Synthesis and antiviral properties of novel indole-based thiosemicarbazides and 4-thiazolidinones

Article abstract of DOI:10.1016/j.bmc.2015.12.008

A novel series of indolylthiosemicarbazides (6a-6g) and their cyclization products, 4-thiazolidinones (7a-7g), have been designed, synthesized and evaluated, in vitro, for their antiviral activity against a wide range of DNA and RNA viruses. Compounds 6a, 6b, 6c and 6d exhibited notable antiviral activity against Coxsackie B4 virus, at EC₅₀ values ranging from 0.4 to 2.1 μg/mL. The selectivity index (ratio of cytotoxic to antivirally effective concentration) values of these compounds were between 9 and 56. Besides, 6b, 6c and 6d also inhibited the replication of two other RNA viruses, Sindbis virus and respiratory syncytial virus, although these EC₅₀ values were higher compared to those noted for Coxsackie B4 virus. The SAR analysis indicated that keeping the free thiosemicarbazide moiety is crucial to obtain this antiviral activity, since the cyclization products (7a-7g) did not produce any antiviral effect.

Full text of DOI:10.1016/j.bmc.2015.12.008

Accepted Manuscript
Synthesis and antiviral properties of novel indole-based thiosemicarbazides and
4-thiazolidinones
Gökçe Cihan-Üstündağ, Elif Gürsoy, Lieve Naesens, Nuray Ulusoy
Güzeldemirci, Gültaze Çapan
PII:
S0968-0896(15)30176-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.12.008
BMC 12701
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 September 2015
20 November 2015
6 December 2015
Please cite this article as: Cihan-Üstündağ, G., Gürsoy, E., Naesens, L., Güzeldemirci, N.U., Çapan, G., Synthesis
and antiviral properties of novel indole-based thiosemicarbazides and 4-thiazolidinones, Bioorganic & Medicinal
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.12.008
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Synthesis and antiviral properties of novel indole-based thiosemicarbazides
and 4-thiazolidinones
Gökçe Cihan-Üstündağa*, Elif Gürsoya, Lieve Naesensb, Nuray Ulusoy Güzeldemircia,
Gültaze Çapana
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul University,
Istanbul, 34116, Turkey
bRega Institute for Medical Research, KU Leuven, Department of Microbiology and Immunology,
B-3000 Leuven, Belgium
*Corresponding author: Tel.: +90 212 4400000; fax: +90 212 4400252.
E-mail address: gokcechn@istanbul.edu.tr (G. Cihan-Üstündağ)
ABSTRACT
A novel series of indolylthiosemicarbazides (6a-6g) and their cyclization products,
4-thiazolidinones (7a-7g), have been designed, synthesized and evaluated, in vitro, for their
antiviral activity against a wide range of DNA and RNA viruses. Compounds 6a, 6b, 6c and
6d exhibited notable antiviral activity against Coxsackie B4 virus, at EC50 values ranging
from 0.4 to 2.1 µg/ml. The selectivity index (ratio of cytotoxic to antivirally effective
concentration) values of these compounds were between 9 and 56. Besides, 6b, 6c and 6d also
inhibited the replication of two other RNA viruses, Sindbis virus and respiratory syncytial
virus, although these EC50 values were higher compared to those noted for Coxsackie B4
virus. The SAR analysis indicated that keeping the free thiosemicarbazide moiety is crucial to
obtain this antiviral activity, since the cyclization products (7a-7g) did not produce any
antiviral effect.
Keywords
Indole; Thiosemicarbazide; 4-Thiazolidinone; Antiviral activity; Coxsackie B4 virus

1. Introduction
Viral diseases continue to cause serious morbidity and mortality worldwide. For several
virus infections, no antiviral medications are presently available. There is a clear need for new
antiviral agents with new mechanisms of action or broad-spectrum activity, to face the issues
of drug-resistant mutant viruses or emerging and neglected viruses.
The Picornaviridae are a family of non-enveloped, single-stranded RNA-viruses for
which no antiviral drugs are yet available. Among these, the Enterovirus genus is of particular
medical importance.1 It contains the Coxsackie viruses which are classified into group A
(serotypes A1 to A24) and group B (serotypes B1 to B6) based on early observations of the
pathogenicity in mice.2 Coxsackie virus group B (CVB) and, to a lesser extent, Coxsackie
virus group A and some other enteroviruses, are the main viral causes of myocarditis and
pericarditis. CVB is also associated with a wide variety of other diseases, including diabetes,
common cold, cardiomyopathy, neurological disorders and inflammation.3 Outbreaks of CVB
occur annually throughout the world. Several synthetic molecules have been reported to be
selective inhibitors of enteroviruses, and some of these have entered into clinical trials.4-7
Unfortunately, neither of these investigational compounds has been formally approved for the
treatment of acute enteroviral (including Coxsackie virus) infections.
The indole core is a ubiquitous substructure in a large number of biologically active
natural and synthetic molecules. Delavirdine (I), arbidol (II) and methisazone (III) (Fig. 1) are
indole derived marketed drugs that have been used to treat viral diseases. Arbidol is an
influenza virus inhibitor which is marketed in Russia, China and a few other countries. It was
reported to have a wide spectrum of antiviral activity against a number of enveloped and non-
enveloped viruses in addition to influenza viruses.8 For instance, arbidol was found to exhibit
potent inhibitory activity against CVB3 and CVB5.3,9 Another indole compound, N-
methylisatin-β-thiosemicarbazone, methisazone, was used in the preventive treatment of
smallpox after 1962. Based on in vitro studies, the antiviral spectrum of methisazone appears
to include diverse DNA and RNA viruses.10,11 A series of investigations on isatin-β-
thiosemicarbazones revealed their antiviral properties against various virus types, such as
herpes simplex virus (HSV),12 Moloney leukemia virus,13 Japanese encephalitis virus14 and
human immunodeficiency virus (HIV).15 In a recent report by Zhang et al,16 an isatin-β-
hydrazone derivative (encoded ID45) was described as a promising antiviral agent against
CVB3. Broad antiviral in vitro activity that includes CVB217 or CVB418 was further reported
for some 2,3,5-trisubstituted indole derivatives synthesized by Giampieri et al17 and

isoindolylureas synthesized by Verma et al.18 Another class are indole-2-carboxamide
derivatives, some of which were revealed to have promising antiviral properties during the
past decade. Compounds with this scaffold were found to inhibit the replication of neurotropic
alphavirus,19 HIV-1,20 HIV-1 wild type or drug-resistant mutant strains, and CVB4.21 Another
relevant scaffold is the 4-thiazolidinone substructure, which is present in several small
synthetic molecules endowed with in vitro antiviral characteristics.22-24
In view of these literature reports, we have focused our work on the design and
synthesis of a new series of indolylthiosemicarbazides (6a-6g) and their cyclization products,
4-thiazolidinones (7a-7g). These new compounds were evaluated for in vitro antiviral activity
against a wide variety of DNA and RNA viruses.
N
CH3
N
NH
CH3
NH
H3C
N
O
S
O
O
N
H
S
CH3
N
I
NH2
CH3
O
NH
O
N
H3C
HO
O
N
S
N
Br
CH3
CH3
III
II
Figure 1. Structures of some indole containing antiviral drugs delavirdine (I), arbidol (II) and
methisazone (III)

