Anti-HIV evaluation of benzo[d]isothiazole hydrazones

Article abstract of DOI:10.1016/j.ejmech.2008.05.030

The synthesis and the anti-HIV-1 activity of novel benzo[d]isothiazole hydrazones are reported. Target compounds tested in MT-4 cells cultures for their anti-HIV properties against wild type HIV-1 and HIV strains carrying clinically relevant mutations (EF

Full text of DOI:10.1016/j.ejmech.2008.05.030

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 1801e1807
http://www.elsevier.com/locate/ejmech
Preliminary communication
Anti-HIV evaluation of benzo[d]isothiazole hydrazones
a
b
b
Paola Vicini a, , Matteo Incerti , Paolo La Colla , Roberta Loddo
*
a Dipartimento Farmaceutico, Universita` degli Studi di Parma, Via G.P. Usberti 27/A, Parma 43100, Italy
b Dipartimento di Scienze e Tecnologie Biomediche, Sez. Microbiologia e Virologia Generale e Biotecnologie Microbiche, Universita` di Cagliari, Cittadella
Universitaria di Monserrato, S.S. 554, Km 4.500, 09042 Monserrato, CA, Italy
Received 1 February 2008; received in revised form 13 March 2008; accepted 23 May 2008
Available online 9 July 2008
Abstract
The synthesis and the anti-HIV-1 activity of novel benzo[d]isothiazole hydrazones are reported. Target compounds tested in MT-4 cells cul-
tures for their anti-HIV properties against wild type HIV-1 and HIV strains carrying clinically relevant mutations (EFVR, Y181C and K103/
Y181C) showed good activity against wild type HIV-1 and against the EFVR mutant. In terms of SAR the relevant result was that, in the class
of benzisothiazole hydrazones, the benzo[d]isothiazol-3(2H )-one moiety (compounds 1 and 4) is an essential structural requirement for the anti-
retroviral activity.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Benzo[d]isothiazole hydrazones; Benzo[d]isothiazol-3-one derivatives; Antiviral activity; Anti-HIV-1 activity
1. Introduction
bioisosters of naturally occurring nucleotides, such as ade-
nine and guanine. Interestingly, compounds sharing a benziso-
thiazole skeleton have shown biological activity [8,9].
Namely, derivatives endowed with antimicrobial [10], anti-
psychotic [11] and anti-human leukocyte elastase [12] activ-
ities have been reported. Moreover, a number of substituted
2-benzisothiazolones also turned out to possess anti-HIV ac-
tivity [13].
We recently studied a new class of benzisothiazole deriva-
tives bearing a hydrazone function at positions 2 or 3 of the
isothiazole nucleus [14]. Many of them showed a very good
antimicrobial activity, and some turned out to be active activ-
ity against penicillin-resistant staphylococci [15,16]. The
whole spectrum of biological activities of hydrazone deriva-
tives has been recently reviewed and examples of compounds
endowed with anti-HIV properties have been reported [17].
Starting from these preliminary remarks, and taking into
account that new leads can emerge from the rational screening
of synthetic heterocyclic compounds, new derivatives belong-
ing to the class of benzisothiazole hydrazones were considered
for anti-HIV activity.
The major drugs for the therapy of acquired immunodefi-
ciency syndrome (AIDS) fall into three families: the nucleo-
side/nucleotide reverse transcriptase inhibitors (NRTIs), the
non-nucleoside reverse transcriptase inhibitors (NNRTIs),
and the protease inhibitors (PIs) [1e3]. Moreover, recently
three new drugs targeted at human immunodeficiency virus
(HIV) entry, cell fusion and integration, have been approved
[4,5]. Highly active antiretroviral therapy (HAART) regimens,
which are based on triple or quadruple combinations of
NRTIs, NNRTIs and PIs, although reducing HIV to very low
levels, are unable to extinguish the infection. Thus, chronic
treatments are necessary, which lead to the emergence of
drug-resistant mutant strains [6,7]. Therefore, new anti-HIV
agents that are effective against the emerging drug-resistant vi-
ral strains are urgently needed.
Our research group has been involved in programs aimed
at more widely exploring the biological activity of benziso-
thiazole derivatives, that can be considered as structural
Herein, we report the synthetic procedures and the results
of cell-based evaluations of cytoxicity and anti-HIV-1 activity
of 94 benzisothiazole hydrazones, 1ae1v, 2ae2u, 3ae3o,
* Corresponding author. Tel.: þ39 521 905051; fax: þ39 521 905006.
E-mail address: paola.vicini@unipr.it (P. Vicini).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.05.030

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P. Vicini et al. / European Journal of Medicinal Chemistry 44 (2009) 1801e1807
4ae4u, 5ae5o, and of two of their starting benzisothiazole
hydrazides, 1 and 2 (Figs. 1 and 2; Schemes 1 and 2).
