Synthesis, molecular modeling and antiviral activity of novel 5-fluoro-1H-indole-2,3-dione 3-thiosemicarbazones

Article abstract of DOI:10.1016/j.bioorg.2020.104202

In this work, novel 5-fluoro-1-methyl/ethyl-1H-indole-2,3-dione 3-[4-(substituted phenyl)-thiosemicarbazones] 6a-n and 7a-n were synthesized. The antiviral effects of the compounds were tested against HSV-1 (KOS), HSV-2 (G) HSV-1 TK^- KOS ACV^r and VV in HEL cell cultures using acyclovir and ganciclovir as standards, and Coxsackie B4 virus in Vero cell cultures using ribavirin and mycophenolic acid as standards. R₂ ethyl substituted 7 derivatives were found effective against viruses tested. R₁ 4-CF₃ substituted 7d, R₁ 4-OCH₃ substituted 7 g and R₁ 3-Cl substituted 7 l showed activity against HSV-1 (KOS), HSV-2 (G) HSV-1 TK^- KOS ACV^r and VV. Whereas only R₁ 4-Br substituted 7n has selective activity against coxsackie B4 virus. Molecular modeling studies of 7d and 7l were performed to determine binding side on HSV-1 glycoprotein B and D, HSV-2 glycoprotein B structures.

Full text of DOI:10.1016/j.bioorg.2020.104202

Bioorganic Chemistry 104 (2020) 104202
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, molecular modeling and antiviral activity of novel 5-fluoro-1H-
indole-2,3-dione 3-thiosemicarbazones
a,b,c,⁎
d
e
a
Zekiye Şeyma Sevinçli
, Gizem Nur Duran , Mehmet Özbil , Nilgün Karalı
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul University, Istanbul 34116, Turkey
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Van Yuzuncu Yil University, Van 65080, Turkey
Department of Pharmaceutical Chemistry, Institute of Health Sciences, Istanbul University, Istanbul 34126, Turkey
Chemistry Department, Faculty of Arts and Sciences, Marmara University, Istanbul 34722, Turkey
b
c
d
e
Department of Biotechnology, Institute of Biotechnology, Gebze Technical University, Kocaeli 41400, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this work, novel 5-fluoro-1-methyl/ethyl-1H-indole-2,3-dione 3-[4-(substituted phenyl)-thiosemicarbazones]
6a-n and 7a-n were synthesized. The antiviral effects of the compounds were tested against HSV-1 (KOS), HSV-2
Synthesis
Antiviral activity
Thiosemicarbazones
-
r
(
G) HSV-1 TK KOS ACV and VV in HEL cell cultures using acyclovir and ganciclovir as standards, and Coxsackie
B4 virus in Vero cell cultures using ribavirin and mycophenolic acid as standards. R
2
3
ethyl substituted 7 deri-
5
-Fluoro-1H-indole-2,3-diones
vatives were found effective against viruses tested. R
1
4-CF
3
substituted 7d, R
1
4-OCH
substituted 7 g and R
1
3-
Molecular modeling
-
r
Cl substituted 7 l showed activity against HSV-1 (KOS), HSV-2 (G) HSV-1 TK KOS ACV and VV. Whereas only
R 4-Br substituted 7n has selective activity against coxsackie B4 virus. Molecular modeling studies of 7d and 7l
1
were performed to determine binding side on HSV-1 glycoprotein B and D, HSV-2 glycoprotein B structures.
1
. Introduction
the pancreas [11–13]. There are studies in the literature on the effect of
thiosemicarbazones on coxsackie B4 virus [14,15]. In 2013, Abbas and
1
H-Indole-2,3-dione (isatin) and its derivatives have a broad spec-
co-workers found that 5-fluoroisatin 3-thiosemicarbazone derivatives
inhibit viral replication [3].
trum of biological properties including antituberculosis, antiviral and
anticancer activities. There are several reports on antiviral activities of
In the present study, 5-fluoro-1-methyl/ ethyl-1H-indole-2,3-dione
3-[4-(substituted phenyl)thiosemicarbazone] derivatives 6a-n and 7a-n
were synthesized by reaction of 4-(substituted phenyl)thiosemicarbazid
derivatives 2a-n with 5-fluoro-1-methyl/ethyl-1H-indole-2,3-dione 4/
5. The structures of all the synthesized compounds were determined by
analytical and spectral methods. The molecular and isomeric structures
of 6h and 6j were determined by X-ray single crystal diffraction ana-
lysis [16,17]. Antiviral activities of 6a-n and 7a-n were evaluated
1
H-indole-2,3-dione 3-thiosemicarbazone derivatives [1–3]. Methisa-
zone (N-methylisatin 3-thiosemicarbazone), synthesized for the vac-
cinia virus causing smallpox, is the first derivative of isatin 3-thiose-
micarbazone [4,5]. Herpes simplex virus-1 (HSV-1), which is a member
of the HSV family and acquired mainly in young ages, causes infections
in the upper half of the body, while herpes simplex virus-2 (HSV-2)
causes genital infections through sexual contact [6,7]. It has been de-
termined that thiocarbonyl group is essential for antiherpetic activity
against HSV-1, HSV-2 and the presence of a 4-substituted phenyl ring at
the N4 position of thiosemicarbazone increases antiviral activity [8,9].
Coxsackie viruses are enteroviruses of the Picornaviridae family which
are classified as group A and group B. Coxsackie virus group B, which
has six members, causes childhood myocarditis, cardiomyopathy, dia-
betes and neurological disorders [10]. There are many reports showing
that Coxsackie B4 virus causes type 1 diabetes by damaging β-cells in
-
against HSV-1 (KOS), HSV-2 (G) HSV-1 TK KOS strain resistant to
r
acyclovir (ACV ) and VV in HEL cell (human embryonic lung fibroblast
cell) cultures, and Coxsackie B4 virus in Vero cell (African green
monkey kidney cell) cultures. Molecular modeling studies of 7d and 7l
were completed on HSV-1 glycoprotein B (gB), HSV-1 glycoprotein D
(gD), and HSV-2 (gD) protein structures.
-
r
-
Abbreviations: HSV-1 (KOS), herpes simplex virus-1 (strain KOS); HSV-1 TK KOS ACV , herpes simplex virus-1 (TK KOS strain resistant to acyclovir); HSV-2, herpes
simplex virus-2 (strain G); VV, vaccinia virus; gB, glycoprotein B; gD, glycoprotein D; EC50, 50% effective concentration; MCC, minimum inhibitory concentration;
HEL cell, human embryonic lung fibroblast cell; Vero cell, African green monkey kidney cell
⁎
Corresponding author at: Department of Pharmaceutical Chemistry, Van Yuzuncu Yil University, 65080 Van, Turkey.
E-mail address: seymasevincli@yyu.edu.tr (Z.Ş. Sevinçli).
https://doi.org/10.1016/j.bioorg.2020.104202
Received 8 June 2020; Received in revised form 11 August 2020; Accepted 18 August 2020
Available online 26 August 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.

Z.Ş. Sevinçli, et al.
Bioorganic Chemistry 104 (2020) 104202
Table 1
Substituents and substituent positions of 6a-n and 7a-n.
Compound
R
1
R
2
Compound
R
1
R
2
6
6
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
3
3
3
3
3
3
3
7a
7b
7c
7d
7e
7f
H
C
C
C
C
C
C
C
C
C
C
C
C
C
C
2
H
2
H
2
H
2
H
2
H
2
H
2
H
2
H
2
H
2
H
2
H
2
H
2
H
2
H
5
5
5
5
5
5
5
5
5
5
5
5
5
5
3-CH
4-CH
3
3
3
3-CH
4-CH
3
3
3
4-CF
4-CF
4-C
2
H
5
3
3
3
3
4-C
2
H
5
3-OCH
3-OCH
3
3
3
g
h
i
4-OCH
7g
7h
7i
4-OCH
4-SCH
4-OCF
3-F
4-SCH
4-OCF
3-F
3
j
7j
k
l
4-F
7k
7l
4-F
3-Cl
4-Cl
4-Br
3-Cl
4-Cl
4-Br
m
n
7m
7n
2
. Results and Discussion
2.1. Chemistry
In this study, hydrazine hydrate was reacted with substituted phenyl
isothiocyanates 1a-n in ethanol to give 4-(substituted phenyl)thiose-
micarbazides 2a-n. A suspension of 5-fluoro-1H-indole-2,3-dione 3
potassium carbonate (K
2
CO ) and potassium iodide (KI) in anhydrous
3
N,N-dimethylformamide (DMF) was added iodomethane (CH
moethane (C Br) to yield 5-fluoro-1-methyl/ethyl-1H-indole-2,3-
dione 4/5. A series of 5-fluoro-1-methyl/ethyl-1H-indole-2,3-dione 3-
3
I)/ bro-
H
2 5
[
4-(substituted phenyl) thiosemicarbazones] 6a-n and 7a-n (Table 1)
Scheme 1. Synthesis of 6a-n and 7a-n. Reagents and conditions: i) Ethanol,
were synthesized by reacting 2a-n with 5-fluoro-1-methyl/ethyl-1H-
indole-2,3-dione 4/5 in ethanol. A catalytic amount of sulfuric acid
stirred, cooled ii) K
2
CO
3
, KI, DMF, CH
3
I/ C
2
5
H Br, strirred, reflux iii) Ethanol or
ethanol and H
2
SO
4
, reflux.
(
H
2
SO ) was added to the reaction to synthesize 6a, 6b, 6d, 6e, 6 l, 7a
4
and 7b. The structures of 6a-n and 7a-n were confirmed by analytical
7
7
.21–7.24 ppm as doublet of doublet whereas it was signaled at δ
.20–7.23 ppm with phenyl C3,5-H in 7c and δ 7.20–7.34 ppm with
1
13
and spectral (IR, H NMR, C NMR, HSQC-2D, HMBC-2D, HRMS-ESI+
and LCMS-ESI+) data (Scheme 1).
indole C
6
-H and phenyl protons as multiplet in 7f and 7k.
IR spectra of 6a-n and 7a-n showed two or three separate absorp-
13
13
C NMR-decoupled run on 6a-d, 6f, 6g, 6i-l, 6n, 7c-e, 7g-n and
−
1
tion bands in the 3354–3196 cm
region resulting from the NH
1
H– C cross correlation run on 6e, 6h, 6m, 7a, 7b and 7f allowed the
complete assignment of the proton and carbon NMR signals. The re-
sonances of indole C , indole C and C=S were detected at δ
30.70–131.76, 160.73–161.40 and 175.62–177.23 ppm, respectively.
Fluorine substituted indole signal was observed at
stretchings of the thioamide groups. The aromatic = CeH C=N
stretching band, aromatic C=C stretching and NeH bending combi-
−1
3
2
nation band were identified at 3101–3010 and 1625–1473 cm
re-
1
gions, respectively. The lactam C=O and thioamide C=S stretchings
C
5
δ
−
1
were observed at 1674–1701 and 1253–1278 cm
regions, respec-
and
1
59.03–159.32 ppm as doublet (J = 238–242 Hz). Mass spectra of 6a-
tively.
d, 6f, 6g, 6i, 6k-n, 6n, 7b-n were taken by Electrospray Ionization
1
H NMR spectra of 6a-n and 7a-n displayed the thioamide N
4
+
positive ionization method (ESI+). In the spectra [M+H] peaks were
N protons of the thiosemicarbazone moiety as two separate singlets at
2
observed which confirmed their molecular weights. The molecular and
isomeric structures of 6h and 6j were determined by X-ray single crystal
diffraction analysis and the stable isomer was found to be in Z config-
uration (Figs. 1 and 2) [16,17].
δ 10.73–11.00 and 12.55–12.73 ppm regions, respectively. The ex-
changeable NH proton signals were further confirmed by deuterium
oxide (D O) exchange experiments of 6c and 7l. Observation of the
2
methyl signals in the spectra of 6a-n, methyl and methylene signals in
the spectra of 7a-n proved 1-alkylation while the lactam NH signal of
the 2-indolinone ring was not observed in the spectra. 2-Indolinone and
phenyl ring proton resonances of new compounds were found in the
expected regions with appropriate coupling constants and integral va-
1
lues. The H NMR spectra of 6a-n and 7a-n, indole C
4
-H usually was
observed at δ 7.63–7.69 ppm as doublet of doublet making the ortho
coupling with fluorine and meta coupling with the indol C
6
-H. At δ
-H gave a multiplet signals with phenyl protons in
j, 6l, 6m, 7l and 7m. The indole C -H usually signaled at δ
.27–7.30 ppm as triplet of doublet making meta coupling with the
-H and ortho coupling with fluorine and indol C -H. At δ
.20–7.35, indole C -H gave a multiplet signals with phenyl protons or
indole C -H in 6a, 6b, 6e, 6f, 6h, 6k, 6l, 7a, 7b, 7e, 7f, 7h and 7k. The
indole C -H signaled at δ 7.14–7.16 ppm as doublet of doublet in 6a-n
making the meta coupling with fluorine and ortho coupling with the
indol C -H. The indole C -H in 7a-n generally was observed at δ
7
6
7
.59–7.68, indole C
4
6
indol C
4
7
7
6
7
7
6
7
Fig. 1. View of the molecular structure of 6h, with the atom labelling16.
2

