Synthesis and evaluation of novel S-benzyl- and S-alkylphthalimide- oxadiazole -benzenesulfonamide hybrids as inhibitors of dengue virus protease

Article abstract of DOI:10.1016/j.bioorg.2020.103567

Direct acting antiviral drugs (DAADs) are becoming therapeutics of choice for the treatment of viral infections. Successful development of anti HIV and HCV drugs by targeting the viral proteases has provided impetus for discovering newer DAADs. Dengue virus (DENV) protease, which is composed of two nonstructural proteins, NS2B and NS3pro, can be likewise exploited for discovering new anti-dengue therapeutics. In this study, we have linked together two pharmaceutically interesting motifs, namely 1,3,4-oxadiazole and benzenesulfonamide in two alternative series to develop novel S-benzylated and S-alkylphthalimidated hybrids. For the first series of hybrids, 4-aminobenzoic acid (1) was reacted with substituted benzenesulfonyl chlorides via its amino group, whereas the carboxylic acid side was elaborated to sulfonamido-1,3,4-oxadiazole-2-thiols (6a/b) in three steps. At this stage, the intermediates 6a/b were bifurcated to either S-alkylphthalimidated (8a-j) or S-benzylated (9a-c) hybrids by reacting with corresponding halides. For the alternative series of hybrids, the carboxylic acid group of probenecid (10) was similarly elaborated to sulfonamido-1,3,4-oxadiazole-2-thiols (13), and diverged to S-alkylphthalimidated (14a-f) and S-benzylated hybrids (15a-e). Bioactivity assays demonstrated that 8g and 8h are the most potent inhibitors among the synthesized analogs, exhibiting the IC₅₀ values of 13.9 μM and 15.1 μM, respectively. Computational assessment predicted the binding of the inhibitors at an allosteric site developed in the open conformation of DENV2 NS2B/NS3pro. Taken together these findings point out that the synthesized hybrid inhibitors possess a great potential for further antiviral drug development.

Full text of DOI:10.1016/j.bioorg.2020.103567

Journal Pre-proofs
Synthesis and evaluation of novel S-benzyl- and S-alkylphthalimide- oxadia-
zole -benzenesulfonamide hybrids as inhibitors of dengue virus protease
Syeda Shamila Hamdani, Bilal Ahmad Khan, Shahid Hameed, Farwa Batool,
Hafiza Nosheen Saleem, Ehsan Ullah Mughal, Muhammad Saeed
PII:
S0045-2068(19)31364-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.103567
YBIOO 103567
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Please cite this article as: S. Shamila Hamdani, B. Ahmad Khan, S. Hameed, F. Batool, H. Nosheen Saleem, E.
Ullah Mughal, M. Saeed, Synthesis and evaluation of novel S-benzyl- and S-alkylphthalimide- oxadiazole -
benzenesulfonamide hybrids as inhibitors of dengue virus protease, Bioorganic Chemistry (2020), doi: https://
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Synthesis and evaluation of novel S-benzyl- and S-alkylphthalimide- oxadiazole -
benzenesulfonamide hybrids as inhibitors of dengue virus protease
Syeda Shamila Hamdania, Bilal Ahmad Khana,*, Shahid Hameedb, Farwa Batoolc, Hafiza
Nosheen Saleemc, Ehsan Ullah Mughald, Muhammad Saeedc,*
a Department of Chemistry, University of Azad Jammu and Kashmir, Muzaffarabad 13100 AJK,
Pakistan
b Department of Chemistry, Quaid-i-Azam University, Islamabad, 45320, Pakistan
c Department of Chemistry and Chemical Engineering, Syed Babar Ali School of Science and
Engineering, Lahore University of Management Sciences, Lahore 54792 Pakistan
d Department of Chemistry, University of Gujrat, Gujrat 50700, Pakistan
Correspondence:
Bilal Ahmad Khan: bkhan@ajku.edu.pk
Muhammad Saeed: muhammad.saeed@lums.edu.pk
1

Abstract
Direct acting antiviral drugs (DAADs) are becoming therapeutics of choice for the treatment of
viral infections. Successful development of anti HIV and HCV drugs by targeting the viral
proteases has provided impetus for discovering newer DAADs. Dengue virus (DENV) protease,
which is composed of two nonstructural proteins, NS2B and NS3pro, can be likewise exploited
for discovering new anti-dengue therapeutics. In this study, we have linked together two
pharmaceutically interesting motifs, namely 1,3,4-oxadiazole and benzenesulfonamide in two
alternative series to develop novel S-benzylated and S-alkylphthalimidated hybrids. For the first
series of hybrids, 4-aminobenzoic acid (1) was reacted with substituted benzenesulfonyl
chlorides via its amino group, whereas the carboxylic acid side was elaborated to sulfonamido-
1,3,4-oxadiazole-2-thiols (6a/b) in three steps. At this stage, the intermediates 6a/b were
bifurcated to either S-alkylphthalimidated (8a-j) or S-benzylated (9a-c) hybrids by reacting with
corresponding halides. For the alternative series of hybrids, the carboxylic acid group of
probenecid (10) was similarly elaborated to sulfonamido-1,3,4-oxadiazole-2-thiols (13), and
diverged to S-alkylphthalimidated (14a-f) and S-benzylated hybrids (15a-e). Bioactivity assays
demonstrated that 8g and 8h are the most potent inhibitors among the synthesized analogs,
exhibiting the IC50 values of 13.9 and 15.1, respectively. Computational assessment
predicted the binding of the inhibitors at an allosteric site developed in the open conformation of
DENV2 NS2B/NS3pro. Taken together these findings point out that the synthesized hybrid
inhibitors possess a great potential for further antiviral drug development.
2

1. Introduction
Recent decades have witnessed a sudden rise in flaviviral infections in tropical and
subtropical regions, with a potential of penetrating into remote areas due to international travel
and trade. Particularly, the infections caused by dengue virus (DENV) and Zika virus (ZIKV)
have raised major public health concerns [1-4]. Epidemiological data suggest that the DENV
infections affect approximately 400 million people each year globally [1]. Clinical symptoms of
DENV infection range from a mild self-limited fibril illness to the fatal dengue hemorrhagic
fever (DHF) and dengue shock syndrome (DSS) [5]. In the recent epidemic outbreaks, four
serotypes of DENV (DENV1-4) have been detected along with the emergence of a fifth one in
Southeast Asia [6].
To date, no specific drug is available for the treatment of DENV infection. Although,
Sanofi-Pasteur has developed a tetravalent dengue vaccine (CYD-TDV, Dengvaxia), which is
licensed for clinical use in several endemic countries [7, 8], yet its effectiveness in general
population has been questioned [9, 10]. Therefore, development of direct-acting antiviral drugs
(DAADs) drugs for the treatment of DENV infection is highly demanded. In this regard, various
viral factors have been proposed as drug targets [11, 12]; amongst them the complex
NS2B/NS3pro is becoming a prominant target for discovering anti-dengue DAA therapeutics[13,
14].
Previous efforts for discovering peptidic, peptidomimetic, and small molecule based
inhibitors of DENV NS2B/NS3pro and closely related flaviviruses have been reviewed recently
[13, 14, 15]. Despite the identification of several potent inhibitors of the protease [16],
unfavorable pharmacokinetics and pharmacodynamic characteristics no inhibitor of DENV
NS2B/NS3pro has hitherto entered the clinical trial. This predicament demands a continuous
effort toward the screening of small organic molecules for discovering newer hits for the
development of potent anti-dengue drug. Carrying on our efforts toward discovering novel
inhibitors of DENV NS2B/NS3pro, we describe here results of newly synthesized oxadiazole-
sulfonamide hybrids.
1,3,4-Oxadiazoles are privileged structural scaffolds with diverse synthetic utility and broad
spectrum of biological activities including anti-cancer [17], anti-mitotic [18], anti-inflammatory
[19-23], antimicrobial [24-28], enzyme inhibitory [29-35] and others [36, 37]. Whereas,
sulfonamides are another useful class of compounds having antibacterial, antimicrobial and
3

anticancer activities [38]. Presence of arylsulfonamide moiety at the P´-position has been found
to enhance the inhibitory potential peptidomimetic inhibitors of hepatitis C virus [39].
Additionally, the replacement of the cleavage site peptide bond with a sulfonamide bond in
peptide-based inhibitors of protease results in enhanced stability of the inhibitors against
proteolytic degradation [40]. Recently, phthalimide containing sulfonamides have been reported
as dengue virus protease inhibitors [41, 42, 43], along with other interesting biological activities
[44-46]. Impressed by the biological profile of these structural scaffolds, we envisioned to
combine these pharmacophores to synthesize novel S-benzyl-oxadiazole-benzene sulfonamides
and S-alkylphthalimide-oxadiazole benzenesufon-amides. This article reports on the synthesis,
bioassay screening against dengue protease (serotype-2), DENV2 NS2B/NS3pro, molecular
docking analyses of these hybrids.
2. Results and discussion
2.1. Chemistry
The syntheses of the target S-alkylphthalimide-oxadiazole benzenesulfonamides (8a-j; 14a-
f) and S-benzyl-oxadiazole benzenesufonamides (9a-c;15a-e) were achieved by following a
multistep synthetic approach as outlined in Scheme 1 and 2. For the synthesis of N-(4-(5-((1,3-
dioxoisoindolin-2-yl)alkylthio)-1,3,4-oxadiazol-2-yl)phenyl)-4-methyl benzenesulfonamide (8a-
j) and N-(4-(5-(trifluoromethyl substituted)benzylthio)-1,3,4-oxadiazol-2-yl)phenyl)-4-methyl
benzene-sulfona-mide (9a-c), a common starting material 4-amino benzoic acid (1) was used.
The reaction of 4-amino benzoic acid (1) with 4-methylbenzene-1-sulfonyl chloride (2a) or 4-
(trifluoromethyl)benzene-1-sulfonyl chloride (2b) in the presence of sodium carbonate provided
4-(tosylamino) benzoic acid (3a) or 4-(4-(trifluoromethyl)phenylsulfonamido) benzoic acid (3b)
in 82% and 86% yield, respectively [47]. The sulfonamide acids 3a, and 3b were esterified in
methanol in the presence of sulfuric acid to obtain methyl 4-(4-methylphenyl-sulfonamido)
benzoate (4a), and methyl 4-(4-(trifluoromethyl)phenylsulfonamido) benzoate (4b) in 83% and
80% yield, respectively. The hydrazinolysis of 4a and 4b with 80% hydrazine hydrate in
methanol under reflux afforded 4- (4-methylphenyl-sulfonamido) benzohydrazide (5a) and 4-(4-
(trifluoromethyl)phenylsulfonamido) benzohydrazide (5b) in 78% and 71% yield, respectively
[48]. The hydrazide 5a and 5b were then treated with carbon disulfide and potassium hydroxide
under reflux in methanol to afford 4-Methyl-N-(4-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-
4

