Facile synthesis and in vitro activity of n-substituted 1,2-benzisothiazol-3(2H)-ones against dengue virus NS2BNS3 protease

Article abstract of DOI:10.3390/pathogens10040464

Several new N-substituted 1,2-benzisothiazol-3(2H)-ones (BITs) were synthesised through a facile synthetic route for testing their anti-dengue protease inhibition. Contrary to the conventional multistep synthesis, we achieved structurally diverse BITs with excellent yields using a two-step, one-pot reaction strategy. All the synthesised compounds were prescreened for drug-like properties using the online Swiss Absorption, Distribution, Metabolism and Elimination (SwissADME) model, indicating their favourable pharmaceutical properties. Thus, the synthesised BITs were tested for inhibitory activity against the recombinant dengue virus serotype-2 (DENV-2) NS2BNS3 protease. Dose–response experiments and computational docking analyses revealed that several BITs bind to the protease in the vicinity of the catalytic triad with IC₅₀ values in the micromolar range. The DENV2 infection assay showed that two BITs, 2-(2-chlorophenyl)benzo[d]isothiazol-3(2H)-one and 2-(2,6-dichlorophenyl)benzo[d]isothiazol-3(2H)-one, could suppress DENV replication and virus infectivity. These results indicate the potential of BITs for developing new anti-dengue therapeutics.

Full text of DOI:10.3390/pathogens10040464

pathogens
Article
Facile Synthesis and In Vitro Activity of N-Substituted
1,2-Benzisothiazol-3(2H)-ones against Dengue Virus
NS2BNS3 Protease
Farwa Batool 1,2 , Muhammad Saeed 1,
* *
, Hafiza Nosheen Saleem 1, Luisa Kirschner 2 and Jochen Bodem 2,
1
Department of Chemistry and Chemical Engineering, Syed Babar Ali School of Science and Engineering,
Lahore University of Management Sciences, Lahore 54692, Pakistan; 15130009@lums.edu.pk (F.B.);
nosheen.saleem@lums.edu.pk (H.N.S.)
Institut für Virologie und Immunbiologie, Versbacher Straße 7, 97078 Würzburg, Germany;
luisa.kirschner@uni-wuerzburg.de
2
*
Correspondence: muhammad.saeed@lums.edu.pk (M.S.); jochen.bodem@uni-wuerzburg.de (J.B.);
Tel.: +92-42-356-08347 (M.S.); +49-931-318-1509 (J.B.)
Abstract: Several new N-substituted 1,2-benzisothiazol-3(2H)-ones (BITs) were synthesised through
a facile synthetic route for testing their anti-dengue protease inhibition. Contrary to the conventional
multistep synthesis, we achieved structurally diverse BITs with excellent yields using a two-step,
one-pot reaction strategy. All the synthesised compounds were prescreened for drug-like properties
using the online Swiss Absorption, Distribution, Metabolism and Elimination (SwissADME) model,
indicating their favourable pharmaceutical properties. Thus, the synthesised BITs were tested for
inhibitory activity against the recombinant dengue virus serotype-2 (DENV-2) NS2BNS3 protease.
Dose–response experiments and computational docking analyses revealed that several BITs bind
to the protease in the vicinity of the catalytic triad with IC50 values in the micromolar range. The
DENV2 infection assay showed that two BITs, 2-(2-chlorophenyl)benzo[d]isothiazol-3(2H)-one and
2-(2,6-dichlorophenyl)benzo[d]isothiazol-3(2H)-one, could suppress DENV replication and virus
infectivity. These results indicate the potential of BITs for developing new anti-dengue therapeutics.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Batool, F.; Saeed, M.;
Saleem, H.N.; Kirschner, L.; Bodem, J.
Facile Synthesis and In Vitro Activity
of N-Substituted 1,2-Benzisothiazol-
3(2H)-ones against Dengue Virus
NS2BNS3 Protease. Pathogens 2021,
10, 464. https://doi.org/10.3390/
pathogens10040464
Keywords: dengue virus; direct-acting antivirals; 1,2-benzisothiazolinone; drug discovery;
infectivity assays
Academic Editor: Lawrence S. Young
1. Introduction
Received: 30 December 2020
Accepted: 9 April 2021
The epidemics of flaviviral diseases have been recognised for more than 200 years.
Among them, mosquito-borne disease prevalence is on the rise in Asia, South America,
Africa and along the Atlantic Gulf coasts of the United States. Dengue virus (DENV) is
transmitted in humans via the bite of the female Aedes aegypti mosquito. It has become
one of the most emerging and prevalent pathogens [1] in tropical and subtropical regions.
The clinical manifestation of DENV infection is dengue fever (DF), which in most cases
is characterised by milder and self-limiting symptoms, such as febrile illness, headache,
Published: 12 April 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
muscular pain and skin rashes [2]. However, a significant number of DENV infected
patients are prone to more advanced and fatal diseases, such as dengue shock syndrome
(DSS) and dengue haemorrhagic fever (DHF) [3].
For more than a decade, several viral structural and nonstructural (NS) proteins have
Copyright:
© 2021 by the authors.
been exploited as drug targets for developing direct-acting antivirals (DAAs) [
the serine protease of DENV, i.e., a complex of two nonstructural viral proteins labelled
NS2BNS3, has become a valuable target for discovering new anti-dengue therapeutics [ ].
The DENV NS2BNS3 protease is an essential viral factor for the co- and post-translational
processing and modification of the dengue virus polyprotein [ ]. Thus, inhibitors of the
4]. Recently,
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This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
5
6
NS2BNS3 protease can be developed into DAAs for the treatment of DENV infections.
The U.S. Food and Drug Administration (FDA) has already approved several protease
Pathogens 2021, 10, 464. https://doi.org/10.3390/pathogens10040464
https://www.mdpi.com/journal/pathogens