2. Results and Discussion
2.1. Chemistry and structural characterization
The synthetic pathways for the preparation of the target products are illustrated in
Scheme 1. The key intermediate 5 was previously synthesized and patented by Ba-Maung and
co-workers as an angiogenesis inhibitor.25 The structures of the new compounds were
established by microanalysis, IR, 1H-NMR, 13C-NMR (proton decoupled and APT), 2D-NMR
(HSQC and HMBC) and electrospray ionization mass spectrometry (ESI-MS). The absolute
stereochemistry of 7d was determined by an X-ray diffraction study.26
The absence of the N-H2 resonance of the intermediate hydrazide (5) at δ 4.48 ppm
together with three new resonances located at about δ 9.68-9.82, 9.35-9.57 and 7.80-8.43 ppm
assigned to the N1H, N2H and N4H protons, supported the synthesis of new
thiosemicarbazides (6a-6g). Observation of new lactam C=O bands (1710-1728 cm-1) besides
C=O amide bands (1636-1670 cm-1) in the IR spectra of 7a-7g provided evidence for ring
closure. New singlets assigned to the methylene protons of 4-thiazolidinone ring (δ 3.95-4.05
1
ppm) in the H-NMR spectra of 7a-7g provided further confirmation. Peaks associated with
the indole subunit were observed in the expected regions and were assigned on the basis of
1H-1H and 1H-19F couplings.
13C-NMR experiments (proton decoupled and APT) run on 5, 6a, 6b, 6e, 6g, 7a-c, 7e
and 2D-NMR experiments (HSQC and HMBC) run on 6c, 6d, 6f, 7d, 7f, 7g allowed
unambiguous assingment of the proton and carbon chemical shifts. The carbocyclic indole
carbons which explicitly showed the 13C-19F couplings of the 5-fluoro indole core were
observed as separate doublets with characteristic coupling constants related to the ipso, ortho,
meta and para positions and allowed definite positional assignment of the C3, C3a, C4-7 and
C7a carbons of 6 and 7. Cross peaks observed in the HMBC spectrum of compound 6c
enabled the definite assignment of C=O (δ161.93-162.04 ppm) and C=S (δ 181.96-182.93
ppm) carbons of the thiosemicarbazide analogs, 6a-6g. The spectra of 7a-7g displayed the
typical carbonyl (δ 171.61-172.94 ppm) and methylene (δ 33.39-33.58 ppm) carbons of the 4-
thiazolidinone skeleton, which further verified the aimed conversion.
Electrospray ionization mass spectrometry (ESI-MS) was employed to confirm the
molecular weights of compounds 6a-6g and 7a-7g. All compounds were analyzed under
negative-ion ESI conditions since none were responsive to the positive-ion mode.

-
Deprotonated [M-H] ions observed in the ESI-MS, verified the calculated molecular weights
of the new compounds.
F
F
F
ii
iii
i
C H
CH
C
6
5
2
+
2
-
NH
N Cl
2
NHN
COOC H
2
5
1
2
3
C H
C H
6
5
6
5
F
F
v
iv
O C H
C
O
C
O
NH NH
2
5
2
N
H
N
H
4
5
F
F
C H
6
5
C H
6
5
S
S
O
vi
R
C
NH
NH
N
H
N
C
O
N
H
C
O
NH
NH
N
R
7a-g
6a-g
6a,7a: R= CH3
6e,7e: R= C4H9
6b,7b: R= C2H5
6c,7c: R= C3H7
6d,7d: R= C3H5
6f, 7f: R= CH2C6H5
6g,7g: R= CH2CH2C6H5
Scheme 1. Synthesis of 6 and 7. Reagents and conditions: (i) 7% NaNO2, EtOH, conc. HCl,
0°C; (ii) ethyl 2-benzyl-3-oxo-butanoate, KOH, EtOH, 0°C; (iii) conc. HCl, reflux, 4h ; (iv)
H2NNH2.H2O, EtOH, reflux, 6h; (v) substituted isothiocyanate, EtOH, reflux, 3h; (vi) ethyl
bromoacetate, fused sodium acetate, abs. EtOH, reflux, 3h.

2.2. Antiviral activity
Antiviral evaluation in cell-based assays revealed that compounds 6a, 6b, 6c and 6d
have interesting activity against Coxsackie B4 virus. This effect, observed in two different
cell lines (HeLa and Vero; Table 1), was quite strong since the antiviral EC50 values were in
the range of 0.4 to 2.1 µg/ml, and the values for the selectivity index (ratio of cytotoxic to
antivirally effective concentration; see values between square brackets in Table 1) were
between 9 and 56. Compound 6b (with an ethyl substituent) was slightly more potent than the
analogues carrying a methyl (6a), propyl (6c) or allyl (6d) moiety; this is also visible from the
dose-response curves shown in Figure 2. Besides, 6b, 6c and 6d also inhibited the replication
of two other RNA viruses, Sindbis virus and respiratory syncytial virus, although these EC50
values were higher compared to those noted for Coxsackie B4 virus. Of note, the analogues
carrying a larger butyl (6e) or aromatic (6f and 6g) substituent at the R position were devoid
of this antiviral activity. The crucial role of the free thiosemicarbazide moiety was evident
from the fact that their cyclized analogues, 4-thiazolidinones (7a-7g), did not produce any
antiviral effect.
Compounds 6a-6g and 7a-7g did not display activity against any of the other RNA-
viruses tested (such as HIV or influenza virus), nor against DNA-viruses (i.e. HSV-1, HSV-2,
feline herpesvirus and vaccinia virus) (Table 2). On the other hand, this broad antiviral testing
allowed to determine the cytotoxicity of the test compounds in diverse mammalian cell lines
(Table 3). This analysis revealed that the 4-thiazolidinone derivatives (7) were consistently
less cytotoxic than the corresponding thiosemicarbazides (6). Within series 6, the compounds
endowed with anti-Coxsackie B4 virus activity (6a-6d) tended to be less cytotoxic than the
antivirally inactive counterparts 6e-6g.

