New thiourea and 1,3-thiazolidin-4-one derivatives effective on the HIV-1 virus

Article abstract of DOI:10.1111/cbdd.13009

Thiourea derivatives have been reported to possess many biological activities, among them antiviral and antitumoral properties. As part of our continuing effort to develop new active compounds, we report the synthesis and the evaluation of new fifteen thiourea derivatives with 1,3-benzothiazole-2-yl moiety, among them a group of biologically active (1–7) also underwent cyclization to 1,3-thiazolidin-4-ones. Molecular structure of four compounds (4, 13, 15 and 3a) was determined by an X-ray crystallography. We here report the evaluation of their cytotoxicity against human leukaemia/lymphoma- and solid tumour-derived cell lines and of their antiviral activity against HIV-1 and representatives of ssRNA and dsDNA viruses. Derivative 5 showed an interesting activity against HIV-1 wild type and against variants carrying clinically relevant mutations. A colorimetric enzyme immunoassay clarified its mode of action as a non-nucleoside inhibitor of the reverse transcriptase.

Full text of DOI:10.1111/cbdd.13009

DR. GIUSEPPINA SANNA (Orcid ID : 0000-0002-3999-4263)
Article type : Research Article
New thiourea and 1,3-thiazolidin-4-one derivatives effective on the HIV-1 virus
Running title: Evaluation of benzothiazol thiourea derivatives
Anna Bielenicaa 1, Giuseppina Sannab 1 *, Silvia Madeddub , Marta Strugac,d, Michał
Jóźwiakd,e, Anna E. Koziołf, Aleksandra Sawczenkof, Ilona B. Materekf, Alessandra Serrab,
Gabriele Gilibertib
a Chair and Department of Biochemistry, Medical University of Warsaw, 02-097 Warszawa, Poland
b
Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria, 09042
Monserrato, Cagliari, Italy
c
Department of Biochemistry and Pharmacogenomics, Faculty of Pharmacy, Medical University of
Warsaw, 02-097 Poland
d Laboratory of Centre for Preclinical Research, Medical University of Warsaw, Banacha 1B, 02-097
Warsaw, Poland
e Department of Biochemistry, Second Faculty of Medicine, Medical University of Warsaw, 02-097
Warsaw, Poland
f Faculty of Chemistry, Maria Curie-Sklodowska University, 20-031 Lublin, Poland
1 Equal contribution
* Corresponding author: Dr. Giuseppina Sanna.
E-mail: g.sanna@unica.it. Phone: +39-070-6754161.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13009
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Keywords: drug discovery; biological screening; thiourea; anti-HIV activity; reverse
transcriptase.
Abstract
Thiourea derivatives have been reported to possess many biological activities, among them
antiviral and antitumoral properties. As part of our continuing effort to develop new active
compounds, we report the synthesis and the evaluation of new fifteen thiourea derivatives
with 1,3-benzothiazol-2-yl moiety, among them a group of biologically active (1-7) also
underwent cyclisation to 1,3-thiazolidin-4-ones. Molecular structure of four compounds (4,
13, 15 and 3a) was determined by an X-ray crystallography. We here report the evaluation of
their cytotoxicity against human leukaemia/lymphoma- and solid tumour-derived cell lines
and of their antiviral activity against HIV-1 and representatives of ssRNA and dsDNA
viruses. Derivative 5 showed an interesting activity against HIV-1 wild type and against
variants carrying clinically relevant mutations. A colorimetric enzyme immunoassay clarified
its mode of action as a non-nucleoside inhibitor of the reverse transcriptase.
Introduction
AIDS represents one of the major health problems worldwide, since that the WHO estimates
for the 2015 approximately 36.7 million people living with Human Immunodeficiency virus
(HIV-1) and 1.1 million people died from AIDS-related causes.
Antiretroviral therapy (HAART), actually based on combinations of drugs belonging to non-
nucleoside reverse transcriptase (NNRTIs), nucleoside reverse transcriptase (NRTIs),
protease (PR) and, more recently, integrase (IN) inhibitors, has improved the life quality and
duration of AIDS patients. However, HIV-1 still remains an interesting biological target due
to the side effects reported for clinically used drugs, to the rapid emergence of new drug-
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resistant strains and to the difficulty to access the existing therapies for people in many
countries [1,2].
