Synthesis, anti-HSV-1, and cytotoxic activities of some new pyrazole- and isoxazole-based heterocycles

Article abstract of DOI:10.1007/s00044-010-9420-4

Abstract Ethyl 2,4-dioxo-4-(p-chloro)phenylbutyrate 2 obtained by condensation of 4-chloroacetophenone with diethyl oxalate was converted to 5-(4-chlorophenyl)-1- phenyl-1H-pyrazole-3-carbohydrazide 5 and 5-(4-chlorophenyl) isoxazole-3-carbohydrazide 6 in

Full text of DOI:10.1007/s00044-010-9420-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:912–919
DOI 10.1007/s00044-010-9420-4
ORIGINAL RESEARCH
Synthesis, anti-HSV-1, and cytotoxic activities of some new
pyrazole- and isoxazole-based heterocycles
•
Kamal M. Dawood Hassan Abdel-Gawad
•
•
Hanan A. Mohamed Farid A. Badria
Received: 21 March 2010 / Accepted: 9 August 2010 / Published online: 4 September 2010
Ó Springer Science+Business Media, LLC 2010
Abstract Ethyl 2,4-dioxo-4-(p-chloro)phenylbutyrate 2
obtained by condensation of 4-chloroacetophenone with
diethyl oxalate was converted to 5-(4-chlorophenyl)-1-
phenyl-1H-pyrazole-3-carbohydrazide 5 and 5-(4-chloro-
Introduction
Herpes viruses are among the most prevalent infectious
agents known and have the ability to infect nearly every
animal species (Roizman and Pellett, 2001). There are
currently eight herpes viruses that infect humans including
herpes simplex 1 and 2 (HSV-1, HSV-2). HSV infection
may affect the face (orofacial herpes), genitalia (genital
herpes), hands (herpes whitlow), eye (herpes keratitis), or
the brain (herpes encephalitis) (Gupta et al., 2007; Kim-
berlin, 2007; McGeoch et al., 2006; Dickerson et al.,
2004). AIDS patients are prone to severe complications
from HSV infections. HSV-1 appeared to be particularly
damaging to the nervous system and increases the risk of
developing Alzheimer’s disease (Wozniak et al., 2009;
Itzhaki et al., 1997; Dobson and Itzhaki, 1999). In the past
few years, the interest in pyrazole and isoxazole derivatives
has increased due to their importance as intermediates in
the preparation of new biological materials. For example,
pyrazoles are known as potential HIV-1 inhibitors (Fahmy
et al., 2002), insecticides (Arrieta et al., 1998), fungicides
(Arrieta et al., 1998), and antiviral (Rashad et al., 2008),
and anticancer (Bouabdallah et al., 2006) agents. Further-
more, isoxazoles possess analgesic, anti-inflammatory
(Habeeb et al., 2001), and antimicrobial (Velikorodov and
Sukhenkol, 2003) activities. Recently, emphasis has been
given to synthesize pyrazoles and isoxazoles having novel
functional groups attached either to C-3 or C-5 of the azole
ring (Wei et al., 2006; Oh et al., 2007; Campagna et al.,
2004; Pommery et al., 2004; Abdel-Wahab et al., 2008;
Abdel-Wahab et al., 2009). Keeping the above results in
view, and in continuation of our research work on the
synthesis of biologically active heterocycles (Dawood,
2005; Dawood et al., 2005, 2006a, b; Abdel-Aziz et al.,
2009), we report herein the preparation of a new series of
phenyl)isoxazole-3-carbohydrazide
6 in good yields.
Treatment of 5 or 6 with phenylisothiocyanate and reaction
of the resulting thiosemicarbazide 7 or 8 with chloroacetic
acid and 4-fluorobenzaldehyde, phenacyl bromides gave
the corresponding 4-thiazolidinone 9 or 10 or 1,3-thiazoles
11 or 12, respectively. While the intramolecular cyclization
of 7 or 8 in concentrated sulfuric acid afforded 1,3,4-thi-
adiazoles 13 or 14. The reactivity of hydrazide 5 or 6
towards fluorinated aldehyde, hydrazonoyl chloride, and
b-ketoester was studied to give fluorinated hydrazones, bis-
hydrazones, and pyrazoles 15–23. The newly synthesized
compounds were screened for their antiviral activity, and
compound 19 reduced the number of viral plaques of
Herpes simplex type-1 (HSV-1) by 69%. The detailed
synthesis and spectroscopic and biological data are
reported.
Keywords Pyrazole ꢀ Isoxazole ꢀ Thiazole ꢀ Thiadiazole ꢀ
Anti-HSV-1 ꢀ Cytotoxic activities
K. M. Dawood
Chemistry Department, Faculty of Science,
Cairo University, Giza 12613, Egypt
H. Abdel-Gawad ꢀ H. A. Mohamed (&)
Applied Organic Chemistry Department,
National Research Centre, Cairo 12622, Egypt
e-mail: Hananm_2004@yahoo.com
F. A. Badria
Pharmacognosy Departments, Faculty of Pharmacy,
Mansoura University, Mansoura 35516, Egypt
123

Med Chem Res (2011) 20:912–919
913
pyrazole- and isoxazole-based heterocycles aiming at
studying their anti-HSV-1 and cytotoxic activities.
et al., 2007; Matysiak et al., 2006; Yar et al., 2006; Wang
et al., 2002). Thus, reaction of the acid hydrazides 5 and 6
with phenyl isothiocyanate in absolute ethanol at refluxing
temperature afforded the corresponding thiosemicarbazides
7 and 8, respectively in high yields. Refluxing a one-pot
mixture of equimolar amounts of the appropriate thiose-
micarbazides 7 or 8 with chloroacetic acid and 4-fluoro-
benzaldehyde in glacial acetic acid in the presence of fused
sodium acetate furnished the corresponding 5-(4-fluorob-
enzylidene)-4-oxo-3-phenylthiazolidines 9 and 10, respec-
tively, as shown in Scheme 2. Thiosemicarbazides 7 and 8
underwent a further smooth condensation with 4-chloroph-
enacyl bromide in refluxing ethanol to afford the corre-
sponding 1,3-thiazole derivatives 11 and 12, respectively, in
good yields (Scheme 2). Furthermore, an intramolecular
cyclization of the thiosemicarbazides 7 and 8 was achieved
when treated with concentrated sulfuric acid at room tem-
perature to afford the 1,3,4-thiadiazoles 13 and 14, respec-
tively (Scheme 2). The structure of the isolated products
7–14 was confirmed from their spectral and elemental
analyses as reported in ‘‘Experimental’’ section.
