Synthesis and antiviral activity of new substituted methyl [2-(arylmethylene-hydrazino)-4-oxo-thiazolidin-5-ylidene]acetates

Article abstract of DOI:10.1002/ardp.201300057

A series of new methyl [2-(arylmethylene-hydrazono)-4-oxo-thiazolidin-5- ylidene]acetates (5a-o) were synthesized via cyclocondensation of thiosemicarbazones (3a-o) with dimethyl but-2-ynedioate (4) in good yields under solvent-free conditions. The environmentally friendly solvent-free protocol overcomes the limitations associated with the customary protracted solution phase synthesis and afforded the title compounds in a few minutes. Compounds 5b-i and 5h-o were evaluated for their antiviral activity against the replication activity of HIV-1 and HIV-2 in MT-4 using the MTT assay. The same compounds were also evaluated in vitro for their selective antiviral activity against hepatitis C virus (HCV) in the Huh 5-2 replicon system (type 1b, Con1 strain).

Full text of DOI:10.1002/ardp.201300057

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
1
Full Paper
Synthesis and Antiviral Activity of New Substituted Methyl
[2-(arylmethylene-hydrazino)-4-oxo-thiazolidin-5-ylidene]acetates
Aamer Saeed1, Najim A. Al-Masoudi2, and Muhammad Latif3
1
Department of Chemistry, Quaid-I-Azam University, Islamabad, Pakistan
Department of Chemistry, College of Science, University of Basrah, Basrah, Iraq
Cancer and Infectious Disease Research Center, Bio-Organic Science Division, Korea Research Institute of
2
3
Chemical Technology, University of Science and Technology, Daejeon, Republic of Korea
A series of new methyl [2-(arylmethylene-hydrazono)-4-oxo-thiazolidin-5-ylidene]acetates (5a–o) were
synthesized via cyclocondensation of thiosemicarbazones (3a–o) with dimethyl but-2-ynedioate (4) in
good yields under solvent-free conditions. The environmentally friendly solvent-free protocol
overcomes the limitations associated with the customary protracted solution phase synthesis and
afforded the title compounds in a few minutes. Compounds 5b–i and 5h–o were evaluated for their
antiviral activity against the replication activity of HIV-1 and HIV-2 in MT-4 using the MTT assay. The
same compounds were also evaluated in vitro for their selective antiviral activity against hepatitis C
virus (HCV) in the Huh 5-2 replicon system (type 1b, Con1 strain).
Keywords: Anti-HCV activity / Anti-HIV activity / Non-nucleoside inhibitors (NNRTIs) / Thiazolidin-4-ones
Received: February 12, 2013; Revised: April 3, 2013; Accepted: April 12, 2013
DOI 10.1002/ardp.201300057
Introduction
rennin inhibitor [17]. In recent years, many research groups
have been engaged in the development of new non-nucleoside
RT inhibitors (NNRTI) having thiazole backbone such as
thiazolo[3,4-a]benzimidazoles (TBZs) and their analogs as
potent anti-HIV agents [18–21], and 1-(2,6-difluorophenyl)-
thiazolo[3,4-a]benzimidazole 2 (Fig. 1) is one example of TBZs
with a highly potential inhibitory of HIV-1-induced cytopathic
effect in a variety of human cell lines, meanwhile it inhibited
the replication of various strains of HIV-1 including
azidovudine resistant strain (G910-6) [21]. Monforte et al.
have reported the synthesis of 2,3-diaryl-1,3-thiazolidin-4-
ones [22] as potent anti-HIV-1 agents, meanwhile Rawal
et al. [23] have synthesized 2-(2,6-dibromophenyl)-3-heteroaryl-
1,3-thiazolidin-4-ones which effectively inhibited HIV-1 repli-
cation at 20–320 nM. Phenethylthiazolethiourea (PTTA)
compounds [24] have been reported as a new class of HIV
RT inhibitors. Further, some derivatives of thiazoles exhibited
remarkable activity against herpes simplex virus (HSV) [25]. A
brief review of thiazoles associated with large number of
biological activities is presented by Siddiqui et al. [26] and
D’hooghe and De Kimpe [27]. In addition, some thiazole
derivatives showed their potency as selective cyclooxygenase-
The global spread and fatal prognosis of human immunode-
ficiency virus (HIV-1) infection, the causative agent in the
transmission and the development of acquired immune
deficiency syndrome (AIDS) [1], emphasize the urgent need for
effective antiretroviral therapies. Combinations of RT nucleo-
side inhibitors (NRTIs), RT non-nucleoside inhibitors (NNRTIs)
and protease inhibitors (PIs), clinically used for the treatment
of HIV infections, dramatically decrease viral load in most
infected persons, but resistance to the currently available
hemotherapeutics invariably emerges [2]. Consequently, there
is a high medical need to develop novel, selective, potent, safe,
inexpensive antiviral agents, also effective against mutant
strains of HIV.
Thiazole and thiazolidines are very useful intermediates/
subunits for the development of molecules of pharmaceutical
or biological interest [3–6] including antibacterial [7–9], anti-
inflammatory [10], antiprotozoal [11, 12], anticonvulsant [13],
anticancer [14, 15] properties, as effective antihypertensive
agent (e.g.: b-(hydroxyethyl)thiazolidines 1; Fig. 1) [16], and
Correspondence: Dr. Najim A. Al-Masoudi, Department of Chemistry,
College of Science, University of Basrah, Basrah, Iraq.