Pathogens 2021, 10, 464
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inhibitors as antiviral drugs for the treatment of HIV and HCV infections; thus, further
supporting the notion of exploiting the DENV NS2BNS3 protease as a drug target [7].
Previous efforts to discover DENV NS2BNS3 protease inhibitors have resulted in the
identification of several peptides [
and cyclopeptides [12 13], as well as small organic molecules, such as synthetic com-
pounds [14 18] and natural products [19 21], as promising leads. However, none of them
8,9], peptidomimetics with electrophilic warheads [10,11]
,
–
–
has reached clinical application due to unfavourable pharmacokinetics and/or pharmaco-
dynamics. Therefore, research to discover new small organic molecules as potent inhibitors
of the DENV protease is still an ongoing endeavour.
1,2-Benzisothiazol-3(2H)-one (BIT) derivatives are an important class of heterocyclic
compounds with a broad range of applications in the field of medicinal chemistry [22
BIT derivatives (Figure 1) have shown a wide range of biological activities, such as anti-
cancer [26], antimicrobial [27], antifungal [28] and antiviral activities [14 29 30]. Recently,
–25].
,
,
combining two pharmacophores into a single molecule is gaining impetus due to the
resulting hybrid’s synergistic effects [16]. For instance, Tiew et al. tethered a BIT scaffold to
a triazole ring (Figure 1A) via an m-aminophenol linker to yield hybrids, which exhibited
inhibitory activities against DENV2 NS2BNS3 at lower micromolar concentrations [14].
Similarly, Lai et al. prepared a series of BIT–oxadiazole hybrids (Figure 1B) and reported
their activities as promising inhibitors of DENV and West Nile virus proteases [29]. Encour-
aged by these findings, we pursued the construction of hybrids of various pharmacophores
with a BIT skeleton and planned to accomplish de novo syntheses of the substituted BIT
derivatives. Several methods for the synthesis of BITs have already been reported in the lit-
erature [31
or require expensive/toxic reagents. Here, we describe an expeditious synthesis of BIT
from thiosalicylic acid ( ) in a one-pot, two-step reaction strategy. The BITs synthesised
–36]; however, these methods involve either multiple steps, are time-consuming
1
in this report were also screened for inhibitory activity against the DENV2 NS2BNS3
protease. The compounds exhibiting antiviral activities in the low-micromolar range were
further analysed using computational modelling to map the BIT–protease interactions at
the atomic level.
S
N
O
H
O
O
O
N
NH
O
N
N
N
N
N
O
S
N
O
O
A
B
O
O
O
S
F
N
S NH2
O
N
N
S
O
S
O
HO
C
D
E
Figure 1. Structures of selected biologically active 1,2-benzisothiazol-3(2H)-ones (BITs): triazole-BIT hybrid (
BIT hybrid ( ), sulphonamide-BIT hybrid ( ) (2S,3S)-3-methyl-2-(3-oxobenzo[d]isothiazol-2(3H)-yl)pentanoic acid (
5-fluoro-2-phenylbenzo[d]isothiazol-3(2H)-one (E).
A
), oxadiazole-
B
C
D
),

Pathogens 2021, 10, 464
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2. Methods
2.1. Chemistry
Commercial chemicals and reagents (Sigma-Aldrich, Taufkirchen, Germany) were
used without any further purification. Ethyl acetate, hexane and dichloromethane (Daejung
Korea) were used for chromatographic analyses after distillation. Purifications were per-
formed using Merck 9385 silica gel (Darmstadt, Germany), pore size 60 Å (70–230 mesh
ASTM). Melting points were measured with a Stuart melting point apparatus (Cole-Parmer,
Staffordshire, UK) and reported without correction. IR spectra were recorded with an ATR-
,
1
IR spectrometer (Bruker, Germany). H and 13C NMR were recorded using a AVANCE
III HD 600 MHz spectrometer (Bruker, Kloten, Switzerland). Chemical shifts were mea-
sured in parts per million using tetramethylsilane (TMS) (Bradbury, Stockport, UK) as
standard. Gas chromatography–mass spectrometry (GC–MS) analyses were performed
using a Thermo Scientific TRACE 1300 Gas Chromatograph (ThermoFischer Scientific,
Waltham, MA, USA).
2.2. General Procedure for the Synthesis of 1,2-Benzisothiazol-2(3H)-ones (5a–5q)
A mixture of thiosalisylic acid (100 mg, 0.65 mmol), thionyl chloride (471.5
µL,
◦
6.5 mmol, 10 equiv.) and a few drops of DMF was treated at 80 C for 12 h. After re-
moving the excess thionyl chloride, the crude mixture was treated directly with amines
(10 equiv.) dissolved in DCM. The resulting solution/suspension was stirred at room
temperature for about 20 h. The reaction mixture was diluted with water and extracted
with ethyl acetate. The organic layer was dried with magnesium sulfate and the solvent
was removed under vacuum to yield the product.
2-Propylbenzo[d]isothiazol-3(2H)-one (5a): Yield 93%; 1H NMR (600 MHz, CDCl3)
δ:
7.99 (d, J = 7.8 Hz, 1 H), 7.55 (t, J = 7.2 Hz, 1 H), 7.35 (t, J = 7.2 Hz, 1 H), 7.51 (d, J = 8.4 Hz,
1 H), 3.82 (t, J = 7.2 Hz, 2 H), 1.75 (m, 1 H), 0.95 (t, J = 7.2 Hz, 3 H); 13C NMR (150 MHz,
CDCl3) δ: 165.37 (C=O), 140.14 (C), 131.63 (CH), 126.65 (CH), 125.41 (CH), 124.82 (C), 120.30
(CH), 45.53 (CH2), 22.90 (CH2), 11.12 (CH3); EI–MS m/z (%): 193 [100, (M+)], 151 (100),
137 (16).
2-Butylbenzo[d]isothiazol-3(2H)-one (5b): Yield 89%; 1H NMR (600 MHz, CDCl3)
δ:
7.99 (d, J = 7.8 Hz, 1 H), 7.55 (t, J = 7.8 Hz, 1 H), 7.35 (t, J = 7.2 Hz, 1 H), 7.51 (d, J = 7.8 Hz,
1 H), 3.82 (t, J = 6.6 Hz, 2 H), 1.75 (m, 2 H), 1.34 (m, 2 H), 0.89 (t, J = 7.2 Hz, 3 H); 13C NMR
(150 MHz, CDCl3) δ: 165.32 (C=O), 140.14 (C), 131.61 (CH), 126.64 (CH), 125.41 (CH), 124.86
(C), 120.29 (CH), 43.67 (CH2), 31.58 (CH2), 19.82 (CH2), 13.65 (CH3); EI–MS m/z: 207 [37,
(M+)], 151 (100), 137 (49).
2-Hexylbenzo[d]isothiazol-3(2H)-one (5c): Yield 87%; 1H NMR (600 MHz, CDCl3)
δ:
8.00 (d, J = 7.8 Hz, 1 H), 7.57 (t, J = 7.2 Hz, 1 H), 7.36 (t, J = 6.6 Hz, 1 H), 7.51 (d, J = 7.8 Hz,
1 H), 3.85 (t, J = 7.2 Hz, 2 H), 1.72 (m, 2 H), 1.35 (m, 2 H), 1.25 (m, 4 H), 0.85 (t, J = 3.6 Hz,
3 H); 13C NMR (150 MHz, CDCl3)
δ: 165.30 (C=O), 140.14 (C), 131.61 (CH), 126.64 (CH),
125.40 (CH), 124.87 (C), 120.29 (CH), 43.98 (CH2), 31.37 (CH2), 29.51 (CH2), 26.26 (CH2),
22.48 (CH2), 14.0 (CH3); EI–MS m/z: 235 [100, (M+)], 151 (88), 137 (29).
2-Dodecylbenzo[d]isothiazol-3(2H)-one (5d): Yield 94%; 1H NMR (600 MHz, CDCl3)
δ
: 8.01 (d, J = 7.8 Hz, 1 H), 7.56 (m, 1 H), 7.36 (t, J = 7.2, 1 H), 7.51 (d, J = 8.4, 1 H), 3.86
(t, J = 7.2 Hz, 1 H), 1.73 (t, J = 7.2, 2 H), 1.21–1.34 (m, 18 H), 0.84 (t, J = 6.6, 3 H); 13C NMR
(150 MHz, CDCl3) : 165.34 (C=O), 140.15 (C), 131.64 (CH), 126.67 (CH), 125.43 (CH), 124.84
δ
(C), 120.28 (CH), 44.04 (CH2), 31.91 (CH2), 30.93 (CH2), 29.71 (CH2), 29.62 (CH2), 29.55
(CH2), 29.46 (CH2), 29.34 (CH2), 29.20 (CH2), 26.60 (CH2), 22.69 (CH2), 14.12 (CH3); EI–MS
m/z: 319 [30, (M+)], 151 (100), 136 (57).
2-(2-Hydroxyethyl)benzo[d]isothiazol-3(2H)-one (5e): Yield 86%, mp 110–112 ◦C; 1H
NMR (600 MHz, CDCl3)
(
δ
: 7.99 (d, J = 8.4 Hz, 1 H), 7.58 (m, 1 H), 7.37 (m, 1 H), 7.51
d, J = 8.4, 1 H), 3.93 (t, J = 5.4 Hz, 2 H), 4.02 (t, J = 4.8 Hz, 2 H), 2.67 (s, 1 H); 13C NMR
(150 MHz, CDCl3) δ: 163.38 (C=O), 139.71 (C), 130.95 (CH), 125.60 (CH), 124.57 (CH), 123.18
(C), 119.21 (CH), 60.91 (CH2), 46.44 (CH2); EI–MS m/z: 195 [88, (M+)], 163 (91), 151 (83), 137
(71), 136 (100).

