Synthesis of new 3,4,5-trisubstituted isothiazoles as effective inhibitory agents of enteroviruses

Article abstract of DOI:10.1016/S0968-0896(98)00237-5

The synthesis and evaluation of 3,4,5-trisubstituted isothiazoles as antiviral agents led to the discovery of several compounds effective in vitro against enteroviruses polio 1 and ECHO 9. Structure-activity relationship studies revealed that a short thioalkyl chain in the 3-position and a methyl ester group in the 4-position are the structural components that, to a large extent, contribute to the positive biological profile in terms of both selectivity and low cytotoxicity. Under one-step growth conditions, methyl 3-methylthio-5-phenyl-4-isothiazolecarboxilate caused the greatest activity if added within 1h after poliovirus adsorption. These data suggest interference with early events of viral replication. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00237-5

Bioorganic & Medicinal Chemistry 7 (1999) 225±230
Synthesis of New 3,4,5-Trisubstituted Isothiazoles as E€ective
Inhibitory Agents of Enteroviruses
Christian C. C. Cutrõ,a Adriana Garozzo,b Maria A. Siracusa,a Maria C. Sarva,a
Angelo Castro,b Ernesto Geremia,c Maria R. Pinizzottob and Francesco Guerreraa,*
a
Á
Dipartimento di Scienze Farmaceutiche, Universita di Catania, Viale A. Doria 6, 95125 Catania, Italy
Á
Á
Istituto di Biologia Generale Universita di Catania, Via Androne 81, 95124 Catania, Italy
b
Dipartimento di Scienze Microbiologiche e Ginecologiche, Universita di Catania, Via Androne 81, 95124 Catania, Italy
c
Received 10 March 1998; accepted 18 June 1998
AbstractÐThe synthesis and evaluation of 3,4,5-trisubstituted isothiazoles as antiviral agents led to the discovery of several com-
pounds e€ective in vitro against enteroviruses polio 1 and ECHO 9. Structure±activity relationship studies revealed that a short
thioalkyl chain in the 3-position and a methyl ester group in the 4-position are the structural components that, to a large extent,
contribute to the positive biological pro®le in terms of both selectivity and low cytotoxicity. Under one-step growth conditions,
methyl 3-methylthio-5-phenyl-4-isothiazolecarboxilate caused the greatest activity if added within 1 h after poliovirus adsorption.
These data suggest interference with early events of viral replication. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
family. The widespread nature of picornavirus illnesses,
the economic consequences and the impracticality of
vaccine development have stimulated our search for new
selective antipicornaviral drugs. Our previous studies
described the synthesis and antiviral activity of 3-mer-
capto-5-aryl-isothiazoles, which were active against
enteroviruses polio 1 and ECHO 9.10±12 Following
works have demonstrated that the presence of a cyano
group in the 4-position of isothiazole ring improved
antiviral activity and broadened the spectrum of action;
in fact, some new 4-isothiazolecarbonitriles were also
e€ective against Coxsackie B1 and measles viruses.13
The most active member of this series, 3-methylthio-5-
phenyl-4-isothiazolecarbonitrile, coded IS-2, exhibited
remarkable viral inhibition against polio 1, with selec-
tivity index of 444, which was higher than those
obtained using the previously studied 3-mercapto-5-
aryl-isothiazoles.11±13
Isothiazole derivatives are known to possess many bio-
logical properties. The isothiazole ring has been incor-
porated into a wide range of established drugs including
sulfonamides,1 thiosemicarbazones,2 amidines3 and
benzimidazoles,4 although this approach does not
appear to have resulted in any major improvements.
However, many isothiazoles, not directly related to
known drugs, have demonstrated biological activity: a
derivative of an isothiazolo[5,4-d]pyrimidine has been
reported to possess antitumoral activity,5 3,5-di-
substituted 4-isothiazolecarbonitriles are known as
herbicide, insecticide and fungicide agents6,7 and iso-
thiazolecarboxylic esters8 have exhibited spasmoly-
tic activities against acetylcholine and histamine as
spasmogens.
So far, few research groups have investigated the
potential antiviral properties of the isothiazole nucleus.
