Inhibition of HIV-1 RT activity by a new series of 3-(1,3,4-thiadiazol-2-yl)thiazolidin-4-one derivatives

Article abstract of DOI:10.1016/j.bmc.2020.115431

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent potent anti-HIV agents targeting HIV-1 reverse transcriptase (RT), a crucial enzyme for the viral life cycle. We have previously identified a series of NNRTIs bearing a 2,3-diaryl-1,3-thiazolidin-4-one core and some compounds proved to be effective in inhibiting HIV-1 replication at micromolar concentration. As a continuation in this research work we report the design, the synthesis and the structure–activity relationship studies of a further series of 3-(1,3,4-thiadiazol-2-yl)thiazolidin-4-one derivatives containing an arylthioacetamide group as pharmacophoric structural requirement for binding to the RT catalytic area. The new compounds proved to be effective to inhibit RT activity at micromolar concentrations. Finally, docking studies were carried out in order to rationalize the biological results of the new synthesized inhibitors.

Full text of DOI:10.1016/j.bmc.2020.115431

Bioorganic&MedicinalChemistry28(2020)115431
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of HIV-1 RT activity by a new series of 3-(1,3,4-thiadiazol-2-yl)
thiazolidin-4-one derivatives
⁎
Maria Rosa Buemia, Rosaria Gittoa, Laura Ielob, Christophe Pannecouquec, Laura De Lucaa,
a Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale Palatucci, 13, I-98168 Messina, Italy
b Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
c Department of Microbiology and Immunology, Lab. of Virology and Chemotherapy, Rega Institute for Medical Research, B-3000 Leuven, Belgium
A R T I C L E I N F O
A B S T R A C T
Keywords:
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent potent anti-HIV agents targeting HIV-1 re-
verse transcriptase (RT), a crucial enzyme for the viral life cycle. We have previously identified a series of
NNRTIs bearing a 2,3-diaryl-1,3-thiazolidin-4-one core and some compounds proved to be effective in inhibiting
HIV-1 replication at micromolar concentration. As a continuation in this research work we report the design, the
synthesis and the structure–activity relationship studies of a further series of 3-(1,3,4-thiadiazol-2-yl)thiazolidin-
4-one derivatives containing an arylthioacetamide group as pharmacophoric structural requirement for binding
to the RT catalytic area. The new compounds proved to be effective to inhibit RT activity at micromolar con-
centrations. Finally, docking studies were carried out in order to rationalize the biological results of the new
synthesized inhibitors.
Non-nucleoside reverse transcriptase inhibitors
Synthesis
HIV-1
Assays
1. Introduction
TZDs, we have discovered several 2,3-diaryl-1,3-thiazolidin-4-one de-
rivatives (Fig. 2) containing different aryl/heteroaryl substituents at C-
The global pandemic of human immunodeficiency virus (HIV) re-
mains an important issue to human health worldwide and type 1 is the
main causative agent of acquired immunodeficiency syndrome (AIDS).
Nowadays, the treatment for HIV/AIDS consists of a combination an-
tiretroviral therapy (ART) including two or more antiretroviral drugs
belonging to different drug classes. Non-nucleoside reverse tran-
scriptase inhibitors (NNRTIs) disrupt the normal functions of HIV-1
reverse transcriptase (RT) enzyme via binding to NNRTI binding pocket
(NNIBP) close to the polymerase active site and are considered essential
components of ART due to their good tolerability, low toxicity and the
high selectivity.1–3 US Food and Drug Administration approved the first
generation six NNRTIs for clinical use: nevirapine (NVP), delavirdine
(DLV), efavirenz (EFV) as first generation NNRTIs and etravirine
(TMC125), rilpivirine (TMC278)4,5 and doravirine (DOR) as second
generation NNRTIs (Fig. 1). Unfortunately, extensive adverse effects
combined with a high mutation rate of HIV-1 RT limit the clinical use of
NNRTIs.6–10 Wherefore, there is still needed a development of a next-
generation of NNRTIs with increased safety and tolerability profile.
Thiazolidinediones (TZDs) exhibit a wide range of pharmacological
activities including antihyperglycemic,11 antimicrobial,12 antiviral,13
antioxidant,14 anticancer,15 anti-inflammatory,16 neuroprotective,17,18
as well as tyrosinase inhibitory effect19,20 Among the large library of
2 and N-3 of the nucleus demonstrating anti-HIV activity as
NNRTIs.21–25
In order to expand this series of promising anti-HIV-agents, we
decided to introduce new chemical moieties thus decorating the 1,3,4-
thiadiazole ring of previously reported 1,3-thiazolidin-4-one deriva-
tives.25–27 Our choice fell on the N-(hetero)arylacetamide “tail” that has
been identified as a structural chemical feature of compounds I-III as
promising NNRTIs (Fig. 3).28–30
In this context, herein we examined the structure-activity relation-
ships (SARs) for 1,3-thiazolidin-4-ones that demonstrated that their
NNRT inhibitory effects were strongly dependent on the nature of the
substituents at C-2 and N-3 of the thiazolidinone ring (see
Fig. 2).25,31,32. Specifically, we speculated that the introduction of a
halogen atom on the C-2 phenyl ring was able to positively influence
the inhibitory activity. This behavior might be explained by considering
the relevant role of hydrophobic interactions with residues Phe227 and
Trp229 on the opening hydrophobic channel of the NNIBP in HIV-1
RT.1,33,34. Furthermore considering the N-(hetero)arylacetamide “tail”
as a structural chemical feature for promising NNRTIs, we combined
the versatile scaffold ‘2-aryl-3-(1,3,4-thiadiazol-2-yl)-1,3-thiazolidine-
4-one’ with S-linked N-arylacetamide.
