Synthesis and anti-rotavirus activity of some nitrogen heterocycles integrated with pyrazole scaffold

Article abstract of DOI:10.1007/s13738-020-01873-7

Herein, the building block synthon 2-cyano-3-(1,3-diphenyl-1H-pyrazol-4-yl)acryloyl chloride 4 was synthesized and utilized for construction of a wide variety of nitrogen heterocycles encompassing a pyrazole scaffold via treatment with some nitrogen nucle

Full text of DOI:10.1007/s13738-020-01873-7

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-020-01873-7
ORIGINAL PAPER
Synthesis and anti‑rotavirus activity of some nitrogen heterocycles
integrated with pyrazole scafold
Eman A. E. El‑Helw1 · Marwa M. Gado2 · Ahmed K. El‑Ziaty1
Received: 25 October 2019 / Accepted: 12 February 2020
© Iranian Chemical Society 2020
Abstract
Herein, the building block synthon 2-cyano-3-(1,3-diphenyl-1H-pyrazol-4-yl)acryloyl chloride 4 was synthesized and utilized
for construction of a wide variety of nitrogen heterocycles encompassing a pyrazole scafold via treatment with some nitrogen
nucleophiles. The seven membered heterocycles 14–16 and 20 were constructed from the reaction of the acid chloride 4 with
1,4-binucleophilic reagents such as 2-aminophenol, 2-aminoaniline, 2-aminothiophenol, and thiosemicarbazide, respectively.
The structures of all the synthesized heterocycles were elucidated from their elemental and spectral analysis. Antiviral activity
screening of products obtained against rotavirus (RV) revealed that compounds 3, 12, 13, 15, and 16 exhibited high reduction
efect on RV titer of ≥2 log10 TCID50. Thus, these compounds can be possible candidates of anti-RV agents.
Keywords Rotavirus · Diarrhea · Pyrazole · Thiazolopyrimidine · Benzothiazepine
Introduction
In spite of over 40 years of clinical and basic research on
rotavirus (RV) following their discovery in 1973, RV infec-
tion continues to be a worldwide health concern in human
[15]. RV, a member of the Reoviridae family, is a major
cause of serious gastroenteritis and diarrhea in young kids
[16] and is responsible for about one-third of all diarrheal
cases requiring hospital admission [17]. RV gastroenteritis
results in >500,000 deaths per year in children <5 years
[18]. Although two approved oral, live, attenuated RV vac-
cines (Rotarix and RotaTeq) are available internationally,
> 90% of rotavirus-mediated childhood mortalities are
recorded within developing countries due to limited access
to these vaccines, sanitation concerns, high cost, and lack
of availability to routine healthcare services [19]. Thus, it is
necessary to develop a new antiviral drug with high safety,
low cost, and high efcacy against RV infection in which
our study is aimed to.
Pyrazoles can be traced in a number of well-established
drugs and different categories with diverse therapeutic
activities and occupy a prime position in medicinal and pes-
ticide chemistry [1–12]. For instance, the antiviral activity of
ethyl 5-amino-1-(4b,5,10,10a-tetrahydronaphtho[2′,3′:4,5]
thieno[2,3-d]pyrimidin-4-yl)-1H-pyrazole-4-carboxylate
(I) (Fig. 1) revealed the highest anti-HAV activity at a con-
centration of 20 µg/105 cells, in comparison with the other
tested compounds [5]. Pyrazole analogues have been found
to be used as building blocks in organic synthesis for design-
ing pharmaceuticals and agrochemicals and as bifunctional
ligands for metal catalysis. The recent pharmaceutical appli-
cations of 2-propenoylamides [13, 14] and other heterocyclic
skeletons have stimulated us to synthesize new derivatives
of these classes of compounds hoping to obtain structures
with possibly enhanced antiviral potency.
Results and discussion
* Eman A. E. El-Helw
eman.abdelrahman@sci.asu.edu.eg
Chemistry
1
Laboratory of Synthetic Organic Chemistry, Chemistry
Department, Faculty of Science, Ain Shams University,
Abbassia, Cairo 11566, Egypt
In continuation of our efforts toward constructing het-
erocyclic compounds and evaluating their pharma-
ceutical efficiency [20–29], the present work investi-
gated the proclivity of high functionality compound:
2
Laboratory of Microbiology, Microbiology
Department, Faculty of Science, Ain Shams University,
Abbassia, Cairo 11566, Egypt
Vol.:(0123456789)
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Fig. 1 Pyrazole derivatives with
diverse therapeutic activities
2-cyano-3-(1,3-diphenyl-1H-pyrazol-4-yl)acryloyl chloride
(4) with some nitrogen nucleophiles. Condensation of pyra-
zole aldehyde 1 with ethyl cyanoacetate aforded the cor-
responding arylidene derivative 2 [30, 31] which was easily
transformed into the corresponding acid 3 via hydrolysis
using ethanolic sodium hydroxide solution followed by acid-
ifcation with dilute hydrochloric acid (20%). Treating the
acid 3 with thionyl chloride acquired the targeted acid chlo-
ride 4 (Scheme 1). The chemical structures of compounds
3 and 4 were elucidated from their analytical and spectral
data. Thus, IR spectrum of the acid 3 revealed the absence of
ester carbonyl group and showed the characteristic absorp-
tion bands for OH and C=O of the carboxylic moiety at ν
3453 and 1697 cm−1, respectively. Also, the IR spectrum of
acid chloride 4 displayed the absence of OH group and the
The proclivity of the acid chloride 4 was studied toward
some N-nucleophiles, such as 1,3-binucleophiles including
2-aminothiazole and thiourea as well as 1,2-binucleophiles
including hydrazine and phenylhydrazine (Scheme 2). Thus,
treatment of the acid chloride 4 with 2-aminothiazole in
refuxing benzene and in the presence of triethylamine as a
base aforded the open-chain compound, the amide deriva-
tive 5 as well as the fused heterocyclic compound, thiazo-
lopyrimidine derivative 6 (Scheme 2). The IR spectrum of
compound 5 retained the stretching absorption band of the
nitrile function, which rules out its cyclization on it. Mean-
while, its 1H-NMR spectrum retained the singlet signal of
the olefnic proton at δ 8.09 ppm. Furthermore, its mass
spectrum strongly confrmed the assigned structure by show-
ing the correct molecular ion peak as the base peak at m/z
397 (100%). IR spectrum of compound 6 lacked to ν C=O
and provided the characteristic absorption bands of cyano
and hydroxyl groups. Also, its 1H-NMR spectrum lacked to
the olefnic proton singlet and displayed a new singlet for
methine proton which supported the cyclization step. Pre-
sumably, the reaction pathway of compound 4 with 2-ami-
nothiazole is illustrated in Scheme 3.