Only isothiazolehydrazides have shown a weak antiviral
activity against polio and herpes simplex viruses.9 As a
result, we have been studying antiviral activity of new
isothiazole derivatives in order to obtain compounds
that are e€ective as antiviral agents. Currently, no drugs
are available for the treatment of diseases caused by
enteroviruses, one of the main genera of the picornavirus
On the basis of these data, we selected IS-2 as the lead
compound for some chemical transformations to estab-
lish the requirements for optimum activity. As a result
of our work, the introduction of substituents on the
phenyl ring or the replacement of the phenyl for other
aromatic and heteroaromatic groups in the 5-position
did not improve antiviral activity.13
As an approach to more extensive structural modi®ca-
tions, in the present study we report the e€ect of repla-
cement of the SCH3 in the 3-position, as well as the CN
conversion into CONH2, COOH or COOMe groups in
*Corresponding author. Tel: +39 0 95 337123; fax: +39 0 95 222239;
e-mail: guerrera@ictuniv.unict.it
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(98)00237-5

Á
C. C. C. Cutrõ et al. / Bioorg. Med. Chem. 7 (1999) 225±230
226
the 4-position. The new 3,4,5-trisubstituted isothiazoles
were screened against both RNA viruses (polio 1,
ECHO 9, Coxsackie B1, EMC, measles) and DNA
viruses (adeno 2 and HSV 1).
Chemistry
3-Alkylthio-5-phenyl-4-isothiazolecarbonitriles 4a±h
were obtained by the reaction of 114 with Na2SÂ9H2O
and suitable alkylating reagents 3a±h (Scheme 1). Reac-
tion of ethanol solutions of 1 with excess dimethylamine
5a, pyrrolidine 5b, piperidine 5c or morpholine 5d
gave the corresponding 3-dialkylamino-5-phenyl-4-
isothiazolecarbonitriles 6a±d by replacement of the 3-
chlorine atom (Scheme 1). A number of 3,5-di-
substituted 4-isothiazolecarboxylic amides, acids and
esters were prepared from the corresponding 4-iso-
thiazolecarbonitriles. 4-Isothiazolecarboxamides 7a,b
were prepared by solution of the appropriate nitrile in
sulfuric acid and subsequent reaction with water. 4-Iso-
thiazolecarboxylic acids 8a,b were prepared by hydro-
lysis with potassium hydroxide in aqueous ethylene
glycol. Methyl esters 9a,b were obtained by re¯uxing
the respective alcoholic solutions of the acids 8a,b
with an excess of gaseous hydrochloric acid, as shown
in Scheme 2.
Scheme 2. Synthesis of compounds 7a,b, 8a,b and 9a,b.
A structure±activity study was initially performed by
maintaining the cyano group and the phenyl ring in the
4- and 5-positions, respectively, and varying the alkyl-
thio chain in the 3-position.
Compounds 4a and 4b, characterized by the replace-
ment of the SCH3 in the 3-position with a longer alkyl-
thio chain, showed in vitro a reduced antiviral activity,
but a cytotoxicity from ®ve (4a) to ten (4b) times lower,
when compared to IS-2 (Table 1). The introduction of a
cyano (4c), hydroxy (4d), carboethoxy (4e) group or a
bromo atom (compounds 4f±h) in the alkyl chain resul-
ted in a diminished activity (compounds 4c,d) or in the
loss of antiviral activity (compounds 4e,f), as reported
in Table 1. More extensive structural modi®cations in
the 3-position gave the 3-dialkylamino derivatives 6a±d,
which were less e€ective than IS-2 against polio 1 and
ECHO 9 (6a,b), only slightly active against polio 1 (6c)
or completely inactive (6d), as reported in Table 1.
Chemical and physical data of new compounds are
reported in Experimental.
Results and Discussion
Table 1 shows the values of test compounds CC50 and
IC50. The 50% cytotoxic dose (CC50) was expressed as
the concentration which inhibited cell growth by 50% as
compared with the control cultures. The compound
concentration required to inhibit virus plaque formation
by 50% was expressed as IC50. The selectivity index (SI)
was determined for the e€ective compounds dividing
CC50 by IC50 (Table 2).
Our next chemical approach was to examine the e€ect
of substitution of the cyano group in the 4-position with
respect to antiviral activity (compounds 7±9a). By far
the greatest enhancement of selectivity was produced by
the 4-carboxymethylester 9a (Table 2), whereas, despite
their low cytotoxicity, the amide and acid derivatives
(7a and 8a, respectively) were inactive against screening
viruses (Table 1). Moreover, the simultaneous replace-
ment of SCH3 and CN groups, which led to the new
alkylthio amide, acid and ester (compounds 7±9b),
caused a diminished (9b) or a loss of antiviral activity
(7±8b), as reported in Table 1. Only few compounds
(4a,b, 4g,h and 9a) were weakly active against EMC
(Table 1).