yl)phenyl) benzenesulfonamide (6a) and N-(4-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-
yl)phenyl)-4-(trifluoromethyl) benzenesulfonamide (6b) in 77% and 70% yield, respectively [49-
51]. Finally, the reaction of 6a and 6b with phthalimido-alkyl bromides 7́a-f or substituted
benzyl bromides 7a-e in the presence of potassium carbonate in acetone at room temperature
resulted in the synthesis of the desired structures 8a-j and 9a-c in decent overall yields [52]
(Scheme 1).
R
R
R
NH2
R
i
ii
iii
+
CO2H
CO2Me
CONHNH2
O2S
O2S
O2S
CO2H
N
N
N
SO2Cl
H
H
H
3a-b
5a-b
1
2a-b
4a-b
2a R = CH3
2b R = CF3
R
iv
R
N
9a: R =CH3, R1 =4'-CF3
9b: R =CH3, R1 =3'-CF3
9c: R =CH3, R1 =2'-CF3
N
v
N
N
S
O
SH
O
ArCH2Br
O2S
N
O2S
7a-c
Reagents and conditions:
H
9a-c
N
R1
H
O
i): Na2CO3 (5 equiv.)/ CHCl3/ RT/ 4h
ii): H2SO4/ MeOH/ Reflux/ 8-12h
iii): N2H4.H2O/ MeOH/ Reflux/ 6-8
iv): CS2/ KOH/ Reflux/ 8-10h
6a-b
Br
8a: R =CH3, n =1
8b: R =CH3, n =2
8c: R =CH3, n =4
8d: R =CH3, n =6
8e: R =CF3, n =1
8f: R =CF3, n =2
8g: R =CF3, n =3
8h: R =CF3, n =4
8i: R =CF3, n =5
8j: R =CF3, n =6
N
v
n
R
7/a-f O
N
N
O
S
O
v): K2CO3/ Acetone/ RT/ 3-4h
N
O
n
O2S
8a-j
N
H
Scheme 1. Synthetic route of 8a-j and 9a-c from 4-aminobenzoic acid (1).
The target compounds and intermediates were purified by recrystallization and the final
products were characterized by 1H & 13C NMR spectroscopy, FT-IR, and CHNS analyses. In the
1HNMR, NH proton of 8a-j and 9a-c displayed a characteristic broad singlet at δ 11.13-10.71
ppm. The twelve aromatic protons of 8a-j and 9a-c were located in the range of δ 8.05-7.06 ppm.
The singlet corresponding to CH3 group on the phenyl ring of 8a-d and 9a-c appeared between δ
2.32-2.31 ppm. The singlet for two protons of S-CH2 group in compounds 8a and 8e was found
at δ 5.28 and 5.31 ppm, respectively whereas, the singlet for S-CH2 protons of 9a-c showed up in
the range of δ 4.68-4.62 ppm. Similarly, in compounds 8b-d and 8f-j a triplet in the range of δ
3.56-3.24 ppm corresponded to two protons of N-CH2. All the remaining methylene groups
5

present between the S-CH2 and N-CH2 group of 8c-d and 8g-j were found as multiplets in the
range of δ 2.21-1.29 ppm. In 13C NMR, the two carbonyl signals of phthalimido group were
located between δ 168-164 ppm. The two oxadiazole carbons were present between δ 160-165
ppm and δ 140-145 ppm. All the aromatic carbons of 8a-j and 9a-c were obtained in the range of
δ 141-118 ppm. All the aliphatic carbons were observed below δ 50 ppm.
For the synthesis of 4-(5-((1,3-dioxoisoindolin-2-yl)alkylthio)-1,3,4-oxadiazol-2-yl)-N,N-
dipropyl benzenesulfonamide (14a-f) and 4-(5-(substituted)benzylthio)-1,3,4-oxadiazol-2-yl)-
N,N-dipropyl benzenesulfonamide (15a-e), we followed the similar synthetic route; however, by
using a different starting material, i.e. probenecid (10) containing an n-propylsulfonamide
moiety. Thus, esterification of 10 was followed by hydrazinolysis of the resulting ester 11 with
80% hydrazine hydrate in methanol under reflux to afford 4-(dipropylsulfamoyl)benzo-hydrazide
(12) in 76% yield. The cyclization of 12 was accomplished with CS2/KOH/MeOH system under
refluxing conditions to afford N,N-dipropyl-4-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)
benzenesulfonamide (13) in 73% yield. The synthetic route was again bifurcated at this stage in
the two series 14a-f and 15a-e by reaction of 13 with phthalimido-alkyl bromides 7́ a-f and
substituted benzyl bromides 7a-e in the presence of potassium carbonate in acetone at room
temperature, in fairly good yields (Scheme 2).
1
The HNMR of compounds 14a-f displayed the characteristic peaks for two isopropyl
groups and two aromatic rings. In addition, the singlet for S-CH2 group of 14a was found at δ
5.42 ppm. The triplets for the S-CH2 and N-CH2 group of 14b-f were present in the range of δ-
3.64-3.31 ppm and δ 4.23-3.69 ppm, respectively. The multiplet for the CH2 group present
between S-CH2 and N-CH2 group of 14c was found at δ 2.34-2.27 ppm. The four protons of two
methylene groups of compound 14d present between S-CH2 and N-CH2 group were obtained as a
multiplet at δ 1.97-1.87 ppm. The other protons of remaining methylene groups of 14e and 14f
13
were found between δ 1.93-1.56 ppm. The C NMR spectrum of 14a-c revealed characteristic
peaks for two carbonyl carbons between δ 166-164 ppm, two oxadiazole carbons at around δ 162
and 143 ppm, aromatic carbons between δ 138- 120 ppm and aliphatic carbons below δ 50 ppm.
6

SH
S
N
N
N
N
R1
O
CO2H
O
CO2Me
CONHNH2
iii
i
ii
iv
ArCH2Br
7a-e
O S O
O S O
N(n-Pr)2
O S O
O S O
N(n-Pr)2
O S O
N(n-Pr)2
N(n-Pr)2
N(n-Pr)2
13
10
11
12
15a-e
O
O
15a: R1 =4'-CF3
15b: R1 =3'-CF3
15c: R1 =2'-CF3
15d: R1 = H
Reagents and conditions:
Br
n
iv
i): H2SO4/ MeOH/ Reflux/ 8-12h
ii): NH2NH2.H2O/ MeOH/ reflux/ 6-8h
iii): CS2/ KOH/ Reflux/ 8-10h
iv): K2CO3/ Acetone/ RT/ 3-4h
15e: R1 = 4'-NO2
7/a-f
14a: n=1
O
N
O
N
14b: n=2
14c: n=3
14d: n=4
14e: n=5
14f: n=6
(n-Pr)2N S
O
O
S
N
n
14a-f
O
Scheme 2. Synthetic route of 14a-f and 15a-e from probenecid (10).
1
In addition to the characteristic signals of the n-propyl group in H NMR spectra,
compounds 15a-e showed peaks for aromatic protons between δ 8.23-7.62 ppm and singlets for
two benzylic protons between δ 4.76-4.58 ppm. Similarly, 13C NMR for compounds 15a-e
showed peaks for carbonyl groups, aromatic carbons, oxadiazole carbons and n-propyl carbons.
The benzylic carbons were found between δ 39-33 ppm.
2.2 DENV2 protease activity
All the synthesized compounds were tested for DENV2 protease inhibitory activity by using
the recombinant Gly4-Ser-Gly4 linked NS2B/NS3pro [53] in the presence of fluorogenic Bz-Nle-
Lys-Arg-Arg-AMC substrate. The assay measures the cleavage of the fluorescent tag, 7-amino-
4-methylcoumarine (AMC) from the substrate as a function of time. A dose of 50 of each of
the sulfonamide-oxadiazol hybrids was incubated with 100nM protease and reduction in the
fluorescence was deemed as the inhibition of the protease. A separate experiment was run to
measure background fluorescence originating from the synthetic compounds or quenching of
AMC by the compounds to rule out the false positive inhibition (data not shown). None of the
7

tested compounds showed any significant background fluorescence or quenching effect under the
conditions used for these experiments. Moreover, the enzymatic reactions of potent inhibitors
were repeated by replacing the zwitterionic detergent with non-ionic detergent to rule out the
possibility of false positive hit due to aggregation of compounds.
The hybrids containing S-alkylphthalimide moiety, i.e. 8 and 14 demonstrated overall a
better inhibition than the S-benzylated analogs, as shown in Fig. 1 (A & C versus B & D).
Presence of trifluoromethyl (CF3) moiety on the benzene ring of sulfonamide moiety in 8e-j
seems to improve the inhibition of the protease as compared to the methyl moiety in 8a-d (Fig 1,
A); however, the inhibitory activity is strictly depended on the length of the alkyl chain flanking
between the phthalimide and oxadiazole moieties. The analogs containing shorter alkyl chain
(n=1, 2) in 8 and 14 showed <50% inhibition at 50 concentration. The most potent inhibition
A
B
C
D
Fig. 1. Inhibition of DENV2 NS2B/NS3pro by S-alkylphthalimide- (A,C) and S-benzyl-
sulfonamide-oxadiazole hybrids (B, D). A dose of 50 M of the hybrids was incubated in the
presence of 100 nM recombinant DENV2 NS2B/NS3pro, 5M tetrapeptide-AMC substrate in
a Tris buffer at pH 9.5. Time dependent release of fluorescent AMC tag was measured for 30
min and compared with the control, which was run in the absence of the hybrid under the
same assay conditions. The results are reported as percentage of inhibition.
8