Z.Ş. Sevinçli, et al.
Bioorganic Chemistry 104 (2020) 104202
was bound through mostly hydrogen bonds (H-bonds). Moreover, Tyr
3
01 creates a halogen bond with F atom of the ligand and Trp 356
creates π-stacking with the ligand. The binding affinity of the ligand
was calculated as −7.9 kcal/mol.
Ligand 7l was bound to the binding site formed by amino acids Gly
3
02, Arg 304, Gln 321, Thr 340, Thr 341, and Trp 356, which is very
similar to the binding site of ligand 7d (Fig. 3).
Due to bulky and polar CF
3
group, compared to smaller Cl group,
the orientation of 7l shifted towards outwards of the protein. Trp 356
and Gly 302 create two small hydrophobic clefts and number of H-
bonds formed between protein and ligands decreased in the case of li-
gand 7l (Table 4). Thus, the binding affinity of the ligand was calcu-
lated as −7.4 kcal/mol, 0.5 kcal/mol lower than that of ligand 7d.
2
.3.2. HSV-1 (gD)
Fig. 2. View of the molecular structure of 6j, with the atom labelling17.
HSV-1 (gD) was modeled as a homodimer according to its biological
assembly. Compound 7d bound to HSV-1 (gD) protein right at the in-
terface of two monomers. The the binding site formed by amino acids
by Asp 139*, Ser 140*, Leu 139, Pro 194, Tyr 234, Ser 235, Ser 235*,
and Ile 238 (Fig. 4). The ligand interacted through H-bonds with amino
acids Asp 139*, Pro 194, and Ser 235. It also created hydrophobic in-
teractions with Ser 140*, Leu 193, Tyr 234, Ser 235*, and Ile 238. These
interactions are listed in Table 4. The binding affinity of compound 7d
was calculated as −7.3 kcal/mol at this binding site. The positioning of
compound 7d might be very critical for disrupting the assembly of
homodimer HSV-1 gD protein.
2
.2. Antiviral Activity
The antiviral effects of the compounds were tested against HSV-1
-
r
(
KOS), HSV-2 (G), HSV-1 TK KOS ACV and VV in HEL cell cultures,
Coxsackie B4 virus in Vero cell cultures. For all derivatives, the 50%
effective concentration (EC50) and minimum cytotoxic concentration
(
MCC) were determined. R
2
ethyl substituted 7d, 7g and 7l were ef-
-
fective and nontoxic against HSV-1 (KOS), HSV-2 (G), HSV-1 TK KOS
r
ACV and VV at low μM doses. 7n showed antiviral effect against
Coxsackie B4 virus at 10 µM. The most effective 7 derivative against
Ligand 7l was bound to the binding site formed by amino acids Ser
HSV-1 and HSV-2 strains was found to be R
1
4-CF substituted 7d. The
3
6
2
8, Leu 70, Glu 146, Leu 152, His 154, Val 233, Trp 241, Gly 243, Pro
44, Ala 246, and Pro 247. This binding site was highly non-polar.
compound is effective at 6.8, 7.6 and 2.3 µM against HSV-1 (KOS), HSV-
-
r
2
7
7
(G) and HSV-1 TK KOS ACV , respectively. While R
m and R 3-F substituted 7j are inactive and toxic, R
1
1
4-Cl substituted
3-Cl substituted
Thus, the ligand was embedded inside the protein, forming mainly
hydrophobic interactions. Moreover, Ser 68 creates a H-bond with O
atom of the ligand and Gly 243 creates H-bond with N of the ligand. The
binding affinity of the ligand was calculated as −7.2 kcal/mol.
1
l was found to be nontoxic and effective at 6.8, 8.9 and 6.8 µM against
-
r
HSV-1 (KOS), HSV-2 (G) and HSV-1 TK KOS ACV , respectively.
Whereas R
1
3-OCH substituted 7f is inactive against all viruses tested.
3
R
1
4-OCH
3
substituted 7g is active against HSV-1 (KOS), HSV-2 (G) and
2
.3.3. HSV-2 (gD)
-
r
HSV-1 TK KOS ACV at 20 µM. 7d, 7g and 7l were found to be high
Molecular docking simulations were only performed on HSV-2 (gD)
-
r
effective against HSV-2 (G) and HSV-1 TK KOS ACV compared to
protein as there is no 3-D structure of HSV-2 (gB) protein available.
Upon molecular docking to HSV-2 (gD) protein, Ligand 7d was bound
to the binding site formed by amino acids Ser 52, Ile 80, Ala 88, Tyr
-
brivudine and also 7d was determined more active against HSV-1 TK
r
KOS ACV compared to acyclovir. 7d and 7l were found to be high
effective against VV compared to brivudin, acyclovir and ganciclovir at
1
20, Ile 170, Asp 172, Trp 173 (Fig. 5). The binding site was formed by
8
.9 µM whereas 7g was determined more active compared to acyclovir
both polar and mostly non-polar amino acids. The ligand interacted
through H-bonds with amino acids Ser 52, Tyr 120, and Asp 172. Two
and ganciclovir at 45 µM. Only R
effect at 10 µM against Coxsackie B4 virus in tested compounds (Tables
and 3). The test results show the importance of the ethyl group in
position 1 of the indole ring and the electron-attracting groups as 4-CF
-OCH , 3-Cl and 4-Br in the phenyl ring of the thiosemicarbazone
1
4-Br substituted 7n had selective
H-bonds were formed between Ser 52 and CF group of the ligand. It
3
2
also created π-stacking with Trp 173. Ile 80, Ala 88, and Ile 170 formed
hydrophobic clefts, where indole group fitted in. The binding affinity of
the ligand was calculated as −7.4 kcal/mol.
3
,
4
3
residue for antiviral effect. Whereas, the R methyl substituted 6 deri-
2
Ligand 7l was bound to the binding site formed by amino acids Pro 51,
Ile 53, Thr 56, Tyr 58, Pro 74, Glu 76, Glu 175, Ile 176, Thr 177, and Leu
vatives were found to be inactive against all viruses tested (Table S1-
S2). Based on these results, more effective 5-fluoro-1-ethyl-2-indolinone
derivatives can be designed and synthesis.
2
52. This binding pocket was buried inside the protein, compared to
binding pocket for ligand 7d. The ligand interacted with the protein
mainly through H-bonds from residues Thr 56, Tyr 58, and Thr 177. It also
created hydrophobic interactions with Pro 51, Ile 53, Pro 74, sidechain
ethyl groups of Glu 76, and Leu 252 amino acids. The Cl group created
strong H-bond with backbone amine H atom of Leu 252. Moreover, indole
group interacted with glutamate ion of Glu 175 through π-anion interac-
tion and F group with Ile 176 through halogen bond (Table 6).
2
.3. Molecular Modeling
Binding of compounds 7d and 7l on HSV-1 (gB), HSV-1 (gD), and
HSV-2 (gD) were evaluated computationally. Lack of 3D structure of
HSV-2 (gB) protein restrained the full comparison. However compar-
isons between two different proteins from the same virus (HSV-1) and
the same protein (gD) from two different types of viruses (HSV-1 and
HSV-2) could be made.
The binding affinity of the ligand was calculated as −7.5 kcal/mol,
0.1 kcal/mol higher than that of ligand 7d. Main reason for this slight
increment was the more buried ligand position for ligand 7l inside the
protein, thus increased number of interactions.
2
.3.1. HSV-1 (gB)
When binding affinities of compounds 7d and 7l are compared on
the same virus, HSV-1, it was clear that binding affinities were higher
for gB protein than they were for gD protein (Tables 4 and 5). Binding
affinity for compound 7d on HSV-1 (gB) protein was calculated as
−7.9 kcal/mol, which was the highest binding affinity in this study.
The binding affinity comparison for compounds 7d and 7l for the same
Molecular docking simulations of 7d to HSV-1 glycoprotein B (gB)
produced the binding pose in Fig. 3. The ligand was bound to the
binding site formed by amino acids Tyr 301, Arg 304, Ser 307, Gln 321,
Thr 341, Asn 343, Trp 356, Lys 359. These interactions are listed in
Table 4. The binding pocket consisted of polar residues, thus, ligand
3

Z.Ş. Sevinçli, et al.
Bioorganic Chemistry 104 (2020) 104202
Table 2
-
r
Antiviral activities of 7 derivatives against HSV-1 (KOS), HSV-2 (G) HSV-1 TK KOS ACV and VV in HEL cell cultures.
EC50 (µM)
-
r
Compound
R
1
R
2
MCC
HSV-1 (KOS)
HSV-2 (G)
HSV-1 TK KOS ACV
VV
7
a
j
H
C
2
2
H
5
5
≥50
> 50
> 50
0.03
> 50
> 50
146
> 50
> 50
250
> 50
> 50
100
7
3-F
C
H
> 50
> 250
> 250
> 100
> 100
> 100
≥20
Brivudin
Acyclovir
Ganciclovir
0.2
0.9
113
> 250
> 100
> 100
> 100
12
0.01
0.02
> 100
> 100
7.6
0.03
> 100
> 100
4
7
7
7
b
3-CH
3
3
3
C
2
C
2
C
2
H
5
H
5
H
5
> 100
> 100
7.6
c
4-CH
d (test 1)
4-CF
(
test 2)
100
6.8
8.9
2.3
8.9
7
7
7
7
7
e
f
4-C
2
H
5
3
3
3
C
2
C
2
C
2
C
2
C
2
H
5
H
5
H
5
H
5
H
5
≥100
≥100
100
> 100
> 100
> 100
> 100
8.9
> 100
> 100
> 100
> 100
12
> 100
> 100
> 100
> 100
7.6
> 100
> 100
> 100
> 100
10
3-OCH
h
i
4-SCH
4-OCF
3-Cl
≥20
l (test 1)
> 100
100
(test 2)
6.8
8.9
6.8
8.9
Brivudin
> 250
> 250
> 100
100
0.08
> 250
0.29
0.3
250
22
Acyclovir
Ganciclovir
3.8
3.5
> 250
> 100
> 100
45
0.11
0.35
20
7
g (test 1)
4-OCH
3
C
2
H
5
> 100
20
20
(test 2)
> 100
4
34
50
7
7
7
k
4-F
C
2
C
2
C
2
H
5
H
5
H
5
> 100
> 100
> 100
0.04
> 100
> 100
> 100
50
> 100
> 100
> 100
50
> 100
> 100
> 100
22
m
n
4-Cl
4-Br
≥4
≥4
Brivudin
> 250
> 250
> 100
Acyclovir
Ganciclovir
0.2
0.2
85
> 250
> 100
0.032
0.06
8.9
a
b
EC50: 50% effective concentration, producing 50% inhibition of virus-induced cytopathic effect, as determined by microscopy.
MCC: minimum inhibitory concentration, or compound concentration causing minimal changes in cell morphology, as assessed by microscopy.
-
Table 3
were found to be effective against HSV-1 (KOS), HSV-2 (G), HSV-1 TK
r
Antiviral activities of 7 derivatives against Coxsackie B4 virus in Vero cell
KOS ACV and VV at low micromolar levels, while 7n showed antiviral
effect against Coxsackie B4 virus at 10 µM. Molecular docking simu-
lations were performed for 7d and 7l on HSV-1 (gB), HSV-1 (gD), and
HSV-2 (gD). The binding affinities of the 7d were calculated as −7.9,
cultures.
EC50 (µM)
Compound
R
1
R
2
MCC
Coxsackie B4 virus
−
7.3 and −7.4 kcal/mol on HSV-1 (gB), HSV-1 (gD), and HSV-2 (gD)
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
H
C
C
C
C
C
C
C
C
C
C
2
2
2
2
2
2
2
2
2
2
H
H
H
H
H
H
H
H
H
H
5
5
5
5
5
5
5
5
5
5
> 50
≥20
≥4
> 50
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 50
> 100
8.9
respectively, while for 7l were calculated as −7.4, −7.2 and
7.5 kcal/mol on HSV-1 (gB), HSV-1 (gD), and HSV-2 (gD).
3-CH
4-CH
3
3
3
−
4-CF
≥4
4-C
2
H
5
3
3
3
≥4
3-OCH
4
4
. Experimental protocols
h
i
4-SCH
4-OCF
3-F
20
≥4
4.1. Chemistry
j
≥10
4
l
3-Cl
DS-10.000 (µg/ml)
Ribavirin
> 100
> 250
> 100
100
Melting points were estimated with a Buchi 540 melting point ap-
> 250
> 100
> 100
> 100
> 100
10
paratus in open capillaries and are uncorrected. Elemental analyses
were performed on a Thermo Finnigan Flash EA 1112 elemental ana-
lyzer. IR spectra were recorded on KBr discs, using a Shimadzu
Mycophenolic acid
7
7
7
7
g
4-OCH
4-F
3
C
2
C
2
C
2
C
2
H
5
H
5
H
5
H
5
k
≥100
≥100
≥100
100
1
13
m
4-Cl
IRAffinity-1 FTIR spectrometer. H NMR, C NMR, HSQC-2D and
HMBC-2D spectra were obtained on VarianUNITY INOVA 500, Varian
Mercury (Agilent) 400 and Varian Mercury 300 NMR spectro-
n (test 1)
4-Br
(
test 2)
10
DS-10.000 (µg/ml)
Ribavirin
> 100
≥250
> 100
1.8
photometers using DMSO‑d . HRMS and LC/MS datas were recorded by
6
> 250
> 100
Mycophenolic acid
Thermo Scientific, TSQ-Quantum Access and Waters 2695 Alliance
Micromass ZQ LC/MS, respectively.
gD protein for HSV-1 and HSV-2 yielded very similar affinities. Affi-
nities were very similar between −7.2 and −7.5 for both proteins.
However, for HSV-1 (gD) protein compound 7d was binding slightly
better than compound 7l, but for HSV-2 (gD) protein compound 7l was
calculated to bind slightly better than compound 7d (Tables 5 and 6).
4
.1.1. The synthesis of 4-(substituted phenyl) thiosemicarbazides (2a-n)
To a solution of hydrazine hydrate (5 mmol) in ethanol (10 mL), a
suspension of (substituted phenyl)isothiocyanates 1a-n (5 mmol) in
ethanol (10 mL) was added dropwise with vigorous stirring and cooling
in an ice bath. The mixture was allowed to stand overnight. The ob-
tained solid molecules were filtered, washed three times with water
3
. Conclusions
(
30 mL), dried, recrystallized from ethanol. The structures of 2a-n in
the literature were confirmed by analytical and spectral data by re-
In summary, while the ethylated derivatives were determined to be
searcher except 2e [18–22].
active, no activity was observed in the methyl derivatives. 7d, 7l and 7g
4