E-mail: najim.al-masoudi@gmx.de
Fax: þ49 7531 34435
Additional correspondence: Dr. Aamer Saeed
E-mail: aamersaeed@yahoo.com
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
A. Saeed et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
group), were synthesized by condensation of thiosemicarba-
zones with dimethyl acetylenedicarboxylate 4. The first
step of the synthesis involved the preparation of a series of
thiosemicarbazones 3a–o by acid catalyzed condensation
of thiosemicarbazide with a range of substituted aromatic
aldehydes. IR spectra of the synthesized hydrazide derivatives
ꢀ1
–
–
revealed the presence of C S stretching at 1602 cm (C N),
–
–
–
1590 (Ar–C C), in addition to stretching at 3385 and
–
3229 cmꢀ1
, indicating the presence of NH2 and NH,
Figure 1. Structures of compounds 1 and 2. 1: 4-(2,4-Dichloro-
phenyl)-3-(6-methylpyridin-2-yl)thiazolidin-2-one. 2: Thiazolobenzi-
midazole (TBZ).
respectively. The 1H NMR spectra of 3a–o showed a singlet
–
at ꢁ d 8.11 ppm, assigned to azomethine (CH N) protons
–
while the singlets at ꢁd 10.40 and 7.50 ppm were attributed
2 [28], as well as bacterial DNA gyrase B [29] inhibitors, while
some organosilicon-containing thiazole derivatives have
been reported as potential lipoxygenase inhibitors and anti-
inflammatory agents [30]. We have recently shown that Schiff
base derivatives of some thiazolidines are potential urease
inhibitors [31].
In continuation of our attempts in searching for new anti-
HIV agents [32] and on the basis of above promising biological
results, we considered thiazolidines and their analogs
particularly interesting to optimize the synthetic approaches
to our antiviral agents. In this study, we prepared a new
series of thiazolidines with evaluation of their anti-HIV and
anti-hepatitis virus (HCV) activity.
for NH and NH2 protons, respectively. In the 13C NMR spectra,
–
the signals at ꢁd 179.0 and 153 ppm were assigned to C S
–
–
–
and C N carbon atoms, respectively (Scheme 1).
Dimethyl acetylenedicarboxylate 4 is a highly electrophilic
reagent and widely employed as a dienophile in cycloaddition
reactions, such as the Diels–Alder reaction and behaving as
Michael acceptor in organic transformations. Next, treatment
of an equimolar mixture of thiosemicarbazones 3a–o with 4,
in the absence of any solvent or catalyst, afforded thiazoli-
nones 5a–o in good to excellent yields (Scheme 2).
The structures of 5a–o were assigned on the basis of their
1H and 13C NMR, and mass spectra. In the 1H NMR spectra of
5a–o, the down-field singlets in the region d 13.02–8.05 ppm
were assigned to NH ring proton, while the azomethine
–
Results and discussion
protons (C2
H N) appeared in the region d 9.35–
–
thiazol
–
7.48 ppm. The olefinic protons (CH C) resonated in the
–
Chemistry
region d 6.72–6.40 ppm, while the singlets in the region
d 3.90–3.78 ppm were attributed to the protons acetoxy
group. In the 13C NMR spectra, carbonyl carbon atoms of the
A new series of thiazole derivatives, incorporating two
important pharmacophores (thiazole and imino/hydrazono
Scheme 1. Synthesis of 1-(substituted benzylidene)thiosemicarbazides 3a–o.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
Anti-HIV Activity of 4-Oxo-thiazolidin-5-ylidene acetate Derivatives
3
Scheme 2. Synthesis of methyl [(2-arylmethylene)-hydrazino)-4-oxo-thiazolidin]-acetates 5a–o.
thiazole ring and the acetoxy group appeared in the regions
d 174.0–170.1 ppm and d 168.5–164.5 ppm, respectively,
whereas C-2 of the thiazole ring resonated in the region d
159.4–156.3 ppm. The resonances in the region d 149.6–
145.2 ppm were assigned to azomethine carbon atoms
–
are summarized in Table 1, in which the data for nevirapine
(BOE/BIRG587) [35, 36] and azidothymidine (DDN/AZT) [37]
were included for comparison. Compound 5i was found to be
the only compound in the series inhibiting HIV-1 and HIV-2
replication in cell cultures with EC50 of >0.088 mM, but no
selectivity was witnessed (SI < 1). However, implantation of
hydroxy and nitro in 2 and 5 positions of the phenyl group
considerably increased the anti-HIV activity, in comparison
to the effectiveness of other functional groups.
attached to the aromatic rings (ArCH N), except those of
–
compounds 5i and 5l which appeared at d 160.3 and
5
–
–
158.4 ppm, respectively. Olefinic carbon atoms (Cthiazol
CH) were observed in the region d 141.9–137.9 ppm, while C-5
of the thiazole ring appeared in the region d 133.6–131.1 ppm.
Aromatic carbon atoms and carbons of the other substituents
were fully assigned (cf. Experimental section).