of the DENV2 NS2B/NS3pro (>90%) was demonstrated by 8g, with a propyl group (n =3)
between them.
The n-propyl sulfonamide-oxadiazol-phthalimide hybrids (14a-f) (Fig. 1, C) also showed the
alkyl-chain dependent inhibitory activity. The analogs 14c-f containing the alkyl chain with n=3-
6 demonstrated similar inhibitions (~ 60 to 70%) of the protease, indicating similar mode of
binding with the protease as exhibited by 8i-j.
Interestingly, the introduction of trifluoromethyl (CF3) moiety in the S-benzyl group in 9a-c
(Fig. 1, B) enhanced the inhibition up to ~80%, regardless the position of CF3 on the aromatic
ring. The inhibitory activity of the S-benzylated analogs was reduced significantly, even in the
presence of the CF3 moiety in the n-propylsulfonamide hybrids 15a-e (Fig. 1, D).
The hybrids showing close to or better than 70% DENV2 NS2B/NS3pro inhibition were further
tested in the dose response experiments. A 3-fold serial dilution of the compounds, ranging from
100
80
60
8d
8g
8h
40
9a
9b
9c
20
0
14d
0
1
2
Log[Conc (uM)]
Fig. 2. Dose response of selected hybrids for the inhibition of DENV2 NS2B/NS3 protease
activity. Three-fold serial dilution of selected hybrids were screen between 150M and
620nM concentration in DENV2 NS2B/NS3pro assay in triplicate.
150 to 620nM was screened in triplicate. The kinetic data were fit into the dose response
model embedded in Graphpad Prism software (version 7.0). Half-maximal inhibitory dose of all
selected hybrids was observed within a range of approximately 14 to 45 concentration. As
9

shown in Table 1 and Fig 2, 8g and 8h demonstrated promising inhibitory profiles with IC50
values of 13.9 ± 1.4 and 15.1 ± 1.3, respectively. When experiments were conducted in
the presence of nonionic detergent Brij35, both compounds 8g and 8h showed IC50 values of
14.1+ and 16.3+1.1, respectively.
Table 1.
Activity and predicted binding free energy of the selected S-alkylphthalimide-oxadiazole-
sulfonamide inhibitor against DENV2 NS2B/NS3pro.
Compound Inhibition (%)a
Docking score (kcal/mol)
IC50 ()
8d
71.7 ± 2.9
99.9 ± 1.3
77.6 ± 2.1
83.9 ± 1.3
80.4 ± 1.7
80.8 ± 1.0
74.8 ± 1.0
~99.8 ± 0.8
44.3 ± 1.5
-6.9
-9.0
-8.8
-8.6
-8.6
-8.6
-6.8
-11.0
8g*
13.9 ± 1.4 (14.1+1.5)
15.1 ± 1.3 (15.7+1.1)
25.2 ± 1.2
8h*
9a
9b
23.9 ± 1.2
9c
24.0 ± 1.6
14d
23.9 ± 1.3
Aprotinin
(control, 5 )
0.026 ± 0.003
aPercentage of inhibition was measured in triplicate by using a single dose (50 ) of the test
compound.
*The IC50 value for the compound 8g and 8h was within 5% error when experiments were conducted
in the presence of nonionic detergent Brij35 as shown in the parenthesis.
10

0.150
0.125
0.100
0.075
0.050
0.025
0.125
0.100
0.075
0.050
0.025
A
B
20 M
10 M
No Inhibitor
20 M
10 M
No Inhibitor
-0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30
-0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30
1/[sub, M]
1/[sub, M]
Fig 3. Mode of inhibition. Lineweaver-Burk plots showing non-competitive inhibition of
DENV2 NS2B/NS3pro in the presence of 8g (A) and 8h (B). Initial rates of enzymatic reactions
were measured at varying concentration of the substrate (4.6-60 ) in the absence () and in
the presence of 10  (x) and 20  (o) of inhibitors. Each data point is a mean of three
replicates and error bars show the standard deviation of the replicates.
Mode of inhibition of the protease by 8g and 8h was determined by measuring the protease
activity at varying concentrations (4.6-60) of the tetrapeptide substrate in the presence of
different concentrations of the inhibitors (0, 10, 20 ). The data obtained were fitted to the
Lineweaver-Burk model and the linear plots corresponding to the different concentrations of 8g
and 8h were found to be passing through different y-intercepts, but intercepting at the same point
on the negative x-axis. This indicated further that no change in the value of KM of the protease
(31.3 ± 4.5 ) occurred in the presence of different amounts of the inhibitors; however the Vmax
was reduced in response to the higher concentration of the inhibitor. Therefore, from the kinetics
analysis, we concluded a non-competitive type inhibition by these inhibitors, with Ki values of
12.3 ± 3.8  and 13.8 ± 5.1 , for 8g and 8h, respectively (Fig 3).
To get an insight into atomic level details of binding of the synthesized inhibitors to DENV2
NS2B/NS3pro, we performed molecular docking assessments by using AutoDock Vina [54]. For
this purpose, we used the recently reported co-crystal structure of DENV2 NS2B/NS3 protease
(PDB ID: 6MO0) containing a bound inhibitor [55]. The docking files for the receptor (protease)
and the selected inhibitors were prepared in AutoDock Tool. As shown in the Fig 4 and Table 1,
11

all of the tested compounds were found to bind with favorable binding energies (between -9.0 to
-6.8 kcal/mol) at an allosteric allosteric cleft between two -sheets in the open conformation of
the protease. The polar sulfonamide and oxadiazol moieties of the docked structures were found
to be pointing away from the cavity in the solvent exposed side. In all of the docked models, the
N-atoms of the Trp-83 and Asn-167 exhibited H-bonding with the O-atoms of the carbonyl of
the phthalimide moiety. However, another dipole-dipole interaction is observed between the
hydroxyl group of Thr-118 and the trifluoromethyl group of 8g. This interaction seems to be
absent in 8h and 8d, most probably due to longer alkyl chains (n=4 in 8h, and n=6 in 8d). A
number of residues in the cleft shown hydrophobic interactions with the non-polar parts of the
inhibitors. The docking data demonstrated the synthesized inhibitors are allosteric in nature and
have potential to bind at the open conformation of DENV2 protease. Similar computational
binding interactions were also observed when other crystal structures of DENV2 NS2B/NS3pro
(e.g., 2FOM) were used in the docking analyses (data not shown). To the best of our knowledge,
all of the reported crystal structures from the DENV2 NS2B/NS3pro have an under-developed
substrate binding site in the so-called open conformation. Therefore, the estimated binding
interactions could be biased toward the allosteric site. To minimize this bias, we also conducted
the docking analyses by using the crystal structure of DENV3 NS2B/NS3pro (3U1I), having a
fully developed substrate binding site (closed conformation). With the exception of a loop
comprising of Gly151 to Glu171 residues and located near the allosteric site, both the structures
depicted by 3U1I and 6MO0 align very well. The loop Gly151-Glu171 seems highly dynamic in
the open conformation of the protease and thus did not show significant electron density in the
crystal structure 6MO0; however, it is very well resolved in the closed conformation (crystal
structure 3U1I). Docking analyses of 8g and 8h by using the closed conformation of 3U1I
showed that both of the inhibitors bind at the same allosteric site with slightly weaker binding
energies (~ -8.0 kcal/mol), probably due to the closeness of the G151-Glu171 loop.
Computational assessment, therefore, indicated that the inhibitors 8g and 8h have higher affinity
towards the open conformation of the protease for binding at the allosteric site, and corroborated
their non-competitive behavior in the enzyme inhibition assays. Interestingly, Yao et al. have
very recently published an article reporting the inhibition and X-ray crystallographic data of
allosteric inhibitors that also bind at the same cleft [55]. This finding demonstrated that our
12

synthesized hybrid inhibitors can be further developed into novel therapeutics for the treatment
of DENV2 infections.
13

Leu149
Gly148
A
B
2.90
Trp83
2.77
3.24
Leu85
Trp83
Asn167
Val147
Gly87
Leu76
Asn167
Ile123
Ile165
Lys74
3.33
Thr118
Thr118
Trp83
C
D
Trp83
Trp83
2.94
Leu76
Gly87
Leu85
Leu149
Glu86
3.18
Asn167
Asn167
Lys74
Ile165
Ile123
Asp75
Thr118
Trp83
E
F
Leu149
Trp83
Leu76
3.13
Leu85
Ile165
Asn167
3.09
Ile123
Asn167
Lys74
Phe116
Thr118
Figure 4. Molecular docking interaction of 8g, 8h, and 8d with DENV2 NS2B/NS3pro.
A,C,E) The protease is shown as blue cartoon, the residues showing hydrophobic interactions
are shown as yellow surface, and those showing H-bonding as magenta colored sticks with the
docked 8g (A), 8h (C), and 8d (E) as stick models. Interactions of the individual residues with
8g, 8h, and 8d are depicted in LigPlot and shown in B, D, and F, respectively.
14

3. Experimental section
3.1. Chemistry
3.1.1. General
All the laboratory grade chemicals and reagents were obtained commercially and used
without further purification. Analytical grade solvents were used without drying/ distillation.
Melting points were recorded in open capillaries using a Gallenkamp melting point apparatus
(MP-D) and are uncorrected. All the reactions were monitored using thin layer chromatography,
which was accomplished on Merck pre-coated plates (silica gel 60 F254, 0.25 mm) and
1
visualized using fluorescence quenching under UV light (254 nm). H NMR and 13C NMR
spectra were recorded on a Bruker AV-300 spectrometer (300 MHz). IR spectra were recorded
on Shimadzu Fourier Transform Infra-Red spectrophotometer model using ATR (Attenuated
Total Reflectance). Mass spectra were recorded on a Fisons VG Autospec X double-focusing
mass spectrometer.
3.1.2. General procedure for the synthesis of 4- (4-alkylphenylsulfonamido)benzoic acid (3a-b)
A 250-mL single necked round bottom flask was charged with 4-aminobenzoic acid 2
(30mmol) dissolved in 40 mL of aqueous sodium carbonate (60 mmol). A solution of respective
sulfonyl chloride 1a-b (31mmol) in 25 mL of chloroform was added slowly and the mixture was
stirred at room temperature for 4 hours. Aqueous layer was acidified with 2N HCl to precipitate
pure 3a and 3b.
3a: Colorless solid; yield: 82%; Rf 0.51 (n-hexane:ethylacetate, 6:4); mp: 232-234°C (lit.
1
m.p. 230-232); H NMR (300 MHz, DMSO-d6  ppm 12.85 (bs, 1H, OH), 10.78 (s, 1H, NH),
7.79 (d, J = 8.7 Hz, 2H, Ar-H), 7.70 (d, J = 8.4 Hz, 2H, Ar-H), 7.35 (d, J = 8.1 Hz, 2H, Ar-H),
7.19 (d, 2H, J = 8.7 Hz, Ar-H), 2.32 (s, 3H, Ar-CH3); IR (cm-1): 3560-3400 (OH), 3320 (N-H),
3025 (C-H, aromatic), 2823 (C-H, aliphatic), 1738 (C=O), 1348, 1158 (O=S=O).
1
3b: Colorless solid; yield: 86%; Rf 0.34 (n-hexane: ethylacetate, 6:4); m.p.: 298-300°C; H
NMR (300 MHz, DMSO-d6  ppm 12.79 (bs, 1H, OH), 11.06 (bs, 1H, NH), 8.04 (d, J = 9 Hz
2H, Ar-H), 7.96 (d, J = 9H, 2H, Ar-H), 7.83 (d, J = 9 Hz, 2H, Ar-H), 7.21 (d, J = 9 Hz, 2H, Ar-
H); 13C NMR (75MHz, DMSO-d6  ppm 167.12, 143.59, 141.83, 133.47, 133.04, 131.31,
128.14, 127.26, 127.23, 127.18, 127.12, 126.62, 125.56, 121.95, 119.09. IR (cm-1): 3400 (OH),
3263 (N-H), 3020 (C-H, aromatic), 2826 (C-H, aliphatic), 1673 (C=O), 1323, 1163 (O=S=O).
15

