Design, synthesis and preliminary antiviral screening of new N-phenylpyrazole and dihydroisoxazole derivatives

Article abstract of DOI:10.1007/s00044-009-9248-y

A new series of N-phenylpyrazoles and dihydroisoxazles was synthesized starting from α,β-unsaturated ketones in basic media using phenyl hydrazine and hydroxylamine HCl, respectively. Antiviral evaluation of the target compounds revealed that the dihydroi

Full text of DOI:10.1007/s00044-009-9248-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:1025–1035
DOI 10.1007/s00044-009-9248-y
ORIGINAL RESEARCH
Design, synthesis and preliminary antiviral screening of
new N-phenylpyrazole and dihydroisoxazole derivatives
•
Adel A. Rashad Osama I. El-Sabbagh
•
•
Mohamed M. Baraka Samy M. Ibrahim
•
•
Christophe Pannecouque Graciela Andrei
•
•
•
Robert Snoeck Jan Balzarini Ahmed Mostafa
Received: 21 March 2009 / Accepted: 26 August 2009 / Published online: 14 October 2009
¨
ꢀ Birkhauser Boston 2009
Abstract A new series of N-phenylpyrazoles and dihydroisoxazles was synthe-
sized starting from a,b-unsaturated ketones in basic media using phenyl hydrazine
and hydroxylamine HCl, respectively. Antiviral evaluation of the target compounds
revealed that the dihydroisoxazole derivatives have promising antiviral activity
against hepatitis A virus and herpes simplex virus type 1.
Keywords Synthesis ꢀ Antiviral activity ꢀ N-phenylpyrazoles ꢀ Dihydroisoxazole ꢀ
Herpes simplex virus type 1 ꢀ Hepatitis A virus
Introduction
Herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2)
belong to the herpes virus family. HSV-1 infects mucocutaneous epithelial cells and
establishes latency in sensory ganglions (Whitley and Roizman, 2001). HSV-2
causes genital lesions. Much research effort has been focused on HSV-1 and HSV-2,
as these viruses have a high incidence rate and a high prevalence (Gudmundsson
et al., 2008). Vesicular stomatitis virus (VSV) is a single-strand RNA virus of the
Rhabdoviridae family. VSV has been isolated from a variety of animals and causes
nonfatal disease of significant economic importance in cattle and swine (Rose and
A. A. Rashad (&) ꢀ O. I. El-Sabbagh ꢀ M. M. Baraka ꢀ S. M. Ibrahim
Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, 44511 Zagazig,
Egypt
e-mail: adelrashadzu@yahoo.com
C. Pannecouque ꢀ G. Andrei ꢀ R. Snoeck ꢀ J. Balzarini
Rega Institute for Medical Research, Faculty of Medicine, K.U. Leuven, Leuven, Belgium
A. Mostafa
Environmental Virology Laboratory, Department of Water Pollution, National Research Centre,
Dokki, Egypt

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Med Chem Res (2010) 19:1025–1035
O
N
O
N
N
OH
O
O
O
Cl
(B)
(A)
Fig. 1 Antiviral compounds used as models for the synthesis of the target compounds
R
R
N
N
O
N
O
O
R
O
O
α,β unsaturated ketone
N -phenyl4,5-dihydro-1H-pyrazoles
4-benzyloxyphenyl-4,5-dihydroisoxazoles
Fig. 2 Synthesis of the target compounds
Whitt, 2007). Hepatitis A is an acute infectious disease of the liver caused by
hepatitis A virus (HAV), which is most commonly transmitted by the fecal-oral
route via contaminated food or drinking water. Every year, approximately
10 million people worldwide are infected with the virus (Vento, 2000).
In the search for antiviral agents, several nonnucleoside derivatives have been
developed (Pauwels, 2004; Rashad et al., 2008), such as 5-(4-chlorophenyl)-3-(1,3-
diphenylpyrazol-4-yl)-methylene-2(3H)-furanone (A), which exerts high antiviral
activities toward HAV (Hashem et al., 2007), while 3-(3-(benzyloxy)phenyl)isox-
azole-5-carboxylic acid (B) shows HIV-1 integrase (IN) inhibitory effect and good
antiviral activity (Zeng et al., 2008) (Fig. 1).
Therefore, in our quest for synthesis of new antiviral agents with an efficacy
similar to or greater than that of the other reported compounds, a series of 4,5-
dihydro-1H-pyrazoles was synthesized bearing N-phenylpyrazole moiety similar to
that of compound A. Moreover, a series of 4-benzyloxyphenyl-4,5-dihydroisoxaz-
oles was synthesized based on the aforementioned structurally similar compound
(B), with the aim to explore their antiviral activity (Fig. 2).
Experimental
Melting points were determined with a Gallenkamp melting-point apparatus and are
uncorrected. IR spectra (KBr, cm-1) were recorded on a Testscan Shimadzu FT
8000 spectrometer. 1H NMR (200- and 500-MHz) and 13CNMR (125-MHz) spectra
were recorded on a Bruker AC 200-MHz and an Avance 500-MHz spectrometer
with DMSO-d6 or CDCl3 as a solvent and tetramethylsilane (TMS) as an internal