The designed compounds were synthesized and screened as
⁎ Corresponding author.
E-mail address: ldeluca@unime.it (L. De Luca).
https://doi.org/10.1016/j.bmc.2020.115431
Received 13 January 2020; Received in revised form 2 March 2020; Accepted 4 March 2020
Availableonline17March2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.

M.R. Buemi, et al.
Bioorganic&MedicinalChemistry28(2020)115431
Fig. 4. Chemical structure of the new designed 3-(1,3,4-thiadiazol-2-yl)-1,3-
thiazolidin-4-ones.
Fig. 1. Chemical structure of first and second generation approved NNRTIs.
Fig. 2. 2,3-Diaryl-1,3-thiazolidin-4-one derivatives with anti-HIV activity
(NNRTIs).
Scheme 1. Reagents and conditions: (i) CH3CH2OH, reflux, 72 h; (ii) K2CO3,
DMF, r.t., 24 h.
thiazolidin-4-one derivatives were synthesized straightforwardly fol-
lowing synthetic strategy depicted in Scheme 1. The required 2-aryl-3-
(5-sulfanyl-1,3,4-thiadiazol-2-yl)thiazolidin-4-ones (1 a-d) were pre-
pared by reaction of commercially available 5-amino-1,3,4-thiadiazole-
2-thiol (5) with 2-mercaptoacetic acid (6) and the appropriate benzal-
dehyde 4a-d.35 In turn, key reagents 1a-d were coupled with N-ar-
ylacetamides 7 and 8, which were prepared by reaction of appropriate
commercially available anilines 9–10 with chloroacetyl chloride (see
Scheme 2). Finally, the coupling reaction between the 2-aryl-3-(5-sul-
fanyl-1,3,4-thiadiazol-2-yl)thiazolidin-4-ones (1 a-d) and obtained 2-
chloro-N-(phenyl)acetamides (7–8) afforded in good yields target
compounds
2-([5-(4-oxo-2-phenyl-1,3-thiazolidin-3-yl)-1,3,4-thia-
diazol-2-yl]sulfanyl)-N-(4-sulfamoyphenyl)acetamide (2 a-d) and N-(4-
methylsulfonylphenyl)-2-[[5-(4-oxo-2-phenyl-thiazolidin-3-yl)-1,3,4-
thiadiazol-2-yl]sulfanyl]acetamide (3 a-d). Analytical and spectral data
(1H NMR) of all synthesized compounds were in full agreement with
the proposed structures.
Fig. 3. Previously reported NNRTIs (I, II, and III) bearing N-(hetero)ar-
ylacetamide “tail”.
inhibitors of polymerase activity of HIV-1 RT in an enzymatic assay and
as inhibitors of HIV-1 replication in MT-4 cell culture. Finally, docking
studies were carried to clarify the binding mode of the newly synthe-
tized compounds.
The newly synthesized compounds were evaluated to assess their
ability to inhibit the polymerase activity of HIV-1 RT as well as HIV-1
replication in MT-4 cell cultures. We collected in Table 1 summarizes
biological data of target compounds 2 a-d and 3 a-d. We also examined
2. Results and discussion
On the basis of above mentioned rational design, we report a series
of compounds obtained for the combination of the 1,3-thiazolidin-4-one
nucleus with the 1,3,4-thiadiazol-2-yl]sulfanyl]-N-arylacetamide por-
tion. Inspired by previously optimized SARs for NNRTIs27 we in-
troduced a sulfonamide or methylsulfonic group in para position of
phenyl ring of N-arylacetamide (Fig. 4).
To avoid false positive results, we preliminary explored possible
assays interference; all designed compounds passed the PAINS (pan-
assay interference compounds) filter investigated by means of the free
web tool (http://www.swissadme.ch/). Therefore, the target
Scheme 2. Reagents and conditions: (i) DIPEA 0 °C, CH2Cl2, r.t., 24 h.
2

M.R. Buemi, et al.
Bioorganic&MedicinalChemistry28(2020)115431
Table 1
Anti-RT and anti-HIV-1 activities, cytotoxicity and selectivity index in MT-4 cells.a
Comp.
R
R1
IC50 (µM)b
EC50 (µM)c
CC50 (µM)d
˃ 423.1
SIe
1a
1b
1c
H
–
> 100
> 100
> 100
> 363
31.99
0.78
13
2-Cl
3-Cl
4-Cl
H
–
˃199.78
˃238.22
> 194.63
> 99.24
> 18.81
> 118.17
≥225.3
> 130.46
≥ 172.9
> 112.88
> 115.5
199.8
18.06
< 1
< 1
< 1
< 1
–
–
238.2
49.76
18.34
15.30
1d
2a
2b
2c
–
194.6
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2CH3
SO2CH3
SO2CH3
SO2CH3
30.56
21.84
19.17
71.95
40.64
11.83
15.75
39.43
1.55
6.12
99.24
2-Cl
3-Cl
4-Cl
H
2.89
3.86
7.0
≥18.81
118.2
11.05
26.70
< 1
–
2d
3a
3b
3c
≥222.5
130.5
6.72
0.3
< 1
–
2-Cl
3-Cl
4-Cl
≥ 172.9
112.9
3.95
13.8
34.00
49.19
< 1
< 1
> 81
3d
NVP
115.5
0.25
0.18
0.03
> 15.02
a
b
c
d
e
Data reported as mean
standard deviations.