Results and discussion
Chemistry
Treatment of an equimolar amounts of 4-chloroacetophe-
none (1) with diethyl oxalate in sodium ethoxide and
anhydrous ethanol at room temperature afforded the Clai-
sen-condensation-type product; ethyl 4-(4-chlorophenyl)-
2,4-dioxobutanoate (2) which reacted subsequently with
phenyl hydrazine in glacial acetic acid at reflux condition
to afford 5-(4-chlorophenyl)-1-phenyl-1H-pyrazole-3-car-
boxylate 3 in good yield according to reported literature
(Wei et al., 2006). Treatment of ethyl pyrazole-3-ester 3
with excess hydrazine hydrate in refluxing ethanol gave the
new hydrazide derivative 5 in 80% yield (Scheme 1).
Compound 2 was converted into the isoxazole ester 4 in
75% yield upon its reaction with hydroxylamine hydro-
chloride in refluxing ethanol as reported in literature (Pei
and Wickham, 1993; Roy and Batra, 2003). Condensation
of the isoxazole-3-ester 4 with excess hydrazine hydrate in
refluxing ethanol furnished 5-(4-chlorophenyl)isoxazole-3-
carbohydrazide (6) in good yield. Structures of the hydra-
zides 5 and 6 were established on the basis of their spectral
data (IR, NMR, Mass) and elemental analyses. In the IR
spectrum of 5, a broad absorption bands at 3139 and
3318 cm-1 appeared due to the presence of NH and NH2
Treatment of the acid hydrazides 5 and 6 with 4-fluo-
robenzaldehyde in refluxing ethanol in the presence of
catalytic amount of glacial acetic acid afforded the corre-
sponding hydrazones 15 and 16 (Scheme 3). The 1H NMR
of 15 showed three singlet signals at d 7.14, 8.48 ppm
attributed to the pyrazole-4-CH, hydrazone-CH-, and 11.81
hydrazone-NH, respectively, in addition to an aromatic
multiplet in the region d 7.25–7.69. Treatment of iso-
xazole-3-carbohydrazide 6 with phthalic anhydride in gla-
cial acetic acid at refluxing conditions furnished the
corresponding phthalimide derivative 17 in 80% yield. The
IR spectrum of 17 showed an absorption band at
3131 cm-1 and two bands at 1739 and 1714 cm-1 due to
two C=O and NH functions, respectively, and its mass
1
functions, and its H NMR spectrum showed a singlet at d
4.52 and 9.64 due to the NH2 and NH protons, respectively.
In addition, the mass spectrum of hydrazide 5 showed a
peak at m/z 312 corresponding to its molecular ion.
In fact, acid hydrazides are considered as key interme-
diates for the synthesis of several heterocyclic systems (Zaki
Ph NH
HN NH
O
O
NH2
NH
S
Ar
O
O
OEt
OEt
O
O
PhNCS / EtOH
O
1. NaOEt / EtOH
2. 10% AcOH
N
+
reflux, 1h
X
Ar
CO2Et
Ar
X
N
Ar
CH3
7 8
,
2
5 6
,
1
ClCH2COOH
4-FC6H4CHO
AcONa
NH2OH. HCl
reflux, 2h
K2CO3
PhNHNH2
AcOH, reflux 2h
Conc. H2SO4
r.t. 24h
Ar`COCH2Br
EtOH,AcONa
reflux
AcOH
Ar
O
O
O
Ar
O
N
Ar
N
Ph
Ar
N
N
N
H
N
Ph
N
3
OEt
O
N
S
X
S
Ph
N
H
OEt
N
Ar
N
X
S
H
N
4
N
13,14
N
F
Ph
X
N2H4.
EtOH
N2H4.
EtOH
11,12
N
Ar'
O
9,10
11, 12 : Ar`= 4-ClC6H4
Ar
N
Ar
HN
NH2
HN
O
NH2
5,7,9,11,13 : X = NPh
6,8,10,12,14 : X = O
5~14 : Ar = 4-ClC6H4
O
N
O
N
Ph
6
5
1~6 : Ar = 4-Cl-C6H4
Scheme 1 Route to the starting materials
Scheme 2 Synthesis of 1,3-thiazoles and 1,3,4-thiadiazoles
123

914
Med Chem Res (2011) 20:912–919
bands at 1693 and 3249 and 3157 cm-1 in the IR spectrum
of the product 19 in addition to three singlet signals at d
2.38, 7.12, 10.26, and 10.54 in its 1H NMR spectrum due to
CH3, pyrazole-4-CH, and two NH supports the assigned
structure 19 for the reaction product.
CHO
Cl
F
F
HN
O
N
EtOH / AcOH,
reflux 6h
X
15 16
N
,
O
O
O
In addition, treatment of the hydrazides 5 and 6 with
ethyl 2-(2-(4-fluorophenyl)hydrazono)-3-oxobutanoate 21
in ethanol in the presence of few drops of acetic acid at
reflux temperature furnished the corresponding pyrazoles
22 and 23 in moderate yields (Scheme 3). The IR spectra
of the isolated products 22 and 23 showed in each case two
C=O absorptions near 1650 and 1720 cm-1 and one NH
O
O
N
N
H
Ar
AcOH, reflux, 6h
O
H
N
O
N
O
17
NH2
O
CH3
H
O
N
N
Cl
N
N
CH3
Ar
X
N
H
Ar
18
F
H
N
5, 6
N
X
Cl
N
EtOH, reflux 3-5h
19,20
absorption around 3150 cm-1. In addition, their H NMR
F
1
O
CH3
H
CH3
N
N
N
O
X
spectra exhibited two singlet signals near d 2.3 and 11
attributed to CH3 and NH protons, respectively.
