1,2,3-Thiadiazole thioacetanilides as a novel class of potent HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.09.055

A novel series of 1,2,3-thiadiazole thioacetanilide (TTA) derivatives have been designed, synthesized and evaluated for its anti-HIV activities in MT-4 cells. Some derivatives proved to be highly effective in inhibiting HIV-1 replication at nanomolar concentrations. Among them, 2-[4-(2,4-dichlorophenyl)-1,2,3-thiadiazol-5-ylthio]-N-(2-nitrophenyl)acetamide 7d2 was identified as the most promising compound (EC₅₀ = 0.059 ± 0.02 μM, CC₅₀ > 283.25 μM, SI > 4883). The structure-activity relationship (SAR) of these novel structural congeners is discussed. Crown Copyright

Full text of DOI:10.1016/j.bmcl.2008.09.055

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5368–5371
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1,2,3-Thiadiazole thioacetanilides as a novel class of potent HIV-1
non-nucleoside reverse transcriptase inhibitors
a
a
b
b,
*
Peng Zhan a, Xinyong Liu a, , Yuan Cao , Yan Wang , Christophe Pannecouque , Erik De Clercq
*
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road, Jinan, Shandong 250012, PR China
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2008
Revised 27 August 2008
Accepted 15 September 2008
Available online 18 September 2008
A novel series of 1,2,3-thiadiazole thioacetanilide (TTA) derivatives have been designed, synthesized and
evaluated for its anti-HIV activities in MT-4 cells. Some derivatives proved to be highly effective in inhib-
iting HIV-1 replication at nanomolar concentrations. Among them, 2-[4-(2,4-dichlorophenyl)-1,2,3-thia-
diazol-5-ylthio]-N-(2-nitrophenyl)acetamide 7d2 was identified as the most promising compound
(EC50 = 0.059 0.02
lM, CC50 > 283.25 lM, SI > 4883). The structure–activity relationship (SAR) of these
novel structural congeners is discussed.
Keywords:
Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
HIV
NNRTIs
Thiadiazole thioacetanilides
1,2,3-Thiadiazole
Synthesis
SAR
Human immunodeficiency virus type-1 (HIV-1) infection affects
close to 40 million individuals worldwide. Since 1981, when the
first case reports of individuals dying from a rare opportunistic
infection were published, 20 million people have died from this
epidemic.1 Although the introduction of highly active anti-retrovi-
ral therapy (HAART) has dramatically decreased the morbidity and
mortality resulting from the infection with HIV, the AIDS preva-
lence remains one of the world’s most serious health problems,
causing millions of deaths each year.1 As one of the components
of the fist line HAART regimen, non-nucleoside reverse transcrip-
tase inhibitors (NNRTIs) have gained a definitive and important
place due to their unique antiviral potency, high specificity and
low toxicity. However, NNRTIs currently in clinical use are found
having a low genetic barrier to resistance and, therefore, the need
for novel NNRTIs active against drug-resistant mutants selected by
current therapies is of paramount importance.2–4
Extensive structural modification and bioactivity research dem-
onstrated that most derivatives showed submicromolar activity in
cell assay and significant in vitro activity against the WT or double
mutant K103N+Y181C strain of HIV-1 RT.7–9 A number of com-
pounds derived from this triazole/tetrazole scaffold are currently
being considered for clinical evaluation. Among them, VRX-
480773 (Fig. 1) was found to inhibit viruses from EFV-resistant
molecular clones and most NNRTI-resistant clinical HIV-1 isolates.
It also has an excellent pharmacokinetic profile, warranting further
clinical development for the treatment of HIV infection in both
NNRTI-naive and -experienced patients.10
In order to further confirm the importance of the five-mem-
bered heterocycle, a novel series of 1,2,3-thiadiazole thioacetani-
lide (TTA) derivatives was designed and synthesized based on the
general principle of bioisosterism in medicinal chemistry.11,12 In
the TTA analogues, the 1,2,3-thiadiazole ring was substituted for
the triazole or tetrazole moiety in the corresponding lead com-
pounds (Fig. 2), the other fragments which were considered to be
necessary for conserving anti-HIV-1 activity, such as the ‘S–CH2–
CO–NH’ linker and the 2-substituted anilides, were left unchanged.
Herein, a new approach for the synthesis of novel TTAs (7a–d), as
well as the evaluation of their inhibitory effects on HIV replication
in MT-4 cell culture, is reported.