Z.Ş. Sevinçli, et al.
Bioorganic Chemistry 104 (2020) 104202
Fig. 3. A) Docking poses for compound 7d
in cyan, 7l in orange, on HSV-1 (gB) pro-
tein. Polar residues were shown in green,
basic residues in blue, and hydrophobic re-
sidues in white. B) Zoomed in view of li-
gand 7d. C) Zoomed in view of ligand 7l.
(
For interpretation of the references to
colour in this figure legend, the reader is
referred to the web version of this article.)
Table 4
reaction was controlled with TLC method. The product was poured onto
ice–water. The solid molecules formed were filtered, dried, re-
crystallized from ethanol. The structures of 4 and 5 in the literature
were confirmed by analytical and spectral data by researcher [23–25].
Binding pockets on HSV-1 (gB) protein.
Compound
Interacting Amino
Interaction Types
Binding Affinity
(kcal/mol)
Acids
7
d
Tyr 301
Arg 304
Ser 307
Gln 321
Thr 341
Asn 343
Trp 356
Lys 359
Gly 302
Arg 304
Gln 321
Thr 340
Thr 341
Trp 356
Halogen Bond
H-Bond
−7.9
4
.1.4. The synthesis of 5-fluoro-1-methyl/ ethyl-1H-indole-2,3-dione 3-[4-
H-Bond
(
substituted phenyl) thiosemicarbazone] derivatives (6a-n and 7a-n)
H-Bond
H-Bond
4-(Substituted phenyl) thiosemicarbazides (2a-n) (3.5 mmol) was
H-Bond
added to a solution of 5-fluoro-1-methyl/ ethyl-1H-indole-2,3-diones 4/
π-Stacking
H-bond
5
(3.5 mmol) in ethanol (20 mL) or containing a catalytic amount of
H
2
SO in ethanol (20 mL) (for 6a, 6b, 6d, 6e, 6l, 7a and 7b). After the
4
7
l
Hydrophobic
H-Bond
−7.4
mixture was refluxed on a water bath. Completion of the reaction was
controlled with TLC method. The product formed after cooling was
filtered and washed with ethanol and recrystallized from ethanol.
H-Bond
H-Bond
Hydrophobic
π-Stacking
4
.1.5. 5-Fluoro-1-methyl-1H-indole-2,3-dione 3-[4-(phenyl) thiosemica-
4
.1.2. 4-(4-Ethylphenyl)thiosemicarbazide (2e)
rbazone] (6a)
White powder (88%): mp 133–135 °C; IR (KBr): υ 3340, 3277, 3190
Red powder (88%): mp 242–247 °C; IR (KBr): υ 3300, 3232 (NH),
1
1
(
NH), 1278 (CeN), 1207 (C=S). H NMR (DMSO‑d
6
/ 300 MHz) ppm:
1691 (C=O), 1276 (C=S). H NMR (DMSO‑d
6
/ 500 MHz) ppm: 3.22
-H),
0
.97 (3H, t, J: 7.6 Hz, CH
), 6.92–6.98 (2H, m, AA′BB′ system phenyl C3,5-H),
.21–7.31 (2H, m, AA′BB′ system, phenyl C2,6-H), 8.85 (1H, s, N -H),
.40 (1H, s, N -H). C NMR (DECOUPLED DMSO‑d / 75 MHz): 16.00
), 27.89 (CH ), 124.03 (phenyl C2,6), 127.58 (phenyl C3,5), 137.07
), 139.93 (phenyl C ), 179.63 (C=S).
2
CH
3
), 2.37 (2H, q, J: 7.6 Hz, CH
2
CH
3
), 4.56
(3H, s, indole N-CH
3
), 7.16 (1H, dd, J: 8.8, 3.9 Hz, indole C
7
(
2H, s, NH
2
7.27–7.32 (2H, m, indole C
6
-H, phenyl C -H), 7.43 (2H, t, J: 7.8 Hz,
4
7
9
4
phenyl C3,5-H), 7.60 (2H, d, J: 7.8 Hz, phenyl C2,6-H), 7.67 (1H, dd, J:
1
3
13
8.0, 2.7 Hz, indole C
4
-H), 10.76 (1H, s, N
4
-H), 12.55 (1H, s, N
2
-H).
C
2
6
(
CH
3
2
NMR (DECOUPLED DMSO‑d
(d, J: 26.1 Hz, indole C ), 111,59 (d, J: 8,4 Hz, indole C
4.0 Hz, indole C ), 121.24 (d, J: 9.6 Hz, indole C3a), 126.20 (phenyl
2,6), 126.82 (phenyl C ), 129.02 (phenyl C3,5), 131,28 (d, J: 2.7 Hz,
indole C ), 138.82 (phenyl C ); 140.53 (indole C ), 159.24 (d, J:
6
/ 125 MHz): 26.46 (indole N-CH
3
), 108.72
(
phenyl C
1
4
4
7
), 117.90 (d, J:
2
6
C
4
4
.1.3. The synthesis of 5-fluoro-1-methyl/ ethyl-1H-indole-2,3-dione (4
3
1
7a
and 5)
237.9 Hz, indole C ), 161.38 (indole C ), 176.84 (C=S). LC/MS (ESI)
5
2
+
+
A suspension of 5-fluoro-1H-indole-2,3-dione 3 (5 mmol), K
2
CO
3
[M+H]
C
H
FN OS: 329.37141; Found ([M+H] : 329.4. Anal.
1
6
14
13
4
(
7 mmol) and KI (1 mmol) in anhydrous DMF (5 mL) was stirred for
Calcd for C
H
FN OS: C, 58.52; H, 3.99; N, 17.06 Found: C, 58.46; H,
1
6
4
3
0 min at room temperature. After addition of CH
3
I or C
H
2 5
Br
4.05; N, 16.72.
(
15 mmol), the mixture was heated at 50 °C for 4 h. Completion of the
5

Z.Ş. Sevinçli, et al.
Bioorganic Chemistry 104 (2020) 104202
Fig. 4. A) Docking pose for compound 7d in cyan, B) Docking pose for compound 7l in orange on HSV-1 (gD) protein. In figure B orientation of protein was rotated
1
80°. Monomers are presented with gold and silver colors. Polar residues were shown in green, basic residues in blue, acidic residues in red, and hydrophobic residues
in white. C) Zoomed in view of ligand 7d. D) Zoomed in view of ligand 7l. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
Fig. 5. A) Docking poses for compound 7d in cyan, 7l in orange, on HSV-2 (gD) protein. Polar residues were shown in green, acidic residues in red, and hydrophobic
residues in white. B) Zoomed in view of ligand 7d. C) Zoomed in view of ligand 7l. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
6