Concentration required to inhibit the in vitro RNA-dependent DNA polymerase activity of recombinant RT by 50%.
Effective concentration required to reduce HIV-1-induced cytopathic effect by 50% in MT-4 cells.
Cytotoxic concentration required to reduce MT-4 cell viability by 50%.
Selectivity index: ratio CC50/EC50
.
the biological effects of synthetic precursors 2-phenyl-3-(5-sulfanyl-
1,3,4-thiadiazol-2-yl)thiazolidin-4-ones 1 a-d as negative control to
establish the role of N-arylacetamide tail. The results were expressed as
IC50 values (anti-RT activity), EC50 values (anti-HIV-1 activity), CC50
(1,3,4-thiadiazol-2-yl)thiazolidin-4-one derivatives. Biological tests
highlighted that the presence of arylacetamide moiety positively in-
fluences the RT enzymatic inhibition. This evidence was supported also
by docking studies that showed the contacts between our ligands and
crucial residues controlling the recognition process in to the enzymatic
cavity RT.
values (cytotoxicity) and SI values (selectivity index, given by the CC50
/
EC50 ratio). The FDA-approved drugs nevirapine (NVP) was used as
reference compound in the same tests.
The preliminary biological testing for all target compounds 2 a-d
and 3 a-d revealed that they were generally lower active inhibitors
when compared with reference compound NVP. Among the two series
of tested compounds 2 a-d and 3 a-d, the most promising molecules
were derivatives 2c, 3b and 3c showing IC50 value of 19.17 μM,
11.83 μM, 15.75 μM, respectively. Interestingly, target compounds 2 a-
d and 3 a-d were more active than their synthetic precursors 2-phenyl-
3-(5-sulfanyl-1,3,4-thiadiazol-2-yl)thiazolidin-4-ones (1 a-d), thus sug-
gesting that the introduction of the arylacetamide substituent positively
influences the RT enzymatic inhibition. Unfortunately, all the designed
derivatives showed low anti-HIV activity in MT-4 cell culture (Table 1).
In order to investigate the binding mode of studied derivatives we
performed docking studies into the non-nucleoside binding site of HIV-
1 RT (PDB code: 3DLG)36 using AutoDock Vina37 following the same
procedure described in our previous papers.30 Fig. 5 displays the
docking results of compounds 1c, 2c and 3c that were selected as
protype for each class of tested inhibitors. By analysis of the docking
results, it was possible to observe that active inhibitors 2c and 3c
shared a similar binding mode by occupying the hydrophobic pocket
made by the residues of Tyr181, Tyr188 and Trp229 (see panels 5B and
5C); moreover they established a favourable hydrogen bond contact
with Lys103 that represents an important residues for RT activity.38,39
On the contrary, the inactive intermediate 1c was not able to form this
crucial hydrogen bond interaction with Lys103. Additionally, com-
pound 1c does not occupy the region lined by Lys101 and Pro236
(panel 5A) as relevant residues for the interaction of well-known active
NNRTIs.30,36,38,40,41
4. Experimental section
4.1. Chemistry
All starting materials and reagents commercially available (Sigma-
Aldrich Milan, Italy; Alfa Aesar Karlsruhe, Germany) were used without
further purification. Melting points were determined on a Buchi B-545
apparatus (BUCHI Labortechnik AG Flawil, Switzerland) and are un-
corrected. Elemental analyses (C, H, N) were carried out on a Carlo Erba
Model 1106 Elemental Analyzer (Carlo Erba Milano, Italy); the results
confirmed a ≥95% purity. Merck silica gel 60 F254 plates were used for
analytical TLC (Merck KGaA, Darmstadt, Germany). Flash
Chromatography (FC) was carried out on a Biotage SP1 EXP (Biotage
AB Uppsala, Sweden). 1H NMR spectra were measured in CDCl3 with a
Gemini 300 spectrometer (Varian Inc. Palo Alto, California USA); che-
mical shifts are expressed in δ (ppm) and coupling constants (J) in Hz.
4.1.1. General procedure to synthesize 2-phenyl-3-(5-sulfanyl-1,3,4-
thiadiazol-2-yl)thiazolidin-4-ones (1 a-d)
To a solution of commercially available 5-amino-1,3,4-thiadiazol-2-
thiol (1 mmol) in ethanol (16 mL) the appropriate benzaldehyde (4 a-d)
(1 mmol) and 2-mercaptoacetic acid (1 mmol, 70 μl) were added. The
mixture was stirred 72 h at 100 °C; the evaporation of the solvent
furnished the crude product that was crystallized from a mixture of
Et2O and EtOH. This synthesis was carried out following a previously
reported procedure with slight modifications and the 1H NMR and 13C
NMR spectral assignments were in good agreement with previous
data.18,35,42 For intermediates 1a, 1b, and 1d registered CAS numbers
have been already assigned.
3. Conclusions
Herein we reported design, synthesis and biological evaluation of 3-
4.1.1.1. Phenyl-3-(5-sulfanyl-1,3,4-thiadiazol-2-yl)thiazolidin-4-one (1a)
3

M.R. Buemi, et al.
Bioorganic&MedicinalChemistry28(2020)115431
C 40.05, H 2.44, N 12.74; Found: C 40.16, H 2.81, N 12.33.