Compound 5e was selected for further NMR studies. In the
gradient-selected HMBC spectrum [33] of 5e, the olefinic
In vitro anti-HCV assay
Compounds 5b–i and 5k–o were evaluated for their in vitro
selective antiviral activity against hepatitis C virus in the Huh
5-2 replicon system (type 1b, Con1 strain). The results are
shown in Table 2.
The establishment of stable HCV subgenomic replicon
systems [38, 39] in the human hepatoblastoma cell line Huh7
has provided a useful system for the development of new
antiviral approaches against HCV [40–43]. The overriding
aims of the new therapeutic strategies are higher efficacy
proton (CH C) at d ¼ 8.14 ppm showed two 2JC,H couplings:
–
–
H
one to C-5 of the thiazole ring at dC 131.4 ppm and the other
coupling was with the carbonyl carbon atom of the ester
group at dC 167.5 ppm, respectively. A 3JC,H between the same
–
proton (CH C) with carbonyl carbon atom of the thiazole ring
–
2
at dC 171.0 ppm was observed. Furthermore, a JC,H coupling
between azomethine proton at dH 6.60 ppm and aromatic
carbon atom (C-1) at dC 136.8 ppm was assigned (Fig. 2).
Mass spectral data and elemental analyses are also in
accordance with the proposed structure of compounds 5a–o.
Bioactivities
In vitro anti-HIV assay
Compounds 5a–i and 5k–o were evaluated for their in vitro
anti-HIV-1 (strain IIIB) and anti-HIV-2 (strain ROD) activity and
monitored by the inhibition of the virus induced cytopathic
effect in the (MT-4) cells, based on MTT assay [34]. The results
Figure 2. JC,H correlations in the HMBC NMR spectrum of 5e.
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4
A. Saeed et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
Table 1. In vitro anti-HIV-1a) and HIV-2b) activity and cytotoxicity of some new 1,3-thiazolidin-4-ones.
HIV-1 (IIIB)
HIV-2 (ROD)
CC50
SIe)
(IIIB)
SIe)
(ROD)
Entry
EC50 (mM)c)
EC50 (mM)c)
(mM)d)
5b
5c
5d
5e
>61.30
>72.10
>78.70
>4.23
>61.30
>72.10
>78.70
>4.23
61.30
72.10
78.70
4.23
<1
<1
<1
<1
<1
<1
<1
<1
5f
5g
5h
5i
5k
5l
5m
5n
>11.15
>52.70
>87.25
>0.088
>14.50
>14.35
>97.45
>13.45
>57.53
0.050
>11.15
>52.70
>87.25
>0.088
>14.50
>14.35
>97.45
>13.45
>57.53
>4.00
11.15
52.70
87.25
0.088
14.50
14.35
97.45
13.45
57.53
>4.00
>25
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
5o
<1
<1
<1
Nevirapine
AZT
>80
>11,363
0.0022
0.00094
>26,596
a)
Anti-HIV-1 activity measured with strain IIIB.
Anti-HIV-2 activity measured with strain ROD.
Compound concentration required to achieve 50% protection of MT-4 cells from the HIV-1 and 2-induced cytopathogenic effect.
Compound concentration that reduces the viability of mock-infected MT-4 cells by 50%.
SI: selectivity index (CC50/EC50).
b)
c)
d)
e)
associated with shortened duration of treatment, favorable
mode of administration, and thus improved tolerability and
adherence.
criteria of a selective inhibitor of virus replication in this
assay (i.e. >70% inhibition at concentrations that do not
elicit an anti-metabolic effect on the host cells).
However, compounds 5d and 5g showed EC50 of 3.15 and
3.26 mM with CC50 of 29.2 mM and >50 mM, resulting in a Conclusion
selectivity index of 9.27 and >15.4, in addition to inhibition
of 89.8 and 77%, respectively.
In conclusion, we report the in vitro antiviral activity against
Even though for many compounds an EC50 was obtained
with selectivity index (SI) up to >15.4 (e.g. compound 5g)
(Table 2), none of the compounds matched the selection
HIV-1 (IIIB strain) and HIV-2 (ROD strain) and HCV (type 1b,
Con1 strain) of methyl [2-(arylmethylene-hydrazono)-4-oxo-
thiazolidin-5-ylidene]-acetates 5a–i and 5j–o. The first results
Table 2. In vitro anti-HCV-1b activity, cytotoxicity, and inhibition% of some thiazolidin-4-ones.
Entry
CC50 mM
EC50 mM
SI
% Inh
at []
34
31.7
12.8
48.9
9.6
20.9
41.6
4.79
31.4
12.7
50
12.8
30.1
5b
5c
5d
5e
5f
5g
5h
5i
5k
5l
5m
5n
5o
>50
¼8.28
¼14.7
¼31.5
¼18
¼6.04
¼2.52
¼9.27
>2.78
¼7.88
>15.4
¼3.47
¼3.01
¼1.18
¼6.11
>3.91
¼9.27
¼1.81
76.1
64.9
89.8
80.2
71.8
77
78.8
64.5
54.1
82.8
81.5
89.9
62.6
¼36.9
¼29.2
>50
¼23.2
¼2.95
¼3.26
¼14.4
¼1.94
¼22.2
¼3.9
>50
>50
¼5.86
¼26.2
¼23.9
>50
¼12.8
¼8.8
¼25.7
¼29.9
¼16.5
CC50 ¼ 50% cytostatic/cytotoxic concentration (concentration at which 50% adverse effect is observed on the host cell).
