Synthesis and HIV-1 RT inhibitory action of novel (4/6-substituted benzo[d]thiazol -2-yl)thiazolidin-4-ones. Divergence from the non-competitive inhibition mechanism

Article abstract of DOI:10.3109/14756366.2011.636362

Reverse transcriptase (RT) inhibitors play a major role in the therapy of human immunodeficiency virus type 1 (HIV-1) infection. Although, many compounds are already used as anti-HIV drugs, research on development of novel inhibitors continues, since drug resistant strains appear because of prolonged therapy. In this paper, we present the synthesis and evaluation of HIV-1 RT inhibitory action of eighteen novel (4/6-halogen/MeO/EtO-substituted benzo[d]thiazol-2-yl) thiazolidin-4-ones. The two more active compounds (IC50 : 0.04 μM and 0.25 μM) exhibited better inhibitory action than the reference compound, nevirapine. Docking analysis supports a stable binding of the most active derivative to the allosteric centre of RT. Kinetic analysis of two of the most active compounds indicate an uncompetitive inhibition mode. This is a desired characteristic, since mutations that affect activity of traditional non-competitive NNRTIs may not affect activity of compounds of this series. Interestingly, the less active derivatives (IC50 > 40 μM) exhibit a competitive mode of action.

Full text of DOI:10.3109/14756366.2011.636362

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(1): 113–122
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.636362
research artIcle
Synthesis and HIV-1 RT inhibitory action of novel
(4/6-substituted benzo[d]thiazol -2-yl)thiazolidin-4-ones.
Divergence from the non-competitive inhibition mechanism
Eleni Pitta1, Athina Geronikaki1, Sofiko Surmava2, Phaedra Eleftheriou2, Vaibhav P. Mehta3, and
Erik V. Van der Eycken3
1Department of Medicinal Chemistry, School of Pharmacy, Aristotle University of essaloniki, Greece,
2Department of Medical Laboratory Studies, School of Health and Medical Care, Alexander Technological
Educational Institute of essaloniki, Greece, and 3Laboratory of Organic Synthesis, Department of Chemistry,
University of Leuven, Belgium
abstract
Reverse transcriptase (RT) inhibitors play a major role in the therapy of human immunodeficiency virus type 1
(HIV-1) infection. Although, many compounds are already used as anti-HIV drugs, research on development of novel
inhibitors continues, since drug resistant strains appear because of prolonged therapy. In this paper, we present
the synthesis and evaluation of HIV-1 RT inhibitory action of eighteen novel (4/6-halogen/MeO/EtO-substituted
benzo[d]thiazol-2-yl)thiazolidin-4-ones. The two more active compounds (IC50 :0.04 µM and 0.25 µM) exhibited
better inhibitory action than the reference compound, nevirapine. Docking analysis supports a stable binding of the
most active derivative to the allosteric centre of RT. Kinetic analysis of two of the most active compounds indicate
an uncompetitive inhibition mode. This is a desired characteristic, since mutations that affect activity of traditional
non-competitive NNRTIs may not affect activity of compounds of this series. Interestingly, the less active derivatives
(IC50 > 40 µM) exhibit a competitive mode of action.
Keywords RT inhibitors, HIV-1, benzo[d]thiazol-2-yl)thiazolidin-4-ones, competitive, uncompetitive
Introduction
effects and the disadvantage of easier development of
During the past two decades, investigations resulted in
development of a great number of potential drugs for
the treatment of AIDS, some of which were approved for
broad use. Among the different categories of anti-HIV
drugs, such as HIV Reverse Transcriptase (RT) inhibi-
tors, Protease Inhibitors, Integrase Inhibitors and Entry
Inhibitors, RT inhibitors play the major role in treatment
of HIV-infected patients. RT inhibitors structurally belong
to two families: the nucleoside/nucleotide RT Inhibitors
(NRTI) and the non-nucleoside RT inhibitors (NNRTI).
Compared to NRTIs, the commercially available NNRTIs
have the advantage of exhibiting less undesired side
mutations driving to resistant strains. Combination ther-
apies are used to delay development of such mutants.
Commercially available NNRTIs are among the ingredi-
ents of combination therapies used as a first treatment of
infected patients. ough all approved NNRTI have dif-
ferent chemical structures, all of them contact the same
site of the molecule of RT. erefore, a mutation provid-
ing resistance to one NNRTI, also provides resistance to
all other NNRTIs (“cross resistance”)1,2. So, the need for
developing novel NNRT inhibitors still exists, and devel-
opment of structures with differences in binding mode
is desired1. Moreover, achievement of lower toxicity
Address for Correspondence: Prof. A. Geronikaki, Department of Medicinal Chemistry, School of Pharmacy, Aristotle University of
essaloniki, essaloniki, 54124, Greece. Tel: +30 2310997616. Fax: +302310997612. E-mail: geronik@pharm.auth.gr; Dr. Phaedra
Eleftheriou, Department of Medical Laboratory Studies, School of Health and Medical Care, Alexander Technological Educational Institute
of essaloniki, essaloniki, 57400, Greece. Tel: +302310997616. E-mail: elfther@mls.teithe.grv
(Received 01 September 2011; revised 26 October 2011; accepted 26 October 2011)
113

114 E. Pitta et al.
treatments may be an additional expectation of novel
agents. Liver toxicity is among the undesired side effects
of currently used NNRTIs like nevirapine and efavirenz
while central nervous system (CNS) toxicity is associated
with treatments containing efavirenz3.
differences in the volume of the cavity formed depending
on the inhibitor.