Table 1
Antiviral activity in HeLa and Vero cell cultures infected with diverse RNA viruses
Compound
Antiviral assays in HeLa cells
Antiviral EC50a value (µg/ml)
Antiviral assays in Vero cells
Antiviral EC50a value (µg/ml)
MCCb
MCCb
Vesicular
stomatitis
Coxsackie Respiratory
(µg/ml)
Para-
influenza-
3 virus
Reovirus- Sindbis Coxsackie Punta
(µg/ml)
syncytial
Toro
B4 virus
1
virus
B4 virus
virus
virus
virus
>100
>100
>100
>100
2.1 ± 0.2
[9]
>100
20
20
20
20
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.7 ± 0.5
[12]
>100
>100
>100
>100
20
20
6a
6b
6c
6d
0.87 ± 0.47
[23]
≥4
[≥5]
3.2 ± 0.8 0.4 ± 0.0
[6] [50]
6.5 ± 2.5 1.8 ± 0.0
1.1 ± 0.3
[18]
≥2.3
[≥9]
100
20
[15]
[56]
1.5 ± 0.1
[13]
≥4
[≥5]
≥4
[≥5]
2.0 ± 0.2
[10]
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
10
10
6e
6f
4
4
10
6g
7a
7b
7c
7d
7e
7f
100
>100
>100
>100
>100
>100
100
>100
20
20
>100
>100
>10
≥10
10
7g
8.0 ± 3.1
[>12]
≥4
[≥25]
5.3 ± 3.3
[>19]
68 ± 32
[>1.5]
40 ± 10
[>2.5]
20 ± 0
[>5]
>100
DS-5000c
23 ± 6
[>11]
61 ± 32
[>4]
15 ± 7
[>17]
>250
>250
>250
250 ± 0
[>1]
>250
50 ± 0
[>5]
>250
Ribavirind

Table 1 (continued-footnotes)
a EC50: 50% effective concentration, producing 50% inhibition of virus-induced cytopathic effect, as determined by microscopy.
b MCC: minimum inhibitory concentration, or compound concentration causing minimal changes in cell morphology, as assessed by microscopy.
c DS-5000: dextran sulfate of MW 5000.
d Data for ribavirin are expressed in µM.
Values shown are the mean ± SEM of three independent tests.
In square brackets, the selectivity index (i.e. ratio of MCC to antiviral EC50) is given.

Table 2
Results for the DNA- and RNA-viruses shown to be insensitive to the synthesized compounds
Compound
Assays in HELa cells
Assays in MT-4b cells
Assays in MDCKc cells
Influenza Influenza B
A
Assays in CRFKd cells
Feline Feline
coronavirus herpesvirus
HSV-1
HSV-2
Vaccinia
HIV-1
HIV-2
virus
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
8.9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
6a
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
6b
6c
6d
6e
6f
6g
7a
7b
7c
7d
7e
7f
7g
Ribavirine
Ganciclovire
Brivudine
Azidothymidine
0.5
0.2
29
>100
22
>100
7.4
0.04
0.0019
0.0018
aHEL: human embryonic lung fibroblast cells;
bMT4: human T-lymphoblast cells;
cMDCK: Madin-Darby canine kidney cells;
dCRFK: Crandell-Rees feline kidney cells.
eFor these compounds, data are expressed in µM. All other concentrations are expressed in µg/ml.

Table 3
Cytotoxic activity in diverse mammalian cell linesa
Compound
MCCb (µg/ml)
CC50c (µg/ml)
CRFK MT4
HEL
20
HeLa Vero MDCK
7.5
2.9
9.7
8.6
1.4
1.9
1.9
18
14
11
13
11
11
11
20
20
20
20
4
20
20
100
20
10
10
10
20
20
20
4
6a
20
6b
20
0.8
4
6c
20
6d
>10
>10
10
10
>10
2
6e
4
6f
4
6g
>100
>100
>100
>100
>10
>10
>10
ND
>100 >125
>100 >125
>100 >125
>100 >125
100
>100
100
100
7a
7b
>100 >100 >100
7c
>100 >100
100
>10
>10
>10
ND
100
ND
ND
7d
>100
>100
17
≥98
38
>100
>100
100
>10
≥10
10
7e
7f
≥108
ND
ND
ND
>25
7g
DS-5000d
Ribavirine
Ganciclovire
Azidothymidine
>100 >100
>250 >250
ND
>250
>100
ND
ND
ND
ND
ND
ND
>100
ND
aHEL: human embryonic lung fibroblast cells; HeLa: human cervix carcinoma cells; Vero: African green monkey
kidney cells; MDCK: Madin-Darby canine kidney cells; CRFK: Crandell-Rees feline kidney cells; MT4: human T-
lymphoblast cells.
bMCC: minimum inhibitory concentration, or compound concentration causing minimal changes in cell morphology,
as assessed by microscopy.
cCC50: 50% cytotoxic concentration, assessed by the spectroscopic MTS cell viability assay.
dDS-5000: dextran sulfate with MW 5000.
eFor these compounds,data are expressed in µM.
ND: not done.

Figure 2. Dose-response curves for inhibition of Coxsackie B4 virus replication.
(Upper panel: assay in HeLa cells; lower panel: assay in Vero cells)

3. Conclusion
We have efficiently synthesized novel thiosemicarbazide (6a-6g) and 4-thiazolidinone (7a-
7g) derivatives with the 5-fluoro-3-phenyl-1H-indole scaffold, and evaluated their in vitro
antiviral activity against a broad range of DNA and RNA viruses. Compounds 6a, 6b, 6c and 6d
exhibited significant and selective inhibitory effect on the replication of Coxsackie B4 virus.
Compounds 6b, 6c and 6d also had weaker antiviral activity against Sindbis virus and respiratory
syncytial virus. The SAR analysis indicated that keeping the free thiosemicarbazide moiety is
crucial for the antiviral activity since the cyclization products (7a-7g) did not produce any
antiviral effect. Also, it was observed that the presence of a bulkier butyl (6e) or aromatic (6f and
6g) substituent at the R position led to a dramatic decrease in biological activity.
In conclusion, the promising anti-Coxsackie B4 virus activity of new
indolylthiosemicarbazide derivatives (6a-6d) makes them interesting lead compounds for further
antiviral drug development. Hence, we have embarked on further structural optimization, besides
mechanistic antiviral experiments, to improve and understand this antiviral activity.
4. Experimental
4.1. Chemistry
4.1.1. General methods
Melting points were determined in open capillary tubes with a Buchi B-540 melting point
apparatus and are uncorrected. Microanalyses were performed on a Thermo Finnigan Flash EA
1112 elemental analyzer. IR spectra were recorded in KBr discs (υmax in cm-1) on a Perkin-Elmer
1600 FTIR. 1H-NMR (DMSO-d6),13C-NMR (Proton decoupled, APT) (DMSO-d6) and
heteronuclearcorrelation 1H-13C (HSQC, HMBC) (DMSO-d6) spectra were run on Bruker AC 200
(200 MHz) and VarianUNITYINOVA (500 MHz) instruments. Chemical shifts are reported as 
(ppm) relative to TMS as internal standard and coupling constants (J) are given in hertz (Hz). MS
(ESI-) were determined on a Finnigan LCQ Advantage Max mass spectrometer. (br.:broad, ind.:
indole, thz.:thiazolidinone)
4.1.2 Ethyl 2-benzyl-2-(4-fluorophenylhydrazono)acetate (3)
To a solution of 1 (0.02 mol) in ethanol (10 mL), water (10 mL) and conc. HCl (6 mL), 7%
aqueous NaNO2 solution (10 mL) was added dropwise at 0oC with stirring. The resulting solution