Given the multitude of enzymes and steps of the virus mul-
tiplication cycle targeted by anti-HIV agents, in order to evi-
dence any potential antiviral activity, we chose to evaluate
our compounds in cell-based, rather than in cell-free enzy-
matic assays; in fact, in our opinion, the former are better
suited for optimization studies of a given lead compound.
With the aim to establish the key structural requirements
for the anti-HIV activity of benzisothiazole hydrazones, our
synthetic strategy was centred on: (1) variation of the alkyli-
dene/arylidene hydrazono group, (2) variation of the substitu-
ent group and position on the benzylidene ring, (3)
modification of the position of the hydrazone moiety on the
isothiazole nucleus and (4) shortening/lengthening of the
bridge between the supporting heterocycle and the hydrazone
function.
were consistent with the assigned structures. In accordance
with our previous experimental observations on analogous
1
compounds, it should be noted that the H NMR spectra of
the benzo[d]isothiazole hydrazones 3o and 5o evidenced reso-
nance signals attributable, in DMSO-d6 solution at room tem-
perature, to the presence of two conformers [18].
3. Biological results and discussion
The activities of the title benzisothiazole hydrazones
against HIV-1 wt and its clinically relevant NNRTI resistant
mutants (EFVR, Y181C and K103/Y181C) were established
by determining their ability to prevent the virus-induced cyto-
pathogenicity in MT-4 cells, as described elsewhere [20]. The
results, detected for compounds of series 1 and 4, are reported
in Table 1. Results were compared with the antiviral activity of
efavirenz, the most active anti-HIV NNRTI used in the clinic.
The results obtained with compounds 2, 3 and 5 are not
shown because none of them displayed antiretroviral activity
at non-cytotoxic concentrations. Interestingly, compounds be-
longing to the above series (2, 3 and 5) showed cytotoxicity for
MT-4 cells at micromolar concentrations; therefore, they had
been investigated as potential antiproliferative agents and their
properties already reported [18].
The results of cell-based assays are expressed as CC50
(compound concentration required to reduce the viability of
mock-infected cells by 50%), or EC50 (compound concentra-
tion required to achieve 50% protection of MT-4 cells from
the HIV-1 induced cytopathogenicity). As shown in Table 1,
among the benzisothiazol-3-one derivatives 1, 1ae1v and
4ae4u, eleven compounds inhibited the cytopatic effect of
HIV-1 wt at non-cytotoxic concentrations. The best EC50
values were 11 and 4.4 mM for 1a and 1d, respectively.
2. Chemistry
The benzisothiazole hydrazones described in this paper
were prepared by following the general procedure outlined
in Schemes 1 and 2. Briefly, all the benzisothiazole hydra-
zones were obtained through condensation of the amino inter-
mediates, namely the cyclic (Scheme 1) or acyclic hydrazides
(Scheme 2) prepared according to the method previously re-
ported by us, with the appropriate aryl- or alkyl- aldehyde
[14e16].
Compounds 1, 1ae1n, 1qe1u, 2, 2ae2t, 3ae3n, 4a, 4ee4l,
and 5ae5n were re-synthesized as previously described by us
[14e16,18]. The new compounds 1o, 1p, 1v, 2u, 3o, 4be4d,
4me4o, 4u and 5o were synthesized in 55e84% yield, through
the same procedure, and then characterized and analysed by el-
emental analysis; their analytical IR and 1H NMR spectral data
O
O
R
R
N
N
R1
N
N
S
S
O
1a-1s (R=H), 4a-4r (R=CH3)
1t (R=H), 4s (R=CH3)
1
a
b
c
d
e
f
R1
4
a
b
c
d
e
f
R1
H
H
O
2-F
2-F
3-F
4-F
N
N
3-F
4-F
S
R
R
2-Cl
2-Cl
3-Cl
3-Cl
g
h
i
4-Cl
g
h
i
4-Cl
1u (R=H), 4t (R=CH3)
2-NO2
3-NO2
4-NO2
2,4-(NO2)2
2-OH
2-NO2
3-NO2
j
j
4-NO2
O
N
k
l
k
l
2,4-(NO2)2
2-OH
10
N
m
n
o
p
q
r
3-OH
4-OH
2,4-(OH)2
3,4-(OH)2
3,4-O-CH2-O
4-OCH3
3-OCH3,4-OH
m
n
o
p
q
r
3-OH
4-OH
S
3,4-(OH)2
3,4-O-CH2-O
4-OCH3
3-OCH3,4-OH
1v (R=H), 4u (R=CH3)
s
Fig. 1. Chemical structures of the benzo[d]isothiazole hydrazones 1ae1v and 4ae4u.