The lead compound IS-2 demonstrated the highest level
of activity against polio 1 and ECHO 9 viruses, with
IC50 values of 0.045 and 0.25 mM, respectively (Table 1).
All the new trisubstituted isothiazoles were ine€ective
against Coxsackie B1, measles, adeno 2 and HSV 1
(Table 1).
Compound 9a was not virucidal, since exposure of
poliovirus to 100 and 500 mM drug concentrations had
no e€ect on virion infectivity. In order to determine
whether 9a inhibited the virus yield during a speci®c
period in the virus cycle, the e€ect of time addition of
this compound was studied for poliovirus type 1.
Results obtained from these experiments clearly
demonstrated that 9a was e€ective to the highest degree
Scheme 1. Synthesis of compounds 4a±h and 6a±d.

Á
C. C. C. Cutrõ et al. / Bioorg. Med. Chem. 7 (1999) 225±230
227
Table 1. Antiviral activity of isothiazoles 4a±h, 6a±d, and 7±9a,b
Compd
R
R1
CC50(mM)a,c
HEp-2 L-929Vero
IC50(mM)b,c
EMC
Polio
ECHO
Coxs.
Measles
Adeno
HSV
IS
4a
4b
4c
4d
4e
4f
4g
4h
6a
6b
2
SCH3
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
20
100
200
10
5
200
25
25
25
15
7
0.045
0.1
15
0.5
0.3
>200
>25
>25
>25
0.2
0.25
0.5
5
0.5
>20
>100
>200
>10
>5
10
20
5
>10
>5
>20
>100
>200
>10
>5
>200
>25
>25
>25
>15
>7
>20
>100
>200
>10
>5
>200
>25
>25
>25
>15
>7
>20
>100
>200
>10
>5
>200
>25
>25
>25
>15
>7
SCH2CH3
S(CH2)2CH3
SCH2CN
S(CH2)2OH
SCH2COOEt
S(CH2)2Br
S(CH2)3Br
S(CH2)4Br
N(CH3)2
0.6
>200
>25
>25
>25
1
>200 >200
>25
>25
>25
>15
>7
>25
2.5
5
>15
>7
0.1
0.3
6c
6d
CN
CN
25
1
2.5
>25
>1
>25
>1
>25
>1
>25
>1
>25
>1
>25
>1
>1
7a
7b
8a
8b
9a
9b
SCH3
S(CH2)2CH3
SCH3
SCH2CH3
SCH3
SCH2CH3
CONH2
CONH2
COOH
COOH
COOMe
COOMe
250
300
500
500
100
50
>250
>300
>500
>500
0.09
>250
>300
>500
>500
0.3
>250 >250
>300 >300
>500 >500
>500 >500
>250
>300
>500
>500
>100
>50
>250
>300
>500
>500
>100
>50
>250
>300
>500
>500
>100
>50
>100
>50
15
>50
0.2
2
aCC50: concentration which inhibited cell growth by 50% as compared with control cultures.
bIC50: concentration which inhibited virus plaque formation by 50%.
cValues are mean‹0.5 S.D. (estimated maximal standard deviation) of three separate assays.
when it was added at the end of adsorption period,
whereas the viral yield was already reduced when 9a was
added 30 min after the adsorption period. Addition
during virus adsorption, and more than 1 h after the
virus adsorption period, did not cause any virus yield
reduction (Fig. 1).
methyl 3-methylthio-5-phenyl-4-isothiazolecarboxilate
9a were demonstrated to be the most potent inhibitors
of members of the picornavirus family (polio 1 and
ECHO 9), with a range of IC50 values of 0.09±0.5 mM,
which were very much lower than CC50 values (100 mM),
as reported in Table 1. In this way, we obtained selec-
tivity indexes for 4a (1000 and 200) and 9a (1111 and
333) for polio 1 and ECHO 9, respectively, which were
higher than those observed for IS-2 (Table 2).