of diazonium salt (2) was poured into a cooled (0oC) mixture of ethyl 2-benzyl-3-oxobutanoate
(0.02 mol), ethanol (10 mL), water (10 mL) and KOH (5.4 g) while stirring. The resulting mixture
was refrigerated overnight. The red oily residue thus obtained was separated, washed with water
and used without further purification.
4.1.3. Ethyl 5-fluoro-3-phenyl-1H-indole-2-carboxylate (4)
A solution of 3 (0.02 mol) in conc. HCl (20 mL) was heated under reflux for 4 h. The crude
product was filtered off, washed with water until tested neutral to litmus and used without further
purification.
4.1.4. 5-Fluoro-3-phenyl-1H-indole-2-carbohydrazide (5)25
A mixture of 4 (0.02 mol), ethanol (20 mL) and H2NNH2.H2O (98%, 8 mL) was heated under
reflux for 6 h. The resulting brown crystals were filtered off and recrystallized from ethanol-
1
chloroform. Mp 222-225 °C; IR(KBr): υmax 3279 (N-H), 1624 (C=O); H-NMR (DMSO-
d6/200MHz):δ 4.48 (s, 2H, NH2), 7.10 (td, 1H, J=9.1, 2.2, H6-ind.), 7.21 (dd, 1H, J=9.8, 2.0,
H4-ind.), 7.34-7.38 (m, 1H, H7-ind.), 7.42-7.61 (m, 5H, 3-C6H5-ind.), 8.86 (s, 1H, CONH), 11.82
(s, 1H, NH); 13C-NMR(Proton decoupled, DMSO-d6/125MHz):δ 104.65 (d, J=23.7, C4-ind.),
112.78 (d, J=26.1, C6-ind.), 114.24 (d, J=10.0, C7-ind.), 117.50 (d, J=4.3, C3-ind.), 127.36 (d,
J=8.7, C3a-ind.), 129.21 (3-C6H5(C4)-ind.), 129.94 (3-C6H5(C3,C5)-ind.), 130.26 (C2-ind.),
130.41 (3-C6H5(C2,C6)-ind.), 132.82 (C7a-ind.), 134.27 (3-C6H5(C1)-ind.), 158.28 (d, J=232.2,
C5-ind.), 162.23 (C=O).
4.1.5. General procedure for the synthesis of 1-((5-fluoro-3-phenyl-1H-indol-2-yl)carbonyl)-
4-substituted-3-thiosemicarbazides (6a-6g)
A mixture of 5 (0.005 mol) and an appropriate isothiocyanate(0.005 mol) was refluxed in 30 mL
abs. ethanol for 3 h and then allowed to cool. The precipitate was filtered and purified by
recrystallization from ethanol.
4.1.5.1. 1-((5-fluoro-3-phenyl-1H-indol-2-yl)carbonyl)-4-methyl-3-thiosemicarbazide (6a)
White powder (81.4%); mp 198-200 °C; IR(KBr): υmax =3479, 3342, 3157 (O-H, N-H), 1636
1
(C=O); H-NMR (DMSO-d6/500MHz):δ 2.90 (3H, d, J=4.4, NH-CH3), 7.15 (1H, td, J=9.3, 2.4,
H6-ind.), 7.23 (1H, dd, J=10.0, 2.2, H4-ind.), 7.34 (1H, t, J=7.3, 3-C6H5(H4)-ind.), 7.44 (2H, t,
J=7.5, 3-C6H5(H3,H5)-ind.), 7.56-7.52 (3H, m, H7, 3-C6H5(H2, H6)-ind.), 7.82 (1H, br. s, N4H),

9.41 (1H, s, N2H), 9.72 (1H, s, N1H), 11.78 (1H, s, NH-ind.);13C-NMR (APT) (DMSO-
d6/125MHz):δ 31.52 (NH-CH3), 104.97 (d, J=23.5, C4-ind.), 113.65 (d, J=26.8, C6-ind.), 114.46
(d, J=9.6, C7-ind.), 119.51 (d, J=5.3, C3-ind.), 127.28 (d, J=9.9, C3a-ind.), 127.55 (3-C6H5(C4)-
ind.), 128.60 (C2-ind.), 129.13 (3-C6H5(C3,C5)-ind.), 130.52 (3-C6H5(C2,C6)-ind.), 132.85 (C7a-
ind.), 133.83 (3-C6H5(C1)-ind.), 158.37 (d, J=233.9, C5-ind.), 161.98 (C=O), 182.93 (C=S); MS
(ESI-) m/z (%):341.1 (M-H-, 100).Anal. Calcd for C17H15FN4OS.H2O (342.39): C, 56.65; H, 4.75;
N, 15.55. Found: C, 57.11; H, 5.12; N, 15.62.
4.1.5.2. 4-Ethyl-1-((5-fluoro-3-phenyl-1H-indol-2-yl)carbonyl)-3-thiosemicarbazide (6b)
White powder (76.9%); mp 189-191 °C; IR(KBr): υmax 3337, 3177 (N-H), 1646 (C=O); 1H-NMR
(DMSO-d6/500MHz):δ 1.07 (3H, t, J=7.3, NH-CH2-CH3), 3.48 (2H, m, NH-CH2-CH3), 7.16 (1H,
td, J=9.2, 2.4, H6-ind.), 7.23 (1H, dd, J=9.8, 2.4, H4-ind.), 7.35 (1H, t, J=7.3, 3-C6H5(H4)-ind.),
7.45 (2H, t, J=7.8, 3-C6H5(H3,H5)-ind.), 7.54-7.57 (3H, m, H7, 3-C6H5(H2, H6)-ind.), 7.81 (1H,
br. t, N4H), 9.36 (1H, s, N2H), 9.68 (1H, s, N1H), 11.82 (1H, s, NH-ind.);13C-NMR (APT)
(DMSO-d6/125MHz):δ 15.18 (NH-CH2-CH3), 56.73 (NH-CH2-CH3), 104.96 (d, J=23.5, C4-ind.),
113.64 (d, J=26.4, C6-ind.), 114.47 (d, J=9.6, C7-ind.), 119.50 (d, J=5.3, C3-ind.), 127.28 (d,
J=9.6, C3a-ind.), 127.56 (3-C6H5(C4)-ind.), 128.58 (C2-ind.), 129.14 (3-C6H5(C3,C5)-ind.),
130.56 (3-C6H5(C2,C6)-ind.), 132.88 (C7a-ind.), 133.85 (3-C6H5(C1)-ind.), 158.37 (d, J=233.8,
C5-ind.), 161.94 (C=O), 181.96 (C=S); MS (ESI-) m/z (%):355.1 (M-H-, 100). Anal. Calcd for
C18H17FN4OS (356.42): C, 60.66; H, 4.81; N, 15.72. Found: C, 60.02; H, 5.06; N, 15.48.
4.1.5.3. 1-((5-fluoro-3-phenyl-1H-indol-2-yl)carbonyl)-4-propyl-3-thiosemicarbazide (6c)
1
White flakes (67.7%); mp 191-193 °C; IR(KBr): υmax 3349, 3290 (N-H), 1656 (C=O); H-NMR
(DMSO-d6/500MHz):δ 0.83 (3H, t, J=7.3, NH-CH2-CH2-CH3), 1.51 (2H, m, NH-CH2-CH2-CH3),
3.41 (2H, br. q, NH-CH2-CH2-CH3), 7.15 (1H, td, J=9.3, 2.4, H6-ind.), 7.23 (1H, dd, J=9.8, 2.0,
H4-ind.), 7.35 (1H, t, J=7.6, 3-C6H5(H4)-ind.), 7.45 (2H, t, J=7.6, 3-C6H5(H3,H5)-ind.), 7.54-7.57
(3H, m, H7, 3-C6H5(H2, H6)-ind.), 7.81 (1H, br. t, N4H), 9.36 (1H, s, N2H), 9.68 (1H, s, N1H),
11.82 (1H, s, NH-ind.);13C-NMR (HMBC) (DMSO-d6/125MHz):δ 11.88 (NH-CH2-CH2-CH3),
22.72 (NH-CH2-CH2-CH3), 45.00 (NH-CH2-CH2-CH3), 104.96 (d, J=23.5, C4-ind.), 113.64 (d,
J=26.4, C6-ind.), 114.49 (d, J=9.6, C7-ind.), 119.48 (d, J=5.3, C3-ind.), 127.30 (d, J=9.6, C3a-
ind.), 127.57 (3-C6H5(C4)-ind.), 128.58 (C2-ind.), 129.14 (3-C6H5(C3,C5)-ind.), 130.55 (3-
C6H5(C2,C6)-ind.), 132.88 (C7a-ind.), 133.85 (3-C6H5(C1)-ind.), 158.37 (d, J=233.9, C5-ind.),