P. Vicini et al. / European Journal of Medicinal Chemistry 44 (2009) 1801e1807
1803
R
R
H
N
H
N
R1
N
N
n
n
O
O
O
N
N
S
S
2s (n=0, R=H), 3m (n=1, R=H), 5m (n=1, R=CH3)
2a-2r (n=0, R=H), 3a-3l (n=1, R=H), 5a-5l (n=1, R=CH3)
R
H
2
a
b
c
d
e
f
R1
3, 5 R1
N
H
a
b
c
d
e
f
H
N
n
2-F
2-Cl
O
N
3-F
3-Cl
S
4-F
4-Cl
2-Cl
2-NO2
3-Cl
3-NO2
2t (n=0, R=H), 3n (n=1, R=H), 5n (n=1, R=CH3)
g
h
i
4-Cl
g
h
i
4-NO2
2-NO2
3-NO2
4-NO2
2,4-(NO2)2
2-OH
2,4-(NO2)2
2-OH
R
H
N
j
j
3,4-O-CH2-O
4-OCH3
3-OCH3,4-OH
k
l
k
l
N
10
n
m
n
o
p
q
r
3-OH
4-OH
3,4-(OH)2
3,4-O-CH2-O
4-OCH3
3-OCH3,4-OH
O
N
S
2u (n=0, R=H), 3o (n=1, R=H), 5o (n=1, R=CH3)
Fig. 2. Chemical structures of the benzo[d]isothiazole hydrazones 2ae2u, 3ae3o and 5ae5o.
Interestingly, nine compounds (1a, 1c, 1g, 1i, 1n, 4d, 4g, 4i,
4m) displayed more potent activity (low micromolar range)
against the EFVR mutant than the wt strain. The same was
true for compound 1i against Y181C, while none of the test
compounds showed better activity against the K183N/Y181C
mutant than against the wt virus. The two starting benzisothia-
zole hydrazides 1 and 2 gave no significantly different results
with respect to their hydrazono derivatives, being inactive at
17 mM (Table 1) and 100 mM (data not shown), respectively.
It is not easy to rule out the influence exerted by the mod-
ulation of the different hydrazono moieties that we have ex-
amined to acquire preliminary SAR information; however, it
resulted clear that, in the class of the benzisothiazole hydra-
zones, the benzo[d]isothiazol-3(2H )-one moiety (series 1
and 4) is an essential structural requirement for the antiretro-
viral activity. On the other hand, the hydrazones of the ben-
zo[d]isothiazol-3-yl- series (2, 3 and 5) display a potential
antiproliferative activity [18]. In accordance with the results
of our previous conformational analysis [18], we suggest
that 1 and 4 (active as antiretroviral compounds), made by
two flat structures connected by three flexible bonds, fit in
the classical NNRTI butterfly model [19]. Our next step
will be of further proving that the RT is the specific molecular
target of the active benzisothiazol-3-one hydrazones and of
exploring the structural requirements aimed at rationally
modifying the chemical scaffold to obtain more potent
derivatives.
4. Conclusion
New analogues of benzisothiazole hydrazones were syn-
thesized and tested in vitro against HIV-1 wt and clinically
relevant NNRTI resistant mutants. A number of compounds
belonging to the benzo[d]isothiazol-3(2H )-one series, 1
and 4, showed antiretroviral activity. Compounds 1a and
1d showed good activity against HIV-1 wild type, while
compounds 1a, 1c, 1g, 1i, 1n, 4d, 4g, 4i and 4m showed
good activity against the EFVR mutant. Only one compound,
1i, showed significant activity against the Y181C mutant.
Unfortunately, no significant improvement in antiretroviral
potency was observed when test compounds were compared
to EFV.
Further studies are in progress to elucidate the mechanism
of action of the active benzisothiazol-3-one hydrazones and to
correlate their molecular structure with their anti-HIV-1
activity.
HN−NH2
R
n
O
O
2a-2u, 3a-3o, 5a-5o
Aryl/Alkyl−CHO
R
N
S
Aryl/Alkyl −CHO
N
NH2
1a-1v, 4a-4u
Aryl = -C6H4-R1, 2-furyl
Alkyl = -CH-CH=CH-C6H5, -(CH2)10-CH3
S
2: R=H, n=0
Aryl = -C6H4-R1, 2-furyl
Alkyl = -CH-CH=CH-C6H5, -(CH2)10-CH3
1: R=H
3: R=H, n=1
4: R=CH3
5: R=CH3, n=1
Scheme 1. The synthesis of the benzo[d]isothiazole hydrazones 1ae1v and
4ae4u. Reaction conditions: EtOH/H2O/HCl/CH3COONa, rt or 70 ꢀC,
60 min.