EC50 ¼ 50% effective concentration (concentration at which 50% inhibition of virus replication is observed).
SI ¼ Selectivity index (CC50/EC50).
[] ¼ Concentration [mM].
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
Anti-HIV Activity of 4-Oxo-thiazolidin-5-ylidene acetate Derivatives
5
–
–
showed that 5i has antiviral properties against HIV with
EC50 ¼ 0.088 mM. Although no selectivity was observed
(SI < 1) for 5i, it was the agent of choice for further
pharmacological evaluation. In addition, 5d and 5g exhibited
anti-HCV activity (EC50 ¼ 3.15 and 3.26 mМ with SI ¼ 9.27
and >15.4, respectively, but none of these compounds
matched the selection criteria of a selective inhibitor of virus
replication in this assay. However, introduction of hydroxyl
and nitro groups in positions 2 and 5 of the phenyl group,
respectively, considerably increased the anti-HIV but left
the anti-HIV activity intact (compound 5i).
(Cthiazol2), 149.2 (ArCH N), 139.2 (C5 CH), 135.4 (Carom1),
–
–
131.1 (Cthiazol5), 121.3 (Carom6), 116.4, (Carom4), 111.5 (Carom2),
56.3 (Carom-OMe), 53.3 (CO2Me). LC–MS [MþH]þ calcd. for
C14H13N3O4S 319.06; found 320.16. Anal. calcd. for C14H13N3O4S
(319.34): C, 52.66; H, 4.10; N, 13.16. Found: C, 52.61; H, 4.17;
N, 13.10.
Methyl 2-(2-(4-methoxybenzylidene)hydrazino)-4-oxo-
thiazolidin-5-ylidene)acetate 5c
From 3c (180 mg). Yield: 218 mg (79%) as a yellow solid, m.p.:
198°C. H NMR (DMSO-d ): d 11.29 (s, 1H, NH), 8.07 (s, 1H, CH N),
1
–
–
6
7.72 (s, 2H, J ¼ 8.5 Hz, Ar-H), 6.93 (d, 2H, J ¼ 8.5 Hz, Ar-H), 6.65
–
–
(s, 1H, CH C), 3.37 (s, 3H, 4-MeO-Ph), 3.81 (s, 3H, s, CO Me);
2
13C NMR (DMSO-d6): d 172.3 (Cthiazol O), 166.7 (CO Me), 158.5
–
–
2
–
–
2
Experimental
–
2
(Cthiazol þ Carom-OMe), 149.1 (ArCH N), 139.4 (C CH), 131.2
–
5
3
(Cthiazol5), 128.2 (Carom1), 127.3 (Carom þ Carom6), 114.5 (Carom
þ
Carom5), 55.3 (Carom-OMe), 54.3 (CO2Me). LC–MS (MþHþ) calcd. for
General
C
14H13N3O4S 319.06; found 320.13; Anal. calcd. for C14H13N3O4S
Melting points are uncorrected and were measured on a digital
Gallenkamp (SANYO) model MPD BM 3.5 apparatus. Nuclear
magnetic resonance (1H NMR and 13C NMR) spectra were recorded
on a Varian Gemini 300 [300 MHz (1H), 75 MHz (13C)] spectro-
meter or Bruker NMR [300 MHz (1H)] spectrometer, using CDCl3
as the solvent or otherwise specified, and chemical shifts are
reported in parts per million downfield from the TMS internal
standard or as described. LCMS were performed on an Agilent
1200 series LC system. All compounds were purified by thick layer
chromatography using silica gel from Merck.
(319.34): C, 52.66; H, 4.10; N, 13.16%. Found: C, 52.80; H, 4.01;
N, 13.01.
Methyl 2-(2-(2-chlorobenzylidene)hydrazino)-4-
oxothiazolidin-5-ylidene)acetate 5d
Form 3d (213 mg). Yield: 290 mg (90%) as a yellow solid,
m.p.: 198°C. 1H NMR (DMSO-d6): d 10.45 (s, 1H, NH), 8.14 (s,
–
–
1H, CH N), 7.78 (d, 2H, J ¼ 7.4 Hz, Ar-H), 7.66–7.70 (m, 2H, Ar-H),
6.60 (s, 1H, CH C), 3.81 (s, 3H, CO Me). 13C NMR (DMSO-d6): d 171.0
–
–
2
2
–
–
–
–
(Cthiazol O), 167.5 (CO Me), 159.3 (C
(C5 CH), 136.8 (C
), 146.5 (ArCH N), 141.9
2
thiazol
–
–
General method for the preparation of methyl 2-(2-
(arylidenehydrazino)-4-oxo-thiazolidin-5-ylidene)acetates
5a–o
A thoroughly mixed uniform mixture of substituted benzylidene
thiosemicarbazides (3a–o) (1.0 mmol) and dimethyl acetylenedi-
carboxylate 4 (142 mg, 1.0 mmol) was stirred for 1–5 min under
MWI. The progress of reaction was monitored by TLC (hexane/
ethyl acetate 4:1). After the reaction was completed, the solution
was diluted with EtOH and the mixture was filtered. The
precipitate was recrystallized from EtOH to afford the esters (5a–
o) in good yields.