4.1.1.3. 2-(3-Chlorophenyl)-3-(5-sulfanyl-1,3,4-thiadiazol-2-yl)
thiazolidin-4-one (1c). Yield 65%; M.p. 240–242 °C. 1H NMR
(DMSO‑d6): (δ) 3.94 (d, 1H, J = 17.0), 4.27 (d, 1H, J = 17.0), 6.50
(s, 1H, CH), 7.33–7.36 (m, 3H, ArH), 7.53 (s, 1H, J = 9.3, ArH), 14.18
(bs, 1H, SH). Anal. Calcd for. (C11H8ClN3OS3): C 40.05, H 2.44, N
12.74; Found: C 40.00, H 2.66, N 12.45.
4.1.1.4. 2-(4-Chlorophenyl)-3-(5-sulfanyl-1,3,4-thiadiazol-2-yl)
thiazolidin-4-one (1d) CAS registry number 2226509–73-3. Yield 67%;
M.p. 248–250 °C. 1H NMR (DMSO‑d6): (δ) 3.93 (d, 1H, J = 17.0), 4.26
(d, 1H, J = 17.0), 6.49 (s, 1H, CH), 7.40 (d, 1H, J = 8.2, ArH), 7.53 (s,
1H, J = 8.2, ArH), 7.62–7.78 (m, 2H, ArH), 14.15 (bs, 1H, SH). Anal.
Calcd for. (C11H8ClN3OS3): C 40.05, H 2.44, N 12.74; Found: C 40.25, H
2.31, N 12.50.
4.1.2. General procedure to synthesize 2-chloro-N-arylacetamides (7–8)
The 2-chloro-N-arylacetamide derivatives 7–8 were synthesized
following a previously reported procedure27 introducing slow mod-
ifications as reported below. To a suspension of appropriate aniline
(9–10) (1 mmol) in DCM (2 mL) were added dropwise EDIPA (1 mmol,
174 μl) and chloroacetyl chloride (1 mmol, 80 μl). The reaction mixture
was stirred at 0 °C for 5 min, and subsequently at room temperature for
24 h. The completion of the reaction was followed by TLC Cyclo-
hexane/EtOAc 50:50. Afterwards, H2O (5 mL) and a saturated solution
of NaHCO3 (5 mL) were added to quench the reaction. The obtained
water layer was extracted with EtOAc (3x10 mL), dried over Na2SO4,
filtered and evaporated in vacuo. Desired intermediates 7–8 were pur-
ified by crystallization from Et2O and MeOH. All spectral data of in-
termediates 7–8 were in good agreement with literature.27
4.1.3. General procedure to synthesize compounds 2-([5-(4-oxo-2-phenyl-
1,3-thiazolidin-3-yl)-1,3,4-thiadiazol-2-yl]sulfanyl)-N-(4-sulfamoyphenyl)
acetamide (2 a-d) and N-(4-methylsulfonylphenyl)-2-[[5-(4-oxo-2-phenyl-
thiazolidin-3-yl)-1,3,4-thiadiazol-2-yl]sulfanyl]acetamide (3 a-d)
To a mixture of appropriate intermediate 1 a-d (1 mmol) and K2CO3
(1 mmol, 138 mg) in DMF (2 mL) the suitable 2-chloro-N-(4-sulfa-
moylphenyl)acetamide (1 mmol, 248 mg, 7) or 2-chloro-N-(4-methyl-
sulfonylphenyl)acetamide (0.5 mmol, 123 mg, 8) was added. The re-
action mixture was stirred at room temperature for 24 h, then quenched
with H2O (5 mL) and solution of NaHCO3 (5 mL). The water layer was
successively extracted with EtOAc (3x10 mL) and the organic layer was
washed with brine (3x5 mL), dried over Na2SO4, filtered and evapo-
rated under reduced pressure. Final products 2 a-d and 3 a-d were
obtained by chromatography on silica gel with DCM/MeOH 98:2 and
subsequent crystallization from Et2O at 0 °C.
4.1.3.1. 2-[[5-(4-Oxo-2-phenyl-thiazolidin-3-yl)-1,3,4-thiadiazol-2-yl]
sulfanyl]-N-(4 sulfamoylphenyl)acetamide (2a). Yield 30%, M.p.: 172 °C
dec. 1H NMR (DMSO‑d6), (δ) 3.41 (d, 1H, J = 17.0); 4.21 (s, 2H, CH2);
4.28 (d, 1H, J = 17.0); 6.68 (s, 1H); 7.26 (bs, 2H, NH2); 7.29–7.33 (m,
5H, ArH); 7.68 (d, 2H, J = 8.8, ArH); 7.74 (d, 2H, J = 8.8, ArH); 10.63
(bs, 1H, NH). Anal. Calcd for.: (C19H17N5O4S4): C 44.96; H 3.38; N
13.80; Found: C 44.80; H 3.15; N 13.60.
Fig. 5. Binding mode of derivatives 1c (A), 2c (B), 3c (C) in complex with
NNIBP (binding affinity values of −8,6 Kcal/mol, −8,8 Kcal/mol and −8,9
Kcal/mol, respectively). The hydrogen bonds are in dotted lines. The figure was
created using PyMOL software.
CAS registry number 138225-69-1. Yield 65%; M.p. 262–264 °C. 1H
NMR (DMSO‑d6): (δ) 3.95 (d, 1H, J = 17.8), 4.23 (d, 1H, J = 17.8),
6.51 (s, 1H, CH), 7.29–7.35 (m, 5H, ArH), 14.09 (bs, 1H, SH). Anal.
Calcd for. (C11H9N3OS3): C 44.72, H 3.07, N 14.22; Found: C 44.54, H
3.20, N 14.00.