N
N
EtO2C
21
F
O
HN
N
EtOH / AcOH,
reflux, 3h
Ar
22,23
Anti-herpes simplex-1 virus
F
15,19,22: X = NPh
16,20,23: X = O
The synthesized compounds were tested in vitro for their
antiviral activity using Herpes simplex type-1 (HSV-1) as a
viral model. The antiviral testing was performed using
Vero cells (cells isolated from the kidneys of green mon-
keys), HSV-1, and Aphidicolin as a positive control (Abou-
Karam and Shier, 1990; El-Subbagh et al., 2000). The viral
culture and test solutions were incubated for 66 h. At the
end of the incubation period, the percentage of reduction in
the number of virus plaques were recorded and compared
to the positive control.
Scheme 3 Reactivity of hydrazides towards different reagents
spectrum showed a peak at m/z 367 due to its molecular
1
ion. Its H NMR revealed two singlets at d 7.64 and 11.83
due to isoxazole-4-CH and NH protons, respectively.
When the hydrazides 5 and 6 were treated with the
hydrazonoyl chloride 18 in refluxing ethanol under neutral
condition they gave the corresponding bis-hydrazones 19
and 20, respectively, in good yields as shown in Scheme 3.
The presence of one carbonyl and two NH absorption
The results in Table 1 indicated that 1,3-thiazoles 9–12
and thiadiazoles 13 and 14 showed no antiviral activity, but
Table 1 Characteristic data of the synthesized compounds
Compd. no.
Mol. formula (M. Wt)
C%
H%
N%
Mp. (°C)
Yield
(%)
Calcd.
Found
Calcd.
Found
Calcd.
Found
5
C16H13ClN4O (312.754)
61.44
50.54
61.67
52.97
64.70
60.18
63.92
59.18
64.25
57.55
65.95
59.40
58.79
58.95
52.55
62.34
56.41
61.39
50.69
61.72
53.04
64.88
60.23
64.11
59.29
64.29
57.61
66.09
59.56
58.86
59.06
52.63
62.46
56.53
4.19
3.39
3.51
3.92
3.56
3.11
3.63
3.18
3.75
3.12
3.85
3.23
2.74
3.76
3.25
3.62
3.08
4.32
3.46
4.14
4.05
3.69
3.27
3.65
3.23
3.78
3.21
3.91
3.39
2.81
3.82
3.31
3.71
3.12
17.91
17.68
15.63
15.03
11.79
10.80
12.02
11.04
16.29
15.79
13.38
12.22
11.43
16.50
16.13
16.78
16.45
17.89
17.78
15.58
15.16
11.85
10.97
11.97
11.20
16.33
15.88
13.39
12.41
11.56
16.56
16.31
16.62
16.56
188–190
217–218
215–217
202–203
221–222
238–240
239–241
277–278
212–213
289–290
211–212
260–261
233–234
220–221
252–253
263–265
[300
80
80
83
79
50
55
63
70
45
50
70
65
80
85
80
40
48
6
C10H8ClN3O2 (237.642)
C23H18ClN5OS (447.94)
C17H13ClN4O2S (372.829)
C32H21ClFN5O2S (594.058)
C26H16ClFN4O3S (518.947)
C31H21Cl2N5OS (582.502)
C25H16Cl2N4O2S (507.391)
C23H16ClN5S (429.925)
C17H11ClN4OS (354.813)
C23H16ClFN4O (418.851)
C17H11ClFN3O2 (343.74)
C18H10ClN3O4 (367.743)
C25H19Cl2FN6O (509.362)
C19H14Cl2FN5O2 (434.251)
C26H18ClFN6O2 (500.912)
C20H13ClFN5O3 (425.8)
7
8
9
10
11
12
13
14
15
16
17
19
20
22
23
123

Med Chem Res (2011) 20:912–919
915
cytotoxicity which demonstrated a selective DNA binding
and consequently a safe antiviral activity.
Table 2 Results of in vitro anti-herpes simplex-1 virus (HSV-1) and
cytotoxicity
c
In conclusion, we described a facile synthesis a series of
new thiazoles, thiadiazoles, hydrazones, pyrazoles deriva-
tives by using pyrazole-3-carbohydrazide or isoxazole-3-
carbohydrazide as precursors. Hydrazide and hydrazone
derivatives showed a significant antiviral activity against
herpes simplex type-1 (HSV-1).
Compd. no.
% Reductiona
MACb
(CD50
)
5
65
0.1
0.1
–
0.02
0.01
0.02
0.02
0.03
0.02
0.02
0.02
0.03
0.02
0.02
0.01
0.01
0.02
0.02
0.02
0.02
0.01
6
43
7
None
19
8
0.1
–
9
None
None
None
None
None
None
67
10
–
11
–
Experimental
12
–
13
–
Chemistry
14
–
15
0.1
–
All melting points were measured using Electrothermal IA
9000 series digital melting point apparatus. Elemental
analytical data (in accord with the calculated values) were
obtained from the microanalytical center, Cairo University,
Giza, Egypt. The IR spectra (KBr) were recorded on a
Shimadzu CVT-04 spectrophotometer. The 1H NMR
spectra were recorded at 270 MHz on a Varian EM-360
spectrometer using TMS as an internal standard. Chemical
shifts (d) values are given in parts per million (ppm). The
mass spectra were performed using a Varian MAT CH-5
spectrometer (70 eV). Ethyl 4-(4-chlorophenyl)-2,4-di-
oxobutanoate 2 (Wei et al., 2006), ethyl 5-(4-chloro-
phenyl)-1-phenyl-1H-pyrazole-3-carboxylate 3 (Wei et al.,
2006), ethyl 5-(4-chlorophenyl)isoxazole-3-carboxylate 4
(Pei and Wickham, 1993; Roy and Batra, 2003), 1-(2-(4-
fluorophenyl)hydrazono)-1-chloropropan-2-one 18 (Biere
et al., 1982), and ethyl 2-(2-(4-fluorophenyl)hydrazono)-3-
oxobutanoate 21 (Mahesh et al., 1980) were prepared
according to the procedures reported in literature.
