 (5), 245 (8), 227 (11), 203 (13), 149 (17), 135 (49), 107 (31), 69 (100); Anal.
Calcd. for C38H29N7OS (631.75): C, 72.25; H, 4.63; N, 15.52; S, 5.08; found: C, 72.25; H, 4.62; N, 15.50;
S, 5.10.
(E)-4-Phenyl-2-(1’-phenyl-3’,5-di-p-tolyl-3,4-dihydro-1’H,2H-[3,4’-bipyrazol]-2-yl)-5-(phenyldiazenyl)-thiazole
◦
15d). Red solid, yield (1.5 g, 45%), mp: 208–209 C; IR (KBr, cm−1): 3142 (=C–H), 2924 (–C–H);
(
1H-NMR:
δ 2.50 (s, 6H, 4-CH3C6H4), 3.36 (q, 1H, pyrazoline-H), 3.63 (q, 1H, pyrazoline-H), 5.30 (q, 1H,
pyrazoline-H), 7.36–9.00 (m, 24H, Ar–H + pyrazole-H-5); MS (m/z): 655 (M+, 1), 498 (10), 339 (10), 281
(17), 243 (51), 242 (62), 210 (12), 171 (32), 156 (25), 73 (35), 71 (76), 41 (26), 39 (14), 27 (19); Anal. Calcd.
for C41H33N7S (655.81): C, 75.09; H, 5.07; N, 14.95; S, 4.89; found: C, 75.10; H, 5.05; N, 14.96; S, 4.88.
(E)-2-(3,5-di(Furan-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-phenyl-5-(phenyldiazenyl)-thiazole (15e). Red solid,
◦
yield (1.3 g, 57%), mp: 181–183 C; IR (KBr, cm−1): 3100 (=C–H); 1H-NMR:
δ 4.00 (m, 2H, pyrazoline-H),
6.00 (q, 1H, pyrazoline-H), 6.46; 6.64; 6.74 (m, 3H, furyl-H), 7.16 (d, 1H, furyl-H), 7.41–8.27 (m, 12H,
Ar–H + 2furyl-H); MS (m/z): 465 (M+, 3), 271 (29), 253 (70), 233 (16), 221 (100), 105 (10); Anal. Calcd.
for C26H19N5O2S (465.53): C, 67.08; H, 4.11; N, 15.04; S, 6.89; found: C, 67.10; H, 4.09; N, 15.05; S, 6.86.
(E)-2-(5-(Furan-2-yl)-1’-phenyl-3’-(p-tolyl)-3,4-dihydro-1’H,2H-[3,4’-bipyrazol]-2-yl)-4-phenyl-5-(phenyldiazenyl)-
thiazole (15f). Orange, yield (1.7 g, 53%), mp: 249–250 ◦C; IR (KBr, cm−1): 3150 (=C–H), 2922 (–C–H);
1H-NMR:
δ 2.37 (s, 3H, 4-CH3C6H4), 3.30 (dd, 1H, pyrazoline-H), 3. 90 (dd, 1H, pyrazoline-H), 6.16 (q,
1H, pyrazoline-H), 6.65 (q, 1H, furyl-H), 7.01 (q, 1H, furyl-H), 7.82–8.10 (m, 21H, Ar–H + pyrazole-H-5
+ 1furyl-H); MS (m/z): 633 (M + 2, 1), 632 (M + 1, 8), 631 (M+, 28), 630 (60), 380 (7), 337 (14), 322 (37), 321
(100), 310 (47), 292 (13), 109 (13), 97 (24), 83 (29), 69 (35); Anal. Calcd. for C38H29N7OS (631.75): C,
72.25; H, 4.63; N, 15.52; S, 5.08; found: C, 72.23; H, 4.63; N, 15.51; S, 5.09.