161.93 (C=O), 182.24 (C=S); MS (ESI-) m/z (%):369.1 (M-H-, 100). Anal. Calcd for
C19H19FN4OS (370.44): C, 61.60; H, 5.17; N, 15.12. Found: C, 61.38; H, 5.76; N, 15.07.
4.1.5.4. 4-Allyl-1-((5-fluoro-3-phenyl-1H-indol-2-yl)carbonyl)-3-thiosemicarbazide (6d)
White needles (69.0%); mp 193-195 °C; IR(KBr): υmax 3366, 3290 (N-H), 1669 (C=O); 1H-NMR
(DMSO-d6/500MHz):δ 4.13 (2H, t, J=5.2, NH-CH2-CH=CH2), 5.05 (1H, dd, J=10.3, 1.5, NH-
CH2-CH=CH2), 5.13 (1H, dd, J=17.3, 1.5, N-CH2-CH=CH2), 5.78-5.86 (1H, m, NH-CH2-
CH=CH2), 7.15 (1H, td, J=9.1, 2.4, H6-ind.), 7.23 (1H, dd, J=9.8, 2.4, H4-ind.), 7.33 (1H, t, J=7.3,
3-C6H5(H4)-ind.), 7.43 (2H, t, J=7.6, 3-C6H5(H3,H5)-ind.), 7.52-7.57 (3H, m, H7, 3-C6H5(H2,
H6)-ind.), 7.99 (1H, t, J=5.7, N4H), 9.47 (1H, s, N2H), 9.75 (1H, s, N1H), 11.80 (1H, s, NH-ind.);
13C-NMR (HSQC) (DMSO-d6/125MHz): δ 46.55 (NH-CH2-CH=CH2), 104.96 (d, J=24.0, C4-
ind.), 113.64 (d, J=26.4, C6-ind.), 114.47 (d, J=9.6, C7-ind.), 116.14 (N-CH2-CH=CH2), 119.56
(d, J=5.2, C3-ind.), 127.31 (d, J=9.6, C3a-ind.), 127.57 (3-C6H5(C4)-ind.), 128.56 (C2-ind.),
129.13 (3-C6H5(C3,C5)-ind.), 130.51 (3-C6H5(C2,C6)-ind.), 132.88 (C7a-ind.), 133.84 (3-
C6H5(C1)-ind.), 135.45 (NH-CH2-CH=CH2), 158.38 (d, J=233.9, C5-ind.), 161.99 (C=O), 182.60
(C=S); MS (ESI-) m/z (%):367.1 (M-H-, 100).Anal. Calcd for C19H17FN4OS (368.43): C, 61.94;
H, 4.65; N, 15.21. Found: C, 61.67; H, 5.11; N, 15.16.
4.1.5.5. 4-Butyl-1-((5-fluoro-3-phenyl-1H-indol-2-yl)carbonyl)-3-thiosemicarbazide (6e)
White crystals (92.8%); mp 194-196 °C; IR(KBr): υmax 3385, 3300, 3169 (N-H), 1670 (C=O); 1H-
NMR (DMSO-d6/500MHz):δ 0.87 (3H, t, J=7.3,CH2-CH3), 1.23-1.30 (2H, m, CH2-CH3), 1.44-
1.50 (2H, quin., J=7.3, NH-CH2-CH2), 3.42-3.45 (2H, m, NH-CH2-CH2), 7.16 (1H, td, J=9.3, 2.4,
H6-ind.), 7.23 (1H, dd, J=9.8, 2.0, H4-ind.), 7.35 (1H, t, J=7.3, 3-C6H5(H4)-ind.), 7.45 (2H, t,
J=7.8, 3-C6H5(H3,H5)-ind.), 7.53-7.57 (3H, m, H7, 3-C6H5(H2, H6)-ind.), 7.80 (1H, br. t, N4H),
9.35 (1H, s, N2H), 9.68 (1H, s, N1H), 11.82 (1H, s, NH-ind.);13C-NMR (APT) (DMSO-
d6/125MHz): δ 14.50 (CH2-CH3), 20.13 (CH2-CH3), 31.62 (NH-CH2-CH2) 44.04 (NH-CH2-CH2),
104.96 (d, J=23.5, C4-ind.), 113.64 (d, J=26.8, C6-ind.), 114.50 (d, J=9.6, C7-ind.), 119.49 (d,
J=5.5, C3-ind.), 127.31 (d, J=9.9, C3a-ind.), 127.57 (3-C6H5(C4)-ind.), 128.57 (C2-ind.), 129.14
(3-C6H5(C3,C5)-ind.), 130.55 (3-C6H5(C2,C6)-ind.), 132.88 (C7a-ind.), 133.86 (3-C6H5(C1)-ind.),
158.37 (d, J=233.9, C5-ind.), 161.94 (C=O), 182.18 (C=S); MS (ESI-) m/z (%):383.1 (M-H-, 100).
Anal. Calcd for C20H21FN4OS (384.47): C, 62.48; H, 5.51; N, 14.57. Found: C, 62.08; H, 5.68; N,
14.35.