Z.Ş. Sevinçli, et al.
Bioorganic Chemistry 104 (2020) 104202
1
Table 5
(NH), 1683 (C=O), 1276 (C=S). H NMR (DMSO‑d
2.31 (3H, s, CH ), 3.21 (3H, s, indole N-CH ), 7.15 (1H, dd, J: 8.6,
.0 Hz, indole C -H); 7.21–7.23 (2H, m, AA′BB′ system, phenyl C3,5-H),
6
/ 400 MHz) ppm:
Binding pockets on HSV-1 (gD) protein.
3
7
3
4
Compound
Interacting Amino
Interaction Types
Binding Affinity
(kcal/mol)
Acids
7.28 (1H, td, J: 9.0, 2.8 Hz, indole C -H), 7.44–4.47 (2H, m, AA′BB′
6
system, phenyl C2,6-H), 7.66 (1H, dd, J = 8.0, 2.8 Hz, indole C
4
-H),
7
d
Asp 139*
Ser 140*
Leu 193
Pro 194
Tyr 234
Ser 235
Ser 235*
Ile 238
H-Bond
−7.3
13
1
0.78 (1H, s, N
4
-H, D
2
O exch.), 12.55 (1H, s, N
2
-H, D
2
O exch). C NMR
Hydrophobic
Hydrophobic
H-Bond
(
DECOUPLED DMSO‑d
CH ), 108.60 (d, J: 26.2 Hz, indole C
117.76 (d, J: 24.1 Hz, indole C ), 121.17 (d, J: 9.6 Hz, indole C3a),
25.96 (phenyl C2,6), 129.36 (phenyl C3,5), 131.08 (d, J: 3.4 Hz, indole
6
/ 100 MHz): 21.08 (CH
3
), 26.34 (indole N-
3
4
), 111.49 (d, J: 8.2 Hz, indole C
7
),
Hydrophobic
H-Bond
6
1
Hydrophobic
Hydrophobic
H-Bond
C
3
), 136.00 (phenyl C
1
), 136.14 (phenyl C ), 140.40 (indole C7a),
4
1
59.12 (d, J: 238.1 Hz, indole C
5
), 161.29 (indole C
2
), 176.70 (C=S).
7
l
Ser 68
−7.2
+
+
Leu 70
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
H-Bond
HRMS (ESI) [M+H]
C
17
H
16FN
4
OS: 343.10234; Found [M+H]
:
Glu 146
Leu 152
His 154
Val 233
Trp 241
Gly 243
Pro 244
Ala 246
Pro 247
343.10239. Anal. Calcd for C17
H
15FN OS: C, 59.63; H, 4.42; N, 16.36
4
Found: C, 59.75; H, 4.34; N, 16.25.
4.1.8. 5-Fluoro-1-methyl-1H-indole-2,3-dione 3-[4-(4-trifluoromethylph-
enyl)thiosemicarbazone] (6d)
Hydrophobic
Hydrophobic
Hydrophobic
Orange powder (86%): mp 217–220 °C; IR (KBr): υ 3278, 3224
1
(
NH), 1681 (C=O), 1278 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
3
.21 (3H, s, indole N-CH
7.30 (1H, td, J: 9.2, 2.8 Hz, indole C
indole C -H), 7.78–7.80 (2H, m, AA′BB′ system, phenyl C2,6-H),
.92–7.94 (2H, m, AA′BB′ system, phenyl C3,5-H), 10.98 (1H, s, N -H),
/ 100 MHz):
), 111.53 (d, J:
), 120.93 (d, J:
.5 Hz, indole C3a), 120.55, 123.25, 125.88, 125.91, 125.95, 125.98,
3
), 7.16 (1H, dd, J: 8.8, 4.4 Hz, indole C
7
-H),
*
Amino acid residues of monomer 2 were shown by stars.
6
-H), 7.65 (1H, dd, J: 8.4, 2.8 Hz,
4
7
4
Table 6
1
3
Binding pockets on HSV-2 (gD) protein.
12.71 (1H, s, N
2
-H). C NMR (DECOUPLED DMSO‑d
6.33 (indole N-CH ), 108.69 (d, J: 26.1 Hz, indole C
.3 Hz, indole C ), 118.01 (d, J: 24.1 Hz, indole C
6
2
8
9
1
3
4
6
Compound
Interacting Amino
Interaction Types
Binding Affinity
(kcal/mol)
Acids
7
7
d
Ser 52
H-Bond
−7.4
26.02, 126.35, 126.67, 126.99, 128.66, 142.41 (phenyl C1,2,3,4,5,6
,
Ile 80
Hydrophobic
Hydrophobic
H-Bond
CF
3
) 131.72 (d, J: 3.3 Hz, indole C
3
), 140.53 (indole C7a), 159.13 (d, J:
Ala 88
Tyr 120
Ile 170
Asp 172
Trp 173
Pro 51
Ile 53
2
38.3 Hz, indole C
5
4
), 161.25 (indole C
2
), 176.68 (C=S). HRMS (ESI)
+
+
Hydrophobic
H-Bond
[M+H]
C
17
H
13
F
N
4
12
OS: 397.07407; Found [M+H] : 397.07419.
Anal. Calcd for C17
H
F
4
N OS: C, 51.51; H, 3.05; N, 14.14 Found: C,
4
π-Stacking
Hydrophobic
Hydrophobic
H-Bond
5
0.94; H, 3.14; N, 14.15.
7
l
−7.5
4
.1.9. 5-Fluoro-1-methyl-1H-indole-2,3-dione 3-[4-(4-ethylphenyl)thiose-
Thr 56
Tyr 58
Pro 74
Glu 76
Glu 175
Ile 176
Thr 177
Leu 252
H-Bond
micarbazone] (6e)
Hydrophobic
Hydrophobic
π-anion
Orange powder (94%): mp 193–196 °C; IR (KBr): υ 3290, 3207
1
(
NH), 1681 (C=O), 1271 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
1
.19 (3H, t, J: 7.6 Hz, CH
2
CH ), 2.62 (2H, q, J: 7.6 Hz, CH
), 7,15 (1H, dd, J: 8.6, 4.1 Hz, indole C
3,5-H, indole C
3
2
CH
3
), 3.21
Halogen Bond
H-Bond
(3H, s, indole N-CH
3
7
6
-H),
-H),
Hydrophobic
7.24–7.31 (3H, m, AA′BB′ system phenyl
C
7
2
.48–7.50 (2H, m, AA′BB′ system, phenyl C2,6-H), 7.66 (1H, dd, J: 8.1,
1
3
.7 Hz, indole C
4
-H), 10.76 (1H, s, N
/ 125 MHz): 16.07 (CH
), 108.61 (d, J: 26.6 Hz, indole C
17.86 (indole C
2,6), 128.18 (phenyl C3,5), 131.14 (indole C
40.45 (indole C7a), 142.35 (phenyl C
), 161.34 (indole C ), 176.70 (C=S). Anal. Calcd for C18
4
-H); 12.56 (1H, s, N
), 26.38 (indole N-CH
), 111.53 (indole C
2
-H). C NMR
4
.1.6. 5-Fluoro-1-methyl-1H-indole-2,3-dione
3-[4-(3-methylphenyl)
(
HSQC-2D DMSO‑d
6
3
3
7
),
thiosemicarbazone] (6b)
2
1
8.22 (CH
2
4
),
Orange powder (97%): mp 208–211 °C; IR (KBr): υ 3307, 3211
6
), 121.20 (d, J: 9.5 Hz, indole C3a), 126.02 (phenyl
1
(
NH), 1681 (C=O), 1278 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
C
3
), 136.36 (phenyl C ),
1
2
.33 (3H, s, CH
phenyl C -H), 7.15 (1H, dd, J: 8.8, 4.0 Hz, indole C
m, indole C -H, phenyl C -H), 7.39 (1H, s, phenyl C
.0 Hz, phenyl C -H), 7.67 (1H, dd, J: 8.0, 2.4 Hz, indole C
3
), 3.21 (3H, s, indole N-CH
3
), 7.09 (1H, d, J: 7.6 Hz,
-H), 7.26–7.32 (2H,
-H), 7.42 (1H, d, J:
-H), 10.78
-H). C NMR (DECOUPLED DMSO‑d /
1
4
), 159.16 (d, J: 237.7 Hz, indole
4
7
C
5
2
H
17FN OS: C,
4
6
5
2
6
0.66; H, 4.81; N, 15.72 Found: C, 60.90; H, 5.09; N, 15.70.
8
6
4
1
3
(
1H, s, N
4
-H), 12.56 (1H, s, N
2
6
4
.1.10. 5-Fluoro-1-methyl-1H-indole-2,3-dione 3-[4-(3-methoxyphenyl)
1
00 MHz): 21.37 (CH
3
), 26.31 (indole N-CH
3
), 108.62 (d, J: 26.2 Hz,
), 117.76 (d, J: 24.2 Hz, indole
), 126.40
), 131.05 (d, J:
), 138.56 (phenyl C ), 140.35
), 161.23 (indole C ),
thiosemicarbazone] (6f)
indole C
4
), 111.42 (d, J: 8.3 Hz, indole C
7
Red powder (74%): mp 202–205 °C; IR (KBr): υ 3302, 3217 (NH),
C
6
), 121.10 (d, J: 9.6 Hz, indole C3a), 123.09 (phenyl C
6
1
1
687 (C=O), 1259 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm: 3.21
(
phenyl C
2
), 127.32 (phenyl C
4
), 128.71 (phenyl C
5
(
3H, s, indole N-CH
3
), 3.77 (3H, s, OCH
3
), 6.85 (1H, dd, J: 8.2, 2.4 Hz,
-H), 7.22 (1H, d, J:
-H, phenyl C2,5-H),
-H), 10.78 (1H, s, N -H), 12.58
-H). C NMR (DECOUPLED DMSO‑d / 100 MHz): 26.29
), 55.69 (OCH ), 108.66 (d, J: 26.2 Hz, indole C
d, J: 8.2 Hz, indole C ), 111.51 (phenyl C ), 112.10 (phenyl C
), 117.92 (phenyl C
), 131.06 (d, J: 3.3 Hz, indole C
3
.2 Hz, indole C
3
), 138.27 (phenyl C
1
3
phenyl C
4
-H), 7.14 (1H, dd, J: 8.6, 4.0 Hz. indole C
-H), 7.26–7.34 (3H, m, indole C
7
6
(
indole C7a), 159.12 (d, J: 238.1 Hz, indole C
5
2
7
7
.8 Hz, phenyl C
6
+
1
76.54 (C=S). HRMS (ESI) [M+H]
C
17
H
16FN OS: 343.10234; Found
4
4
.67 (1H, dd, J: 8.0, 2.6 Hz, indole C
4
4
+
[
M+H] : 343.10223. Anal. Calcd for C17
H15FN
OS: C, 59.63; H, 4.42;
13
(
(
(
1H, s, N
2
6
N, 16.36 Found: C, 60.02; H, 4.48; N, 16.28.
indole N-CH
3
3
4
), 111.39
7
2
4
),
4
.1.7. 5-Fluoro-1-methyl-1H-indole-2,3-dione
3-[4-(4-methylphenyl)
117.76 (d, J: 24.2 Hz, indole C
6
6
), 121.04 (d, J:
thiosemicarbazone] (6c)
9.6 Hz, indole C3a), 129.62 (phenyl C
139.77 (phenyl C
5
3
),
Orange powder (88%): mp 224–227 °C; IR (KBr): υ 3319, 3232
1
), 140.33 (indole C7a), 159.12 (d, J: 238.2 Hz, indole
7