Pathogens 2021, 10, 464
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◦
2-Benzylbenzo[d]isothiazol-3(2H)-one (5f): Yield 77% mp 87–89 C; 1H NMR (600 MHz,
CDCl3) : 8.05 (d, J = 7.8 Hz, 1 H), 7.56 (t, J = 7.8 Hz, 1 H), 7.38 (t, J = 7.2 Hz, 1 H), 7.46
(d, J = 7.8 Hz, 1 H), 5.04 (s, 2 H), 7.34–7.29 (m, 5 H); 13C NMR (150 MHz, CDCl3)
: 165.35
δ
δ
(C=O), 140.42 (C), 136.19 (C), 131.87 (CH), 128.86 (CH), 128.46 (CH), 128.31 (CH), 126.84
(CH), 125.53 (CH), 124.50 (C), 120.42 (CH), 47.59 (CH2); EI–MS m/z: 241 [100, (M+)], 136
(79), 91 (88).
2-(4-Butylphenyl)benzo[d]isothiazol-3(2H)-one (5g): Yield 92%, mp 168–172 ◦C; 1H
NMR (600MHz, CDCl3)
7.45 (d, J = 7.2 Hz, 1 H), 7.40 (d, J = 7.2 Hz, 2 H), 7.13 (d, J = 8.4 Hz, 2 H), 2.57 (t, J = 7.8 Hz
2 H), 1.57 (m, 2 H), 1.33 (m, 2 H), 0.91 (t, J = 7.2 Hz, 3 H); 13C NMR (150 MHz, CDCl3)
: 163.23 (C=O), 141.28 (C), 133.53 (C), 131.22 (C), 128.29 (CH), 127.86 (CH), 126.12 (CH),
δ: 7.76 (d, J = 8.4 Hz, 1 H), 7.31 (t, J = 7.2 Hz, 1 H), 7.55 (m, 1 H),
,
δ
124.75 (CH), 123.76 (CH), 119.26 (C), 119.03 (CH), 32.46 (CH2), 28.68 (CH2), 21.29 (CH2),
12.92 (CH3); EI–MS (EI) m/z: 283 [44, (M+)], 282 (77), 240 (100).
2-(2-Nitrophenyl)benzo[d]isothiazol-3(2H)-one (5h): Yield 78%, mp 199–200 ◦C; 1H
NMR (600 MHz, CDCl3)
δ
: 8.07 (d, J = 4.8 Hz, 2 H), 7.71 (m, 2 H), 7.63 (m, 1 H), 7.55 (m, 1 H),
: 164.65 (C=O), 146.84
7.53 (m, 1 H), 7.44 (t, J = 7.2, 1 H); 13C NMR (150 MHz, CDCl3)
δ
(C), 141.02 (C), 133.86 (CH), 132.91 (CH), 130.03 (CH), 129.71 (C), 129.33 (CH), 127.61 (CH),
126.13 (CH), 125.84 (CH), 122.97 (C), 120.35 (CH); EI–MS m/z: 272 [35, (M+)], 152 (100).
2-(4-Nitrophenyl)benzo[d]isothiazol-3(2H)-one (5i): Yield 82%, mp 126–129 ◦C; 1H
NMR (600 MHz, CDCl3)
(
δ
: 8.09 (d, J = 7.8 Hz, 1 H), 7.70 (m, 1 H), 7.46 (m, 1 H), 7.59
: 164.32
d, J = 7.8 Hz, 1 H), 8.02 (m, 2 H), 8.31 (m, 2 H); 13C NMR (150 MHz, CDCl3)
δ
(C=O), 133.36 (CH), 127.58 (CH), 126.41 (CH), 124.97 (CH), 124.51 (C), 122.90 (CH), 120.19
(CH); EI–MS m/z: 272 [100, (M+)], 271 (36), 242 (31).
2-(2-Methyl-4-nitrophenyl)benzo[d]isothiazol-3(2H)-one (5j): Yield 81%, mp 135–138 ◦C
;
1H NMR (600 MHz, CDCl3)
H), 7.48 (t, J = 7.2, 1 H), 7.46 (d, J = 8.4, 1 H), 8.10 (d, J = 8.4, 1 H), 8.23 (s, 1 H), 2.35 (s, 3 H);
13C NMR (150 MHz, CDCl3)
: 164.56 (C=O), 146.71 (C), 145.99 (C), 141.12 (C), 135.80 (C),
δ: 7.05 (d, J = 7.8 Hz, 1 H), 7.70 (t, J = 7.2, 1 H), 7.56 (d, J = 7.8, 1
δ
132.81 (CH), 132.08 (CH), 127.44 (CH), 126.20 (CH), 124.72 (CH), 124.17 (CH), 123.15 (CH),
120.46 (C), 18.62 (CH3); EI–MS m/z: 286 [78, (M+)], 270 (29), 269 (100).
◦
2-(4-Bromophenyl)benzo[d]isothiazol-3(2H) (5k): Yield 84%, mp 138–140 C; 1H NMR
(600 MHz, CDCl3) δ: 8.07 (d, J = 7.8 Hz, 1 H), 7.65 (t, J = 7.8, 1 H), 7.43 (t, J = 7.8, 1 H), 7.58
(m, 5 H); 13C NMR (150 MHz, CDCl3)
δ: 163.07 (C=O), 138.58 (C), 135.37 (C), 131.59 (CH),
131.42 (CH), 126.26 (CH), 124.98 (CH), 124.82 (CH), 123.61 (C), 119.31 (C), 119.11 (CH);
EI–MS m/z (%): 305 [62, (M+)], 307 (100), 306 (46), 304 (72), 226 (82).
2-(2-Hydroxyphenyl)benzo[d]isothiazol-3(2H)-one (5l): Yield 77%, mp 198–200 ◦C;
1H NMR (600 MHz, CDCl3)
δ: 8.11 (d, J = 7.8 Hz, 1 H), 7.48 (m, 1 H), 7.69 (m, 1 H), 7.61
(
d, J = 7.2, 1 H), 7.36 (dd, J = 7.9, 1 H), 7.24 (m, 1 H), 6.95 (m, 1 H), 7.01 (dd, J = 8.2, 1 H);
13C NMR (150 MHz, CDCl3)
δ
: 165.03 (C=O), 151.69 (C), 141.77 (C), 132.70 (CH), 129.56
(CH), 127.20 (CH), 126.23 (CH), 125.37 (CH), 123.89 (CH), 121.39 (C), 121.33 (CH), 120.09
(CH); EI–MS m/z: 243 [95, (M+)], 136 (100).
◦
2-(2-Chlorophenyl)benzo[d]isothiazol-3(2H)-one (5m): Yield 68%, mp 117–119 C;
1H NMR (600 MHz, CDCl3)
(
δ
: 8.10 (d, J = 7.8 Hz, 1 H), 7.66 (m, 1 H), 7.38 (m, 1 H), 7.57
d, J = 7.8 Hz, 1 H), 7.53 (dd, J = 5.4Hz, 1 H), 7.39 (m, 2 H), 7.42 (dd, J = 6.0Hz, 1 H); 13
C
NMR (150 MHz, CDCl3) δ: 163.80 (C=O), 140.36 (C), 133.08 (C), 132.66 (C), 131.45 (CH),
130.07 (CH), 129.72 (CH), 129.68 (CH), 126.80 (CH), 126.38 (CH), 124.73 (CH), 122.32 (C),
119.27 (CH); EI–MS m/z: 261 [69, (M+)], 226 (100).
2-(2,6-Dichlorophenyl)benzo[d]isothiazol-3(2H)-one (5n): Yield 92%, mp 188–190 ◦C;
1H NMR (600 MHz, CDCl3)
δ
: 8.12 (d, J = 7.8 Hz, 1 H), 7.68 (m, 1-H), 7.35 (t, J = 7.8 Hz, 1H),
7.59 (d, J = 7.8 Hz, 1 H), 7.45 (m, 3 H); 13C NMR (150 MHz, CDCl3)
δ
: 163.53 (C=O), 140.87
(C), 135.75 (CH), 131.65 (CH), 130.55 (C), 130.21 (CH), 127.92 (CH), 126.58 (CH), 124.67
(CH), 122.0 (C), 119.53 (CH); EI–MS m/z: 295 [53, (M+)], 260 (100).
2-(4-Methoxyphenyl)benzo[d]isothiazol-3(2H)-one (5o): Yield 89%, mp 146–148 ◦C;
1H NMR (600 MHz, CDCl3)
(
δ
: 8.07 (d, J = 7.8 Hz, 1 H), 7.63 (m, 1 H), 7.53 (m, 3 H), 7.41
t, J = 7.2 Hz, 1 H), 6.96 (d, J = 9.0 Hz, 2 H), 3.82 (s, 3 H); 13C NMR (150 MHz, CDCl3)
δ:

