Evaluation of some pyrazoloquinolines as inhibitors of herpes simplex virus type 1 replication

Article abstract of DOI:10.1002/ardp.200400930

Three structurally related aminopyrazoloquinoline derivatives were evaluated for their antiviral activity against Herpes Simplex virus type 1. These compounds were examined for their in vitro antiviral activity by two different bioassays, namely; crystal violet staining and tetrazolium dye (MTS) measurement. The antiviral role of these compounds was confirmed by enumerating the infectious particles with plaque assay. The acute toxicity values of the biologically active compounds were determined prior to their screening as antiviral agents.

Full text of DOI:10.1002/ardp.200400930

74
DOI 10.1002/ardp.200400930
Arch. Pharm. Chem. Life Sci. 2005, 338, 74−77
Evaluation of Some Pyrazoloquinolines as Inhibitors
of Herpes Simplex Virus Type 1 Replication
Adnan A. Bekhita, Ola A. El-Sayeda, Hassan Y. Aboul-Eneinb*, Yunus M. Siddiquic,
and Mohammed N. Al-Ahdalc
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria,
Egypt
b Pharmaceutical Analysis and Drug Development Laboratory, Biological and Medical Research Depart-
ment, King Faisal Specialist Hospital Research Centre, Riyadh, Saudi Arabia
c Molecular Virology and Infectious Diseases Unit, Biological and Medical Research Department,
King Faisal Specialist Hospital Research Centre, Riyadh, Saudi Arabia
Three structurally related aminopyrazoloquinoline derivatives were evaluated for their antiviral activity
against Herpes Simplex virus type 1. These compounds were examined for their in vitro antiviral
activity by two different bioassays, namely; crystal violet staining and tetrazolium dye (MTS) measure-
ment. The antiviral role of these compounds was confirmed by enumerating the infectious particles
with plaque assay. The acute toxicity values of the biologically active compounds were determined
prior to their screening as antiviral agents.
Keywords: Pyrazoloquinolines; Herpes Simplex virus type 1; Acute toxicity
Received: August 4, 2004; Accepted: January 13, 2005 [FP930]
Introduction
severity and duration of the initial infection [7]. Although
herpes is usually a mild disease in immunocompetent indi-
viduals, mucocutaneous herpetic infections are troublesome,
especially for patients with frequent episodes. Moreover,
immunocompromised (HIV or cancer) patients have an
increased risk of developing severe and more frequent
herpetic infections.
Herpes simplex virus (HSV) infection is an ancient disease,
with descriptions of orolabial herpes appearing in records
from the fifth century BC [1]. HSV remains a common
cause of ulcerative mucocutaneous diseases today [2Ϫ4].
Due to the high prevalence of HSV infection worldwide,
most persons infected with HIV are also infected with one
or both HSV types [5] Initial infection results in painful
ulcerative mucosal and cutaneous lesions. Herpes simplex
virus (HSV) type 1 (HSV-1) has the ability to become latent
in sensory ganglia and to induce recurrent infections follow-
ing reactivation [6]. The frequencies of recurrent herpetic
infections in the U.S. population are estimated to be 50 to
70% for HSV-1 and 23% for HSV-2 [7]. Mucosal or skin
surfaces are the usual sites of primary infection. Recurrent
herpes labialis and herpes genitalis represent the most com-
mon clinical manifestations associated with HSV-1 and
HSV-2 infections, respectively. Most recurrences are
asymptomatic infections, and the shedding of herpes virus
under these conditions represents the most common form
of transmission of this disease. Recurrences are associated
with physical or emotional stress, fever, exposure to UV
light, tissue damage, and immune suppression. The fre-
quency of recurrences has also been correlated with the
During the past several decades, acyclovir has been the drug
of choice for the treatment of herpetic infections. However,
the emergence of acyclovir-resistant HSV isolates has been
reported for immunocompromised patients [8] as well as for
organ and bone marrow recipients [9, 10]. Recurrent acyclo-
vir-resistant genital herpes has also been described for an
immunocompetent host [11]. Foscarnet (trisodium phos-
phonoformate) has a broad antiviral spectrum and in vitro
activity against all human viruses of the herpes virus family,