Interestingly, besides non-competitive inhibitors,
NNRTIs with different mode of action were also men-
tioned. e bis(heteroaryl)piperazine inhibitor (BHAP),
U-90152E, acts as a mixed inhibitor with respect to the
template: primer and dNTP binding sites for both the
RNA- and DNA-directed DNA polymerase domains of
the enzyme25 while chloroxoquinolinic ribonucleoside,
6-chloro-1,4-dihydro-4-oxo-1-(beta-D-ribofuranosyl)
quinoline-3-carboxylic acid, was found to inhibit RT with
an uncompetitive and a non-competitive mode of action
with respect to dTTP incorporation and to RNA:primer
template respectively26.
During the last years, flexible molecules or molecules
with unusual conformation or mode of inhibitory action
attracted the interest of the scientists since there is
increased probability that these molecules retain capa-
bility to inhibit RT mutants resistant to commercially
available, traditional NNRTIs.
iazolyl/thiazolidinone derivatives with various
biological activities have been synthesized by many
investigators during the last decades. Analgestic,
anti-inflammatory, COX, LOX inhibitory action and
antimicrobial activity are among the pharmaceuti-
cally interesting properties that were detected in dif-
ferent thiazolyl/thiazolidinone derivatives during the
past years27–29. A number of thiazolidinone derivatives
with anti-HIV activity have been found by different
investigators14,15,30–34. In 2007, Rawal et al. synthesised and
tested a number of 2-aryl-3-heteroaryl-1,3-thiazolidin-
4-ones with anti-HIV-1 RT inhibitory action34. Most
recently, two 3-[4-(2-adamantyl)-1,3-thiazol-2-yl]-2-
(dihalogenphenyl)-1,3-thiazolidin-4-ones with uncom-
mon mode of inhibitory action were synthesized by our
team21. e compounds showed uncompetitive or mixed
mechanism of inhibition for RNA/primer template and
competitive mechanism for dNTPs21.
In this study, we present the synthesis and evalua-
tion of HIV-1 RT inhibitory activity of nineteen 2-(2,6-
dihalophenyl)-3-(4/6-substituted benzo[d]thiazol-2-yl)
thiazolidin-4-ones. Eighteen of them are novel com-
pounds, while one of them, the 3-(benzo[d]thiazol-2-
yl)-2-(2-chloro-6-fluorophenyl)thiazolidin-4-one had
been previously synthesized by Rawal et al and found to
have low anti-HIV-1 RT activity34. is compound is also
tested in the present study for comparison reasons, since
the novel compounds have the same main scaffold with
it but contain a substituted benzo[d]thiazol-2-yl moiety.
e novel compounds differentiate with each other in the
substituents of the benzo[d]thiazol-2-yl moiety as well
as in the halogens of the 2,6-dihalophenyl moiety. e
substituted compounds are divided into two categories:
4/6-halogen substituted benzo [d]thiazol-2-yl deriva-
tives and 4/6-MeO/EtO substituted benzo[d]thiazol-2-yl
derivatives. Kinetic studies and docking analysis were
performed for some of the tested compounds.
Commercially available NNRTIs are complex com-
pounds bearing a variety of heterocyclic rings such as
benzoxazin-2-one (efavirenz), dipyrido[1,4]diazepin-6-
one (nevirapine), pyrimidine (efavirenz)4, piperazine and
indolyl moieties (delavirdine)5. Apart from molecules
having received FDA acceptance, many compounds have
been found to exhibit RT inhibitory action among which
derivatives of (hydroxyethoxy-methyl)-6-(phenyl thio)
thymine (HEPT)6, tetrahydro-imidazo[4,5,1-jk][1,4]-ben-
zodiazepine-2(1H)-one and−thione (TIBO)7, dipyrido-
diazepinone like nevirapine8, 3-[[(1,3-benzoxazol-2-yl)
methyl]amino]-5-ethyl-6-methylpyridin-2(1H)-one9,
bis(heteroaryl)piperazines (BHAPs) like delavirdine10,
alpha-anilinophenylacetamide (alpha-APA)11, 1H,3H-
thiazolo[3,4-a]benzimidazole (TBZs)12, diarylpyrimidin
(DAPY)13 like rilpivirine and 2,3-diaryl-1,3-thiazolidin-4-
ones14,15. Reverse trancriptase inhibition potency differs
among inhibitors. Two commonly used and effective
NNRTIs, nevirapine and efavirenz, strongly differ in IC50
value with nevirapine exhibiting an indicative IC50 of 0.28
μΜ and efavirenz having an IC50 of 47 nm16,17
.
