Novel thiazolidin-4-ones as potential non-nucleoside inhibitors of HIV-1 reverse transcriptase

Article abstract of DOI:10.3390/molecules24213821

Background: HIV is the causative agent of Acquired Immunodeficiency Syndrome (AIDS), an infectious disease with increasing incidence worldwide. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) play an important role in the treatment of AIDS. Although, many compounds are already being used as anti-HIV drugs, research for the development of new inhibitors continues as the virus develops resistant strains. Methods: The best features of available NNRTIs were taken into account for the design of novel inhibitors. PASS (Prediction of activity spectra for substances) prediction program and molecular docking studies for the selection of designed compounds were used for the synthesis. Compounds were synthesized using conventional and microwave irradiation methods and HIV RT inhibitory action was evaluated by colorimetric photometric immunoassay. Results: The evaluation of HIV-1 RT inhibitory activity revealed that seven compounds have significantly lower IC₅₀ values than nevirapine (0.3 μM). It was observed that the activity of compounds depends not only on the nature of substituent and it position in benzothiazole ring but also on the nature and position of substituents in benzene ring. Conclusion: Twenty four of the tested compounds exhibited inhibitory action lower than 4 μM. Seven of them showed better activity than nevirapine, while three of the compounds exhibited IC₅₀ values lower than 5 nM. Two compounds 9 and 10 exhibited very good inhibitory activity with IC₅₀ 1 nM.

Full text of DOI:10.3390/molecules24213821

molecules
Article
Novel Thiazolidin-4-ones as Potential
Non-Nucleoside Inhibitors of HIV-1
Reverse Transcriptase
Anthi Petrou 1 , Phaedra Eleftheriou 2, Athina Geronikaki 1,*, Melpomeni G. Akrivou 3 and
Ioannis Vizirianakis 3
1
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki,
Thessaloniki 54124, Greece; anthi.petrou.thessaloniki1@gmail.com
Department of Biomedical Sciences, School of Health Sciences, International Hellenic University, Nea
2
Moudania 57001, Greece; eleftheriouphaedra@gmail.com
Department of Pharmacology and Pharmacognosy, School of Pharmacy, Aristotle University of Thessaloniki,
3
Thessaloniki 54124, Greece; akrivoumg@pharm.auth.gr (M.G.A.); vizir@pharm.auth.gr (I.V.)
*
Correspondence: geronik@pharm.auth.gr; Tel.: +30-2310997616
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 30 August 2019; Accepted: 22 October 2019; Published: 23 October 2019
Abstract: Background: HIV is the causative agent of Acquired Immunodeficiency Syndrome (AIDS),
an infectious disease with increasing incidence worldwide. Non-nucleoside reverse transcriptase
inhibitors (NNRTIs) play an important role in the treatment of AIDS. Although, many compounds
are already being used as anti-HIV drugs, research for the development of new inhibitors continues
as the virus develops resistant strains. Methods: The best features of available NNRTIs were taken
into account for the design of novel inhibitors. PASS (Prediction of activity spectra for substances)
prediction program and molecular docking studies for the selection of designed compounds were
used for the synthesis. Compounds were synthesized using conventional and microwave irradiation
methods and HIV RT inhibitory action was evaluated by colorimetric photometric immunoassay.
Results: The evaluation of HIV-1 RT inhibitory activity revealed that seven compounds have
significantly lower IC50 values than nevirapine (0.3
µM). It was observed that the activity of
compounds depends not only on the nature of substituent and it position in benzothiazole ring but
also on the nature and position of substituents in benzene ring. Conclusion: Twenty four of the tested
compounds exhibited inhibitory action lower than 4 µM. Seven of them showed better activity than
nevirapine, while three of the compounds exhibited IC50 values lower than 5 nM. Two compounds 9
and 10 exhibited very good inhibitory activity with IC50 1 nM.
Keywords: thiazolidin-4-ones; NNRTIs; AIDS; HIV-1 reverse transcriptase; molecular docking
1. Introduction
Acute Immunodeficiency Syndrome (AIDS) is a result of the infection and destruction of
T-lymphocytes by the human immunodeficiency virus (HIV). Over 60 million people are infected with
the virus placing AIDS as the fourth cause of death worldwide [1–3].
Non-nucleoside reverse transcriptase inhibitors, as a part of Highly Active Antiretroviral Therapy
(HAART), remain in the first line of fighting against AIDS. However, research for the development of
novel NNRT inhibitors still continues as the virus develops resistant strains, limiting their use.
Among the approved by FDA compounds are several heterocyclic compounds such as
benzoxazin-2-one (etravirine), dipirydo [
(delaverdine) moieties [ ]. Except of these drugs many other molecules have been found to display RT
inhibitory action [ 13], among which are: benzothiazine dioxides [14], N1,N3-disubstituted uracils [15],
1,4] diazepine-6-one (etravirin) [4], piperazine and indolyl
5,6
7
–
Molecules 2019, 24, 3821; doi:10.3390/molecules24213821
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3821
2 of 29
6-arylmethyl-substituted S-DABOs [16], adamantyl-substituted thiazolidine-4-ones [17
,18] and many
others [19–27]. The 2,3-diaryl-thiazolidin-4-one scaffold appeared as a selective NNRTI [28,29].
Modeling studies on the HIV-1 RT inhibitory activity of 2,3-diaryl-thiazolidin-4-ones showed the
importance of overall hydrophobicity of the analogues and the presence of a heteroaryl system over
the 3-aryl moiety for better HIV-1 RT inhibitory activity [30–34].
Computational methods are an important part of the drug design process and they are presently
used to get a deeper understanding into the drug-enzyme interactions.
In this study, taking into account the best features of available NNRTIs a large number of novel
2,3-diaryl-thiazolidin-4ones were designed. The most promising compounds were selected with the
aid of Molecular docking studies and prediction of spectra of biological activity for compounds by
computer program PASS, synthesized and evaluated.
2. Results and Discussion
2.1. Rational Design of the Compounds
It is known that HIV-1 reverse transcriptase (HIV-1 RT) has a flexible structure that contains two
binding domains. The active site characterized by the catalytic triad of Asp110, Asp185 and Asp186
residues, where the deoxynucleoside triphosphates are bound by the physiological process, and an
allosteric site of NNRTIs about 10 Å away from the active site.
The NNRTIs binding pocket is hydrophobic, as most of its amino acids are hydrophobic (Pro59,
Leu100, Val106, Val179, Leu234 and Pro236) and five of them are aromatic (Tyr181, Tyr188, Phe227,
Trp229 and Tyr232). Certain hydrophilic amino acids such as Lys101, Lys103, Ser105, Asp132 and
Glu224 are also present [35]. For the binding of NNRTIs to the reverse transcriptase enzyme, their
ability to adopt a configuration characterized as a “butterfly orientation” is needed [36]. Aromatic rings
are usually present in both wings of “batterfly” conformation. A heeterocyclic ring or other hydrogen
donor/acceptor groups stabilized the complex via hydrogen bond formation, usually with Lys101.
The design of the compounds was based on the presence of the above characteristics and consist
of a benzothiazole ring connected via thiazolidinone moiety with a phenyl ring. The benzothiazole and
the phenyl ring serve as the wings of the ‘’butterfly” confirmation, forming hydrothobic interactions,
while the thiazolidinone moiety involves in the formation of hydrogen bond interactions especially
with Lys101 (Figure 1). Different substituents at the benzothiazole and phenyl rings were used to
determinate the compounds with the best predicted activity.
Figure 1. Structure and Basic Characteristics of the Designed Compounds.

Molecules 2019, 24, 3821
3 of 29
2.2. Computational Prediction of Anti-HIV Activity
PASS prediction of anti-HIV RT inhibitory activity was performed for several dozen designed
molecules, from which thirty two were selected for synthesis and testing. Prediction was carried out
using PASS online [37] version. For all selected compounds, anti-HIV inhibitory activity was predicted
with Pa values in range of 0.297–0.704 (Table 1). The calculated Pa values for most of compounds were
less than 0.5, indicating their relative novelty compared to the structures of the compounds from the
PASS training set [38,39].
2.3. Molecular Docking Prediction
For the docking studies the enzyme of RT in complex with the inhibitor TMC 125, the most widely
used in the literature, was selected (PDB code: 3MEC). At first stage, a validation test was performed
to certify the program’s reliability. Firstly, the ligand TMC 125 (etravirine), in the conformation found
in the crystal structure, was extracted and docked back into the corresponding binding pocket to
determine the ability of Autodock to reproduce the orientation and position of the inhibitor observed in
the crystal structure. According to docking results, the orientation of the docked TMC 125 inhibitor was
very close to that found in the crystal structure (Figure 2). The low RMS deviation of 0.58 Å between
the docked and the crystal ligand is an indication of the very good alignment of the experimental and
calculated positions.
Figure 2. Conformation of TMC 125-crystal Structure (green) as Compared to the Docked Conformation
of TMC 125 (yellow).
All designed compounds and the reference drug nevirapine were docked to the HIV-1 reverse
transcriptase enzyme using the same parameters (Table 1). Both the (S) and (R) isomer of each
compound were docked separately. It was observed that the (R) isomer exhibited proportionaly
decreased free binding energy for each compound [40–42].
The three-dimensional docked structures of the studied compounds were compared with the
X-ray crystallographic structure of TMC 125 (Figure 3). Detailed analysis of the binding mode found
in the docked compounds-RT and other NNRTIs-RT crystal complexes showed that the most of the
compounds could bind to RT in a butterfly-like conformation. Like TMC 125 (Figure 3), most of the
compounds are involved in the formation of a hydrogen bond with Lys101, which was observed in the
X-ray crystallographic structure of the TMC 125-RT complex [43].

Molecules 2019, 24, 3821
4 of 29
Figure 3. Docked Conformation of TMC125(etravirine) (A) and Nevirapine (B) in the Allosteric Center
of HIV-1 RT.
From the 132 compounds designed, the 32 showed very good binding scores and taking into
account also the prediction results by PASS, were chosen for the synthesis (Tables 1 and 2).
It was observed that these compounds bind to the allosteric center of the enzyme occupying
a similar configuration to TMC 125 inhibitor (Figure 4a, best docked conformations of compounds
1
,
9,
14
,
27). Comparison of the X-ray structure of TMC 125 with compound
9
(−14.63 kcal/mol)
(Figure 4b), showed that this compound occupies similar configuration to TMC 125 inhibitor and
forms a hydrogen bond with Lys101. Specifically, the CH2-S moiety of 4-thiazolidinone is oriented
towards a channel formed between the residues Lys101, Val179 (p66 subunit) and Glu138 (p51 subunit).
The nitrogen atom of Lys101 interacts with the oxygen atom of the 4-thiazolidinone forming a hydrogen
bond. The substituted benzothiazole ring interacts hydrophobically with the residues Leu100, Val179,
Glu138 and Leu234. These hydrophobic interactions contribute significantly to the stabilization of
the compound-RT complex. As shown in Figure 4b, the 4-hydroxyphenyl ring is located inside the
cavity surrounded by the residues Pro95, Thr139, Glu138, and Ile180. It is important to mention that
the presence of the hydrogen bond with Lys101 is characteristic of many NNRTIs inhibitors. This
phenomenon is also observed in the crystallographic TMC 125-RT and 9-Cl TIBO-RT complexes [43].
Compounds with higher docking scores exhibit a different binding mode from those with lower
docking scores. In this type of binding, the substituted phenyl ring is oriented to a “channel” formed
by Leu100, Glu138, Ile180 and Val179. This orientation results in the CH2-S moiety of 4-thiazolidinone
being removed away from Lys101 and thus, being not able to interact and form a hydrogen bond with
it. This fact contributes significantly to the ability of these compounds to form a stable complex with
the enzyme and, potentially, to their intended action.

Molecules 2019, 24, 3821
5 of 29
Table 1. Molecular docking scores and PASS prediction of all the designed compounds.
Free Binding
Energy
(kcal/mol)
S-(−)
Free Binding
Energy
(kcal/mol)
R-(+)
Free Binding
Energy
(kcal/mol) -(−)
Free Binding
Energy
(kcal/mol) R-(+)
N
R1
R2
Pa
N
R1
R2
Pa
a1 (1)
a2 (2)
b1 (3)
b2 (4)
b3 (5)
b4 (6)
b5 (7)
b6
b7
b8
c1 (8)
c2
c3
c4
c5 (9)
c6
c7
c8
d1 (10)
d2 (11)
d3 (12)
d4 (13)
d5 (14)
d6
7-Cl
7-Cl
6-F
6-F
6-F
6-F
6-F
6-F
6-F
6-F
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
2,6-di-F
2-F, 6-Cl
4-F
4-NO2
4-Cl
4-OCH3
4-OH
4-Br
2,3-di-Cl
2,4-di-Cl
4-F
2-Cl
3-Cl
3-Br
4-OH
4-Br
2,3-di-Cl
2,4-di-Cl
4-F
4-NO2
4-Cl
4-OCH3
4-OH
4-Br
2,3-di-Cl
2,4-di-Cl
4-F
4-NO2
4-Cl
4-OCH3
4-OH
4-Br
−10.21
−11.42
−10.95
−8.02
−8.15
−7.14
−8.27
−6.17
−6.53
−6.05
−10.13
−6.28
−6.55
−6.01
−11.08
−6.01
−6.25
−5.92
−11.16
−8.45
−8.73
−9.11
−10.58
−6.17
−6.63
−7.14
−9.93
−10.05
−9.92
−8.76
−10.15
−6.58
−13.37
−14.10
−13.21
−9.11
−9.02
−8.70
−9.27
−6.54
−6.71
−6.41
−12.30
−6.85
−6.93
−7.11
−13.63
−7.11
−7.59
−6.42
−14.59
−9.28
−10.26
−11.69
−13.72
−7.76
−7.15
−7.93
−11.51
−11.58
−11.23
−10.97
−11.02
−7.46
0.651
0.704
0.472
0.374
0.398
0.360
0.415
0.401
0.353
0.349
0.426
0.372
0.285
0.402
0.492
0.419
0.411
0.419
0.469
0.393
0.480
0.378
0.436
0.422
0.424
0.404
0.353
0.374
0.347
0.380
0.379
0.329
i7
i8
6-Br
6-Br
6-CN
6-CN
6-CN
6-CN
6-CN
6-CN
6-CF3
6-CF3
6-CF3
6-CF3
6-CF3
6-CF3
6-Ad
6-Ad
6-Ad
6-Ad
6-Ad
6-Ad
6-Ad
6-Ad
6-Ad
6-Ad
2,3-di-Cl
2,4-di-Cl
3-Cl
3-Br
3-F
2,6-di-Cl
2,3-di-Cl
2,4-di-Cl
2,6-di-Cl
3-Cl
3-Br
4-Br
2,3-di-Cl
2,4-di-Cl
3-Cl
−3,25
−5,37
−5.29
−4.18
−6.32
−6.58
−6.45
−6.93
−6.44
−5.41
−5.07
−5.09
−4.05
−6.41
−5.12
−5.96
−6.25
−7.14
−7.02
−6.82
−5.17
−6.79
−6.49
−4.18
−4.26
−4.13
−7.03
−6.58
−6.93
−6.74
−5.08
−6.83
−4.07
−6.43
−6.57
−5.66
−703
0.352
0.341
0.353
0.393
0.287
0.498
0.447
0.366
0.448
0.332
0.254
0.378
0.336
0.332
0.273
0.280
0.428
0.289
0.285
0.325
0.226
0.319
0.295
0.333
0.226
0.280
0.507
0.280
0.276
0.313
0.202
0.307
k1
k2
k3
k4
k5
k6
l1
l2
l3
l4
l5
−7.84
−7.16
−7.18
−7.12
−6.27
−5.71
−5.82
−4.70
−6.91
−6.74
−6.89
−7.04
−8.05
−7.94
−7.88
−6.75
−7.55
−7.28
−5.94
−5.97
−5.62
−8.12
−7.89
−7.91
−7.59
−6.65
−7.94
l6
m1
m2
m3
m4
m5
m6
m7
m8
m9
m10
n1
n2
n3
n4
n5
n6
n7
n8
3-Br
2,6-di-F
2,3-di-Cl
2,4-di-Cl
4-F
4-NO2
4-Cl
4-OCH3
4-OH
3-Cl
d7
d8
4-CH3, 6-Ad
4-CH3, 6-Ad
4-CH3, 6-Ad
4-CH3, 6-Ad
4-CH3, 6-Ad
4-CH3, 6-Ad
4-CH3, 6-Ad
4-CH3, 6-Ad
4-Cl
3-Br
e1 (15)
e2 (16)
e3 (17)
e4 (18)
e5 (19)
e6
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
2-F, 6-Cl
2,3-di-Cl
2,4-di-Cl
4-F
4-NO2
4-Cl