Some thiourea compounds were reported to act as NNRTIs [3]. Their inhibitory activity is the
result of binding into an allosteric hydrophobic pocket located near to the DNA polymerase
active site, with the distortion of essential aspartyl residues [4]. Phenylethylthiourea
compounds, such as the prototype thiazolylthiourea LY 73497, the its close derivative
Trovirdine (TRV) and the new vaginal microbicide HI-443 are important representatives of
this group (Figure 1) [5,6]. It was revealed that substitution of 2-pyridyl ring, as well as the
presence of an ethyl spacer between phenyl and thiourea group are are important for the anti-
HIV activity [7,8]. Another drug-candidate, an imidoylthiourea (ITU), in contrary to the
voluminous and rigid structures of first-generation NNRTIs, has a flexible chemical
arrangment based on an open chain. Thus it can adopt “horseshoe” or “U” conformation in
the receptor binding site [9-11]. This torsional freedom enables to bind to a mutated NNRTI
binding pocket (NNIBP), compensating the effects of these mutations. Since that the NNRTIs
potency is considerably reduced by the development of viral strains with a mutated form of
RT, the effective chemicals with high affinity for the mutated NNIBP are extensively
designed. The thiourea derivatives are also recently discovered as HIV-1 capsid binders [12]
or as inhibitors of the HIV-1 gp120-CD4 binding [13], offering potential additional targets to
the combination chemotherapy.
Many other structural changes of 1,3-disubstituted (thio)ureas have been developed for novel
potential antiviral agents. Bis(aryl)thioureas with amide functionality were considered as
inhibitors of herpesviruses such as HSV, CMV and VZV [14]. A thiourea analogue with a
pyridine-2-carboxamide moiety (ACH-806) has exhibited potent activity against the
replication in vivo of genotype 1 HCV and in a proof-of-concept clinical trial [15]. The 2-
methylbenzimidazol-1-ylthiourea derivative (MBZM-N-IBT) was found to inhibit the
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Chikungunya virus (CHIKV) replication [16]. A benzimidazoleurea Frentizole (Figure 1) has
been reported as immunosuppressive and antiviral agent [17]. Thiourea derivatives with
substituted 1,2,4-triazole-3-thione group significantly inhibit Coxsackievirus B4 [18].
It was proved that the presence of (hetero)aromatic rings such as pyrazole [19],
chloroquinoline [20], thiazole [21], acridine [22] or podophyllotoxin [23] at different
positions of the thiourea branch, enhances cytotoxicity and anticancer properties of a
compound. Recently, the combination of a halogeno-substituted benzothiazole with the
thiourea moiety, as well as its oxidative cyclisation to N-bis-benzothiazoles, have delivered
new potent antitumor agents, effective against human cancer cell lines [24].
1,3-Thiazolidinones, the products of the thiourea branch cyclisation, are a promising group of
bioactive compounds, that exhibit diverse applications in medicinal chemistry, acting as anti-
HIV [25], antimicrobial [26,27], antimalarial [28], antinflammatory [29] and anticancer
agents [30,31]. The mechanism of anti-HIV action of studied 2,3-diaryl-1,3-thiazolidin-4-one
derivatives is attributed to the inhibition of HIV-1 RT, with a simultaneous minimal toxicity
to MT-4 cells [32,33]. Substituted 2-imino-4-thiazolidinones, derivatives of benzothiazole,
are known for antiproliferative activity through their ability to inhibit heat shock protein 70
(Hsp70) [30]. Thiophene ring-containing thiazolidinones show anticancer properties via
induction of apoptosis or contribution in cell cycle arrest in melanoma cells [31].
These findings encouraged us to go further with our ongoing studies on the thiourea
derivatives, since that many biological properties have been already discovered by our team
among this group of compounds [34-39]. In the present study we have synthesized new
thiourea and thiazolidinone derivatives with 1,3-benzothiazol-2-yl scaffold. All synthesized
compounds were tested for their anti-HIV activity, some of them also for other antiviral
activities. Antiproliferative potential of compounds was also examined against human
leukaemia/lymphoma- and solid tumour-derived cell lines.
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Methods and Materials
Chemistry
The starting amine was commercially available (Koch-Light Laboratories Ltd).
Isothiocyanates were supplied from Alfa Aesar or Sigma Aldrich. Organic solvents
(acetonitrile, chloroform and methanol) were supplied from POCh (Polskie Odczynniki
Chemiczne). Prior usage, dried acetonitrile was kept in crown cap bottles over anhydrous
phosphorus pentoxide (Carl Roth). The NMR spectra were recorded on Varian VNMRS 300
Oxford NMR spectrometer, operating at 300 MHz (1H NMR) and 75.4 MHz (13C NMR).