4.1.3.2. 2-[[5-[2-(2-Chlorophenyl)-4-oxo-thiazolidin-3-yl]-1,3,4-
thiadiazol-2-yl]sulfanyl]-N-(4-sulfamoylphenyl)acetamide
(2b). Yield
52%, M.p.: 178 °C dec. 1H NMR (DMSO‑d6), (δ) 4.04 (d, 1H,
J = 16.4); 4.17–4.23 (m, 3H); 6.79 (s, 1H); 7.26 (bs, 2H, NH2);
7.27–7.33 (m, 4H, ArH); 7.67 (d, 2H, J = 8.8, ArH); 7.75 (d, 2H,
J = 8.8, ArH); 10.63 (bs, 1H, NH). Anal. Calcd for.: (C19H16ClN5O4S4):
C 42.10; H 2.98; N 12.92; Found: C 42.00; H 2.66; N 12.70.
4.1.1.2. 2-(2-Chlorophenyl)-3-(5-sulfanyl-1,3,4-thiadiazol-2-yl)
thiazolidin-4-one (1b) CAS registry number 106146–16-1. Yield 68%;
M.p. 240–242 °C. 1H NMR (DMSO‑d6): (δ) 3.96 (d, 1H, J = 17.2), 4.15
(d, 1H, J = 17.2), 6.62 (s, 1H, CH), 7.26–7.36 (m, 3H, ArH), 7.52 (d,
1H, J = 9.3, ArH), 14.17 (bs, 1H, SH). Anal. Calcd for. (C11H8ClN3OS3):
4.1.3.3. 2-[[5-[2-(3-Chlorophenyl)-4-oxo-thiazolidin-3-yl]-1,3,4-
thiadiazol-2-yl]sulfanyl]-N-(4-sulfamoylphenyl)acetamide
(2c). Yield
4

M.R. Buemi, et al.
Bioorganic&MedicinalChemistry28(2020)115431
98%, M.p.: 215 °C dec. 1H NMR (DMSO‑d6), (δ) 4.01 (d, 1H, J = 17.0);
4.22 (s, 2H, CH2); 4.32 (d, 1H, J = 17.0); 6.67 (s, 1H); 7.25 (bs, 2H,
NH2); 7.26–7.33 (m, 3H, ArH); 7.67 (s, 1H, ArH); 7.70–7.76 (m, 4H,
ArH); 10.63 (bs, 1H, NH). Anal. Calcd for.: (C19H16ClN5O4S4): C 42.10;
H 2.98; N 12.92; Found: C 42.38; H 2.64; N 12.80.
tray wells. Five days after infection, the viability of mock-and HIV-in-
fected cells was examined spectrophotometrically using the MTT assay.
The MTT assay is based on the reduction of yellow colored 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Acros
Organics) by mitochondrial dehydrogenase activity in metabolically
active cells to a blue-purple formazan that can be measured spectro-
photometrically. The absorbances were read in an eight-channel com-
puter-controlled photometer (Infinite M1000, Tecan), at two wave-
lengths (540 and 690 nm). All data were calculated using the median
absorbance value of three wells. The 50% cytotoxic concentration
(CC50) was defined as the concentration of the test compound that re-
duced the absorbance (OD540) of the mock-infected control sample by
50%. The concentration achieving 50% protection against the cyto-
pathic effect of the virus in infected cells was defined as the 50% ef-
fective concentration (EC50).
4.1.3.4. 2-[[5-[2-(4-Chlorophenyl)-4-oxo-thiazolidin-3-yl]-1,3,4-
thiadiazol-2-yl]sulfanyl]-N-(4-sulfamoylphenyl)acetamide
(2d). Yield
88%, M.p.: 225 °C dec. 1H NMR (DMSO‑d6), (δ) 4.01 (d, 1H,
J = 17.0); 4.22 (s, 2H, CH2); 4.26 (d, 1H, J = 17.0); 6.68 (s, 1H);
7.25 (bs, 2H, NH2); 7.33–7.40 (m, 4H, ArH); 7.71–7.77 (m, 4H, ArH);
10.63 (bs, 1H, NH). Anal. Calcd for.: (C19H16ClN5O4S4): C 42.10; H
2.98; N 12.92; Found: C 42.42; H 2.77; N 12.63.
4.1.3.5. N-(4-Methylsulfonylphenyl)-2-[[5-(4-oxo-2-phenyl-thiazolidin-3-
yl)-1,3,4-thiadiazol-2-yl]sulfanyl]acetamide (3a). Yield 98%, M.p.:
222 °C dec. 1H NMR (DMSO‑d6), (δ) 3.14 (s, 3H, CH3); 4.00 (d, 1H,
J = 17.0); 4.22 (s, 2H, CH2); 4.26 (d, 1H, J = 17.0); 6.68 (s, 1H);
7.28–7.33 (m, 5H, ArH); 7.76 (d, 2H, J = 8.8, ArH); 7.84 (d, 2H,
J = 8.8, ArH); 10.74 (bs, 1H, NH). Anal. Calcd for.: (C20H18N4O4S4): C
47.41; H 3.58; N 11.06; Found: C 47.12; H 3.26; N 11.37.