Our results indicate that new derivatives endowed with
lower cytotoxicity and a higher selectivity index can be
obtained starting from the lead compound IS-2. In
fact, 3-ethylthio-5-phenyl-4-isothiazolecarbonitrile 4a and
In conclusion, considering the general formula (Table 1)
of the new antiviral agents described, the structural fea-
tures which ensure the best biological pro®le seem to be:
(1) a short thioalkyl chain in the 3-position, (2) a cyano
or methylester group in the 4-position, and (3) a not
substituted phenyl ring in the 5-position. In fact, the
presence of an aryl group or of other aromatic (naphthyl)
or heteroaromatic (pyridyl or thienyl) rings in the 5-
position, as we previously reported,13 the introduction
of polar groups (CONH2 or COOH) in the 4-position,
as well as the presence of substituents in the alkyl chain
of the 3-position or its replacement with a dialkylamino
function have always reduced or eliminated antiviral
activity.
Table 2. Selectivity Index (SI) of e€ective compounds for polio 1 and
ECHO 9
SIa
Compd
Polio
ECHO
IS
4a
4b
4c
4d
6a
6b
6c
9a
9b
2
444
1000
13
20
17
75
70
10
80
200
40
20
8
15
23
1
1111
250
333
25
On the basis of the results of these studies, IS-2 and its
derivatives (compounds 4a and 9a) are strong candidates
for in vivo evaluation as systemic agents for the treat-
ment of enterovirus infections.
aSelectivity Index (SI) was determined for the e€ective compounds
dividing CC50 by IC50
.

Á
C. C. C. Cutrõ et al. / Bioorg. Med. Chem. 7 (1999) 225±230
228
Figure 1. E€ect of time of 9a (10 mM) addition on virus inhibition from single-round replication of polio 1. Time 0=post 2 h absorption period at
4 ꢀC. All values are mean ‹S.D. of three separate assays.
Experimental
3-Propylthio-5-phenyl-4-isothiazolecarbonitrile (4b). Yield
52%; mp 66±68 ꢀC (petroleumether 40±60 ꢀC); IR (KBr)
2212 (CN) cm 1; MS m/e 260 (M), 218, 77.
Melting points were determined on a Buchi 510 appa-
ratus and are not corrected. Elemental analyses for all
new compounds were performed on a C. Erba Model
1106 elemental analyzer and the data of C, H, N and S
are within ‹0.3% of calculated values. Thin-layer
chromatography (TLC) was used to monitor reactions.
IR spectra were recorded as KBr pellets using a Perkin±
Elmer 281 spectrophotometer. Mass spectra (MS) data
were run on a C. Erba/Kratos Ms.
3-Cyanomethylthio-5-phenyl-4-isothiazolecarbonitrile (4c).
Yield 55%; mp 160±162 ꢀC (cyclohexane); IR (KBr)
2251 (CN), 2219 (CN) cm 1; MS m/e 258 (M+1), 231, 32.
3-(2-Hydroxy-ethylthio)-5-phenyl-4-isothiazolecarbonitrile
(4d). Yield 65%; mp 107±110 ꢀC (cyclohexane); IR
(KBr) 3274 (br, OH), 2221 (CN) cm 1; MS m/e 262
(M), 231, 218.
Chemistry
3-Carboxyethylmethylthio-5-phenyl-4-isothiazolecarbo-
nitrile (4e). Yield 51%; mp 123±125 ꢀC (ligroin); IR (KBr)
2218 (CN), 1738 (CˆO) cm 1; MS m/e 304 (M), 231, 77.
General procedure for synthesis of 3-alkylthio-5-phenyl-
4-isothiazolecarbonitriles 4a±h (Scheme 1). A solution
of Na2SÂ9H2O (4.54 mmol) in a mixture of methanol
(50 mL) and water (5 mL) was re¯uxed for 15 min. A
solution of 114 (4.5 mmol) in methanol (20 mL) was
added dropwise and the mixture was re¯uxed for 3 h.
After cooling, the solution was partitioned between
water and diethyl ether and an excess of suitable alky-
lating reagents 3a±h was added to the aqueous layer.
The mixture reaction was stirred at room temperature
for 12 h to yield the compounds 4a±h. The products
obtained were ®ltered, washed with H2O and puri®ed
by crystallization. The following compounds were
obtained:
3-(2-Bromo-ethylthio)-5-phenyl-4-isothiazolecarbonitrile
(4f). Yield 40%; mp 105±106 ꢀC (hexane); IR (KBr)
2210 (CN) cm 1; MS m/e 326 (M+1), 245, 218.