1), 133.8 (Carom–Cl), 133.8 (Carom4), 131.2
arom
(Cthiazol5), 129.8, 129.1, 122.6 (3 ꢂ Carom), 52.7 (CO2Me). LC–MS
[MþH]þ calcd. for C13H10BrN3O3S 323.01; found 324.12. Anal.
calcd. for C13H10ClN3O3S (368.21): C, 42.41; H, 2.74; N, 11.41.
Found: C, 42.80; H, 2.69; N, 11.21.
Methyl 2-(2-(4-chlorobenzylidene)hydrazino)-4-
oxothiazolidin-5-ylidene)acetate 5e
Form 3e (200 mg). Yield: 220 mg (73%) as a yellow solid, m.p.:
198°C. H NMR (DMSO-d ): d 9.35 (s, 1H, NH), 8.10 (s, 1H, CH N),
1
–
–
6
7.80 (d, 2H, J ¼ 7.5 Hz, Ar-H), 7.70 (d, 2H, J ¼ 7.5 Hz, Ar-H), 6.70
(s, 1H, CH C), 3.80 (s, 3H, CO Me). 13C NMR (DMSO-d6): d 171.2
–
–
2
2
–
–
–
Methyl (2-(2-benzylidenehydrazino)-4-oxo-thiazolidin-5-
ylidene)acetate 5a
From 3a (179 mg). Yield: 268 mg (93%) as a yellow solid, m.p.:
(Cthiazol O), 167.5 (CO Me), 159.4 (C
), 146.5 (ArCH N),
–
2
thiazol
1
141.8 (C5 CH), 137.8 (Carom–Cl), 133.2 (Carom þ Cthiazol5), 129.8,
–
–
122.6 (4 ꢂ Carom), 53.7 (CO2Me). LC–MS [MþH]þ calcd. for
C13H10BrN3O3S 323.03; found 324.10. Anal. calcd. for
C13H10ClN3O3S (368.21): C, 42.41; H, 2.74; N, 11.41. Found: C,
42.80; H, 2.69; N, 11.21.
1
–
203°C. H NMR (DMSO-d ): d 9.05 (s, 1H, NH), 8.42 (s, 1H, CH N),
–
6
7.51–7.80 (m, 5H, Ar-H), 6.60 (s, 1H, CH C), 3.80 (s, 3H, CO Me). 13C
–
–
2
–
d 170.1 (Cthiazol O), 165.7 (CO2Me), 156.3
–
NMR (DMSO-d6):
2
–
–
–
–
(Cthiazol. ), 145.2 (ArCH N), 140.2 (C CH), 135.2 (C
1), 132.2
arom
5
(Cthiazol5), 131.2, 128.3, 126.5 (Carom), 53.3 (CO2Me). LC–MS [MþH]þ
calcd. for C14H13N3O4S 289.05, found 290.08. Anal. calcd. for
C13H11N3O3S (289.31): C, 53.97; H, 3.83; N, 14.52. Found: C, 54.03;
H, 3.87; N, 14.61.
Methyl 2-(2-(3-bromobenzylidene)hydrazino)-4-oxo-
thiazolidin-5-ylidene)acetate 5f
From 3f (258 mg). Yield: 301 mg (82%) as a yellow solid, m.p.:
201°C. H NMR (DMSO-d ): d 11.55 (s, 1H, NH), 8.24 (s, 1H, CH N),
1
–
–
6
7.73 (d, 2H, J ¼ 7.4 Hz, Ar-H), 7.61 (d, 1H, J ¼ 7.5 Hz, Ar-H), 7.39
Methyl 2-(2-(3-methoxybenzyledine)hydrazino)-4-oxo-
–
(d, 1H, J ¼ 7.3 Hz, Ar-H), 6.61 (s, 1H, CH C), 3.83 (s, 3H, CO Me).
–
2
–
thiazolidin-5-ylidene)acetate 5b
13C NMR (DMSO-d6): d 174.0 (Cthiazol O), 168.5 (CO Me), 158.3
–
2
2
(Cthiazol ), 149.5 (ArCH N), 139.9 (C CH), 138.8 (C
1), 133.9
–
–
From 3b (209 mg). Yield: 298 mg (93%) as a yellow solid, m.p.:
–
–
5
arom
(Carom4), 133.9 (Carom2), 131.8 (Cthiazol5), 129.8 (2 ꢂ Carom), 122.6
(Carom-Br), 52.1 (CO2Me). LC–MS [MþH]þ calcd. for C13H10BrN3O3S
366.96; found 367.59. Anal. calcd. for C13H10BrN3O3S (368.21):
C, 42.41; H, 2.74; N, 11.41. Found: C, 42.80; H, 2.69; N, 11.21.