Med Chem Res (2010) 19:1025–1035
1027
standard (chemical shift in d, ppm). Mass spectra were determined using a GCMS-
QP1000EX Shimadzu spectrometer with an ionization energy of 70 eV. Elemental
analyses were determined using Heraeus and Vario EL-III (Elemntar; Germany)
CHNS analyzers at the Microanalytical Center, Faculty of Science, University of
Cairo. All results of elemental analyses corresponded to the calculated values within
experimental error. TLC was performed on silica gel G (Fluka) and spots were
visualized by iodine vapors or irradiation with UV light (254 nm). All chemicals
were purchased from Sigma Aldrich.
3-(4-(Benzyloxy)phenyl)-1-(4-substituted phenyl)prop-2-en-1-ones (1–4)
The key chalcone intermediates (1–4) were synthesized through base-catalyzed
Claisen–Schmidt condensation of equimolar amounts of acetophenone and its
derivatives with 4-benzyloxybenzaldehyde by stirring the reactants in an aqueous
ethanolic solution containing 20% NaOH at room temperature for 24 h in
accordance with the method described in the literature (Li et al., 2005; Robinson
et al., 2005).
General procedure for synthesis of 5-(4-(benzyloxy)phenyl)-3-(4-substituted
phenyl)-1-phenyl-4,5-dihydro-1H-pyrazoles (5–8)
A mixture of the corresponding chalcone (1–4; 0.01 mol), phenyl hydrazine
(0.01 mol), and NaOH (0.025 mol) was refluxed in ethanol for 4 h. The mixture was
cooled and the precipitated product was filtered, washed, and crystallized from
dioxane/H2O
5-(4-(Benzyloxy)phenyl)-1,3-diphenyl-4,5-dihydro-1H-pyrazole (5)
1
Yield, 50%; m.p., 135–136ꢁC. H NMR (500 MHz, DMSO-d6): d = 3.05–3.10
(two d, J = 6.3, 6.35 Hz, 1H, C4–H of pyrazole), 3.85–3.91 (two d, J = 12.15,
12.15 Hz, 1H, C4–H of pyrazole), 5.05 (s, 2H, benzylic H), 5.41–5.45 (two d,
J = 6.3, 6.25 Hz, 1H, C5–H of pyrazole), 6.70–7.76 (m, 19H, ArH) ppm. 13C NMR
(DMSO): d = 157.61 (C–O), 147.14 (C = N, pyrazole), 144.24 (N–C) 136.99–
112.99 (phenyl-C), 69.19 (OCH2), 62.63 (CH of pyrazole), 42.95(CH2 of pyrazole)
ppm. Analysis for C28H24N2O. Calcd: C, 83.14; H, 5.98; N, 6.93. Found: C, 83.29;
H, 5.81; N, 7.00.
5-(4-(Benzyloxy)phenyl)-3-(4-bromophenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (6)
Yield, 65%; m.p., 158–159ꢁC. 1H NMR (500 MHz,DMSO-d6): d = 3.08–3.13 (two
d, 1H, J = 7.2, 7.25 Hz, C4–H of pyrazole), 3.77–3.83 (two d, J = 12.35, 12.35 Hz,
1H, C4–H of pyrazole), 5.05 (s, 2H, benzylic H), 5.25–5.29 (two d, J = 7.2,
7.15 Hz, 1H, C5–H of pyrazole), 6.84–7.61 (m, 18H, ArH) ppm. EI (70 eV) m/z
(%): (M??1, 485, 17.4), (M?, 484, 70.6), 391 (15.6), 298 (13.6), 91 (100.0).
Analysis for C28H23BrN2O. Calcd: C, 69.57; H, 4.80; N, 5.80. Found: C, 69.74; H,
4.82; N, 5.58.