Chemical shifts (δ) were expressed in parts per million relative to tetramethylsilane used as
the internal reference. Mass spectral ESI measurements were carried out on Waters ZQ
Micro-mass instruments with quadruple mass analyzer. The spectra were performed in the
negative or positive ion mode at a declustering potential of 40–60 V. The sample was
previously separated on a UPLC column (C18) using UPLC ACQUITYTM system by
Waters connected with DPA detector. Flash chromatography was performed on Merck silica
gel 60 (230–400 mesh) using chloroform eluent.
Analytical TLC was carried out on silica gel F254 (Merck) plates (0.25 mm thickness).
The diffraction data for 4, 13 and 15 were collected at 120(2) K and at room temperature for
3a on a SuperNova diffractometer (Oxford Diffraction) equipped with the microfucus X-ray
source (Cu Kα, λ = 1.54184 Å) and CCD detector. The CRYSALIS program system was
used for data collection, cell refinement and data reduction. The data were corrected for
Lorentz and polarization effects. A multi-scan absorption correction was applied. The
structure was solved using direct methods implemented in the SHELXS-97, and refined by
the full-matrix least-squares on F2 with the SHELXL-97 program [40]. All non-H atoms were
refined with anisotropic displacement parameters. The H-atoms attached to carbon were
positioned geometrically and refined using the riding model with Uiso(H) = a · Ueq(C), where
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a = 1.2, except for –CH3 groups in 13 (a = 1.3), and in 4 and 3a (a = 1.5). The nitrogen
bonded H-atoms were found in the difference-Fourier map and refined with isotropic
displacement parameters. The final structural parameters can be retrieved from the Cambridge
Structural Database (CSD) (deposition numbers: CCDC).
Preparation of 1-(1,3-benzothiazol-2-yl)-3-thiourea derivatives (1-15).
A solution of 1,3-benzothiazol-2-amine (0.0027 mol, 0.40 g) in dry acetonitrile (20 mL) was
treated with appropriate isothiocyanate (mol. 1:1). The mixture was heated under reflux
condenser for 30 h. After solvent evaporating, the residue was either crystallized from
acetonitrile or purified by column chromatography (chloroform). The details of the synthesis
of each compound are provided in the “Supporting information”.
Preparation of 2-(1,3-benzothiazol-2-ylimino)-1,3-thiazolidin-4-ones (1a–7a).
A solution of an appropriate thiourea derivative 1-7 (0.001 mol) was heated with
chloroacetamide (0.003 mol) in dried acetonitrile (20 mL) for 36h. After the solvent has been
evaporated, the solid residue was purified by column chromatography (chloroform). The
details of the synthesis of each compound are provided in the “Supporting information”.
Cells and viruses
Cell lines were purchased from American Type Culture Collection (ATCC). Cell lines
supporting the multiplication of RNA and DNA viruses were the following: CD4+ human T-
cells containing an integrated HTLV-1 genome (MT-4); Baby Hamster Kidney (BHK-21)
[ATCC CCL 10 (C-13) Mesocricetus auratus] and Monkey kidney (Vero 76) [ATCC CRL
1587 Cercopithecus Aethiops]. Human Immunodeficiency Virus type-1 (HIV-1) IIIB
laboratory strain was obtained from the supernatant of the persistently infected H9/IIIB cells
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(NIH 1983). Viruses representative of ssRNA+ were: i) Flaviviridae: yellow fever virus
(YFV) [strain 17-D vaccine (Stamaril Pasteur J07B01)], ii) Picornaviridae: enterovirus B
[coxsackie type B5 (CV-B5), strain Ohio-1 (ATCC VR-29)], and enterovirus C [poliovirus
type-1 (Sb-1), Sabin strain Chat (ATCC VR-1562)]. Viruses representative of ssRNA- were:
iii) Paramyxoviridae: human respiratory syncytial virus (RSV) [strain A2 (ATCC VR-1540)];
iv) Rhabdoviridae: vesicular stomatitis virus (VSV) [lab strain Indiana (ATCC VR 1540)].