including cytomegalovirus, HSV, and varicella-zoster virus
[12, 13]. This drug is also effective against acyclovir-resistant
HSV and varicella-zoster virus [14-18]. Moreover, acyclovir-
resistant HSV strains that become resistant to foscarnet may
once again be susceptible to acyclovir [19]. Because the in-
travenous administration of foscarnet is limited by the oc-
currence of nephrotoxic reactions, the development of top-
ical formulations represents an attractive approach for the
treatment of mucocutaneous herpetic infections, especially
for those caused by acyclovir-resistant strains.
Correspondence: Hassan Y. Aboul-Enein, Pharmaceutical Analysis
and Drug Development, Biological and Medical Research Depart-
ment (MBC 03-65), King Faisal Specialist Hospital Research Cen-
tre, P.O. Box 3354, Riyadh 11211, Saudi Arabia. Phone:ϩ966 1 442-
78-59, Fax: ϩ966 1 442-78-58, e-mail: enein@kfshrc.edu.sa
Topical formulations currently available for the treatment
of mucocutaneous herpetic infections include 5% acyclovir
ointment (Zovirax) and penciclovir cream formulation (Vec-
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 74−77
Pyrazoloquinolines as Inhibitors of Herpes Simplex virus
75
sugar derivatives of pyrazoloquinoline [32]. The study re-
sulted in two aldopentose derivatives 1 and 2 possess inhibi-
tory activity against virus growth, Figure 1. In the present
work, the activity of animopyrazoloquinoline precursors of
the already reported aldehydo-sugar derivatives were evalu-
ated for their inhibitory activity against herpes simplex type
1 replication.
Results and discussion
Chemistry
The tested aminopyrazoloquinoline derivatives were syn-
thesised according our reported procedure [33, 34] as shown
in Scheme 1.
Figure 1. Structures of aldehydo-sugar pyrazoloquinoline
derivatives.
tavir cold sore cream or Denavir cream in the United
States). The currently available treatment, either topical or
systemic, has only limited efficacy, particularly against
symptomatic recurrent herpes. Treatment of recurrent her-
pes with topical acyclovir demonstrated no or only limited
clinical benefit [20Ϫ23]. Wallin et al. demonstrated a limited
but significant effect of topical foscarnet cream on time to
healing for recurrent genital herpes [24]. Conversely, no sig-
nificant improvements in time to healing or loss of symp-
toms were observed for recurrent genital herpes in two other
clinical trials [25, 26]. Patients who received treatment in the
prevesicular stage had a slightly reduced number of days
with lesions [27]. Treatment of herpes labialis in immuno-
competent patients with penciclovir cream was reported ben-
eficial for treatment started in the prodrome and erythema
stages as well as in the papule and vesicle lesion stages [28].
Biological screening
The plaque assays repeatedly demonstrated that the com-
pound 5 had the maximum antiviral activity against HSV.
Proper controls included the diluent and the well-known
anti-HSV guanosine analog, Acyclovir. Previously reported
compounds 1 and 2 were also included in this study and
shown to have antiviral activity by the plaque assay. With-
out the sugar moiety, compounds 3 and 4 were restricting
the replication of HSV while compound 5 clearly had simi-
lar or more anti-HSV activity than Acyclovir as shown in
Figure 2.
Nevertheless, a high medical need exists for improved anti-
herpetic drugs for the treatment of severe disease. Encepha-
litis in newborns, for example, results in 15% mortality, and
only 29% of survivors develop normally after acyclovir
therapy [29]. Also, for patients with less severe disease, an
agent that will achieve a better reduction of lesion duration
with episodic treatment beyond the 1 to 2 days’ reduction
achieved with current medications is urgently required [30].
Furthermore, a drug which continues to show profound effi-
cacy when given at later stages of herpetic disease would be
a new and highly desired standard in the treatment of herpes
[31]. We have already reported the synthesis and herpes sim-
plex virus type 1 replication inhibitory activity of aldehydo-
Figure 2. Effect of Pyrazoloquinolines on HSV replication.
Scheme 1. Synthesis of the aminopyrazoloquinoline derivatives that were tested in this study.
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