Scheme 2. The synthesis of the benzo[d]isothiazole hydrazones 2ae2u, 3ae
3o and 5ae5o. Reaction conditions: EtOH/H2O/HCl/CH3COONa, rt or
70 ꢀC, 60 min.

1804
P. Vicini et al. / European Journal of Medicinal Chemistry 44 (2009) 1801e1807
Table 1
Cytotoxicity and anti-HIV activity of compounds 1, 1ae1v and 4ae4ua
`
Universita di Parma, on a ThermoQuest (Italia) FlashEA
1112 Elemental Analyser, for C, H, N, and S. The values
found for C, H, N, S were always ꢁ0.4% of the theoretical
ones. IR spectra, such as KBr pellets, were recorded on
a Jasco FT-IR 300E spectrophotometer (Jasco Ltd., Tokyo,
b
Compound CC50 [mM]
c
EC50 [mM]
MT-4 Wt
EFVRd
Y181C
K103N/Y181C
1
17
17
>17
11
>17
8.6
>17
>17
>20
>15
>17
26
>17
>17
>20
>15
>17
>47
ꢃ100
>26
33
Japan); wave numbers in the IR spectra are given in cmꢂ1
.
1a
1b
1c
1d
1e
1f
1H NMR spectra of the newly synthesized compounds, in
DMSO-d6 solutions, were recorded on a Bruker AC 300 in-
strument at 298 K. Chemical shifts are reported as d (ppm)
relative to TMS as internal standard; coupling constants J
are expressed in Hertz. The reactions were followed by
TLC on F254 silica-gel precoated sheets (Merck) and the pu-
rified compounds each showed a single spot. Solvents, unless
otherwise specified, were of analytical reagent grade or of the
highest quality commercially available. Synthetic starting ma-
terial, reagents and solvents were purchased from Aldrich
Chemical Co.
20
15
ꢃ11
>15
>20
9
17
47
4.4
ꢃ17
24
20
100
>26
19
>100
26
68
68
ꢃ100
ꢃ11
20
1g
1h
1i
2.9
20
11
>100
>100
>100 >100
36
13
29
57
36
1j
45
33
1k
1l
>100
>18
>15
8.4
>100
>18
>15
>18
>21
>44
>17
>19
>15
>18
>18
>17
ꢃ33
>18
>30
>15
100
>100
>18
>15
>18
>21
>44
>17
>19
>15
>18
>18
>17
>44
>18
>30
>15
>100
>59
33
18
15
>18
>15
>18
>21
>44
>17
>19
>15
>18
>18
>17
ꢃ33
>18
>30
>15
82
1m
1n
1o
1p
1q
1r
1s
18
21
>21
>44
>17
>19
>15
>18
>18
>17
ꢃ33
>18
>30
7.3
44
17
5.1.1. General procedure for synthesis of new benzisothiazole
hydrazones 1o, 1p, 1v, 2u, 3o, 4be4d, 4me4o, 4u, 5o
The appropriate hydrazide (5 mmol) was poured into water
(70 mL), under stirring, and hydrochloric acid was added up to
acidic pH; the suspension was then buffered with sodium ac-
etate, and ethanol (20 mL) was added. The suitable aldehyde
(5.7 mmol), dissolved in ethanol (10 mL), was dropped into
the mixture and the reaction was left, while stirring, at room
temperature (60 min) for compounds 4be4d, 4me4o, or
heated at 70 ꢀC (60 min), and then cooled at room tempera-
ture, for compounds 1o, 1p, 1v, 2u, 3o, 4u and 5o. The result-
ing crude product was filtered, washed with water and
recrystallised.
19
15
1t
18
18
1u
1v
4a
4b
4c
4d
4e
4f
17
44
18
30
15
>100
59
57
79
25
>59
24
>59
22
4g
4h
4i
11
>100 >100
>100 48
>100
18
72
>100
32
>100
83
4j
>100 >100
>100 >100
ꢃ100
>100
>30
>16
>23
>49
33
>100
>100
>30
>16
>23
>49
47
4k
4l
>100
>30
8.4
5.1.1.1. 2-(2,4-Dihydroxybenzylideneamino)-benzo[d]isothiazol-
3(2H )-one (1o). Yield: 68%; mp 274e276 ꢀC dec. (EtOH).
TLC: eluent ¼ CH2Cl2/MeOH 9/1. IR(KBr) n: 3328 (OeH),
3070e3035 (aromatic CeH) 2920, 2842 (aliphatic CeH),
30
16
>30
>16
>23
>49
33
4m
4n
4o
4p
4q
4r
4s
23
49
ꢃ23
>49
20
1668 (C]O), 1602 (N]C) cmꢂ1 1H NMR (300 MHz,
.