3.1.3. General procedure for the synthesis of carboxylate esters (4a-b & 11)
The carboxylic acid 3a, 3b and 10 (0.02 mol) was dissolved in 30 mL of methanol and
concentrated sulfuric acid (0.5 mL) and stirred at reflux temperature for 8-10 hours. The reaction
mixture was concentrated at reduced pressure. The reaction mixture was neutralized with
saturated aqueous sodium bicarbonate solution (150 mL) and extracted with ethyl acetate (3 x 50
mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to obtain pure
products 4a, 4b and 11.
4a: Colorless solid; yield: 83%; Rf 0.83 (n-hexane: ethylacetate, 6:4), m.p.: 204-206°C (lit.
1
mp 202-204); H NMR (300 MHz, DMSO-d6  ppm 10.81 (s, 1H, NH), 7.81 (d, J = 8.7 Hz,
2H, Ar-H), 7.70 (d, J = 8.1 Hz, 2H, Ar-H), 7.35 (d, J = 8.1 Hz, 2H, Ar-H), 7.20 (d, J = 9 Hz, 2H,
Ar-H), 3.77 (s, 3H, OCH3), 2.32 (s, 3H, Ar-CH3); IR (cm-1): 3300 (NH), 3059, 3025 (C-H,
aromatic), 2874 (C-H, aliphatic), 1691 (C=O), 1338 (asym), 1158 (sym) (O=S=O).
1
4b: Colorless solid; yield: 80%; Rf: 0.90 (n-hexane: ethylacetate, 6:4); m.p.: 162-163°C; H
NMR (300 MHz, DMSO-d6  ppm 11.10 (bs, 1H, NH), 8.02 (d, J = 9 Hz, 2H, Ar-H), 7.96 (d, J
13
= 9 Hz,2H, Ar-H), 7.86-7.83 (m, 2H, Ar-H), 7.26-7.22 (m, 2H, Ar-H), 3.77 (s, 3H, OCH3); C
NMR (75 MHz, DMSO-d6  ppm 166.02, 143.52, 142.22, 133.51, 133.08, 131.21, 128.14,
127.27, 127.24, 127.19, 127.13, 125.55, 125.38, 119.09, 52.43; IR (cm-1): 3400 (NH), 3066, (C-
H, aromatic), 2950 (C-H, aliphatic), 1692 (C=O), 1349 (asym), 1162 (sym) (O=S=O).
1
11: Colorless solid; yield: 84%; Rf 0.77 (n-hexane: ethylacetate, 9:1); m.p.: 65-66°C; H
NMR (300 MHz, chloroform-d  ppm 8.18-8.15 (m, 2H, Ar-H), 7.90-7.87 (m, 2H, Ar-H), 3.97
(s, 3H, OCH3), 3.14-3.09 (m, 4H, N(CH2)2), 1.62-1.49 (m, 4H, N(CH2CH2CH3)2), 0.88 (t, J = 15
Hz, 6H, (CH2CH3)2); 13C NMR (75 MHz, chloroform-d  ppm 165.76, 144.27, 133.41, 130.22,
127.00, 52.61, 49.89, 21.91, 11.15; IR (cm-1): 3100, 3051 (C-H, aromatic), 2935, 2873 (C-H,
aliphatic), 1726 (C=O), 1341 (asym), 1156 (sym) (O=S=O).
3.1.4. Synthesis of Carboxylic Acid Hydrazides (5a, 5b & 12)
To the solution of carboxylate esters 4a, 4b and 11 (0.02 mol) in 30 mL of methanol,
hydrazine hydrate (80%, 0.06 m0l) was added. The reaction mixture was subjected to reflux for
6-8 hours. The mixture was brought to the room temperature and cold water was added. The
precipitated solid was filtered, dried and recrystallized from methanol to obtain pure compound.
16

1
5a: Colorless solid; yield: 78%, Rf: 0.43 (Chloroform: acetone, 9:1); m.p.: 270-272°C; H
NMR (300 MHz, DMSO-d6  ppm 10.58 (bs, 1H, NH), 9.60 (s, 1H, NH), 7,68 (dd, J = 8.4, 3.9
Hz, 4H, Ar-H), 7.34 (d, J = 8.4 Hz 2H, Ar-H), 7.12 (d, J = 8.7 Hz, 2H, Ar-H), 4.43 (bs, 2H,
NH2), 2.32 (s, 3H, Ar-CH3); IR (cm-1): 3340, 3290 (NH2), 3220, 3100 (N-H), 3048, 2940 (C-H,
aromatic.), 2874 (C-H, aliphatic), 1650 (C=O), 1333 (asym.), 1153 (sym.) O=S=O.
5b: Colorless solid; yield: 71%; Rf: 0.58 (Chloroform: methanol, 9:1); m.p: 236-238°C.
1HNMR (300 MHz, DMSO-d6  ppm: 10.59 (bs, 1H, NH), 9.63 (bs, 1H, NH), 8.02-7.95 (m,
13
4H, Ar-H), 7.70 (d, J = 9 Hz, 2H, Ar-H), 7.15 (d, J = 9 Hz, 2H, Ar-H), 4.45(bs, 2H, NH2); C
NMR (75 MHz, DMSO-d6  ppm 165.64, 143.69, 140.18, 133.39, 132.96, 129.39, 128.76,
128.12, 127.14, 119.36; IR (cm-1): 3320, 3304 (NH2), 3130 (N-H), 3050, (C-H, aromatic.), 2936,
2877 (C-H, aliphatic), 1666 (C=O), 1322 (asym.), 1159 (sym.) O=S=O.
12: Colorless solid; yield: 76%; Rf: 6.50 (n-hexane: ethyl acetate 6:4); m.p.: 116-118°C; 1H
NMR (300 MHz, DMSO-d6  ppm 10.00 (s, 1H, NH), 7.98 (d, J = 9 Hz, 2H, Ar-H), 7.86 (d, J
= 9 Hz, 2H, Ar-H), 4.59 (s, 2H, NH2), 3.04 (m, J = 15 Hz, 4H, N(CH2)2), 1.52-1.42 (m, 4H,
(CH2CH2CH3)2), 0.80 (6H, t, J = 15 Hz (CH2CH3)2); 13C NMR (75 MHz, DMSO-d6  ppm
164.94, 142.06, 137.31, 128.42, 127.29, 50.09, 22.09, 11.42. IR (cm-1): 3297, 3211 (NH2), 3086
(N-H), 3036 (C-H aromatic.), 2964, 2873 (C-H, aliphatic), 1658 (C=O), 1328 (asym.), 1155
(sym.) O=S=O.
3.1.5. Synthesis of 5-aryl-1,3,4-oxadiazole-2-thiols/thiones (6a-b & 13)
Hydrazide 5a, 5b & 12 (0.02 mol) were dissolved in methanol and a 20-mL of methanolic
solution of potassium hydroxide (0.03 mole) was added. Carbon disulfide (0.06 mol) was added
dropwise after 10 mints. The yellowish reaction mixture was subjected to reflux for 12-14 hours.
The mixture was cooled to room temperature and concentrated than placed in ice cold water.
Crude solid product precipitated out on treatment with dilute HCl up to pH 2. The precipitate
was filtered and washed with warm water and recrystallized from methanol to afford pure
oxadiazoles 6a, 6b and 13.
1
6a: Colorless solid; yield: 77%; Rf: 0.45 (Chloroform: methanol, 9:1); m.p.: 221-223°C; H
NMR (300 MHz, DMSO-d6  ppm 14.68 (s, 1H, NH), 10.83 (s, 1H, NH), 7.73 (m, 4H, Ar-H),
7.36 (d, J = 6 Hz, 2H, Ar-H), 7.27 (d, J = 9 Hz, 2H, Ar-H), 2.32 (s, 3H, Ar-CH3); 13C NMR (75
MHz, DMSO-d6  ppm 177.65, 160.65, 144.21, 141.89, 136.79, 130.35, 127.93, 119.40,
17