Molecules 2019, 24, 3821
6 of 29
Table 1. Cont.
Free Binding
Energy
(kcal/mol)
S-(−)
Free Binding
Energy
(kcal/mol)
R-(+)
Free Binding
Energy
(kcal/mol) -(−)
Free Binding
Energy
(kcal/mol) R-(+)
N
R1
R2
Pa
N
R1
R2
Pa
e7
e8
4-OCH3
4-OCH3
6-OCH3
6-OCH3
6-OCH3
6-OCH3
6-OCH3
6-OCH3
6-OCH3
6-OCH3
6-OCH2CH3
6-OCH2CH3
6-OCH2CH3
6-OCH2CH3
6-OCH2CH3
6-OCH2CH3
6-OCH2CH3
6-OCH2CH3
6-OCF3
6-OCF3
6-OCF3
6-OCF3
6-OCF3
6-OCF3
6-OCF3
6-OCF3
6-OCF3
2,3-di-Cl
2,4-di-Cl
4-F
4-NO2
4-Cl
4-OCH3
3-Br
4-Br
2,3-di-Cl
2,4-di-Cl
4-F
4-NO2
4-Cl
4-OCH3
4-OH
4-Br
2,3-di-Cl
2,4-di-Cl
2,6-di-Cl
2,6-di-F
3-Cl
2,3-di-Cl
3-Br
4-F
4-NO2
4-Cl
3-F
4-OH
4-F
4-NO2
4-Cl
4-OCH3
4-OH
4-Br
−5.14
−5.79
−10.87
−7.14
−9.88
−9.91
−5.03
−5.17
−5.61
−6.24
−6.97
−7.14
−9.84
−9.10
−10.05
−4.28
−5.84
−5.33
−9.87
−7.77
−5.96
−7.23
−5.17
−6.94
−8.45
−6.89
−5.22
−7.97
−7.31
−6.88
−7.19
−5.35
−7.22
−5.11
−6.03
−6.48
−12.17
−8.96
−11.00
−11.49
−6.11
−6.13
−6.92
−7.32
−8.35
−9.02
−11.35
−10.98
−11.56
−5.43
−6.47
−6.21
−11.03
−8.85
−6.88
−9.31
−6.33
−8.74
−9.07
−8.71
−6.19
−8.51
−8.10
−7.95
−8.02
−6.42
−8.02
−5.84
0.317
0.322
0.393
0.346
0.365
0.420
0.302
0.367
0.303
0.325
0.373
0.339
0.376
0.356
0.387
0.358
0.286
0.320
0.388
0.424
0.315
0.300
0.242
0.345
0.315
0.332
0.330
0.442
0.301
0.326
0.393
0.285
0.409
0.405
n9
n10
o1
o2
o3
o4
o5
o6
o7
o8
p1
p2
p3
p4
p5
p6
p7
p8
q1
q2
q3
q4
q5
q6
q7
q8
r1
4-CH3, 6-Ad
4-CH3, 6-Ad
5,6-di-CH3
5,6-di-CH3
5,6-di-CH3
5,6-di-CH3
5,6-di-CH3
5,6-di-CH3
5,6-di-CH3
5,6-di-CH3
6-NO2
6-NO2
6-NO2
6-NO2
6-NO2
6-NO2
6-NO2
6-NO2
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
4-OCH3
4-OH
4-F
4-NO2
4-Cl
4-OCH3
4-OH
−6.37
−4.16
−6.95
−5.18
−5.21
−3.28
−4.19
−3.05
−6.17
−6.14
−6.84
−5.11
−5.20
−3.15
−3.47
−3.01
−6.18
−6.29
−7.01
−5.10
−5.03
−4.85
−4.08
−3.17
−6.14
−6.33
−7.00
−5.02
−5.01
−4.77
−4.05
−3.12
−6.13
−6.10
−7.17
−5.93
−7.56
−6.05
−6.17
−4.56
−5.13
−4.27
−7.01
−6.86
−6.92
−6.01
−6.15
−4.42
−4.18
−4.15
−6.81
−6.98
−7.82
−6.12
−5.85
−5.16
−5.49
−4.55
−6.80
−6.86
−7.81
−5.93
−5.83
−5.12
−5.40
−4.52
−6.78
−6.64
0.287
0.321
0.433
0.328
0.432
0.327
0.351
0.355
0.275
0.286
0.390
0.225
0.382
0.348
0.398
0.284
0.341
0.337
0.345
0.326
0.335
0.290
0.353
0.338
0.390
0.382
0.337
0.315
0.327
0.284
0.345
0.330
0.377
0.372
f1 (20)
f2 (21)
f3 (22)
f4 (23)
f5
f6
f7
f8
g1 (24)
g2 (25)
g3 (26)
g4 (27)
g5 (28)
g6
g7
g8
h1 (29)
h2
h3
h4 (30)
h5
h6
h7 (31)
h8 (32)
h9
h10
i1
i2
i3
4-Br
2,3-di-Cl
2,4-di-Cl
4-F
4-NO2
4-Cl
4-OCH3
4-OH
4-Br
2,3-di-Cl
2,4-di-Cl
4-F
4-NO2
4-Cl
4-OCH3
4-OH
4-Br
2,3-di-Cl
2,4-di-Cl
4-F
4-NO2
4-Cl
4-OCH3
4-OH
4-Br
6-OCF3
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
r2
r3
r4
r5
r6
r7
r8
i4
i5
i6
2,3-di-Cl
2,4-di-Cl
Etravirine
−11.25
Nevirapine
−11.95

Molecules 2019, 24, 3821
7 of 29
Table 2. Molecular docking scores and PASS prediction of the selected compounds.
Free
Binding
Energy
(kcal/mol)
S-(−)
Free
Binding
Energy
(kcal/mol)
R-(+)
Predicted
Anti-HIV
Activity (Pa)
Amino
Acids
Hydrophobic
Interactions
Halogen
Interact.
N
1
H Bonds
Pi Interact.
Pro95,
Leu100,
Lys103,
Glu138,
Val179,
Tyr181
Thr139,
Lys172,
Gly190
0.651
0.704
−10.21
−13.37
−14.10
1
Lys101
Lys101
Ile180
Glu28, Gly99,
Leu100,
Lys103,
Asn136,
Tyr181,
Ile382,
Trp383
Glu138,
Ile135,
Val179,
Tyr319,
Pro321
2
3
−11.42
1
2
Glu138
Leu100,
Lys103,
Glu138,
Val179,
Tyr181,
Tyr188,
Trp229,
Leu234
Leu100,
Val106,
Glu138
Val106,
Gly190,
Leu234
Lys101
Trp229
0.472
−10.95
−13.21
-
-
Glu131,
Thr132
4
5
0.374
0.398
−8.02
−8.15
−9.11
−9.02
1
-
Lys101
-
Glu138,
Tyr181,
Tyr188,
Gly190,
Phe227,
Tyr318
Leu100,
Lys101,
Val106,
Val179
Leu234
Pro236
Leu100,
Glu138,
Val179,
Tyr181
Ser127,
Glu131
6
7
0.360
0.415
−7.14
−8.27
−8.70
−9.72
1
-
Lys103
-
Pro95,
Lys101,
Lys103,
Tyre181,
Gly190,
Phe117,
Trp229,
His235,
Pro236,
Tyr318
Leu100,
Val106,
Glu138,
Val179,
Tyr188,
Leu234
-
Glu138
Glu23,
Ser127,
Glu131,
Thr132,
Pro176,
Ile178, Ile180
Pro95,
Lys103,
Lys172,
Tyr181,
Leu234
Ile128,
Arg172,
Val179
Lys101
Pro126
8
9
0.426
0.492
−10.13
−11.08
−12.30
−14.63
2
2
-
-
Leu100,
Glu138,
Val179
Lys101
Thr139
Leu100,
Lys103,
Val106,
Glu138,
Val179,
Tyr188,
Leu234
Ile180,
Tur181,
Gly190,
Trp229
Lys101
His235
Pro236
10
0.469
−11.16
−14.59
1
Tyr318

Molecules 2019, 24, 3821
8 of 29
Table 2. Cont.
Free
Binding
Energy
(kcal/mol)
S-(−)
Free
Binding
Energy
(kcal/mol)
R-(+)
Predicted
Anti-HIV
Activity (Pa)
Amino
Acids
Hydrophobic
Interactions
Halogen
Interact.
N
H Bonds
Pi Interact.
Lys103,
Val106,
Ile180
Leu100,
Val179,
Tyr181
Leu100,
Glu138,
Val179
11
12
0.393
0.480
−8.45
−8.73
−9.28
1
1
Lys101
Lys101
-
Lys103,
Ile180
−10.26
Leu100,
Glu138,
Val179,
Tyr181
Leu100,
Lys103,
Val179,
Tyr181
Leu100,
Lys103,
Glu138,
Val179,
Tyr181
Leu100,
Glu138,
Val179,
Tyr181
Leu100,
Lys103,
Glu138,
Val179,
Tyr188,
Tyr318
Lys103,
Lys172,
Ile180
Lys101
Thr139
13
14
0.378
0.436
−9.11
−11.69
−13.72
2
3
-
-
Lys101
Glu138
Thr139
Val106,
Leu234
−10.58
Pro95,
Val106,
Tyr188,
Leu234
Lys101
Trp229
15
16
0.353
0.374
−9.93
−11.51
−11.58
2
2
-
-
Pro95,
Val106,
Tyr188
−10.05
Lys101
Lys101
Pro95, Ile180,
Tyr181,
Gly190,
Trp229,
His235
17
18
0.347
0.380
−9.92
−8.76
−11.23
−10.97
1
2
-
-
Pro95,
Lys101,
Lys102,
Tyr181,
Tyr188,
Gly190,
Trp229,
His235
Lys103,
Ile180,
Tyr181,
Gly190,
Trp229,
Pro236,
Tyr318,
His235
Pro95,
Leu100,
Val106,
Glu138,
Val179,
Leu234
Glu138
Tyr318
Leu100,
Val106,
Glu138,
Val179,
Tyr188,
Leu234
19
20
0.379
0.393
−10.15
−11.02
−12.17
1
2
Lys101
-
-
Leu100,
Lys103,
Lys172,
Ile180,
Glu138,
Val179,
Tyr181,
Trp229
Lys101
Thr139
10.87
Tyr188
Leu100,
Glu138,
Val179
Pro95, Ile180,
Tyr188
21
22
0.346
0.365
−7.14
−9.88
−8.96
1
2
Lys101
-
-
Pro95,
Lys172,
Ile180,
Tyr188
Lys102,
Lys103,
Ile180,
Tyr188,
Phe225,
Pro234
Leu100,
Glu138,
Val179
Lys101
Thr132
−11.00
Leu100,
Val106,
Val179,
Trp227,
Leu232,
Tyr316
Lys101
Tyr181
23
0.420
−9.91
−11.49
2
-

Molecules 2019, 24, 3821
9 of 29
Table 2. Cont.
Free
Binding
Energy
(kcal/mol)
S-(−)
Free
Binding
Energy
(kcal/mol)
R-(+)
Predicted
Anti-HIV
Activity (Pa)
Amino
Acids
Hydrophobic
Interactions
Halogen
Interact.
N
H Bonds
Pi Interact.
Pro95,
Lys103,
Ile180,
Leu100,
Glu138,
Val179
24
25
0.373
0.339
−6.97
−7.14
−8.35
−9.02
1
1
Lys101
Lys101
-
-
Tyr181
Pro95, Gly99,
Thr139,
Ile180,
Leu100,
Glu138,
Val179
Tyr181
Pro95, Gly99,
Lys103,
Thr139,
Lys172,
Ile180,
Leu100,
Glu138,
Val179,
Trp229,
Leu234
26
0.376
−9.84
−11.35
1
Lys101
Lys101
-
Tyr181
Pro95, Pro97,
Lys103,
Thr139,
Lys172,
Ile180,
Leu100,
Glu138,
Val179,
Trp229,
Leu234
27
28
0.356
0.387
−9.10
−10.98
−11.56
1
2
-
-
Tyr181
Leu100,
Glu138,
Val179,
Trp229,
Leu234
Leu100,
Lys103,
Val106,
Val179,
Tyr188,
Leu234
Pro95, Pro97,
Lys103,
Lys172,
Lys101
Thr139
−10.05
Ile180
Pro95,
Tyr181,
Gly190,
Phe227,
Pro236,
Tyr318
Lys101,
Tyr181,
Gly190,
His235
Pro95,
Glu138
Gly99
29
30
0.388
0.300
−9.87
−7.23
−11.03
−9.31
1
1
Lys101
Leu100,
Val179,
Tyr188
Lys101
Lys101
-
Leu100,
Glu138,
Val179,
Lys103,
Lys172,
Leu234
31
32
0.315
0.332
−8.45
−6.89
−9.07
−8.71
1
2
-
-
Glu138,
Val179,
Pro321
Lys103
Thr132
Pro95,
Tyr181
Glu28, Gly99,
Leu100,
Lys101,
Lys103,
Val179,
Lys101,
Glu138,
Ile135,
Tyr319,
Trp383
Lys101,
Glu138
Nevirapine
Etravirine
−11.95
2
2
-
-
Ile382
Pro95,
Leu100,
Lys103,
Val106,
Val179,
Tyr181,
Thr229,
Leu234
Lys102,
Val108,
Ty188,
Pro225,
Phe227,
Pro236
Lys101,
Glu138
−11.25

Molecules 2019, 24, 3821
10 of 29
Figure 4.
(
a) Docking of TMC125 (yellow) and Compounds
1
(green), (blue), 14 (light blue) and 27
9
(purple) to the Allosteric Site of HIV-1 RT. (b) Docking of Compound 9 (green) in the Binding Site
of HIV-1RT.
2.4. Prediction of Toxicity
According to Lasar model throughout OpenTox, all the compounds found to be at the category IV
with LD50 between 500 mg/kg and 1000 mg/kg and they are saved for use (Table 3).