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5-(4-(Benzyloxy)phenyl)-3-(4-chlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (7)
1
Yield, 59%; m.p., 148–149ꢁC. H NMR (500 MHz,CDCl3): d = 3.08–3.13 (two d,
J = 7.2, 7.2 Hz, 1H, C4–H of pyrazole), 3.77–3.83 (two d, J = 12.3, 12.35 Hz, 1H,
C4–H of pyrazole), 5.05 (s, 2H, benzylic H), 5.26–5.30 (two d, J = 7.2, 7.15 Hz,
1H, 5-pyrazole H), 6.76–7.88 (m, 18H, ArH) ppm. 13C NMR (CDC13): d = 158.28
(C–O), 145.54 (C = N, pyrazole), 144.62 (N–C) 136.86–104.62 (phenyl–C), 70.07
(OCH2), 64.11 (CH of pyrazole), 43.46 (CH2 of pyrazole) ppm. Analysis for
C28H23ClN2O. Calcd: C, 76.61; H, 5.28; N, 6.38. Found: C, 76.74; H, 5.14; N, 6.18.
5-(4-(Benzyloxy)phenyl)-1-phenyl-3-(4-methylphenyl)-4,5-dihydro-1H-pyrazole (8)
Yield, 60%; m.p., 154–155ꢁC. IR (KBr, cm-1): t = 3026 (CH, aromatic), 2916
1
(CH, aliphatic), 1596 (C = N), 1510 (C = C) cm-1. H NMR (500 MHz, DMSO-
d6): d = 2.34 (s, 3H, CH3), 3.03–3.08 (two d, J = 6.3, 6.35 Hz, 1H, C4–H of
pyrazole), 3.83–3.88 (two d, J = 12.05, 12.15 Hz, 1H, C4–H of pyrazole), 5.05 (s,
2H, benzylic H), 5.37–5.41(two d, J = 6.35, 6.3 Hz, 1H, C5–H of pyrazole), 6.69–
7.65 (m, 18H, ArH) ppm. 13C NMR (DMSO) d = 157.59 (C–O), 147.27 (C = N,
pyrazole), 144.39 (N–C) 138.24–112.93 (phenyl–C), 69.19 (OCH2), 62.57 (CH of
pyrazole), 43.07 (CH2 of pyrazole), 20.91 (CH3) ppm. Analysis for C29H26N2O.
Calcd: C, 83.22; H, 6.26; N, 6.69. Found: C, 83.79; H, 6.36; N, 6.43.
General procedure for synthesis of 5-(4-(benzyloxy)phenyl)-3-(4-substituted
phenyl)-4,5-dihydroisoxazole (9–12)
A mixture of the corresponding chalcone (1–4; 0.01 mol), hydroxylamine HCl
(0.01 mol), and NaOH (0.025 mol) was refluxed in ethanol for 8 h. The mixture was
cooled and the precipitated product was filtered, washed, and crystallized from an
appropriate solvent.
5-(4-(Benzyloxy)phenyl)-3-phenyl-4,5-dihydroisoxazole (9)
Yield, 56%; m.p., 143–144ꢁC; crystallized from ethanol. 1H NMR (200 MHz,
DMSO-d6): d = 3.31–3.39 (two d, J = 3.2, 3.25 Hz, 1H, C4–H of dihydroisox-
azole), 3.71–3.79 (two d, J = 6, 6.12 Hz, 1H, C4–H of dihydroisoxazole), 5.10 (s,
2H, benzylic H), 5.38–5.48 (trip, 1H, C5–H of dihydroisoxazole), 7.00–7.64 (m,
14H, ArH) ppm. EI (70 eV) m/z (%): (M??1, 330, 2.24), (M?, 329, 8.59), 281
(2.6), 256 (6.85), 57 (100). Analysis for C22H19NO2. Calcd: C, 80.22; H, 5.81; N,
4.25. Found: C, 80.05; H, 6.20; N, 4.15.
5-(4-(Benzyloxy)phenyl)-3-(4-bromophenyl)-4,5-dihydroisoxazole (10)
Yield, 63%; m.p., 197–198ꢁC; crystallized from dioxane/H2O. 1H NMR (200 MHz,
DMSO-d6): d = 3.32–3.41 (two d, J = 4.2, 4 Hz, 1H, C4–H of dihydroisoxazole),
3.72–3.86 (two d, J = 6, 6.15 Hz, 1H, C4–H of dihydroisoxazole), 5.11 (s, 2H,