Kinetic studies revealed that currently used NNRT
inhibitors have a non-competitive mode of action
18,19
against both RNA:primer hybrid and dNTPs
and
most of the NNRTIs discovered till now also exhibit the
same way of action8,9,19. Crystallographic studies revealed
that non-competitive NNRTIs bind to an allosteric cen-
tre that is located near the RNA depended polymerase
active site of the enzyme on subunit p6620,21. Aromatic
aminoacids Tyr181, Tyr188, Phe227, Trp229, Tyr318 and
hydrophobic aminoacids Pro95, Leu100, Val106, Val108,
Val179, Leu234, Pro236 form the hydrophobic cleft of the
allosteric binding site22. Aromatic π→π interactions and
hydrophobic interactions are essential for RT-inhibitor
complex stabilization. Hydrogen bonds with Lys101 or
Lys103 are present in many cases. e strong involvement
of Lys103 in complex stabilization may be responsible for
the high frequency (>50%) of mutations of this residue
in resistant strains. Crystallographic studies of first gen-
eration NNRTIs showed that a butterfly conformation of
the molecules favoured binding and was mandatory for
effective compounds19,22. However, inhibitors adapting
different conformations, such as the 4-dihydroquinox-
alin-2(1H)-thione derivative, HBY097, were found to
strongly interact with the active site as well23. Moreover,
flexible molecules with the capability to acquire multiple
conformations, like etravirine, were found to present
inhibition activity against more mutated strains24. e
allosteric centre is not present in pure RT and is created
after interaction with the inhibitor16. Moreover, although,
the same aminoacids are involved in enzyme-compound
interaction of all studied complexes, there are slight
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and HIV-1 RT inhibitory action 115
1610, 1590, 1096.1HNMR (300 MHz, DMSO): δ= 8.02 (d,
J= 8.2 Hz, 1H), 7.63 (d, J= 9.1 Hz, 1H), 7.43− 7.31 (m, 3H),
7.16− 7.06 (m, 3H), 4.32− 4.16 (m, 2H). 13C NMR (75 MHz,
DMSO): δ= 171.5, 161.8, 161.7, 158.5, 156.3, 158.0, 147.8,
131.5, 131.2, 126.4, 124.8, 122.3, 121.6, 117.1, 112.8, 112.5,
54.0, 33.4, 31.0. HRMS (EI): m/z calcd. for C16H10ON2S2F2
[M+]: 348.0203; found 348.0217. Anal C16H10ON2S2F2: C, H,
N. Calc.: C: 55.16%, H: 2.89,% N: 8.04%. Found: C: 54.74%,
H: 2.98%, N: 7.99%.
Methods
Chemistry
Melting points of the compounds were determined and
are uncorrected. IR spectra were recorded on Perkin
Elmer Spectrum BX and DR-8001 Shimadzu. 1H, 13C
NMR spectra were recorded on 300 MHz instrument.
1
e H chemical shifts are reported in ppm relative to
tetramethylsilane. High-resolution mass spectra were
recorded by using ion source temperature 150–250°C
as required. High-resolution EI-mass spectra were per-
formed with a resolution of 10,000. e purity of com-
pounds was proved by combustion method. Elemental
analyses were obtained on an acceptable range ( /0.4%)
in a Perkin-/Elmer 240B CHN analyzer (e Perkin-/
Elmer Corporation Ltd., Lane Beaconsfield, Bucks, UK).
For thin-layer chromatography, analytical TLC plates SIL
G/UV254 and 70–230 mesh silica gel were used. Reagents
were used without further purification.
3-(Benzo[d]thiazol-2-yl)-2-(2-chloro-6-fluorophenyl)thiazoli-
din-4-one (2). Yield: 59%, m.p. 174-6 °C, Rf: 0.62 (petro-
leum ether/ethylacetate 8:2). IR (Nujol), cm−1:1720, 1600,
1
1520, 1086. HNMR (300 MHz, DMSO): δ= 8.01 (d, J= 8.2
Hz, 1H), 7.62− 7.56 (m, 1H), 7.46− 7.31 (m, 4H), 7.20− 7.14
(m, 2H), 4.31− 4.16 (m, 2H). 13C NMR (75 MHz, DMSO):
δ= 171.7, 156.4, 147.8, 132.9, 131.1, 126.8, 126.4, 124.8,
122.3, 121.6, 116.4, 116.1, 58.1, 31.0. HRMS (EI): m/z
calcd for C16H10ON2S2ClF [M+]: 363.9907; found 363.9901.