1
appearance of a carbonyl group at ν 1754 cm−1. The H-
NMR spectra were completely consistent with the assigned
structures. Furthermore, the mass spectrum of 4 supported
its structure as it exhibited the correct molecular ion peak
at m/z 333.20 (52.03%) in addition to [M.+.+2] peak at m/z
335.11 (15.44%) attributable for the presence of chlorine
atom (cf. “Experimental” section).
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Journal of the Iranian Chemical Society
Scheme 1 Synthesis of the
target acid chloride 4
Scheme 2 Reaction of acid chloride 4 with some nitrogen nucleophiles
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Journal of the Iranian Chemical Society
On the other hand, thiourea reacted with acid chloride 4
to furnish a mixture of thiourea derivative 7 as canary yellow
crystals and tetrahydropyrimidinethione derivative 8 as deep
yellow crystals which were separated by fractional recrystal-
lization (cf. “Experimental” section). The spectral data were
ft with the assigned structures. The 1H-NMR spectrum of
compound 7 provided exchangeable singlet signals for NH
and NH2 protons.
with acid chloride 4 to obtain the corresponding acrylamide
derivative bearing benzotriazole moiety 12 as yellow crys-
tals (cf. Scheme 2). Its IR spectrum displayed the absorption
bands for C≡N and C=O groups. The 1H-NMR spectrum
showed signals for the olefnic and aromatic protons. Fur-
thermore, the mass spectrum exhibited the highest recorded
peak at m/z 416 (10.56%) attributable for the molecular ion
peak, as well as some of the abundant peaks (cf. “Experi-
mental” section).
It was interesting that hydrazinolysis of the acid chlo-
ride 4 at 0 °C aforded a mixture of diheterylazine 9 as
pale yellow crystals and 5-hydroxy-1′,3′-diphenyl-1′H,2H-
[3,4′-bipyrazole]-4-carbonitrile (10) as yellow crystals
(Scheme 2). The chemical structure of the azine 9 was evi-
denced from its spectral data and supported by direct com-
parison with an authentic sample prepared from refuxing
ethanolic solution of the pyrazole aldehyde 1 with hydrazine
[3]. The 1H-NMR spectrum of pyrazole derivative 10 dis-
played the disappearance of the olefnic hydrogen, and the
presence of two labile hydrogen atoms corresponds to both
NH and OH protons. The hydrazinolysis of acid chloride 4
is displayed in Scheme 4.
Condensation of acid chloride 4 with some 1,4-binucle-
ophilic reagents, namely 2-aminophenol, 2-aminoaniline,
2-aminothiophenol, 1,2-diaminoethane, 2-aminoethanol, and
thiosemicarbazide, is displayed in Scheme 6. Thus, treat-
ing 4 with 2-aminophenol aforded a mixture of acrylamide
derivative 13 and benzoxazepine derivative 14. The IR spec-
trum of amide 13 exhibited phenolic OH absorption band
which disappeared in that of compound 14. The 1H-NMR
spectrum of amide 13 retained singlet signal for olefnic pro-
ton at δ 8.19 ppm and provided two exchangeable singlet
signals for amide NH and phenolic OH protons at δ 10.13
and δ 7.37 ppm, respectively. Such a reaction, treating the
acid chloride 4 with 2-aminoaniline or 2-aminothiophenol,
acquired the benzodiazepine 15 and benzothiazepine 16,
respectively. In the 1H-NMR spectra of compounds 14–16,
the absence of the olefnic singlet confrmed the cyclization
step. The reaction could be visualized to occur via elimi-
nation of HCl molecule to aford the corresponding amide
derivative which underwent cyclization followed by dehy-
drogenation to construct benzoxazepine, benzodiazepine and
benzothiazepine derivatives 14–16 (Scheme 7).
In turn, reaction of phenylhydrazine with acid chloride
4 led to the construction of pyranedione derivative 11 as
brown crystals (cf. Scheme 2). Its IR spectrum displayed
the absorption bands for lactone C=O at ν 1734 cm−1
,
ketone C=O at ν 1687 cm−1, CN at ν 2240 cm−1 and NH
at ν 3139 cm−1 groups. Perhaps, formation of pyranedione
11 could be explained as depicted in Scheme 5. From syn-
thetic and biological point of views, benzotriazole reacted
In turn, treatment of acid chloride 4 with 1,2-diami-
noethane afforded N,N′-(ethane-1,2-diyl)bis(2-cyano-
3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamide) (17) as
yellow crystals. Its IR spectrum displayed the absorption
bands for NH at ν 3319 cm−1, C≡N at ν 2206 cm−1 and
C=O at ν 1672 cm−1. Also, the 1H-NMR spectrum showed
singlet signal for NH, olefnic and methylene protons as
well as signals for aromatic protons (cf. “Experimental”
section). In case of ethanolamine, the reaction prompted
the acrylamide derivative 18 as pale yellow crystals. IR
spectrum of compound 18 showed bands for OH, NH,
C≡N and C=O groups. Meanwhile, the 1H-NMR spectrum
displayed two exchangeable singlet signals for NH and OH
protons, two triplet signals for CH2–CH2 protons, as well
as signals for olefnic and aromatic protons. Furthermore,
the mass spectrum provided its molecular ion peak as
the base peak at m/z 358.28 (100%). It was fortunate that
thiosemicarbazide reaction with acid chloride 4 produced
a mixture of the open-chain amide 19 in addition to the
cyclic structure, triazepine derivative 20. The IR spectra
exhibited absorption bands for NH, C≡N, C=O and C=S
1
groups. Full analysis of the H-NMR and mass spectra
Scheme 3 Formation of thiazolopyrimidine 6
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Scheme 4 Suggested pathways for hydrazinolysis of acid chloride 4
strongly confrmed the proposed structures (cf. “Experi-
should be totally selective for specific viral processes
with no or little impacts on cellular metabolism [32].