2-(5-(Furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-phenylthiazole (16). A mixture of 5-(furan-2-yl)-
3-(p-tolyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (1.42 g, 5 mmol) and
ω-bromo-acetophenone
(0.99 g, 5 mmol) in ethanol (30 mL) was heated under reflux for 2 h to give 2-(5-(Furan-2-yl)-3-(p-tolyl)-
4,5-dihydro-1H-pyrazol-1-yl)-4-phenylthiazole (15) as a white precipitate which was washed with water
◦
and recrystallized from glacial acetic acid a white solid, yield (1.9 g, 71%), mp: 221 C; IR (KBr,
cm−1): 3100; 3041 (=C–H), 2943 (–C–H), 1717 (–C=O amide); 1H-NMR:
δ 2.45 (s, 3H, 4-CH3C6H4), 3.77
(dd, 1H, pyrazoline-H), 3.93 (dd, 1H, pyrazoline-H), 6.34 (q, 1H, pyrazoline-H), 6.70–7.91 (m, 13H,
Ar–H + 3furyl-H + 1thiazole H-5); MS (m/z): 385 (M+, 7%), 381 (3%), 378 (10%), 374 (23%), 369 (51%),
263 (13%), 262 (100%), 260 (12%), 223 (12%), 216 (20%), 215 (12%), 77 (33%), 76 (16%); Anal. Calcd. for
C23H19N5O2S (385.48): C, 71.66; H, 4.97; N, 10.90; S, 8.32; found: C, 71.63; H, 4.98; N, 10.92; S, 8.32.
4.1.5. (2-Phenylhydrazono)thiazol-4(5H)-one Derivatives 18a–e
Method A
Equimolar amounts of the appropriate thioamides 13a–d, 13f and ethyl 2-chloro-2-(2-
phenylhydrazono)acetate with triethylamine (5 mmol) in ethanol (25 mL) were refluxed for 2 h. The
solid so formed was collected and crystallized from glacial acetic acid to afford 2-phenylhydrazono)
thiazol-4(5H)-one 18a–e, respectively.
Method B
Benzenediazonium chloride (5 mmol), which prepared from aniline (0.45 mL, 5 mmol),
hydrochloric acid (6 N, 6 mL), and sodium nitrite (0.35 g, 5 mmol), was added dropwise with stirring
to a cold solution of 19 (5 mmol) and sodium acetate trihydrate (1.3 g, 10 mmol) in ethanol (50 mL).
The reaction mixture was stirred in ice bath for 3 h. The resulting solid was collected and crystallized
to give a product identical in all aspects (mp, mixed mp, and spectra) with 18a.

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(E)-2-(5-(Furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-5-(2-phenylhydrazono)-thiazol-4(5H)-one (18a).
◦
Yellow solid, yield (1.4 g, 67%), mp: 247–248 C; IR (KBr, cm−1): 3432 (N–H), 2970; 2921 (–C–H),
1717 (–C=O amide); 1H-NMR:
δ
2.39 (s, 3H, 4-CH3C6H4), 3.63 (dd, 1H, pyrazoline-H), 4.04 (dd, 1H,
pyrazoline-H), 5.96 (q, 1H, pyrazoline-H), 6.44 (q, 1H, furyl-H), 6.54 (d, 1H, furyl-H), 6.9–7.78 (m, 10H,
Ar–H + 1furyl-H), 10.4 (s, 1H, N–H); MS (m/z): 431 (M + 2, 1), 430 (M + 1, 29), 429 (M+, 100), 413 (15),
346 (15), 345 (40), 316 (10), 264 (23), 263 (11), 248 (10), 228 (58), 227 (25), 226 (16), 214 (44), 212 (12), 200
(12), 198 (11), 196 (16), 186 (14), 172 (11), 170 (13), 158 (14), 157 (10), 156 (13), 146 (17), 144 (18), 130 (16),
115 (10); Anal. Calcd. for C23H19N5O2S (429.49): C, 64.32; H, 4.46; N, 16.31; S, 7.47; found: C, 64.33; H,
4.46; N, 16.32; S, 7.44.
(Z)-2-(3’,5-di(Furan-2-yl)-1’-phenyl-3,4-dihydro-1’H,2H-[3,4’-bipyrazol]-2-yl)-5-(2-phenylhydrazono)-thiazol-
4(5H)-one (18b). Yellow solid, yield (1.7 g, 63%), mp: 284–285 ◦C; IR (KBr, cm−1): 3407 (N–H), 3044
(=C–H), 2852 (–C–H), 1635 (C=O); 1H-NMR:
δ 3.08 (dd, 1H, pyrazoline-H), 3.90 (dd, 1H, pyrazoline-H),
6.19 (dd, 1H, pyrazoline-H), 6.66–7.90 (m, 17H, Ar–H + 1N–H + 6furyl-H), 8.09 (s, 1H, pyrazole-H-5);
MS (m/z): 549 (M + 2, 1), 548 (M + 1, 6), 547 (M+, 16), 374 (100), 329 (7), 254 (19), 227 (61), 212 (17), 173
(77), 91 (9), 77 (7); Anal. Calcd. for C29H21N7O3S (547.59): C, 63.61; H, 3.87; N, 17.91; S, 5.86; found: C,
63.61; H, 3.88; N, 17.93; S, 5.80.