Molecules 2019, 24, 3821
11 of 29
Table 3. Predicted toxicity with program PROTOX.
Predicted
Toxicity
Class
Predicted
LD50
‘E.
Hepatotoxicity
Carcinogenicity Immunotoxicity
Mutagenicity
Cytotoxicity
1
2
3
4
5
6
7
8
500 mg/kg
500 mg/kg
500 mg/kg
1000 mg/kg
500 mg/kg
500 mg/kg
500 mg/kg
500 mg/kg
500 mg/kg
500 mg/kg
1000 mg/kg
500 mg/kg
500 mg/kg
500 mg/kg
500 mg/kg
1000 mg/kg
500 mg/kg
500 mg/kg
500 mg/kg
500 mg/kg
1000 mg/kg
500 mg/kg
500 mg/kg
500 mg/kg
1000 mg/kg
500 mg/kg
500 mg/kg
500 mg/kg
500 mg/kg
500 mg/kg
1000 mg/kg
500 mg/kg
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
Inactive 0.56
Inactive 0.56
Inactive 0.57
Inactive 0.55
Inactive 0.57
Inactive 0.60
Inactive 0.63
Inactive 0.57
Inactive 0.60
Inactive 0.57
Inactive 0.52
Inactive 0.54
Inactive 0.57
Inactive 0.60
Inactive 0.59
Inactive 0.52
Inactive 0.56
Inactive 0.50
Inactive 0.52
Inactive 0.59
Inactive 0.52
Inactive 0.56
Inactive 0.50
Inactive 0.52
Inactive 0.53
Inactive 0.52
Inactive 0.53
Inactive 0.50
Inactive 0.61
Inactive 0.61
Inactive 0.56
Inactive 0.61
Inactive 0.57
Inactive 0.57
Inactive 0.55
Inactive 0.68
Inactive 0.55
Inactive 0.58
Inactive 0.59
Inactive 0.55
Inactive 0.58
Inactive 0.55
Inactive 0.65
Inactive 0.55
Inactive 0.58
Inactive 0.58
Inactive 0.60
Inactive 0.65
Inactive 0.60
Inactive 0.62
Inactive 0.64
Inactive 0.60
Inactive 0.65
Inactive 0.60
Inactive 0.62
Inactive 0.57
Inactive 0.63
Inactive 0.56
Inactive 0.62
Inactive 0.62
Inactive 0.56
Inactive 0.56
Inactive 0.64
Inactive 0.56
Inactive 0.94
Inactive 0.95
Inactive 0.99
Inactive 0.99
Inactive 0.99
Inactive 0.98
Inactive 0.99
Inactive 0.99
Inactive 0.99
Inactive 0.99
Inactive 0.99
Inactive 0.99
Inactive 0.99
Inactive 0.99
Inactive 0.98
Inactive 0.99
Inactive 0.98
Inactive 0.98
Inactive 0.99
Inactive 0.98
Inactive 0.99
Inactive 0.98
Inactive 0.98
Inactive 0.96
Inactive 0.97
Inactive 0.98
Inactive 0.97
Inactive 0.99
Inactive 0.91
Inactive 0.91
Inactive 0.81
Inactive 0.97
Inactive 0.67
Inactive 0.67
Inactive 0.66
Inactive 0.66
Inactive 0.67
Inactive 0.65
Inactive 0.66
Inactive 0.67
Inactive 0.68
Inactive 0.67
Active 0.91
Inactive 0.68
Inactive 0.66
Inactive 0.68
Inactive 0.66
Inactive 0.66
Inactive 0.67
Inactive 0.61
Inactive 0.60
Inactive 0.66
Inactive 0.66
Inactive 0.67
Inactive 0.61
Inactive 0.64
Inactive 0.66
Inactive 0.65
Inactive 0.61
Inactive 0.63
Inactive 0.66
Inactive 0.66
Inactive 0.66
Inactive 0.66
Inactive 0.76
Inactive 0.76
Inactive 0.80
Inactive 0.73
Inactive 0.81
Inactive 0.66
Inactive 0.70
Inactive 0.81
Inactive 0.68
Inactive 0.81
Inactive 0.74
Inactive 0.82
Inactive 0.64
Inactive 0.68
Inactive 0.66
Inactive 0.74
Inactive 0.65
Inactive 0.63
Inactive 0.63
Inactive 0.66
Inactive 0.74
Inactive 0.65
Inactive 0.63
Inactive 0.62
Inactive 0.62
Inactive 0.59
Inactive 0.61
Inactive 0.63
Inactive 0.64
Inactive 0.64
Inactive 0.64
Inactive 0.65
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
2.5. Chemistry
Synthesis of the compounds was performed by one pot method according to Scheme 1 [44] and for
some compounds by microwave assisted one-pot procedures (Scheme 1). According to conventional
method, the mixture of the suitable aminobenzothiazole, substituted benzaldehyde and mercaptoacetic
acid was refluxed in toluene for 18–32 h. All products were obtained as racemates in low yield. Instead
using microwave assisted one-pot synthetic procedure the reaction time was reduced to 30 min. The
final products were obtained in good yield. All synthesized compounds were characterized by TLC
and spectroscopic methods (IR, 1H NMR, 13C-NMR and MS for some compounds).
In IR spectra stretching absorption bands at 1700 cm−1 (strong) of C=O, 1600 and 1540 cm−1
of -C–C- and 3200 of –OH were detected. In 1H-NMR spectra signal at 8.10–6.89 ppm as well as at
4.35–4.10 ppm are attributed to aromatic protons and proton of the position 2 of thiazolidinone moiety
respectively. The rest of the protons appeared at the expected chemical shifts. In 13C-NMR spectra
peaks were observed for C=O group at
δ 172–170 ppm, for C-2 of benzothiazole ring at δ 161–165
ppm and for C-2 and C-5 of thiazolidinone moiety at 53–60 ppm and at 30–34 ppm respectively (see
experimental).

Molecules 2019, 24, 3821
12 of 29
Ν
R1
R2
Ν
R1
R2
Ν
R1
R2
1
7-Cl 2′,6-‘di-F 12
2′-F,
4-Cl
4′-Cl
23
6-OCH3
4′-OCH3
2
7-Cl
13
4-Cl
4′-OCH3 24
6-OCH2CH3
4′-F
6′-Cl
4′-F
3
4
5
6
7
8
9
6-F
6-F
14
15
16
17
18
19
20
21
22
4-Cl
4′-OH
4′-F
4′-NO2
4′-Cl
4′-OCH3 29
4′-OH
4′-F
4′-NO2
4′-Cl
25
26
27
28
6-OCH2CH3
6-OCH2CH3
6-OCH2CH3
6-OCH2CH3
6-OCF3
4′-NO2
4′-Cl
4′-OCH3
4′-OH
2′,6′-di-Cl
2′,3′-di-Cl
4′-NO2
4′-Cl
4′-NO2
4′-Cl
4′-OCH3
4′-OH
4′-F
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
6-OCH3
6-OCH3
6-OCH3
6-F
6-F
6-F
6-Cl
30
31
32
6-OCF3
6-OCF3
6-OCF3
6-Cl
4′-OH
4′-F
10 4-Cl
11 4-Cl
4′-NO2
Scheme 1. General synthetic procedure. Reagents and conditions: (a) Conventional method: toluene,
reflux for 18–32 h, (b) microwave-assisted technic: 100 ◦C, power 100 W, 30 min.
2.6. HIV-1 RT Inhibitory Action
The results of the evaluation of RT inhibitory activity are presented in Table 4. All compounds at
the concentration of 4 µM exhibited inhibitory action varying from 21% to 83%.
Seven compounds were found to have significantly lower IC50 than nevirapine (0.3
of which displayed IC50 value lower than 1 M. The order of the compound’s potency can be presented
as follows: 10 ≥ 9 14 > nevirapine > 20
18 22 21. Nine compounds, the 11
greater than 4 µM.
µM), the best
µ
>
2
=
>
>
3
>
1
>
8
>
27
24
>
13
,
>
28
>
15
31
>
16
> 23 > 26 > 17 > 19 >
>
>
4
>
7
=
12
5,
6,
,
,
25
29
,
30
,
,
32, exhibited IC50 values
The study of structure-activity relationships revealed that the activity of compounds depends
not only on the nature of substituent and its position in benzothiazole ring but also on the nature and
position of substituents in benzene ring. The presence of 6-Cl substitution in the benzothiazole moiety
in combination with the presence of hydroxyl group at position 40 of the benzene ring (
9) appeared to
be beneficial for the HIV RT inhibitory action. The same effect was observed in case of 4-Cl substitution
in the benzothiazole moiety combined with the presence of 40-F group in benzene ring (10).
The replacement of 40-F substitution in compound 10, one of the two most active compounds,
by 40-OH (14) remarkably decreased the activity although compound 14 still remained a very active
compound being more active than nevirapine. On the other hand, compound
7-Cl substituent in benzothiazole moiety and 20-F, 60-Cl (
) substitution in benzene ring was the third
most active compound. The replacement of 60-Cl (compound ) by 60-F in benzene ring (
) significantly
decreased activity compared to compound . While introduction of 6-F in benzothiazole moiety and
40-F substituent in benzene ring (
) increased the activity compared to compound , the replacement
2 with the presence of
2
2
1
2
3
1

Molecules 2019, 24, 3821
13 of 29
of 6-F by 6-Cl led to compound
8
with decreased activity in comparison with compounds and
1
3.
However, despite that compounds
1,
3
and were less active compared to compounds 10, 2 and 14
8
9
,
they still appeared to be more active than the reference compound.
Table 4. HIV-1 RT inhibitory action.
Inhibition
% (4 µM)
IC50
(µM)
Inhibition
% (4 µM)
IC50
(µM)
Inhibition
% (4 µM)
IC50
(µM)
N
N
N
1
2
3
4
5
6
7
8
9
70
72
72
54
44
35
50
83
56
63
40
0.21
0.0047
0.15
3.8
>4
>4
12
13
14
15
16
17
18
19
20
21
22
50
65
83
69
68
67
72
71
55
50
72
4
23
24
25
26
27
28
29
30
31
32
73
36
44
73
76
62
49
46
46
46
2.39
>4
>4
2.48
1.93
2.00
4.28
>4
1.99
0.01
2.08
2.20
2.60
2.69
2.62
1.51
4
4
0.26
0.001
0.001
>4
>4
>4
0.3
10
11
2.76
Nevirapine
The replacement of 40-F substituent by nitro-, chloro-, methoxy and hydroxyl group respectively
resulted to compounds with decreased activity ( ). The 6-F benzothiazole substituted derivatives
M. On the contrary, the 6-Cl benzothiazole substituted derivatives and
exhibited good inhibitory action with compound being one of the most active compounds. It seems
).
14) it appeared that the presence of the
hydrogen acceptor 40-F group was the most beneficial (10) followed by the hydrogen donor 40-OH
14) and 40-OMe group (13). The rest of compounds exhibited IC50 values
M. It is interesting to
mention that 4-OCH3 as well as 6-OCH3 benzothiazole substituted derivatives showed activity with
IC50 values between 1.5 and 3 M, showing small differentiation in concern to phenyl substituents.
An exception was observed only for the 40-nitrophenyl derivatives with 6-methoxy group in the
benzothiazole moiety (21, IC50
M) being the less active compound of the group. In general, 40-nitro
4–7
4
9
–
7
showed IC50 values
≥
4
µ
8
9
that in this group of compounds the presence of the hydrogen donor 40-OH group is beneficial (
9
In case of the 4-Cl benzothiazole substituted derivatives (10
–
(
≥
4 µ
µ
4
µ
substitution in benzene ring was detrimental for all groups of compounds with exception of the
derivatives with methoxy group in benzothiazole moiety.
The derivatives 26–
28 (40-Cl, 40-OMe, 40-OH) with 6-ethoxy group in benzothiazole moiety
displayed activity almost similar to that of 6-methoxy substitution (R1) with IC50 1.93–2.48 µM.
Regarding the 40-fluoro- and 40-nitrophenyl derivatives (24
, 25) IC50 values higher than 4 µM
were observed.
In conclusion, it is interesting to notice that the presence of chlorine in position 40 (R2) of benzene
ring was negative for all derivatives with halogen substituted benzothiazole moiety independent of
its position. The presence of a chloro- substituent at the benzothiazole ring (R1) at positions
significantly increases the inhibitory effect ( 10 14). On the other hand, the introduction
of -OCF3 substituent into the benzothiazole system further reduces the activity as compared to the
4, 6 or
7
1
,
2
,
8,
9,
,
chloro-substituted derivatives, with most of the compounds of this group (30
values higher than 4 µM.
–32) displaying IC50
2.7. Docking Analysis
Docking analysis effectively predicted RT inhibitory action in most cases as shown in Figure 5.