1
–
192°C. H NMR (DMSO-d ): d 8.05 (s, 1H, NH), 7.48 (s, 1H, CH N),
–
6
–
–
7.41 (d, 2H, J ¼ 8.5 Hz, Ar-H), 7.01 (s, 1H, Ar-H), 6.70 (s, 1H, CH C),
3.91 (s, 3H, 3-MeO-Ph), 3.85 (s, 3H, CO2Me). 13C NMR (DMSO-d6):
–
d 172.3 (Cthiazol O), 166.7 (CO Me), 160.2 (Carom-OMe), 158.4
–
2
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
A. Saeed et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
2
Methyl 2-(2-(2-chloro-6-fluorobenzylidene)hydrazino)-4-
oxo-thiazolidin-5-ylidene)acetate 5g
From 3g (232 mg). Yield: 270 mg (79%) as a yellow solid, m.p.:
134.9 (Carom1), 134.2 (Cthiazol5), 103.5, 102.1 (Carom þ Carom6), 57.3
(2 ꢂ Carom-OMe), 53.3 (CO2Me). LC–MS [MþH]þ calcd. for C15H15N3O5S
365.36; found 366.09. Anal. calcd. for C15H15N3O6S (365.36): C, 49.31;
H, 4.14; N, 11.50. Found. C, 50.01; H, 4.19; N, 11.41.
1
–
211°C. H NMR (DMSO-d ): d 11.70 (s, 1H, NH), 8.29 (s, 1H, CH N),
–
6
7.36–7.45 (m, 3H, Ar-H), 6.72 (s, 1H, CH C), 3.78 (s, 3H, CO Me). 13C
–
–
2
–
–
NMR (DMSO-d6): d 174.0 (Cthiazol O), 165.7 (CO Me þ Carom-F),
Methyl 2-(2-(2-(benzyloxy)benzylidene)hydrazino)-4-oxo-
2
158.3 (C2
), 149.6 (ArCH N), 139.9 (C CH), 138.8 (C
1),
arom
–
–
–
–
thiazol
5
thiazolidin-5-ylidene)acetate 5l
133.9 (Carom4), 133.9 (Carom2), 131.9 (Cthiazol5), 129.8 (2 ꢂ Carom),
From 3l (105 mg). Yield: 98 mg (67%) as a yellow solid, m.p.: 203°C.
H NMR (DMSO-d ): d 12.73 (s, 1H, NH), 8.55 (s, 1H, CH N), 8.11 (s, 1H,
122.6 (Carom–Br), 52.1 (CO2Me). LC–MS [MþH]þ calcd. for
1
–
–
6
C
13H9ClFN3O3S 341.00; found 342.13. Anal. calcd. for
Ar-H), 7.87 (s, 1H, Ar-H), 7.36–7.49 (m, 5H, Ar-H), 7.10–6.98 (m, 2H,
Ar-H), 6.67 (s, 1H, CH C), 5.23 (s, 2H, OCH ), 3.82 (s, 3H, CO Me). 13C
C13H9ClFN3O3S (341.5): C, 45.69; H, 2.65; N, 12.30. Found: C,
45.71; H, 2.60; N, 12.17.
–
–
2
2
2),
arom
–
–
NMR (DMSO-d6): d 173.5 (Cthiazol O), 165.8 (CO Me), 162.3 (C
2
2
5), 136.3,
–
–
–
–
158.4 (Cthiazol þ ArCH N), 139.7 (C CH), 134.7 (C
5
thiazol
Methyl 2-(2-(3-(dimethylamino)benzylidene)hydrazino)-4-
oxo-thiazolidin-5-ylidene)acetate 5h
From 3h (150 mg). Yield: 170 mg (76%) as a yellow solid, m.p.:
132.3, 128.9, 127.7, 127.1, 123.7, 121.1, 114.3 (Carom), 72.3 (OCH2),
53.1 (CO2Me). LC–MS [(MþH]þ calcd. for C20H17N3O4S 395.09; found
396.19. Anal. calcd. for C20H17N3O4S (395.43): C, 60.75; H, 4.33;
N, 10.63. Found: C, 60.77; H, 4.31; N, 10.67.
1
–
215°C. H NMR (DMSO-d ): d 8.34 (s, 1H, s, CH N), 7.62 (d, 2H,
–
6
–
J ¼ 8.4 Hz, Ar-H), 6.78 (d, 2H, J ¼ 8.4 Hz, Ar-H), 6.61 (s, 1H, CH C),
–
3.79 (s, 3H, CO2Me), 3.01 (s, 6H, NMe2). 13C NMR (DMSO-d6): d 172.5
Methyl 2-(2-(4-methoxybenzylidene)hydrazino)-4-oxo-
2), 151.8 (Carom4-NMe2),
–
(Cthiazol O), 166.5 (CO Me), 156.4 (C
–
2
thiazol
thiazolidin-5-ylidene)acetate 5m
2
–
–
–
–
147.4 (ArCH N), 138.7 (C CH), 134.9, 134.5 (C
þ Carom6),
5
arom
From 3m (120 mg). Yield:137 mg (82%) as a yellow solid, m.p.: 217°C.
H NMR (DMSO-d): d 13.02 (s, 1H, NH), 8.72 (s, 1H, CH N), 8.63 (s, 1H,
Ar-H), 8.51 (s, 1H, Ar-H), 8.15–8.35 (m, 5H, Ar-H), 7.72–7.83 (m, 2H,
133.6 (Cthiazol5), 118.4 (Carom1), 111.3 (2 ꢂ Carom), 55.1 (CO2Me),
41.4 (NMe2). LC–MS [MþH]þ calcd. for C15H16N4O3S 332.09; found
333.23. Anal. calcd. for C15H16N4O3S (332.38): C, 54.20; H, 4.85;
N, 16.86. Found: 54.08; H, 4.92; N, 16.91.