(E)-2-(3’-(Furan-2-yl)-1’-phenyl-5-(p-tolyl)-3,4-dihydro-1’H,2H-[3,4’-bipyrazol]-2-yl)-5-(2-phenylhydrazono)-
thiazol-4(5H)-one (18c). Yellow solid, yield (1.5 g, 54%), mp: 245–247 ◦C; IR (KBr, cm−1): 3396 (N-H),
3140 (=C–H), 2990 (–C–H), 1715 (–C=O); 1H-NMR:
δ 2.34 (s, 3H, 4-CH3C6H4), 3.18 (dd, 1H,
pyrazoline-H), 3.90 (dd, 1H, pyrazoline-H), 6.17 (dd, 1H, pyrazoline-H), 6.65–8.08 (m, 19H, Ar–H +
3furyl-H + 1N–H + 1pyrazole-H-5); MS (m/z): 571 (M+, 2), 569 (4), 553 (10), 398 (12), 370 (15), 248 (51),
235 (100), 155 (12), 107 (10), 91 (27), 77 (2), 55 (9); Anal. Calcd. for C32H25N7O2S (571.65): C, 67.23; H,
4.41; N, 17.15; S, 5.61; found: C, 67.25; H, 4.41; N, 17.13; S, 5.60.
(E)-2-(1’-Phenyl-3’,5-di-p-tolyl-3,4-dihydro-1’H,2H-[3,4’-bipyrazol]-2-yl)-5-(2-phenylhydrazono)-thiazol-
4(5H)-one (18d). Yellow solid, yield (1.6 g, 55%), mp: 278–279 ◦C; IR (KBr, cm−1): 3431 (N–H), 3140
(=C–H), 2919 (–C–H), 1715 (–C=O); 1H-NMR:
δ 2.34 (s, 3H, 4-CH3C6H4), 2.36 (s, 3H, CH3), 3.30 (dd,
1H, pyrazoline-H), 3.85 (dd, 1H, pyrazoline-H), 6.10 (dd, 1H, pyrazoline-H), 7.24–8.08 (m, 20H, 18Ar-H
+ 1N–H + pyrazole-H-5); MS (m/z): 595 (M+, 1), 578 (11), 554 (6), 397 (10), 340 (7), 279 (100), 263 (4),
236 (29), 193 (28), 91 (1); Anal. Calcd. for C35H29N7OS (595.72): C, 70.57; H, 4.91; N, 16.46; S, 5.38;
found: C, 70.58; H, 4.92; N, 16.49; S, 5.32.
(E)-2-(5-(Furan-2-yl)-1’-phenyl-3’-(p-tolyl)-3,4-dihydro-1’H,2H-[3,4’-bipyrazol]-2-yl)-5-(2-phenylhydrazono)-
thiazol-4(5H)-one (18e). Yellow solid, yield (2.1 g, 75%), mp: 294–296 ◦C; IR (KBr, cm−1): 3433 (N-H),
3137 (=C–H), 2921 (–C–H), 1708 (–C=O); 1H-NMR:
δ 2.37 (s, 3H, 4-CH3C6H4), 3.40 (dd, 1H,
pyrazoline-H), 4.10 (dd, 1H, pyrazoline-H), 5.90 (q, 1H, pyrazoline-H), 6.72 (q, 1H, furyl-H), 6.90–7.99
(m, 16H, Ar–H + 2furyl-H), 8.52 (s, 1H, pyrazole-H-5), 10.33(s, 1H, N-H); MS (m/z): 571 (M+, 2),
553 (10), 398 (12), 370 (15), 248 (51), 235 (100), 155 (12), 107 (10), 91 (27), 71 (4); Anal. Calcd. for
C32H25N7O2S (571.65): C, 67.23; H, 4.41; N, 17.15; S, 5.61; found: C, 67.20; H, 4.41; N, 17.13; S, 5.64.