Molecules 2019, 24, 3821
14 of 29
-7
-9
-0.6
0.4
1.4
2.4
3.4
4.4
-11
-13
-15
-17
IC50
R² = 0.8264
Figure 5. Correlation of Predicted Free Binding Energy of the Compounds in the Allosteric Center of
the Enzyme with IC50 of the Compounds.
According to docking results, most of the compounds are placed within the NNRTIs binding
pocket between the place equipped by the classic butterfly shaped compound, nevirapine, and the
binding site of etravirine or within the etravirine binding pocket (Figure 6).
Figure 6. (Left) Docking of Etravirine (yellow), Nevirapine (blue) and Compounds 8 (magenta), 20 (light
green) and 10 (orange) in the Binding Site of HIV-1RT. (Right) Docked Conformations of Compounds
(black), 5 (dark purple), 7 (light blue) and 10 (orange) in the Binding Site of HIV-1RT.
3
Interestingly, the most active compounds structurally belong to three different groups and adopt
three different orientations.
An orientation most close to the nevirapine binding place is adopted by compound 2
, the 20-F,60-Cl
derivative of the subgroup of compounds bearing a Cl substituent at the 7-position of the benzothiazolyl
moiety (Figure 7A,B). More precisely, the benzothiazole and thiazolidinone moieties occupy positions
similar with the “wings” of nevirapine. The benzothiazolyl moiety is placed, in vicinity to the amino
acids Trp383 and Ile135 with which it forms pi interactions. The thiazolidinone ring is placed in the
area of Lys103, while the phenyl ring is placed near Glu138, Tyr181 and Lys101, forming pi interactions
with Glu138. Halogen and hydrogen bonds interactions are also formed between the F atom and the
amino acids Glu138 and Lys101, respectively, stabilizing the complex.
A different orientation is adapted by the 20,60-di-fluoro derivative of the same subgroup, compound
1 (Figure 7A,C). In this case, the thiazolidinone ring is placed in vicinity to Lys 101, which forms a
hydrogen bond with the CO group of this moiety. The benzothiazolyl group is placed in the same

Molecules 2019, 24, 3821
15 of 29
area where the central aromatic ring of etravirine is bound, towards Tyr181, Leu100 and Val179 with
which it participates in pi interactions. The two fluoro-atoms at the o-positions of the phenyl moiety
participate in halogen bond interactions, an intramolecular one with CO of the thiazolidinone ring
stabilizing the conformation of the molecule and a second one with Ile180.
Figure 7.
Etravirine (yellow) at the Allosteric Site of HIV-1 RT. (
of HIV-1 RT. (C) Docking of Compound 1 at the Allosteric Site of HIV-1 RT.
(
A
) Docking of Compounds
1
(dark green),
2
(light green), Nevirapine (dark blue) and
B
) Docking of Compound
2
at the Allosteric Site
A similar orientation is adopted by several active compounds like compounds 8, 9, the two more
active compounds of the subgroup bearing a Cl-atom at position 6 of the benzothiazolyl moiety (R1)
(Figure 8A,B,D) and compound 14, the most active compound of the subgroup bearing a Cl-atom at the
4-position of the benzothiazolyl moiety (Figure 8A,D). Among these, compounds
the four more potent out of the thirty-two tested compounds with IC50 values at the nanomolar range.
In case of compounds and 14, a hydrogen bond is formed between the CO group of the thiazolidinone
9 and 14 belong in
9
ring and Lys101 while a second hydrogen bond is formed between hydrogen acceptor substituents of
the 4-position of the phenyl ring and Thr139. An additional hydrogen bond between the H-atom of the
40-OH substituent and the Glu138 is also formed in compound 14. Pi interactions between the rings of
the benzothiazolyl moieties and the amino acids Leu100 and Val179 were observed in all these cases.
An orientation enabling pi interactions between the aromatic rings of the benzothiazolyl moiety
and the amino acids Leu100 and Val179, as well as hydrogen bond formation between Lys101 and the
CO group of the thiazolidinone ring were also observed in the active compounds of the 4-OMe, 6-OMe
and 6-OEt subgroups such as compounds 18, 20, 27 and 28 (Figure 9).

Molecules 2019, 24, 3821
16 of 29
Figure 8.
(dark blue) and etravirine (yellow) at the allosteric site of HIV-1 RT. (
allosteric site of HIV-1 RT. ( ) Docking of compound 10 at the allosteric site of HIV-1 RT. (
of compound 14 at the allosteric site of HIV-1 RT.
(
A
) Docking of compounds
8
(magenta),
9
(light blue), 10 (orange), 14 (cyan), nevirapine
) Docking of compound at the
) Docking
B
9
C
D
On the contrary, the presence of the strongly electro-negative 40-F (R2) substituent at compound
10 (Figure 8A) leads to a totally different orientation placing the compound with the phenyl moiety in
vicinity to Tyr318 with which the F-atom forms a hydrogen bond and close to Pro236 and His235 forming
halogen bonds. The differentiation of this compound from the other derivatives and most known
NNRTIs where hydrogen bond interactions with Lys101 play a crucial role in complex stabilization
is an interesting characteristic. However, the benzothiazolyl moiety is still placed in the area of
Lys101, Val179 and Leu100 forming pi interactions with the last two amino acids (Figure 8A,C). In this
conformation, the phenyl and the thiazolidinone moieties occupy the places occupied by the edge
aromatic rings of etravirine while the benzothiazolyl moiety is placed in the area of the central ring.
Similar orientations, with the phenyl or benzothiazole moiety placed towards Tyr 318 are also
observed in other cases (Figure 6).
In general, the presence of a Cl substituent at the 7-, 6- or 4- position of the benzothiazolyl
moiety facilitates an orientation which enables complex stabilization via pi-interactions and hydrogen
bond formation involving either the CO group of the thiazolidinone ring or hydrogen acceptor/donor
substituents of the phenyl ring. In case of subgroups bearing hydrogen acceptor substituents at the
benzothiazole moiety, involvement of these substituents in hydrogen bond formation also favors
complex stabilization such as in case of compound
3 of the 6F-subgroup, 18 of the 4-OMe subgroup,
23 of the -OEt subgroup and 33 of the -CF3 subgroup.

Molecules 2019, 24, 3821
17 of 29
Figure 9. 2D docking diagrams of compounds 19, 20, 27, and 28 at the allosteric site of HIV-1 RT.
2.8. Cytotoxicity Assessment
For the evaluation of cellular cytotoxicity of the compounds, the MRC-5 normal cells were
incubated for 48 h separately with each compound at the concentration of 10 M. This concentration
is much higher than IC50 values of all tested compounds (IC50 0.001 (compounds 10) and 0.01
µ
9
,
(compound 14). The evaluation revealed that compounds exhibited no evidence of cytotoxicity against
this normal cell line employed, since no statistically significant change was observed with the cell
growth to be 80% as compared to control-untreated culture (Figure 10a). Importantly, no significant
≥
dead cells accumulation was observed in the treated cultures, since their maximum number did not
exceed that of 3.3% (Figure 10b).

Molecules 2019, 24, 3821
18 of 29
Figure 10. Assessment cell growth (
a
) and cell death (
b
) of MRC-5 cells exposed to various compounds.
The MRC-5 cell cultures were incubated separately with each of the compounds at 10−5 M (10
µM) for
48 h. The cell growth was determined by measuring the cell number in cultures under the microscope
using the Neubauer counting chamber, as described in the “Materials and Methods”. Moreover, the
dead cells accumulated in these cultures were also evaluated using the Trypan-blue exclusion-dye
method, as presented in “Materials and Methods”. The results shown above indicate the mean
numbers
±
SD of two independent biological experiments. The diagrams shown above and the t-test
statistical analysis were carried out using the GraphPad Prism 6.0 program. Notably, no statistical
significance between the control-untreated culture with each one of compounds-treated cultures is seen
(p values > 0.05).
3. Materials and Methods
3.1. Computer Simulation Methods
For docking calculations, the software AutoDock 4.2 was used [45]. The free energy of binding
(∆G) of HIV-1 Reverse Transcriptase complex with the tested compounds was generated using this
molecular docking program.
The crystal structure of HIV-1 Reverse Transcriptase in Complex with TMC125 (PDB code: 3MEC)
was downloaded from the Protein Data Bank [46]. For RT preparation, water molecules were deleted,
polar hydrogens were added and Kollman United Atom charges were assigned. For the preparation of
ligand structures, 2D structure was sketched in chemdraw12.0 software (Chemical Structure Drawing
Standard; Perkin Elmer Informatics, Waltham, MA, USA) and converted to three-dimensional structures,
mol2 format, for each ligand. Hydrogens were added to the structures and used for docking. The Grid

Molecules 2019, 24, 3821
19 of 29
center was calculated: 9.7037 12.7492 16.914 (xyz-coordinates), for HIV-1 Reverse Transcriptase. The
grid size was set to 110 110 110 xyz points with grid spacing of 0.375 Å. All parameters used in
×
×
docking were default. A primary blind docking was performed in order to discriminate the preferential
binding sites of the ligand to the receptor. The ligand TMC125, as it is binded in the crystal structure
was extracted and docked back into the analogous binding pocket to determine the ability of Autodock
to replicate the position and orientation of the inhibitor in the crystal structure. Control docking
showed that Autodock was able to determine the orientation, position and the same interactions of the
crystallographic TMC125. The number of docking runs was 100. After docking, the 100 solutions were
clustered into groups with RMS lower than 1.0 E. The resulting poses and interactions were visualized
in Discovery studio 4.1 silent.
3.2. PASS Prediction
For the prediction of activity spectra of the designed compounds the program PASS was used.
PASS, as already described in our paper [47] predicts the biological activity spectrum of a compound
based on the analysis of structure–activity relationships of more than 1 million of known compounds.
These compounds possess over eight thousand biological activities. The average accuracy of prediction
is about 96%.
3.3. Prediction of Toxicity
For the prediction of toxicity of the designed compounds the Lazar model over the Open Tox
Predict Program was used. OpenTox is a free online program, that provides access to experimental
toxicity data, in Silico models (including (Q)SAR), and validation/reporting procedures. Lazar model
predicts the toxicity of the compounds based on their structure by searching for similar compounds.
Lazar is a k-nearest-neighbor approach to predict chemical endpoints from a training set based on
structural fragments. It uses a SMILES file and precomputed 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 uses activity-specific similarity (i.e., each
fragment contributes with its significance for the target activity) that is the basis for predictions and
confidence index for every single prediction [48,49].
3.4. Chemistry
3.4.1. General Procedure for the Synthesis of Thiazolidinones by Conventional Method
The appropriate heteroaromatic amine (1.0 mmol) and substituted benzaldehyde (1.2 mmol) were
stirred in dry toluene under reflux condition followed by addition of mercatoacetic acid (2.0 mmol).
The reaction mixture was refluxed for 18–32 h and then concentrated to dryness under reduce pressure.
The residue was diluted in ethyl acetate and the organic layer was washed with 5% aq citric acid,
water and 5% aq sodium hydrogen carbonate. The organic layer was dried over sodium sulfate and
concentrated under reduced pressure to give a clear product. All the synthesized compounds were
characterized by TLC, 1H-NMR, 13C-NMR and MS.
3.4.2. Microwave Irradiation Experiments
All microwave irradiation experiments were carried out in CEM-Discover monomode microwave
device, operating at a frequency of 2.45 GHz. Substituted aminobenzothiazole, equimolar amount of
substituted benzaldehyde (1.5 mmol) and mercaptoacetic acid in absolut ethanol (3 mL) were placed in
a 10 mL reaction vial containing a stirring bar. The vial was sealed with a Teflon septum and placed
into the microwave cavity. It was irradiated at 100 ◦C using 100 W as maximum power for 30 min.
at the end of the reaction the mixture was rapidly cooled with gas jet cooling to room temperature.
The clean product was obtained after filter under reduced pressure.

Molecules 2019, 24, 3821
20 of 29
3.4.3. Synthesis of 3-(7-Chloro-benzo[d]thiazol-2-yl)-2-(2,6-difluorophenyl)thiazolidin-4-one (1)
Yield: 39%, m.p. 200–202 ◦C, Rf: 0.66 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 1705
1
(C=O), 1541, 1102. H-NMR (500 MHz, CHCl3-d):
δ
= 3.86 (d, J = 16.38 Hz, 1H, CH2), 4.10 (d, J =
-S), 7.21 (t, J = 8.56 Hz, 2H, Ar-C18, C20), 7.06–7.15
(m, 1H, Ar-C5), 7.61–7.72 (m, 3H, Ar-C5, C6, C19), 8.05 (d, J = 8.02 Hz, 1H, Ar-C4). 13C-NMR (500
16.63 Hz, 1H, CH2), 6.83 (d, J = 18.59 Hz, 1H, N-C
H
MHz, CHCl3-d):
δ = 33.12, 60.42, 113.05, 119.34, 123.02, 124.15, 125.03, 126.11, 126.79, 129.01, 130.00,
135.65 150.42, 162.16, 164.17, 171.12. MS: m/z = 382 (M+, 100%), 353 (33.3%), 372 (36%), 363 (28%), 348
(21%), 242 (72%).
3.4.4. Synthesis of 3-(7-Chloro-benzo[d]thiazol-2-yl)-2-(2-chloro-6-fluorophenyl)thiazolidin-4-one (2)
Yield: 48%, m.p. 159–160 ◦C, Rf: 0.52 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 1708
1
(C=O), 1541, 1101, 721. H-NMR (500 MHz, CHCl3-d):
δ
= 3.85 (d, J = 16.38 Hz, 1H, C
H2), 4.12 (d, J =
16.63 Hz, 1H, CH2), 6.71 (d, J = 18.59 Hz, 1H, N-C
H-S), 7.07 (t, J = 8.56 Hz, 1H, Ar-C19), 7.30–7.55 (m,
4H, Ar-C5, C-6, C18, C19), 8.06 (d, J = 8.02 Hz, 1H, Ar-C4). 13C-NMR (500 MHz, CHCl3-d): δ = 32.88,
63.15, 111.07, 112.25, 119.11, 123.02, 126.53, 128.02, 128.25, 150.44, 162.13, 164.12, 171.02. MS: m/z = 400
(M+, 39%), 363 (100%), 321 (32%), 289 (88%), 254 (13%), 227 (10%), 215 (22%), 169 (23%), 153 (66%), 107
(18%).
3.4.5. Synthesis of 3-(6-Fluoro-benzo[d]thiazol-2-yl)-2-(4-fluorophenyl)thiazolidin-4-one (3)
Yield: 21%, m.p. 192–193 ◦C, Rf: 0.71 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 1705
1
(C=O), 1571, 1231, 1105. H-NMR (500 MHz, CHCl3-d):
δ
= 3.85 (d, J = 16.38 Hz, 1H, C
-S), 7.02 (t, J = 8.56 Hz, 1H, Ar-C5), 7.06–7.15 (m,
1H, Ar-C5), 7.27–7.36 (m, 4H, Ar-C17, C18, C20, C21), 7.48 (d, J = 8.02 Hz, 1H, Ar-C7). 13C-NMR (500
MHz, CHCl3-d): = 32.79, 63.09, 109.98, 113.94, 115.84, 120.83, 127.49, 127.55, 130.01, 133.32, 146.77,
156.13, 161.98, 163.52, 170.87 (1C,
=O). MS: m/z = 349 (M+, 7%), 288 (3%), 272 (25%), 245 (14%), 244
H2), 4.11 (d, J =
16.63 Hz, 1H, CH2), 6.77 (d, J = 18.59 Hz, 1H, N-CH
δ
C
(100%), 240 (11%), 200 (7%), 163 (5%), 130 (6%).
3.4.6. Synthesis of 3-(6-Fluoro-benzo[d]thiazol-2-yl)-2-(4-nitrophenyl)thiazolidin-4-one (4)
Yield: 55%, m.p. 204–205 ◦C, Rf: 0.44 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 1706
1
(C=O), 1572, 1235. H-NMR (500 MHz, CHCl3-d):
δ
= 4.04 (d, J = 16.63 Hz, 1H, CH2), 4.29 (d, J =
-S), 7.22 (t, J = 8.43 Hz, 1H, Ar-C5), 7.62 (br s, 1H, Ar-C4), 7.68
(d, J = 7.34 Hz, 2H, Ar-C17, C21), 7.92 (br d, J = 8.80 Hz, 1H, Ar-C7), 8.17 (d, J = 8.31 Hz, 2H, Ar-C18,
16.63 Hz, 1H, CH2), 6.97 (s, 1H, N-CH
C20). 13C-NMR (500 MHz, CHCl3-d):
133.32, 145.78, 146.77, 156.12, 163.56, 170.96. MS: m/z = 376 (M+, 100%), 342 (25%), 272 (41%), 150 (38%),
δ = 32.77, 63.08, 109.95, 113.97, 117.94, 120.83, 123.32, 127.49(2C),
115 (17%), 73 (11%).
3.4.7. Synthesis of 3-(6-Fluoro-benzo[d]thiazol-2-yl)-2-(4-chlorophenyl)thiazolidin-4-one (5)
Yield: 74%, m.p. 175–176 ◦C, Rf: 0.67 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 1708
1
(C=O), 1554, 1231, 721. H-NMR (500 MHz, CHCl3-d):
δ
= 3.86 (d, J = 16.38 Hz, 1H, CH2), 4.10 (br d, J
-S), 7.06–7.19 (m, 1H, Ar-C5), 7.22–7.40 (m, 4H, Ar-C17, C18,
C20, C21), 7.48 (br d, J = 7.83 Hz, 1H, Ar-C4), 7.56–7.79 (m, 1H, Ar-C7). 13C-NMR (500 MHz, CHCl3-d):
= 16.63 Hz, 1H, CH2), 6.73 (s, 1H, N-C
H
δ
= 32.79, 63.09, 109.98, 113.94, 117.94, 127.94, 130.32, 131.31, 132.35, 137.38, 146.77, 156.13, 163.52,
170.87. MS: m/z = 365 (M+, 100%), 343 (47%), 320 (7%), 291 (70%), 279 (34%), 259 (32%), 225 (28%).
3.4.8. Synthesis of 3-(6-Fluoro-benzo[d]thiazol-2-yl)-2-(4-methoxyphenyl)thiazolidin-4-one (6)
Yield: 29%, m.p. 198–200 ◦C, Rf: 0.63 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 1706
1
(C=O), 1572, 1230, 1213. H-NMR (500 MHz, CHCl3-d):
δ
= 3.76 (s, 3H, O-C
-S), 6.84 (d, J = 8.80 Hz, 2H, Ar-C18, C20),
7.12 (t, J = 8.56 Hz, 1H, Ar-C5), 7.23–7.35 (m, 1H, Ar-C4), 7.46–7.55 (m, 2H, Ar-C17, C21), 7.58–7.79
H3), 3.91 (d, J = 16.38 Hz,
1H, CH2), 4.10 (d, J = 16.63 Hz, 1H, CH2), 6.73 (s, 1H, N-CH