1
–
–
–
–
Ar-H), 6.70 (s, 1H, CH C), 5.20 (s, 2H, OCH ), 3.80 (s, 3H, CO Me);
2
2
13C NMR (DMSO-d6): d 172.5 (Cthiazol O), 165.8 (CO Me), 162.3 (C
4),
–
–
2
arom
2
–
–
–
–
157.4 (Cthiazol ), 146.4 (ArCH N), 140.7 (C CH), 136.4 (Carom), 131.3
5
Methyl 2-((2-(2-hydroxy-4-nitrobenzylidene)hydrazino)-4-
(Cthiazol5), 129.4, 128.9, 127.7, 127.1, 126.3, 114.3 (Carom), 70.3 (OCH2),
54.1 (CO2Me). LC–MS [MþH]þ calcd. for C20H17N3O4S 395.05; found
396.15. Anal. calcd. for C20H17N3O4S: C, 60.75; H, 4.33; N, 10.63.
Found: C, 60.83; H, 4.29; N, 10.67.
oxo-thiazolidin-5-ylidene)acetate 5i
From 3i (100 mg). Yield: 112 mg (77%), as a yellow solid, m.p.:
1
–
226°C. H NMR (DMSO-d ): d 12.40 (s, 1H, NH), 8.72 (s, 1H, CH N),
–
6
8.58 (s, 1H, Ar-OH), 7.66 (d, 2H, J ¼ 5.4 Ar-H), 7.10 (s, 1H, Ar-H), 6.72
(s, 1H, CH C), 3.90 (s, 3H, CO Me). 13C NMR (DMSO-d6): d 173.5
–
–
Methyl 2-(2-(2-bromobenzylidene)hydrazino)-4-oxo-
2
2
–
–
(Cthiazol O), 164.5 (CO Me), 161.3 (C
-OH), 160.3 (ArCH N),
–
–
–
2
arom
thiazolidin-5-ylidene)acetate 5n
158.4 (Cthiazol2), 151.7 (Carom3-NO2), 139.8 (C5 CH), 131.5 (C
5),
–
thiazol
From 3n (137 mg). Yield: 150 mg (77%) as a yellow solid, m.p.:
124.6 (Carom5), 124.6 (Carom6), 118.4 (Carom1), 111.3 (Carom3), 54.1
(CO2Me). LC–MS [MþH]þ calcd. for C13H10N4O6S 350.03; found
351.09. Anal. calcd. for C13H10N4O6S (350.31): C, 44.57; H, 2.88; N,
15.99. Found: 44.61; H, 2.92; N, 16.01.
1
–
203°C. H NMR (DMSO-d ): d 11.55 (s, 1H, N), 8.24 (s, 1H, CH N),
–
6
7.73 (d, 2H, J ¼ 7.4 Hz, Ar-H), 7.61 (d. 1H, J ¼ 7.5 Hz, Ar-H), 7.39
–
(d, 1H, J ¼ 7.3 Hz, Ar-H), 6.61 (s, 1H, CH C), 3.83 (s, 3H, CO Me).
–
2
13C NMR (DMSO-d6): d 173.0 (Cthiazol O), 168.5 (CO Me), 158.3
–
–
2
2
1), 133.8
–
–
(Cthiazol ), 149.5 (ArCH N), 139.9 (C CH), 135.4 (C
–
–
5
arom
(Carom3), 131.8 (Cthiazol5), 130.2, 127.8, 127.1 (3 ꢂ Carom), 121.6
(Carom-Br), 52.1 (CO2Me). Anal. calcd. for C13H10BrN3O3S: C, 42.41;
H, 2.74; N, 11.41. Found: C, 42.38; H, 2.76; N, 11.31.
Methyl 2-(-2-(4-nitrobenzylidene)hydrazino)-4-
oxothiazolidin-5-ylidene)acetate 5j
Form 3j (224 mg). Yield: 270 mg (81%), as a yellow solid, m.p.:
1
–
210°C. H NMR (DMSO-d ): d 9.35 (s, 1H, CH N), 7.60 (d, 2H,
–
6
–
Methyl 2-(2-(cyclohexylmethylene)hydrazino)-4-oxo-
thiazolidin-5-ylidene)acetate 5o
From 3o (98 mg). Yield: 110 mg (70%) as a yellow solid, m.p.: 225°C.
J ¼ 8.4 Hz, Ar-H), 6.78 (d, 2H, J ¼ 8.5 Hz, Ar-H), 6.60 (s, 1H, CH C),
–
3.79 (s, 3H, CO2Me). 13C NMR (DMSO-d6): d 170.5 (Cthiazol O), 167.5
–
–
(CO2Me), 159.4 (Cthiazol2), 151.8 (Carom -NO ), 149.4 (ArCH N),
3
–
–
2
1), 131.8 (Cthiazol5), 134.9, 134.5, 127.5, 123.3
1
–
–
137.9 (C5 CH þ C
–
arom
H NMR (DMSO-d ): d 8.50 (s, 1H, NH), 7.80 (s, 1H, CH N), 6.40 (s, 1H,
–
6
(Carom), 53.1 (CO2Me). LC–MS [MþH]þ calcd. for C13H10N4O5S
334.03; found 335.13. Anal. calcd. for C13H10N4O5S (334.31): C,
46.71; H, 3.02; N, 16.76. Found: C, 47.01; H, 3.12; N, 16.82.