>100
55
16
>55
>16
>18
>19
>19
>55
>16
>18
>19
>19
3
>55
>16
>18
>19
>19
>55
>16
>18
>19
>19
0.3
DMSO-d6, d ppm): 10.55 (s, 1H, OH); 10.10 (s, 1H, OH);
8.38 (s, 1H, CH); 8.02 (d, 1H, J ¼ 8.1 Hz, H-4); 7.95 (d,
1H, J ¼ 7.9 Hz, H-7); 7.76 (td, 1H, J ¼ 0.9 Hz, J ¼ 7.9 Hz
H-5); 7.58 (d, 1H, J ¼ 8.1 Hz, H-60); 7.49 (td, 1H,
J ¼ 0.9 Hz, J ¼ 7.9 Hz H-6); 6.40e6.37 (m, 2H, H-30, H-50).
Anal. Calcd. for C14H10N2O3S (286.31): C, 58.73; H, 3.52;
N, 9.78; S, 11.20. Found: C, 58.68; H, 3.60; N, 9.52; S,
10.91.
18
19
4t
4u
EFV
19
30
0.002
0.008
Bold values refer to the most significant results. They can be converted in plain
style values.
a
Data represent mean values for the independent determinations.
Compound dose required to reduce the viability of mock-infected cells by
b
50% as determined by the MTT method.
c
5.1.1.2. 2-(3,4-Dihydroxybenzylideneamino)-benzo[d]isothiazol-
3(2H )-one (1p). Yield: 83%; mp 251e252 ꢀC (EtOH). TLC:
eluent ¼ CH2Cl2/MeOH 9/1. IR(KBr) n: 3531 (OeH),
3320e3010 (bonded OeH), 2920, 2852 (aliphatic CeH),
Compound dose required to achieve 50% protection of MT-4 cells from
HIV-1 induced cytopathogenicity, as determined by the MTT method.
d
EFVR ¼ K103 þ V179D þ P225H mutant.
5. Experimental
1666 (C]O), 1598 (N]C) cmꢂ1 1H NMR (300 MHz,
.
DMSO-d6, d ppm): 9.43 (br s, 1H, OH); 9.36 (br s, 1H,
OH); 8.31 (s, 1H, CH); 8.01 (d, 1H, J ¼ 8.7 Hz, H-4); 7.95
(d, 1H, J ¼ 8.7 Hz, H-7); 7.76 (t, 1H, J ¼ 8.2 Hz, H-5); 7.49
(t, 1H, J ¼ 8.0 Hz, H-6); 7.34 (s, 1H, J ¼ 1.8 Hz, H-20); 7.13
(dd, 1H, J ¼ 1.8 Hz, J ¼ 7.9 Hz, H-60); 6.83 (d, 1H,
J ¼ 8.1 Hz, H-50). Anal. Calcd for C14H10N2O3S (286.31):
5.1. Chemistry
Melting points (ꢀC) were determined with a Buchi 512 ap-
paratus and are uncorrected. New compounds were analysed
in the analytical laboratory of the Dipartimento Farmaceutico,

P. Vicini et al. / European Journal of Medicinal Chemistry 44 (2009) 1801e1807
1805
C, 58.73; H, 3.52; N, 9.78; S, 11.20. Found: C, 58.47; H, 3.81;
N, 9.38; S, 10.81.
(EtOH). TLC: eluent ¼ CH2Cl2/EtOH 98/2. IR(KBr) n: 3068
(aromatic CeH), 2980e2845 (aliphatic CeH), 1691 (C]O),
1
1601 (N]C) cmꢂ1. H NMR (300 MHz, DMSO-d6, d ppm):
5.1.1.3. 2-(Dodecylideneamino)-benzo[d]isothiazol-3(2H )-one
(1v). Yield: 72%; mp 38e40 ꢀC (Ligroin). TLC:
eluent ¼ Petroleum ether/AcOEt 1/1. IR(KBr) n: 3062 (aro-
matic CeH) 2930, 2858 (aliphatic CeH), 1675 (C]O),
8.29 (s, 1H, CH); 7.91 (d, 1H, J ¼ 8.1 Hz, H-7); 7.81 (s, 1H,
H-4); 7.71 (d, 1H, J ¼ 7.5 Hz, H-60); 7.66e7.51 (m, 3H, H-
20, H-40, H-6); 7.33 (t, 1H, J ¼ 8.4 Hz, H-50); 2.43 (s, 3H,
CH3). Anal. Calcd. for C15H11FN2OS (286.33): C, 62.92; H,
3.87; N, 9.78; S, 11.20. Found: C, 63.00; H, 4.15; N, 9.48;
S, 11.42.