117.75, 21.43; IR (cm-1): 3232 (NH), 3061 (C-H, aromatic), 2945 (C-H, aliphatic), 1615, 1511
(C=N), 1320 (C-S), 1175 (O=S=O) [42-44].
1
6b: Colorless solid; yield: 70%; Rf: 2.25 (n-hexane:ethylacetate 6:4); m.p.: 259-260°C; H
NMR (300 MHz, DMSO-d6  ppm 14.70 (s, 1H, NH), 11.28 (s, 1H, NH), 8.00 (dd, J = 18, 9
Hz, 4H, Ar-H), 7.77 (d, J = 9 Hz, 2H, Ar-H), 7.30 (d, J = 9 Hz, 2H, Ar-H); 13C NMR (75 MHz,
DMSO-d6  ppm 177.69, 160.53, 143.51, 141.16, 133.30, 128.15, 128.08, 127.29, 127.25,
119.99, 118.42; IR (cm-1): 3198 (NH), 3071 (C-H, aromatic), 1612, 1516 (C=N), 1322 (C-S),
1165 (O=S=O).
1
13: Colorless solid; yield: 73%; Rf: 0.47 (n-hexane:ethylacetate, 6:4); m.p.: 177-180°C; H
NMR (300 MHz, DMSO-d6  ppm 14.92 (s, 1H, NH), 8.06 (2H, d, J = 9Hz, Ar-H), 7.97 (2H, d,
J = 9Hz, Ar-H), 3.06 (t, 4H, J = 15Hz, N(CH2)2), 1.46 (quin, 4H, J = 15 Hz, CH2CH2CH3), 0.80
(t, 6H, J = 15 Hz, CH3); 13C NMR (75 MHz, DMSO-d6,   ppm 178.12, 159.79, 142.78,
128.24, 127.49, 126.44; IR (cm-1) 3068 (C-H, aromatic), 2968 (C-H, aliphatic), 1612, 1592
(C=N), 1340 (C-S), 1158 (O=S=O).
3.1.6. General method for the synthesis of S-alkyl 1,3,4- oxadiazole (8a-j) & (9a-c)
S-alkylation of the products was achieved by following a modified method [38]. The
oxadiazoles (6a, 6b, 13) (0.02, mol) and K2CO3 (0.022, mol) were stirred in 10 mL of acetone
for 20 mints followed by the addition of alkyl bromides 7a-c and 7d-m (0.022, mol). The
reaction mixture was stirred for 4-6 hours at room temperature. The reaction mixture was
concentrated under vacuum and crude solid gained was recrystallized from methanol to get pure
products.
1
8a: Colorless solid, 55% yield, Rf: 0.55 (chloroform: acetone, 9:1), m.p. 224-226°C. H
NMR (300 MHz, DMSO-d6  ppm 10.71 (s, 1H, NH), 7.91-7.84 (m, 4H, Ar-H), 7.64 (dd, 4H, J
= 18, 9Hz, Ar-H), 7.28 (d, 2H, J = 9 Hz, Ar-H), 7.06 (d, 2H, J = 9Hz, Ar-H), 5.28 (s, 2H,
13
SCH2N), 2.31 (s, 3H, Ar-CH3); C NMR (75 MHz, DMSO-d6  ppm 167.02, 166.74, 160.01,
144.00, 142.03, 140.01, 135.44, 131.76, 129.72, 127.89, 126.94, 124.06, 119.95, 118.11, 43.00,
21.37; IR (cm-1) 3260 (N-H), 3025 (C-H, aromatic), 2948 (C-H, aliphatic), 1716 (C=O), 1602
(C=N), 1308, 1176 (O=S=O); Anal. Calcd for C24H18N4O5S2: C, 56.91; H, 3.58; N, 11.06; O,
15.79; S, 12.66 Found: C, 56.98; H, 3.61; N, 11.11; O, 15.79; S, 12.72.
18

1
8b: Colorless solid; yield; 70 %, Rf: 0.33 (Chloroform: acetone, 9:1); m.p.; 214-216°C; H
NMR (300 MHz, DMSO-d6  ppm 10.81 (1H, s, NH), 7.78-7.71 (m, 8H, Ar-H), 7.37 (d, 2H, J
= 8.1 Hz, Ar-H), 7.26 (d, 2H, J = 9 Hz, Ar-H), 4.02 (t, 2H, J = 6Hz, N-CH2), 3.56 (t, 2H, J =
13
6Hz, S-CH2), 2.32 (s, 3H, Ar-CH3); C NMR (75 MHz, DMSO-d6  ppm 168.00, 165.00,
163.21, 144.13, 141.72, 136.81, 134.84, 131.83, 130.35, 128.12, 127.21, 123.50, 119.44, 118.36,
37.34, 31.12, 21.40; IR (cm-1): 3268 (N-H), 3027 (C-H, aromatic), 2948 (C-H, aliphatic), 1714
(C=O), 1609, 1579 (C=N), 1331, 1152 (O=S=O); Anal. Calcd for C25H20N4O5S2: C, 57.68; H,
3.87; N, 10.76; O, 15.37; S, 12.32 Found: C, 57.70; H, 3.88; N, 10.82; O, 15.38; S, 12.34.
1
8c: Colorless solid; yield: 78%; Rf: 0.28 (Chloroform: acetone, 9:1), m.p.: 220-223 °C; H
NMR (300 MHz, DMSO-d6  ppm 10.78 (s, 1H, NH), 7.83-7.70 (m, 8H, Ar-H), 7.35 (d, 2H, J
= Hz, Ar-H), 7.26 (d, 2H, J = 9 Hz, Ar-H), 3.59 (t, 2H, J = 12 Hz, NCH2), 3.28 (t, 2H, J = 12 Hz,
S-CH2), 2.31 (s, 3H, Ar-CH3), 1.75-173 (m, 4H, CH2CH2CH2CH2); 13C NMR (75 MHz, DMSO-
d6  ppm 168.41, 165.13, 163.66, 144.14, 141.67, 136.85, 134.76, 132.01, 130.32, 128.13,
127.20, 123.41, 119.52, 118.40,37.27, 3212, 27.24, 28.18, 21.42; IR (cm-1): 3240 (N-H), 3027
(C-H, aromatic), 2942 (C-H, aliphatic), 1700 (C=O), 1611, 1595 (C=N), 1338, 1192 (O=S=O);
Anal. Calcd for C27H24N4O5S2. C, 59.11; H, 4.41; N, 10.21; O, 14.58; S,11.69 Found: C, 59.30;
H, 4.54; N, 10.31; O, 14.62; S, 11.71.
1
8d: Colorless solid; yield: 75%; Rf: 0.37 (Chloroform: acetone, 9:1); m.p.; 162-164 °C; H
NMR (300 MHz, DMSO-d6  ppm 10.79 (s, 1H), 7.85-7.78 (m, 6H, Ar-H), 7.70 (d, 2H, J = 9
Hz, Ar-H), 7.34 (d, 2H, J = 9 Hz, Ar-H), 7.25 (d, 2H, J = 9 Hz, Ar-H), 3.54 (t, 2H, J = 15 Hz,
NCH2), 3.24 (t, 2H, J = 15 Hz, SCH2), 2.31 (s 3H, Ar-CH3), 1.73-1.66 (m, 2H, NCH2CH2), 1.60-
13
1.53 (m, 2H, SCH2CH2), 1.43-1.29 (m 4H, CH2CH2CH2CH2); C NMR (75 MHz, DMSO-d6
 ppm 168.40, 165.16, 163.75, 143.95, 142.19, 137.13, 134.79, 132.06, 130.26, 128.10, 127.17,
123.43, 119.58, 118.10, 37.71 32.43, 29.23, 28.21, 27.80, 26.09, 21.41; IR (cm-1) 3211 (N-H),
3025 (C-H, aromatic), 2929 (C-H, aliphatic), 1705 (C=O), 1611, 1568 (C=N), 1331, 1157
(O=S=O); Anal. Calcd for C29H28N4O5S2 C, 60.40; H, 4.89; N, 9.72; O, 13.87; S, 11.12 Found:
C, 60.45; H, 4.94; N, 9.78; O, 13.82; S, 11.18.
1
8e: Colorless solid; yield: 65 %; Rf: 0.34 (Chloroform: acetone, 9:1); m.p.: 201-203°C; H
NMR (300 MHz, DMSO-d6  ppm 11.136 (s, 1H, NH), 8.06 (d, 2H, J = 8.4 Hz, Ar-H), 8.7 (d,
2H, J = 9 Hz, Ar-H), 7.89-7.83 (m, 6H, Ar-H), 7.30 (d, 2H, J = 9 Hz, Ar-H), 5.31 (s, 2H,
SCH2N); 13C NMR (75 MHz, DMSO-d6  ppm 166.78, 166.15, 161.20, 143.51, 141.21,
19

135.44, 133.56, 131.72, 128.47, 128.18, 127.28, 127.24, 125.55, 124.04, 120.04, 118.90, 40.69;
IR (cm-1) 3241 (N-H), 3064 (C-H, aromatic), 2923 (C-H, aliphatic), 1711 (C=O), 1614, 1503
(C=N), 1350, 1165 (O=S=O); Anal. Calcd for C24H15F3N4O5S2 C, 51.43; H, 2.70; F, 10.17; N,
10.00; O, 14.27; S, 11.44 Found: C, 51.54; H, 2.78; F, 10.82; N, 10.21; O, 14.32; S, 11.51.
1
8f: Colorless solid; yield: 74%; Rf: 0.23 (Chloroform: acetone, 9:1) m.p.; 176-179°C; H
NMR (300 MHz, DMSO-d6  ppm 11.09 (s,1H, NH), 8.05 (d, 2H, J = 9 Hz, Ar-H), 7.99 (d,
2H, J = 9 Hz, Ar-H), 7.81-7.70 (m, 6H, Ar-H), 7.30 (d, 2H, J = 9Hz, Ar-H), 4.02 (t, 2H, J =
13
12Hz, NCH2), 3.56 (t, 2H, J = 12 Hz, SCH2); C NMR (75 MHz, DMSO-d6  ppm 168.05,
164.96, 163.40, 153.52, 140.95, 134.82, 133.52, 133.09, 131.84, 128.26, 128.19, 127.29, 127.24,
123.51, 120.06, 118.91, 37.31, 31.12; IR (cm-1) 3391, 3218 (N-H), 2927 (C-H, aliphatic), 1713
(C=O), 1614, 1503 (C=N), 1366, 1166 (O=S=O); Anal. Calcd for C25H17F3N4O5S2 C, 52.26; H,
2.98; F, 9.92; N, 9.75; O, 13.92; S, 11.16 Found: C, 52.31; H, 3.10; F, 9.98; N, 9.81; O, 13.96; S,
11.74.
1
8g: Colorless solid; yield: 75%; Rf: 0.25 (Chloroform: acetone, 9:1); m.p.170-173 °C; H
NMR (300 MHz, DMSO-d6  ppm 11.08 (s, 1H, NH), 8.03 (d, 2H, J = 9 Hz, Ar-H), 7.97 (d,
2H, J = 9 Hz, Ar-H), 7.86-7.79 (m, 6H, Ar-H), 7.29 (d, 2H, J = 9 Hz, Ar-H), 3.71 (t, 2H, J = 12
Hz, NCH2), 3.30 (t, 2H, J = 12 Hz, SCH2), 2.13-2.06 (m, 2H, SCH2CH2); 13C NMR (75 MHz,
DMSO-d6  ppm 168.50, 165.05, 163.79, 143.54, 140.91, 134.75, 133.50, 133.07, 132.18,
128.28, 128.16, 127.26, 127.21, 125.56, 123.43, 121.94, 120.11, 119.08, 36.58, 30.09, 28.67; IR
(cm-1): 3200, 3400 (N-H), 2930 (C-H, aliphatic), 1713 (C=O), 1608, 1503 (C=N), 1321, 1159
(O=S=O); Anal. Calcd for C26H19F3N4O5S2: C, 53.06; H, 3.25; F, 9.68; N, 9.52; O, 13.59; S,
10.90. Found C, 53.12; H, 3.32; F, 9.82; N, 9.62; O, 13.64; S, 10.95.
1
8h: Colorless solid; yield: 76%; Rf: 0.26 (Chloroform: acetone, 9:1); m.p.; 210-213 °C; H
NMR (300 MHz, DMSO-d6  ppm 11.08 (s, 1H, NH), 8.04 (d, 2H, J = 8.4 Hz, Ar-H), 7.99 (d,
2H, J = 9 Hz, Ar-H), 7.83-7.75 (m, 6H, Ar-H), 7.30 (d, 2H, J = 9 Hz, Ar-H), 3.59 (t, 2H, J = 12
Hz, NCH2), 3.28 (t, 2H, J = 12 Hz, SCH2), 1.75-1.73 (m, 4H, SCH2CH2CH2); 13C NMR (75
MHz, DMSO-d6  ppm 168.43,165.03, 163.81, 143.54, 140.93, 134.77, 133.49, 132.02, 128.28,
128.17, 127.27, 123.42, 120.10, 119.07, 37.26, 32.12, 27.23, 26.79; IR (cm-1): 3218 (N-H), 2942
(C-H, aliphatic), 1699 (C=O), 1612, 1503 (C=N), 1343, 1165 (O=S=O); Anal. Calcd for
C27H21F3N4O5S2. C, 53.81; H, 3.51; F, 9.46; N, 9.30; O, 13.28; S, 10.64 Found C, 53.88; H, 3.57;
F, 9.51; N, 9.35; O, 13.32; S, 10.73.
20