4.1.5.6. 4-Benzyl-1-((5-fluoro-3-phenyl-1H-indol-2-yl)carbonyl)-3-thiosemicarbazide (6f)
White flakes (82.3%); mp 202-204 °C; IR(KBr): υmax 3346, 3323, 3294, 3248 (N-H), 1670 (C=O);
1H-NMR (DMSO-d6/500MHz):δ 4.77 (2H, d, J=5.9, NH-CH2), 7.15 (1H, td, J=9.3, 2.4, H6-ind.),
7.22-7.25 (2H, m, CH2-C6H5(H4), H4-ind.), 7.28-7.31 (4H, m, CH2-C6H5(H2,H3,H5,H6), 7.33
(1H, t, J=7.3, 3-C6H5(H4)-ind.), 7.42 (2H, t, J=7.8, 3-C6H5(H3,H5)-ind.), 7.54-7.56 (3H, m, H7, 3-
C6H5(H2,H6)-ind.), 8.43 (1H, br. t, N4H), 9.57 (1H, s, N2H), 9.82 (1H, s, N1H), 11.80 (1H, s, NH-
ind.);13C-NMR (HSQC) (DMSO-d6/125MHz):δ 47.41 (NH-CH2), 104.98 (d, J=24.0, C4-ind.),
113.67 (d, J=26.8, C6-ind.), 114.50 (d, J=9.6, C7-ind.), 119.61 (d, J=5.3, C3-ind.), 127.29 (d,
J=9.6, C3a-ind.), 127.43, 127.54, 127.70 (CH2-C6H5(C2-6), 3-C6H5(C4)-ind.), 128.50 (C2-ind.),
128.83 (CH2-C6H5(C2,C3,C5,C6)), 129.12 (3-C6H5(C3,C5)-ind.), 130.52 (3-C6H5(C2,C6)-ind.),
132.86 (C7a-ind.), 133.82 (3-C6H5(C1)-ind.), 139.79 (CH2-C6H5(C1)), 158.37 (d, J=233.9, C5-
ind.), 162.04 (C=O), 182.91 (C=S); MS (ESI-) m/z (%):417.1 (M-H-, 100).Anal. Calcd for
C23H19FN4OS (418.49): C, 66.01; H, 4.58; N, 13.39. Found: C, 66.33; H, 4.53; N, 13.24.
4.1.5.7. 1-((5-fluoro-3-phenyl-1H-indol-2-yl)carbonyl)-4-phenethyl-3-thiosemicarbazide (6g)
White flakes (84.3%); mp 201-202 °C; IR(KBr): υmax 3343, 3296, 3265, 3159 (N-H), 1663 (C=O);
1H-NMR (DMSO-d6/500MHz):δ 2.81 (2H, t, J= 7.8, CH2-C H ), 3.63-3.67 (2H, m, NH-CH2),
6
5
7.16 (1H, td, J=9.0, 2.4, H6-ind.), 7.19-7.28 (6H, m, CH2-C6H5, H4-ind.), 7.35 (1H, t, J=7.3, 3-
C6H5(H4)-ind.), 7.44 (2H, t, J=7.8, 3-C6H5(H3,H5)-ind.), 7.52 (2H, d, J=6.8, 3-C6H5(H2, H6)-
ind.), 7.56 (1H, dd, J=8.8, 4.4, H7-ind.), 7.90 (1H, br. t, N4H), 9.49 (1H, s, N2H), 9.69 (1H, s,
N1H), 11.83 (1H, s, NH-ind.);13C-NMR (APT) (DMSO-d6/125MHz):δ 35.61 (CH2-C H5),45.91
6
(NH-CH2), 104.97 (d, J=23.9, C4-ind.), 113.69 (d, J=26.6, C6-ind.), 114.50 (d, J=10.0, C7-ind.),
119.53 (C3-ind.), 126.87 (CH2-C6H5(C4)), 127.31 (d, J=10.0, C3a-ind.), 127.60 (3-C6H5(C4)-
ind.), 128.50 (C2-ind.), 129.11, 129.15, 129.36 (CH2-C6H5(C2,C3,C5,C6), 3-C6H5(C3,C5)-ind.),
130.57 (3-C6H5(C2,C6)-ind.), 132.90 (C7a-ind.), 133.85 (3-C6H5(C1)-ind.), 139.84 (CH2-
C6H5(C1)), 158.37 (d, J=234.3, C5-ind.), 182.32 (C=S); MS (ESI-) m/z (%):431.1 (M-H-,
100).Anal. Calcd for C24H21FN4OS (432.51): C, 66.65; H, 4.89; N, 12.95. Found: C, 66.77; H,
4.95; N, 12.74.