Z.Ş. Sevinçli, et al.
Bioorganic Chemistry 104 (2020) 104202
C
5
), 159.65 (phenyl C
3
), 161.19 (indole C
2
), 176.36 (C=S). HRMS (ESI)
7.43–7.51 (2H, m, phenyl C5,6-H), 7.59–7.65 (2H, m, indole C
4
-H,
+
+
13
[
M+H]
C
17
H
16FN
4
O
2
S: 359.09725; Found [M+H] : 359.09711.
phenyl C
2
-H), 10.86 (1H, s, N
4
-H), 12.62 (1H, s, N
2
-H). C NMR
Anal. Calcd for C17
H
15FN
4
O
2
S: C, 56.97; H, 4.22; N, 15.63 Found: C,
(DECOUPLED DMSO‑d
6
/ 125 MHz): 26.48 (indole N-CH
), 111.67 (d, J: 8.4 Hz, indole C
), 113.40 (d, J: 26.0 Hz, phenyl C ), 118.08 (d, J:
3
), 108.79 (d, J:
5
6.53; H, 4.26; N, 15.77.
26.2 Hz, indole C
4.9 Hz, phenyl C
21.8 Hz, indole C
), 130.57 (d, J: 9.5 Hz, phenyl C
140.50 (d, J: 10.8 Hz, phenyl C ), 140.66 (indole C7a), 159.25 (d, J:
238.3 Hz, indole C ), 161.40 (indole C
), 176.71 (C=S). Anal. Calcd for C16
16.18 Found: C, 55.26; H, 3.41; N, 16.18.
4
2
7
4
), 112.80 (d, J:
2
4
.1.11. 5-Fluoro-1-methyl-1H-indole-2,3-dione 3-[4-(4-methoxyphenyl)
6
), 121.14 (d, J: 9.5 Hz, indole C3a), 121.81 (phenyl
thiosemicarbazone] (6 g)
C
6
5
), 131.67 (d, J: 3.2 Hz, indole C ),
3
Orange powder (73%): mp 248–250 °C; IR (KBr): υ 3286, 3217
1
1
(
NH), 1687 (C=O), 1257 (C=S). H NMR (DMSO‑d
6
/ 500 MHz) ppm:
5
2
H
), 162.18 (d, J: 242.7 Hz, phenyl
3
.22 (3H, s, indole N-CH
AA′BB′ system, phenyl C3,5-H), 7.16 (1H, dd, J: 8.5, 4.1 Hz, indole C
H), 7.30 (1H, td, J: 9.3, 2.9 Hz, indole C -H), 7.45–7.47 (2H, m, AA′BB′
system, phenyl C2,6-H), 7.66 (1H, dd, J: 7.8, 2.4 Hz, indole C -H), 10.76
-H). C NMR (DECOUPLED DMSO‑d
), 55.75 (OCH ), 108.53 (d, J: 26.2 Hz,
), 111.38 (d, J: 8.2 Hz, indole C ), 114.08 (phenyl C3,5),
), 121.15 (d, J: 9.5 Hz, indole C3a),
3
), 3.78 (3H, s, OCH
3
), 6.97–7.00 (2H, m,
C
3
12
2
F N
4
OS: C, 55.48; H, 3.49; N,
7
-
6
4
4
.1.15. 5-Fluoro-1-methyl-1H-indole-2,3-dione
3-[4-(4-fluorophenyl)
1
3
(
1H, s, N
4
-H), 12.55 (1H, s, N
2
6
/
thiosemicarbazone] (6k)
1
00 MHz): 26.29 (indole N-CH
3
3
7
Orange powder (76%): mp 258–262 °C; IR (KBr): υ 3327, 3302,
indole C
4
1
3
201 (NH), 1681 (C=O), 1271 (C=S). H NMR (DMSO‑d
6
/ 400 MHz)
1
1
17.66 (d, J: 24.2 Hz, indole C
6
ppm: 3.21 (3H, s, indole N-CH
3
), 7.15 (1H, dd, J: 8.6, 4.2 Hz, indole C -
7
27.58 (phenyl C2,6), 130.85 (d, J: 3.2 Hz, indole C ), 131.53 (phenyl
3
H), 7.23–7.31 (3H, m, AA′BB′ system phenyl C3,5-H, indole C
6
-H),
C
1
), 140.29 (indole C7a), 157.92 (phenyl C
4
), 159.13 (d, J: 238.2 Hz,
7
2
.58–7.61 (2H, m, AA′BB′ system, phenyl C2,6-H), 7.64 (1H, dd, J: 8.0,
+
indole C
5
), 161.21 (indole C
2
), 176.87 (C=S). HRMS (ESI) [M + H]
13
.8 Hz, indole C
4
-H), 10.82 (1H, s, N
/ 125 MHz): 26.45 (indole N-CH
), 111.61 (d, J: 8.1 Hz, indole C
0.0 Hz, phenyl C3,5), 117.94 (d, J: 23.9 Hz, indole C
.5 Hz, indole C3a), 128.48 (d, J: 8.4 Hz, phenyl C2,6), 131.39 (d, J:
.1 Hz, indole C ), 135.16 (d, J: 2.6 Hz, phenyl C ), 140.56 (indole C7a),
59.22 (d. J: 238.6 Hz. indole C ), 160.63 (d. J: 243.2 Hz. phenyl C ),
61.37 (indole C ), 177.23 (C=S). LC/MS ESI [M+H] OS:
47.36187; Found [M+H] : 347.4. Anal. Calcd for C16 OS: C,
5.48; H, 3.49; N, 16.18 Found: C, 55.52; H, 3.57; N, 16.16.
4
-H), 12.59 (1H, s, N
2
-H). C NMR
+
C
17
H
16FN
4
O
2
4
S: 359.09725; Found [M + H] : 359.09711. Anal. Calcd
(
DECOUPLED DMSO‑d
6
3
), 108.65 (d, J:
for C17
H
15FN
O S: C, 56.97; H, 4.22; N, 15.63 Found: C, 57.08; H, 4.25;
2
2
2
9
3
1
1
3
5
6.6 Hz, indole C
4
7
), 115.76 (d, J:
), 121.20 (d, J:
N, 15.70.
6
4
.1.12. 5-Fluoro-1-methyl-1H-indole-2,3-dione 3-[4-(4-methylthiophenyl)
3
1
thiosemicarbazone] (6h)
5
4
Red crystal (94%): mp 235–238 °C; IR (KBr): υ 3269, 3226 (NH),
+
2
C
16
12
H
13
F
2 4
N
1
1
681 (C=O), 1274 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm: 2.49
+
H
F N
2 4
(
3H, s, SCH
3
), 3.21 (3H, s, indole N-CH
3
), 7.15 (1H, dd, J: 8.6, 4.1 Hz,
-H, AA′BB′ system phenyl
indole C
7
-H), 7.26–7.31 (3H, m, indole C
6
C
3,5-H), 7.54–7.56 (2H, m, AA′BB′ system, phenyl C2,6-H), 7.64 (1H, dd,
4
.1.16. 5-Fluoro-1-methyl-1H-indole-2,3-dione
3-[4-(3-chlorophenyl)
J: 8.1, 2.7 Hz, indole C
4
-H), 10.81 (1H, s, N
4
-H), 12.57 (1H, s, N
/ 125 MHz): 15.36 (SCH ), 26.36 (indole
), 111.51 (d, J: 8.6 Hz, indole
), 121.14 (d, J: 9.5 Hz, indole C3a),
26.34 (phenyl C2,6), 126.57 (phenyl C3,5), 131.18 (d, J: 3.9 Hz, indole
), 135.77 (phenyl C ), 136.34 (phenyl C ), 140.44 (indole C7a),
), 161.29 (indole C ), 176.62 (C=S).
: C, 54.53; H, 4.04; N, 14.96 Found: C,
2
-H).
1
3
thiosemicarbazone] (6l)
C NMR (HMBC-2D DMSO‑d
6
3
Orange powder (94%): mp 238–240 °C; IR (KBr): υ 3336, 3219
N-CH
3
), 108.62 (d, J: 25.7 Hz, indole C
4
1
(
NH), 1695 (C=O), 1273 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
C
7
), 117.82 (d, J: 23.7 Hz, indole C
6
3
.21 (3H, s, indole N-CH
.28–7.35 (2H, m, indole C
-H), 7.63–7.66 (2H, m, phenyl C
-H), 10.88 (1H, s, N
NMR (DECOUPLED DMSO‑d / 100 MHz): 26.35 (indole N-CH
d, J: 26.4 Hz, indole C ), 111.54 (d, J: 8.6 Hz, indole C ), 117.97 (d, J:
4.0 Hz, indole C ), 120.99 (d, J: 9.7 Hz, indole C3a), 124.39 (phenyl
), 125.42 (phenyl C
31.57 (d, J: 3.2 Hz, indole C
3
), 7.16 (1H, dd, J: 8.6, 4.0 Hz, indole C
-H, phenyl C -H), 7.45 (1H, t, J: 8.0 Hz,
-H, indole C -H), 7.77 (1H, t,
-H), 12.64 (1H, s, N
), 108.66
7
-H),
1
7
6
4
6
C
3
1
4
phenyl C
5
4
1
59.16 (d, J: 237.7 Hz, indole C
5
2
1
3
J: 2.0 Hz, phenyl C
2
4
2
-H).
C
Anal. Calcd for C17
H
15FN
4
OS
2
6
3
5
4.24; H, 4.09; N, 14.99.
(
4
7
2
6
4
.1.13. 5-Fluoro-1-methyl-1H-indole-2,3-dione 3-[4-(4-trifluoromethoxy-
C
6
2
), 126.40 (phenyl C
4
), 130.48 (phenyl C
), 132.97 (phenyl C ), 140.16 (phenyl
), 161.27
13ClFN OS:
5
),
phenyl)thiosemicarbazone] (6i)
1
3
3
Orange powder (81%): mp 214–217 °C; IR (KBr): υ 3317, 3224
1
C
1
), 140.53 (indole C7a), 159.13 (d, J: 238.2 Hz, indole C
5
(
NH), 1674 (C=O), 1276 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
+
(
indole C
2
), 176.65 (C=S). HRMS (ESI) [M+H]
C
16
H
4
3
.21 (3H, s, indole N-CH
3
), 7.16 (1H, dd, J: 8.6, 4.4 Hz, indole C
7
-H),
+
3
63.04771; 365.04476 Found [M+H] : 363.04761; 365.04465. Anal.
7
.29 (1H, td, J: 9.2, 2.8 Hz, indole C
6
-H), 7.41–7.43 (2H, m, AA′BB′
Calcd for C16
H
12ClFN OS: C, 52.97; H, 3.33; N, 15.44 Found: C, 53.16;
4
system, phenyl C3,5-H), 7.63 (1H, dd, J: 8.4, 2.8 Hz, indole C
4
4
-H),
-H),
H, 3.40; N, 15.53.
7
1
2
8
2
1
.72–7.76 (2H, m, AA′BB′ system, phenyl C2,6-H), 10.89 (1H, s, N
13
2.64 (1H, s, N
6.35 (indole N-CH
.3 Hz, indole C ), 117.96 (d, J: 24.2 Hz, indole C
56.2 Hz, OCF ), 121.04 (d, J: 9.5 Hz, indole C3a), 121.57 (phenyl C2,6),
27.83 (phenyl C3,5), 131.58 (d, J: 3.3 Hz, indole C ), 137.87 (phenyl
2
-H). C NMR (DECOUPLED DMSO‑d
6
/ 100 MHz):
3
), 108.60 (d, J: 26.1 Hz, indole C
4
6
), 111.57 (d, J:
4.1.17. 5-Fluoro-1-methyl-1H-indole-2,3-dione
thiosemicarbazone] (6m)
3-[4-(4-chlorophenyl)
7
), 120.53 (d, J:
3
Red powder (85%): mp 269–273 °C; IR (KBr): υ 3315, 3230 (NH),
1
3
1681 (C=O), 1278 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm: 3.21
C
4
), 140.55 (indole C7a), 146.38 (q, J: 1.7 Hz, phenyl C
1
), 159.12 (d, J:
(3H, s, indole N-CH
3
), 7.15 (1H, dd, J: 8.6, 4.1 Hz, indole C
-H), 7.46–7.50 (2H, m, AA′BB′ system,
-H, AA′BB′ system phenyl
7
-H), 7.29
2
38.2 Hz, indole C
5
), 161.30 (indole C
2
), 176.98 (C=S). HRMS (ESI)
(1H, td, J: 9.4, 2.7 Hz, indole C
6
+
+
[
M+H]
C
17
H
13
F N
4 4
O
2
S: 413.06899; Found [M+H] : 413.06900.
phenyl C3,5-H), 7.62–7.67 (3H, m, indole C
4
1
3
Anal. Calcd for C17
H
12
F
4
N
4
O
2
S: C, 49.52; H, 2.93; N, 13.59 Found: C,
C
2,6-H), 10.85 (1H, s, N
4
-H), 12.63 (1H, s, N
2
-H). C NMR (HSQC-2D
), 108.69 (d, J: 30.5 Hz,
), 121.03 (d, J: 9.5 Hz,
4
9.58; H, 2.89; N, 13.67.
DMSO‑d
6
/ 125 MHz): 26.36 (indole N-CH
3
indole C
4
), 111.58 (indole C
7
), 118.13 (indole C
6
4
.1.14. 5-Fluoro-1-methyl-1H-indole-2,3-dione
3-[4-(3-fluorophenyl)
indole C3a), 127.81 (phenyl C2,6), 128.90 (phenyl C3,5), 130.79 (phenyl
thiosemicarbazone] (6j)
C
4
), 131.63 (d, J: 2.9 Hz, indole C
3
), 137.68 (phenyl C
1
), 140.54 (indole
Orange crystal (82%): mp 237–243 °C; IR (KBr): υ 3288, 3226 (NH),
C7a), 159.15 (d, J: 238.7 Hz, indole C
5
), 161.34 (indole C
2
), 176.89
1
+
1
695 (C=O), 1276 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm: 3.20
(C=S). LC/MS ESI [M+H]
C
16
H
13ClFN
4
OS: 363.81647; Found [M
+
(
3H, s, indole N-CH
3
), 7.09–7.16 (1H, m, phenyl C
4
-H), 7.14 (1H, dd, J:
+H] : 363.3; 365.6. Anal. Calcd for C16
H
12ClFN OS: C, 52.97; H, 3.33;
4
8
.7, 3.9 Hz, indole C
7
-H), 7.28 (1H, td, J: 9.0, 2.7 Hz, indole C -H),
6
N, 15.44 Found: C, 52.96; H, 3.34; N, 15.42.
8