16
56
17
None
69
0.1
0.1
0.1
–
19
20
51
22
None
None
100
23
Aphidicolind
–
0.005
– Not detected
a
Percent (%) reduction in the number of viral plaques
Minimum antiviral concentration (in mg/ml)
b
c
Cytotoxicity (compound concentration caused 50% loss of the
monolayer present around the viral plaques) (in mg/ml)
d
Positive antiviral control
bis-hydrazones 19 and 20 and Schiff bases 15 and 16 are
the best among the tested compounds. Also hydrazides 5
and 6 show significant antiviral activities. It is appeared
from Table 1 that, compounds including pyrazole nucleus
show antiviral activity than iso-oxazole compounds.
Compound 19 showed the highest activity among the
tested compounds and reduced the number of HSV-1 pla-
ques by 69 while compounds 15, 5, 20, 16, 6, and 8 showed
67, 56, 51, 43, and 19% reduction. All other compounds
proved to lack any antiviral activity (Table 2).
Synthesis of carbohydrazides 5 and 6
Hydrazine hydrate (1.0 g, 20 mmol) was added to ethyl 5-
(4-chlorophenyl)-1-phenyl-1H-pyrazole-3-carboxylate
3
(3.27 g, 10 mmol) or to ethyl 5-(4-chlorophenyl)isoxazole-
3-carboxylate 4 (2.51 g, 10 mmol) in absolute ethanol
(40 ml). The reaction mixture was heated under reflux for
4 h and then cooled. The solid precipitate was filtered off
and recrystallized from ethanol.
Cytotoxicity assay
Cultured mammalian cells have been utilized to determine
the response of tumor cells isolated from cancer patients to
various chemotherapeutic agents (Kocan et al., 1985;
Hafford et al., 1991) as well as the antiviral potency of the
synthesized compounds using Herpes Simplex as a viral
model (El-Subbagh et al., 2000). CC50 (the compound
concentration that cause 50% reduction in cell density) was
recorded and compared to the positive control. Regarding
the cytotoxicity of the tested compounds, the results
obtained revealed that these compounds have weak
5-(4-Chlorophenyl)-1-phenyl-1H-pyrazole-3-
carbohydrazide (5)
IR (KBr) mmax/cm-1 1687 (C=O), 3139 (NH), 3318, 3254
1
(NH2); H NMR (DMSO-d6) d 4.52 (s, 2H, NH2), 6.95 (s,
1H, pyrazole-4-CH), 7.07–7.45 (m, 9H, ArH), 9.64 (s, 1H,
NH); MS m/z (%) 314 (M? ? 2, 19), 313 (M? ? 1, 15),
312 (M?, 62), 281 (100).
123

916
Med Chem Res (2011) 20:912–919
5-(4-Chlorophenyl)isoxazole-3-carbohydrazide (6)
7.24–7.70 (m, 18H, ArH), 8.48 (s, 1H, CH), 11.83 (s, H,
NH).
IR (KBr) mmax/cm-1 1666 (C=O), 3145 (NH), 3320, 3250
1
5-(4-Chlorophenyl)-N0-[5-(4-fluorobenzylidene)-4-oxo-3-
phenylthiazolidin-2-ylidene]-isoxazole-3-carbohydrazide
(10)
(NH2); H NMR (DMSO-d6) d 4.65 (s, 2H, NH2), 7.40 (s,
1H, isoxazole-4-CH), 7.61 (d, 2H, J = 8.1 Hz), 7.94 (d,
2H, J = 8.1 Hz), 10.11 (s, 1H, NH); MS m/z (%) 239
(M? ? 2, 60), 238 (M? ? 1, 22), 237 (M?, 100).
1
IR (KBr) mmax/cm-1 1626, 1672 (2C=O), 3176 (NH); H
Synthesis of thiosemicarbazides 7 and 8
NMR (DMSO-d6) d 7.31 (s, 1H, isoxazole-4-CH),
7.34–7.82 (m, 13H, ArH), 8.51 (s, 1H, CH), 12.38 (s, H,
NH); MS m/z (%) 520 (M? ? 2, 21), 519 (M? ? 1, 8),
518 (M?, 66), 237 (100).
To a solution of the appropriate carbohydrazide derivative
5 or 6 (5 mmol) in absolute ethanol (30 ml), phenyliso-
thiocyanate (0.67 g, 5 mmol) was added. The reaction
mixture was heated under reflux for 1 h and then left to
cool to room temperature. The formed solid was filtered off
and recrystallized from EtOH/DMF to give the thiose-
micarbazides 7 and 8, respectively.
Synthesis of thiazolidene derivatives 11 and 12
To a mixture of the appropriate thiosemicarbazide deriva-
tive 7 or 8 (1 mmol) and 4-chlorophenacyl bromide
(0.23 g, 1 mmol) in absolute ethanol (30 ml), anhydrous
sodium acetate (0.33 g, 4 mmol) was added. The mixture
was then heated at refluxing temperature for 6 h. The
reaction mixture was then cooled and the so-formed solid
product was filtered off, washed with water, dried, and
finally recrystallized from EtOH/DMF.