1
1605 (N]C) cmꢂ1. H NMR (300 MHz, DMSO-d6, d ppm):
7.97 (d, 1H, J ¼ 8.2 Hz, H-4); 7.92 (d, 1H, J ¼ 7.8 Hz, H-7);
7.74 (t, 1H, J ¼ 7.4 Hz, H-5); 7.58 (t, 1H, J ¼ 5.0 Hz, CH);
7.46 (t, 1H, J ¼ 7.4 Hz, H-6); 2.43e2.36 (m, 2H, CH2);
1.55e1.22 (m, 18H, 9 CH2); 0.82 (t, 3H, CH3). Anal. Calcd.
for C19H28N2OS (332.50): C, 68.63; H, 8.49; N, 8.42; S,
9.64. Found: C, 69.04; H, 8.86; N, 8.10; S, 9.98.
5.1.1.8. 2-(4-Fluorobenzylideneamino)-5-methylbenzo[d]iso-
thiazol-3(2H )-one (4d). Yield: 63%; mp 193e194 ꢀC
(EtOH). TLC: eluent ¼ CH2Cl2/EtOH 95/5. IR(KBr) n:
3070e3052 (aromatic CeH), 2973, 2918, 2860 (aliphatic
CeH), 1666 (C]O), 1603 (N]C) cmꢂ1
.
1H NMR
5.1.1.4. N0-Dodecylidenebenzo[d]isothiazole-3-carboxydrazide
(2u). Yield: 55%; mp 65e67 ꢀC (Ligroin). TLC:
eluent ¼ Petroleum ether/AcOEt 1/1. IR(KBr) n: 3190 (Ne
H), 3055 (aromatic CeH) 2918, 2850 (aliphatic CeH), 1662
(300 MHz, DMSO-d6, d ppm): 8.30 (s, 1H, CH); 7.95e7.90
(m, 3H, H-20, H-60, H-7); 7.80 (s, 1H, H-4); 7.62 (d, 1H,
H-6); 7.37e7.31 (m, 2H, H-30, H-50); 2.43 (s, 3H, CH3).
Anal. Calcd. for C15H11FN2OS (286.33): C, 62.92; H, 3.87;
N, 9.78; S, 11.20. Found: C, 63.02; H, 4.08; N, 9.46; S,
11.38.
1
(C]O), 1623 (N]C) cmꢂ1. H NMR (300 MHz, DMSO-
d6, d ppm): 11.90 (s, 1H, NH); 8.71 (d, 1H, J ¼ 8.1 Hz, H-
4); 8.29 (d, 1H, J ¼ 8.1 Hz, H-7); 7.88 (t, 1H, J ¼ 5.5 Hz,
CH); 7.68e7.56 (m, 2H, H-5, H-6); 2.24 (td, 2H,
J ¼ 7.5 Hz, J ¼ 5.5 Hz CH2); 1.44 (quint, 2H, J ¼ 6.9 Hz
CH2); 1.24e1.19 (m, 16H, 8 CH2); 0.80 (t, 3H, J ¼ 6.6 Hz
CH3). Anal. Calcd. for C20H29N3OS (359.53): C, 66.81; H,
8.13; N, 11.69; S, 8.92. Found: C, 67.16; H, 8.42; N, 11.46,
S, 9.24.
5.1.1.9. 2-(3-Hydroxybenzylideneamino)-5-methylbenzo[d]iso-
thiazol-3(2H )-one (4m). Yield: 65%; mp 228e230 ꢀC (Diox-
ane). TLC: eluent ¼ CH2Cl2/MeOH 95/5. IR(KBr) n: 3182
(OeH), 2918, 2843 (aliphatic CeH), 1664 (C]O), 1610
1
(N]C) cmꢂ1. H NMR (300 MHz, DMSO-d6, d ppm): 9.68
(s, 1H, OH); 8.18 (s, 1H, CH); 7.89 (d, 1H, J ¼ 8.1 Hz, H-
7); 7.80 (s, 1H, H-4); 7.62 (d, 1H, J ¼ 8.1 Hz, H-6); 7.32e
7.26 (m, 3H, H-6’, H-20, H-50); 6.90e6.87 (m, 1H, H-40);
2.43 (s, 3H, CH3). Anal. Calcd. for C15H12N2O2S (284.33):
C, 63.36; H, 4.25; N, 9.85; S, 11.28. Found: C, 63.17; H,
4.32; N, 9.55; S, 11.19.
5.1.1.5. 2-(Benzo[d]isothiazol-3-yl)-N0-dodecylideneacetohy-
drazide (3o). Yield: 83%; mp 119e120 ꢀC (Ligroin-EtOH).