1
8i: Off-white solid; yield: 77%; Rf: 0.31 (Chloroform: acetone, 9:1); m.p.; 180-181 °C; H
NMR (300 MHz, DMSO-d6  ppm 11.08 (s 1H, NH), 8.03 (d, 2H, J = 9 Hz, Ar-H), 7.97 (d,
2H, J = 9 Hz, Ar-H), 7.86-7.78 (m, 6H, Ar-H), 7.30 (d, 2H, J = 9 Hz, Ar-H), 3.56 (t, 2H, J = 15
Hz, NCH2), 3.24 (t, 2H, J = 15 Hz, SCH2), 1.76 (quin, 2H, J = 15, 9 Hz, NCH2CH2), 1.62 (2H,
13
quin, J = 15, 9 Hz, SCH2CH2), 1.39 (quin, 2H, , J = 15, 9 Hz, SCH2CH2CH2); C NMR (75
MHz, DMSO-d6  ppm 168.40, 165.00, 163.90, 143.53, 140.88, 134.79, 133.06, 132.03,
128.29, 128.16, 127.26, 127.21, 123.43, 120.12, 119.14, 37.61, 32.32, 28.94, 27.81, 25.52; IR
(cm-1): 3400, 3200 (N-H), 3137 (C-H, aromatic), 2945 (C-H, aliphatic), 1714 (C=O), 1606, 1569
(C=N), 1353, 1159 (O=S=O); Anal. Calcd for C28H23F3N4O5S2: C, 54.54; H, 3.76; F, 9.24; N,
9.09; O, 12.97; S, 10.40 Found C, 54.58; H, 3.82; F, 9.34; N, 9.13; O, 13.01; S, 10.68.
1
8j: Colorless solid; yield: 78%; Rf: 0.38 (Chloroform: acetone, 9:1); m.p.; 168-171°C; H
NMR (300 MHz, DMSO-d6  ppm 11.07 (s, 1H, NH), 8.02 (d, 2H, J = 8.4 Hz, Ar-H), 7.96 (d,
2H, J = 9 Hz, Ar-H), 7.85-7.79 (m, 6H, Ar-H), 7.29 (d, 2H, J = 9 Hz, Ar-H), 3.54 (t, 2H, J = 15
Hz, NCH2), 3.24 (t, 2H, J = 15 Hz, S-CH2), 1.71 (quin, 2H, J = 15, 6 Hz, NCH2CH2), 1.57 (quin,
2H, J = 15, 6 Hz, SCH2CH2), 1.46-1.29 (m, 4H, CH2CH2CH2CH2); 13C NMR (75 MHz, DMSO-
d6  ppm 168.40, 165.00, 163.96, 143.57, 140.94, 134.79, 132.06, 128.28, 128.15, 127.21,
125.56, 123.43, 120.13, 119.09, 37.71, 32.43, 29.22, 28.21, 27.80 26.08; IR (cm-1): 3238 (N-H),
2941 (C-H, Aliph.), 1711 (C=O), 1611, 1504 (C=N), 1335, 1186 (O=S=O); Anal. Calcd for
C29H25F3N4O5S2: C, 55.23; H, 4.00; F, 9.04; N, 8.88; O, 12.68; S, 10.17. Found C, 55.29; H,
4.10; F, 9.08; N, 8.91; O, 12.71; S, 10.21.
1
9a: Off-white solid; yield: 71%; Rf: 0.50 (Chloroform: acetone, 9:1); m.p. 164-165 °C; H
NMR (300 MHz, DMSO-d6  ppm 10.81 (s, 1H, NH), 7.78 (d, 2H, J = 9 Hz, Ar-H), 7.70 (d,
6H, J = 9 Hz, Ar-H), 7.35 (d, 2H, J = 9 Hz, Ar-H), 7.27 (d, 2H, J = 9 Hz, Ar-H), 4.62 (s, 2H,
SCH2), 2.32 (s, 3H, Ar-CH3); 13C NMR (75 MHz, DMSO-d6  ppm 165.44, 162.96, 144.17,
142.35, 141.75, 136.79, 130.30, 128.83, 128.41, 128.20, 127.20, 125.90, 125.85, 119.46, 118.25,
35.58, 21.41; IR (cm-1) 3175 (N-H), 2950 (C-H, aliphatic), 1609 (C=N), 1347 (C-S). 1322, 1159
(O=S=O); Anal. Calcd for C23H18F3N3O3S2: C, 54.64; H, 3.59; F, 11.27; N, 8.31; O, 9.49; S,
12.69 Found C, 54.68; H, 3.62; F, 11.81; N, 8.38; O, 9.53; S, 12.74.
1
9b: Colorless solid; yield: 74%; Rf: 0.41 (Chloroform: acetone, 9:1); m.p.: 160-161 °C; H
NMR (300 MHz, DMSO-d6:  ppm 10.80 (s, 1H, NH), 7.85 (s, 1H, Ar-H), 7.78 (d, 3H, J = 9
Hz, Ar-H), 7.70 (d, 2H, J = 6 Hz, Ar-H), 7.63 (d, 1H, J = 6 Hz, Ar-H), 7.57 (t, 1H, J = 15 Hz,
21

Ar-H), 7.35 (d, 2H, J = 9 Hz, Ar-H), 7.26 (d, 2H, J = 9 Hz, Ar-H), 4.62 (s, 2H, SCH2), 2.32 (s,
13
3H, Ar-CH3); C NMR (75 MHz, DMSO-d6  ppm 165.43, 162.99, 144.17, 141.73, 139.10,
136.77, 133.65, 130.31, 130.11, 129.78, 129.36, 128.18, 127.20, 126.31, 126.16, 126.11, 124.89,
124.84, 122.69, 119.47, 118.28, 35.54, 21.40; IR (cm-1): 3166 (N-H), 2926 (C-H, aliphatic),
1609 (C=N), 1327 (C-S). 1302, 1155 (O=S=O); Anal. Calcd for C23H18F3N3O3S2: C, 54.64; H,
3.59; F, 11.27; N, 8.31; O, 9.49; S, 12.69 Found C, 54.68; H, 3.62; F, 11.81; N, 8.38; O, 9.53; S,
12.74.
1
9c: Colorless solid; yield: 73%; Rf: 0.56 (Chloroform: acetone, 9:1); m.p.: 143-144°C; H
NMR (300 MHz, DMSO-d6  ppm 10.82 (s, 1H, NH), 7.81-7.29 (m, 12, Ar-H), 4.68 (s, 2H,
13
SCH2), 2.31 (s, 3H, Ar-CH3); C NMR (75 MHz, DMSO-d6  ppm 165.63, 162.60, 144.18,
141.81, 136.78, 134.80, 133.53, 132.53, 130.33, 129.21, 128.26, 127.21, 126.79, 126.51, 119.46,
118.24, 33.59, 21.41; IR (cm-1): 3205 (N-H), 2940 (C-H, aliphatic), 1611 (C=N), 1345 (C-S).
1303, 1151 (O=S=O); Anal. Calcd for C23H18F3N3O3S2: C, 54.64; H, 3.59; F, 11.27; N, 8.31; O,
9.49; S, 12.69 Found C, 54.68; H, 3.62; F, 11.81; N, 8.38; O, 9.53; S, 12.74.
14a: Off-white solid; yield: 81%; Rf: 0.46 (Chloroform: acetone, 9:1); m.p.: 154-155 °C; 1H
NMR (300 MHz, chloroform-d  ppm 8.20 (d, 2H, J = 9 Hz, Ar-H), 7.95 (d, 2H, J = 9 Hz, Ar-
H), 7.91-7.87 (m, 2H, Ar-H), 7.82-7.77 (m, 2H, Ar-H), 5.42 (s, 2H, SCH2N), 3.17-3.11 (m, 4H,
NCH2), 1.69-1.54 (m, 4H, NCH2CH2CH3), 0.90 (t, 6H, J = 15 Hz, NCH2CH2CH3); 13C NMR (75
MHz, DMSO-d6  ppm 166.45, 165.51, 162.41, 143.29, 134.75, 131.70, 127.75, 127.49,
126.77, 123.97, 49.97, 39.79, 21.99, 11.20; IR (cm-1): 3025 (C-H, aromatic), 2960 (C-H,
aliphatic), 1723 (C=O), 1606, 1568 (C=N), 1327, 1154 (O=S=O); Anal. Calcd for
C23H24N4O5S2: C, 55.18; H, 4.83; N, 11.19; O, 15.98; S, 12.81 Found C, 55.90; H, 4.88; N,
11.24; O, 16.01; S, 12.84.
14b: Colorless solid; yield: 78%; Rf: 0.49 (Chloroform: acetone, 9:1); m.p.: 131-132 °C; 1H
NMR (300 MHz, chloroform-d  ppm 8.15 (dd, 2H, J = 6.9, 5.4 Hz, Ar-H), 7.95-7.92 (m, 2H,
Ar-H), 7.88-7.82 (m, 2H, Ar-H), 7.76-7.72 (m, 2H, Ar-H), 4.23 (t, 2H, J = 12 Hz, NCH2), 3.64
(t, 2H, J = 15 Hz, SCH2), 3.15-3.10 (m, 4H, NCH2CH2), 1.63-1.50 (m, 4H, NCH2CH2CH3), 0.88
(t, 6H, J = 5 Hz, NCH2CH2CH3); 13C NMR (75 MHz, chloroform-d  ppm 168.01, 164.67,
164.25, 143.05, 134.27, 131.79, 127.69, 127.24, 126.84,123.52, 49.89, 36.62, 30.90, 21.92,
11.18; IR (cm-1): 3050 (C-H, aromatic), 2968 (C-H, aliphatic), 1703 (C=O), 1614, 1549 (C=N),
22