2-(5-(Furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-thiazol-5(4H)-one (19) Equimolar amounts of
5-(furan-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (13a, 1.42 g, 5 mmol) and ethyl
chloroacetate (0.61 g, 5 mmol) in ethanol (25 mL) were heated under reflux for 2 h, then allowed to
cool at room temperature, the solid so formed was collected and recrystallized from dioxane to give 18
as a pale yellow solid, yield (1.2 g, 72%), mp: 244–245 ◦C; IR (KBr, cm−1): 3143 (=C–H aromatic), 3039
(=C–H), 2991 (–C–H), 1697 (C=O); 1H-NMR:
δ 2.44 (s, 3H, 4-CH3C6H4), 3.61 (dd, 1H, pyrazoline-H),
3.92 (s, 2H, thiazole-H), 3.95 (dd, 1H, pyrazoline-H), 5.85 (q, 1H, pyrazoline-H), 6.42–7.76 (m, 7H,
4Ar-H + 3furyl-H); MS (m/z): 327 (M + 2, 1), 326 (M + 1, 10), 325 (M+, 50), 308 (47), 293 (100), 275 (51),
101 (35), 77 (40), 69 (67); Anal. Calcd. for C17H15N3O2S (325.38): C, 62.75; H, 4.65; N, 12.91; S, 9.85;
found: C, 62.74; H, 4.67; N, 12.92; S, 9.81.

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4.1.6. General Procedure for the Synthesis of Compounds 20 and 27
Equimolar amounts of the appropriate pyrazole aldehyde 1a or 2b (10 mmol), N,N0-
dimethylbarbituric acid (1.56 g, 10 mmol), thiourea (0.76 g, 10 mmol) and conc. hydrochloric acid
(5 mL) in ethanol (25 mL) were heated under refluxed for 1 h. The reaction mixture was allowed to cool
to room temperature and the precipitate was filtered and crystallized from dioxane to give compounds
20 and 27, respectively.
1,3-Dimethyl-5-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-7-thioxo-5,6,7,8-tetrahydro-pyrimido[4,5-d]pyrimidine-
2,4(1H,3H)-dione (20). Yellow solid, yield (4.3 g, 94%), mp: 267–268 ◦C; IR (KBr, cm−1): 3434 (N–H),
3175 (=C-H aromatic), 3019 (C=H), 2921 (–C–H), 1669 (C=O); 1H-NMR:
δ 2.45 (s, 3H, 4-CH3C6H4), 3.40
(s, 3H, N–CH3), 3.44 (s, 3H, N–CH3), 7.27–7.93 (m, 11H, Ar-H + pyrimidine-H-5 + pyrazole-H-5), 8.62
(s, 1H, N–H), 9.88 (s, 1H, N–H); MS (m/z): 460 (M + 2, 2), 459 (M + 1, 12), 458 (M+, 36), 426 (73), 398
(33), 394 (28), 383 (52), 368 (38), 367 (100), 365 (16), 305 (10), 289 (34); Anal. Calcd. for C24H22N6O2S
(458.54): C, 62.86; H, 4.84; N, 18.33; S, 6.99; found: C, 62.86; H, 4.85; N, 18.34; S, 6.98.
5-(3-(Furan-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1,3-dimethyl-7-thioxo-5,6,7,8-tetrahydropyrimido[4,5-d]pyrimidine-
2,4(1H,3H)-dione (27). Brown solid, yield (3.6 g, 85%), mp: 186–187 ◦C; IR (KBr, cm−1): 3439 (N-H),
3170 (=C–H), 2954 (–C–H), 1664 (C=O); 1H-NMR:
δ 3.44 (s, 6H, 2N–CH3), 6.59–7.90 (m, 10H, ArH +
3furyl-H + pyrimidine-H+ pyrazole-H-5), 9.13 (s, 1H, N–H), 9.85 (s, 1H, N–H); MS (m/z): 434 (M+1, 3),
433 (M+, 5), 310 (100), 296 (51), 238 (49), 168 (28), 166 (34), 150 (17), 139 (59), 138 (42), 125 (74), 99 (11),
83 (14), 43 (22); Anal. Calcd. for C21H18N6O3S (434.47): C, 58.05; H, 4.18; N, 19.34; S, 7.38; found: C,
58.01; H, 4.19; N, 19.36; S, 7.38.