Recently, from high-throughput screening (HTS) of compound
libraries, several interesting sulfanyltriazole- and sulfanyltetraz-
ole-type leads (A and B) were identified as novel potent HIV-1
NNRTIs, which have a simple, yet distinctively different chemical
structure from the other HIV-1 NNRTIs reported in the literature
(Fig. 1).5,6
The synthesis of the 1,2,3-thiadiazole thioacetanilides 7 was
straightforward and is depicted in Scheme 1. 2-Halo-1-(substituted
phenyl)ethanones (2), synthesized by direct halogenation of
acetophenone or by Friedel–Crafts reaction of substituted
benzene with 2-chloroacetyl chloride, were reacted with ethyl
* Corresponding authors. Tel.: +86 531 88380270; fax: +86 531 88382731 (X.L.);
tel.: +32 16 337341; fax: +32 16 337340 (E.D.C.).
E-mail addresses: xinyongl@sdu.edu.cn (X. Liu), erik.declercq@rega.kuleuven.
ac.be (E. De Clercq).
0960-894X/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.055

P. Zhan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5368–5371
5369
Cl
Cl
N
N
N
N
N
N
H
H
H
N
N
N
N
S
S
S
Br
N
N
N
O
O
O
SO2NH2
A
B
VRX-480773
EC50 = 2.053 μM
EC50 = 0.14 μM (WT)
WT RT Pol(IC50)=5 nM
Figure 1. Sulfanyltriazole- and sulfanyltetrazole-type NNRTIs.
X
N
N
H
N
N
S
N
O
Y
X
R
N
S
H
N
N
S
O
X
N
N
Y
H
R
N
S
R'
N
O
Y
R
Leads
Target compounds
Figure 2. The newly designed 1,2,3-thiadiazole thioacetanilide (TTA) scaffold.
O
O
Br
(i)
R1
O
O
S
1a
2a
(iv)
R1
(iii)
O
O
S
O
S
R1
NH
R2
R1
O
N
O
Cl
(ii)
O
R2
R2
R2
2b-d
3a-d
4a-d
1b: R1=H,R2=OCH3
1c: R1=R2=F
1d: R1=R2=Cl
N
N
X
N
S
N
S
N
N
S
H
N
O
OH
S
S
(v)
S
(vi)
(vii)
R1
R1
R1
O
O
O
Y
7a1-7a7
R2
R2
R2
7b1-7b7
7c1-7c5
7d1-7d6
5a-d
6a-d
Scheme 1. Reagents and conditions: (i) Br2, AlCl3; (ii) AlCl3, ClCH2COCl, DCM; (iii) Na2CO3, HSCH2CO2Et, EtOH, rt; (iv) p-tosyl hydrazine, toluene, reflux; (v) excess SOCl2; (vi)
KOH–EtOH–H2O, 50–70 °C; (vii) PCl5, aniline, DCM, rt, 1–2 h.
2-mercaptoacetate in EtOH at ambient temperature for several
hours to obtain the ethyl 2-(1-phenyl ethanone-2-thio)acetates
(3). Condensation of an appropriately substituted 3 with p-tosyl
hydrazine in refluxing toluene for 5–8 h, followed by recrystallisa-
tion from EtOH or by column chromatography on silica gel affor-
ded the corresponding ethyl 2-(1-phenylethanone-2-thio)acetate

5370
P. Zhan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5368–5371
Table 1
hydrazones (4), which are the key intermediates for the prepara-
tion of the substituted 1,2,3-thiadiazole heterocycles 5. The ring
closure reaction of compounds 4 was carried out via the reaction
with thionyl chloride according to the method reported by Hurd
and Mori.13–15 The thioacetic acid derivatives 6 were produced
by saponification of compounds 5 in KOH–EtOH–H2O solution.
The final 1,2,3-thiadiazole thioacetanilides 7 were synthesized by
reaction of 6 with substituted anilines in the presence of 1.1 equiv
mole of PCl5. Both analytical and spectral data of all the newly syn-
thesized compounds are in full agreement with the proposed
structures.16
Anti-HIV activity in MT-4 cells of 1,2,3-thiadiazole thioacetanilides (7a–d)
N
X
S
H
N
N
S
R1
O
Y
R2
The mechanism of the Hurd–Mori ring closure reaction has
been investigated and discussed in detail.13,17 In brief, an interme-
diate thiadiazoline-1-one II is formed by condensation of I with the
excess thionyl chloride, which readily aromatizes to form the 1,2,3-
thiadiazoles IV, possibly through a Pummerer-typed rearrange-
ment18 in concomitance with the cleavage of the PhSO2À group
through the transition state III (Fig. 3).