76
Aboul-Enein et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 74−77
Biological screening
The MTT and crystal violet assays are based on the integ-
rity of the eukaryotic cells either at the presence of intact
cytoplasmic membrane or the cellular enzymatic level. In
certain cases where the reagents interfere with the spectro-
photometric analysis, the interpretation of result becomes
difficult. With the previously reported result, this inter-
ference was negligible and a proper correlation was ob-
tained when the antiviral effect of compound 1 and 2 was
reported [32]. With these pyrazoloquinolines, the spectro-
photometric observation was difficult and, therefore, the
plaque assay was used in this study.
Antiviral assay
Reagents needed for the MTT cytotoxicity test, Crystal Violet stain-
ing and the compound dilutions are described in our previous com-
munication [32]. Cell culture and the viral plaque assay reagents
were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Acyclovir (for intravenous injection) was obtained from Faulding
Pharmaceutical Co. (Paramus, N.J. USA).
The antiviral activity of these pyrazoloquinolines was determined
as follows. Twelve-well cluster tissue culture plates (Nunc,
Denmark) were seeded with African green monkey kidney (Vero)
cells. After 24 h of incubation the medium was aspirated and the
HSV inoculum was added at a multiplicity of infection of 10 and
left for 90 min for adsorption at 37°C. Unadsorbed virus particles
were removed with phosphate buffered saline (PBS) containing 2%
fetal bovine serum. The stock solution of these chemical com-
pounds were suspended in the maintenance medium (EMEM con-
taining 2% fetal bovine serum [FBS]) and added wells in duplicate
to give a working concentration of 100 and 50 μM. Cells were lysed
with two cycles of freezing and thawing after 24Ϫ36 h of incubation.
Lysed cell suspensions were collected in separate tubes and kept
frozen at Ϫ80°C until used. Before conducting the plaque assay, the
cell lysates were centrifuged at 1500g for 5 min, and log dilutions
of the supernatant were made in PBS containing 2% FBS. The
plaque assay for the enumeration of infectious HSV particle has
been reported earlier [38]. Briefly, the 60-mm tissue culture dishes
(Nunc, Denmark) were seeded with Vero cells and after 48 h of
incubation medium was aspirated and virus dilutions were added in
triplicate. After 90 min of adsorption at 37°C, 3 mL of PBS-FBS
was added to wash away unadsorbed virus particle. Agar overlay in
a volume of 5ml was added to each dish and the dishes were trans-
ferred to incubator after the agar was solidified. Dishes were incu-
bated for 3-5 days, inoculated with 3 mL of neutral red solution
(0.033%) made in PBS, and incubated for 4 h. Excess stain was
decanted and the plaques were counted over a light box.
Acute toxicity
Compounds 3, 4 and 5 were further evaluated for their oral
acute toxicity in male mice using a literature method [35,
36]. The results indicated that the tested compounds proved
to be non-toxic and well tolerated by the experimental ani-
mals up to 200 mg/kg, although no mortality was recorded
at 350 mg/kg. The oral LD50 of compounds 3, 4 and 5 were
found to be 364, 378 and 356 mg/kg respectively. Moreover,
these compounds were tested for their toxicity through par-
enteral route [37]. The results revealed that all the test com-
pounds were non-toxic up to 80 mg/kg.
It can be safely concluded from repeated experiments, that
compound 5 outperformed Acyclovir at equimolar ratio. We
intend to continue this study at the molecular level to under-
stand the mechanism of action of compounds 1, 2 and 5.
Such compounds would represent a fruitful matrix for the
development of a new class of antiherpetic agent with good
safety margin.
Acute toxicity
Compounds 3, 4 and 5 were further investigated for their acute
toxicity in male mice [35, 36]. Eight groups of mice each consisting
of six animals, were used. The compounds were given orally in doses
of 1, 10, 100, 200, 250, 500 mg/kg, respectively. Twenty-four hours
later, the% mortality in each group and for each compound was
recorded. The LD50 values were calculated using the method de-
scribed by Litchfield and Wilcoxon [36].
Experimental
Chemistry
Moreover, these compounds were tested for their toxicity through
parenteral route [37]. Groups of mice each consisting of six animals
were used. The compounds or their vehicle, propylene glycol (con-
trol) were given by intraperitoneal injection in doses of 10, 25, 50,
80, 100 mg/kg, respectively. Survival was followed up to 7 days.
All chemicals were purchased from E. Merck (Darmstadt, Ger-
many), Fluka AG (Buchs, Switzerland) and Aldrich (St. Louis, MO,
USA) companies. Melting points were determined in open glass
capillaries using a Thomas capillary melting point apparatus and
are uncorrected. Follow up of the reactions and checking the purity
of the compounds were made by TLC on silica gel-protected alumi-
num sheets (Type 60 F254, Merck) and the spots were detected by
exposure to UV-lamp at 254 nm for few seconds.
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