DNA virus representatives were: v) Poxviridae: vaccinia virus (VV) [vaccine strain Elstree-
Lister (ATCC VR-1549)]; vi) Herpesviridae: human herpes 1 (HSV-1) [strain KOS (ATCC
VR-1493)].
Mutants carrying NNRTI mutations used: Y181C mutant (NIH N119) of HIV-1 derives from
an AZT-sensitive clinical isolate passaged initially in CEM and then in MT-4 cells, in the
presence of Nevirapine (up to 10 µM); K103N + Y181C mutant (NIH A17) derives from an
IIIB strain passaged in H9 cells in the presence of BI-RG 587 (up to 1 µM); K103R + V179D
+ P225H mutant (EFVR) derives from an IIIB strain passaged in MT-4 cells in the presence
of Efavirenz (up to 2 µM). Mutants carrying NRTI mutations used: AZTR strain (67N, 70R,
215F, 219Q); MDR strain (74V, 41L, 106A, 215Y).
Cytotoxicity assays
Exponentially growing MT-4 cells were seeded at an initial density of 4x105 cells/ml in 96-
well plates in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 100
units/mL penicillin G and 100 μg/mL streptomycin. BHK cells were seeded in 24-well plates
at an initial density of 6x105 and 1x106 cells/mL, respectively, in Minimum Essential
Medium with Earle’s salts (MEM-E), L-glutamine, 1mM sodium pyruvate and 25mg/L
kanamycin, supplemented with 10% fetal bovine serum (FBS). Vero-76 cells were seeded in
24-well plates at an initial density of 4x105 cells/mL, in Dulbecco’s Modified Eagle Medium
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(D-MEM) with L-glutamine and 25mg/L kanamycin, supplemented with 10% FBS. Cell
cultures were then incubated at 37 °C in a humidified, 5% CO2 atmosphere, in the absence or
presence of serial dilutions of test compounds. Cell viability was determined after 48-96 hrs
at 37 °C by MTT method for MT-4 and BHK [41]. Cell viability was determined after 48-96
hrs at 37 °C by the crystal violet staining method for Vero-76.
Antiviral assays
Compound’s activity against HIV-1 was based on inhibition of virus-induced
cytopathogenicity in exponentially growing MT-4 cell acutely infected with a multiplicity of
infection (m.o.i.) of 0.01. Briefly, 50 μL of RPMI containing 1x104 MT-4 cells were added to
each well of flat-bottom microtitre trays, containing 50 μL of RPMI without or with serial
dilutions of test samples. Then, 20 μL of a HIV-1 suspension containing 100 CCID50 were
added. After a 4-day incubation at 37 °C, cell viability was determined by the MTT method.
Compound’s activity against YFV was based on inhibition of virus-induced
cytopathogenicity in BHK-21 cells acutely infected with a m.o.i. of 0.01. Briefly, BHK cells
were seeded in 96-well plates at a density of 5x104 cells/well and were allowed to form
confluent monolayers by incubating overnight in growth medium at 37 °C in a humidified
CO2 (5%) atmosphere. Cell monolayers were then infected with 50 μL of a proper virus
dilution in maintenance medium [MEM-Earl with L-glutamine, 1mM sodium pyruvate and
0.025g/L kanamycin, supplemented with 0.5% inactivated FBS]. At the same time, 50 μL of
maintenance medium, without or with serial dilutions of test compounds, were added. After a
3 day incubation at 37°C, cell viability was determined by the MTT method. Compound’s
activity against CV-B5, Sb-1, VV, VSV, HSV-1 and RSV was determined by plaque
reduction assays in infected cell monolayers. Briefly, Vero-76 monolayers were infected for 2
hours with 250 μL of proper virus dilutions; following removal of unadsorbed virus, 500 μL
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of maintenance medium containing 0.75% methyl-cellulose, without or with serial dilutions
of test compounds, were added. Cultures were incubated at 37°C for 2 (Sb-1 and VSV), 3
(CV-B5, VV and HSV-1) or 5 days (RSV) and then fixed with PBS containing 50% ethanol
and 0.8% crystal violet, washed and air-dried. Plaques were then counted. The extent of cell
growth/viability and viral multiplication, at each sample concentration tested, were expressed
as percentage of untreated controls. Concentrations resulting in 50% inhibition (CC50 or
EC50) were determined by linear regression analysis.
Reverse transcriptase Assay
The colorimetric Reverse Transcriptase Assay (Roche, Cat. 11468120910) was used for the
quantitative determination of retroviral reverse transcriptase activity, using efavirenz as
reference compound.