Pathogens 2021, 10, 464
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164.31 (C=O), 158.74 (C), 140.06 (C), 132.16 (CH), 129.69 (C), 127.14 (CH), 126.84 (CH),
125.74 (CH), 124.66 (C), 120.08 (CH), 114.61 (CH), 55.57 (CH3); EI–MS m/z: 257 [83, (M+)],
257 (83), 242 (100).
2.3. Computational Assessment of the Absorption, Distribution, Metabolism and Elimination
(ADME) Properties of BITs
Please see the supporting information for the computational measurements of the
ADME parameters.
2.4. Biological Evaluation
Inhibition of the DENV2 NS2BNS3 Protease by BITs
The DENV NS2BNS3 activity inhibition assays were conducted in a 96-well plate
containing 100
50 mM Tris, pH 9.0, 10 mM NaCl, 25% glycerol, 50
1 mM CHAPS in the presence of BITs (200 g/mL). The reaction, in triplicates, was initiated
µL total reaction mixture. The reaction was conducted in a buffer containing
µM Bz-Nle-Lys-Arg-Arg-AMC and
µ
by the addition of 100 nM DENV2 NS2B-NS3, and the release of fluorogenic AMC was
measured at 60 s intervals using a spectrofluorometer for a maximum reaction time of
30 min. A solvent (DMSO) control was run for each assay plate by measuring the activity
of DENV NS2BNS3 in the absence of BITs (DMSO control). The effect of background
fluorescence was measured by preparing a calibration curve, which in turn was obtained
by spiking known concentrations of free AMC in the same volume of the reaction mixtures
containing the buffer, enzyme and compounds, and comparing the slope of this curve with
that obtained using free AMC concentrations in reaction mixtures containing the buffer,
enzyme, no compound and no substrate. The percentage of inhibition was calculated using
the measurement of the initial rates of reaction in the presence (vexp), or absence (vpro) of
BIT according to the following formula:
ꢀ
ꢁ
vexp
vpro
Inhibition (%) = 100 −
× 100 .
The significance of the observed inhibitions was determined using Student’s t-test by
comparing the negative control with the values obtained with a single compound.
For the dose–response experiments, the assays were conducted as described above in
the presence of threefold serial dilutions of BITs, ranging from 0.076–500 µM concentrations
in DMSO. The final concentration of DMSO in each reaction mixture was less than 5%. IC50
values were obtained by plotting the residual activity of the DENV protease vs. log of the
concentration of BITs by using GraphPad Prism v.7 software (San Diego, CA USA). For
the positive control, aprotinin was used in a 5
conducted in triplicates.
µM concentration. All experiments were
2.5. Cytotoxicity Measurements Using an MTS Assay
Vero cells were seeded in a 96-well plate (2000 cells/well in 100 µL DMEM) and placed
in an incubator maintained at 37 ◦C in a 5% CO2 environment for overnight incubation.
◦
The cells were treated with 30
µM (2
µ
L) of BITs for 2 days at 37 C in the incubator. Control
wells received an equivalent amount of DMSO without the test compound. After two
days, each well was directly treated with 50 L of the CellTiter 96 (Promega, Mannheim,
µ
Germany) and incubated for 1–2 h. The absorbance was recorded at 490 nm with a 96-well
plate reader (Berthold, Bad Wildbad, Germany).
2.6. RNA Isolation and Quantification of DENV
Based on the manufacturer’s manual, viral RNA was isolated from 200
µL cell culture
supernatants using the Roche HP Viral Nucleic Acid Kit (Roche, Mannheim, Germany).
Relative viral genome copy numbers were determined using 5 µL of the eluted RNA
and RT-qPCR using the LightMix® Modular Dengue kit (TIB MOLBIOL, Germany) in
combination with the LightCycler RNA Process Control Kit (Roche, Mannheim, Germany).