Anal. C16H10ClFN2OS2: C, H, N. Calc.: C: 52.67%, H: 2.76%,
N: 7.68%. Found:C: 52.44%, H: 2.42%, N: 7.62%.
General procedure for the synthesis of thiazolidinones
by conventional method15,21,31,33
e appropriate (hetero) aromatic amine (1.0 mmol) and
2,6-dihalosubstituted benzaldehyde or monosubstituted
benzaldehyde (1.2 mmol) were stirred in dry toluene
under reflux condition followed by addition of mercatoa-
cetic acid (2.0 mmol). e reaction mixture was refluxed
for an additional 20–26h, concentrated to dryness under
reduce pressure and the residue was taken up in ethyl ace-
tate. e organic layer was washed with 5% aq citric acid,
water, 5% aq sodium hydrogen carbonate, and finally with
brine. e organic layer was dried over sodium sulfate and
removed under reduced pressure to give a crude product.
e later was purified by column chromatography on
silica-gel using hexane-ethyl acetate (8:2) as eluent. e
structures of synthesized compounds were characterized
by TLC, 1H-NMR, 13C-NMR and HRMS.
Evaluation of RT inhibitory action35
HIV-1 Reverse Transcriptase Activity was measured
using the colorimetric photometric immunoassay kit
provided by Roche. Estimation of RT activity is achieved
by quantification of the DNA synthesized by RT in a
known period of time. Incubation was performed for 2 h
at 37°C. PolyA:oligodT RNA template:primer hybrid at
a concentration of 2.2 μg/ml was incubated with 20nM
of purified recombinant HIV-1 reverse transcriptase in
a 50 μL incubation mixture containing 50mM Tris-HCl
pH 7.8, 160 mM KCl, 11 mM MgCl2, 5.3 mM DTT, 0.5 mM
EDTA, 0.3% Triton-X 100 and 10 μM dTTP (unless
otherwise mentioned) in the presence of digoxigenin-
conjugated dUTP (dUTP-DIG) and biotin-conjugated
dUTP (dUTP-biotin). In a following step, the synthesised
biotin-DIG-labelled DNA was bound to the surface of
streptavidin-coated ELISA-plate wells. After washing out
the unbound material, an antibody to digoxigenin, con-
jugated to peroxidase (anti-DIG-POD), was added and
bound to the digoxigenin-labelled nucleotides. After a
washing step and addition of the appropriate substrate,
peroxidase catalyzed the transformation of the substrate,
ABTS, to a yellow coloured reaction product. 15min after
ABTS addition, the coloured product was measured
using a microtitre plate (ELISA) reader. For the estima-
tion of inhibitory action of the compounds, reverse tran-
scriptase was incubated in the presence of the potential
inhibitor for 45 min at room temperature before addition
of its substrates (pre-incubation). e activity measured
was compared with the total activity of the enzyme in
the absence of inhibitor. For total-activity measurement,
equal volume of the compound solvent was added in the
reaction mixture. Incubation in the absence of enzyme
was performed and used as blank for the determination
of enzyme activity. e absorbance of the blank was
Microwave irradiation experiments
All microwave irradiation experiments were carried out in
a dedicated CEM-Discover monomode microwave appa-
ratus, operating at a frequency of 2.45GHz with continu-
ous irradiation power from 0 to 300 W with utilization of
the standard absorbance level of 300W maximum power.
e reagents, aminobenzothiazole, equimolar amount
of 2,6-dihalosubstituted benzaldehyde (1.5 mmol) and
mercaptoacetic acid were placed in a 10 mL reaction vial
containing a stirring bar. e vial was kept under argon
for 30 s and then sealed tightly with a Teflon septum and
placed into the microwave cavity. It was irradiated at a
required ceiling temperature of 80–100°C using 100W as
maximum power for 10–60 min. en the reaction mix-
ture was rapidly cooled with gas jet cooling to ambient
temperature.
3-(Benzo[d]thiazol-2-yl)-2-(2,6-difluorophenyl)thiazolidin-4-
-one (1). Yield: 43%, m.p. 143-4 °C, Rf: 0.62 (petroleum
ether/ethylacetate 8:2). IR (Nujol), cm−1:1700 (C = 0),
© 2013 Informa UK, Ltd.

116 E. Pitta et al.
abstracted from the absorbance values obtained from the
rest of the samples. Reaction velocity, V, was expressed in
arbitary units. 1 Unit equals to product formation which
yields 1 O.D. at 405 nm under the testing conditions.
Different inhibitor concentrations were added for the
calculation of IC50 values. For kinetic studies, incubation
with different concentrations of dNTPs was performed.
Enzfitter for windows 32 bit version was used for the
calculations of kinetic analysis results36. All experiments
were performed in triplicate. Values obtained using nevi-
rapine were used for comparison reasons.
of the allosteric pocket presented certain differences
compared to the previously used 9-TIBO-RT complex.