Z.Ş. Sevinçli, et al.
Bioorganic Chemistry 104 (2020) 104202
4
.1.18. 5-Fluoro-1-methyl-1H-indole-2,3-dione
3-[4-(4-bromophenyl)
(CH
3
), 34.68 (indole N-CH
2
CH
3
7
), 108.79 (d, J: 26.3 Hz, indole C
4
),
thiosemicarbazone] (6n)
111.56 (d, J: 8.4 Hz, indole C
), 117.80 (d, J: 23.9 Hz, indole C
6
),
Red powder (86%): mp 248–251 °C; IR (KBr): υ 3309, 3296, 3230
121.39 (d, J: 9.5 Hz, indole C3a), 125.93 (phenyl C2,6), 129.38 (phenyl
1
(
NH), 1681 (C=O), 1278 (C=S). H NMR (DMSO‑d
6
/ 500 MHz) ppm:
C
3,5), 131.09 (d, J: 3.2 Hz, indole C
3
), 135.99 (phenyl C
1
), 136.17
3
.22 (3H, s, indole N-CH
3
), 7.16 (1H, dd, J: 8.5, 4.1 Hz, indole C
7
-H),
-H),
(phenyl C
4
), 139.32 (indole C7a), 159.06 (d, J: 238.2 Hz, indole C
5
),
+
7
.30 (1H, td, J: 9.3, 2.9 Hz, indole C
6
-H), 7.59–7.62 (4H, m, AA′BB′
160.93 (indole C
2
), 176.69 (C=S). LC/MS ESI [M+H]
C
18
H
18FN
4
OS:
+
system, phenyl C2,3,5,6-H), 7.65 (1H, dd, J: 8.0, 2.7 Hz, indole C
4
357.42457; Found [M+H] : 357.5. Anal. Calcd for C18
H17FN
4
OS: C,
1
3
1
0.85 (1H, s, N
DMSO‑d / 125 MHz): 26.48 (indole N-CH
indole C ), 111.65 (d, J: 7,2 Hz, indole C ), 118.03 (d, J: 22.0 Hz, indole
), 119.11 (phenyl C ), 121.16 (d, J: 9.5 Hz, indole C3a), 128.10
4
-H), 12.64 (1H, s, N
2
-H). C NMR (DECOUPLED
60.66; H, 4.81; N, 15.72 Found: C, 60.85; H, 4.82; N, 15.60.
6
3
), 108.72 (d, J: 26.2 Hz,
4
7
4.1.22. 1-Ethyl-5-fluoro-1H-indole-2,3-dione 3-[4-(4-trifluoromethylphe-
nyl)thiosemicarbazone] (7d)
C
6
4
(
phenyl C2,6), 131.60 (d, J: 3.3 Hz, indole C
3
), 131.89 (phenyl C3,5),
Orange powder (78%): mp 199–202 °C; IR (KBr): υ 3282, 3211
1
1
38.24 (phenyl C
1
), 140.64 (indole C7a), 159.23 (d, J: 238.2 Hz, indole
(NH), 1695 (C=O), 1271 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
+
C
C
5
), 161.40 (indole
13BrFN
C
2
), 176.82 (C=S). LC/MS ESI [M+H]
1.19 (3H, t, J: 7.2 Hz, indole N-CH
N-CH CH ), 7.23 (1H, dd, J: 8.7, 4.2 Hz, indole C
9.0, 2.7 Hz, indole C -H), 7.67 (1H, dd, J: 8.0, 2.6 Hz, indole C
7.78–7.80 (2H, m, AA′BB′ system, phenyl C2,6-H), 7.92–7.94 (2H, m,
AA′BB′ system, phenyl C3,5-H), 11,00 (1H. s, N -H), 12.73 (1H, s, N -H).
NMR (DECOUPLED DMSO‑d
CH ), 34.69 (indole N-CH CH ), 108.90 (d, J: 26.0 Hz, indole C
2
CH
3
), 3.78 (2H, q, J: 7.2 Hz, indole
-H), 7.30 (1H, td, J:
-H),
+
16
H
4
OS: 408.26747; Found [M+H] : 407.2; 409.3. Anal.
2
3
7
Calcd for C16
H12BrFN
4
OS: C, 47.19; H, 2.97; N, 13.76 Found: C, 47.22;
6
4
H, 3.11; N, 13.64.
4
2
1
3
4
.1.19. 1-Ethyl-5-fluoro-1H-indole-2,3-dione
3-[4-(phenyl)thiosemicar-
C
6
/
100 MHz): 12.96 (indole N-
bazone] (7a)
CH
2
3
2
3
4
6
),
Yellow powder (99%): mp 203–205 °C; IR (KBr): υ 3292, 3209 (NH),
111.59 (d, J: 8.1 Hz, indole C ), 118.04 (d, J: 24.1 Hz, indole C
7
),
1
1
683 (C=O), 1271 (C=S). H NMR (DMSO‑d
6
/ 500 MHz) ppm: 1.20
121.13 (d, J: 9.5 Hz, indole C3a), 120.54, 123.25, 125.83, 125.89,
(
3H, t, J: 7.3 Hz, indole N-CH
2
CH
3
), 3.79 (2H, q, J: 7.3 Hz, indole N-
-H), 7.26–7.30 (2H, m,
-H), 7.43 (2H, t, J: 7.8 Hz, phenyl C3,5-H), 7.61
125.93, 125.97, 126.01, 126.33, 126.65, 126.97, 128.65, 142.40
CH
2
CH
3
), 7.22 (1H, dd, J: 8.5, 4.1 Hz, indole C
7
(phenyl C1,2,3,4,5,6, CF
3
), 131.76 (d, J: 3.3 Hzj indole C
3
), 139.46 (indole
), 176.64
OS: 411.08972; Found [M
OS: C, 52.68; H, 3.44; N,
indole C
6
-H, phenyl C
4
C
7a), 159.04 (d, J: 238.4 Hz, indole C
5
), 160.88 (indole C
2
+
(
2H, d, J: 8.3 Hz, phenyl C2,6-H), 7.69 (1H, dd, J: 8.1, 2.7 Hz, indole C
4
-
(C=S). HRMS (ESI) [M+H]
C
18
H
15
F
4 4
N
1
3
+
H), 10.84 (1H, s, N
DMSO‑d
4
-H), 12.62 (1H, s, N
2
-H). C NMR (HSQC-2D
CH ), 34.70 (indole N-
), 111.59 (indole C ), 117.87
), 121.37 (d, J: 9.5 Hz, indole C3a), 126.09
phenyl C2,6), 126.73 (phenyl C ), 128.95 (phenyl C3,5), 131.24 (d, J:
.9 Hz, indole C ), 138.73 (phenyl C ), 139.37 (indole C7a), 159.08 (d,
), 160.95 (indole C ), 176.75 (C=S). Anal. Calcd
OS: C, 59.63; H, 4.42; N, 16.36 Found: C, 59.86; H, 4.66;
+H] : 411.08969. Anal. Calcd for C18
H
14
F
4 4
N
6
/ 125 MHz): 13.03 (indole N-CH
2
3
13.65 Found: C, 52.59; H, 3.58; N, 13.81.
CH
2
CH
3
), 108.82 (d, J: 26.7 Hz, indole C
4
7
(
(
d, J: 24.7 Hz, indole C
6
4.1.23. 1-Ethyl-5-fluoro-1H-indole-2,3-dione 3-[4-(4-ethylphenyl)thiosem-
icarbazone] (7e)
4
3
3
1
Orange powder (99%): mp 124–127 °C; IR (KBr): υ 3348, 3197
1
J: 237.8 Hz, indole C
5
2
(NH), 1695 (C=O), 1271 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
for C17
H
15FN
4
1.18 (3H, t, J: 7.6 Hz, CH
CH CH ), 2.62 (2H, q, J: 7.6 Hz, CH
N-CH CH ), 7.21 (1H, dd, J: 8.6, 4.2 Hz, indole C
m, AA′BB′ system phenyl C3,5-H, indole C -H), 7.48–7.50 (2H, m,
AA′BB′ system, phenyl C2,6-H), 7.67 (1H, dd, J: 8.0, 2.6 Hz, indole C -
2
CH
3
), 1.19 (3H, t, J: 7.2 Hz, indole N-
CH ), 3.78 (2H, q, J: 7.2 Hz, indole
-H), 7.24–7.30 (3H,
N, 16.36.
2
3
2
3
2
3
7
4
.1.20. 1-Ethyl-5-fluoro-1H-indole-2,3-dione
3-[4-(3-methylphenyl)
6
thiosemicarbazone] (7b)
4
1
3
Orange powder (91%): mp 181–184 °C; IR (KBr): υ 3315, 3215
H), 10.77 (1H, s, N
DMSO‑d / 100 MHz): 13.01 (indole N-CH
(CH ), 34.66 (indole N-CH CH ), 108.78 (d, J: 26.0 Hz, indole C
111.49 (d, J: 8.1 Hz, indole C ), 117.76 (d, J: 24.1 Hz, indole C
4
-H), 12.58 (1H, s, N -H). C NMR (DECOUPLED
2
1
(
NH), 1678 (C=O), 1273 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
6
2
CH
3
), 16.04 (CH
3
), 28.20
1
.19 (3H, t, J: 7.2 Hz, indole N-CH
CH ), 7.09 (1H, d, J: 7.5 Hz, phenyl C
-H), 7.25–7.32 (2H, m, indole C
-H), 7.40 (1H, s, phenyl C -H), 7.42 (1H, d, J: 7.7 Hz, phenyl
-H), 10.79 (1H, s, N -H),
-H). C NMR (HSQC-2D DMSO‑d / 125 MHz): 13.02
CH ), 21.38 (CH ), 34.70 (indole N-CH CH ), 108.84 (d,
J: 26.6 Hz, indole C ), 111.57 (indole C ), 117.86 (d, J: 24.8 Hz, indole
), 121.37 (d, J: 9.5 Hz, indole C3a), 123.10 (phenyl C ), 126.43
phenyl C ), 127.31 (phenyl C ), 128.74 (phenyl C ), 131.17 (d, J:
), 138.61 (phenyl C ), 139.35
), 160.95 (indole C ),
2
CH
3
), 2.33 (3H, s, CH
3
), 3.78 (2H, q,
-H), 7.22
-H,
2
2
3
4
6
),
J: 7.2 Hz, indole N-CH
2
3
4
7
),
(
1H, dd, J: 8.7, 4.2 Hz, indole C
7
6
121.33 (d, J: 9.5 Hz, indole C3a), 125.93 (phenyl C2,6), 128.13 (phenyl
phenyl C
5
2
C
3,5), 131.02 (d, J: 3.3 Hz, indole C
3
), 136.33 (phenyl C
1
), 139.26
), 159.04 (d, J: 238.1 Hz, indole C ),
+
C
6
-H), 7.69 (1H, dd, J: 8.1, 2.6 Hz, indole C
4
4
(indole C7a), 142.27 (phenyl C
4
5
1
3
1
2.58 (1H, s, N
2
6
160.88 (indole
C
2
), 176.59 (C=S). HRMS (ESI) [M
+
H]
+
(
indole N-CH
2
3
3
2
3
C
19
for C19
H
20FN
4
OS: 371.13364; Found [M + H] : 371.13361. Anal. Calcd
4
7
H
19FN OS: C, 61.60; H, 5.17; N, 15.12 Found: C, 61.93; H, 5.15;
4
C
6
6
N, 15.10.
(
2
4
5
3
.9 Hz, indole C
3
), 138.31 (phenyl C
1
3
4.1.24. 1-Ethyl-5-fluoro-1H-indole-2,3-dione
thiosemicarbazone] (7f)
3-[4-(3-methoxyphenyl)
(
indole C7a), 159.08 (d, J: 238.8 Hz, indole C
5
4
2
+
1
76.59 (C=S). LC/MS ESI [M+H]
C
18
H
18FN
OS: 357.42457; Found
Orange powder (90%): mp 159–162 °C; IR (KBr): υ 3302, 3217
+
1
[
M+H] : 357.5. Anal. Calcd for C18
H
17FN
4
OS: C, 60.66; H, 4.81; N,
(NH), 1681 (C=O), 1276 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
1
5.72 Found: C, 60.65; H, 4.47; N, 15.42.
1.19 (3H, t, J: 7.2 Hz, indole N-CH
q, J: 7.2 Hz, indole N-CH CH ), 6.85 (1H, ddd, J: 8.3, 2.5, 0.8 Hz,
phenyl C -H), 7.20–7.34 (5H, m, indole C6,7-H, phenyl C2,5,6-H), 7.69
(1H, dd, J: 8.1, 2.6 Hz, indole C -H), 10.79 (1H, s, N -H), 12.61 (1H, s,
-H). C NMR (HSQC-2D DMSO‑d / 125 MHz): 13.23 (indole N-
CH CH ), 34.90 (indole N-CH CH ), 55.92 (OCH ), 109.10 (d, J:
25.7 Hz, indole C ), 111.51 (phenyl C ), 111.74 (indole C ), 112.33
(phenyl C ), 117.68 (phenyl C ), 118.14 (indole C ), 121.54 (d, J:
9.5 Hz, indole C3a), 129.86 (phenyl C ), 131.44 (indole C ), 139.58
),
), 176.63 (C=S). LC/MS ESI [M
2
CH
3
), 3.77 (3H, s, OCH ), 3.78 (2H,
3
2
3
4
.1.21. 1-Ethyl-5-fluoro-1H-indole-2,3-dione
3-[4-(4-methylphenyl)
4
thiosemicarbazone] (7c)
4
4
1
3
Orange powder (85%): mp 159–161 °C; IR (KBr): υ 3311, 3226
N
2
6
1
(
NH), 1685 (C=O), 1273 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
2
3
2
3
3
1
.19 (3H, t, J: 7.2 Hz, indole N-CH
2
CH
3
), 2.31 (3H, s, CH
3
), 3.78 (2H, q,
-H, AA′BB′
-H),
.45–7.47 (2H, m, AA′BB′ system, phenyl C2,6-H), 7.67 (1H, dd, J: 8.0,
4
2
7
J: 7.2 Hz, indole N-CH CH ), 7.20–7.23 (3H, m, indole C
2
3
7
4
6
6
system, phenyl C3,5-H), 7.28 (1H, td, J: 9.0, 2.8 Hz, indole C
6
5
3
7
2
(phenyl C
1
), 140.02 (indole C7a), 159.28 (d, J: 238.7 Hz, indole C
5
1
3
.8 Hz, indole C
4
-H), 10.78 (1H, s, N
4
-H), 12.58 (1H, s, N
2
-H). C NMR
159.90 (phenyl C
3
), 161.15 (indole C
2
+
+
(
DECOUPLED DMSO‑d
6
/ 125 MHz): 13.04 (indole N-CH
2
CH ), 21.09
3
+H]
C
18
H17FN
4 2
O S: 373.42397; Found [M+H] : 373.4. Anal. Calcd
9