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All reactions were performed in triplicates on Roche LightCycler96 or Roche LightCycler480
II qPCR machines (Roche, Mannheim, Germany). The relative RNA concentration was
determined using an RNA standard curve and the respective cycler software. The quality
of the RT-qPCRs was ensured by following the Minimum Information for Publication
of Quantitative Digital PCR Experiments guidelines, i.e., the recursion coefficient of the
relative standard curve (r2) was >0.95, and the triplicate assays with a standard deviation of
>0.5 were excluded from the analyses. The resulting genome copy numbers were expressed
as genome copies per millilitre of the cell culture supernatants. The statistical significance
of the reduction in viral titres was calculated using Student’s t-test by comparing the viral
load of a single compound with the DMSO control.
2.7. Immunofluorescence Staining of Vero Cells Infected with DENV
The inhibition of viral replication was analysed using immunofluorescence staining.
Vero cells were preincubated with selected BITs at 30 µM. The cells were subsequently
infected with DENV-2 (multiplicity of infection (MOI) of 0.5). Cellular supernatants were
collected after 2 days, centrifuged at 2000 rpm for 5 min to remove the detached cells,
and titrated on Vero cells. These cells were fixed with 4% paraformaldehyde 3 days
after infection. We performed immunofluorescence staining to visualise the infected cells
with monoclonal anti-DENV-2 E antibodies. Bound antibodies were detected with a Cy3-
conjugated donkey anti-mouse antibody (Dianova, Hamburg, Germany) (1:500). Stained
infected cells were counted using a fluorescence microscope, and the viral titres were
calculated. The experiment was performed in triplicates. The statistical significance of the
reduction of infected cells was calculated using Student’s t-test by comparing numbers of
infected cells incubated with a compound with the DMSO control.
2.8. Molecular Docking
Molecular docking studies were performed using AutoDock Vina designed and imple-
mented by Dr.Oleg Trott in the Molecular Graphics Lab at The Scripps Research Institute,
San Diego, CA, USA [37]. 2D structures of BITs were drawn in ChemDraw, version 12.0,
PerkinElmer Massachusetts, USA and converted into 3D structures using Avogadro, ver-
sion 1.2.0, Pittsburgh, PA, USA. The optimisation of the geometry and minimisation of
the energy was accomplished using the MMFF94 force field in Avogadro and then saved
in the pdb format. Coordinates of the reported co-crystal structure of DENV3 NS2NS3
(PDB ID: 3U1I) were downloaded from the protein databank (rcsb.org) on 22 June 2019 and
then processed with Autodock Tool, where polar hydrogen atoms and Kollman charges
were added. The pdbqt files of both DENV3 BS2BNS3 and BITs were prepared using
Autodock Tools and submitted for molecular docking by using a volume grid defined
by 54 × 58 × 48 Å along the x, y and z sides, respectively, with the central point defined
as center_x = 23.463, center_y =
most negative docking scores (binding energy in kcal/mol) were selected to investigate the
binding interactions using PyMol and Ligplot.
−
8.442 and center_z = 17.732. The top models with the
3. Results and Discussion
3.1. Synthesis of BITs
The synthetic method of Grivas, using thiosalicylic acid (
was the method of choice for the preparation of the BIT derivatives (Scheme 1) [35]. In
this multistep approach, was first converted to the disulfide ( ) with I2 in MeOH, and
then both the carboxylate groups in were chlorinated with thionyl chloride. Treatment of
the generated bisthiosalicylyl chloride ( ) with elemental halogen (Cl2 or Br2) in CCl4 or
sulfuryl chloride resulted in the cleavage of the –S–S– bond to yield 2-chlorosulfenylbenzoyl
chloride ( ). Finally, a double nucleophilic displacement reaction with primary amines
afforded the synthesis of the target 1,2-benzisothiazol-3(2H)-one (5) in four steps total.
1) as the starting material,
1
2
2
3
4

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Scheme 1. Multistep approach towards the synthesis of BITs.
For the synthesis of a library of 2-mercaptobenzamides, we needed 2-mercaptobenzoyl
chloride ( , X = H) as precursors, which could have been prepared from the reaction of
with thionyl chloride [38 39]. Surprisingly, the reaction of n-propylamine with the crude
reaction mixture, prepared from and thionyl chloride, resulted in the formation of the
BIT 5a as a major (93%) product, rather than the formation of
(Scheme 2). A 1H NMR
6
1
,
1
7
investigation showed the absence of both NH and S–H chemical shifts and the EI-MS
analysis indicated a molecular ion peak at m/z = 193. Additionally, the spectroscopic and
physical data (bp = 126–128 ◦C) matched those previously reported for BIT 5a. A quick
literature search on the predation of
6
(X = H) from
1
using thionyl chloride retrieved
only two references [38 39]. In both references, crude
,
6 was used in the next step without
a detailed structural characterisation. Further chemical investigation on this reaction is
underway and will be reported elsewhere. Nevertheless, we speculate that the reagent
thionyl chloride (SOCl2) may contain traces of Cl2 or sulfuryl chloride (SO2Cl2), causing
the chlorination of
2-chlorosulfenylbenzoyl chloride (
may have taken place via an entirely different mechanism. Altogether, the reaction of
1
at both the carboxylate and the sulfhydryl (–SH) site to produce
4), as shown in the Scheme 2. Alternatively, the reaction
1
with thionyl chloride produced an intermediate, which reacted with primary amines or
anilines, leading to the formation of BITs in excellent yields.
Scheme 2. Two-step, one-pot synthesis of BIT 5a depicting the proposed formation of 2-chlorosulfenyl benzoylchloride (
as an intermediate.
4)
Following this methodology, we reacted various primary amines and anilines with the
crude chlorination mixture and prepared a library of 15 derivatives of 1,2-benzisothiazol-
3(2H)-ones, as described in Scheme 3.