Several techniques have been developed for investigat-
ing the antiviral activities of compounds in cell culture.
Evaluation of cytotoxicity in vitro is usually performed by
cell viability assays, such as the dye uptake by non-viable
cells after breakdown of the permeability of the cellular
barrier (e.g., eosin Y, trypan blue, etc.) or function of
mitochondria (e.g., XTT or MTT assay), but other meth-
ods, such as changes in cell culture morphology under
microscopic investigation, have also been applied as
mental” section).
Antiviral screening
In the present study, anti-RV activities were carried out
for the synthetic ten compounds. First of all, cytotoxicity
assay was made. Evaluation of cytotoxicity is clearly a
significant step of the assessment of a potential antiviral
drug since a useful drug should show either long-term
toxicity or chronic against the host. Such a compound
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Scheme 5 Plausible pathway for the formation of pyranedione 11
indicators of compound toxicity [33]. Our results demon-
strated that the CC50 of the tested compounds ranged from
750 to 1500 µg/mL as summarized in Table 1 and Fig. 2.
Safe concentrations of the synthetic compounds were
then evaluated against RV infection using TCID50 meas-
urement. The tested compounds showed reduction effect
in the virus titer ranging from 0 to 3.25 log10 TCID50.
Two compounds (8 and 11) did not show antiviral activi-
ties against RV infection. Week antiviral activities with
reduction in virus titers < 1 log10 TCID50 were shown
from 4, 6, and 10 compounds against RV infection. Strong
antiviral activities with reduction in virus titers ≥ 2 log10
TCID50 were observed from compounds 3, 12, 13, 15, and
16. These five compounds are therefore regarded to be
powerful anti-rotavirus drug candidates (Fig. 3).
Viral life cycle involves various steps: attachment, pen-
etration, replication of viral proteins and genetic materi-
als, assembly, and viral exit from infected cells. These
steps can be used as targets of anti-rotavirus SA-11 agents
[34]. Our results demonstrated that our tested compounds
might be affected on the RV infections via reacting with
viral capsid, preventing the virus attachment with the cell
host. Further studies on the antiviral activities of these
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Scheme 6 Behavior of acid chloride 4 toward some 1,4-binucleophiles
Scheme 7 A suggested mecha-
nism for synthesis of com-
pounds 13–16
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Table 1 Results of antiviral
activity of compounds on
RV SA11 by TCID50/0.1 mL
measurement
Sample ID
CC50 (µg/mL)
Virus titers without
compounda
Virus titers with
compoundb
Reduction
value of virus
titersc
3
4
6
8
10
11
12
13
15
16
850
900
1400
1500
850
1200
750
800
106.25
106
103
103.25
100.75
100.5
0
105.25
105.75
106
106.25
106
106.25
106.25
106.25
106.25
106.25
106
106
100.25
0
106.25
106
102.25
102.25
102
104
900
1200
104.25
103.25
102.75
aTiter of positive control
bTiter of rotavirus when incubated with the compound prior to infection
cReduction of virus titer was calculated as “virus titer without extract–virus titer with extract”
1600
1400
1200
Fig. 2 CC50 of the tested com-
1500
pounds
1400
1200
1200
1000
800
600
400
200
0
900
900
850
850
800
750
3
4
6
8
10
11
12
13
15
16
Sample ID
3.5
3
Fig. 3 Reduction efect in the
3.25
virus titer
2.75
2.5
2
2.25
2.25
2
1.5
1
0.75
0.5
0.5
0
0.25
0
0
3
4
6
8
10
11
12
13
15
16
Sample ID
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Journal of the Iranian Chemical Society
compounds in cell culture to investigate whether these
compounds can have a effect on the virus via other repli-
cation steps are required.
2‑Cyano‑3‑(1,3‑diphenyl‑1H‑pyrazol‑4‑yl)acrylic
acid (3)
To a solution of ethyl 2-cyano-3-(1,3-diphenyl-1H-pyrazol-
4-yl)acrylate (2) [30, 31] (3 mmol, 1 g) in ethanol (15 mL),
sodium hydroxide (3 mmol, 0.11 g) in ethanol (5 mL) was
added. The reaction mixture was stirred at room tempera-
ture for 1 h. The reaction mixture was poured onto crushed
ice and acidifed with cold dilute HCl. The separated solid
was fltered of, washed with water, dried, and recrystal-
lized from ethanol to give the acid 3 as pale yellow crystals,
mp. 298–300 °C, yield 93%. IR (KBr, ν, cm−1): 3453 (OH),
Conclusion
Synthesis of some nitrogen heterocycles such as thiazolopy-
rimidine, pyrimidinethione, hydroxypyrazole, pyranedione,
benzotriazole, benzoxazepine, benzodiazepine, benzothiaze-
pine, and triazepinethione derivatives encompassing pyrazole
scafold starting with treatment of acryloyl chloride derivative
4 with some nitrogen nucleophiles has been reported. The anti-
viral screening of the synthesized products showed promising
anti-RV activities of fve compounds (3, 12, 13, 15, and 16)
which can be considered as novel anti-rotavirus drugs.