Antiproliferative assays
Cell lines derived from human haematological tumours were: CD4+ human T-cells
containing an integrated HTLV-1 genome (MT-4); CD4+ human acute T-lymphoblastic
leukaemia (CCRF-CEM); human splenic B-lymphoblastoid cells (WIL-2NS); human acute
B-lymphoblastic leukaemia (CCRF-SB). Cell lines derived from human solid tumours were:
human lung carcinoma (SK-MES-1); prostate carcinoma (DU-145); human cervix
adenocarcinoma (Hela). Normal tissues foreskin fibroblasts (CRL-7065) were also used.
Cells were seeded at an initial density of 1×105 cells/ml in 96 well plates in RPMI-1640
medium supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin G and 100
μg/ml streptomycin. Cell cultures were then incubated at 37 °C in a humidified, 5% CO2
atmosphere in the absence or presence of serial dilutions of test compounds. Cell viability
was determined after 96 hrs at 37 °C by the MTT method.
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Results
The synthetic route for the preparation of title derivatives of 1,3-benzothiazol-2-amine is
shown in the Figure 2. First, the starting compound was heated with appropriate
isothiocyanates in acetonitrile medium to give 1-(1,3-benzothiazol-2-yl)thiourea derivatives
1-15. To provide structural diversity, isothiocyanates with aryl (1-4, 6, 7, 9-12, 15), alicyclic
(8), alkyl (13) or alkylaryl (5, 14) substituents were introduced. In the second step, previously
obtained biologically active (cytotoxic) compounds 1-7 was directly cyclized to the
corresponding iminothiazolidinones 1a-7a by treating these intermediates with
chloroacetamide in acetonitrile. According to literature data [42-45], this synthetic route
usually gives a mixture of two isomeric forms, that differ in substitutions at thiazolidinone
N2 and N3 nitrogen atoms (Figure 3). Under reaction conditions proposed in this paper, the
cyclization led to the formation of one product only, the 2-iminothiazolidin-4-one derivative.
This regioselectivity could be explained by the use of the polar aprotic solvent (acetonitrile)
and by the reactive halo acetic acid derivative as a substrate, as well as high temperature of
the reaction. Finally, the nature of both terminal functionalities at the nitrogen atoms
determined the type of the product - bulky polar benzothiazole ring prefers substitution at N2
of the 2-imino-4-thiazolidinone [43].
The structures of newly synthesized compounds were verified by spectral analyses, and
molecular structure of the molecules 4, 13 and 15, and of the 2-imino-4-thiazolidinone
derivative 3a was confirmed by an X-ray crystallography (Figures 4 and 5). The
conformation adopted by the molecules of 4, 13 and 15 is stabilized by the intramolecular N-
H...N hydrogen bond between the Z oriented benzothiazole and thiourea fragments (see Table
1S of Supporting information). This hydrogen bonding causes coplanarity of thiourea and
thiazole fragments, and exposes the N1-H, S1 and S2 atoms for intermolecular contacts, e.g.
hydrogen bonds with receptors.
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The antiviral activity of newly synthesized thiourea and thiazolidinone derivatives was
evaluated against HIV-1 in cell based assay. In parallel the cytotoxicity was investigated
against the cell line supporting the multiplication of HIV-1, the CD4+ human T-cells derived
from human haematological tumours, containing an integrated HTLV-1 genome (MT-4).
Some derivatives were also tested their antiviral potency against representative of several
RNA and DNA virus families. Results are reported in the Table 1.
The 3-(1-phenylethyl)thiourea (5) showed anti-HIV-1 activity (with EC50 value of 1.0 µM),
with a moderate cytotoxicity against the MT-4 cells. Since a critical issue in clinical
management of HIV-1 disease is the development of drug resistant strains, compound 5 was
further tested against a set of viruses possessing mutations that confer resistance to NRTI and
NNRTI inhibitors, and that often appear during HAART therapy, reducing its effectiveness
[46]. Interestingly, the activity against AZTR and MDR strains is comparable with those of
HIV-1 wild type, while it resulted less or not active against NNRTI-resistant mutants (Table
2). Results suggest its mode of action as an inhibitor of the reverse transcriptase.
The derivative 5 was tested in a colorimetric enzyme immunoassay to evaluate its ability to
inhibit the reverse transcriptase of HIV-1. Results confirmed it as a good inhibitor of the
reverse transcriptase, with a 60% of inhibition at 10μM and a 85% at 50μM (Figure 5).