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Scheme 3. Synthesis of the N-substituted BITs with their associated yields.
The structures of all BITs synthesised in this report were elucidated using various
spectroscopic techniques. Additionally, the spectral data of the previously reported BITs
1
matched with the ones synthesised via this short route. The H NMR spectra of com-
pounds 5a–o displayed the characteristic chemical shifts, ranging from δ 7.10 to 8.30 ppm
for the aromatic ring of 1,2-benzisothiazol-3(2H)-one, as well as the chemical shifts of the
N-substituted R groups (aliphatic and aromatic) in their respective parts per million range.
1,2-Benzisothiazol-3(2H)-ones with aliphatic substitutions had their aliphatic region peaks
in the range of
peaks overlapping the benzene ring peaks of 1,2-benzisothiazol-3(2H)-one moiety. The
13C spectra of compounds 5a
displayed the characteristic chemical shifts for carbonyl
groups, ranging from 163 to 165 ppm. The compounds with aliphatic substitutions dis-
played chemical shifts ranging from 11 to 47 ppm, and those with aromatic substitutions
displayed chemical shifts ranging from δ 119 to 151 ppm.
δ 0.8–3.9 ppm, while those with the aromatic substitutions had some of the
–
o
δ
δ

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3.2. Pharmacokinetics Studies: Predictions of the ADME
The potential of a molecule to exhibit pharmacological or therapeutic properties
depends on various physicochemical characteristics of the molecule. Nowadays, computa-
tional ADME modelling is used to estimate the lead/drug likeliness of desired molecules
and predict whether the molecule should initially be placed in the approved drug molecule
section. Lipinski’s rule is considered a criterion for the synthesised molecules’ expec-
tation to be an orally active drug. It consists of 5 tenets: the molecular weight of the
compound being <500; the number of hydrogen bond acceptors and donors being <10
and <5, respectively; a lipophilicity threshold <4.15; the partition coefficient ClogP < 5.
Violating any of these criteria would render the drug inappropriate for oral intake due to
bioavailability problems.
In the current study, we evaluated several parameters to predict the drug-likeness
of the synthesised BITs. All the BITs were estimated for the following in silico physico-
chemical studies: number of rotatable bonds (nROTB), hydrogen bond acceptor (HBA),
hydrogen bond donor (HBD), lipophilicity (iLogP) and topological polar surface area
(TPSA). The in silico percentage absorption was calculated using the reported formula
[(%ABS = 109 – (0.345 × TPSA)]. The percentage absorption of the BITs was found to be
in the range of 76–92%. Compounds 5a–d, 5f, 5g, 5k, 5m and 5n showed the highest
absorption, while compounds 5h–j showed the lowest in silico absorption.
All the synthesised BITs were observed to follow the Lipinski rule of five. The molec-
ular weight ranged from 193 to 319 (<500), the HBA ranged from 1 to 3 (
ranged from 0 to 1 ( 5), iLogP (lipophilicity) was found to be in the range of 1.92 to 4.51
5) and MLogP was found to be in the range of 1.08 to 4.59 ( 4.5), which suggests that
≤
10), the HBD
≤
(
≤
≤
these compounds possess good drug likeliness properties upon administration (Table 1).
All the compounds exhibited high gastrointestinal absorption and brain permeability,
except 5d, 5e and 5h–j. Furthermore, all the compounds, except compound 5d, showed
nROTB in the range 1–5 (<10), suggesting good bioavailability. The TPSA of the synthesised
BITs ranged between 50.24 and 96.06 Å2 (<140 Å2), indicating good intestinal absorption.
From these parameters, it was concluded that the synthesised BITs would exhibit good
drug-like properties (Table 1).
Table 1. Pharmacokinetics/ADME (absorption, distribution, metabolism and elimination) predictions of the target BITs.
Lipinski Parameters
MW a
HBD c
Violations nROTB e
%ABS g
HBA b
iLogP d
TPSA f
BBB h
GI ABS i
Compounds
5a
5b
5c
5d
5e
5f
5g
5h
5i
193
207
235
319
195
241
283
272
272
286
306
243
262
296
257
1
1
1
1
2
1
1
3
3
3
1
2
1
1
2
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
2.49
2.72
3.19
4.51
1.92
2.64
3.52
2.21
2.31
2.45
2.96
2.44
2.76
2.84
2.88
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
2
3
5
11
2
2
4
2
2
2
1
1
1
1
2
50.24
50.24
50.24
50.24
70.47
50.24
50.24
96.06
96.06
96.06
50.24
70.47
50.24
50.24
59.47
e
91.67
91.67
91.67
91.67
84.69
91.67
91.67
75.86
75.86
75.86
91.67
84.69
91.67
91.67
88.49
Yes
Yes
Yes
No
No
Yes
Yes
No
No
No
Yes
Yes
Yes
Yes
Yes
f
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
5j
5k
5l
5m
5n
5o
a
b
c
d
Molecular weight. Hydrogen bond acceptor. Hydrogen bond donor. Partition coefficient. Number of rotatable bonds. Topological
g
h
i
polar surface area. Absorption percentage. Blood–brain barrier. Gastro-intestinal absorption.
The standard parameter that is used to describe lipophilicity is the partition coefficient
between n-octanol and water (logPo/w) and is considered to be a useful web-based tool for
evaluating pharmacokinetics. Multiple computational techniques for logPo/w estimation