The preliminary activity and cytotoxicity of the newly designed
and synthesized 1,2,3-thiadiazole thioacetanilides were tested in
MT-4 cells for inhibition of HIV-1 (strain IIIB) and HIV-2 (strain
ROD) by the MTT method.19,20 The compounds nevirapine (NVP),
delaviridine (DLV), efavirenz (EFV) and zidovudine (azidothymi-
dine, AZT) were used as the reference drugs. The sulfanyltriazole
VRX-480773 was used as reference compound for comparative
purposes. The experimental results indicated that most of the com-
pounds exhibited potent inhibitory activity against HIV-1 (Table 1)
and none of the compounds was active against HIV-2.
Code
R1 R2
X
Y
HIV-1(IIIB)
EC50
(
l
M)a
CC50
(l
M)b
SIc
7a1
7a2
7a3
7a4
7a5
7a6
7a7
7b1
7b2
7b3
7b4
7b5
7b6
7b7
7c1
7c2
7c3
7c4
7c5
7d1
7d2
7d3
7d4
7d5
7d6
NVP
DLV
EFV
AZT
VRX-480773e
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
Cl
NO2
F
Br
Br
Br
Br
H
H
H
H
26.11 5.28 >345.4
>13
NAd
NA
>14
>22
P45
>53
>121
P283
NA
P54.23
>361.8
P246.72
>361.8
22.42 0.27 >307.64
13.73 3.83 >297.37
CH3
CO2C2H5 5.21 0.38
CO2CH3
H
H
H
H
CH3
CO2C2H5 3.01 0.33
CO2CH3
H
H
H
H
CH3
H
H
P232.03
>269.19
>318.97
P280.78
>332.94
>286.46
249.24 11.95 49
>245.86
195.40 7.38
178.08 38.63 146
164.62 8.86 40
147.88 26.90 27
144.47 9.29
>273.92
5.10 1.34
2.63 0.97
0.99 0.17
>332.94
3.19 0.07
5.06 0.84
OMe Cl
OMe NO2
OMe
F
OMe Br
OMe Br
OMe Br
OMe Br
F
F
F
F
F
>90
>82
97
2.00 1.25
1.23 0.10
4.14 1.08
5.45 1.36
2.26 0.16
1.45 0.22
Cl
NO2
F
Br
Br
Cl
NO2
F
F
F
F
F
As shown in Table 1, most of the compounds inhibited HIV-1
replication in the lower micromolar concentration range. Among
143
>189
Cl Cl
Cl Cl
Cl Cl
Cl Cl
Cl Cl
Cl Cl
0.118 0.03 111.43 93.95 943
them, analogues of the 7d series showed an EC50 value <0.2
lower than the reference drugs of NVP and DLV. The most potent
HIV-1 inhibitors were 7d2 (EC50 = 0.059 M, CC50 > 283.25 M,
SI > 4883), 7d6 (EC50 = 0.099 M, CC50 > 228.41 M, SI > 2298),
and 7d1 (EC50 = 0.118 M, CC50 = 111.43 M, SI = 943). The EC50
lM,
0.059 0.02 >283.25
0.135 0.13 19.53 15.62
0.149 0.01 P146.88
0.204 0.02 >255.50
>4883
145
P981
>1265
>2298
>72
H
H
CH3
l
l
Br
Br
Br
l
l
CO2C2H5 0.099 0.01 >228.41
l
l
0.208
0.320
0.00440
0.0151
0.14
>15.02
>3.827
>6.336
>93.55
NDf
values of these two compounds were lower than that of the lead
compound VRX-480773, which indicates that the 1,2,3-thiadiazole
is an acceptable isosteric replacement for the triazole/tetrazole in
the lead compounds.
>12
>1434
>6192
ND
In all series (7a–d), we observed that p-methoxy substitution on
the phenyl of the thiadiazole ring increased the antiviral activity
compared to the unsubstituted phenyl series (7a). The 2,4-dihal-
ophenyl substituted compounds (7c and 7d) were also found to
be essential for keeping the potent activities. Interestingly, replac-
ing the 2,4-difluoro (7c series) by 2,4-dichloro phenyl group (7d
series) led to even superior anti-HIV-1 activities. No substantial
differences in antiviral activity against HIV-1 were observed be-
tween the 7b and 7c series. From the structure–activity relation-
ship (SAR) results, we find that the antiviral potency of the 1,2,3-
thiadiazole thioacetanilides is closely related to the electronic or
spatial characteristics of the aryl linked to the 1,2,3-thiadiazole
core.
a
EC50: concentration required to protect the cell against viral cytopathogenicity
by 50% in MT-4 cells.
b
CC50: concentration that reduces the MT-4 cell viability by 50%.