4.1.6. General procedure for the synthesis of 5-fluoro-N’-(4-oxo-3-substituted-1,3-
thiazolidinon-2-ylidene)-3-phenyl-1H-indol-2-carbohydrazides (7a-7g)
A mixture of 6 (0.0025 mol), ethyl bromoacetate (0.0025 mol) and fused sodium acetate (0.01
mol) in absolute ethanol (15 ml) was heated under reflux for 3 h. The solid thus obtained was
filtered, dried and purified by recrystallization from ethanol/chloroform.
4.1.6.1.
5-Fluoro-N’-(3-methyl-4-oxo-1,3-thiazolidinon-2-ylidene)-3-phenyl-1H-indol-2-
carbohydrazide (7a) Beige crystals (97.9%); mp 270-273 °C; IR(KBr): υmax 3318, 3232 (N-H),
1
1723, 1674 (C=O); H-NMR (DMSO-d6/500MHz):δ 3.07 (3H, s, N-CH3), 3.99 (2H, s, S-CH2),
7.11 (1H, td, J=9.0, 2.4, H6-ind.), 7.15 (1H, br. d, H4-ind.), 7.37 (1H, br. s, 3-C6H5(H4)-ind.),
7.47-7.50 (5H, m, H7, 3-C6H5(H2, H6, H3, H5)-ind.), 9.68 (1H, s, NH), 11.90 (1H, s, NH-
ind.);13C-NMR (APT) (DMSO-d6/125MHz): δ 29.89 (N-CH3), 33.58 (S-CH2), 104.73 (d, J=23.5,
C4-ind.), 113.25 (d, J=26.8, C6-ind.), 114.42 (d, J=9.6, C7-ind.), 118.20 (C3-ind.), 127.52 (br. d,
C3a-ind.), 127.82 (3-C6H5(C4)-ind.), 129.47 (C2-ind.), 129.58 (3-C6H5(C3,C5)-ind.), 130.49 (3-
C6H5(C2,C6)-ind.), 132.98 (C7a-ind.), 134.03 (3-C6H5(C1)-ind.), 158.31 (d, J=233.8, C5-ind.),
159.06, 159.37 (C=N, C=O), 172.94 (C=O-thz.);MS (ESI-) m/z (%):381.1 (M-H-, 100).Anal.
Calcd for C19H15FN4O2S(382.41): C, 59.67; H, 3.95; N, 14.65. Found: C, 59.61; H, 4.37; N,
14.62.
4.1.6.2.
N’-(3-ethyl-4-oxo-1,3-thiazolidinon-2-ylidene)-5-fluoro-3-phenyl-1H-indol-2-
carbohydrazide (7b) White crystals (69.5%); mp 267-270 °C; IR(KBr): υmax 3310, 3237 (N-H),
1710, 1654 (C=O); 1H-NMR (DMSO-d6/500MHz):δ 1.13 (3H, br. s, N-CH2-CH3), 3.67 (2H, br. s,
N-CH2-CH3), 4.00 (2H, s, S-CH2), 7.12 (1H, td, J=9.3, 2.4, H6-ind.), 7.15 (1H, br. d, J=9.3, H4-
ind.), 7.37 (1H, br. t, 3-C6H5(H4)-ind.), 7.48-7.51 (5H, m, H7, 3-C6H5(H2, H6, H3, H5)-ind.), 9.78
(1H, s, NH), 11.89 (1H, s, NH-ind.); 13C-NMR (APT) (DMSO-d6/125MHz): δ 12.80 (N-CH2-
CH3), 33.49 (S-CH2), 38.23 (N-CH2-CH3), 104.72 (d, J=23.5, C4-ind.), 113.22 (d, J=27.3, C6-
ind.), 114.41 (d, J=9.6, C7-ind.), 118.20 (C3-ind.), 127.51 (C3a-ind.), 127.76 (3-C6H5(C4)-ind.),
129.52 (3-C6H5(C3,C5)-ind.), 130.50 (3-C6H5(C2,C6)-ind.), 132.94 (C7a-ind.), 134.04 (3-
C6H5(C1)-ind.), 158.31 (d, J=233.9, C5-ind.), 159.16 (C=N, C=O), 171.76 (C=O-thz.);MS (ESI-)
m/z (%): 395.2 (M-H-, 100).Anal. Calcd for C20H17FN4O2S(396.44): C, 60.59; H, 4.32; N, 14.13.
Found: C, 60.24; H, 4.87; N, 14.07.

4.1.6.3.
5-Fluoro-N’-(4-oxo-3-propyl-1,3-thiazolidinon-2-ylidene)-3-phenyl-1H-indol-2-
carbohydrazide (7c) White crystals (91.4%); mp 254-256 °C; IR(KBr): υmax 3308, 3253 (N-H),
1
1712, 1658 (C=O); H-NMR (DMSO-d6/500MHz):δ 0.83 (3H, br. s, NH-CH2-CH2-CH3), 1.60
(2H, br. s, NH-CH2-CH2-CH3), 3.61 (2H, br. s, NH-CH2-CH2-CH3), 4.02 (2H, s, S-CH2), 7.11
(1H, td, J=9.2, 2.4, H6-ind.), 7.16 (1H, br. d, J=9.3, H4-ind.), 7.37 (1H, br. t, 3-C6H5(H4)-ind.),
7.48-7.51 (5H, m, H7, 3-C6H5(H2,H6,H3,H5)-ind.), 9.80 (1H, s, NH), 11.87 (1H, s, NH-ind.);13C-
NMR (APT) (DMSO-d6/125MHz): δ 11.75 (NH-CH2-CH2-CH3), 20.47 (NH-CH2-CH2-CH3),
33.39 (S-CH2), 44.67 (NH-CH2-CH2-CH3), 104.72 (d, J=23.5, C4-ind.), 113.21 (d, J=26.4, C6-
ind.), 114.40 (d, J=10.1, C7-ind.), 118.21 (d, J=4.8, C3-ind.), 127.50 (C3a-ind.), 127.73 (3-
C6H5(C4)-ind.), 129.49 (3-C6H5(C3,C5)-ind.), 130.50 (3-C6H5(C2,C6)-ind.), 132.94 (C7a-ind.),
134.07 (3-C6H5(C1)-ind.), 158.31 (d, J=233.4, C5-ind.), 159.15, 159.81 (C=N, C=O), 172.11
(C=O-thz.);MS (ESI-) m/z (%):409.1 (M-H-, 100).Anal. Calcd for C21H19FN4O2S(410.46): C,
61.45; H, 4.67; N, 13.65. Found: C, 61.29; H, 5.24; N, 13.63.
4.1.6.4.
N’-[(2Z)-3-allyl-4-oxo-1,3-thiazolidinon-2-ylidene]-5-fluoro-3-phenyl-1H-indol-2-
carbohydrazide (7d)26 White crystals (67.3%); mp 262-265 °C; IR(KBr): υmax 3309, 3247 (N-H),
1
1716, 1654 (C=O); H-NMR (DMSO-d6/500MHz):δ 4.05 (2H, s, S-CH2), 4.24 (2H, br. s, NH-
CH2-CH=CH2), 5.12 (2H, m, NH-CH2-CH=CH2), 5.81 (1H, br. s, NH-CH2-CH=CH2), 7.11 (1H,
td, J=9.1, 2.4, H6-ind.), 7.15 (1H, br. d, J=9.3, H4-ind.), 7.36 (1H, br. t, 3-C6H5(H4)-ind.), 7.46-
7.49 (5H, m, H7, 3-C6H5(H2,H6,H3,H5)-ind.), 9.78 (1H, s, NH), 11.87 (1H, s, NH-ind.);13C-NMR
(HSQC) (DMSO-d6/125MHz): δ 33.42 (S-CH2), 45.02 (NH-CH2-CH=CH2), 104.71 (d, J=23.5,
C4-ind.), 113.21 (d, J=25.9, C6-ind.), 114.39 (d, J=10.1, C7-ind.), 117.99 (NH-CH2-CH=CH2),
118.29 (br. d, C3-ind.), 127.51 (br. d, C3a-ind.), 127.75 (3-C6H5(C4)-ind.), 129.49 (3-
C6H5(C3,C5)-ind.), 130.49 (3-C6H5(C2,C6)-ind.), 131.72 (NH-CH2-CH=CH2), 132.96 (C7a-ind.),
134.05 (3-C6H5(C1)-ind.), 158.31 (d, J=233.4, C5-ind.), 159.01, 159.18 (C=N, C=O), 171.61
(C=O-thz.);MS (ESI-) m/z (%):407.1 (M-H-, 100).Anal. Calcd for C21H17FN4O2S(408.45): C,
61.75; H, 4.20; N, 13.72. Found: C, 61.84; H, 4.87; N, 13.69.
4.1.6.5.
N’-(3-butyl-4-oxo-1,3-thiazolidinon-2-ylidene)-5-fluoro-3-phenyl-1H-indol-2-
carbohydrazide (7e) White crystals (74.5%); mp 243-245 °C; IR(KBr): υmax 3292, 3265 (N-H),
1
1728, 1667 (C=O); H-NMR (DMSO-d6/500MHz):δ 0.87 (3H, br. s, CH2-CH3), 1.26 (2H, br. d,
J=5.9, CH2-CH3), 1.56 (2H, br. s, NH-CH2-CH2), 3.64 (2H, br. s, NH-CH2-CH2), 4.01 (2H, s, S-
CH2), 7.10-7.17 (2H, m, H4, H6-ind.), 7.37 (1H, br. t, 3-C6H5(H4)-ind.), 7.49-7.52 (5H, m, H7, 3-