Regarding the other tested viruses, no relevant activities were found.
With the exception of 5, none of the other tested compounds showed selective anti-HIV-1
activity; however compounds 1, 2, 3, 4, 6, 7 turned out cytotoxic for exponentially growing
MT4 cells in the low micromolar range (CC50 ≤ 10 μM). The antiproliferative activity against
CD4+ human T cell line derived from a haematological human tumor, prompted us to
evaluate the antiproliferative activity of them also for a panel of other human haematological
and solid tumours and for cell lines derived from normal human tissues (Table 1).
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Interestingly, all tested compounds showed antiproliferative activity with CC50 values
comparable to that obtained with MT-4 cells. Particularly derivatives 1, 2, 6 and 7 showed
more interesting values, but they did not result highly selective, showing cytotoxic also
against the “normal” CRL7065 cell lines, even if with values slightly lower.
Discussion
Novel 1,3-benzothiazol-2-yl derivatives of thiourea and their 2-iminothiazolidin-4-one
analogs have been designed and synthesized. The 3-(1-phenylethyl)thiourea (5) showed a
potent anti-HIV-1 activity, targeting the reverse transcriptase of HIV-1, while other
derivatives (1, 2, 3, 4, 6, 7) turned out cytotoxic for the exponentially growing MT4 cells.
They also showed antiproliferative properties against cells derived from other human
haematological and solid tumours. This could be a good starting point for the development of
novel and more active compounds for the treatment of AIDS, for which there is a continued
need.
A close overview of the structures of presented 2-aminobenzothiazoles (Table 1, Figure 2)
revealed that their biological potency is strongly bound to the nature of the substituent of the
thiourea. The anti-HIV activity of the compound 5 is directly influenced by its flexibility due
to the presence of an alkylphenyl spacer, attached to the heteroaryl core. From this point of
view, the compound is an analog of other known NNRTIs such as (hetero)aryletylthioureas
LY 73497, Trovirdine , HI-443, as well as the (phenyl)thiazole connections in the mentioned
LY 73497 or Frentizole (Figure 1). On the contrary, the higher rigidity of the other
synthesized benzothiazoles resulted in their inactivity in antiviral tests.
A remarkable cytotoxicity was observed within the group of substituted phenylthioureas
bearing electron-withdrawing (1, 2, 2a, 6, 7) or weakly electron-donating (3, 4)
functionalities on the benzene ring. On the contrary, the introduction of electronegative atoms
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such as chlorine or fluorine (compounds 9, 10, 15) or the adding of strongly electron-
donating groups to a phenyl ring (11) led to the reduction of cytotoxicity of 2-
aminobenzothiazole derivatives. The same effect was achieved by connection of the thiourea
branch with an alkyl (13, 14) or alicyclic (8) group.
The cyclization of the compounds 1-7 resulted in an overall decrease of the biological
activities. Indeed, anti-HIV activity of 5 is totally lost in the derivative 5a and the compounds
1a-7a have lost their cytotoxicity on the MT4 cells. Only 2a remains cytotoxic even if with a
lower value (CC50 from 1.6 to 9 μM). Compound 2a seems to be less cytotoxic also in the
other antiproliferative assays, when compared with others and with its linear analog 2;
interestingly it showed no cytotoxicity against the normal cells CRL7065.
Obtained results are in contrary to our previous reports on this class of compounds [31, 34],
in which the highest activity was observed for mono- and dihalogenothioureas.
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Supporting information
A supporting information file is available and contains: the synthetic details of the
preparation of 1-(1,3-benzothiazol-2-yl)-3-thiourea derivatives (1-15) and of 2-(1,3-
benzothiazol-2-ylimino)-1,3-thiazolidin-4-ones (1a–7a); the geometry of the intramolecular
N-H...N hydrogen bonds in molecules 4, 13 and 15 (Table 1S); the 1H NMR and 13C NMR
spectra of all newly synthesized derivatives.
Figure legends
Figure 1. Chemical structures of (thio)urea derivatives with antiviral activity.
Figure 2. Scheme of the synthesis of thioureas 1-15 and 1,3-thiazolidin-4-one derivatives 1a-
7a.
Figure 3. Regioselectivity of the 2-iminothiazolidin-4-one synthesis.