3-(3-Bromo-propylthio)-5-phenyl-4-isothiazolecarbonitrile
(4g). Yield 42%; mp 65±67 ꢀC (hexane); IR (KBr) 2210
(CN) cm 1; MS m/e 340 (M+1), 259, 232.
3-(4-Bromo-butylthio)-5-phenyl-4-isothiazolecarbonitrile
(4h). Yield 42%; mp 64±67 ꢀC (hexane); IR (KBr) 2214
(CN) cm 1; MS m/e 354 (M+1), 273, 218.
3-Ethylthio-5-phenyl-4-isothiazolecarbonitrile (4a). Yield
67%; mp 78±79 ꢀC (petroleumether 40±60 ꢀC); IR (KBr)
2210 (CN) cm 1; MS m/e 246 (M), 231, 77.
General procedure for synthesis of 3-dialkylamino-5-
phenyl-4-isothiazolecarbonitriles 6a±d (Scheme 1). An
ethanol solution of 1 (1.3 mmol) was heated under re¯ux

Á
C. C. C. Cutrõ et al. / Bioorg. Med. Chem. 7 (1999) 225±230
229
with an excess of dialkylamine 5a±d for 2±4 h and
poured into water. The precipitated solid was collected
on a ®lter, washed with ethanol and water to remove
excess amine, and dried to yield the compounds 6a±d.
The following compounds were obtained:
gaseous hydrochloric acid was passed into a re¯uxing
solution of 4-isothiazolecarboxylic acid 8a,b (1.6 mmol)
in absolute methanol for 12 h. Concentration to a small
volume and dilution with diethyl ether gave the pro-
ducts 9a,b. The following compounds were obtained:
3-N,N-Dimethylamino-5-phenyl-4-isothiazolecarbonitrile
(6a). Yield 70%; mp 75 ꢀC (petroleumether 40±60 ꢀC);
IR (KBr) 2205 (CN) cm 1; MS m/e 229 (M), 77, 44.
Methyl 3-methylthio-5-phenyl-4-isothiazolecarboxylate
(9a). Yield 56%; mp 62±63 ꢀC (ligroin); IR (KBr) 1698
(CˆO) cm 1; MS m/e 265 (M), 129, 59.
3-(1-Pyrrolidinyl)-5-phenyl-4-isothiazolecarbonitrile (6b).
Yield 84%; mp 106 ꢀC (petroleumether 40±60 ꢀC); IR
(KBr) 2210 (CN) cm 1; MS m/e 255 (M), 77, 70.
Methyl 3-ethylthio-5-phenyl-4-isothiazolecarboxylate
(9b). Yield 59%; mp 104±105 ꢀC (cyclohexane); IR
(KBr) 1702 (CˆO) cm 1; MS m/e 279 (M), 77, 59.
3-(1-Piperidinyl)-5-phenyl-4-isothiazolecarbonitrile (6c).
Yield 75%; mp 74 ꢀC (ethanol); IR (KBr) 2215 (CN)
cm 1; MS m/e 269 (M), 84, 77.
Biology
Viruses and cells. Poliovirus 1 (Brunhilde strain), echo-
9 (Hill strain), coxsackievirus B1, measles
virus
3-(4-Morpholinyl)-5-phenyl-4-isothiazolecarbonitrile (6d).
Yield 75%; mp 129 ꢀC (ethanol); IR (KBr) 2215 (CN)
cm 1; MS m/e 271 (M), 86, 77.
(Edmonston strain) and adenovirus type 2 were pur-
chased from the American Type Culture Collection
(ATCC) and propagated in human epidermoid carci-
noma larynx cells (HEp-2). Encephalomyocarditis
(EMC strain) and Herpes simplex type 1 (F strain) were
purchased from the ATCC and propagated in mouse
connective tissue cells (L-929) and African green mon-
key kidney cells (Vero), respectively. Cells were kept in a
humidi®ed 5% carbon dioxide atmosphere at 37 ꢀC and
grown in Dulbecco modi®ed Eagle's Minimum Essential
medium (DMEM) supplemented with 6% heat inacti-
vated fetal calf serum (FCS), 200 mg mL 1 of streptomycin
and 200 units mL 1 of penicillin G. For all viruses tested
working stocks were prepared as cellular lysates using
DMEM without FCS (maintenance medium).