TLC: eluent ¼ Petroleum ether/AcOEt 1/1. IR(KBr) n: 3218
(NeH), 3080 (aromatic CeH) 2923, 2862 (aliphatic CeH),
1663 (C]O), 1575 (N]C) cmꢂ1 1H NMR (300 MHz,
.
DMSO-d6, d ppm): 11.39 and 11.13 (2 s, 1H, NH); 8.19e
8.14 (m, 1H, H-4); 8.07 (d, 1H, J ¼ 8.1 Hz, H-7); 7.61e7.53
(m, 1H, H-5); 7.53e7.45 (m, 2H, H-6, CH); 7.33 (t, 1H,
J ¼ 5.2 Hz, CH); 4.35 and 4.04 (2 s, 2H, CH2); 2.20e2.09
(m, 2H, CH2); 1.43e1.36 (m, 2H, CH2); 1.22e1.20 (m,
16H, 8 CH2); 0.84e0.80 (m, 3H, CH3). Anal. Calcd. for
C21H31N3OS (373.56): C, 67.52; H, 8.36; N, 11.25; S, 8.58.
Found: C, 67.16; H, 8.62; N, 11.48; S, 8.92.
5.1.1.10. 2-(4-Hydroxybenzylideneamino)-5-methylbenzo[d]i-
sothiazol-3(2H )-one (4n). Yield: 70%; mp 242e243 ꢀC (Diox-
ane). TLC: eluent ¼ CH2Cl2/EtOH 95/5. IR(KBr) n: 3181
(OeH), 3015 (aromatic CeH), 2963, 2888, 2850 (aliphatic
CeH), 1658 (C]O), 1596 (N]C) cmꢂ1
.
1H NMR
(300 MHz, DMSO-d6, d ppm): 10.05 (s, 1H, OH); 8.19 (s,
1H, CH); 7.87 (d, 1H, J ¼ 8.1 Hz, H-7); 7.78 (s, 1H, H-4);
7.69 (d, 2H, J ¼ 8.4 Hz, H-20, H-60); 7.49 (d, 1H, J ¼ 8.1 Hz,
H-6); 6.86 (d, 2H, J ¼ 8.7 Hz, H-30, H-50); 2.43 (s, 3H, CH3).
Anal. Calcd. for C15H12N2O2S (284.33): C, 63.36; H, 4.25; N,
9.85; S, 11.28. Found: C, 63.06; H, 4.25; N, 9.82; S, 11.18.
5.1.1.6. 2-(2-Fluorobenzylideneamino)-5-methylbenzo[d]iso-
thiazol-3(2H )-one (4b). Yield: 68%; mp 172e174 ꢀC
(EtOH). TLC: eluent ¼ CH2Cl2/EtOH 95/5. IR(KBr) n:
3065e3015 (aromatic CeH) 2960, 2920, 2845 (aliphatic
5.1.1.11. 2-(3,4-Dihydroxybenzylideneamino)-5-methylbenzo[-
d]isothiazol-3(2H )-one (4o). Yield: 62%; mp 240e243 ꢀC
(Dioxane). TLC: eluent ¼ CH2Cl2/EtOH 9/1. IR(KBr) n:
3459 (OH) 3350e3002 (bonded OeH), 2955, 2920, 2843 (al-
CeH), 1675 (C]O), 1601 (N]C) cmꢂ1
.
1H NMR
(300 MHz, DMSO-d6, d ppm): 8.28 (s, 1H, CH); 7.99e7.89
(m, 2H, H-60, H-7); 7.80 (s, 1H, 4); 7.63e7.52 (m, 2H, H-
30, H-50); 7.39e7.32 (m, 2H, H-40, H-6); 2.43 (s, 3H, CH3).
Anal. Calcd. for C15H11FN2OS (286.33): C, 62.92; H, 3.87;
N, 9.78; S, 11.20. Found: C, 62.58; H, 4.11; N, 9.38; S,
11.60.
1
iphatic CeH), 1664 (C]O), 1586 (N]C) cmꢂ1. H NMR
(300 MHz, DMSO-d6, d ppm): 9.51 (s, 1H, OH); 9.35 (s,
1H, OH); 8.09 (s, 1H, CH); 7.86 (d, 1H, J ¼ 8.1 Hz, H-7);
7.78 (d, 1H, J ¼ 1.2 Hz, H-4); 7.49 (dd, 1H, J ¼ 1.2 Hz,
J ¼ 8.1 Hz, H-6); 7.34 (d, 1H, J ¼ 2.1 Hz, H-20); 7.17 (dd,
1H, J ¼ 1.8 Hz, J ¼ 8.1 Hz, H-60); 6.81 (d, 1H, J ¼ 8.1 Hz,
H-50); 2.43 (s, 3H, CH3). Anal. Calcd. for C15H12N2O3S
5.1.1.7. 2-(3-Fluorobenzylideneamino)-5-methylbenzo[d]iso-
thiazol-3(2H )-one (4c). Yield: 56%; mp 190e191 ꢀC

1806
P. Vicini et al. / European Journal of Medicinal Chemistry 44 (2009) 1801e1807
(300.33): C, 59.99; H, 4.03; N, 9.33; S, 10.68. Found: C,
59.51; H, 4.25; N, 8.91; S, 10.29.