2-[5-(4-Chlorophenyl)-1-phenyl-1H-pyrazole-3-carbonyl]-
N-phenylhydrazine-carbothioamide (7)
IR (KBr) mmax/cm-1 1259 (C=S), 1644 (C=O), 3287–3139
1
(3NH); H NMR (DMSO-d6) d 6.40 (s, 1H, pyrazole-4-
CH), 7.08–7.60 (m, 14H, ArH), 9.45 (s, H, NH), 9.88 (s, H,
NH), 10.21 (s, H, NH); MS m/z (%) 449 (M? ? 2, 28), 448
(M? ? 1, 12), 447 (M?, 53), 281 (100).
5-(4-Chlorophenyl)-N0-(4-(4-chlorophenyl)-3-
phenylthiazol-2(3H)-ylidene)-1-phenyl-1H-pyrazole-3-
carbohydrazide (11)
2-[5-(4-Chlorophenyl)isoxazole-3-carbonyl]-N-
phenylhydrazinecarbothioamide (8)
1
IR (KBr) mmax/cm-1 1633 (C=O), 3136 (NH); H NMR
(DMSO-d6) d 6.96 (s, 1H, thiazole-5-CH), 6.99 (s, 1H,
pyrazole-4-CH), 6.22–7.57 (m, 18H, ArH), 10.76 (s, H,
NH); MS m/z % 520 (M? ? 2, 21), 582 (M? ? 1, 2), 581
(M?, 16), 185 (100).
IR (KBr) mmax/cm-1 1259 (C=S), 1644 (C=O), 3287–3139
(3NH); H NMR (DMSO-d6) d 7.32 (s, 1H, isoxazole-4-
CH), 7.62–7.92 (m, 9H, ArH), 9.53 (s, H, NH), 9.98 (s, H,
NH), 10.45 (s, H, NH); MS m/z (%) 374 (M? ? 2, 30), 373
(M? ? 1, 13), 372 (M?, 66), 237 (100).
1
5-(4-Chlorophenyl)-N0-[4-(4-chlorophenyl)-3-
phenylthiazol-2(3H)-ylidene]isoxazole-3-carbohydrazide
(12)
Synthesis of thiazolidinone derivatives 9 and 10
1
IR (KBr) mmax/cm-1 1620 (C=O), 3123 (NH); H NMR
To a mixture of the appropriate thiosemicarbazide deriva-
tive 7 or 8 (1 mmol), chloroacetic acid (0.1 g, 1 mmol) and
4-fluorobenzaldehyde (0.12 g, 1 mmol) in glacial acetic
acid (20 ml), anhydrous sodium acetate (0.33 g, 4 mmol)
was added. The reaction mixture was heated under reflux
for 6 h and then left to cool to room temperature. The
formed solid was filtered off, washed with water, dried, and
finally recrystallized from EtOH.
(DMSO-d6) d 7.03 (s, 1H, thiazole-5-CH), 7.34 (s, 1H,
isoxazole-4-CH), 7.37–8.03 (m, 13H, ArH), 11.00 (s, H,
NH); MS m/z % 520 (M? ? 2, 21), 507 (M? ? 1, 0.8),
506 (M?, 2), 338 (100).
Synthesis of 1,3,4-thiadiazole derivatives 13 and 14
A suspension of the appropriate thiosemicarbazide deriv-
ative 7 or 8 (1 mmol) in concentrated sulfuric acid (5 ml)
was stirred in an ice bath for 1 h. The reaction mixture was
then left to stir at room temperature overnight, then poured
into cooled water and neutralized with 2 N NaOH. The
solid formed was filtered off, dried, and recrystallized from
ethanol.
5-(4-Chlorophenyl)-N0-[5-(4-fluorobenzylidene)-4-oxo-3-
phenylthiazolidin-2-ylidene]-1-phenyl-1H-pyrazole-3-
carbohydrazide (9)
IR (KBr) mmax/cm-1 1661, 1693 (2C=O), 3197 (NH);
1H NMR (DMSO-d6) d 7.15 (s, 1H, pyrazole-4-CH),
123

Med Chem Res (2011) 20:912–919
917
5-[5-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-3-yl]-N-
phenyl-1,3,4-thiadiazol-2-amine (13)
isoxazole-4-CH), 7.67–8.03 (m, 8H, ArH), 11.83 (s, H,
NH); MS m/z % 369 (M? ? 2, 8), 368 (M? ? 1, 20), 367
(M?, 65), 206 (100).
1
IR (KBr) mmax/cm-1 3193 (NH); H NMR (DMSO-d6) d
6.97 (s, 1H, pyrazole-4-CH), 7.19–7.60 (m, 14H, ArH),
10.53 (s, H, NH); MS m/z % 430 (M? ? 1, 24), 429 (M?,
51), 428 (100).
Synthesis of the bis-hydrazones 19 and 20
A mixture of the appropriate carbohydrazide 5 or 6
(1 mmol) and the hydrazonoyl chloride 18 (0.21 g,
1 mmol) in absolute ethanol (30 ml) was refluxed for 4 h.
The formed solid was filtered off, washed with ethanol,
dried, and then recrystallized from EtOH/DMF (2:1).
5-[5-(4-Chlorophenyl)isoxazol-3-yl]-N-phenyl-1,3,4-
thiadiazol-2-amine (14)
1
IR (KBr) mmax/cm-1 3188 (NH); H NMR (DMSO-d6) d
7.12 (s, 1H, isoxazole-H), 7.23–7.54 (m, 9H, Ar–H), 11.33
(s, H, NH); MS m/z % 356 (M? ? 2, 10), 355 (M? ? 1,
8), 354 (M?, 20), 149 (100).