General procedure for synthesis of 3-alkylthio-5-phenyl-
4-isothiazolecarboxamides 7a,b (Scheme 2). 3,5-Di-
substituted 4-isothiazolecarboxamides 7a,b were pre-
pared from the corresponding nitriles (1.6 mmol), IS-2
and 4b, respectively, in concentrated sulfuric acid,
warming the solution in a steam bath for 20 min and
allowing the mixture to stand 18 h at room temperature.
The products were precipitated by dilution in ice-water.
The following compounds were obtained:
3-Methylthio-5-phenyl-4-isothiazolecarboxamide (7a).
Yield 92%; mp 208±209 ꢀC (ethyl acetate); IR (KBr)
3382, 3190 (br, NH),1645 (CˆO) cm 1; MS m/e 250
(M), 129, 44.
Test compounds. Compounds were dissolved in DMSO
and diluted in maintenance medium to achieve the ®nal
concentration needed. Dilution of test compounds con-
tained a maximum concentration of 0.01% DMSO,
which was not toxic to our cell lines.
3-Propylthio-5-phenyl-4-isothiazolecarboxamide (7b).
Yield 92%; mp 191±192 ꢀC (cyclohexane); IR (KBr)
3380, 3187 (br, NH), 1645 (CˆO) cm 1; MS m/e 278
(M), 77, 44.
Cell viability. The cytotoxicity of the test compounds
was evaluated by measuring the e€ect produced on cell
morphology and cell growth. Cell monolayers were
prepared in 24-well tissue culture plates and exposed to
various concentrations (mM) of the compounds. Plates
were checked by light microscopy after 12, 24 and 48 h.
Cytotoxicity was scored as morphological alterations
(rounding up, shrinking, detachment). The viability of
the cells was determined by the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method.
Brie¯y, HEp-2, L-929 and Vero cells were prepared in
96-well tissue culture plates and serial concentrations of
the compounds were added. After incubation for 48 h at
37ꢀC, MTT (0.5 mg mL 1) in DMEM without phenol red
was replaced in each well. After 90 min incubation at
37 ꢀC the overlay was removed and isopropanol
(100 mL) was added; plates were then mixed twice to
dissolve the dark blue crystals. The optical density (OD)
was read at 540 and 690 nm on a Titertek Multiscan
MCC/340, within 15 min of adding the isopropanol.15,16
The absorbance at 690 nm was automatically subtracted
from the absorbance at 540 nm, so as to eliminate the
e€ect of non speci®c absorption. Cell viability values
General procedure for synthesis of 3-alkylthio-5-phenyl-
4-isothiazolecarboxylic acids 8a,b (Scheme 2). A mixture
of 4-isothiazolecarbonitriles (1.6 mmol), IS-2 or 4a, of
potassium hydroxide (3.6 mmol), 0.36 mL of water and
5 mL of ethylene glycol was re¯uxed for 48 h. After
cooling, the reaction mixture was poured into 10 mL of
water. A small amount of insoluble material was ®ltered
o€ and the ®ltrate was acidi®ed with dilute hydrochloric
acid to separate the crude acids 8a,b. The following
compounds were obtained:
3-Methylthio-5-phenyl-4-isothiazolecarboxylic acid (8a).
Yield 86%; mp 160 ꢀC (cyclohexane); IR (KBr) 3450
(br, OH), 1693 (CˆO) cm 1; MS m/e 251 (M), 129, 45.
3-Ethylthio-5-phenyl-4-isothiazolecarboxylic acid (8b).
Yield 86%; mp 134±136 ꢀC (cyclohexane); IR (KBr) 3400
(br, OH), 1705 (CˆO) cm 1; MS m/e 265 (M), 77, 45.
General procedure for synthesis of methyl 3-alkylthio-5-
phenyl-4-isothiazolecarboxylates 9a,b (Scheme 2). Dry

Á
C. C. C. Cutrõ et al. / Bioorg. Med. Chem. 7 (1999) 225±230
230
obtained in the presence of the compounds were
expressed as the percentage of those obtained in
untreated controls and were calculated by the following
formula:
E€ect of time of addition of 9a on virus inhibition.
Monolayers of HEp-2 cells were grown to con¯uence
in 24-well plates and inoculated with polio 1 at a MOI
(multiplicity of infection) of 0.1. The plates were incu-
bated for 2 h at 4 ꢀC to ensure synchronous replication
of the viruses, with or without compound 9a (10 mM),
for the adsorption period. The compound was removed
or added at various times after the adsorption period,
respectively, as indicated in Figure 1. The plates were
incubated at 37 ꢀC for 8 h and were then frozen. Virus
yield was determined by plaque assay.