Pathogens 2021, 10, 464
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have been developed with varying results on different chemical sets. SwissADME pro-
vides access to five free predictive models for lipophilicity, i.e., XLOGP3 (atomic method),
WLOGP (atomistic method based upon Wildman and Crippen’s fragmentation method),
MLOGP (archetypal topology tool based on a linear relation with 13 molecular descrip-
tors), SILICOS-IT (a hybrid system with 27 fragments and seven topological descriptors)
and iLOGP (a physics-based method that uses free solvation energies of n-octanol and
water, calculated using the Generalized Born and solvent accessible surface area (GB/SA)
model). The logPo/w values estimated for each BIT followed similar trends. The values
were significantly low in the case of compounds 5e, 5h and 5i. This may be accounted
for by the presence of the hydroxyl group and nitro groups in these compounds, which
increased the polarity of the compounds. The compound 5d showed exceptionally high
values due to a long aliphatic chain in the molecule. All the remaining compounds showed
lipophilicities in a similar range, indicating that the replacement of 4-carbon aliphatic
chains with substituted phenyl groups did not significantly alter the lipophilicity. In this
study, the logPo/w values for the designed compounds were found in the range of +0.77
to +7.04 (Figure 2). These positive values showed that the BITs were highly lipophilic and
adhered to the essential criteria for drug-like molecules.
Figure 2. Predicted lipophilicity (logP values) of the compounds that were obtained using different calculation methods.
3.3. Screening of BITs for Anti-DENV NS2BNS3 Protease Inhibition
The synthesised BITs were tested for their inhibitory activity against the DENV2
NS2B/NS3 protease assay using the previously reported 96-well plate method [16
Briefly, the reaction mixtures (200 L) containing 50 M test compound were incubated
with 100 nM protease and 5 M tetrapeptide substrate Bz-Ile-Lys-Arg-Arg-AMC. A reduc-
–20].
µ
µ
µ
tion in the cleavage of the 4-amino-7-methylcoumarine (AMC) measured in terms of the
reduction of fluorescence was deemed to show inhibition of the protease and is reported as
a percentage inhibition compared with the solvents’ control reaction. An experiment was
conducted to measure the background fluorescence from the organic compounds and the
ability of the BITs to quench the AMC fluorescence and lay off the false-positive inhibition
(data not shown). No significant background fluorescence and quenching behaviour were
observed by any of the tested compounds under the conditions used in these experiments.
Furthermore, the enzymatic reactions of the potent inhibitors were repeated by replacing
the zwitterionic detergent (CHAPS) with non-ionic (Brij-35) detergent to remove the false-
positive results arising due to the possible aggregation of compounds in the experiments.
Aprotinine was used as the positive control, as described previously. [18]

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Among all the tested BITs, the ones containing an N-substituted aliphatic residue (5a–e
showed an overall weaker inhibition (<50%) at 50 M as compared to the ones containing
aromatic substitutions (5f ) at the N-atom, as shown in Figure 3A. The compounds 5j 5m
)
µ
–
o
,
and 5n showed the highest percentage (>90%) of inhibition at the tested dose. Although
the presence of an electronegative Cl atom (5m and 5n) or O atom (5l) at the ortho-position
of the N-substituted benzene ring favoured the inhibition of the DENV-2 protease (
and above), the presence of a NO2 group at the same position in 5h or the para position
in 5i showed relatively weaker inhibition ( 70%). Interestingly, the presence of a methyl
group at the ortho position, albeit with a NO2 group athe para position in 5i, enhanced the
inhibition of DENV2 NS2BNS3 up to 31%, indicating that the weakly electron-donating
≈
90%
≈
≈
substituents at the ortho position of the N-substituted aromatic ring were preferable for
enhanced inhibition by BITs.
Figure 3. Inhibition and dose–response of DENV NS2BNS3 protease against the BITs. (
centration of 50 M BITs was incubated in the presence of 100 nM recombinant DENV2 NS2BNS3
protease and 5 M tetrapeptide-AMC substrate in a Tris buffer at pH 9.5. The time-dependent release
of fluorescent AMC tag was measured for 30 min and compared with the solvent control ( ). The
results are displayed as the percentage of inhibition. Aprotinine (5 M) was used as a positive control.
Each bar represents a mean value of three replicates. The observed inhibition with all compounds
was significant with p < 0.05 for 5a and p < 0.01 for 5e ) Three-fold serial dilutions of the
selected BITs were screened between 150 M and 200 nM concentration in the DENV2 NS2BNS3
protease assay in triplicate. The dashed horizontal line indicates the 50% activity level.
A) A con-
µ
µ
−
µ
–
d
–o (B
µ
The BITs exhibiting more than 65% inhibition at the 50
investigated for the dose-dependent inhibition of the DENV2 protease. A threefold serial
dilution of the selected BITs, ranging from 150 M to about 200 nM, were screened in tripli-
µM concentration were further
µ
cate experiments, as described above. The kinetic data were obtained and the protease’s
residual activities in the presence of the tested BITs were inserted into the dose–response
model rooted in the GraphPad Prism software (version 7.0). The half-maximal inhibitory
(IC50) dose of the selected compounds was observed within a concentration range of ap-
proximately 2–53
positions or a methoxy group at the para position (5o) of the benzene ring demonstrated the
highest IC50 values of 41.80 1.17 M, 52.70 3.23 M and 26.50 4.32 M, respectively.
) showed lower IC50 values ranging between 2 and
µM. Two BITs containing the NO2 group at the ortho (5h) and para (5i)
±
µ
±
µ
±
µ
On the other hand, five BITs (5j
–n
8.24 µM (Table 2).

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Table 2. Percent inhibition and IC50 values of the selected BITs.
Inhibition a
b
IC50
Compound
#
(%)
(µM)
1
2
3
4
5
6
7
8
5h
5i
5j
5k
5l
5m
5n
5o
72
67
92
80
89
94
94
58
41.80 ± 1.17
52.70 ± 3.23
2.56 ± 1.03
8.24 ± 1.16
6.66 ± 2.17
2.01± 0.98
5.28 ± 1.89
26.50 ± 4.32
a
Inhibition of the protease at a single dose (50
µ
M) of BIT. b IC50 values are the concentrations at which the DENV
protease is inhibited by 50%. Bold values: the most potent inhibitors of all BITs.
3.4. Molecular Docking of Compounds Clustered in the Substrate-Binding Site
The BITs exhibiting promising inhibitory activities in the enzymatic assay were further
assessed computationally for an insight into the ligand–receptor binding interactions. For
this purpose, the protein sequence of the recombinant DENV-2 protease was submitted
for the homology model, which was constructed using the reported structure of DENV-
2 protease (PDB ID: 2M9P) as the folding template. Hence, the homology model was
folded in the active/closed conformation of the protease and was used for computational
modelling using AutoDock Vina.
The structures of 5j, 5m and 5n were drawn in ChemDraw and then imported into
Avogadro for conversion into 3D models. The energies were minimised (and the geometries
were optimised) by using the MMFF94 force field. The pdbqt files of the protein and ligands
were prepared by AutoDock Tools Software and finally submitted for molecular docking
using a grid incorporating the whole protein structure.
The docked structures were clustered in the substrate-binding site with the binding
energies of
−
7.6,
−
6.2 and
−
6.4 kcal/mol for 5j, 5m and 5n, respectively, as shown in
Figure 4A. The amide oxygen of compound 5j was docked in the vicinity of the catalytic
triad of the NS3 domain and was found to exhibit H-bonding with His-112 and Ser-196
at distances of 3.3 Å and 3.0 Å, respectively. The aromatic rings of 5j were found to be
surrounded by several nonpolar (e.g., Val-113, Ile-97, Val-215, Val-216 and Pro-193) and
aromatic (Tyr-211 and Tyr-222) side chains of the substrate-binding site and contributed
towards the favourable hydrophobic and π−π interactions, as shown in Figure 4B. Anal-
ogous to the BIT 5j, the compounds 5m and 5n were also docked in the same place with
docking scores of
−
6.2 kcal/mol and
−
6.4 kcal/mol, respectively. Each of them was also
involved in H-bonding with the His-112 and Ser-196 of the catalytic triad at distances of
3.5 Å and 2.9 Å, respectively.
Since 5j, 5m and 5n were found to dock at the same location, their benzisothiazolinone
ring and the aromatic ring of the N-substituents were assumed to be surrounded by
identical residues. The difference in the values of the predicted docking scores and the
measured IC50 values could be assigned to the nature and orientation of substituents on
the aromatic rings. For instance, 5j containing a polar nitro group demonstrated additional
interactions with Ser-224 and Tyr-211 residues, thus lowering its binding energy compared
to 5m and 5n, whereas these interactions were missing in the latter molecules. Nevertheless,
the order of the IC50 values of these compounds was found to be 2.01
µ
M for 5m < 2.56
M for 5n, which is in contradiction with the predicted order of the binding
7.6 kcal/mol for 5j 6.4 kcal/mol M for 5n 6.2 kcal/mol for 5m.
µM
for 5j < 5.28
µ
energies as
−
<
−
µ
<
−
This may be accounted for by the presence of a suitably oriented and negatively charged
nitro group in compound 5j, which can enhance favourable binding interactions once at
the docking site, but also has to pay a desolvation penalty due to the shedding of the
surrounded shell of water molecules in the aqueous environment of the bioassay. This fact
was further substantiated in the in vitro studies, where 5j did not show any activity against
DENV2, potentially due to its low uptake by the Vero cells.