Molecules 2019, 24, 3821
21 of 29
(m, 1H, Ar-C7). 13C-NMR (500 MHz, CHCl3-d):
δ
= 32.78, 58.01, 63.08, 109.98, 113.89, 114.13, 117.94,
129.49, 131.98, 132.35, 137.38, 146.73, 156.1, 158.01, 163.51, 171.03. MS: m/z = 361 (M+, 15%), 288 (9%),
272 (25%), 244 (100%), 243 (10%), 153 (13%), 135 (7%).
3.4.9. Synthesis of 3-(6-Fluoro-benzo[d]thiazol-2-yl)-2-(4-hydroxyphenyl)thiazolidin-4-one (7)
Yield: 21%, m.p. 230–231 ◦C, Rf: 0.37 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 3258
1
(-OH), 2361, 1706 (C=O), 1573, 1230. H-NMR (500 MHz, CHCl3-d): 4.04 (d, J = 16.63 Hz, 1H, C
H
2),
-S), 7.22 (t, J = 8.43 Hz, 1H, Ar-C5),
7.62 (br s, 1H, Ar-C4), 7.68 (d, J = 7.34 Hz, 2H, Ar-C18, C20), 7.92 (br d, J = 8.80 Hz, 1H, Ar-C7), 8.16 (d,
4.29 (d, J = 16.63 Hz, 1H, CH2), 5.35 (br s, 1H, -OH), 6.97 (s, N-CH
J = 8.31 Hz, 2H, Ar-C17, C21). 13C-NMR (500 MHz, CHCl3-d):
δ = 32.81, 63.11, 109.94, 113.91, 115.12,
117.94, 130.00, 131.24, 131.53, 146.68, 156.09, 158.04, 163.52, 171.05. MS: m/z = 347 (M+, 42%), 309 (92%),
279 (40%), 263 (70%), 243 (72%).
3.4.10. Synthesis of 3-(6-Chloro-benzo[d]thiazol-2-yl)-2-(4-fluorophenyl)thiazolidin-4-one (8)
Yield: 51%, m.p. 196–197 ◦C, Rf: 0.76 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2363,
1
1704 (C=O), 1568, 721. H-NMR (500 MHz, CHCl3-d):
δ
= 3.85 (d, J = 16.38 Hz, 1H, CH2), 4.11 (d, J =
-S), 7.10–7.18 (m, 2H, Ar-C18, C20), 7.27–7.36 (m, 2H, Ar-C17,
C21), 7.56 (t, J = 8.56 Hz, 1H, Ar-C5), 7.61 (d, J = 8.02 Hz, 1H, Ar-C4), 8.05 (s, 1H, Ar-C7). 13C-NMR (500
16.63 Hz, 1H, CH2), 6.75 (s, 1H, N-C
H
MHz, CHCl3-d):
δ = 32.86, 63.10, 115.20, 120.82, 122.73, 126.76, 127.73, 133.30, 136.08, 146.90, 156.05,
163.49, 171.11. MS: m/z = 364 (M+, 100%), 343 (10%), 299 (10%), 290 (100%), 279 (9%), 184 (11%), 115
(80%), 88 (9%), 73 (94%).
3.4.11. Synthesis of 3-(6-Chloro-benzo[d]thiazol-2-yl)-2-(4-hydroxyphenyl)thiazolidin-4-one (9)
Yield: 24%, m.p. 236–237 ◦C, Rf: 0.40 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2359
1
(-OH), 2361, 1701 (C=O), 1573. H-NMR (500 MHz, CHCl3-d):
δ
= 3.86 (d, J = 16.38 Hz, 1H, CH2), 4.10
(d, J = 16.63 Hz, 1H, C
H
2), 5.35 (br s, 1H, -O
H
), 6.73 (s, 1H, N-C
H
-S), 7.26–7.43 (m, 5H, Ar-C5, C17, C18,
C20, C21), 7.76 (br d, J = 8.80 Hz, 1H, Ar-C4), 8.13 (s, 1H, Ar-C7). 13C-NMR (500 MHz, CHCl3-d):
δ =
33.12, 63.71, 115.75, 118.23, 121.55, 125.14, 129.02, 130.01, 131.52, 132.87, 151.68, 156.71, 163.77, 171.12.
MS: m/z = 363 (M+, 100%), 288 (88%), 272 (9%), 225 (33%), 184 (48%), 146 (7%), 136 (21%), 115 (18%), 73
(17%).
3.4.12. Synthesis of 3-(4-Chloro-benzo[d]thiazol-2-yl)-2-(4-fluorophenyl)thiazolidin-4-one (10)
Yield: 33%, m.p. 215–216 ◦C, Rf: 0.74 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1702 (C=O), 1573, 1101. H-NMR (500 MHz, CHCl3-d):
δ
= 3.93 (d, J = 16.63 Hz, 1H, CH2), 4.21 (d, J =
-S), 7.02 (br t, J = 8.56 Hz, 2H, Ar-C18, C20), 7.15–7.24 (m, 1H,
Ar-C6), 7.41 (d, J = 7.34 Hz, 1H, Ar-C5), 7.50 (br dd, J = 8.31, 5.38 Hz, 2H, Ar-C17, C21), 7.68 (d, J = 7.83
16.63 Hz, 1H, CH2), 6.81 (s, 1H, N-C
H
Hz, 1H, Ar-C7). 13C-NMR (500 MHz, CHCl3-d):
130.71, 132.45, 134.38, 149.11, 161.33, 164.02, 171.05. MS: m/z = 364 (M+, 72%), 343 (35%), 232 (31%), 290
δ = 32.81, 63.87, 115.02, 119.33, 120.77, 121.13, 125.76,
(100%), 292 (79%), 279 (33%), 242 (64%), 225 (65%), 184 (77%), 174 (44%).
3.4.13. Synthesis of 3-(4-Chloro-benzo[d]thiazol-2-yl)-2-(4-nitrophenyl)thiazolidin-4-one (11)
Yield: 41%, m.p. 203–204 ◦C, Rf: 0.45 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1701 (C=O), 1573. H-NMR (500 MHz, CHCl3-d):
δ
= 3.93 (d, J = 16.63 Hz, 1H, CH2), 4.21 (d, J = 16.63
-S), 7.21–7.30 (d, J = 7.34 Hz, 2H, Ar-C17, C21), 7.40 (br d, J = 8.80 Hz,
1H, Ar-C6), 7.80 (br d, J = 8.80 Hz, 1H, Ar-C5), 8.20 (d, J = 8.31 Hz, 3H, Ar-C7, C18, C20). 13C-NMR (500
Hz, 1H, CH2), 6.85 (s, 1H, N-CH
MHz, CHCl3-d):
δ = 32.86, 63.35, 119.32, 121.14, 121.58, 123.69, 125.81, 129.66, 132.11, 145.38, 146.37,
149.72, 164.01, 171.02. MS: m/z = 373 (M+, 33%), 349(14%), 343 (46%), 332 (40%), 304 (29%), 292 (92%),
290 (100%), 279 (18%), 259 (7%).

Molecules 2019, 24, 3821
22 of 29
3.4.14. Synthesis of 3-(4-Chloro-benzo[d]thiazol-2-yl)-2-(4-chlorophenyl)thiazolidin-4-one (12)
Yield: 56%, m.p. 184–185 ◦C, Rf: 0.73 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1704 (C=O), 1568, 721. H-NMR (500 MHz, CHCl3-d):
δ
= 3.94 (d, J = 16.63 Hz, 1H, CH2), 4.21 (d, J =
-S), 7.21–7.31 (m, 3H, Ar-C6, C17, C21), 7.39–7.43 (m, 3H, Ar-C5,
C18, C20), 7.68 (d, J = 7.83 Hz, 1H, Ar-C7). 13C-NMR (500 MHz, CHCl3-d):
= 32.83, 63.12, 119.89,
16.63 Hz, 1H, CH2), 6.79 (s, 1H, N-CH
δ
121.23, 121.59, 125.83, 128.77, 130.77, 132.31, 132.59, 145.38, 137.36, 149.71, 163.95, 171.03. MS: m/z = 383
(M+, 13%), 381 (43%), 379 (61%), 346 (11%), 341 (11%), 337 (18%), 269 (46%), 227 (32%).
3.4.15. Synthesis of 3-(4-Chloro-benzo[d]thiazol-2-yl)-2-(4-methoxyphenyl)thiazolidin-4-one (13)
Yield: 67%, m.p. 223–224 ◦C, Rf: 0.54 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1700 (C=O), 1571, 1232. H-NMR (500 MHz, CHCl3-d):
δ
= 3.75 (s, 3H, O-CH3), 3.92 (d, J = 16.38 Hz,
-S), 6.84(d, J = 8.80 Hz, 2H, Ar-C18, C20),
7.19 (t, J = 8.56 Hz, 1H, Ar-C6), 7.25 (s, 1H, Ar-C5), 7.41–7.45 (m, 2H, Ar-C17, C21), 7.68 (d, J = 7.83
1H, CH2), 4.20 (d, J = 16.63 Hz, 1H, CH2), 6.81 (s, 1H, N-C
H
Hz, 1H, Ar-C7). 13C-NMR (500 MHz, CHCl3-d):
125.86, 129.79, 131.28, 132.38, 149.49, 159.00, 163.52, 171.04. MS: m/z = 377 (M+, 100%), 362 (15%), 353
δ = 32.85, 55.81, 63.32, 114.21, 119.31, 121.18, 121.57,
(31%), 326 (27%), 290 (40%), 288 (82%), 136 (36%), 115 (17%) 73 (14%).
3.4.16. Synthesis of 3-(4-Chloro-benzo[d]thiazol-2-yl)-2-(4-hydroxyphenyl)thiazolidin-4-one (14)
Yield: 17%, m.p. 258–259 ◦C, Rf: 0.39 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 3205
1
(-OH), 2361, 1701 (C=O), 1569. H-NMR (500 MHz, CHCl3-d):
δ
= 3.93 (d, J = 16.63 Hz, 1H, C
-S), 7.02 (br t, J = 8.56 Hz, 2H, Ar-C18,
C20), 7.15–7.24 (m, 1H, Ar-C6), 7.41 (d, J = 7.34 Hz, 1H, Ar-C5), 7.50 (br dd, J = 8.31, 5.38 Hz, 2H,
H2), 4.21
(d, J = 16.63 Hz, 1H, CH2), 5.32 (br s, 1H, -OH), 6.81 (s, 1H, N-CH
Ar-C17, C21), 7.68 (d, J = 7.83 Hz, 1H, Ar-C7). 13C-NMR (500 MHz, CHCl3-d):
δ = 33.02, 63.31, 115.18,
119.23, 121.55, 121.89, 125.15, 130.03, 131.52, 132.17, 149.68, 156.73, 163.79, 171.08. MS: m/z = 363 (M+,
75%), 346 (28%), 310 (22%), 290 (21%), 272 (100%), 184 (19%), 136 (22%), 115 (51%), 88 (27%), 73 (15%).
3.4.17. Synthesis of 3-(4-Methoxy-benzo[d]thiazol-2-yl)-2-(4-flurophenyl)thiazolidin-4-one (15)
Yield: 51%, m.p. 251–252 ◦C, Rf: 0.73 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1968 (C=O), 1587, 1272, 1100. H-NMR (500 MHz, DMSO-d6):
δ
= 3.68–3.86 (m, 3H, O-CH3), 3.91–4.09
-S), 7.06 (br d, J = 10.27 Hz, 1H,
Ar-C5), 7.26 (br dd, J = 10.03, 8.07 Hz, 1H, Ar-C6), 7.55 (br d, J = 10.27 Hz, 1H, Ar-C7), 7.61–7.76 (m, 2H,
Ar-C17, C21), 8.08–8.26 (m, 2H, Ar-C18, C20). 13C-NMR (500 MHz, DMSO-d6):
= 32.86, 55.82, 63.18,
(m, 1H, CH2), 4.25 (br d, J = 9.78 Hz, 1H, CH2), 6.88 (s, 1H, N-C
H
δ
108.26, 114.11, 115.43, 121.18, 130.33, 131.99, 135.59, 142.31, 150.23, 161.51, 163.91, 171.08. MS: m/z = 361
(M+, 100%), 343 (42%), 321 (36%), 290 (72%), 288 (31%), 279 (39%), 225 (24%).
3.4.18. Synthesis of 3-(4-Methoxy-benzo[d]thiazol-2-yl)-2-(4-nitrophenyl)thiazolidin-4-one (16)
Yield: 63%, m.p. 298–299 ◦C, Rf: 0.48 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1705 (C=O), 1585, 1273. H-NMR (500 MHz, DMSO-d6):
δ
= 3.77 (s, 3H, O-C
-S), 7.05 (s, 1H, Ar-C5), 7.26 (s, 1H, Ar-C6),
7.56 (d, J = 7.83 Hz, 2H, Ar-C17, C21), 7.67 (d, J = 8.80 Hz, 1H, Ar-C7), 8.17 (d, J = 8.32 Hz, 2H, Ar-C18,
H3), 4.03 (d, J = 17.12 Hz,
1H, CH2), 4.20 (d, J = 17.12 Hz, 1H, CH2), 6.93 (s, 1H, N-C
H
C20). 13C-NMR (500 MHz, DMSO-d6):
131.97, 142.31, 145.26, 146.32, 150.21, 163.95, 171.02. MS: m/z = 388 (M+, 100%), 288 (73%), 272 (3%), 225
δ = 32.85, 55.85, 63.21, 108.25, 114.12, 121.18, 123.84, 129.66,
(42%), 184 (76%), 136 (42%), 115 (31%), 73 (28%).
3.4.19. Synthesis of 3-(4-Methoxy-benzo[d]thiazol-2-yl)-2-(4-chlorophenyl)thiazolidin-4-one (17)
Yield: 43%, m.p. 218–219 ◦C, Rf: 0.53 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1708 (C=O), 1589, 1276, 1154. H-NMR (500 MHz, DMSO-d6):
δ
= 3.80 (s, 3H, O-CH3), 3.99 (br d, J =
2), 6.93 (s, 1H, N-C -S), 6.91–6.97 (m, 1H, Ar-C5),
7.26 (br t, J = 8.07 Hz, 1H, Ar-C4), 7.39 (br d, J = 9.29 Hz, 4H, Ar-C17, C18, C20, C21), 7.54 (br d, J = 7.83
17.12 Hz, 1H, C
H
2), 4.23 (br d, J = 16.63 Hz, 1H, C
H
H