Selectivity index: ratio CC50/EC50, a higher SI means a more selective compound.
NA: not active.
Ref. 10.
c
d
e
f
ND: not determined. The symbol ‘>’ in the CC50 column is the highest concen-
tration at which the compounds were tested and still found to be non-cytotoxic.
Average EC50 and CC50 values for at least two separate experiments are presented.
substituents, but the introduction of a fluorine atom in this posi-
tion caused a substantial decrease in potency. However, substitu-
ents at the para position of the anilide almost did not influence
the antiviral potency in the same series.
Substitution at the phenyl ring of the anilide moiety revealed
that nitro and halogen at the ortho position were the preferred
H
HOH
H
Ar
Ar
Ar
Ar
N
S
N
S
N
N
S
Ph
Ph
Ph
SOCl2
N
N
O
S
HN
S
N S
O
O
O
O
O
S
S
S
S
OH
O
R'
R'
R'
R'
H+
I
II
III
IV
Figure 3. The mechanism of the Hurd–Mori reaction.

P. Zhan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5368–5371
2. De Clercq, E. Nat. Rev. Drug Disc. 2007, 6, 1001.
5371
From the SAR studies, we found that the SAR features of the 4-
substituted phenyl and anilide moieties of the TTAs were highly
consistent with the previously observed sulfanyltriazole/tetra-
zole-type NNRTIs.5–9 In addition, the TTA analogues proved active
against HIV-1, but not HIV-2. Taking together these data we can as-
sume that the novel TTAs most probably act as genuine NNRTIs.
Experiments for inhibition of HIV-1 RT, activity against NNRTI-
resistant strains and docking studies are in progress, and further
results will be reported in due course.
3. Jochmans, D. Virus Res. 2008, 134, 171.
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In summary, the bioassay results show that our ‘bioisosterism’
based approach has led to the discovery of novel TTA-based anti-
HIV agents. Some derivatives proved to be highly effective in inhib-
iting HIV-1 replication at nanomolar concentrations. Among them,
compound 7d2 was identified as the most promising candidate
(EC50 = 0.059 0.02 lM, SI > 4883). Additional structural modifica-
tions of the TTA derivatives may further increase their potency as
anti-HIV-1 agents.
8. OMeara, J. A.; Jakalian, A.; La Plante, S.; Bonneau, P. R.; Coulombe, R.; Faucher,
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Acknowledgments
16. Selected data for compound 7d1: 1H NMR (600 MHz, CDCl3, ppm) d: 8.53
(s, 1H, NH), 8.27 (d, 1H, J = 8.4 Hz, PhH), 7.57 (d, 1H, J = 2.4 Hz, PhH), 7.44–
7.10 (m, 5H, PhH), 3.84 (s, 2H, S-CH2); 13C NMR (150 MHz, CDCl3, ppm) d:
163.73 (C@O), 156.84 (thiadiazole), 148.28 (thiadiazole), 136.78, 135.12,
133.44, 132.80, 130.12, 129.19, 127.91, 127.51, 127.45, 125.67, 123.23,
Financial support of this work by the National Natural Science
Foundation of China (NSFC No. 30371686, No. 30772629), Key
Project of The International Cooperation, Ministry of Science and
Technology of China (2003DF000033) and Research Fund for the
Doctoral Program of Higher Education of China (070422083), is
gratefully acknowledged.
121.53, 41.64 (S-CH2); IR (KBr, cmÀ1): 3355
(
t
NH), 1698
C@N), 1528 (dNH). EI-MS: m/z 430.2 (M+), 432.2 (M+2), 434.2 (M+4).
C16H10Cl3N3OS2 (MW: 430.76).
17. Stanetty, P.; Kremslehner, M. Heterocycles 1998, 48, 259.
(tC@O), 1592
(t
18. Veerapen, N.; Taylor, S. A.; Walsby, C. J.; Pinto, B. M. J. Am. Chem. Soc. 2006, 128,
227.
References and notes
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20. Pannecouque, C.; Daelemans, D.; De Clercq, E. Nat. Protocols 2008, 3, 427.
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