Pathogens 2021, 10, 464
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Figure 4. Molecular docking and the predicted mode of binding of BITs with the DENV2 NS2BNS3 protease. The catalytic
domain NS3 of the protease is shown in salmon red color, the cofactor NS2B as a pale-green color and the substrate-binding
site as surface ( ) containing the superposed docked poses of 5j 5m and 5n. The location of the catalytic triad—His112,
Asp-136 and Ser196—is depicted in red. ( ) Individual docked poses of 5j (shown in the green tube model), 5m (magenta)
A
,
B–D
and 5n (off-white) exhibiting the H-bonding interactions with His-112, Ser-196, Ser-224 and Tyr-211 (shown in yellow). The
residues showing the hydrophobic interaction are also marked.
3.5. In Vitro Testing of the Inhibitory Activity of BITs against the DENV Replication Assay
The BITs were tested for cytotoxicity in the Vero cells before their evaluation in cell
culture assays (Figure S1). All the BITs showed no cytotoxicity at 30 µM. The BITs showing
weaker DENV2 NS2BNS3 protease activity in the biochemical assay were excluded in
the in vitro evaluations. To analyse the inhibition of viral replication, Vero cells were pre-
incubated for 1 h with the test compounds at a concentration of 30 µM and subsequently
infected with DENV2. All infections were performed in triplicate. After two days, the
cellular supernatants were collected and the viral RNAs were isolated and quantified using
real-time quantitative polymerase chain reaction (RT-qPCR). Among the tested compounds,
only 5m (p = 1.1
significant level (Figure 5).
×
10−5) and 5n (p = 0.01) were able to suppress DENV replication at a

Pathogens 2021, 10, 464
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(A)
(B)
A) Vero cells were infected with
Figure 5. 5m inhibited DENV replication and viral infectivity. (
DENV2 and incubated in the presence of the compounds. After incubation, viral RNA was isolated
and quantified using RT-qPCR. ( ) Viral infectivity of the compounds 5m and 5n was determined
B
using immunofluorescence (IF) staining.
To further analyse whether the compounds 5m and 5n suppressed the viral gene
infectivity, Vero cells were pretreated with the compounds and subsequently infected with
DENV2 for two days. Cellular supernatants were uses to infect the Vero cells. After two
additional days, the infected cells were visualised using immunofluorescence with anti-
DENV antibodies. The stained cells were counted (Figure 5). In this assay, 5m significantly
(p = 0.003) reduced the viral infectivity by 1.4 orders of magnitude, while the reduction by
5n was about sixfold (p = 0.005).
Furthermore, to analyse the potential of BITs 5k–n against other flaviviruses, similar
experiments were performed with the Zika virus, and none of the tested BITs yielded
positive results regarding the Zika virus replication. This observation may lie in the fact
that the organisational behaviour is different for both viruses inside of host cells [18,40].
Both DENV and ZIKV viruses have been shown to form dissimilar replication assemblies
containing RNA and NS3 such that the ZIKV replication assemblies are additionally
surrounded by intermediate filaments in cage-like moieties [20]. This additional support
could lead to powerful shielding of the ZIKV protease from inhibitors compared to DENV
protease. Therefore, it seemed very likely that the cell-based and in vitro assays’ differences
could be traced back to the cellular metabolism and that compounds 5m and 5n may serve
as lead compounds in flaviviral protease drug discovery.
4. Conclusions
In summary, a library of BITs was synthesised using a one-pot, two-step method in a
very fast way and tested for their potential against the DENV2 NS2BNS3 protease. The
compounds exhibiting high inhibition at 50
µM were investigated for their IC50 values,
which were found in the range of 2 to 8.25 M. The BITs demonstrating a lower IC50 were
µ
further screened by the in vitro viral (DENV and ZIKV) assays, and compound 5m was
found to reduce the viral infectivity by a log value of 1. Computational assessments were
also performed, which indicated that these compounds bound at the catalytic site of the
NS2BNS3 protease. These preliminary results indicate the potential of BITs for their further
development into anti-dengue therapeutics.

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Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/pathogens10040464/s1. Figure S1: MTS viability assays with the compounds. Vero cells were
incubated with the compounds at a concentration of 30 µM for 72h. The MTS substrate was added
and the cell were further incubated for 1 h. The absorbance was measured at 490 nm.
Author Contributions: Formal analysis, F.B.; Funding acquisition, M.S. and J.B.; Investigation, F.B.,
H.N.S., L.K. and J.B.; Methodology, H.N.S. and J.B.; Supervision, M.S. and J.B.; Validation, J.B.;
Writing—original draft, F.B., M.S. and J.B. All authors have read and agreed to the published version
of the manuscript.
Funding: We acknowledge the financial support from the Higher Education Commission (HEC),
Pakistan, under the National Research Programme for Universities (NRPU#5912) and from Lahore
University of Management Sciences (LUMS) under the Faculty Initiative fund mechanism to M.S.
We acknowledge the Deutscher Akademischer Austauschdienst (DAAD) for providing a short-term
scholarship to F.B. to accomplish in vitro viral assays in the laboratory of J.B. in Germany.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The assistance and support of Heike Oberwinkler and Melissa Zimniak in
designing and executing the in-vitro studies are greatly acknowledged.
Conflicts of Interest: No potential conflict of interest was reported by the authors.
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