Med Chem Res (2010) 19:1025–1035
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benzylic H), 5.41–5.49 (trip, 1H, C5–H of dihydroisoxazole), 7.01–7.66 (m, 13H,
ArH) ppm. Analysis for C22H18BrNO2. Calcd: C, 64.72; H, 4.44; N, 3.43. Found: C,
64.88; H, 4.94; N, 3.19.
5-(4-(Benzyloxy)phenyl)-3-(4-chlorophenyl)-4,5-dihydroisoxazole (11)
1
Yield, 62%; m.p., [300ꢁC; crystallized from dioxane/H2O. H NMR (200 MHz,
DMSO-d6): d = 3.32–3.41 (two d, J = 4.15, 4.2 Hz, 1H, C4–H of dihydroisox-
azole), 3.72–3.86 (two d, J = 6, 6.2 Hz, 1H, C4–H of dihydroisoxazole), 5.11 (s,
2H, benzylic H), 5.59–5.72 (trip, 1H, C5–H of dihydroisoxazole), 7.01–7.74 (m,
13H, ArH) ppm. Analysis for C22H18ClNO2, Calcd: C, 72.62; H, 4.99; N, 3.85.
Found: C, 73.00; H, 4.50; N, 3.43.
5-(4-(Benzyloxy)phenyl)-3-(4-methylphenyl)-4,5-dihydroisoxazole (12)
Yield, 48%; m.p., 154-155ꢁC; crystallized from ethanol; 1H NMR (200 MHz,
DMSO-d6): d = 2.35 (s, 3H, CH3), 3.30–3.37 (two d, J = 3.3, 3.35 Hz, 1H, C4–H
of dihydroisoxazole), 3.72–3.81 (two d, J = 6.2, 6.25 Hz, 1H, C4–H of dihydrois-
oxazole), 5.12 (s, 2H, benzylic H), 5.39–5.47 (trip, 1H, C5–H of dihydroisoxazole),
7.02–7.66 (m, 13H, ArH) ppm. MS EI (70 eV) m/z (%): (M??1, 344, 0.0), (M?,
343, 3.83), 284 (2.65), 256 (7.82), 55 (100). Analysis for C23H21NO2. Calcd: C,
80.44; H, 6.16. N, 4.08; Found: C, 80.00; H, 6.36; N, 3.88.
Antiviral activity evaluation
Antiviral assays for compounds 5–8
The antiviral activity of the new compounds (5–8) was determined using
cytopathicity (CPE) assay (Gazivoda et al., 2007; Pauwels et al., 1988) against
HSV-1 (KOS), HSV-2 (G), vaccinia virus (VV), VSV, HSV-1 TK– KOS ACVr in
HEL cell cultures; VSV, coxsackie virus B4, respiratory syncytial virus in HeLa cell
cultures; parainfluenza-3 virus, reovirus-1, sindbis virus, coxsackie virus B4, punta
toro virus in Vero cell cultures. Stock solutions of the test compounds were prepared
in DMSO at a concentration of 10 mg/ml. Cells, grown to confluency in 96-well
plates, were infected with 100 CCID50 of virus, one CCID50 being the 50% cell
culture infective dose. After an adsorption period of 2 h at 37ꢁC, virus was removed
and serial dilutions of the compounds were added. Cultures were further incubated
at 37ꢁC for 3 days, until complete CPE was observed in the infected and untreated
virus control. Determination of the anti-HIV activity of the compounds was based
on virus-induced cytopathicity (destruction) of HIV-infected MT-4 cells, measured
at day 5 post virus infection by the MTT colorimetric method (Pauwels et al., 1988).
Results are expressed as the 50% effective concentration (EC50) as reported in
Tables 1, 2 and 3. The 50% effective antiviral concentration (EC50) was defined as
the compound concentration required to protect 50% of the virus-infected cells
against viral cytopathogenicity. The symbol ‘‘[’’ is used to indicate the highest