ꢀOD†
ꢀOD†
c
t
 100
ꢀOD†
c
where (OD)c and (OD)t indicated the absorbances of the
untreated cell control and of the test sample, respec-
tively. The 50% cytotoxic dose (CC50), calculated by
dose±response curves and linear regression, was expres-
sed as the concentration of the compound that reduced
the absorbance of the control sample by 50%.
Acknowledgements
Antiviral activity. The antiviral activity was assessed
using a plaque reduction assay. Con¯uent cells were
grown in 6-well tissue culture plates and infected with
300 plaque forming units (PFU) of the virus stock per
well. During and after 1 h of virus adsorption at 37 ꢀC
(30 min for picornavirus), overlay medium containing
1% of methylcellulose with or without the test com-
pound at doses below CC50 was added. After 24 h of
incubation at 37 ꢀC, when the plaques appeared clearly
in virus controls, the overlay was removed and cells
were stained with 1% crystal violet in methanol. The
number of visible plaques was then counted under light
microscopy. The antiviral activity of each compound
was determined as the percentage decrease in the num-
ber of plaques, which was calculated by the following
formula:
We are grateful to the Italian M.U.R.S.T. for ®nancial
support and to Dr. Pucci for the mass spectra.
References and Notes
1. Adams, A.; Freeman, W. A.; Holland, A.; Hossack, D.;
Inglis, J.; Parkinson, J.; Reading, H. W.; Slack, R.; Suther-
land, R.; Wien, R. Nature London 1960, 186, 221.
2. Slack, R.; Wooldridge, K. R. H.; McFadzean, J. A.;
Squires, S. Nature London 1964, 204, 4958.
3. Jones, D. H.; Wooldridge, K. R. H. J. Chem. Soc. 1968,
550.
4. US Patent 3, 055, 907. 1962. Chem. Abstr. 1962, 58, 15517.
5. Machon, Z. Diss. Pharm. Pharmacol. 1969, 21, 325.
6. US Patent 3, 155, 678. 1964. Chem. Abstr. 1965, 62, 2778.
7. US Patent 3, 375, 161. 1968. Chem. Abstr. 1968, 68, 113581.
8. Naik, S. K.; Meher, S. S.; Nayak, A. J. Indian Chem. Soc.
1983, 60, 975.
9. Kuczynski, L.; Kuriata, M.; Ciupka, B. Pol. J. Pharmacol.
Pharm. 1984, 36, 485.
10. Condorelli, P.; Pappalardo, G.; Tornetta, B. Ann. Chim.
(Rome) 1967, 57, 471.
11. Pinizzotto, M. R.; Garozzo, A.; Furneri, P. M.; Castro,
A.; Geremia, E. J. Chemoth. 1991a, 3 (Suppl 4), 486.
12. Pinizzotto, M. R.; Garozzo, A.; Guerrera, F. ; Castro, A.;
La Rosa, M. G.; Furneri, P. M.; Geremia, E. Antiviral Res.
1992, 19, 29.
13. Cutrõ, C. C. C.; Garozzo, A.; Siracusa M. A.; Sarva M. C.;
Tempera, G.; Geremia, E.; Pinizzotto, M. R.; Guerrera, F.
Bioorg. Med. Chem. 1998, 6, 2271.
14. Nakagawa, S.; Okumura, J.; Sakai, F.; Hoshi, H.; Naito,
T. Tetrahedron Lett. 1970, 42, 3719.
15. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
16. Denizot, F.; Lang, R. J. Immunol. Methods 1986, 89, 271.
No: of plaques ꢀcontrol† No: of plaques ꢀtest†
 100
No: of plaques ꢀcontrol†
The compound concentration required to inhibit virus
plaque formation by 50% was expressed as IC50 and
calculated by dose±response curves and linear regres-
sion.
Virucidal activity. To test possible virucidal activity,
equal volumes (0.5 mL) of poliovirus suspension (con-
taining 107 PFU mL 1) and DMEM containing com-
pound 9a (100 and 500 mM) were mixed and incubated
for 2 h at 37 ꢀC. Infectivity was determined by plaque
assay after dilution of the virus below the inhibitory
concentration.