–
CH C), 3.77 (s, 3H, CO Me), 2.40 (m, 1H, C
1), 1.13–1.49
–
2
cyclohexane
(m, 10H, CH2-cyclohexane). 13C NMR (DMSO-d6): d 172.0 (Cthiazol O),
–
–
–
–
166.5 (CO Me), 163.5 (cyclohexan-CH N), 158.4 (C
2), 138.9
2
thiazol
–
–
(C5 CH), 131.5 (C
5), 29.8, 25.6 (5 ꢂ CH2-cyclohexane), 54.1
thiazol
(CO2Me). LC–MS [MþH]þ calcd. for C13H17N3O3S 295.09; found
296.59. Anal. calcd. for C13H17N3O3S (295.36): C, 52.86; H, 5.80;
N, 14.23. Found: C, 52.91; H, 5.81; N, 14.17.
Methyl 2-(2-(3,5-dimethoxybenzylidene)hydrazino)-4-oxo-
thiazolidin-5-ylidene)acetate 5k
From 3k (100 mg). Yield: 121 mg (85%) as a yellow solid, m.p.: 196°C
1
–
(dec.). H NMR (DMSO-d ): d 9.03 (s, 1H, NH), 7.52 (s, 1H, CH N), 7.11
–
6
Antiviral assays
In vitro anti-HIV assay
Evaluation of the antiviral activity of the compounds 6–15 and
16–19 against HIV-1 strain IIIB and HIV-2 strain (ROD) in MT-4 cells
–
(s, 1H, Ar-H), 6.53 (s, 1H, Ar-H), 6.40 (s, 1H, CH C), 3.81 (s, 6H, 2ꢂ
–
Carom-OMe), 3.70 (s, 3H, CO2Me), 2.90 (br s., 1H, OH). 13C NMR (DMSO-
5
–
2
d6): d 173.3 (Cthiazol O), 167.6 (CO Me), 162.2 (C
OMe), 157.4 (Cthiazol ), 147.4 (ArCH N), 140.8 (C CH þ C
3-OMe þ Carom
-
–
2
–
–
arom
–
4-OH),
arom
–
5
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8
Anti-HIV Activity of 4-Oxo-thiazolidin-5-ylidene acetate Derivatives
7
was performed using the MTT assay as previously described [35,
36, 44]. Briefly, stock solutions (10ꢂ final concentration) of test
compounds were added in 25 mL volumes to two series of
triplicate wells so as to allow simultaneous evaluation of their
effects on mock and HIV-infected cells at the beginning of
each experiment. Serial 5-fold dilutions of test compounds were
made directly in flat-bottomed 96-well microtiter trays using a
Biomek 3000 robot (Beckman instruments). Untreated control
HIV and mock-infected cell samples were included for each
sample. HIV-1(IIIB) [45] or HIV-2 (ROD) [46] stock (50 mL) at 100–
300 CCID50 (50% cell culture infectious dose) or culture medium
was added to either the infected or mock-infected wells of the
microtiter tray. Mock-infected cells were used to evaluate the
effect of test compound on uninfected cells in order to assess
the cytotoxicity of the test compound. Exponentially growing
MT-4 cells [47] were centrifuged for 5 min at 1000 rpm and the
supernatant was discarded. The MT-4 cells were resuspended at
6 ꢂ 105 cells/mL and 50 mL volumes were transferred to the
microtiter tray wells. Five days after infection, the viability
of mock and HIV-infected cells was examined spectrophoto-
metrically by the MTT assay.
the concentration at which the metabolic activity of the cells is
reduced by 50% as compared to untreated cells.
A concentration of compound is considered to elicit a genuine
antiviral effect in the HCV replicon system when the anti-replicon
effect is well above the 70% threshold at concentrations where
no significant anti-metabolic activity is observed [48].
Cells and HCV viruses
The Huh-5-2 and Huh 9-13 HCV subgenomic replicon-containing
cells were provided by Prof R Bartenschlager (University of
Heidelberg, Heidelberg, Germany).
We thank Prof. C. Pannecouque and Dr. P. Leyssen of Rega Institute for
Medical Research, Katholieke Universiteit Leuven, Belgium, for the anti-
HIV and anti-HCV screening, respectively. Mr. U. Haunz and Miss A.
Frimel of the Chemistry Department, University of Konstanz, Germany,
are acknowledged for the NMR experiments. Prof. Al-Masoudi would
like to thank Basrah University (Iraq) for sabbatical leave.
The authors have declared no conflict of interest.
Anti-HCV assay
Huh 5.2 cells, containing the hepatitis C virus genotype 1b
I389luc-ubi-neo/NS3-30/5.1 replicon [48] were sub-cultured in
DMEM supplemented with 10% FCS, 1% non-essential amino
acids, 1% penicillin/streptomycin and 2% geneticin at a ratio of
1:3 to 1:4, and grown for 3–4 days in 75 cm2 tissue culture flasks.
One day before addition of the compound, cells were harvested
and seeded in assay medium (DMEM, 10% FCS, 1% non-essential
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