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Table 1 Cytotoxicity and antiviral activity of compounds 5-8 in HEL cell cultures
Compound
Minimum
cytotoxic
concentrationa
(lg/ml)
ECb50 (lg/ml)
Herpes
simplex
virus-1
(KOS)
Herpes
Vaccinia Vesicular
virus
virus
Herpes simplex
simplex
virus-2
(G)
stomatitis virus-1 TK-
KOS ACVr
5
100
100
[20
[20
[20
[20
0.08
[250
3
[20
[20
[20
[20
126
[20
[20
[20
[20
10
[20
[20
[20
[20
[20
[20
[250
[250
5
6
7
100
[20
8
100
[20
Brivudin (lM)
Ribavirin (lM)
Cidofovir (lM)
[250
[250
[250
[250
[250
[250
[100
[250
5
146
10
Ganciclovir (lM) [100
0.08
0.08 [100
6
a
Required to cause a microscopically detectable alteration of normal cell morphology
Required to reduce virus-induced cytopathogenicity by 50%
b
Table 2 Cytotoxicity and antiviral activity of compounds 5–8 in HeLa cell cultures
Compound
Minimum cytotoxic
concentrationa (lg/ml)
ECb50 (lg/ml)
Vesicular
stomatitis virus
Coxsackie
virus B4
Respiratory
syncytial virus
5
100
100
[20
[20
[20
[20
[100
[250
29
[20
[20
[20
[20
9
[20
[20
[20
[20
0.8
6
7
C20
8
100
DS-5000
[100
[250
[250
(S)-DHPA (lM)
Ribavirin (lM)
a
[250
146
[250
10
Required to cause a microscopically detectable alteration of normal cell morphology
b
Required to reduce virus-induced cytopathogenicity by 50%
concentration at which the compounds were tested and found not to be antiviral
active.
Antiviral bioassays for compounds 5–12
African green monkey kidney-derived cells (Vero cell culture) were propagated in
Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal
bovine serum and 1% antibiotic/antimycotic mixture. The pH was adjusted to 7.2–7.4
with a 7.5% sodium bicarbonate solution. HSV-1 and HAV (MBB cell culture
adapted strain) were obtained from Environmental Virology Laboratory, Department
of Water Pollution Research, National Research Centre, Cairo.

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Table 3 Cytotoxicity and antiviral activity of compounds 5–8 in Vero cell cultures
Compound
Minimum
cytotoxic
ECb50 (lg/ml)
Para-influenza-3 virus Reovirus-1 Sindbis Coxsackie Punta Toro
virus
concentration
(lg/ml)a
virus
virus B4
5
100
C20
100
[20
[20
[20
[20
[100
[250
45
[20
[20
[20
[20
[20
[20
59
[20
[20
[20
[20
[20
[20
100
6
7
[20
[20
8
100
[20
[20
DS-5000
[100
[100
[250
[250
[100
[250
[250
(S)-DHPA (lM) [250
[250
[250
[250
146
Ribavirin (lM) [250
a
Required to cause a microscopically detectable alteration of normal cell morphology
Required to reduce virus-induced cytopathogenicity by 50%
b
Plaque infectivity reduction assay
Preparation of synthetic compounds for bioassay
The tested compounds were dissolved at 100 mg each in 1 ml of 10% DMSO in
water. The final concentration was 100 lg/ml (stock solution). The dissolved stock
solutions were sterilized by the addition of a 50 lg/ml antibiotic-antimycotic
mixture (10,000 U) penicillin G sodium, 10,000 mg streptomycin sulfate, and
250 lg amphotericin B.
Plaque reduction assay
A six-well plate was cultivated with Vero cell culture (105 cell/ml) and incubated
overnight at 37ꢁC. HSV-1 and HAV were diluted to give a 104 plaque forming unit
(PFU)/ml final concentration for each virus, mixed with the tested compound at the
previous concentration, and incubated overnight at 4ꢁC. Growth medium was
removed from the multiwell plate and the virus compound mixture was inoculated
(100 l1/well). After 1 h of contact, the inoculum was aspirated and 3 ml of MEM
with 1% agarose was overlaid on cell sheets. Plates were left to solidify and
incubated at 37ꢁC until the development of virus plaques. Cell sheets were fixed in
10% formalin solution for 2 h, then stained with crystal violet stain. Control virus
and cells were treated identically without chemical compounds. Virus plaques were
counted and the percentage reduction was calculated (Tebas et al., 1998).
Cytotoxicity assays
The cytotoxicity of the compounds was evaluated in parallel with their antiviral
activity in uninfected cells and is expressed as the minimum cytotoxic concentration
(MCC) that causes a microscopically detectable alteration of normal cell
morphology (HEL, HeLa, and Vero cells).