5.2.2. HIV titration
Titration of HIV was performed in C8166 cells by the
standard limiting dilution method (dilution 1:2, four replica
wells per dilution) in 96-well plates. The infectious virus ti-
ter was determined by light microscope scoring of syncytia
after 4 days of incubation. Virus titers were expressed as
CCID50/mL.
5.1.1.12. 2-(Dodecylideneamino)-5-methylbenzo[d]isothiazol-
3(2H )-one (4u). Yield: 84%; mp 77e78 ꢀC (Ligroin).
TLC: eluent ¼ Petroleum ether/AcOEt 1/1. IR(KBr) n:
3073 (aromatic CeH) 2952, 2858 (aliphatic CeH), 1665
1
(C]O), 1604 (N]C) cmꢂ1. H NMR (300 MHz, DMSO-
d6, d ppm): 7.85 (d, 1H, J ¼ 8.1 Hz, H-7); 7.74 (s, 1H, H-
4); 7.58e7.54 (m, 2H, H-6, CH); 2.50 (m, 2H, CH2); 2.41
(s, 3H, CH3); 1.58e1.23 (m, 18H, 9 CH2); 0.84 (t, 3H,
CH3). Anal. Calcd. for C20H30N2OS (346.53): C, 69.32; H,
8.73; N, 8.08; S, 9.25. Found: C, 69.70; H, 9.08; N, 7.65;
S, 9.19.
5.2.3. Anti-HIV assays
The activity of test compounds against multiplication of
wild type HIV-1, Y181C, K103N þ Y181C and
K103R þV179D þ P225H (EFVR) in acutely infected cells
was based on inhibition of virus-induced cytopathogenicity
in MT-4 cells. Briefly, an amount of 50 mL of culture medium
containing 1 ꢄ 104 cells was added to each well of flat-bottom
microtiter trays containing 50 mL of culture medium with or
without various concentrations of test compounds. Then, an
amount of 20 mL of HIV suspensions containing the appropri-
ate amount of CCID50 to cause complete cytopathogenicity at
day 4, was added. After incubation at 37 ꢀC, cell viability was
determined by the 3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) method [20]. The cytopathogenic-
ity of test compounds was evaluated in parallel with their
antiviral activity and was based on the viability of mock-in-
fected cells, as monitored by the MTT method.
5.1.1.13. 2-(5-Methylbenzo[d]isothiazol-3-yl)-N0-dodecylide-
neacetohydrazide (5o). Yield: 80%; mp 112e113 ꢀC (Li-
groin). TLC: eluent ¼ Petroleum ether/AcOEt 1/1. IR(KBr)
n: 3205 (NeH), 3064 (aromatic CeH) 2918, 2850 (aliphatic
CeH), 1668 (C]O), 1560 (N]C) cmꢂ1
.
1H NMR
(300 MHz, DMSO-d6, d ppm): 11.37 and 11.12 (2 s, 1H,
NH); 8.06e8.01 (m, 1H, H-7); 7.94 and 7.86 (2 s, 1H, H-4);
7.52 (t, 1H, J ¼ 5.4 Hz, CH); 7.46e7.41 (m, 1H, H-6); 7.33
(t, 1H, J ¼ 5.1 Hz, CH); 4.31 and 4.00 (2 s, 2H, CH2); 2.49
and 2.46 (2s, 3H, CH3); 2.22e2.10 (m, 2H, CH2); 1.43e
1.36 (m, 2H, CH2); 1.23e1.20 (m, 16H, 8 CH2); 0.84e0.80
(m, 3H, CH3). Anal. Calcd. for C22H33N3OS (387.58): C,
68.18; H, 8.58; N, 10.84; S, 8.27. Found: C, 68.54; H, 8.97;
N, 10.46; S, 8.56.
Acknowledgements
Financial support from Italian MUR (Ministero dell’Uni-
`
versita e della Ricerca) is gratefully acknowledged. We are
grateful to CIM (Centro Interdipartimentale Misure) of the
University of Parma for NMR instrumental facilities.
5.2. Antiviral assay procedures
Compounds were solubilized in DMSO at 100 mM and
then diluted in culture medium.
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