1337, 1154 (O=S=O); Anal. Calcd for C24H26N4O5S2: C, 56.01; H, 5.09; N, 10.89; O, 15.54; S,
12.46 Found C, 56.05; H, 5.13; N, 10.92; O, 15.62; S, 12.51.
1
14c: Colorless solid; yield: 79%; Rf: 0.45 (Chloroform: acetone, 9:1); m.p.: 157-158°C; H
NMR (300 MHz, chloroform-d  ppm 8.13 (d, 2H, J = 9 Hz, Ar-H), 7.93 (d, 2H, J = 9 Hz, ar-
H), 7.89-7.84 (m, 2H, Ar-H), 7.78-7.74 (m, 2H, Ar-H), 3.91 (t, 2H, J = 12 Hz, NCH2), 3.37 (t,
2H, J = 15 Hz, SCH2), 3.13 (t, 4H, J = 15 Hz, NCH2CH2), 2.34-3.27 (m, 2H, NCH2CH2), 1.63-
1.50 (4H, m), 0.89 (t, 6H, J = 15 Hz, NCH2CH2CH3); 13C NMR (75 MHz, chloroform-d  ppm
168.41, 165.18, 164.55, 143.00, 134.19, 131.94, 127.71, 127.17, 126.91, 123.42, 49.88, 36.49,
29.98, 28.90, 21.92, 11.19; IR (cm-1): 3025 (C-H, aromatic), 2937 (C-H, aliphatic), 1708 (C=O),
1620, 1545 (C=N), 1337, 1151 (O=S=O); Anal. Calcd for C25H28N4O5S2: C, 56.80; H, 5.34; N,
10.60; O, 15.13; S, 12.13 Found C, 56.84; H, 5.38; N, 10.66; O, 15.18; S, 12.16.
14d: Off-white solid; yield: 83%; Rf: 0.51 (Chloroform: acetone, 9:1); m.p.; 117-119 °C; 1H
NMR (300 MHz, chloroform-d  ppm 8.15-8.11 (m, 2H, Ar-H), 7.95-7.91 (m, 2H, Ar-H), 7.86-
7.82 (m, 2H, Ar-H), 7.76-7.71 (m, 2H, Ar-H), 3.76 (t, 2H, J = 12 Hz, NCH2), 3.38 (t, 2H, J = 12
Hz, SCH2), 3.15-3.10 (m, 4H, NCH2CH2), 1.97-1.87 (m, 4H, NCH2CH2CH2CH2S), 1.63-1.50
(m, 4H, NCH2CH2CH3),0.88 (t, 6H, J = 15 Hz, NCH2CH2CH3); 13C NMR (75 MHz, chloroform-
d,  ppm168.38, 165.34, 164.47, 142.99, 134.05, 132.01, 127.70, 127.15, 126.94, 123.30, 49.88,
37.14, 31.99, 27.52, 26.52, 21.92, 11.18; IR (cm-1): 3088 (C-H, aromatic), 2970 (C-H, aliphatic),
1703 (C=O), 1617, 1543 (C=N), 1332, 1160 (O=S=O); Anal. Calcd for C26H30N4O5S2: C, 57.54;
H, 5.57; N, 10.32; O, 14.74; S, 11.82 Found C, 57.63; H, 5.65; N, 10.38; O, 14.79; S, 11.88.
1
14e: Colorless solid; yield: 80%; Rf: 0.53 (Chloroform: acetone, 9:1); m.p.: 106-109°C; H
NMR (300 MHz, chloroform-d  ppm 8.15-8.12 (m, 2H, Ar-H), 7.93 (dd, 2H, J = 6, 3 Hz, Ar-
H), 7.87.7.82 (m, 2H, Ar-H), 7.76-7.71 (m, 2H, Ar-H), 3.72 (t, 2H, J = 15 Hz, NCH2), 3.32 (t,
2H, J = 15 Hz, SCH2), 3.15-3.10 (m, 4H, NCH2CH2CH3), 1.93 (2H, quin, J = 15, 9 Hz,
NCH2CH2CH2CH2CH2S), 1.76 (2H, quin, J = 15, 9 Hz, NCH2CH2CH2CH2CH2S), 1.63-1.50 (m,
13
6H, NCH2CH2CH2CH2CH2S and NCH2CH2CH3), 0.88 (t, 6H, J = 15 Hz, NCH2CH2CH3); C
NMR (75 MHz, chloroform-d  ppm 168.45, 165.55, 164.41, 142.93, 134.00, 132.06, 127.70,
127.15, 126.98, 123.25 ,49.85, 37.62, 32.36, 28.75, 28.06, 25.81, 21.92, 11.19; IR (cm-1): 3088
(C-H, aromatic), 2947 (C-H, aliphatic), 1712 (C=O), 1617, 1550 (C=N), 1336, 1177 (O=S=O).
Anal. Calcd for C27H32N4O5S2: C, 58.25; H, 5.79; N, 10.06; O, 14.37; S, 11.52 Found C, 58.31;
H, 5.84; N, 10.72; O, 14.42; S, 11.58.
23

1
14f: Colorless solid; yield: 82%; Rf: 0.55 (Chloroform: acetone, 9:1); m.p.; 124-126°C; H
NMR (300 MHz, chloroform-d:  ppm 8.13 (dd, 2H, J = 6, 3 Hz, Ar-H), 7.94-7.92 (m, 2H, Ar-
H), 7.83 (dd, 2H, J = 6, 3 Hz, Ar-H), 7.71 (dd, 2H, J = 6, 3 Hz, Ar-H), 3.69 (t, 2H, J = 15 Hz,
NCH2), 3.31 (t, 2H, J = 15 Hz, SCH2), 3.15-3.1 (m, 4H, NCH2CH2CH3), 1.86-1.84 (m, 2H,
NCH2CH2), 1.73-1.68 (m, 2H, CH2CH2S), 1.60-1.52 (m, 4H, NCH2CH2CH3), 1.43-1.41 (m, 4H,
NCH2CH2CH2CH2CH2CH2S), 0.88 (t, 6H, J = 15 Hz, NCH2CH2CH3); 13C NMR (75 MHz,
chloroform-d  ppm 168.46, 165.64, 164.38, 142.93, 133.94, 132.09, 127.69, 127.13, 126.99,
123.21, 49.88, 37.77, 32.50, 29.03, 28.40, 28.09, 26.25, 21.92, 11.18; IR (cm-1): 3070 (C-H,
aromatic), 2931 (C-H, aliphatic), 1704 (C=O), 1614, 1553 (C=N), 1340, 1153 (O=S=O). Anal.
Calcd for C28H34N4O5S2: C, 58.93; H, 6.00; N, 9.82; O, 14.02; S, 11.24 Found C, 58.98; H, 6.07;
N, 9.89; O, 14.12; S, 11.31.
1
15a: Colorless solid; yield: 78%; Rf: 0.68 (Chloroform: acetone, 9:1); m.p.: 120-121°C; H
NMR (300 MHz, chloroform-d  ppm 8.1 (d, 2H, J = 12 Hz, Ar-H), 7.93 (d, 2H, J = 9 Hz, Ar-
H), 7.62 (s, 4H, Ar-H), 4.58 (s, 2H, SCH2), 3.15-3.10 (m, 4H, NCH2CH2CH3), 1.62-1.5 (m, 4H,
NCH2CH2CH3), 0.88 (t, 6H, J = 15 Hz, NCH2CH2CH3); 13C NMR (75 MHz, chloroform-
d  ppm 164.80, 164.41, 143.22, 139.77, 130.58, 130.14, 129.57, 127.73, 127.16, 126.72,
125.79, 125.74, 122.10, 49.87, 35.97, 21.90, 11.16; IR (cm-1): 3100 (C-H, aromatic), 2969 (C-H,
aliphatic), 1617 (C=N), 1372 (C-S). 1325, 1153 (O=S=O); Anal. Calcd for C22H24F3N3O3S2: C,
52.89; H, 4.84; F, 11.41; N, 8.41; O, 9.61; S, 12.84 Found C, 52.91; H, 4.88; F, 11.47; N, 8.46;
O, 9.67; S, 12.88.
1
15b: Colorless solid; yield: 80%; Rf: 0.68 (Chloroform: acetone, 9:1); m.p.: 93-94°C; H
NMR (300 MHz, chloroform-d  ppm 8.11 (d, 2H, J = 9 Hz, Ar-H), 7.93 (d, 2H, J = 9 Hz, Ar-
H), 7.74 (d, 1H, J = 6 Hz, Ar-H), 7.70 (s, 1H, Ar-H), 7.58 (d, 1H, J = 9 Hz, Ar-H), 7.49 (t, 1H, J
= 18 Hz, Ar-H), 4.59 (s, 2H, SCH2), 3.13 (t, 4H, J = 15 Hz, NCH2CH2CH3), 1.62-1.50 (m, 4H,
NCH2CH2CH3), 0.88 (t, 6H, J = 15 Hz, NCH2CH2CH3); 13C NMR (75 MHz, chloroform-
d  ppm 164.78, 164.41, 143.20, 136.69, 132.64, 131.42, 130.99, 129.36, 127.73, 127.18,
126.74, 125.91, 125.86, 125.08, 125.03, 122.01, 49.86, 36.08, 21.90, 11.16; IR (cm-1): 2970 (C-
H, aliphatic), 1613 (C=N), 1370 (C-S). 1328, 1153 (O=S=O); Anal. Calcd for C22H24F3N3O3S2:
C, 52.89; H, 4.84; F, 11.41; N, 8.41; O, 9.61; S, 12.84 Found C, 52.91; H, 4.88; F, 11.47; N,
8.46; O, 9.67; S, 12.88.
24