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Med Chem Res (2010) 19:1025–1035
Results and discussion
Chemistry
The synthetic route used to synthesize the title compounds is outlined in Scheme 1.
The key a,b-unsaturated ketones 3-(4-(benzyloxy)phenyl)-1-(4-substituted phenyl)-
2-propen-1-one intermediates (1–4) were prepared by base-catalyzed Claisen–
Schmidt condensation of the acetophenone and its derivatives with 4-benzyloxy-
benzaldehyde in accordance with the method described in the literature (Li et al.,
2005; Robinson et al., 2005). In basic media, N-phenylpyrazole derivatives (5–8)
were prepared by heating at reflux the corresponding a,b-unsaturated ketones (1–4)
with phenyl hydrazine in ethanol for 4 h. 4,5-Dihydroisoxazole derivatives (9–12)
were obtained by heating at reflux equimolar amounts of hydroxylamine HCl and
the corresponding a,b-unsaturated ketones (1–4) in hot ethanolic NaOH solution for
8 h. Chemical structures of the target compounds were determined on the basis of
1
spectral data analysis, such as IR, H NMR, 13C NMR, and mass spectrometry.
1H NMR of the new 4,5-dihydro-1H-pyrazoles revealed the presence of two
peaks at a chemical shift (d = *3.1 and *3.7) for the two magnetically
nonequivalent protons at the 4-position of the pyrazoline ring. The CH proton at C5
also appears as a doublet of doublets in the region of 5.25–5.45 ppm due to vicinal
coupling with two nonmagnetically equivalent geminal protons of the C4 carbon.
The signals obtained from 13C NMR spectra further confirmed the proposed
structures; the C4 and C5 carbons of the pyrazoline ring resonate at 42.95–43.46 and
62.57–64.11 ppm, respectively. All compounds showed signals at 145–147 ppm,
1
which was assignable to the azomethine carbon of the pyrazoline ring. H NMR
spectra of the 4,5-dihydroisoxazole derivatives revealed the presence of two peaks
at a two different chemical shifts (d = *3.3 and *3.8) for protons at C4 and the
presence of a peak around d = 5.4 for CH proton at C5, confirming the formation of
compounds.
Biological evaluation
Compounds 5–8 were evaluated against a group of viruses, namely, HSV-1 KOS,
HSV-2 G, VV, VSV, and thymidine kinase-deficient HSV- 1 TK- KOS ACVr in
HEL cell cultures; VSV, Coxsackie virus B4, and respiratory syncytial virus in
HeLa cell cultures; para-influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie
virus B4, and Punta Toro virus in Vero cell cultures; and HIV-1 (IIIB) and HIV-2
(ROD) in MT-4 cell cultures.
The activities of the compounds were compared with reference antiviral drugs,
namely, (E)-5-(2-bromovinyl)-20-deoxyuridine (brivudin), 1-(b-D-ribofuranosyl)-
1H-1,2,4-triazole-3-carboxamide (ribavirin), 1-[(S)-3-hydroxy-2-(phosphonometh-
oxy)propyl]-cytosine (cidofovir), 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine
(ganciclovir), 9-(1,3-dihydroxypropyl)adenine [(S)-DHPA], DS-5000, and nevira-
pine (Tables 1, 2 and 3).
Compounds 5–12 were further evaluated using two viruses, HAV and HSV-1 in
Vero cell cultures. The plaque infectivity reduction assay was applied (Tebas et al.,