Molecules 2019, 24, 3821
23 of 29
Hz, 1H, Ar-C7). 13C-NMR (500 MHz, DMSO-d6):
δ
= 32.82, 55.88, 63.27, 108.25, 114.11, 121.32, 128.73,
130.12, 131.93, 132.75, 137.36, 142.32, 150.22, 164.03, 171.00. MS: m/z = 377 (M+, 41%), 272 (23%), 244
(100%), 242 (63%), 165 (17%), 136 (23%).
3.4.20. Synthesis of 3-(4-Methoxy-benzo[d]thiazol-2-yl)-2-(4-methoxyphenyl)thiazolidin-4-one (18)
Yield: 52%, m.p. 196–197 ◦C, Rf: 0.63 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1701 (C=O), 1556, 1273. H-NMR (500 MHz, CHCl3-d):
δ
= 33.76 (s, 3H, O-CH3benz), 3.94 (d, J = 16.63
3), 4.13 (d, J = 16.63 Hz, 1H, C 2), 6.81–6.85 (m, 2H, Ar-C18, C20), 7.93
-S), 7.25 (br d, J = 9.29 Hz, 3H, Ar-C5, C6, C7), 7.39 (br d, J = 7.83 Hz, 2H, Ar-C17, C21).
Hz, 1H, C
(s, 1H, N-C
13C-NMR (500 MHz, CHCl3-d):
H
2), 3.95 (s, 3H, O-C
H
H
H
δ
= 32.86, 55.83, 63.35, 108.25, 114.11, 114.76, 121.82, 129.75, 131.55,
131.96, 142.34, 150.23, 160.82, 164.06, 171.11. MS: m/z = 373 (M+, 76%), 272 (33%), 243 (56%), 244 (100%),
242 (8%), 165 (5%), 164 (7%), 135 (11%).
3.4.21. Synthesis of 3-(4-Methoxy-benzo[d]thiazol-2-yl)-2-(4-hydroxyphenyl)thiazolidin-4-one (19)
Yield: 29%, m.p. 261–262 ◦C, Rf: 0.72 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 3205
1
(-OH), 2361, 1707 (C=O), 1582, 1271. H-NMR (500 MHz, DMSO-d6):
δ
= 3.77 (s, 3H, O-C
), 6.93 (d, J = 7.83 Hz, 2H,
-S), 7.26 (br t, J = 8.56 Hz, 1H, Ar-C5), 7.56 (d, J = 7.83 Hz, 1H, Ar-C6),
7.67 (d, J = 8.80 Hz, 1H, Ar-C7), 8.17 (d, J = 8.32 Hz, 2H, Ar-C17, C21). 13C-NMR (500 MHz, CHCl3-d):
H3), 4.03 (d, J
= 17.12 Hz, 1H, CH2), 4.20 (d, J = 17.12 Hz, 1H, CH2), 5.32 (br s, 1H, -O
H
Ar-C18, C20), 7.05 (s, 1H, N-C
H
δ
= 32.88, 55.82, 63.33, 108.21, 114.13, 115.81, 121.73, 130.13, 131.52, 131.96, 142.31, 150.22, 156.95, 164.13,
171.04. MS: m/z = 359 (M+, 82%), 344 (30%), 328 (64%), 189 (27%), 244 (100%), 165 (54%), 135 (21%).
3.4.22. Synthesis of 3-(6-Methoxy-benzo[d]thiazol-2-yl)-2-(4-fluorophenyl)thiazolidin-4-one (20)
Yield: 52%, m.p. 242–243 ◦C, Rf: 0.62 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1701 (C=O), 1531, 1225. H-NMR (500 MHz, DMSO-d6):
δ
= 3.76 (s, 3H, O-CH3), 3.99 (br d, J = 16.63
-S), 6.97 (dd, J = 8.80, 2.45 Hz,
1H, Ar-C5), 7.13 (br t, J = 8.80 Hz, 2H, Ar-C18, C20), 7.44 (br dd, J = 8.31, 5.38 Hz, 2H, Ar-C17, C21),
Hz, 1H, CH2), 4.26 (br d, J = 16.63 Hz, 1H, CH2), 6.84 (s, 1H, N-C
H
7.49–7.71 (m, 2H, Ar-C4, C7). 13C-NMR (500 MHz, DMSO-d6):
115.81, 121.73, 130.13, 131.52, 131.96, 142.31, 150.22, 156.95, 164.13, 171.04. MS: m/z = 361 (M+, 14%),
δ = 32.96, 55.82, 63.33, 108.21, 114.13,
288 (8%), 272 (25%), 260 (5%), 258 (8%), 244 (100%), 243 (8%), 153 (13%), 165 (4%), 135 (7%), 109 (8%).
3.4.23. Synthesis of 3-(6-Methoxy-benzo[d]thiazol-2-yl)-2-(4-nitrophenyl)thiazolidin-4-one (21)
Yield: 41%, m.p. 220–221 ◦C, Rf: 0.49 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1701 (C=O), 1532, 1226. H-NMR (500 MHz, DMSO-d6):
δ
= 3.76 (s, 3H, O-CH3), 4.03 (br d, J = 16.63
-S), 7.51 (br d, J = 9.29
Hz, 1H, Ar-C5), 7.60 (br s, 1H, Ar-C7), 7.67 (br d, J = 7.34 Hz, 2H, Ar-C17, C21), 8.17 (br d, J = 8.31 Hz,
Hz, 1H, CH2), 4.27 (br d, J = 16.63Hz, 1H, CH2), 6.89–7.06 (m, 2H, Ar-C5, N-C
H
2H, Ar-C18, C20). 13C-NMR (500 MHz, DMSO-d6):
125.96, 131.95, 145.13, 145.51, 146.79, 156.81, 164.03, 170.92. MS: m/z = 388 (M+, 62%), 288 (100%), 225
δ = 32.87, 55.86, 63.32, 104.12, 114.33, 117.25, 124.38,
(29%), 184 (61%), 136 (27%), 115 (23%), 107 (27%), 73 (18%).
3.4.24. Synthesis of 3-(6-Methoxy-benzo[d]thiazol-2-yl)-2-(4-chlorophenyl)thiazolidin-4-one (22)
Yield: 52%, m.p. 179–180 ◦C, Rf: 0.53 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1706 (C=O), 1536, 1219, 1108. H-NMR (500 MHz, DMSO-d6):
δ
= 3.77 (s, 3H, O-C
-S), 6.97 (dd, J = 8.80, 2.45 Hz, 1H,
Ar-C5), 7.32–7.47 (m, 4H, Ar-C17, C18, C20, C21), 7.47–7.65 (m, 2H, Ar-C4, C7). 13C-NMR (500 MHz,
H3), 3.99 (d, J = 17.12
Hz, 1H, CH2), 4.26 (d, J = 16.63 Hz, 1H, CH2), 6.84 (s, 1H, N-C
H
DMSO-d6):
δ = 32.76, 55.85, 63.31, 104.99, 114.61, 118.23, 127.81(2C), 129.16, 131.93, 132.15, 137.33,
145.52, 156.74, 164.03, 170.85. MS: m/z = 377 (M+, 100%), 288 (82%), 272 (33%), 225 (21%), 136 (35%),
115 (20%), 73 (18%).

Molecules 2019, 24, 3821
24 of 29
3.4.25. Synthesis of 3-(6-Methoxy-benzo[d]thiazol-2-yl)-2-(4-methoxyphenyl)thiazolidin-4-one (23)
Yield: 33%, m.p. 225–226 ◦C, Rf: 0.62 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1700 (C=O), 1540, 1225. H-NMR (500 MHz, CHCl3-d):
δ
= 3.76 (s, 3H, O-CH3benz), 3.94 (d, J = 16.63
-S), 6.93 (br d, J =
8.31 Hz, 2H, Ar-C18, C20), 7.51 (br d, J = 9.29 Hz, 1H, Ar-C5), 7.25 (br d, J = 9.29 Hz, 2H, Ar-C4, C7,
Hz, 1H, CH2), 3.98 (s, 3H, O-CH3), 4.13 (d, J = 16.63 Hz, 1H, CH2), 6.84 (s, 1H, N-C
H
C21), 7.39 (br d, J = 7.83 Hz, 1H, Ar-C17). 13C-NMR (500 MHz, CHCl3-d):
δ = 32.81, 55.84, 63.33, 104.92,
114.81, 115.01, 118.22, 127.79, 131.91, 132.28, 145.55, 156.72, 159.85, 163.92, 170.93. MS: m/z = 373 (M+,
78%), 299 (10%), 344 (10%), 272 (33%), 244 (100%), 242 (7%), 165 (5%), 164 (7%), 150 (9%), 135 (3%).
3.4.26. Synthesis of 3-(6-Ethoxy-benzo[d]thiazol-2-yl)-2-(4-fluorophenyl)thiazolidin-4-one (24)
Yield: 63%, m.p. 144–145 ◦C, Rf: 0.44 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2316,
1701 (C=O), 1531, 1225. 1H-NMR (500 MHz, CHCl3-d):
δ
= 1.25 (t, J = 6.85 Hz, 3H, -OCH2CH3),
3.82–3.85 (m, 3H, CH2,-OCH2CH3), 4.10 (d, J = 16.63 Hz, 1H, CH2), 6.79 (s, 1H, N-C -S), 6.97 (dd, J =
8.80, 2.45 Hz, 2H, Ar-C18, C20), 7.25 (s, 1H, Ar-C5), 7.30–7.33 (m, 2H, Ar-C17, C21), 7.59 (d, J = 8.80 Hz,
H
2H, Ar-C4, C7). 13C-NMR (500 MHz, CHCl3-d):
δ = 14.13, 32.35, 55.89, 63.12, 104.52, 115.13, 115.52,
115.61, 130.02, 131.51, 134.03, 144.00, 153.62, 161.13, 164.05, 171.21. MS: m/z = 375 (M+, 100%), 288
(28%), 272 (36%), 225 (18%), 197 (19%), 136 (20%), 115 (16%), 73 (16%).
3.4.27. Synthesis of 3-(6-Ethoxy-benzo[d]thiazol-2-yl)-2-(4-nitrophenyl)thiazolidin-4-one (25)
Yield: 72%, m.p. 155–156 ◦C, Rf: 0.58 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2316,
1
1701 (C=O), 1527, 1225. H-NMR (500 MHz, CHCl3-d):
δ = 1.42 (t, J = 6.85 Hz, 3H, -OCH2CH3), 3.89
(d, J = 16.63 Hz, 1H, CH2), 4.10 (d, J = 16.63 Hz, 1H, CH2), 4.25–4.32 (q, J = 6.68 Hz, 2H, -OCH2CH3),
6.80 (s, 1H, N-C -S), 6.97 (dd, J = 8.80, 2.45 Hz, 1H, Ar-C5), 7.19–7.33 (m, 1H, Ar-C7), 7.42–7.59 (m, 3H,
Ar-C4, C17, C21), 8.19 (d, J = 8.80 Hz, 2H, Ar-C18, C20). 13C-NMR (500 MHz, CHCl3-d):
= 14.12,
H
δ
32.31, 55.97, 63.17, 104.52, 115.25, 117.31, 122.15, 129.75, 131.21, 142.02, 143.85, 145.06, 153.72, 164.02,
170.75. MS: m/z = 402 (M+, 100%), 382 (49%), 373 (42%), 357 (21%), 348 (18%), 115 (12%), 73 (7%).
3.4.28. Synthesis of 3-(6-Ethoxy-benzo[d]thiazol-2-yl)-2-(4-chlorophenyl)thiazolidin-4-one (26)
Yield: 82%, m.p. 180–181◦C, Rf: 0.66 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1696 (C=O), 1546, 1227, 698. H-NMR (500 MHz, CHCl3-d):
δ
= 1.43 (t, J = 6.85 Hz, 3H, -OCH2CH3),
-S), 6.98 (dd,
J = 8.80, 2.45 Hz, 1H, Ar-C5), 7.25–7.30 (m, 5H, Ar-c4, C17, C18, C20, C21), 7.59 (d, J = 8.80 Hz, 1H,
3.83 (d, J = 16.63 Hz, 1H, CH2), 4.04–4.10 (m, 3H, CH2, -OCH2CH3), 6.74 (s, 1H, N-C
H
Ar-C7). 13C-NMR (500 MHz, CHCl3-d):
130.05, 131.24, 132.74, 137.52, 144.86, 145.06, 153.59, 164.07, 170.69. MS: m/z = 391 (M+, 100%), 363
δ = 14.12, 32.31, 55.92, 63.17, 104.83, 114.77, 117.81, 128.72,
(68%), 272 (88%), 244 (67%), 215 (26%), 153 (79%).
3.4.29. Synthesis of 3-(6-Ethoxy-benzo[d]thiazol-2-yl)-2-(4-methoxyphenyl)thiazolidin-4-one (27)
Yield: 55%, m.p. 210–211◦C, Rf: 0.63 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1700 (C=O), 1531, 1227. H-NMR (500 MHz, CHCl3-d):
δ = 1.42 (t, J = 6.85 Hz, 3H, -OCH2CH3), 3.89
(d, J = 16.63 Hz, 1H, CH2), 4.03–4.10 (m, 4H, CH2, -OCH3), 4.25–4.32 (q, J = 6.68 Hz, 2H, -OCH2CH3),
6.80 (s, 1H, N-C -S), 6.97 (dd, J = 8.80, 2.45 Hz, 2H, Ar-C18, C20), 7.19–7.33 (m, 1H, Ar-C5), 7.42–7.59
(m, 2H, Ar-C4, C7), 8.19 (d, J = 8.80 Hz, 2H, Ar-C17, C21). 13C-NMR (500 MHz, CHCl3-d):
= 14.15,
H
δ
32.36, 55.81, 55.96, 63.17, 105.26, 114.21, 114.75, 117.82, 129.85, 131.58, 144.82, 153.56, 159.35, 164.15,
170.57. MS: m/z = 387 (M+, 21%), 344 (16%), 313 (15%), 299 (8%), 272 (37%), 244 (100%), 242 (15%), 165
(35%), 164 (9%), 150 (23%), 135 (8%).
3.4.30. Synthesis of 3-(6-Ethoxy-benzo[d]thiazol-2-yl)-2-(4-hydroxyphenyl)thiazolidin-4-one (28)
Yield: 81%, m.p. 191–192◦C, Rf: 0.37 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 3200
1
(-OH), 2361, 1706 (C=O), 1530, 1225. H-NMR (500 MHz, DMSO-d6):
δ = 1.33 (br t, J = 6.60 Hz, 3 H),