1
15c: Colorless solid; yield: 81%; Rf: 0.89 (Chloroform: acetone, 9:1); m.p.: 99-102°C; H
NMR (300 MHz, chloroform-d  ppm 8.12 (dd, 2H, J = 6.9, 1.5 Hz, Ar-H), 7.94 (dd, 2H, J =
6.9, 1.5 Hz, Ar-H), 7.82 (d, 1H, J = 8.4 Hz, Ar-H), 7.70 (d, 1H, J = 7.8 Hz, Ar-H), 7.55 (t, 1H, J
= 15 Hz, Ar-H), 7.45 (t, 1H, J = 15 Hz, Ar-H), 4.76 (s, 2H, SCH2), 3.16-3.11 (m, 4H,
13
NCH2CH2CH3), 1.65-1.50 (m, 4H, NCH2CH2CH3), 0.88 (t, 6H, J = 15 Hz, NCH2CH2CH3); C
NMR (75 MHz, chloroform-d  ppm 165.02, 164.79, 143.17, 134.19, 132.46, 132.06,
128.93,128.50, 127.73, 127.18, 126.79, 126.53, 126.45, 126.01, 122.38, 49.88, 33.25, 21.92,
11.17; IR (cm-1): 3170 (C-H, aromatic), 2969 (C-H, aliphatic), 1615 (C=N), 1370 (C-S); 1344,
1156 (O=S=O); Anal. Calcd for C22H24F3N3O3S2: C, 52.89; H, 4.84; F, 11.41; N, 8.41; O, 9.61;
S, 12.84 Found C, 52.91; H, 4.88; F, 11.47; N, 8.46; O, 9.67; S, 12.88.
1
15d: Colorless solid; yield: 73%; Rf: 0.82 (Chloroform: acetone, 9:1); m.p.: 88-90 °C; H
NMR (300 MHz, chloroform-d  ppm 8.15 (d, 2H, J = 9 Hz, Ar-H), 7.99 (d, 2H, J = 9 Hz, AR-
H), 7.49 (d, 2H, J = 6 Hz, Ar-H), 7.38-7.29 (m, 3H, Ar-H), 4.61 (s, 2 H, SCH2), 3.07 (t, 4H, J =
15 Hz, NCH2CH2CH3), 1.54-1.41 (m, 4H, NCH2CH2CH3), 0.81 (t, 6H, J = 15 Hz,
NCH2CH2CH3); 13C NMR (75 MHz, chloroform-d  ppm 164.75, 164.69, 142.69, 136.95,
129.55, 129.08 128.29, 127.80, 126.88, 50.05, 36.35, 22.05, 11.43; IR (cm-1): 3100 (C-H,
aromatic), 2964 (C-H, aliphatic), 1613 (C=N), 1373 (C-S). 1335, 1152 (O=S=O); Anal. Calcd
for C21H25N3O3S2: C, 58.44; H, 5.84; N, 9.74; O, 11.12; S, 14.86 Found C, 52.91; C, 58.48; H,
5.89; N, 9.77; O, 11.18; S, 14.90.
1
15e: Colorless solid; yield: 78%; Rf: 0.66 (Chloroform: acetone, 9:1); m.p.: 107-108°C; H
NMR (300 MHz, chloroform-d  ppm 8.23-8.20 (m, 2H, Ar-H), 8.13 (d, 2H, J = 9 Hz, Ar-H),
7.99 (d, 2H, J = 9 Hz, Ar-H), 7.78 (d, 2H, J = 9Hz, Ar-H), 4.73 (s, 2H, SCH2), 3.06 (t, 4H, J = 5
Hz, NCH2CH2CH3), 1.51-1.41 (m, 4H, NCH2CH2CH3), 0.80 (t, 6H, J = 15 Hz, NCH2CH2CH3);
13C NMR (75 MHz, chloroform-d  ppm 164.86, 164.35, 147.38, 145.38, 142.73, 130.85,
128.28, 127.83, 126.83, 124.14, 50.03,35.38, 22.03, 11.42; IR (cm-1): 3170 (C-H, aromatic),
2971 (C-H, aliphatic), 1605 (C=N), 1373 (C-S). 1338, 1150 (O=S=O); Anal. Calcd for
C21H24N4O5S2: C, 52.93; H, 5.08; N, 11.76; O, 16.79; S, 13.46 Found C, 52.98; H, 5.14; N,
11.81; O, 16.80; S, 13.53.
3. 2. DENV-2 NS2BNS3pro assay
25

Recombinant DENV2 NS2B/NS3pro was prepared and characterized as described elsewhere
[56]. Protease assays were performed at 37 oC in opaque 96 well plates containing a total volume
of 100 L/well of the reaction mixture. The reaction mixtures were prepared in Tris buffer (50
, pH 9.0) containing 10 mM NaCl, 25% glycerol, 1 mM CHAPS, and 50 mM tetrapeptide
substrate, Bz-NLe-Lys-Arg-Arg-AMC. Reactions were initiated by the addition of 100nM
DENV2 NS2B/NS3pro and the cleavage of the fluorophore tag, AMC was measured after every
minute for a total 30 min incubation time by using a spectrofluorometer (Molecular Devices).
The excitation and emission wavelengths were set at 380 and 460 nm, respectively. To avoid
false-positive hits, the assays were repeated under the same conditions after replacing the
CHAPS (zwitterionic detergent) by 0.005% Brij 35 (nonionic detergent). Background
fluorescence of the mixture, prepared in the absence of enzyme, was measured and subtracted
from those of the reaction mixtures to obtain net change in fluorescence per unit time.
3.2.1. Inhibition of DENV2 NS2B/NS3pro by the synthetic hybrids
The reaction mixture 100L were prepared as described above in the presence of 50 
solution (DMSO) of the test compound. Total concentration of DMSO was not more than 2.5%.
Reactions were initiated by the addition of 100nM recombinant DENV2 NS2B/NS3pro and
cleavage of fluorophore AMC was measured after every minute for a total 30 min. The rate of
change of fluorescence (RFU/sec) in the presence of the test compound (vexp)was compared
with that in the absence of the compound (vcon) to calculate percent inhibition according to the
following equation.
푣푒푥푝
퐼푛ℎ푖푏푖푡푖표푛 = 100 ―
× 100
(
)
[
]
푣푐표푛
For the determination of IC50 values, a three-fold serial dilutions from 150 to 620nM of
the test compounds were prepared in the reaction mixture the enzyme activity was observed as
described above. The residual activity was plotted against the concentration of the test compound
in Graphpad Prism (version 7) by using the non-linear regressions. For the determination of the
mode of inhibition, the enzymatic assays were performed under the same conditions as described
above, by varying concentration (4.6-60 ) of the substrate in the presence (10  and 20 )
and absence of the selected inhibitors. The enzyme kinetics were observed for 30 min, and the
26

data were linearly fit into the Lineweaver-Burk model by using Graphpad Prism software
(version 7)
3.3. Molecular Docking
To investigate the binding mode of the inhibitors with the DENV2 NS2B/NS3 protease,
docking studies were performed by using the reported co-crystal of DENV2 NS2/NS3pro
containing an allosteric inhibitor (PDB ID: 6MO0) and DENV3 NS2B/NS3pro containing a
covalently bound inhibitor at the substrate binding site (3U1I). All Het-atoms and water
molecules were removed, and polar hydrogen atoms were added by using Autodock Tool. The
structures of the hybrid molecules were drawn in Avogadro software and optimized by energy
minimization by using MMFF94 force field. Finally, the pdbqt files were prepared for both
receptor and the ligand and run on Autodock vina software. The top models (with the most
negative energy) were selected for the investigation of the binding interactions by using PyMol
and Ligplot.
4 Conclusion
In summary, two different series of S-benzylated and S-alklyphthalimidated hybrid structures
of 1,3,4-oxadiazole and sulfonamide were synthesized in very efficient way and their potential
against DENV2 NS2B/NS3pro was tested. Initial investigations have identified 8g and 8h are
potential inhibitors of the dengue protease, demonstrating IC50 values as 13.9 and 15.1,
respectively. Computational assessment indicated that these hybrids bind in an allosteric cleft of
the conformatinally open structure of the protease. Additionally, the phthalimide-moeity of the
hybrid structure exhibit important hydrogen bonding interactions with Trp-83 and Asn-167 and
several hydrophobic interactions with the residues in the cavity. Further work is needed to refine
the binding interactions and medicinal properties before these compounds can be further
developed into antiviral therapeutics.
Emails of Authors and Contributions
S.S.H.: shamilahamdani@hotmail.com, executed the syntheses and wrote the initial draft on the synthesis
of the hybrids; B.A.K.: bkhan@ajku.edu.pk, conceived the idea, supervised the study and edited the
chemistry part of manuscript; S.H.: shameed@qau.edu.pk, provided the lab facility and co-supervised
SSH for conducting the organic syntheses; F.B.: 15130009@lums.edu.pk, computationally assessed the
molecular docking data, prepared the docking figures, and wrote the initial draft of the docking section;
27

H.N.S.:
nosheen.saleem@lums.edu.pk,
executed
the
dengue
bioassays;
M.S.:
muhammad.saeed@lums.edu.pk, interpreted the bioassay and molecular docking data and edited the
whole manuscript. E.U.M.: ehsanmughal@gmail.com, helped in the synthesis.
All authors declare no conflict of interest.
Acknowledgements
M.S. acknowledges the Higher Education Commission (HEC) of Pakistan for providing funds to
conduct the dengue protease assays under the National Research Program for Universities
(NRPU grant #5912).
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