4.1.7. General procedure for the Synthesis of Hexahydropyrimido[4,5-d][1,2,4]triazolo[4,3-a]
pyrimidine Derivatives 26a–c and 28a,d
Equimolar amounts of 20 (2.29 g, 5 mmol) or 27 (2.17 g, 5 mmol) and the appropriate hydrazonyl
halides 5a–d (5 mmol) in ethanol (25 mL) containing 5 drops of triethylamine was heated under reflux
for 20 h. The reaction mixture was evaporated under reduced pressure and the resulting solid was
washed several times with water and ether. The precipitates formed, were filtered and recrystallized
to give:
Ethyl 7,9-dimethyl-6,8-dioxo-1-phenyl-5-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1,5,6,7,8,9-hexahydropyrimido
[4,5-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (26a). White solid from glacial acetic acid, yield (2.4 g,
77%), mp: 250–251 ◦C; IR (KBr, cm−1): 3108 (=C–H aromatic), 3038 (=C–H), 2965; 2916 (–C–H), 1696
(C=O); 1H-NMR:
δ 1.28 (t, 3H,–CH2CH3), 2.34 (s, 3H, 4-CH3C6H4), 2.82 (s, 3H, N–CH3), 2.86 (s, 3H,
N–CH3), 4.30 (q, 2H,-OCH2CH3), 5.59 (s, 1H, pyrimidine-H), 6.88–7.7 (m, 14H, Ar–H), 7.77 (s, 1H,
pyrazole-H-5); MS (m/z): 614 (M+, 9), 563 (6), 379 (10), 323 (54), 311 (30), 295 (100), 284 (11), 267 (19),
239 (4), 111 (12), 96 (22), 85 (18), 71 (26); Anal. Calcd. for C34H30N8O4 (614.65): C, 66.44; H, 4.92; N,
18.23; found: C, 66.43; H, 4.91; N, 18.21.
3-Benzoyl-7,9-dimethyl-1-phenyl-5-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-7,9-dihydropyrimido[4,5-d][1,2,4]
triazolo[4,3-a]pyrimidine-6,8(1H,5H)-dione (26b). White solid from toluene, yield (1.6 g, 50%), mp:
1
211–213 ◦C; IR (KBr, cm−1): 3049 (=C–H), 2920 (–C–H), 1695 (–C=O); H-NMR:
δ 2.43 (s, 3H,
4-CH3C6H4), 2.80 (s, 3H, N–CH3), 2.88 (s, 3H, N–CH3), 5.85 (s, 1H, pyrimidine-H), 6.86–7.68 (m, 19H,
Ar-H), 8.32 (s, 1H, pyrazole-H-5); MS (m/z): 646 (M+, 3), 645 (6), 662 (5), 406 (13), 389 (54), 256 (12), 239
(14), 195 (11), 168 (13), 151 (100), 135 (29), 106 (17), 85 (30); Anal. Calcd. for C38H30N8O3 (646.70): C,
70.58; H, 4.68; N, 17.33; found: C, 70.56; H, 4.68; N, 17.34.
7,9-Dimethyl-6,8-dioxo-N,1-diphenyl-5-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1,5,6,7,8,9-hexahydropyrimido
[4,5-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxamide (26c). White solid from glacial acetic acid, yield (2 g,
60%), mp: 258–259 ◦C; IR (KBr, cm−1): 3364 (N–H), 3136 (=C–H aromatic), 3054 (=C–H), 2921 (–C–H),
1690 (C=O); 1H-NMR:
δ
2.42 (s, 3H, 4-CH3C6H4), 2.80 (s, 3H, N–CH3), 2.87 (s, 3H, N–CH3), 5.76 (s, 1H,
pyrimidine-H), 6.86–7.68 (m, 19H, Ar-H), 7.78 (s, 1H, pyrazole-H-5), 8.55 (s, 1H, N–H); MS (m/z): 661

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(M + 1, 3), 660 (M+, 7), 603 (9), 441 (11), 401 (1), 315 (29), 204 (100), 189 (46), 161 (12), 147 (56), 121 (3);
Anal. Calcd. for C38H31N9O3 (661.71): C, 68.97; H, 4.72; N, 19.05; found: C, 68.93; H, 4.73; N, 19.07.
Ethyl 5-(3-(furan-2-yl)-1-phenyl-1H-pyrazol-4-yl)-7,9-dimethyl-6,8-dioxo-1-phenyl-1,5,6,7,8,9-hexahydropyrimido
[4,5-d][1,2,4]triazolo◦[4,3-a]pyrimidine-3-carboxylate (28a). White solid from benzene, yield (2.2 g,
75%), mp: 249–251 C; IR (KBr, cm−1): 3038 (=C–H), 2969 (–C–H), 1696 (C=O); 1H-NMR:
δ 1.28 (t,
3H, –CH2CH3), 2.82 (s, 3H, N–CH3), 2.85 (s, 3H, N–CH3), 4.27 (q, 2H, –OCH2 CH3), 5.58 (s, 1H,
pyrimidine-H), 6.87–7.69 (m, 13H, Ar–H + 3furyl-H), 7.75 (s, 1H, pyrazole-H-5); MS (m/z): 590 (M+, 1),
511 (2), 397 (10), 340 (7), 279 (100), 236 (29), 193 (28); Anal. Calcd. for C31H26N8O5 (590.59): C, 63.04; H,
4.44; N, 18.97; found: C, 63.01; H, 4.46; N, 18.91.