2-{2-[5-(4-Chlorophenyl)-1-phenyl-1H-pyrazole-3-
carbonyl]hydrazono}-N0-(4-fluoro-
phenyl)propanehydrazonoyl chloride (19)
1
IR (KBr) mmax/cm-1 1693 (C=O), 3249, 3157 (2NH); H
Synthesis of 4-fluorobenzylidene-hydrazides 15 and 16
NMR (DMSO-d6) d 2.38 (s, 3H, CH3), 7.12 (s, 1H, pyra-
zole-4-CH), 7.18–7.49 (m, 13H, ArH), 10.26 (s, 1H, NH),
10.54 (s, 1H, NH); MS m/z % 510 (M? ? 2, 5), 509
(M? ? 1, 4), 508 (M?, 3), 279 (100).
A mixture of the appropriate carbohydrazide 5 or 6
(1 mmol) and 4-fluorobenzaldehyde (0.12 g, 1 mmol) in
absolute ethanol (30 ml) in the presence of few drops of
glacial acetic acid was refluxed for 4 h. The solid product
was filtered off, washed with ethanol, dried, and recrys-
tallized from EtOH/DMF (3:1) to give the corresponding
benzylidene derivatives 15 and 16.
2-{2-[5-(4-Chlorophenyl)isoxazole-3-
carbonyl]hydrazono}-N0-(4-fluorophenyl)-
propanehydrazonoyl chloride (20)
1
IR (KBr) mmax/cm-1 1700 (C=O), 3311, 3138 (2NH); H
5-(4-Chlorophenyl)-N0-(4-fluorobenzylidene)-1-phenyl-1H-
pyrazole-3-carbohydrazide (15)
NMR (DMSO-d6) d 2.32 (s, 3H, CH3),7.13 (s, 1H, isox-
azole-4-CH), 7.18–8.08 (m, 8H, ArH), 11.13 (s, 1H, NH),
11.69 (s, 1H, NH); MS m/z % 435 (M? ? 2, 21), 434
(M? ? 1, 25), 433 (M?, 40), 397 (100).
1
IR (KBr) mmax/cm-1 1662 (C=O), 3199 (NH); H NMR
(DMSO-d6) d 7.14 (s, 1H, pyrazole-4-CH), 7.25–7.69 (m,
13H, ArH), 8.48 (s, 1H, CH), 11.81 (s, H, NH); MS m/z% 420
(M? ? 2, 12), 419 (M? ? 1, 18), 418 (M?, 20), 299 (100).
Synthesis of pyrazolone derivatives 22 and 23
5-(4-Chlorophenyl)-N0-(4-fluorobenzylidene)isoxazole-3-
carbohydrazide (16)
To a mixture of the appropriate carbohydrazide 5 or 6
(1 mmol) in ethanol (20 ml), ethyl 2-(2-(4-fluoro-
phenyl)hydrazono)-3-oxobutanoate 21 (0.25 g 1 mmol),
few drops of glacial acetic acid were added. The reaction
mixture was refluxed for 5 h, and the reaction solvent was
then evaporated to its half then the concentrated reaction
mixture was kept at room temperature overnight. The solid
that separated was filtered off, washed with cold ethanol,
dried, and recrystallized from ethanol.
1
IR (KBr) mmax/cm-1 1679 (C=O), 3173 (NH); H NMR
(DMSO-d6) d 7.35 (s, 1H, isoxazole-4-CH), 7.43–7.64 (m,
8H, ArH), 8.89 (s, 1H, CH), 11.56 (s, H, NH); MS m/z %
345 (M? ? 2, 10), 344 (M? ? 1, 8), 343 (M?, 43), 149
(100).
5-(4-Chlorophenyl)-N-(1,3-dioxoisoindolin-2-yl)isoxazole-
3-carboxamide (17)
1-[5-(4-Chlorophenyl)-1-phenyl-1H-pyrazole-3-carbonyl]-
4-[2-(4-fluorophenyl)-hydrazono]-3-methyl-1H-pyrazol-
5(4H)-one (22)
A mixture of the carbohydrazide derivative 6 (0.24 g,
1 mmol) and phthalic anhydride (0.15 g, 1 mmol) in gla-
cial acetic acid (20 ml) was refluxed for 8 h. The precipi-
tate so-formed was filtered off, washed with ethanol, dried,
and recrystallized from acetic acid/H2O (3: 1 v/v) to give
17 in 80% yield. IR (KBr) mmax/cm-1 1739, 1714, 1626
1
IR (KBr) mmax/cm-1 1727, 1657 (2C=O), 3176 (NH); H
NMR (DMSO-d6) d 2.23 (s, 3H, CH3), 7.08 (s, 1H, pyra-
zole-4-CH), 7.11–7.73 (m, 13H, ArH), 10.30 (s, H, NH);
MS m/z % 502 (M? ? 2, 2), 501 (M? ? 1, 12), 500 (M?,
18), 280 (100).
1
(3C=O), 3131 (NH); H NMR (DMSO-d6) d 7.64 (s, 1H,
123

918
Med Chem Res (2011) 20:912–919
1-[5-(4-Chlorophenyl)isoxazole-3-carbonyl]-4-[2-(4-
fluorophenyl)hydrazono]-3-methyl-1H-pyrazol-5(4H)-one
(23)
NJ) by suspending Vero cells in medium following tryp-
sin–EDTA treatment, counting the suspension with a
hemocytometer, diluting in medium containing 10% calf
serum to 2 9 104 cells per 200 ml culture, aliquoting into
each well of a tray and culturing until confluent.
1
IR (KBr) mmax/cm-1 1737, 1626 (2C=O), 3149 (NH); H
NMR (DMSO-d6) d 2.49 (s, 3H, CH3), 7.55 (s, 1H, isox-
azole-4-CH), 7.64–8.01 (m, 8H, ArH), 11.07 (s, H, NH);
MS m/z % 427 (M? ? 2, 0.8), 426 (M? ? 1, 0.3), 425
(M?, 1.6), 294 (100).