1
2226 (CN), 1697 (C=O). H-NMR (400 MHz, DMSO-
d6): δ 13.87 (br.s, 1H, OH, exchangeable), 9.19 (s, 1H, CH
pyrazole), 8.10 (s, 1H, CH=), 7.95–7.93 (d, 2H, N-phenyl,
J = 7.92 Hz), 7.66–7.56 (m, 7H, Ar–H), 7.49–7.45 (t, 1H,
C4–H, N-phenyl, J=7.36 Hz). EIMS m/z (%): 315.38 (M+·,
40.82), 263.58 (100.00), 170.05 (80.13), 104.19 (68.96),
41.70 (85.17). Anal. Calcd. for C19H13N3O2 (315.33): C,
72.37; H, 4.16; N, 13.33. Found: C, 72.11; H, 4.02; N,
13.36%.
Experimental
Chemistry
Chemicals and solvents are of commercial grade and obtained
from Sigma-Aldrich, Merck, Fluka, and El-Nasr pharmaceuti-
cal chemicals companies and were purifed and dried by stand-
ard techniques. Melting points were measured on a Gallen-
kamp electric melting point apparatus and were uncorrected.
The infrared spectra were recorded using potassium bromide
disks on a FTIR Thermo Electron Nicolet iS10 (USA) infra-
red spectrometer and expressed in wave number (ν, cm−1) at
the Chemistry Department, Faculty of Science, Ain Shams
University. The 1H-NMR spectra were run at 400 MHz on a
Bruker NMR spectrometer using tetramethylsilane (TMS) as
an internal standard in deuterated dimethyl sulfoxide (DMSO-
d6) at Faculty of Pharmacy, Cairo University, Giza, Egypt.
Chemical shifts (δ) are quoted in ppm. The abbreviations used
are as follows: s, singlet; d, doublet; t, triplet; and m, multiplet.
All coupling constant (J) values are given in hertz. The mass
spectra were recorded on a Shimadzu GC–MS-QP-1000 EX
mass spectrometer operating at 70 eV at the regional center
for Mycology and Biotechnology (RCMB) of Al-Azhar Uni-
versity, Nasr City, Cairo, Egypt. Elemental analyses were car-
ried out at the microanalytical unit, Faculty of Science, Ain
Shams University. The reactions were monitored by thin-layer
chromatography using Merck Kieselgel 60 F254 obtained from
Fluka. The antiviral activity was performed at Microanalytical
Center of Ain Shams University, Egypt. The starting pyrazole
aldehyde 1 and arylidene derivatives, namely ethyl 2-cyano-
3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylate (2), were previously
prepared [30, 31].
2‑Cyano‑3‑(1,3‑diphenyl‑1H‑pyrazol‑4‑yl)acryloyl
chloride (4)
A mixture of 3 (5 mmol) and thionyl chloride (5 mL) was
heated on a water bath at 65 °C for 4 h. The excess thio-
nyl chloride was distilled under reduced pressure. The
obtained yellowish green solid was collected and used
without further purifcation, mp. 164–166 °C, yield 84%.
1
IR (KBr, ν, cm−1): 2228 (C≡N), 1754 (C=O). H-NMR
(400 MHz, DMSO-d6): 9.19 (s, 1H, CH pyrazole), 8.12
(s, 1H, CH=), 7.94–7.92 (d, 2H, N-phenyl, J = 7.92 Hz),
7.66–7.50 (m, 7H, phenyl), 7.48–7.45 (t, 1H, C4–H,
N-phenyl, J = 7.32 Hz). EIMS m/z (%): 333.20 (M+·,
52.03), 298.23 (100.00), 270.00 (52.78), 241.06 (10.31),
77.04 (86.33). Anal. Calcd. for C19H12ClN3O (333.78):
C, 68.37; H, 3.62; N, 12.59. Found: C, 68.04; H, 3.40; N,
12.61%.
Reaction with 2‑aminothiazole
To a solution of 4 (2 mmol, 0.7 g) in dry benzene (20 mL)
containing two drops of triethylamine, 2-aminothiazole
(2 mmol, 0.21 g) was added. The reaction mixture was
heated under refux for 2 h and then cooled to room tem-
perature. The precipitated solid was collected and found
to be a mixture of two compounds 5 (recrystallized from
dioxane) and 6 (recrystallized from ethanol).
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Journal of the Iranian Chemical Society
2H, NH2, exchangeable). Anal. Calcd. for C20H15N5OS
(373.43): C, 64.33; H, 4.05; N, 18.75. Found: C, 64.19; H,
3.92; N, 18.78%.
2‑Cyano‑3‑(1,3‑diphenyl‑1H‑pyra‑
zol‑4‑yl)‑N‑(thiazol‑2‑yl)acrylamide (5)
Pale yellow crystals, mp. 288–290 °C, yield 30%. IR (KBr,
ν, cm−1): 3150 (NH), 2215 (C≡N), 1672 (C=O amide).
1H-NMR (400 MHz, DMSO-d6): (E- and Z-isomers, 1:1)
δ 13.51, 13.48 (br.s, 1H, NH, exchangeable), 9.21, 9.19 (s,
1H, CH pyrazole), 8.28, 8.09 (s, 1H, CH=), 7.95–7.93 (d,
2H, N-phenyl, J=7.92 Hz), 7.72–7.70 (d, 1H, C4–H thia-
zole, J=6.38 Hz), 7.6–7.51 (m, 7H, phenyl), 7.49–7.45 (t,
1H, C4–H, N-phenyl, J=7.36 Hz), 7.21–7.19 (d, 1H, C5–H
thiazole, J=6.35 Hz). EIMS m/z (%): 397.24 (M+·, 100.00),
298.22 (19.92), 77.15 (18.19), 51.15 (4.14). Anal. Calcd. for
C22H15N5OS (397.46): C, 66.48; H, 3.80; N, 17.62. Found:
C, 66.23; H, 3.68; N, 17.60%.