4.3. Reverse transcriptase assay
Recombinant wild type p66/p51 HIV-1 RT was expressed and pur-
ified as previously described.43 The RT assay is performed with the
EnzCheck Reverse Transcriptase Assay kit (Molecular Probes, In-
vitrogen), as described by the manufacturer. The assay is based on the
dsDNA quantitation reagent PicoGreen. This reagent shows a pro-
nounced increase in fluorescence signal upon binding to dsDNA or
RNA-DNA heteroduplexes. Single-stranded nucleic acids generate only
minor fluorescence signal enhancement when a sufficiently high dye:
base pair ratio is applied.44 This condition is met in the assay. A poly
(rA) template of approximately 350 bases long, and an oligo(dT)16
primer, are annealed in a molar ratio of 1:1.2 (60 min. at room tem-
perature). Fifty-two ng of the RNA/DNA is brought into each well of a
96-well plate in a volume of 20 µl polymerization buffer (60 mMTris-
HCl, 60 mM KCl, 8 mM MgCl2, 13 mM DTT, 100 µM dTTP, pH 8.1). Five
µl of RT enzyme solution, diluted to a suitable concentration in enzyme
dilution buffer (50 mM Tris-HCl, 20% glycerol, 2 mM DTT, pH 7.6), is
added (final RT enzyme concentration in the reaction mixture is
25 nM). To test the activity of compounds against RT, 1 µl of compound
in DMSO is added to each well before the addition of RT enzyme so-
lution. Control wells without compound contain the same amount of
DMSO (3.85% of the total reaction volume of 26 µl). The reactions are
incubated at 25 °C for 40 min and then stopped by the addition of EDTA
(15 mM fc). Heteroduplexes are then detected by addition of PicoGreen.
Signals are read using an excitation wavelength of 490 nm and emission
detection at 523 nm using a spectrofluorometer (Safire 2, Tecan). Re-
sults are expressed as relative fluorescence i.e. the fluorescence signal of
the reaction mix with compound divided by the signal of the same re-
action mix without compound.
4.1.3.6. 2-[[5-[2-(2-Chlorophenyl)-4-oxo-thiazolidin-3-yl]-1,3,4-
thiadiazol-2-yl]sulfanyl]-N-(4-methylsulfonylphenyl)acetamide
(3b). Yield 31%, M.p.: 195 °C dec. 1H NMR (DMSO‑d6), (δ) 3.12 (s, 3H,
CH3); 4.00–4.22 (m, 4H), 6.77 (s, 1H); 7.13–7.31 (m, 3H, ArH);
7.48–7.82 (m, 5H); 10.74 (bs, 1H, NH). Anal. Calcd for.:
(C20H17ClN4O4S4): C 44.39; H 3.17; N 10.35; Found: C 44.18; H 3.00;
N 10.49.
4.1.3.7. 2-[[5-[2-(3-Chlorophenyl)-4-oxo-thiazolidin-3-yl]-1,3,4-
thiadiazol-2-yl]sulfanyl]-N-(4-methylsulfonylphenyl)acetamide
(3c). Yield 98%, M.p.: 190–192 °C. 1H NMR (DMSO‑d6), (δ) 3.14 (s,
3H, CH3); 4.01 (d, 1H, J = 17.0); 4.24 (s, 2H, CH2); 4.33 (d, 1H,
J = 17.0); 6.77 (s, 1H); 7.25–7.33 (m, 3H, ArH); 7.51 (s, 1H, ArH); 7.76
(d, 2H, J = 8.8, ArH); 7.84 (d, 2H, J = 8.8, ArH); 10.74 (bs, 1H, NH).
Anal. Calcd for.: (C20H17ClN4O4S4): C 44.39; H 3.17; N 10.35; Found: C
44.30; H 3.00; N 10.17.
4.1.3.8. 2-[[5-[2-(4-Chlorophenyl)-4-oxo-thiazolidin-3-yl]-1,3,4-
thiadiazol-2-yl]sulfanyl]-N-(4-methylsulfonylphenyl)acetamide
(3d). Yield 34%, M.p.: 195 °C dec. 1H NMR (DMSO‑d6), (δ) 3.14 (s, 3H,
CH3); 3.98–4.32 (m, 4H); 6.67 (s, 1H); 7.27–7.39 (m, 4H, ArH);
7.78–7.84 (m, 4H, ArH); 10.74 (bs, 1H, NH). Anal. Calcd for.:
(C20H17ClN4O4S4): C 44.39; H 3.17; N 10.35; Found: C 44.15; H 3.00;
N 10.49.
4.2. In vitro anti-HIV assay
4.4. Docking studies
Evaluation of the antiviral activity of the compounds against HIV in
MT-4 cells was performed using the MTT assay as previously de-
scribed.43 Stock solutions (10 × final concentration) of test compounds
were added in 25 µl volumes to two series of triplicate wells so as to
allow simultaneous evaluation of their effects on mock- and HIV-in-
fected cells at the beginning of each experiment. Serial 5-fold dilutions
of test compounds were made directly in flat-bottomed 96-well mi-
crotiter trays using a Biomek 3000 robot (Beckman instruments, Full-
erton, CA). Untreated HIV- and mock-infected cell samples were in-
cluded as controls. HIV stock (50 µl) at 100–300 CCID50 (50% cell
culture infectious doses) 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 effects of test compound on uninfected
cells in order to assess the cytotoxicity of the test compounds. Ex-
ponentially growing MT-4 cells were centrifuged for 5 min at 220 g and
the supernatant was discarded. The MT-4 cells were resuspended at
6 × 105 cells/ml and 50 µl volumes were transferred to the microtiter
The crystal structure of HIV-1 reverse transcriptase in complex with
GW564511 inhibitor was retrieved from the RCSB Protein Data Bank
(PDB code 3DLG).35 The protein and ligands 1–3 a-d were prepared by
Discovery Studio 2.5.5. The ligands were also minimized using
CHARMm forcefield and used for docking by AutoDock Vina following
the same protocol described in previous papers.27,30
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The technical assistance of Kristien Erven and of Kris Uyttersprot for
the HIV experiments and HIV RT polymerase assays is gratefully
5

M.R. Buemi, et al.