In particular, the residues Val106, Pro225, Phe227,
Leu234, His235, Pro236 and Tyr318 appeared to be
moved, making the binding pocket larger and capable to
accept bulkier compounds, allowing them to obtain the
butterfly conformation. A test docking calculation was
also carried out to validate the docking protocol and to
confirm that it could be used to reproduce the binding
mode of our ligands. e ligand TMC125, in the confor-
mation found in the crystal structure was extracted and
docked back into the corresponding binding pocket to
determine the ability of Autodock to reproduce the ori-
entation and position of the inhibitor observed in the
crystal structure. e results of control docking showed
that Autodock was able to determine the orientation and
the interactions pattern of the crystallographic inhibitor
with a root-mean square deviations (RMSD) of 0.58 Å
Docking analysis
Based on previously reported molecular docking stud-
ies on di-aryl-substituted thiazolidin-4-ones37, we first
used the X-ray crystallographic complex of RT with
9-Cl-TIBO which adopts the common butterfly struc-
ture (PDB code 1REV)38. Structures of inhibitors were
built using Maestro 3D-sketcher39 minimized with
OPLS_2005 force field40 using the Polak-Ribiere conju-
gated gradient method (0.001 kJ/Å mol convergence)
and finally docked in the protein. As none of the struc-
tural waters were conserved in the RT-9-Cl-TIBO com-
plex, all the water molecules were removed from the
allosteric site in all the docking simulations. Docking
simulations were carried out using the Autodock 4.241.
e genetic algorithm-local search (GA-LS) method
was used with the following settings: initial population
of 300 randomly placed individuals, a maximum num-
ber of 2.5 millions energy evaluations and a maximum
number of 27,000 generations. e translation step was
1.0 Å, the quaternion and torsion step were set to 5.0
degrees. A mutation rate of 0.02 and a crossover rate of
0.8 were used. Results from Autodock calculations were
clustered using an RMSD tolerance of 1.5 Å and the low-
est energy conformer of the most populated cluster (the
lowest energy cluster in most cases) was selected as the
most probable binding conformer. For each compound,
200 docked conformations were generated. To visual-
ize the highest scored conformations into the binding
site, we used the Pymol Molecular Graphic System. In a
preliminary step, the reliability of the docking protocol
was checked by simulations of the binding modes of
9-Cl-TIBO and a comparison of the modelled complex
with the available 3D structure derived by X-ray crys-
tallography (1REV) was done. e obtained binding
mode of 9-Cl-TIBO was very close to that of the origi-
nal orientation found in the crystal. e RMS deviation
between the docked and crystal ligand coordinates was
0.89 Å. Since, none of the tested compounds obtained
the butterfly conformation and it is well known that the
binding site of NNRTIs is very flexible6; we decided to
use another crystallographic complex. Etravirine com-
plex with HIV-1 RT (TMC125, PDB:1SUQ) was selected
as the most appropriate for the second phase of the
study, since it enables relatively bulky compounds to be
freely oriented inside the binding pocket. In case of the
TMC125-RT complex (PDB code 1SUQ)24, the structure
Prediction of toxicity42
Prediction of toxicity of the tested compounds was
made using lazar model through the Open Tox Predict
Program. OpenTox is an open source web-service-based
framework, that provides unified access to experimental
toxicity data, in Silico models (including (Q)SAR), and
validation/reporting procedures. e Program uses vari-
ous models to predict toxicity of compounds based on
their structure. Lazar model derives predictions from tox-
icity databases by searching for similar compounds. It is a
k-nearest-neighbour approach to predict chemical end-
points from a training set based on structural fragments.
Scheme 1. General synthetic procedure for benzothiazolyl-4-
thiazolidinones. Reagents and conditions: (a) Conventional
method: toluene, reflux for 20–26h, (b) microwave-assisted
technic: 80–100 °C, power 100W, 10–60min.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and HIV-1 RT inhibitory action 117
It uses a SMILES file and pre-computed fragments with
occurrences as well as target class information for each
compound as training input. It also features regression,
in which case the target activities consist of continuous
values. Lazar model uses activity-specific similarity (i.e.
each fragment contributes with its significance for the
target activity) that is the basis for predictions and confi-
dence index for every single prediction.
traditional method. All synthesized compounds were
characterized by elemental analysis and spectroscopic
methods (1H NMR, 13C-NMR, HRMS).
e IR spectra showed >C=O,−C-C- and C-Cl, C-F
stretching absorption band at 1700 cm−1 (strong) 1600
and 1540 cm−1 as well as 1096–1089 cm−1, respectively.