6‑(1,3‑Diphenyl‑1H‑pyrazol‑4‑yl)‑4‑oxo‑2‑thi‑
oxo‑1,2,3,4‑tetrahydropyrimidine‑5‑carbonitrile (8)
Deep yellow crystals, yield 71%, mp. 190–192 °C [Lit. [35,
36], mp. 190–192 °C].
Reaction with hydrazine hydrate
To a solution of 4 (2 mmol, 0.7 g) in dry toluene (20 mL)
in the presence of two drops of triethylamine, hydrazine
hydrate (2 mmol, 0.15 mL, 80%) was added dropwise at
0 °C, and the reaction mixture was stirred for 1 h. The sep-
arated solid was collected by fltration and found to be a
mixture of two compounds 9 and 10 which was fractionally
recrystallized from ethanol to aford the azine 9. The insol-
uble part was recrystallized from ethanol/dioxane mixture
(1:1) to give compound 10.
5‑(1,3‑Diphenyl‑1H‑pyrazol‑4‑yl)‑7‑hy‑
droxy‑5H‑thiazolo[3,2‑a]pyrimidine‑6‑carbonitrile
(6)
Pale yellow crystals, mp. 292–294 °C, yield 70%. IR (KBr,
ν, cm−1): 3333 (OH), 2216 (C≡N), 1614 (C=N). 1H-NMR
(400 MHz, DMSO-d6): δ 13.33 (br.s, 1H, OH, exchangea-
ble), 9.21 (s, 1H, CH pyrazole), 7.95–7.93 (d, 2H, N-phenyl,
J=7.80 Hz), 7.72–7.70 (d, 1H, C4–H thiazole, J=6.68 Hz),
7.67–7.51 (m, 7H, phenyl), 7.58–7.56 (d, 1H, C5–H thia-
zole, J = 6.70 Hz), 7.49–7.46 (t, 1H, C4–H, N-phenyl,
J=7.40 Hz), 7.20 (s, 1H, C4–H pyrimidine). EIMS m/z (%):
397.29 (M+·, 16.48), 298.21 (17.43), 77.13 (100.00), 45.09
(54.21). Anal. Calcd. for C22H15N5OS (397.46): C, 66.48; H,
3.80; N, 17.62. Found: C, 66.19; H, 3.62; N, 17.58%.
(1E,2E)‑1,2‑Bis((1,3‑diphenyl‑1H‑pyrazol‑4‑yl)meth‑
ylene)hydrazine (9)
Pale yellow crystals, mp. 236–238 °C [Lit. [3], mp.
240–242 °C].
5‑Hydroxy‑1′,3′‑diphenyl‑1′H,2H‑[3,4′‑bipyrazole]‑4‑
carbonitrile (10)
Yellow crystals, mp. 306 °C (decomp.), yield 49%. IR
(KBr, ν, cm−1): 3441 (OH), 3185 (NH), 2212 (C≡N), 1618
1
(C=N). H-NMR (400 MHz, DMSO-d6): δ 10.67 (br.s,
Reaction with thiourea
2H, NH + OH, exchangeable), 9.23 (s, 1H, CH pyrazole),
7.97–7.95 (d, 2H, N-phenyl, J=7.84 Hz), 7.77–7.48 (m, 7H,
phenyl), 7.42–7.38 (t, 1H, C4–H, N-phenyl, J = 7.24 Hz).
EIMS m/z (%): 327.35 (M+·, 47.19), 317.70 (80.49), 251.12
(100), 183.21 (45.53), 138.35 (90.52), 55.39 (54.74). Anal.
Calcd. for C19H13N5O (327.35): C, 69.71; H, 4.00; N, 21.39.
Found: C, 69.52; H, 3.83; N, 21.38%.
A mixture of 4 (2 mmol, 0.7 g) in dry benzene (20 mL)
containing few drops of triethylamine and thiourea (2 mmol,
0.16 g) was heated under refux for 2 h. The solvent was
evaporated under vacuum. The residue was found to be a
mixture of compounds 7 (recrystallized from ethanol) and
8 (recrystallized from benzene).
3‑((1,3‑Diphenyl‑1H‑pyrazol‑4‑yl)methylene)‑2,4‑di‑
oxo‑6‑(2‑phenylhydrazinyl)‑3,4‑dihy‑
dro‑2H‑pyran‑5‑carbonitrile (11)
N‑Carbamothioyl‑2‑cyano‑3‑(1,3‑diphenyl‑1H‑pyra‑
zol‑4‑yl)acrylamide (7)
Canary yellow crystals, mp. 254–256 °C, yield 29%. IR
To a solution of 4 (2 mmol, 0.7 g) in dry dioxane (20 mL)
containing few drops of triethylamine, phenylhydrazine
(2 mmol, 0.21 g) was added, and the reaction mixture was
stirred at room temperature for 1 h. The reaction mixture
was poured onto ice/water and acidifed with dilute HCl.
The precipitated solid was fltered of and recrystallized
from benzene to give 11 as brown crystals, mp. 260–262 °C,
(KBr, ν, cm−1): 3321, 3290 (NH, NH2), 2218 (C≡N), 1709
1
(C=O), 1639 (C=N), 1222 (C=S). H-NMR (400 MHz,
DMSO-d6): δ 11.66 (br.s, 1H, NH, exchangeable), 9.25
(s, 1H, CH pyrazole), 8.13 (s, 1H, CH=), 7.98–7.96 (d,
2H, N-phenyl, J = 7.72 Hz), 7.72–7.54 (m, 7H, phenyl),
7.51–7.49 (t, 1H, C4–H, N-phenyl, J=7.60 Hz), 7.47 (br.s,
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Journal of the Iranian Chemical Society
yield 72%. IR (KBr, ν, cm−1): 3139 (NH), 2240 (C≡N), 1734
(d, 1H, C6–H hydroxyphenyl, J = 7.80 Hz), 7.71–7.69
(d, 2H, phenyl, J = 6.8 Hz), 7.64–7.56 (m, 5H, phenyl),
7.50–7.46 (t, 1H, N-phenyl, J=7.40 Hz), 7.37 (br.s, 1H, OH,
exchangeable), 7.05–7.01 (dd, 1H, C4–H hydroxyphenyl,
J=7.96 and 7.36 Hz), 6.95–6.93 (d, 1H, C7–H hydroxyphe-
nyl, J=7.92 Hz), 6.85–6.81 (dd, 1H, C5–H hydroxyphenyl,
J=8.00 and 7.44 Hz). EIMS m/z (%): 406.18 (M+·, 100.00),
343.20 (3.28), 298.14 (74.94), 80.14 (69.57). Anal. Calcd.
for C25H18N4O2 (406.45): C, 73.88; H, 4.46; N, 13.78.