Bioorganic&MedicinalChemistry28(2020)115431
acknowledged. This work was in part supported by University of
transcriptase inhibitors. Curr Med Chem. 2012;19:2026–2037.
23. Chimirri A, Grasso S, Monforte AM, Monforte P, Zappala M. Anti-HIV agents. I:
Synthesis and in vitro anti-HIV evaluation of novel 1H,3H-thiazolo[3,4-a]benzimi-
dazoles. Farmaco. 1991;46:817–823.
Messina Research
&
Mobility 2015 Project (project code
RES_AND_MOB_2015_DE_LUCA).
24. Barreca ML, Chimirri A, De Luca L, et al. Discovery of 2,3-diaryl-1,3-thiazolidin-4-
ones as potent anti-HIV-1 agents. Bioorg Med Chem Lett. 2001;11:1793–1796.
25. Barreca ML, Balzarini J, Chimirri A, et al. Design, synthesis, structure-activity re-
lationships, and molecular modeling studies of 2,3-diaryl-1,3-thiazolidin-4-ones as
potent anti-HIV agents. J Med Chem. 2002;45:5410–5413.
References
1. Kudalkar SN, Ullah I, Bertoletti N, et al. Structural and pharmacological evaluation of
a novel non-nucleoside reverse transcriptase inhibitor as a promising long acting
nanoformulation for treating HIV. Antiviral Res. 2019;167:110–116.
2. Kohlstaedt LA, Wang J, Friedman JM, Rice PA, Steitz TA. Crystal structure at 3.5 A
resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science.
1992;256:1783–1790.
26. Ravichandran V, Jain A, Kumar KS, Rajak H, Agrawal RK. Design, synthesis, and
evaluation of thiazolidinone derivatives as antimicrobial and anti-viral agents. Chem
Biol Drug Des. 2011;78:464–470.
27. Monforte AM, De Luca L, Buemi MR, Agharbaoui FE, Pannecouque C, Ferro S.
Structural optimization of N1-aryl-benzimidazoles for the discovery of new non-
nucleoside reverse transcriptase inhibitors active against wild-type and mutant HIV-1
strains. Bioorg Med Chem. 2018;26:661–674.
3. Kohlstaedt LA, Steitz TA. Reverse transcriptase of human immunodeficiency virus
can use either human tRNA(3Lys) or Escherichia coli tRNA(2Gln) as a primer in an in
vitro primer-utilization assay. Proc Natl Acad Sci USA. 1992;89:9652–9656.
4. Rawal RK, Murugesan V, Katti SB. Structure-activity relationship studies on clinically
relevant HIV-1 NNRTIs. Curr Med Chem. 2012;19:5364–5380.
28. Zhan P, Chen X, Li D, Fang Z, De Clercq E, Liu X. HIV-1 NNRTIs: structural diversity,
pharmacophore similarity, and implications for drug design. Med Res Rev.
2013;33(Suppl 1):E1–72.
5. Prajapati DG, Ramajayam R, Yadav MR, Giridhar R. The search for potent, small
molecule NNRTIs: a review. Bioorg Med Chem. 2009;17:5744–5762.
6. Mehellou Y, De Clercq E. Twenty-six years of anti-HIV drug discovery: where do we
stand and where do we go? J Med Chem. 2010;53:521–538.
29. Li W, Li X, De Clercq E, Zhan P, Liu X. Discovery of potent HIV-1 non-nucleoside
reverse transcriptase inhibitors from arylthioacetanilide structural motif. Eur J Med
Chem. 2015;102:167–179.
30. Ferro S, Buemi MR, De Luca L, Agharbaoui FE, Pannecouque C, Monforte AM.
Searching for novel N1-substituted benzimidazol-2-ones as non-nucleoside HIV-1 RT
inhibitors. Bioorg Med Chem. 2017;25:3861–3870.
7. Das K, Arnold E. HIV-1 reverse transcriptase and antiviral drug resistance. Part 2.
Curr Opin Virol. 2013;3:119–128.
8. Das K, Arnold E. HIV-1 reverse transcriptase and antiviral drug resistance. Part 1.
Curr Opin Virol. 2013;3:111–118.
31. Rao A, Balzarini J, Carbone A, et al. Synthesis of new 2,3-diaryl-1,3-thiazolidin-4-
ones as anti-HIV agents. Farmaco. 2004;59:33–39.
9. Croxtall JD. Etravirine: a review of its use in the management of treatment-experi-
enced patients with HIV-1 infection. Drugs. 2012;72:847–869.
32. Murugesan V, Tiwari VS, Saxena R, et al. Lead optimization at C-2 and N-3 positions
of thiazolidin-4-ones as HIV-1 non-nucleoside reverse transcriptase inhibitors. Bioorg
Med Chem. 2011;19:6919–6926.
10. Asahchop EL, Wainberg MA, Sloan RD, Tremblay CL. Antiviral drug resistance and
the need for development of new HIV-1 reverse transcriptase inhibitors. Antimicrob
Agents Chemother. 2012;56:5000–5008.
33. Ren J, Stammers DK. Structural basis for drug resistance mechanisms for non-nu-
cleoside inhibitors of HIV reverse transcriptase. Virus Res. 2008;134:157–170.