Z.Ş. Sevinçli, et al.
Bioorganic Chemistry 104 (2020) 104202
for C18
H
17FN
4
O
2
S: C, 58.05; H, 4.60; N, 15.04 Found: C, 57.88; H, 4.79;
1681 (C=O), 1273 (C=S). 1H NMR (DMSO‑d
/ 400 MHz) ppm: 1.19
6
N, 15.12.
(3H, t, J: 7.2 Hz, indole N-CH
2
CH ), 3.78 (2H, q, J: 7.2 Hz, indole N-
3
CH
2
CH
3
), 7.09–7.14 (1H, m, phenyl C
4
-H), 7.22 (1H, dd, J: 8.7, 4.2 Hz,
-H), 7.43–7.51 (2H,
-H), 7.67 (1H,
-H), 12.67 (1H, s, N
4
.1.25. 1-Ethyl-5-fluoro-1H-indole-2,3-dione
3-[4-(4-methoxyphenyl)
indole C
7
-H), 7.29 (1H, td, J: 9.3, 2.2 Hz, indole C
6
thiosemicarbazone] (7g)
m, phenyl C5,6-H), 7.62 (1H, dt, J: 10.9, 2.2 Hz, phenyl C
2
Orange powder (98%): mp 175–177 °C; IR (KBr): υ 3354, 3196
dd, J: 8.0, 2.6 Hz, indole C
4
-H), 10.88 (1H, s, N
4
2
-
1
13
(
NH), 1681 (C=O), 1253 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
H). C NMR (DECOUPLED DMSO‑d
6
/ 100 MHz): 12.99 (indole N-
1
.19 (3H, t, J: 7.2 Hz, indole N-CH
q, J: 7.2 Hz, indole N-CH CH ), 6.96–6.98 (2H, m, AA′BB′ system,
phenyl C3,5-H), 7.21 (1H, dd, J: 8.8, 4.4 Hz, indole C -H), 7.27 (1H, td,
J: 8.8, 2.4 Hz, indole C -H), 7.45–7.47 (2H, m, AA′BB′ system, phenyl
2,6-H), 7.67 (1H, dd, J: 7.8, 2.4 Hz, indole C -H), 10.73 (1H, s, N -H),
2.56 (1H, s, N -H). C NMR (DECOUPLED DMSO‑d
3.01 (indole N-CH CH ), 34.66 (indole N-CH CH ), 55.74 (OCH
08.73 (d, J: 26.1 Hz, indole C ), 111.49 (d, J: 8.2 Hz, indole C
14.07 (phenyl C3,5), 117.72 (d, J: 24.1 Hz, indole C
2
CH
3
), 3.77 (3H, s, OCH
3
), 3.78 (2H,
CH
111.56 (d, J: 8.2 Hz, indole C
113.21 (d, J: 20.9 Hz, phenyl C
121.19 (d, J: 9.5 Hz, indole C3a), 121.58 (d, J: 2.9 jHz, phenyl C
130.41 (d, J: 9.3 Hz, phenyl C ), 131.52 (d, J: 3.3 Hz, indole C
2
CH
3
), 34.69 (indole N-CH
2
CH
), 112.57 (d, J: 24.8 Hz, phenyl C
), 117.96 (d, J: 24.1 Hz, indole C
3
), 108.87 (d, J: 26.1 Hz, indole C
4
2
6
6
),
),
),
),
2
3
7
7
4
6
C
4
4
5
3
), 139.41
1
3
1
1
1
1
9
2
6
/ 100 MHz):
(indole C7a), 140.38 (d, J: 10.7 Hz, phenyl C ), 159.04 (d, J: 238.3 Hz,
1
2
3
2
3
3
7
),
),
indole C
5
), 161.40 (indole C
2
), 162.06 (d, J: 242.6 Hz, phenyl C
3
),
+
4
176.51 (C=S). HRMS (ESI) [M+H]
C
17
H
15
H
F
2
14
N OS: 361.09291;
4
+
6
), 121.37 (d, J:
Found [M+H] : 361.09262. Anal. Calcd for C17
F
2
N OS: C, 56.66;
4
.5 Hz, indole C3a), 127.56 (phenyl C2,6), 130.93 (d, J: 3.2 Hz, indole
H, 3.92; N, 15.55 Found: C, 56.62; H, 3.99; N, 15.63.
C
3
), 131.53 (phenyl C
1
), 139.25 (indole C7a), 157.91 (phenyl C
4
),
1
59.04 (d, J: 238.2 Hz, indole C
5
), 160.89 (indole C
2
), 176.86 (C=S).
4.1.29. 1-Ethyl-5-fluoro-1H-indole-2,3-dione
thiosemicarbazone] (7k)
3-[4-(4-fluorophenyl)
+
+
HRMS (ESI) [M+H]
C
18
H
18FN
4
O
2
4
S: 373.11290; Found [M+H]
:
3
73.11279. Anal. Calcd for C18
H
17FN
O
2
S: C, 58.05; H, 4.60; N, 15.04
Orange powder (67%): mp 207–210 °C; IR (KBr): 3336, 3280, 3224
1
Found: C, 58.23; H, 4.76; N, 15.22.
(NH), 1681 (C=O), 1271 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
1
.20 (3H, t, J: 7.2 Hz, N-CH
CH CH ), 7.23–7.33 (4H, m, indole C6,7-H, AA′BB′ system, phenyl C3,5
H), 7.58–7.63 (2H, m, AA′BB′ system, phenyl C2,6-H), 7.66 (1H, dd, J:
2
CH
3
), 3.80 (2H, q, J: 7.2 Hz, indole N-
4
.1.26. 1-Ethyl-5-fluoro-1H-indole-2,3-dione 3-[4-(4-methylthiophenyl)
2
3
-
thiosemicarbazone] (7h)
1
3
Orange powder (80%): mp 171–173 °C; IR (KBr): 3302, 3228 (NH),
7.8, 2.6 Hz, indole C
NMR (DECOUPLED DMSO‑d
34.69 (indole N-CH CH ), 108.76 (d, J: 26.0 Hz, indole C
J: 8.1 Hz, indole C ), 115.61 (d, J: 22.7 Hz, phenyl C3,5), 117.85 (d, J:
24.1 Hz, indole C ), 121.28 (d, J: 9.5 Hz, indole C3a), 128.26 (d, J:
8.4 Hz, phenyl C2,6), 131.27 (d, J: 3.3 Hz, indole C ), 135.06 (d, J:
2.8 Hz, phenyl C ), 139.34 (indole C7a), 159.04 (d. J: 238.3 Hz. indole
4
-H), 10.87 (1H, s, N
/ 100 MHz): 12.98 (indole N-CH
), 111.52 (d,
4
-H), 12.63 (1H, s, N
2
-H).
C
1
1
681 (C=O), 1273 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm: 1.19
6
2
CH
3
),
(
3H, t, J: 7.2 Hz, indole N-CH
2
CH
3
), 2.49 (3H, s, SCH
3
), 3.78 (2H, q, J:
2
3
4
7
7
7
2
.2 Hz, indole N-CH
2
CH
3
), 7.22 (1H, dd, J: 8.6, 4.2 Hz, indole C
7
-H),
7
6
.26–7.31 (3H, m, indole
C
6
-H, AA′BB′ system phenyl
C
3,5-H),
.54–7.56 (2H, m, AA′BB′ system, phenyl C2,6-H), 7.66 (1H, dd, J: 8.0,
3
1
3
.6 Hz, indole C
4
-H), 10.81 (1H, s, N
/ 100 MHz): 13.01 (indole N-CH
), 34.68 (indole N-CH CH ), 108.79 (d, J: 26.0 Hz, indole C
11.52 (d, J: 8.1 Hz, indole C ), 117.81 (d, J: 24.1 Hz, indole C
4
-H), 12.61 (1H, s, N
2
-H). C NMR
1
(
(
DECOUPLED DMSO‑d
6
2
CH
3
), 15.37
C
5
), 160.50 (d. J: 243.3 Hz. phenyl C
4
), 160.88 (indole C ), 177.09
2
+
SCH
3
2
3
7
4
6
),
),
(C=S). HRMS (ESI) [M+H]
C
17
H
15
F
2
N OS: 361.09291; Found [M
4
+
1
1
+H] : 361.09280. Anal. Calcd for C17
H
14
F
2
N
4
OS: C, 56.66; H, 3.92; N,
21.30 (d, J: 9.5 Hz, indole C3a), 126.31 (phenyl C2,6), 126.47 (phenyl
15.55 Found: C, 56.63; H, 4.02; N, 15.59.
C
3,5), 131.17 (d, J: 3.3 Hz, indole C
3
), 135.75 (phenyl C
1
), 136.27
(
phenyl C
4
), 139.31 (indole C7a), 159.04 (d, J: 238.2 Hz, indole C
5
),
4.1.30. 1-Ethyl-5-fluoro-1H-indole-2,3-dione
thiosemicarbazone] (7l)
3-[4-(3-chlorophenyl)
+
1
60.89 (indole
C
2
), 176.56 (C=S). HRMS (ESI) [M+H]
+
C
18
H
18FN
4
OS
2
OS
: 389.09006; Found [M+H] : 389.09003. Anal. Calcd
Orange powder (95%): mp 194–197 °C; IR (KBr): υ 3304, 3197
1
for C18
H
17FN
4
2
: C, 55.65; H, 4.41; N, 14.42 Found: C, 55.38; H, 4.52;
(NH), 1681 (C=O), 1269 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
N, 14.50.
1.20 (3H, t, J: 7.2 Hz, indole N-CH
N-CH CH ), 7.22 (1H, dd, J: 8.6, 4.2 Hz, indole C
9.2, 2.7 Hz, indole C
H), 7.45 (1H, t, J: 8.1 Hz, phenyl C
indole C -H), 7.78 (1H, t, J: 2.0 Hz, phenyl C
O exch), 12.67 (1H, s, N
CDCl / 100 MHz): 12.70 (indole N-CH
108.45 (d, J: 25.7 Hz, indole C ), 110.06 (d, J: 8.0 Hz, indole C
117.79 (d, J: 24.3 Hz, indole C
121.78 (phenyl C ), 123.66 (phenyl C
(phenyl C ), 130.70 (d, J: 3.5 Hz, indole C
2
CH
3
), 3.78 (2H, q, J: 7.2 Hz, indole
-H), 7.28 (1H, td, J:
2
3
7
4
.1.27. 1-Ethyl-5-fluoro-1H-indole-2,3-dione 3-[4-(4-trifluoromethoxyphe-
6
-H), 7.33 (1H, ddd, J: 8.1, 2.1, 1.0 Hz, phenyl C
4
-H,
-
nyl)thiosemicarbazone] (7i)
5
-H), 7.64–7.68 (2H, m, phenyl C
6
4
Yellow powder (94%): mp 150–153 °C; IR (KBr): 3278, 3236 (NH),
4
2
-H), 10.89 (1H, s, N
-H,
1
13
1
701 (C=O), 1267 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm: 1.20
D
2
2
-H, D
2
O exch). C NMR (DECOUPLED
(
3H, t, J: 7.2 Hz, indole N-CH
2
CH
), 7.22 (1H, dd, J: 8.6, 4.2 Hz, indole C
-H), 7.41–7.43 (2H, m, AA′BB′ system, phenyl C3,5-H),
-H), 7.72–7.76 (2H, m, AA′BB′
3
), 3.78 (2H, q, J: 7.2 Hz, indole N-
3
2
CH
3
), 34.74 (indole N-CH
2
CH
3
),
7
),
CH
2
CH
3
7
-H), 7.28 (1H, td, J: 9.0,
4
6
2
.4 Hz, indole C
6
), 120.62 (d, J: 8.9 Hz, indole C3a),
7
.64 (1H, dd, J: 8.0, 2.8 Hz, indole C
4
6
2
), 126.33 (phenyl C
4
), 129.78
1
3
system, phenyl C2,6-H), 10.89 (1H, s, N
4
-H), 12.66 (1H, s, N
/ 100 MHz): 12.97 (indole N-CH
), 108.79 (d, J: 26.1 Hz, indole C ), 111.59 (d,
), 117.95 (d, J: 24.1 Hz, indole C ), 120.53 (q, J:
), 121.22 (d, J: 9.5 Hz, indole C3a), 121.54 (phenyl C2,6),
27.75 (phenyl C3,5), 131.54 (d, J: 3.3 Hz, indole C ), 137.86 (phenyl
2
-H).
C
5
3
), 134.42 (phenyl C
3
),
NMR (DECOUPLED DMSO‑d
4.69 (indole N-CH CH
6
2
CH
3
),
138.65 (phenyl C ), 138.94 (d, J: 2.0 Hz, indole C7a), 159.32 (d, J:
1
3
2
3
4
242.3 Hz, indole C
5
), 160.73 (indole C
2
), 175.62 (C=S). LC/MS ESI [M
+
+
J: 8.1 Hz, indole C
7
6
+H]
C
17
H
15ClFN
4
OS: 377.84305; Found [M+H] : 377.5; 379.7.
2
56.3 Hz, OCF
3
Anal. Calcd for C17
H
14ClFN OS: C, 54.18; H, 3.74; N, 14.87 Found: C,
4
1
3
53.69; H, 3.85; N, 14.48.
C
4
), 139.42 (indole C7a), 146.36 (q, J: 1.8 Hz, phenyl C ), 159.04 (d, J:
1
2
38.3 Hz, indole C
5
), 160.90 (indole C
2
), 176.92 (C=S). HRMS (ESI)
4.1.31. 1-Ethyl-5-fluoro-1H-indole-2,3-dione
thiosemicarbazone] (7m)
3-[4-(4-chlorophenyl)
+
+
[
[M+H]
C
18
H
15
F
4
N
4
O
2
S: 427.08464; Found [M+H] : 427.08472.
Anal. Calcd for C18
H
14
4
F N
4
O
2
S: C, 50.70; H, 3.31; N, 13.14 Found: C,
Orange powder (69%): mp 212–215 °C; IR (KBr): υ 3300, 3221
1
5
0.53; H, 3.36; N, 13.23.
(NH), 1691 (C=O), 1271 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
1
.20 (3H, t, J: 7.2 Hz, indole N-CH
N-CH CH ), 7.24 (1H, dd, J: 8.6, 4.2 Hz, indole C
8.8, 2.4 Hz, indole C -H), 7.49–7.52 (2H, m, AA′BB′ system, phenyl
3,5-H), 7.65–7.68 (3H, m, AA′BB′ system, phenyl C2,6-H, indole C -H)),
2
CH
3
), 3.79 (2H, q, J: 7.2 Hz, indole
4
.1.28. 1-Ethyl-5-fluoro-1H-indole-2,3-dione
3-[4-(3-fluorophenyl)
Red powder (90%): mp 205–207 °C; IR (KBr): 3304, 3242 (NH),
2
3
7
-H), 7.30 (1H, td, J:
thiosemicarbazone] (7j)
6
C
4
10