Found: C, 73.61; H, 4.27; N, 13.81%.
1
(C=O lactone), 1687 (C=O ketone). H-NMR (400 MHz,
DMSO-d6): δ 13.64 (br.s, 1H, NHPh, exchangeable), 9.19
(s, 1H, CH pyrazole), 8.09 (s, 1H, CH=), 7.95–7.93 (d,
2H, N-phenyl, J = 7.84 Hz), 7.66–7.55 (m, 12H, phenyl),
7.49–7.47 (t, 1H, C4–H, N-phenyl, J=7.40 Hz), 6.38 (br.s,
1H, NH, exchangeable). EIMS m/z (%): 447.40 ([M+ −CN·],
24.65), 378.30 (75.56), 277.48 (87.46), 174.59 (64.96),
117.98 (100.00). Anal. Calcd. for C28H19N5O3 (473.49):
C, 71.03; H, 4.04; N, 14.79. Found: C, 70.96; H, 3.91; N,
14.81%.
2‑(1,3‑Diphenyl‑1H‑pyrazol‑4‑yl)‑4‑oxo‑4,5‑dihydro
benzo[b] [1,4] oxazepine‑3‑carbonitrile (14)
2‑(1H‑Benzo[d] [1,2,3] tria‑
zole‑1‑carbonyl)‑3‑(1,3‑diphenyl‑1H‑pyrazol‑4‑yl)
acrylonitrile (12)
Pale yellow crystals, mp. 290–292 °C, yield 45%. IR
(KBr, ν, cm−1): 3360 (NH), 2212 (C≡N), 1656 (C=O).
1H-NMR (400 MHz, DMSO-d6): δ 10.12 (br.s, 1H, NH,
exchangeable), 9.20 (s, 1H, CH pyrazole), 7.97–7.95 (d,
2H, N-phenyl, J=8.08 Hz), 7.88–7.86 (d, 1H, C9–H oxaz-
epine, J=7.92 Hz), 7.71–7.69 (d, 2H, phenyl, J=7.36 Hz),
7.64–7.54 (m, 5H, phenyl), 7.50–7.46 (t, 1H, N-phe-
nyl, J = 7.60 Hz), 7.05–7.01 (dd, 1H, C6–H oxazepine,
J=7.76 and 7.60 Hz), 6.95–6.93 (d, 1H, C8–H oxazepine,
J=8.00 Hz), 6.85–6.81 (dd, 1H, C7–H oxazepine, J=7.76
and 7.56 Hz). EIMS m/z (%): 404.03 (M+·, 4.98), 298.24
(76.34), 108.18 (8.98), 80.13 (67.15), 77.12 (100.00). Anal.
Calcd. for C25H16N4O2 (404.43): C, 74.25; H, 3.99; N,
13.85. Found: C, 74.01; H, 3.73; N, 13.80%.
A solution of 4 (2 mmol, 0.7 g) and benzotriazole (2 mmol,
0.25 g) in dry benzene (20 mL) was heated under refux
for 1 h in the presence of two drops of triethylamine. The
separated solid after cooling was collected and recrystallized
from ethanol to give 12 as yellow crystals, mp. 234–236 °C,
yield 71%. IR (KBr, ν, cm−1): 2220 (C≡N), 1686 (C=O).
1H-NMR (400 MHz, DMSO-d6): (E- and Z-isomers,
53:47%) δ: 9.41, 9.19 (s, 1H, CH pyrazole), 8.64, 8.09 (s,
1H, CH=), 8.33–8.30, 8.25–8.23 (d, 1H, C4–H benzotria-
zole, J=8.06 Hz), 8.01–7.99, 7.95–7.93 (d, 1H, C7–H ben-
zotriazole, J=7.90 Hz), 7.85–7.82 (dd, 1H, C5–H benzo-
triazole, J=8.04 and 7.36 Hz), 7.77–7.75 (d, 2H, N-phenyl,
J = 6.04 Hz), 7.68–7.46 (m, 8H, phenyl), 7.51–7.48 (dd,
1H, C6–H benzotriazole, J=7.88 and 7.37 Hz). EIMS m/z
(%): 416.79 (M+·, 10.56), 291.58 (44.76), 163.35 (34.74),
119.48 (37.66), 54.70 (100.00). Anal. Calcd. for C25H16N6O
(416.44): C, 72.10; H, 3.87; N, 20.18. Found: C, 71.97; H,
3.71; N, 20.15%.
4‑(1,3‑Diphenyl‑1H‑pyrazol‑4‑yl)‑2‑oxo‑2,5‑dihy‑
dro‑1H‑benzo[b] [1,4] diazepine‑3‑carbonitrile (15)
A solution of 4 (2 mmol, 0.7 g) and 2-aminoaniline (2 mmol,
0.22 g) in dry dioxane or benzene (20 mL) containing two
drops of triethylamine was refuxed for 1 h. The precipitated
solid was collected and recrystallized from dioxane to aford
15 as yellow crystals, mp. >360 °C, yield 83%. IR (KBr,
ν, cm−1): 3351 (NH), 2209 (C≡N), 1686 (C=O). 1H-NMR
(400 MHz, DMSO-d6): δ 9.91 (br.s, 1H, NHCO, exchange-
able), 9.21 (s, 1H, CH pyrazole), 8.22 (br.s, 1H, NH,
exchangeable), 7.96–7.94 (d, 2H, N-phenyl, J = 8.00 Hz),
7.69–7.67 (d, 2H, phenyl, J=7.28 Hz), 7.64–7.57 (m, 6H,
phenyl), 7.54–7.31 (m, 4H, Ar–H, diazepine). EIMS, m/z
(%): 403.53 (M+·, 21.63), 291.42 (64.22), 178.51 (62.62),
126.73 (100.00), 73.70 (44.85). Anal. Calcd. for C25H17N5O
(403.45): C, 74.43; H, 4.25; N, 17.36. Found: C, 74.19; H,
4.08; N, 17.39%.