Med Chem Res (2010) 19:1025–1035
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O
CHO
+
H3C
O
R
R= H, Br, Cl, CH3
20 % NaOH, ethanol
Stirring, rt, 24 h
O
O
R
1-4
NH2NHC6H5
NH2OH.HCl
NaOH, ethanol
reflux 4 h
NaOH, ethanol
reflux 8 h
N
O
N
N
R
R
O
O
5,9 : R= H
6,10 : R= Br
7,11 : R= Cl
8,12 : R= CH3
9-12
5-8
Scheme 1 Synthesis of N-phenylpyrazoles (5–8) and 4,5-dihydroisoxazoles (9–12)
1998). The two known drugs commonly utilized for therapeutic treatments of HAV
and HSV-1 are amantadine and acyclovir, respectively. So, these two drugs were
considered as references. Results obtained from antiviral evaluation of the tested
compounds are listed in Table 4.
According to Tables 1, 2 and 3, compounds 5–8 showed appreciable antiviral
activity against HSV-1 KOS, HSV-2 G, VV, VSV, and HSV-1 TK-KOS ACVr, with
an EC50 [ 20 lg/ml, which was more than five times lower than its cytotoxic
concentration (100 lg/ml). Regarding HeLa cell cultures, the active concentration
of compound 7 equaled its cytotoxic concentration (C20 lg/ml) therefore it is
considered inactive. In Vero cell cultures compound 6 was active at a concentration
which caused microscopically detectable alteration of normal cell morphology at
concentrations C20 lg/ml, which is equal to its EC50. In MT-4 cell cultures,
unfortunately, none of the compounds showed activity against HIV-1 (IIIB) or HIV-
2 (ROD).
The results presented in Table 4 reveal the following.
1. 4,5-Dihydroisoxazole 12 showed the best anti-HSV-1 activity compared with
the other tested compounds, especially at a concentration of 20 lg/ml (79%
inhibition).

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Table 4 Cytotoxicity and antiviral activity of compounds 5–12 in Vero cell cultures
Compound
Minimum cytotoxic
concentration (lg/ml)a
Percentage reduction
HSV-1
HAV
10 lg/ml
20 lg/ml
10 lg/ml
20 lg/ml
5
100
C20
0
30
50
43
58
15
67
60
79
7
2
23
16
21
39
2
6
13.3
19
7
100
0
8
100
31
14
0
9
[100
C20
3
10
11
12
a
26.1
38
7.3
10
27
21
58
46
[100
[100
45
Required to cause a microscopically detectable alteration of normal cell morphology
2. 4,5-Dihydroisoxazoles 10 and 11 exhibited moderate activity toward HSV-1
(67% and 60% inhibition, respectively); compound 10 was cytotoxic at the
active concentration 20 lg/ml, therefore, it is considered as inactive.
3. Compound (11) exerted the highest activity toward HAV (58% inhibition)
compared with the other tested compounds.
4. Other compounds tested showed weak to moderate activity, especially at a
concentration of 20 lg/ml.
The structure–activity relationship of the 4,5-dihydroisoxazole derivatives
reveals the following:
•
•
Substitution at the phenyl ring at C3 with an electron donating group increases
the activity toward HSV-1 as in compound 12.
Substitution at the same position with electron withdrawing groups slightly
decreases the activity against HSV-1 and increases the activity toward HAV as
in the case of compound 11 but may produce cytotoxic compounds as in the case
of 5-(4-(benzyloxy)phenyl)-3-(4-bromophenyl)-4,5-dihydroisoxazole (10),
which showed cytotoxicity to Vero cell cultures at a concentration C20 lg/ml.
Nonsubstituted phenyl derivatives showed weak activity toward HSV-1 as well
as HAV. It would therefore be of interest to further explore other substituents on
the phenyl ring in the ortho, meta, and para positions to reveal whether antiviral
activity can be further improved.
•
•
The benzyloxyphenyl-substituted dihydroisoxazole ring is essential for the
activity, which matches with the aforementioned compound B (Zeng et al., 2008).
Conclusion
In summary, eight new derivatives of 4,5-dihydro-1H-pyrazole and 4,5-dihydrois-
oxazole were synthesized and exhibited a range of significant antiviral activities,

Med Chem Res (2010) 19:1025–1035
1035
whereas compounds 11 [5-(4-(benzyloxy)phenyl)-3-(4-chlorophenyl)-4,5-dihyd-
roisoxazole] and 12 [5-(4-(benzyloxy)phenyl)-3-(4-methylphenyl)-4,5-dihydroisox-
azole] exhibited the best activity against HAV and HSV-1, respectively. Three
derivatives, 8, 11, and 12, did not show toxicity to any of the tested cell cultures.
Thus, these derivatives may possibly be used as lead compounds for developing new
antiviral agents. The mechanism of antiviral activity is unknown and requires
further study.
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