1
In H-NMR spectra in DMSO the δ values range (8.10–
7.00 ppm) is in agreement with the theoretical presence
of aromatic protons while the signal at 4.40–4.10ppm is
attributed to a proton of the position 2 (CH) of thiazolidi-
none moiety. e other protons appeared at the expected
results and discussion
13
Chemistry
chemical shifts. Furthermore in C-NMR spectra peaks
Synthesis of the compounds was performed both by the
conventional one pot method previously described15,21
and/or by microwave assisted one-pot synthesis
(Scheme 1). According to one-pot classical procedure,
reaction of the suitable aminobenzothiazole with equi-
molar amount of 2,6-dihalosubstituted benzaldehyde
was performed in the presence of excess amount of
mercaptoacetic acid in reflux toluene for 20–26h. When
classical procedure was used, products were obtained
as racemates in low to moderate yields. Using micro-
wave assisted one-pot synthetic procedure the reaction
time was reduced from 20–26 h needed for the classical
method to 10–60 min, depending on compounds. e
final products were obtained as pure crystals in satisfac-
tory to good yields, higher than those achieved by the
were observed at δ 172–170 ppm due to the presence
of >C=O group as well as peaks at δ 161–165ppm, that
attributed to C-2 of benzothiazole ring. Signals at 158–
141 ppm were accounted for C-F of aromatic ring. e
presence of signals at 53–60 ppm, as well as at 30–34 ppm
were attributed to C-2 and C-5 of thiazolidinone moiety
respectively.
HIV-1 RT inhibitory action
e main observation obtained from the measurement of
HIV-1 RT inhibitory action (Table 1) is that the presence
of the appropriate halogen or alkoxy-substituent at posi-
tions 4 or 6 of the benzyl ring of the benzothiazolyl moi-
ety may convert a practically inactive non-substituted
derivative to a relatively strong inhibitor. So, while the
Table 1. HIV-1 RT Inhibitory action.
Inhibition % (4 μM)
IC50a (μM)
Kmb (μΜ)
Vmaxb
R
X1
F
X2
F
1
4-H, 6-H
19
12
30
33
34
26
83
30
68
32
34
36
39
25
57
21
40
48
32
>40
>40
>40
>40
2
4-H, 6-H
F
Cl
Cl
Cl
F
3
4-H, 6-Cl
4-H, 6-Cl
4-H, 6-Cl
4-Cl, 6-H
4-Cl, 6-H
4-Cl, 6-H
4-H, 6-F
Cl
F
4
5
F
6
Cl
F
Cl
Cl
F
>40
0.04
>40
7
5.5 0.3
0.196 0.016
0.465 0.042
8
F
42.4 3.6
9
Cl
F
Cl
Cl
F
0.25
10
4-H, 6-F
11
4-H, 6-F
F
12
4-H, 6-MeO
4-H, 6-MeO
4-H, 6-MeO
4-MeO, 6-H
4-MeO, 6-H
4-H, 6-EtO
4-H, 6-EtO
4-H, 6-EtO
Cl
F
Cl
Cl
F
>40
>40
>40
3.0
13
14
F
15
Cl
F
Cl
Cl
Cl
Cl
F
16
>40
17
Cl
F
18
3.4
>40
0.3
7.7 0.7
0.285 0.023
0.470 0.055
19
F
25.7 2.5
nevirapine
-
12.5 0.5
0.473 0.025
aValues are means of three determinations and deviation from the mean is <10% of the mean value. adNTP concentration: 10 μM. bEach
experiment was done in triplicate. Enzfitter for windows 32 bit version was used for the calculations. V was expressed in arbitary units. 1
Unit equals to product formation which yields 1 O.D. at 405nm under the testing conditions.
© 2013 Informa UK, Ltd.

118 E. Pitta et al.
non-substituted 3-(benzo[d]thiazol-2-yl)-2-(2-chloro-
6-fluorophenyl)thiazolidin-4-one, 2, exhibits practically
no activity under experimental conditions, as also men-
tioned by Rawal et al.34, its 4-Cl-benzo[d]thiazol-2-yl ana-
logue, 7, is a potent RT inhibitor with an IC50 value of 0.04
μM (0.016 μg/ml). Compound 18, the 6-EtO-benzo[d]
thiazol-2-yl analogue of compound 2, also exhibits con-
siderable activity with an IC50 value of 3.4 μM (1.39 μg/
ml). Although, both, halogen or alkoxy-substitution of
the benzo[d]thiazol-2-yl moiety seems to have a positive
influence on inhibitory action in all cases, this influ-
ence is not substantial for most of the compounds. Only
four of the tested compounds exhibited considerable
inhibition activity under the testing conditions (IC50:
0.04− 3.4 μM). Two halogen substituted derivatives, the
3-(4-Cl-benzo[d]thiazol-2-yl)-2-(2-chloro-6-fluoro-
phenyl)thiazolidin-4-one, 7, and the 3-(6-F-benzo[d]thi-
azol-2-yl)-2-(2,6-dichloro-phenyl)thiazolidinone-4-one,
9, were the most active compounds, followed by two
alkoxy-substituted derivatives, the 3-(4-MeO-benzo[d]
thiazol-2-yl)-2-(2,6-dichloro-phenyl)thiazolidin-4-one,
15, and the 3-(6-EtO-benzo[d]thiazol-2-yl)-2-(2-chloro-
6-fluoro-phenyl)thiazolidin-4-one, 18.