Reaction with 2‑aminophenol
A solution of 4 (2 mmol, 0.7 g) and 2-aminophenol (2 mmol,
0.22 g) in dry dioxane or benzene (20 mL) containing two
drops of triethylamine was refuxed for 1 h. The precipitated
solid was collected and found to be a mixture of two com-
pounds 13 (recrystallized from benzene) and 14 (recrystal-
lized from ethanol).
2‑Cyano‑3‑(1,3‑diphe‑
nyl‑1H‑pyrazol‑4‑yl)‑N‑(2‑hydroxyphenyl)acryla‑
mide (13)
2‑(1,3‑Diphenyl‑1H‑pyrazol‑4‑yl)‑4‑oxo‑4,5‑dihydro
benzo[b] [1,4] thiazepine‑3‑carbonitrile (16)
Pale yellow crystals, mp. 270–272 °C, yield 55%. IR (KBr,
ν, cm−1): 3365 (OH), 3242 (NH), 2211 (C≡N), 1660 (C=O).
1H-NMR (400 MHz, DMSO-d6): δ 10.13 (br.s, 1H, NH,
exchangeable), 9.21 (s, 1H, CH pyrazole), 8.19 (s, 1H,
CH=), 7.98–7.96 (d, 2H, N-phenyl, J=8.00 Hz), 7.87–7.85
A solution of 4 (2 mmol, 0.7 g) and 2-aminothiophenol
(2 mmol, 0.26 g) in dry benzene (20 mL) containing two
1 3

Journal of the Iranian Chemical Society
drops of triethylamine was heated under refux for 1 h. The
precipitated solid upon cooling was collected and recrystal-
lized from benzene to furnish benzothiazepine 16 as beige
crystals, mp. 176–178 °C, yield 68%. IR (KBr, ν, cm−1):
3324 (NH), 2202 (C≡N), 1689 (C=O). 1H-NMR (400 MHz,
DMSO-d6): δ 10.69 (br.s, 1H, NH, exchangeable), 8.57 (s,
1H, CH pyrazole), 7.95–7.93 (d, 2H, N-phenyl, J=7.68 Hz),
7.88–7.86 (d, 2H, phenyl, J=7.84 Hz), 7.76–7.54 (m, 5H,
phenyl), 7.51–7.48 (t, 1H, N-phenyl, J=8.32 Hz), 7.37–7.26
(m, 4H, Ar–H, thiazepine). EIMS, m/z (%): 420.09 (M+·,
54.07), 383.79 (100.00), 327.44 (79.95), 271.94 (64.86),
138.89 (58.72), 76.00 (91.76). Anal. Calcd. for C25H16N4OS
(420.49): C, 71.41; H, 3.84; N, 13.32. Found: C, 71.26; H,
3.75; N, 13.21%.
J=5.76 Hz). EIMS, m/z (%): 358.28 (M+·, 100.00), 298.19
(56.63), 77.08 (65.88), 51.11 (10.60). Anal. Calcd. for
C21H18N4O2 (358.40): C, 70.38; H, 5.06; N, 15.63. Found:
C, 70.21; H, 4.89; N, 15.60%.
Reaction with thiosemicarbazide
A mixture of 4 (2 mmol, 0.7 g) in dry benzene (20 mL)
containing few drops of triethylamine and thiosemicarbazide
(2 mmol, 0.19 g) was heated under refux for 2 h. The solvent
was evaporated under vacuum. The residue was found to be
a mixture of compounds 19 (recrystallized from benzene)
and 20 (recrystallized from ethanol/dioxane mixture, 1:1).
2‑(2‑Cyano‑3‑(1,3‑diphenyl‑1H‑pyrazol‑4‑yl)acry‑
loyl)hydrazine‑1‑carbothioamide (19)
Reaction of acid chloride 4 with 1,2‑diaminoethane
or ethanolamine
Yellow crystal, mp. 220–222 °C, yield 44%. IR (KBr, ν,
cm−1): 3333, 3285, 3195 (NH, NH2), 2214 (C≡N), 1686
To a stirred solution of acid chloride 4 (2 mmol, 0.7 g) in
dry dioxane (10 mL) containing two drops of triethylamine,
1,2-diaminoethane (2 mmol, 0.14 mL) or 2-aminoethanol
(2 mmol, 0.13 mL) was added, and stirring was continued
for 10 min. The precipitated solid was collected and recrys-
tallized from the suitable solvents to aford compounds 17
and 18, respectively.
1
(C=O), 1229 (C=S). H-NMR (400 MHz, DMSO-d6): δ
10.49 (br.s, 1H, NHCO, exchangeable), 9.93 (br.s, 2H, NH2,
exchangeable), 9.19 (br.s, 1H, NH, exchangeable), 9.18 (s,
1H, CH pyrazole), 8.09 (s, 1H, CH=), 7.95–7.93 (d, 2H,
N-phenyl, J = 7.88 Hz), 7.67–7.37 (m, 8H, Ar–H). EIMS,
m/z (%): 388.04 (M+·, 13.20), 321.67 (100.00), 277.69
(77.43), 218.51 (54.06), 107.90 (39.32). Anal. Calcd. for
C20H16N6OS (388.45): C, 61.84; H, 4.15; N, 21.64. Found:
C, 61.72; H, 3.94; N, 21.67%.