Z.Ş. Sevinçli, et al.
Bioorganic Chemistry 104 (2020) 104202
1
3
1
0.89 (1H, s, N
DMSO‑d / 100 MHz): 12.99 (indole N-CH
CH CH ), 108.81 (d, J: 26.0 Hz, indole C ), 111.56 (d, J: 8.1 Hz, indole
), 117.92 (d, J: 24.1 Hz, indole C ), 121.22 (d, J: 9.5 Hz, indole C3a),
27.61 (phenyl C2,6), 128.81 (phenyl C3,5), 130.66 (phenyl C ), 131.44
), 139.38 (indole C7a),
), 160.88 (indole C ), 176.73 (C=S).
OS: 377.06336; 379.06041; Found:
14ClFN OS: C,
4
-H), 12.67 (1H, s, N
2
-H). C NMR (DECOUPLED
stayed in the simulation box. The box was filled with single point
charge (SPC) water molecules [35] and some of them were displaced
during the addition of sodium and chloride ions to neutralize the
system. At the beginning of simulations, starting systems were subse-
quently energy-minimized using the steepest descent method for
50,000 steps. Then, energy-minimized structures were taken for the
production phase. MD simulations without any constraints were carried
out using a constant number of particles (N), pressure (P), and tem-
perature (T), i.e. NPT ensemble. The SETTLE algorithm was utilized to
constrain the bond length and bond angle of the water molecules [36],
while the LINCS algorithm was used to constrain the bond length of the
peptide [37]. Long-range electrostatic interactions were calculated by
particle-mesh Ewald (PME) method [38]. A constant pressure of 1 bar
was applied with a coupling constant of 1.0 ps and water molecules/
chloride ions were coupled separately to a bath at 303 K with a cou-
pling constant of 0.1 ps. The equation of motion was integrated at 2 fs
time steps using a leap-frog algorithm [39]. The tools available in the
GROMACS and VMD 1.9.1. software [40] were utilized to analyze
trajectories. Clustering tool of GROMACS (g_cluster) was used to obtain
the most representative structures of 10 ns long trajectories.
6
2
CH ), 34.68 (indole N-
3
2
3
4
C
7
6
1
4
(
d, J: 3.3 Hz, indole C
3
), 137.68 (phenyl C
1
1
59.03 (d, J: 238.3 Hz, indole C
5
2
+
HRMS (ESI) [M+H]
C
17
H
15ClFN
4
+
[
M+H] : 377.06332; 379.06015. Anal. Calcd for C17
H
4
5
4.18; H, 3.74; N, 14.87 Found: C, 53.63; H, 3.57; N, 15.30.
.1.32. 1-Ethyl-5-fluoro-1H-indole-2,3-dione 3-[4-(4-bromophenyl)
4
thiosemicarbazone] (7n)
Orange powder (79%): mp 213–216 °C; IR (KBr): υ 3300, 3226
1
(
NH), 1689 (C=O), 1271 (C=S). H NMR (DMSO‑d
6
/ 400 MHz) ppm:
1
.20 (3H, t, J: 7.2 Hz, indole N-CH
N-CH CH ); 7.24 (1H, dd, J: 8.4; 4.4 Hz, indole C
.8; 2.4 Hz, indole -H); 7.60–7.66 (4H, m, AA′BB′ system,
phenylC2,3,5,6-H); 7.67 (1H, dd, J: 8.2; 2.6 Hz, indole C -H); 10.87 (1H,
s, N -H); 12.67 (1H, s, N -H). C NMR (DECOUPLED DMSO‑d
00 MHz): 12.98 (indole N-CH CH ), 34.70 (indole N-CH CH ), 108.82
), 111.57 (d, J: 8.1 Hz, indole C ), 117.94 (d, J:
), 118.93 (phenyl C ), 121.23 (d, J: 9.5 Hz, indole
3a), 127.86 (phenyl C2,6), 131.47 (d, J: 3.3 Hz, indole C ), 131.73
2
CH
3
); 3.79 (2H, q, J: 7.2 Hz, indole
2
3
7
-H); 7.30 (1H, td, J:
8
C
6
4
1
3
4
2
6
/
1
2
3
2
3
Root mean square deviation (RMSD) values of all MD simulations
showed that 10 ns long simulations were enough to equilibrate systems.
With this methodology it was made sure that any structural anomalies
and defects from x-ray and electron microscopy structures were over-
come. Thus, average structures obtained for each protein were used to
dock ligands in the next step, molecular docking simulations.
(
d, J: 26.0 Hz, indole C
4
7
2
4.1 Hz, indole C
6
4
C
3
(
phenyl C3,5), 138.14 (phenyl C ), 139.40 (indole C7a), 159.05 (d, J:
1
2
38.3 Hz, indole C
5
), 160.89 (indole C
2
), 176.68 (C=S). HRMS (ESI)
+
+
[
M+H]
C
17
H
15BrFN
4
OS: 421.01285; 423.01080; Found [M+H]
:
4
3
21.01276; 423.01053. Anal. Calcd for C17
H
14BrFN
4
OS: C, 48.47; H,
4
.3.2. Molecular Docking Simulations
.35; N, 13.30 Found: C, 48.27; H, 3.17; N, 13.75.
All docking simulations were performed utilizing AutoDock Vina
.1.2 software [41]. Protein structures were put into a 3 × 3 × 3 nm
1
4.2. Antiviral Activity
grid box to occupy the whole ligand–protein systems and the spacing
was kept at 1.00 Å, a standard value for AutoDock Vina. Each docking
trial produced 20 poses with the exhaustiveness value of 20.
Structures of ligands were optimized by using YASARA software
with YAMBER force field [42,43]. Binding poses were clustered and one
binding pocket for each ligand, which produced the highest binding
affinity, were assessed. Clustering criteria were bearing the highest
binding affinity and the highest population of hits. Docking simulations
for each ligand were repeated twice as AutoDock Vina uses a stochastic
optimization algorithm. Repetition of simulations produced very si-
milar binding results with very similar binding affinities.
Antiviral effects of the compounds were determined using cyto-
pathic effect (CPE) reduction assays. HEL cells were used to study HSV-
-
1
strain KOS, a thymidine kinase-deficient (TK ) HSV-1 KOS strain re-
sistant to acyclovir, HSV-2 strain G and VV (Lederle strain). Vero cells
were used for Coxsackie B4 virus [26,27].
The assays were performed in 96-well plates containing semicon-
fluent cell cultures. At the same time with the virus, serial dilutions of
the test or reference compounds (brivudin, acyclovir and ganciclovir for
HSV-1 and HSV-2 strains; DS-10.000, ribavirin and mycophenolic acid
for Coxsackie B4 virus) were added. After the plates were incubated at
Binding poses with the highest energies and more hits (number of
poses, in which the ligands bind to the same binding site with different
orientations) were identified as the best poses. Thus, for each ligand
only the best poses were discussed.
3
7 °C for 3–6 days, the antiviral activities [expressed as 50% effective
concentration (EC50)] and cytotoxicity [expressed as minimum cyto-
toxic concentration (MCC)] of the compounds were determined by
microscopy.
4.3. Molecular Modeling
Declaration of Competing Interest
4
.3.1. Molecular Dynamics Simulations
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Starting structures for molecular modeling were taken from rcsb
protein data bank. For HSV-1 (gB) protein electron microscopy struc-
ture (PDB ID: 5fz2) [28] from strain KOS, for HSV-1 (gD) protein for
HSV-1 (gD) protein X-ray diffraction structure (PDB ID: 1l2g, resolu-
tion: 2.85 Å) [29], and for HSV-2 (gD) protein X-ray diffraction struc-
ture (PDB ID: 3w9e, resolution: 2.3 Å) [30] from strain HG52, were
obtained. HSV-1 (gB) and HSV-2 (gD) proteins were modeled as
monomers, HSV-1 (gD) protein as dimer because of structural integrity
between two monomers in the crystal structure.
Acknowledgments
We thank Prof. Lieve Naesens from Rega Institute for Medical
Research, Leuven, Belgium for antiviral screening of the compounds.
This work was supported by Scientific and Technological Research
Council of Turkey (TÜBİTAK) (grant number: 1003- 215S011).
All proteins were subjected to 10 ns long classical molecular dy-
namics (MD) simulations, utilizing GROMACS 5.0.0 program [31–33],
which was utilized with GROMOS force field GROMOS96 54A7 [34].
Proteins were stripped off their water molecules and they were placed
Appendix A. Supplementary data
in
truncated,
rectangular
boxes
with
dimensions
of
8
.0 × 8.0 × 14.0 nm and 8.0 × 8.0 × 10.0 nm, respectively. These
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104202.
dimensions ensured that at any point of the simulation the proteins
11

Z.Ş. Sevinçli, et al.
Bioorganic Chemistry 104 (2020) 104202
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