Procedure
Microtiter trays with confluent monolayer cultures of Vero
cells were inverted, the medium shaken out, and replaced
with serial dilutions of sterile compounds in triplicate in
100 ll medium followed by titered virus in 100 ll medium
containing 10% (v/v) calf serum in each well. In each tray,
the last row of wells was reserved for controls that were not
treated with compounds or not treated with virus. The trays
were cultured for 66 h. The trays were inverted onto a pad
of paper towels, the remaining cells rinsed carefully with
medium, and fixed with 3.7% (v/v) formaldehyde in saline
for at least 20 min. The fixed cells were rinsed with water,
and examined visually. Antiviral activity is identified as
confluent, relatively unaltered monolayers of stained Vero
cells treated with HSV-1. Cytotoxicity was estimated as the
concentration that caused approximately 50% loss of the
monolayer present around the plaques caused by HSV-1.
Antiviral and cytotoxicity assays
Sample preparation
Samples were prepared for assay by dissolving in DMSO
(50 ml) and diluting aliquots into sterile culture medium at
0.4 mg/ml. These solutions were diluted to 0.02 mg/ml in
sterile medium and the two solutions used as stocks to test
samples at 100, 50, 20, 10, 5, 2, and 1 mg/ml in triplicate in
the wells of microliter plates.
Virus used in assay
The tested compounds were tested for antiviral activity
against Herpes simplex type 1 (HSV-1) grown on Vero
African green monkey kidney cells.
References
Culture
Abdel-Aziz HA, Mekawey AAI, Dawood KM (2009) Convenient
synthesis and antimicrobial evaluation of some novel
2-substituted-3-methylbenzofuran derivatives. Eur J Med Chem
44:3637–3644
Abdel-Wahab BF, Abdel-Aziz HA, Ahmed EM (2008) Convenient
synthesis and antimicrobial activity of new 3-substituted
5-(benzofuran-2-yl)-pyrazole derivatives. Arch Pharm 341:
734–739
Abdel-Wahab BF, Abdel-Aziz HA, Ahmed EM (2009) Synthesis and
antimicrobial evaluation of some new 1,3-thiazole; 1,3,4-thiadi-
azole; 1,2,4-triazole and [1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
derivatives including 5-(benzofuran-2-yl)-1-phenyl-pyrazole
moiety. Monatsh Chem 140:601–605
Abou-Karam M, Shier WT (1990) A simplified plaque reduction
assay for antiviral agents from plants. Demonstration of frequent
occurrence of antiviral activity in higher plants. J Nat Prod
53:340–345
Herpes simplex type I (HSV-I) was the gift of Dr. R.G.
Hughes, Roswell Park Memorial Institute, Buffalo, NY.
Virus stocks were prepared as aliquots of culture medium
from Vero cells infected at multiplicity of 1 virion per 10
cells and cultured 3 days. They were stored at -80°C.
Working stocks were prepared by titering virus by serial
dilution in culture medium and assayed in triplicate on
Vero monolayers in the wells of microtiter trays. Virus
suspensions that gave about 30 plaques per well were
stored at 4°C until used. Vero African green monkey kid-
ney cells were purchased from Viromed Laboratories,
Minnetonka, MN, and grown in Dulbecco’s modified
Eagle’s medium supplemented with 10% (v/v) calf serum
(Hyclone Laboratories, Ogden, UT), 60 mg/ml Penicillin G
and 100 mg/ml streptomycin sulfate maintained at 37°C in
a humidified atmosphere containing about 15% (v/v) CO2
in air. All medium components were obtained from Sigma
Chemical Co., St Louis, MO, unless otherwise indicated.
Vero stocks were maintained at 34°C in culture flasks filled
with medium supplemented with 1% (v/v) calf serum.
Subcultures for virus titration or antiviral screening were
grown in the wells of microtiter trays (Falcon Microtest III
96-wells trays, Becton–Dickinson Labware, Lincolin Park,
´
´
Arrieta A, Carrillo JR, Cossıo FP, Ortiz AD, Gomez-Escalonilla MJ,
de la Hoz A, Langa F, Moreno A (1998) Efficient tautomeriza-
tion hydrazone-azomethine imine under microwave irradiation.
Synthesis of [4,30] and [5,30]bipyrazoles. Tetrahedron 54:
13167–13180
Biere H, Schroeder E, Ahrens H, Kapp JF (1982) Non-steroidal
antiinflammatory agents. 5. Antiinflammatory pyrazole acetic
acids. I. Eur J Med Chem 17:27–34
Bouabdallah I, M’barek LA, Zyad A, Ramadan A, Zidane I, Melhaoui
A (2006) Anticancer effect of three pyrazole derivatives. Nat
Prod Res 20:1024–1030
Campagna F, Palluotto F, Carotti A, Maciocco E (2004) Synthesis,
central and peripheral benzodiazepine receptor affinity of
123

Med Chem Res (2011) 20:912–919
919
pyrazole and pyrazole-containing polycyclic derivatives. Farma-
co 59:849–856
Dawood KM (2005) Synthesis of spiro-pyrazole-3, 30-thiopyrano
[2,3-b]pyridines and azolo[a]pyrido[20,30:5,6]thiopyrano[3,4-
d]pyrimidines as new ring systems with antifungal and antibac-
terial activities. J Heterocycl Chem 42:221–225
Dawood KM, Farag AM, Abdel-Aziz HA (2005) Synthesis and
antimicrobial evaluation of some 1,2,4-triazole, 1,3,4-oxa(thia)
diazole, and 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine derivatives.
Heteroat Chem 16:621–627
Matysiak J, Nasulewicz A, Pelczynska M, Switalska M, Jaroszewicz
I, Opolski A (2006) Synthesis and antiproliferative activity of
some 5-substituted 2-(2,4-dihydroxyphenyl)-1,3,4-thiadiazoles.