Molecules 2019, 24, 3821
25 of 29
3.37–3.50 (m, 4 H), 3.51–3.68 (m, 1 H), 4.04 (q, J = 6.68 Hz, 2 H), 5.24 (s, 1 H), 6.71 (m, J = 7.83 Hz, 2 H),
6.99 (br d, J = 8.80 Hz, 1 H), 7.20 (m, J = 7.83 Hz, 2 H), 7.52 (br s, 1 H), 7.60 (br d, J = 8.31 Hz, 1 H), 9.55
(br s, 1 H). 13C-NMR (500 MHz, DMSO-d6):
δ = 14.11, 32.31, 55.93, 63.19, 104.97, 114.67, 115.32, 117.82,
130.05, 130.87, 132.56, 144.81, 154.59, 156.73, 164.07, 170.63. MS: m/z = 373 (M+, 9%), 288 (17%), 272
(29%), 244 (100%), 243 (8%), 197 (11%), 109 (4%).
3.4.31. Synthesis of 3-(6-Trifluoromethoxy-benzo[d]thiazol-2-yl)-2-(2,6-dichlorophenyl)
thiazolidin-4-one (29)
Yield: 73%, m.p. 164–165◦C, Rf: 0.53 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1700 (C=O), 1532, 1209, 1134, 721. H-NMR (500 MHz, CHCl3-d):
δ
= 3.99 (d, J = 16.63 Hz, 1H, CH2),
-S), 6.97 (dd, J = 8.80, 2.45 Hz, 1H, Ar-C5), 7.42–7.46
(m, 3H, Ar-C18, C19, C20), 7.49–7.71 (m, 2H, Ar-C4, C7).13C-NMR (500 MHz, CHCl3-d):
= 32.86,
4.26 (d, J = 16.63 Hz, 1H, CH2), 6.84 (s, 1H, N-C
H
δ
63.33, 104.97, 114.22, 118.71, 126.82, 128.53, 129.27, 130.05, 135.11, 139.11, 145.91, 156.74, 164.13, 171.17.
MS: m/z = 446 (M+, 47%), 340 (21%), 336 (16%), 321 (44%), 293 (100%), 292 (11%), 204 (46%), 165 (21%).
3.4.32. Synthesis of 3-(6-Trifluoromethoxy-benzo[d]thiazol-2-yl)-2-(2,3-dichlorophenyl)
thiazolidin-4-one (30)
Yield: 84%, m.p. 142–143 ◦C, Rf: 0.81 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 2361,
1
1700 (C=O), 1532, 1202, 1131, 721. H-NMR (500 MHz, CHCl3-d):
δ = 3.94 (d, J = 16.63 Hz, 1H, CH2),
4.11 (d, J = 16.63 Hz, 1H, CH2), 6.83 (s, 1H, N-CH-S), 6.91–7.01 (m, 2H, Ar-C5, C17), 7.10–7.18 (m, 1H,
Ar-C18), 7.49–7.53 (m, 2H, Ar-C4, C7), 7.60 (dd, J = 8.80, 2.45 Hz, 1H, Ar-C19). 13C-NMR (500 MHz,
CHCl3-d):
δ = 32.85, 63.34, 103.85, 104.92, 114.66, 118.97, 127.95, 128.05, 128.77, 129.23, 129.56, 131.81,
132.05, 145.52, 156.73, 164.13, 170.93. MS: m/z = 466 (M+, 3%), 355 (6%), 272 (18%), 244 (36%), 228 (15%),
226 (22%), 165 (100%).
3.4.33. Synthesis of 3-(6-Trifluoromethoxy-benzo[d]thiazol-2-yl)-2-(4-nitrophenyl)thiazolidin-4-one (31
)
◦
Yield: 33%, m.p. 158–159 C, Rf: 0.62 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1
:
1
1706(C=O), 1539, 1209, 1135. H-NMR (500 MHz, CHCl3-d):
δ
= 3.85 (d, J = 16.63 Hz, 1H, CH2), 4.14
-S), 7.02 (d, J = 7.83 Hz, 1H, Ar-C5), 7.41–7.45 (m, 4H,
Ar-C4, C7, C17, C21), 8.05 (d, J = 7.83 Hz, 2H, Ar-C18, C20). 13C-NMR (500 MHz, CHCl3-d):
= 32.76,
(d, J = 16.63 Hz, 1H, CH2), 6.76 (s, 1H, N-C
H
δ
63.09, 104.92, 114.02, 118.27 123.88, 129.12, 129.93, 131.95, 145.62, 145.94, 149.79, 156.71, 164.19, 170.92.
MS: m/z = 442 (M+, 56%), 395 (51%), 386 (19%), 272 (100%), 244 (22%), 165 (7%).
3.4.34. Synthesis of 3-(6-Trifluoromethoxy-benzo[d]thiazol-2-yl)-2-(4-chlorophenyl)thiazolidin-4-one (32
)
Yield: 35%, m.p. 243–244 ◦C, Rf: 0.77 (petroleum ether/ethyl acetate 8:2). IR (Nujol), cm−1: 1698
1
(C=O), 1528, 1209, 1198, 1131, 721. H-NMR (500 MHz, CHCl3-d):
δ
= 3.85 (d, J = 16.63 Hz, 1H, CH2),
4.10 (d, J = 16.63 Hz, 1H, CH2), 6.74 (s, 1H, N-C
H-S), 7.24–7.31(m, 5H, Ar-C5, C17, C18, C20, C21),
7.66–7.71 (m, 2H, Ar-C4, C7). 13C-NMR (500 MHz, CHCl3-d):
δ = 32.75, 63.05, 104.93, 114.01, 120.11,
128.27, 129.65, 129.81, 131.74, 132.92, 137.33, 145.95, 156.63, 161.62, 164.27, 170.94. MS: m/z = 431 (M+,
46%), 395 (27%), 378 (34%), 377 (100%), 373 (18%), 363 (55%).
3.5. Evaluation of RT Inhibitory Action
For the evaluation of the HIV-1 Reverse Transcriptase activity, the colorimetric photometric
immunoassay kit provided by Roche was used. A two hours incubation at 37 ◦C was performed, as
previously described in our paper [44] following a pre-incubation step in which reverse transcriptase
was incubated in the presence of the potential inhibitor for 45 min at room temperature before the
addition of the substrate. The total-activity was measured in the absence of inhibitors (compounds)
with the addition of an equal volume of the solvent of the compounds to the reaction mixture. For the
determination of enzyme activity, incubation in the absence of enzyme was used as blank. Different
inhibitor concentrations were added for the calculation of IC50 values.

Molecules 2019, 24, 3821
26 of 29
3.6. Toxicity
Cell culture and cytotoxicity assessment.
The normal MRC-5 cell line is stored and used in our laboratory in a routine manner. MRC-5
cells were grown in culture (37 ◦C, humidified atmosphere containing 5% v/v CO2) in DMEM medium
supplemented with 10% v/v FBS, 100
under evaluation were dissolved in DMSO and stored in 4 ◦C (DMSO concentration
cytotoxicity of the 1, 2, 3, 8, 9, 10, 14 compounds were evaluated in MRC-5 cell cultures. In particular
cells were seeded in a 24- well plate at an initial concentration of 1
105 cells/mL and allowed to stand
µg/mL penicillin and 100
µg/mL streptomycin. The compounds
≤
0.2% v/v). The
×
overnight before the addition of the compounds at high concentration of 10−5 M. Note that DMSO
concentration in culture was 0.2% v/v, where this concentration exhibits no cytotoxicity at all [50]. To
estimate the cytotoxicity of each compound, cells were allowed to grow for additional 48h before being
harvested by trypsinization and counted with the aid of the optical microscope (Neubauer counting
chamber). Cell growth in each treated culture is expressed as a percentage compared to that seen for
the untreated control cells. Moreover, cell death was also determined using the Trypan-blue method,
as previously described [51,52]. Statistical t-test analysis was performed via the use of GraphPad Prism
6.0 program.
4. Conclusions
Thirty two compounds out of a hundred thirty two compounds designed were selected based on
PASS prediction results and molecular docking for the synthesis and evaluation of HIV-1 RT inhibitory
activity. Twenty-four out of the thirty-two tested compounds exhibited inhibitory action equal or lower
than 4 µM. Seven of them showed better activity than nevirapine under experimental condition, while
three of the compounds exhibited IC50 values lower than 5 nM. Two compounds
very good inhibitory activity with IC50 1 nM.
9 and 10 exhibited
According to docking results, most of the compounds are placed within the NNRTIs binding
pocket between the place equipped by the classic butterfly shaped compound, nevirapine and the
binding site of etravirine or within the etravirine binding pocket.
It is interesting to mention that, the most active compounds structurally belong to three different
groups and adopt three different orientations.
In general, according to docking studies and in vitro results, it seems that the presence of a Cl
substituent at the 7-, 6- or 4-position of the benzothiazolyl moiety facilitates an orientation which
enables complex stabilization via pi-interactions and hydrogen bond formation involving either the CO
group of the thiazolidinone ring or hydrogen acceptor/donor substituents of the phenyl ring. In case of
subgroups bearing hydrogen acceptor substituents at the benzothiazole moiety, their involvement in
hydrogen bond formation also is favorable for complex stabilization.
The absence of the traditional hydrogen bond interactions with Lys101, in case of one of the most
active compounds, 10, is an interesting characteristic in concern of a probable use of such structures in
future drugs, since mutations in Lys101 are common in resistant mutants.
Finally, it should be mentioned that no statistically significant changes were observed in cell
growth in normal cell line treated with the tested compounds compared to control.
Author Contributions: A.P.—synthesis and characterization, molecular modeling; P.E.—docking, original
draft preparation, data curation; A.G.—PASS, data curation, review and editing, supervision, M.G.A. and
I.V.—evaluation of cytotoxicity.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Ravichandran, S.; Veerasamy, F.; Raman, S.; Krishnan, P.N.; Agrawal, R.K. An overview on HIV-1 reverse
transcriptase inhibitors. Dig. J. Nanomater. Biostructures 2008, 3, 171–187.