Different combination of halogens at positions 2- and
6- of the 2-6-dihalophenyl moiety resulted in remarkable
difference in inhibitory action. is had been observed
in the series of compounds tested previously by Rawal
et al., as well. Moreover, it was found that the presence
of different halogen substituents at the same position of
the benzyl ring of the benzo[d]thiazol-2-yl moiety also
resulted in great difference in activity as concluded by
comparison of the 6-F-benzo[d]thiazol-2-yl derivative, 9
(IC50: 0.25 μM), with the 6-Cl-benzo[d]thiazol-2-yl ana-
logue, 3 (IC50 > 40 μM).
Figure 1. Docking of (A) compounds 7 (magenta) and etravirine
(yellow) and (B) compounds 6 (green) and 8 (blue) in the allosteric
centre of HIV-1 RT.
Docking analysis was performed for some of the com-
pounds in order to elucidate the differences observed in
IC50 values.
Among the different available crystallographic com-
plexes of NNRT Inhibitors with HIV-1 RT, the etravirine
complex (TMC125, PDB:1SUQ) was selected as the most
appropriate, since it enables relatively bulky compounds
to be freely oriented inside the binding pocket.
Docking analysis of selected compounds
Although, only crystallographic results may provide a
certain prove about the binding of the compounds to the
enzyme, docking analysis may give an indication of prob-
able binding mode and help us to propose an explana-
tion of the results.
Using this docking system, docking analysis of the most
active derivative, 7, (Figure 1A) showed that this com-
pound adopts a conformation similar to that of etravirine
(TMC125) in the PDB:1SUQ complex. More precisely,
the 2-Cl-6-F-phenyl ring of compound 7 is oriented in
the pocket consisted by the aminoacids Tyr181, Pro95,
Tyr188 and Trp229 while the substituted benzothiazolyl
moiety interacts with the aminoacids Leu234, Tyr318 and
His235. ese hydrophobic interactions may contrib-
ute to complex stabilization. e part of thiazolidinone
moiety containing the sulfur atom is placed in a cleft
consisted of the aminoacids Lys101 and Val179 of the p66
subunit and Glu138 of p51 subunit of RT while oxygen
of the carbonyl group of the thiazolidinone ring forms a
hydrogen bond (Hb) with hydrogen of the amino group
of the backbone of Lys101. Hb formation with Lys101 is
a common characteristic of NNRTIs although CO of the
All synthesized compounds posses the thiazolidinone
heterocyclic ring as well as a second benzo[d]thiazolyl
heterocyclic moiety offering the possibility of hydrogen
bond formation with Lys101 or Lys103 which stabi-
lizes most NNRTI complexes. Moreover, they have two
hydrophobic moieties, the dihalogen substituted phenyl
group at position 2 of the thiazolidin-4-one ring and the
benzothiazolyl moiety. Both these moieties offer to the
molecule the possibility to occupy the hydrophobic cleft
of Trp229, Tyr181 and Pro95 present at the allosteric site
where traditional NNRTIs bind. However, only some of
the compounds can take the orientation allowing these
interactions.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and HIV-1 RT inhibitory action 119
main-chain of the amino acid is usually involved in the
bond, as also observed in case of ertavirine (Figure 1A)43.
Lys103 which is involved in Hb formation in most cases
of traditional NNRTIs and is mutated in most resistant
strains does not seem to be involved in complex stabili-
zation of compound 7.
e Hb with Lys101, present in case of derivative
7, is not observed in docking results of the less active
compounds 6 and 8 (Figure 1B). Compound 8, seems to
obtain a totally different orientation in the RT allosteric
centre. e 2,6-difluoro-phenyl ring is placed in vicinity
to aminoacids Val179, Tyr188 and Tyr181 while the ben-
zothiazolyl moiety interacts with the aminoacids Phe227
and Tyr318. e part of thiazolidinone moiety contain-
ing the sulfur atom is placed in a cavity consisted of the
aminoacids Trp227, Tyr188 and Tyr181. is orientation
does not favour Hb formation.
Compound 6, the 2,6-dichloro-phenyl analogue, can
be placed in the allosteric centre in a way very similar to
that of derivative 7. However, a slight change in the ori-
entation also disables the Hb formation, thus leading to a
less stable complex.
e absence of Hbs in the complexes of the less active
inhibitors may explain the difference in activity. is
probable explanation is not supported by the differ-
ences in binding energies (compound 6:-9.3 kcal/mol,
compound 7:-9.32 kcal/mol, compound 8:-9.0 kcal/
mol) which are very similar. is implies that the enzyme
probably obtains a much different and more convenient
conformation in case of binding with compound 7 which
can not be approached by the structures obtained by
crystallographic data of etravirine.