Eur J Med Chem 41:475–482
McGeoch DJ, Rixon FJ, Davison AJ (2006) Topics in herpesvirus
genomics and evolution. Virus Res 117:90–104
Oh LM, Wang H, Shilcrat SC, Herrmann RE, Patience DB, Spoors P,
Sisko
J (2007) Development of a scalable synthesis of
GSK183390A, a PPAR a/c agonist. Org Process Res Dev 11:
1032–1042
Dawood KM, Abdel-Gawad H, Ellithey M, Mohamed HA, Hegazi B
(2006a) Synthesis, anticonvulsant and anti-inflammatory activ-
ities of some new benzofuran-based heterocycles. Arch Pharm
339:133–140
Dawood KM, Abdel-Gawad H, Rageb EA, Ellithey M, Mohamed HA
(2006b) Synthesis, anticonvulsant, and anti-inflammatory eval-
uation of some new benzotriazole and benzofuran-based hetero-
cycles. Bioorg Med Chem 14:3672–3680
Dickerson FB, Boronow JJ, Stallings C (2004) Infection with herpes
simplex virus type 1 is associated with cognitive deficits in
bipolar disorder. Biol Psychiatry 55:588–593
Dobson CB, Itzhaki RF (1999) Herpes simplex virus type 1 and
Alzheimer’s disease. Neurobiol Aging 20:457–465
Pei Y, Wickham BOS (1993) Regioselective syntheses of
3-aminomethyl-5-substituted isoxazoles: a facile and chemose-
lective reduction of azide to amine by sodium borohydride using
1,3-propanedithiol as a catalyst. Tetrahedron Lett 34:7509–7512
Pommery N, Taverne T, Telliez A, Goossens L, Charlier C, Pommery
´
J, Goossens JF, Houssin R, Durant F, Henichart JP (2004) New
COX-2/5-LOX inhibitors: apoptosis-inducing agents potentially
useful in prostate cancer chemotherapy. J Med Chem 47:
6195–6206
Rashad AE, Hegab MI, Abdel-Megeid RE, Micky JA, Abdel-Megeid
FME (2008) Synthesis and antiviral evaluation of some new
pyrazole and fused pyrazolopyrimidine derivatives. Biorg Med
Chem 16:7102–7106
El-Subbagh HI, Abu-Zaid SM, Mahran MA, Badria FA, Al-obaid M
(2000) Synthesis and biological evaluation of certain a,b-
unsaturated ketones and their corresponding fused pyridines as
antiviral and cytotoxic agents. J Med Chem 43:2915–2921
Fahmy HTY, Rostom SAF, Bekhit AA (2002) Synthesis and
antitumor evaluation of new polysubstituted thiazole and derived
thiazolo[4,5-d]pyrimidine systems. Arch Pharm 335:213–222
Gupta R, Warren T, Wald A (2007) Genital herpes. Lancet 370:
2127–2137
Habeeb GA, Rao PNP, Kanus EE (2001) Design and synthesis of 4,5-
diphenyl-4-isoxazolines: novel inhibitors of cyclooxygenase-2
with analgesic and antiinflammatory activity. J Med Chem
44:2921–2927
Roizman B, Pellett PE (2001) In: Knipe DM, Howley PM (eds) Fields
virology, vol 2. Lippincott Williams and Wilkins, Philadelphia,
PA, p 2381
Roy AK, Batra S (2003) Facile Baylis–Hillman reaction of substituted
3-isoxazolecarbaldehydes: the impact of a proximal heteroatom
within a heterocycle on the acceleration of the reaction.
Synthesis 15:2325–2330
Velikorodov AV, Sukhenkol LT (2003) Synthesis and antimicrobial
activity of 3,5-disubstituted isoxazolines and isoxazoles with
carbamate groups. Pharm Chem J 37:22–24
Wang X, Li Z, Da Y, Ma Y (2002) Synthesis of 5,50-diaryloxymethyl-
2,20-(1,4-phenylenedioxydiacetylamido)-bis(1,3,4-thiadiazoles).
Synth Commun 32:1121–1127
Hafford LD, Badria FA, Abou-Karam M, Sheir WT, Rogers RD
(1991) Preparation, characterization, and antiviral activity of
microbial metabolites of stemodin. J Nat Prod 54:1543–1552
Itzhaki RF, Lin WR, Shang D, Wilcock GK, Faragher B, Jamieson
GA (1997) Herpes simplex virus type 1 in brain and risk of
Alzheimer’s disease. Lancet 349:241–244
Wei F, Zhao BX, Huang B, Zhang L, Sun CH, Dong WL, Shin DS,
Miao JY (2006) Design, synthesis, and preliminary biological
evaluation of novel ethyl 1-(20-hydroxy-30-aroxypropyl)-3-aryl-
1H-pyrazole-5-carboxylate. Bioorg Med Chem Lett 16:
6342–6347
Wozniak MA, Mee AP, Itzhaki RF (2009) Herpes simplex virus type
1 DNA is located within Alzheimer’s disease amyloid plaques.
J Pathol 217:131–138
Kimberlin DW (2007) Herpes simplex virus infections of the
newborn. Semin Perinatol 31:19–25
Kocan RM, Sabo KM, Landolt ML (1985) Cytotoxicity/genotoxicity:
the application of cell culture techniques to the measurement of
marine sediment pollution. Aquatic Toxicol 6:165–177
Mahesh VK, Goyal RN, Gupta R, Sharma R (1980) Synthesis of some
Yar MS, Siddiqui AA, Ali MA (2006) Synthesis and evaluation of
phenoxy acetic acid derivatives as anti-mycobacterial agents.
Bioorg Med Chem Lett 16:4571–4574
Zaki MEA, Yousef EAA, Hassanien A-ZA (2007) Fatty acid
hydrazides in heterocyclic synthesis: synthesis of 1,2-diazepine
and pyridazine derivatives. Heteroat Chem 18:259–264
pyrimidinylpyrazoles
J Indian Chem Soc 57:1037–1038
and
pyrimidinyl-2-pyrazolin-5-ones.
123