Figure 4. Perspective view of molecules 4 (two molecules in the asymmetric unit, A) and 13
(B).
Figure 5. Perspective view of molecules 15 (A) and 3a (B).
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Figure 6. % of Reverse Transcriptase inhibition of derivative 5 and efavirenz (as reference
compound), used at different concentrations in a colorimetric enzyme immunoassay. Results
represent the mean ± S.D. of three independent determinations.
Acknowledgements
This paper was supported by Medical University of Warsaw, 1WK/N/2016
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Table 1. Antiviral activity of derivatives against RNA and DNA viruses and cytotoxicity against: the cell lines used in the assays;
human leukaemia/lymphoma-derived cell lines; solid tumour-derived cell lines.
CV-B5, Sb-1,
RSV, VSV, VV,
HSV-1
MT-4
cells
BHK
cells
Vero-76
cells
CCRF-
CEM
WIL-
2NS
CCRF-
SB
SK-
MES1
CRL70
65
HIV-1
YFV
DU145
Hela
a
b
c
d
e
f
g
EC50
CC50
EC50
CC50
EC50
CC50
CC50
8.9 ±
0.7
9.0 ±
0.8
6.6 ±
0.6
7.2 ±
0.6
8.0 ±
0.9
6.8 ±
0.9
>8.4
>1.6
>8.6
>9.7
8.4 ± 0.9
1.6 ± 0.1
8.6 ± 0.5
9.7 ± 1.0
38
>100
>100
>76
>12
-
76
12
14
11
11
25
1
2
3
4
5
6
7
2.8 ±
0.3
2.3 ±
0.3
1.7 ±
0.2
8.0 ±
0.7
2.8 ±
0.2
9.9 ±
1.1
>15
-
15
-
9.6 ±
0.9
8.9 ±
1.0
-
10
13
11
18
11
13
26
22
8.7 ±
0.8
>9
9 ± 0.8
>100
>100
-
>12
>100
>100
-
12
11
1.0 ±
0.1
>100
>100
-
>100
>100
-
4.0 ±
0.6
9.0 ±
1.0
4.8 ±
0.5
6.6 ±
0.9
>9.5
>5.8
9.5 ± 0.9
5.8 ± 0.6
15
14
28
20
9.0 ±
0.8
9.0 ±
1.4
7.4 ±
0.6
8.5 ±
0.8
6.8 ±
0.7
8.6 ±
0.5
>46
>14
>15
>68
46
14
15
68
-
-
-
-
8
9
-
-
-
-
>100
>100
2.0 ±
0.1
80
>100
>100
>100
>100
>100
>100
10
11
>100
>100
>100
>100
>2
12
>100
>67
100
67
>100
>100
>80
13
14
15
1a
-
-
-
-
-
-
-
-
-
-
>27
27
-
>100
100
-
9.5 ±
1.5
>9.0
9.0 ± 0.9
-
-
-
-
25 ± 3.0 17 ± 2.5
53
50
53
>100
2a
>86
>100
>84
>29
>67
86
>100
84
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3a
4a
5a
6a
7a
29
67
References
Efavir
enz
0.002 ±
0.0002
40
1.9 ±
0.1
2’-C-methyl-guanosine
>10
0.003 ±
0.005
0.003 ±
0.005
0.002 ±
0.003
0.01 ±
0.002
0.01 ±
0.002
0.07 ±
0.005
Vincristine
>20
Data represent mean values for three independent determinations. Standard deviations are reported for the more active compounds. Also for the others values the variation
was less than 15%.
b
aCompound concentration (µM) required to achieve 50% protection of MT-4 cells from HIV-1 induced cytopathogenicity, determined by the MTT method. Compound
concentration (µM) required to reduce the proliferation of mock-infected MT-4 cells by 50%, determined by the MTT method. cCompound concentration (µM) required to
achieve 50% protection of BHK cells from YFV-induced cytopathogenicity, determined by the MTT method. d,fCompound concentration (µM) required to reduce the
viability of mock-infected BHK(d) and Vero-76(f) cells by 50%, determined by the MTT method. eCompound concentration (µM) required to reduce the plaque number of
CV-B5, Sb-1, RSV, VSV, VV, HSV-1 by 50% in VERO-76 monolayers. gCompound concentration (µM) required to reduce cell proliferation by 50%, as determined by
the MTT method, under conditions allowing untreated controls to undergo at least three consecutive rounds of multiplications.
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