Figure 2. Michaelis-Menten and double reciprocal curves in the
absence of inhibitor (●ꢀ) and in the presence of 4μM of compound
7 (■), compound 18 (▲), compound 8 (□)and compound 19 (△).
Moreover, compounds 9, 15 and 18 which are also
active, do not adopt an orientation that favours Hb for-
mation or ensures a high binding capacity to the allos-
teric centre. is also implies that another binding mode
or structural alteration of the allosteric centre may exist
in the presence of these inhibitors.
In order to explore this hypothesis, we proceeded to
kinetic studies of two of the active compounds, deriva-
tives 7 and 18 and two of the practically inactive com-
pounds, derivatives 8 and 19.
ese structural changes may enable a more stable
interaction between RT allosteric centre and active
inhibitors of this series, although only crystallographic
studies may confirm this hypothesis. One more exam-
ple of this kind of RT inhibition by a quinolinic deriva-
tive which can bind to the allosteric site of NNRTIs in a
different than the classic way, was mentioned recently
by Souza et al.26.
However, inhibitors 8 and 19 probably fail to stably
associate to the allosteric centre of the enzyme and com-
pete with dNTPs for binding to the active centre. Perhaps
this difference in inhibition mechanism may explain the
great differences observed in IC50 values of the studied
molecules.
eKmvalueofRTintheabsenceofinhibitorwasfound
to be 12.5 μΜ under the testing conditions (Table 1). is
is higher than the values calculated when other methods
were used but is close to the values referred by investi-
gators who used the same method44. e presence and
partial incorporation of conjugated-dUTPs according to
the method may be responsible for the relatively high
Km value obtained in the absence of inhibitor, since the
presence of modified dNTPs in the reaction mixture was
found to increase Km values in many cases20,21,45–48. In the
presence of compounds 7 and 18 which showed uncom-
petitive inhibition mode, Km values as well as Vmax
Kinetic properties of selected compounds
RT activity of compounds 7, 18, 8 and 19 was measured
in the presence of stable concentrations of RΝΑ:primer
substrate and increasing concentrations of dNTP (dTTP)
in the absence and in the presence of stable concentra-
tion (4 μM) of inhibitors.
None of the tested compounds exhibited the com-
mon non-competitive inhibition mechanism. e more
active compounds (7 and 18) follow an uncompetitive
inhibition mode while the less active compounds (8
and 19) adopt a competitive inhibition mechanism
(Figure 2, Table 1). is means that, under the testing
conditions, compounds 7 and 18 bind exclusively to
the enzyme after dNTP binding. It is well known that
the structure of RT is modified after binding of dNTPs43.
© 2013 Informa UK, Ltd.

120 E. Pitta et al.
were decreased (compound 7: Km = 5.5, Vmax = 0.196;
compound 18: Km = 7.7, Vmax = 0.285). Vmax remained
practically the same, while Km values were increased
in the presence of compounds 8 and 19 (compound
8: Km = 42.4, Vmax = 0.465; compound 19: Km = 25.7,
Vmax = 0.470).
Kinetic results show that compound 7 probably binds
after dNTPs binding to the active site. is necessary
condition implies that change in enzyme structure after
dNTPs binding is a requirement for stable binding of
compound 7. is may explain the inability of docking
studies based on the structure of etravirine to support the
experimental results.
active compounds probably need structural characteris-
tics of the allosteric centre that can be acquired only after
dNTPs binding. So, they bind at a binding site structur-
ally altered and different of the commonly used NNRTIs
like etravirine. e results differentiate these compounds
from the traditional non-competitive NNRTIs, which is a
desired characteristic, since mutations that affect activ-
ity of non-competitive NNRTIs may not affect activity of
compounds of this series.
Interestingly, the less active derivatives (IC50 > 40 μM)
exhibit a competitive mode of action which probably
explains the much lower activity of these compounds.
Declaration of interest
Toxicity prediction
e authors report no declarations of interest.
According to Lasar model, the predicted LC50 val-
ues for the most active compounds, derivatives 7 and
9, are 0.11945591mM (confidence: 0.11945591) and
0.11945591mM (confidence: 0.105261415) respectively.
Predicted LC50 values for nevirapine and efavirenz, two
currently in use NNRTIs are 0.8378014mM (confidence:
0.17544146)and0.015711168mM(confidence:0.1532123),
respectively. Predicted toxicity of the tested compounds
is lower than that of efavirenz, but higher than nevirap-
ine. Prediction results only give a first indication about
probable toxicity and cannot replace in vitro and in vivo
experiments. However, prediction programs are highly
recommended in order to reduce in vivo experiments.
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