2‑Cyano‑N‑(2‑(2‑cyano‑3‑(1,3‑diphenyl‑1H‑pyra‑
zol‑4‑yl)acrylamido)ethyl)‑3‑(1,3‑diphenyl‑1H‑pyra‑
zol‑4‑yl)acrylamide (17)
5‑(1,3‑Diphenyl‑1H‑pyrazol‑4‑yl)‑7‑oxo‑3‑thi‑
oxo‑2,3,4,7‑tetrahydro‑1H‑1,2,4‑triazepine‑6‑car‑
bonitrile (20)
Yellow crystals, mp. 292–294 °C (dioxane), yield 83%. IR
(KBr, ν, cm−1): 3319 (NH), 2206 (C≡N), 1672 (C=O). 1H-
NMR (400 MHz, DMSO-d6): δ 9.15 (s, 2H, CH pyrazole),
8.53 (br.s, 2H, 2NHCO, exchangeable), 8.06 (s, 2H, CH=),
7.95–7.93 (d, 4H, N-phenyl, J = 7.96 Hz), 7.65–7.52 (m,
14H, phenyl), 7.49–7.45 (t, 2H, N-phenyl, J = 7.12 Hz),
2.89 (s, 4H, 2CH2). EIMS, m/z (%): 403.01 (28.94), 258.49
(39.25), 217.38 (75.42), 149.55 (92.51), 55.63 (100.00).
Anal. Calcd. for C40H30N8O2 (654.73): C, 73.38; H, 4.62;
N, 17.11. Found: C, 73.29; H, 4.54; N, 17.09%.
Pale yellow crystals, mp. > 300 °C, yield 56%. IR (KBr,
ν, cm−1): 3216 (NH), 2222 (C≡N), 1684 (C=O), 1228
(C=S). 1H-NMR (400 MHz, DMSO-d6): δ 9.24 (br.s, 1H,
NHCO, exchangeable), 9.21 (s, 1H, CH pyrazole), 8.28
(br.s, 1H, NHCS, exchangeable), 7.96–7.94 (d, 2H, N-phe-
nyl, J = 7.56 Hz), 7.72 (s, 1H, HNNHCS, exchangeable),
7.70–7.56 (m, 7H, Ar–H), 7.50–7.46 (t, 1H, N-phenyl,
J=7.28 Hz). EIMS, m/z (%): 386.06 (M+·, 30.82), 322.07
(17.77), 197.73 (52.5), 90.25 (59.30), 42.23 (100.00). Anal.
Calcd. for C20H14N6OS (386.43): C, 62.16; H, 3.65; N,
21.75. Found: C, 62.01; H, 3.49; N, 21.70%.
2‑Cyano‑3‑(1,3‑diphe‑
nyl‑1H‑pyrazol‑4‑yl)‑N‑(2‑hydroxyethyl)acrylamide
(18)
Pale yellow crystals, mp. 204–206 °C (benzene), yield 65%.
IR (KBr, ν, cm−1): 3467 (OH), 3352 (NH), 2215 (C≡N),
Antiviral bioassays
1
1669 (C=O). H-NMR (400 MHz, DMSO-d6): δ 9.15 (s,
1H, CH pyrazole), 8.32 (br.s, 1H, NH, exchangeable), 8.07
(s, 1H, CH=), 7.95–7.93 (d, 2H, N-phenyl, J = 8.2 Hz),
7.67–7.55 (m, 7H, Ar–H), 7.49–7.45 (t, 1H, N-phenyl,
J=7.16 Hz), 4.75 (br.s, 1H, OH, exchangeable), 3.51–3.47
(t, 2H, CH2O, J = 5.76 Hz), 3.31–3.26 (t, 2H, CH2N,
Virus and cells
A MA 104 cell line was obtained from VACSERA, Cairo,
Egypt, and used for antiviral assays. Cells were propagated
in DMEM (Gibco, BR), supplemented with fetal bovine
1 3

Journal of the Iranian Chemical Society
carrying out the experimental part in its laboratory and Microbiology
Department, Faculty of Science, Ain Shams University, for helping in
carrying out the antiviral activity.
serum and antibiotic. Rotavirus RV SA-11 was kindly pro-
vided by Dr. Mohamed N. Shaheen, NRC, Giza, Egypt.
Cytotoxicity assay
Compliance with ethical standards
Different concentrations of the tested compounds were
prepared in DMEM containing 2% antibiotic and 2% FBS.
The cytotoxic efect of the extracts on MA 104 cell lines
was determined by using 3-(4,Z-5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) method [37].
This colorimetric assay is based on the conversion of the
yellow tetrazolium bromide (MTT) to a purple formazan
derivative by mitochondrial succinate dehydrogenase in
viable cells. Briefy, MA 104 cells were grown in 96-well
microtiter plates at concentration of 5×103 cells/well. After
24 h at 37 °C in a 5% CO2-humidifed atmosphere, the cul-
ture medium was removed and the diferent concentrations
of the tested extract were added to wells and incubated
with cell lines for an additional 48 h at 37 °C. The culture
medium containing the tested extracts was removed, and
100 μL of MTT solution (5 mg/mL) was added each well.
The plates were further incubated for 4 h at 37 °C to allow
MTT formazan formation. After the removal of MTT, 50 μL
dimethyl sulfoxide (DMSO) was added to each well. The
plates were further incubated for 30 min at 37 °C for solubi-
lization of formazan crystals. The absorbance was read on a
multi-well ELISA reader at a test wavelength of 540 nm. The
percentage of cytotoxicity was calculated as [A−B/A]×100,
where A and B are the mean of three optical densities of
untreated and treated cells, respectively. The 50% cytotoxic
concentration (CC50) was defned as the concentration that
can reduce the cell viability by 50%.
Conflict of interest All authors declare that they have no confict of
interest.
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