Molecules 2019, 24, 3821
27 of 29
2.
Asahchop, E.L.; Wainberg, M.A.; Sloan, R.D.; Tremblay, C.L. Antiviral drug resistance and the need for
development of new HIV-1 reverse transcriptase inhibitors. Antimicrob. Agents Chemother. 2012, 56, 5000–5008.
[CrossRef] [PubMed]
3.
4.
Reeves, J.D.; Doms, R.W. Human immunodeficiency virus type 2. J. Gen. Virol. 2002, 83, 1253–1265.
[CrossRef] [PubMed]
Andries, K.; Azijn, H.; Thielemans, T.; Ludovici, D.; Kukla, M.; Heeres, J.; Janssen, P.; De Corte, B.;
Vingerhoets, J.; Pauwels, R.; et al. TMC125, a novel next-generation nonnucleoside reverse transcriptase
inhibitor active against nonnucleoside reverse transcriptase inhibitor-resistant human immunodeficiency
virus type 1. Antimicrob. Agents Chemother. 2004, 48, 4680–4686. [CrossRef]
5.
6.
7.
Kaushik, N.K.; Kaushik, N.; Attri, P.; Kumar, N.; Kim, C.H.; Verma, A.K.; Choi, E.H. Biomedical importance
of indoles. Molecules 2013, 18, 6620–6662. [CrossRef]
Kadow, J.F.; Regueiro-Ren, A.; Xue, Q.M. Indole, Azaindole and Related Heterocyclic Sulfonylureido
Piperazine Derivatives. US Patent 6,900,206, 9 June 2003.
Pauwels, R.; Andries, K.; Debyser, Z.; Van Daele, P.; Schols, D.; Stoffels, P.; De Vreese, K.; Woestenborghs, R.;
Vandamme, A.M.; Janssen, C.G. Potent and highly selective human immunodeficiency virus type 1 (HIV-1)
inhibition by a series of alpha-anilinophenylacetamide derivatives targeted at HIV-1 reverse transcriptase.
Proc. Nat. Acad. Sci. USA 1993, 90, 1711–1717. [CrossRef]
8.
9.
Pauwels, R.; Andries, K.; Desmyter, J.; Schols, D.; Kukla, M.J.; Breslin, H.J.; Raeymaeckers, A.; VanGelder, J.;
Woestenborghs, R.; Heykants, J.; et al. Potent and selective inhibition of HIV-1 replication in vitro by a novel
series of TIBOderivatives. Nature 1990, 343, 470–474. [CrossRef]
Debyser, Z.; Pauwels, R.; Andries, K.; Desmyter, J.; Kukla, M.; Janssen, P.A.; De Clercq, E.
An antiviral target on reverse transcriptase of human immunodeficiency virus type 1 revealed
bytetrahydroimidazo-[4,5,1-jk][1,4]benzodiazepin-2 (1H)-one and -thione derivatives. Proc. Nat. Acad. Sci.
USA 1991, 88, 1451–1455. [CrossRef]
10. Le Van, K.; Cauvin, C.; Walque, S.; Georges, B.; Boland, S.; Martinelli, V.; Demonté, D.; Durant, F.; Hevesi, L.;
Van Lint, C. New pyridinone derivatives as potent HIV-1 nonnucleoside reverse transcriptase inhibitors. J.
Med. Chem 2009, 52, 3636–3643. [CrossRef]
11. Tanaka, H.; Takashima, H.; Ubasawa, M.; Sekiya, K.; Inouye, N.; Baba, M.; Shigeta, S.;
Walker, R.T.; De Clercq, E.; Miyasaka, T.J. Synthesis and antiviral activity of 6-benzyl analogs of
1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT)as potent and selective anti-HIV-1 agents.
Med. Chem. 1995, 38, 2860–2865. [CrossRef]
12. Penta, A.; Ganguly, S.; Murugesan, S. Design and synthesis of tetrahydrophthalimide derivatives as inhibitors
of HIV-1 reverse transcriptase. Org. Med. Chem. Lett. 2013, 3, 8. [CrossRef] [PubMed]
13. Rawal, R.K.; Tripathi, R.; Kulkarni, S.; Paranjape, R.; Katti, S.B.; Pannecouque, C.; De Clercq, E.
2-(2,6-Dihalo-phenyl)-3-heteroaryl-2-ylmethyl-1, 3-thiazolidin-4-ones: Anti-HIVagents. Chem. Biol. Drug
Des. 2008, 72, 147–154. [CrossRef] [PubMed]
14. Khalid, Z.; Aslam, S.; Ahmad, M.; Munawar, A.; Montero, K.; Detorio, M.; Parvez, M.; Schinazi, R.F. Anti-HIV
activity of new pyrazolobenzothiazine 5,5-dioxide-based acetohydrazides. Med. Chem. Res. 2015, 24,
3671–3680. [CrossRef]
15. Novikov, M.S.; Valuev-Elliston, V.T.; Babkov, D.A.; Paramonova, M.P.; Ivanov, A.V.; Gavryushov, S.A.;
Khandazhinskaya, A.L.; Kochetkov, S.N.; Pannecouque, C.; Andrei, G.; et al. N1,N3-disubstituted uracils as
nonnucleosides inhibitors of HIV-1 reverse transcriptase. Bioorg. Med. Chem. 2013, 21, 1150–1158. [CrossRef]
[PubMed]
16. Wang, Y.P.; Chen, F.-E.; De Clercq, E.; Balzarini, J.; Pannecouque, C. Synthesis and in vitroanti-HIV evaluation
of a new series of 6-arylmethyl-substituted S-DABOs as potentialnon-nucleoside HIV-1 reverse transcriptase
inhibitors. Eur. J. Med. Chem. 2009, 41, 1016–1023. [CrossRef]
17. Pitta, E.; Tsolaki, E.; Geronikaki, A.; Petrovic´, J.; Glamocˇlija, J.; Sokovic´, M.; Crespan, E.; Maga, G.; Bhunia, S.S.;
Saxena, A.K. 4-Thiazolidinone derivatives as potent antimicrobial agents: Microwave-assisted synthesis,
biological evaluation and docking studies. MedChemComm. 2015, 6, 319–326. [CrossRef]
18. Zahid, M.; Yasin, K.A.; Akhtar, T.; Rama, N.H.; Hameed, S.; Al-Masoudi, N.A.; Loddo, R.; Colla, P. Synthesis
and in vitro antiproliferative activity of new adamantylthiazolyl-1,3,4-oxadiazoles. ARKIVOC 2009, xi, 85–93.
19. La Regina, G.; Coluccia, A.; Piscitelli, F.; Bergamini, A.; Sinistro, A.; Cavazza, A.; Maga, J.; Samuele, A.;
Zanoli, S.; Novellino, E.; et al. Indolyl aryl sulfones as HIV-1 non-nucleoside reverse transcriptase inhibitors:

Molecules 2019, 24, 3821
28 of 29
Role of two halogen atoms at the indole ring in developing new analogues with improved antiviral activity.
J. Med. Chem. 2007, 50, 5034–5038. [CrossRef]
20. Akkouh, O.; Ng, T.B.; Singh, S.S.; Yin, C.; Dan, X.; Chan, Y.S.; Pan, W.; Cheung, R.C.F. Lectins with Anti-HIV
activity: A review. Molecules 2015, 20, 648–668. [CrossRef]
21. Famiglini, V.; Coluccia, A.; Brancale, A.; Pelliccia, S.; La Regina, G.; Silvestri, R. Arylsulfone-based HIV-I
non-nucleoside reverse transcriptase inhibitors. Future. Med. Chem. 2013, 5, 2141–2156. [CrossRef]
22. De Clercq, E. Dancing with chemical formulae of antivirals: A personal account. Biochem. Pharm. 2013, 86,
711–725. [CrossRef] [PubMed]
23. Veljkovic, N.; Glisic, S.; Prljic, J.; Perovic, V.; Veljkovic, V. Simple and general criterionfor “in silico” screening
of candidate HIV drugs. Curr. Pharm. Biotechnol. 2013, 14, 561–569. [CrossRef] [PubMed]
24. Li, D.; Zhan, P.; Liu, H.; Pannecouque, C.; Balzarini, J.; De Clercq, E. Synthesis and biological evaluation of
pyrazine derivatives as novel HIV-1 NNRTIs. Bioorg. Med. Chem. 2013, 21, 2128–2134. [CrossRef] [PubMed]
25. La Regina, G.; Coluccia, A.; Brancale, A.; Piscitelli, F.; Gatti, V.; Maga, G.; Sa-muele, A.; Pannecouque, C.;
Schols, D.; Balzarini, J.; et al. Indolylarylsulfones as HIV-1 non-nucleoside reverse transcriptase inhibitors:
New cyclic substituents at indole-2-carboxamide. J. Med. Chem. 2011, 54, 1587–1598. [CrossRef] [PubMed]
26. La Regina, G.; Coluccia, A.; Brancale, A.; Piscitelli, F.; Famiglini, V.; Cosconati, S.; Maga, G.; Samuele, A.;
Gonzales, E.; Clotet, B.; et al. New nitrogen containing substituents at the indole-2-carboxamide yield high
potent and broad spectrum indolylarylsulfone HIV-1 non-nucleoside reverse transcriptase inhibitors. J. Med.
Chem. 2012, 55, 6634–6638. [CrossRef] [PubMed]
27. Gao, P.; Cheng, X.; Sun, L.; Song, S.;
Álvarez, M.; Luczkowiak, J.; Pannecouque, C.; De
Clercq, E.; Men ndez-Arias, L.; Zhan, P.; et al. Design, synthesis and biological evaluation of
é
3-hydroxyquinazoline-2,4(1H,3H)-diones as dual inhibitors of HIV-1 reverse transcriptase-associated RNase
H and integrase. Bioorg. Med. Chem. 2019, 27, 3836–3845. [CrossRef]
28. Tian, Y.; Zhan, P.; Rai, D.; Zhang, J.; Clercq, E.D.; Liu, X. Recent advances in the research of
2,3-diaryl-1,3-thiazolidin-4-one derivatives as potent HIV-1 non-nucleoside reverse transcriptase inhibitors.
Curr. Med. Chem. 2012, 19, 2026–2037. [CrossRef]
29. Suryawanshi, R.; Jadhav, S.; Makwana, N.; Desai, D.; Chaturbhuj, D.; Sonawani, A.; Idicula-Thomas, S.;
Murugesan, V.; Katti, S.; Tripathy, S.; et al. Evaluation of 4-thiazolidinone derivatives as potential reverse
transcriptase inhibitors against HIV-1 drug resistant strains. Bioorg. Chem. 2017, 71, 211–218. [CrossRef]
30. Rao, A.; Balzarini, J.; Carbone, A.; Chimirri, A.; De Clercq, E.; Monforte, A.; Monforte, P.; Pannecouque, C.;
Zappala, M. 2-(2,6-Dihalophenyl)-3-(pyrimidin-2-yl)-1,3-thiazolidin-4-ones as non-nucleoside HIV-1 reverse
transcriptase inhibitors. Antivir. Res. 2004, 63, 79–84. [CrossRef]
31. Chen, H.; Guo, Z.; Yin, Q.; Duan, X.; Gu, Y.; Li, X. Design, synthesis and HIV-RT inhibitory activity of novel
thiazolidin-4-one derivatives. Front. Chem. Sci. Eng. 2011, 5, 231–237. [CrossRef]
32. Rawal, K.; Prabhakar, Y.; Katti, B.V. Molecular surface features in modeling the HIV-1 RT inhibitory activity
of 2-(2,6-disubstituted phenyl)-3-(substituted pyrimidin-2-yl)-thiazolidin-4-ones. QSAR Comb. Chem. Sci.
2007, 26, 398. [CrossRef]
33. Kumar, S.; Purohit, D.; Pandey, P. Molecular docking and its application towards modern drug discovery.
World J. Pharm. Pharm. Sci. 2019, 6, 691–696.
34. Eweas, A.F.; Maghrabi, I.A.; Namarneh, A.I. Advances in molecular modeling and docking as a tool for
modern drug discovery. Der Pharma Chem 2014, 6, 211–228.
35. Zhou, Z.; Lin, X.; Madura, J.D. HIV-1 RT Nonnucleoside inhibitors and their interaction with RT for antiviral
drug development. Infect. Disord. Drug Targets 2006, 6, 391–413. [CrossRef]
36. De Clercq, E. Where rilpivirine meets with tenofovir, the start of a new anti-HIV drug combination era.
Biochem. Pharm. 2012, 84, 241–248. [CrossRef] [PubMed]
37. Filimonov, D.A.; Lagunin, A.A.; Gloriozova, T.A.; Rudik, A.V.; Druzhilovskiy, D.S.; Pogodin, P.V.; Poroikov, V.V.
Prediction of the biological activity spectra of organic compounds using the PASS online web resource. Chem.
Heterocycl. Compd. 2014, 50, 444–457.
38. Geronikaki, A.; Dearden, J.C.; Filimonov, D.; Galaeva, I.; Garibova, T.L.; Gloriozova, T.; Kraineva, V.;
Lagunin, A.; Macaev, F.Z.; Molodavkin, G.; et al. Design of new cognition enhancers: From computer
prediction to synthesis and biological evaluation. J. Med. Chem. 2004, 47, 2870–2876. [CrossRef] [PubMed]

Molecules 2019, 24, 3821
29 of 29
39. Geronikaki, A.; Babaev, E.; Dearden, J.; Dehaen, W.; Filimonov, D.; Galaeva, I.; Kraineva, V.; Lagunin, A.;
Macaev, F.; Molodavkin, G.; et al. Design, synthesis, computational and biological evaluation of new
anxiolytics. Bioorg. Med. Chem. 2004, 12, 6559–6568. [CrossRef] [PubMed]
40. Famiglini, V.; Silvestri, R. Focus on chilarity of HIV-1 non-nucleoside reverse transcriptase inhibitors.
Molecules 2016, 21, 221. [CrossRef]
41. Balzarini, J.; Orzeszko, B.; Maurin, J.; Orzerszko, A. Synthesis and anti-HIV studies of 2-adamantyl-substituted
thiazolidin-4-ones. Eur. J. Med. Chem. 2007, 42, 993–1003. [CrossRef]
42. Chimirri, A.; Grasso, S.; Molica, C.; Monforte, A.-M.; Monforte, P.; Zappalà, M.; De Clercq, E.
Structural features and anti-human immunodeficiency virus (HIV) activity of the isomers of
1-(20,60-difluorophenyl)-1H,3H-thiazolo[3,4-a]benzimidazole, a potent non-nucleoside HIV-1 reverse
transcriptase inhibitor. Antivir. Chem. Chemother. 1997, 8, 363–370. [CrossRef]
43. Prajapati, D.; Ramajayam, R.; Ram Yadar, M.; Giridhar, R. The search for potent, small molecule NNRTIs: A
review. Biorg. Med. Chem. 2009, 17, 5744–5762. [CrossRef] [PubMed]
44. Pitta, E.; Geronikaki, A.; Surmava, S.; Eleftheriou, P.; Mehta, V.P.; Van der Eycken, E.V. 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. J. Enzyme Inhib. Med. Chem. 2013, 8, 113–122. [CrossRef]
45. Kouatly, O.; Eleftheriou, P.; Petrou, A.; Hadjipavlou-Litina, D.; Geronikaki, A. Docking assisted design of
novel 4-adamantanyl-2-thiazolylimino-5-arylidene-4-thiazolidinones as potent NSAIDs. SAR QSAR Environ.
Res. 2018, 29, 83–101. [CrossRef] [PubMed]
46. Available online: https://www.rcsb.org/structure/3MEC (accessed on 9 November 2016).
47. Nadaraia, N.S.; Amiranashvili, L.S.; Merlani, M.; Kakhabrishvili, M.L.; Barbakadze, N.N.; Geronikaki, A.;
Petrou, A.; Poroikov, V.; Ciric, A.; Glamoclija, J.; et al. Novel antimicrobial agents’ discovery among the
steroid derivatives. Steroids 2019, 144, 52–65. [CrossRef] [PubMed]
48. Helma, C. Lazy structure-activity relationships (lazar) for the prediction of rodent carcinogenicity and
Salmonella mutagenicity. Mol. Divers. 2006, 10, 147–158. [CrossRef] [PubMed]
49. Maunz, A.; Helma, C. Prediction of chemical toxicity with local support vector regression and activity-specific
kernels. Sar. Qsar Environ. Res. 2008, 19, 413–431. [CrossRef] [PubMed]
50. Vizirianakis, I.S.; Tsiftsoglou, A.S. Blockade of murine erythroleukemia cell differentiation by hypomethylating
agents causes accumulation of discrete small poly(A)- RNAs hybridized to 30-end flanking sequences of
beta(major) globin gene. Biochim. Biophys. Acta 2005, 1743, 101–114. [CrossRef]
51. Akrivou, M.G.; Demertzidou, V.P.; Theodoroula, N.F.; Chatzopoulou, F.M.; Kyritsis, K.A.; Grigoriadis, N.;
Zografos, A.L.; Vizirianakis, I.S. Uncovering the pharmacological response of novel sesquiterpene derivatives
that differentially alter gene expression and modulate the cell cycle in cancer cells. Int. J. Oncol. 2018, 53,
2167–2179. [CrossRef]
52. Tseligka, E.D.; Rova, A.; Amanatiadou, E.P.; Calabrese, G.; Tsibouklis, J.; Fatouros, D.G.; Vizirianakis, I.S.
Pharmacological Development of Target-Specific Delocalized Lipophilic Cation-Functionalized Carboranes
for Cancer Therapy. Pharm. Res. 2016, 33, 1945–1958. [CrossRef]
Sample Availability: Samples of the compounds are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).