5-(3-(Furan-2-yl)-1-phenyl-1H-pyrazol-4-yl)-7,9-dimethyl-6,8-dioxo-N,1-diphenyl-1,5,6,7,8,9-hexahydropyrimido
[4,5-d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxamide (28b). White solid from toluene, yield (1.7 g,
55%), mp: 145–146 ◦C; IR (KBr, cm−1): 3398 (N–H), 3130 (=C-H aromatic), 3028 (=C–H), 2949 (–C–H);
1H-NMR:
δ 2.84 (s, 3H, N–CH3), 3.35 (s, 3H, N–CH3), 6.03 (s, 1H, pyrimidine-H), 6.53 (q, 1H, furyl-H),
6.91–7.67 (m, 17H, Ar–H + 2furyl-H), 8.73 (s, 1H, pyrazole-H-5), 8.54 (s, 1H, N–H); MS (m/z): 637 (M+,
7), 633 (17), 620 (24), 528 (11), 436 (25), 418 (34), 380 (24), 278 (18), 263 (66), 232 (34), 219 (100), 203 (57),
159 (89); Anal. Calcd. for C35H27N9O4 (637.65): C, 65.93; H, 4.27; N, 19.77; found: C, 65.90; H, 4.28;
N, 19.73.
4.2. Antimicrobial Activity Assay
Chemical compounds under investigation were individually tested against a panel of
Gram-positive and Gram-negative bacteria pathogens, and fungi. Antimicrobial tests were carried out
using the agar well-diffusion method [46]. After the media had cooled and solidified, wells (6 mm in
diameter) were made in the solidified agar, after that microbial inoculum was uniformly spread using
sterile cotton swab on a sterile Petri dish containing nutrient agar (NA) medium or Sabouraud dextrose
agar (SDA) media for bacteria and fungi, respectively. A 100 µL solution was prepared from 1 mL of
DMSO by dissolving 1 mg of the compound. The inoculated plates were then incubated for 24 h at
37 ◦C for bacteria and yeast, 48 h at 28 ◦C for fungi. Negative controls were prepared using DMSO
employed for dissolving the tested compound. Amphotericin B (1 mg/mL), ampicillin (1 mg/mL), and
gentamicin (1 mg/mL) were used as standards for bacteria and fungi, respectively. After incubation,
antimicrobial activity was evaluated by measuring the zone of inhibition against tested microorganisms.
Antimicrobial activity was expressed as inhibition diameter zones in millimeters (mm). The experiment
was carried out in triplicate and the average zones of inhibition ± S.D were calculated.
5. Conclusions
New series of novel functionalized 1,3,4-thiadiazoles, 1,3-thiazoles, and pyrimido[4,5-d][1,2,4]
triazolo[4,3-a]pyrimidines containing pyrazole moieties were synthesized using hydrazonoyl halides
as precursors and evaluated for their in vitro antibacterial, and antifungal activities. From the screening
results, it can be seen Aspergillus fumigatus was susceptible to compounds 12b, 13b, 14b, 15b, and 15c
when compared to the amphotericin B standard. Candida albicans was susceptible to compounds 12b
and 13b when compared to the amphotericin B standard. Streptococcus pneumoniae was susceptible
to compounds 12b
susceptible to compounds 13b
aeruginosa was susceptible to compounds 12b
,
13b, and 14b when compared to an ampicillin standard. Bacillus subtilis was
14b, and 15b when compared to ampicillin standard. Pseudomonas
13b 15a, and 26d when compared to Gentamicin
,
,
,
standard. Escherichia coli was susceptible to compounds 10a, 12b, 13b, 14b, 15f, and 28a when
compared to the gentamicin standard.
Acknowledgments: The authors would like to thank the Chemistry Department, Faculty of Science, Cairo
University and National Center for Social and Criminological Research for their financial support to facilitate the
publication of this study.

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Author Contributions: A.O.A. designed research and analyzed the data. M.N.M. performed experiments.
All authors contributed wrote and approved the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the synthesized compounds are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).