34. Das K, Bauman JD, Clark Jr AD, et al. High-resolution structures of HIV-1 reverse
transcriptase/TMC278 complexes: strategic flexibility explains potency against re-
sistance mutations. Proc Natl Acad Sci USA. 2008;105:1466–1471.
35. Dadlani VG, Somani RR, Tripathi PK. Design, synthesis and in-silico study of novel
series of 2-phenyl-3-(5-sulfanyl-1,3,4-thiadiazol-2-Yl)-1,3-thiazolidin-4-one deriva-
tives with potential anti-tubercular activity. Int J Pharm Sci Res. 2019;10:2565–2576.
36. Ren J, Chamberlain PP, Stamp A, et al. Structural basis for the improved drug re-
sistance profile of new generation benzophenone non-nucleoside HIV-1 reverse
transcriptase inhibitors. J Med Chem. 2008;51:5000–5008.
11. Day C. Thiazolidinediones: a new class of antidiabetic drugs. Diabet Med.
1999;16:179–192.
12. Tuncbilek M, Altanlar N. Synthesis of new 3-(substituted phenacyl)-5-[3'-(4H-4-oxo-
1-benzopyran-2-yl)-benzylidene]-2,4-thiazolidinediones and their antimicrobial ac-
tivity. Arch Pharm (Weinheim). 2006;339:213–216.
13. Bahare RS, Ganguly S, Choowongkomon K, Seetaha S. Synthesis, HIV-1 RT in-
hibitory, antibacterial, antifungal and binding mode studies of some novel N-sub-
stituted 5-benzylidine-2,4-thiazolidinediones. Daru. 2015;23:6.
14. Reddy KA, Lohray BB, Bhushan V, et al. Novel euglycemic and hypolipidemic agents:
Part-2. Antioxidant moiety as structural motif. Bioorg Med Chem Lett.
1998;8:999–1002.
37. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with
a new scoring function, efficient optimization, and multithreading. J Comput Chem.
2010;31:455–461.
15. Patil V, Tilekar K, Mehendale-Munj S, Mohan R, Ramaa CS. Synthesis and primary
cytotoxicity evaluation of new 5-benzylidene-2,4-thiazolidinedione derivatives. Eur J
Med Chem. 2010;45:4539–4544.
38. Wensing AM, Calvez V, Gunthard HF, et al. 2017 update of the drug resistance
mutations in HIV-1. Top Antivir Med. 2017;24:132–133.
16. Prabhakar C, Madhusudhan G, Sahadev K, et al. Synthesis and biological activity of
novel thiazolidinediones. Bioorg Med Chem Lett. 1998;8:2725–2730.
17. Heneka MT, Landreth GE. PPARs in the brain. Biochim Biophys Acta.
2007;1771:1031–1045.
39. Hsiou Y, Ding J, Das K, et al. The Lys103Asn mutation of HIV-1 RT: a novel me-
chanism of drug resistance. J Mol Biol. 2001;309:437–445.
40. Ren J, Milton J, Weaver KL, Short SA, Stuart DI, Stammers DK. ‘Structural basis for
the resilience of efavirenz (DMP-266) to drug resistance mutations in HIV-1 reverse
transcriptase’. Structure (London, England: 1993). 2000;8:1089–1094.
41. Das K, Ding J, Hsiou Y, et al. Crystal structures of 8-Cl and 9-Cl TIBO complexed with
wild-type HIV-1 RT and 8-Cl TIBO complexed with the Tyr181Cys HIV-1 RT drug-
resistant mutant. J Mol Biol. 1996;264:1085–1100.
18. Chimirri A, Grasso S, Monforte AM, Zappala M, De Sarro A, De Sarro GB. Synthesis
and anticonvulsant properties of 3-(1,3,4-thiadiazol-2-yl) thiazolidin-4-ones.
Farmaco. 1991;46:935–943.
19. Ha YM, Park YJ, Lee JY, et al. Design, synthesis and biological evaluation of 2-
(substituted phenyl)thiazolidine-4-carboxylic acid derivatives as novel tyrosinase
inhibitors. Biochimie. 2012;94:533–540.
42. Ragab FA, Heiba HI, El-Gazzar MG, Abou-Seri SM, El-Sabbagh WA, El-Hazek RM.
Synthesis of novel thiadiazole derivatives as selective COX-2 inhibitors.
Medchemcomm. 2016;7:2309–2327.
20. Ha YM, Park YJ, Kim JA, et al. Design and synthesis of 5-(substituted benzylidene)
thiazolidine-2,4-dione derivatives as novel tyrosinase inhibitors. Eur J Med Chem.
2012;49:245–252.
43. Pannecouque C, Daelemans D, De Clercq E. Tetrazolium-based colorimetric assay for
the detection of HIV replication inhibitors: revisited 20 years later. Nat Protoc.
2008;3:427–434.
21. Zhan P, Liu X, Li Z, et al. Novel 1,2,3-thiadiazole derivatives as HIV-1 NNRTIs with
improved potency: synthesis and preliminary SAR studies. Bioorg Med Chem.
2009;17:5920–5927.
44. Singer VL, Jones LJ, Yue ST, Haugland RP. Characterization of PicoGreen reagent
and development of a fluorescence-based solution assay for double-stranded DNA
quantitation. Anal Biochem. 1997;249:228–238.
22. Tian Y, Zhan P, Rai D, Zhang J, De Clercq E, Liu X. Recent advances in the research of
2,3-diaryl-1,3-thiazolidin-4-one derivatives as potent HIV-1 non-nucleoside reverse
6