13
C6H5(H2,H6,H3,H5)-ind.), 9.79 (1H, s, NH), 11.88 (1H, s, NH-ind.); C-NMR (APT) (DMSO-
d6/125MHz): δ 14.27 (CH2-CH3), 20.09 (CH2-CH3), 29.21(NH-CH2-CH2), 33.39 (S-CH2), 42.85
(NH-CH2-CH2), 104.72 (d, J=23.9, C4-ind.), 113.21 (d, J=27.2, C6-ind.), 114.40 (d, J=8.7, C7-
ind.), 118.19 (d, J=4.8, C3-ind.), 127.51 (C3a-ind.), 127.73 (3-C6H5(C4)-ind.), 129.49 (3-
C6H5(C3,C5)-ind.), 130.51 (3-C6H5(C2,C6)-ind.), 132.95 (C7a-ind.), 134.07 (3-C6H5(C1)-ind.),
158.31 (d, J=233.9, C5-ind.), 159.14, 159.79 (C=N, C=O), 172.07 (C=O-thz.);MS (ESI-) m/z
(%):423.2 (M-H-, 100).Anal. Calcd for C22H21FN4O2S(424.49): C, 62.25; H, 4.99; N, 13.20.
Found: C, 62.27; H, 4.87; N, 13.15.
4.1.6.6.
N’-(3-benzyl-4-oxo-1,3-thiazolidinon-2-ylidene)-5-fluoro-3-phenyl-1H-indol-2-
carbohydrazide (7f) White crystals (80.0%); mp 268-271 °C; IR(KBr): υmax 3285, 3105 (N-H),
1
1726, 1655 (C=O); H-NMR (DMSO-d6/500MHz):δ 4.01 (2H, s, S-CH2), 4.84 (2H, s, N-CH2),
7.12 (1H, td, J=9.3, 2.4, H6-ind.), 7.16 (1H, br. d, J=8.8, H4-ind.), 7.26-7.37 (6H, m, CH2-C6H5,
3-C6H5(H4)-ind.), 7.47-7.51 (5H, m, H7, 3-C6H5(H2, H6, H3, H5)-ind.), 9.86 (1H, s, NH), 11.91
(1H, s, NH-ind.);13C-NMR (HSQC) (DMSO-d6/125MHz): δ 33.48 (S-CH2), 46.21 (N-CH2),
104.74 (d, J=23.5, C4-ind.), 113.23 (d, J=26.4, C6-ind.), 114.39 (d, J=9.2, C7-ind.), 118.24 (d,
J=4.8, C3-ind.), 127.52 (d, J=8.7, C3a-ind.), 127.72 (3-C6H5(C4)-ind.), 128.23, 128.63, 128.95,
129.06 (CH2-C6H5(C2-6), C2-ind.), 129.49 (3-C6H5(C3,C5)-ind.), 130.50 (3-C6H5(C2,C6)-ind.),
132.97 (C7a-ind.), 134.06 (3-C6H5(C1)-ind.), 136.52 (CH2-C6H5(C1)), 158.32 (d, J=233.4, C5-
ind.), 159.07, 159.17 (C=N, C=O), 172.06 (C=O-thz.);MS (ESI-) m/z (%):457.1 (M-H-,
100).Anal. Calcd for C25H19FN4O2S(458.51): C, 65.49; H, 4.18; N, 12.22. Found: C, 64.99; H,
4.19; N, 11.83.
4.1.6.7.
5-Fluoro-N’-(4-oxo-3-phenethyl-1,3-thiazolidinon-2-ylidene)-3-phenyl-1H-indol-2-
carbohydrazide (7g) White flakes (78.0%); mp 245-247 °C; IR(KBr): υmax 3312, 3238 (N-H),
1719, 1672 (C=O); 1H-NMR (DMSO-d6/500MHz):δ 2.92 (2H, br. s, N-CH2-CH2), 3.85 (2H, br. s,
N-CH2-CH2), 3.95 (2H, s, S-CH2), 7.12 (1H, br. t, J=8.9, H6-ind.), 7.17-7.28 (6H, m, CH2-C6H5,
H4-ind.), 7.38 (1H, br. s, 3-C6H5(H4)-ind.), 7.50-7.54 (5H, m, H7, 3-C6H5(H2, H6, H3, H5)-ind.),
13
9.84 (1H, s, NH), 11.95 (1H, s, NH-ind.); C-NMR (HMBC) (DMSO-d6/125MHz): δ 32.73 (N-
CH2-CH2), 33.46 (S-CH2), 44.29 (N-CH2-CH2), 104.75 (d, J=23.5, C4-ind.), 113.25 (d, J=26.8,
C6-ind.), 114.41 (d, J=9.1, C7-ind.), 118.22 (C3-ind.), 127.16 (CH2-C6H5(C4)), 127.52 (br. d,
C3a-ind.), 127.77 (3-C6H5(C4)-ind.), 129.13, 129.37 (CH2-C6H5(C2,C3,C5,C6), C2-ind.), 129.53

(3-C6H5(C3,C5)-ind.), 130.52 (3-C6H5(C2,C6)-ind.), 132.99 (C7a-ind.), 134.07 (3-C6H5(C1)-ind.),
138.78 (CH2-C6H5(C1)), 158.33 (d, J=233.4, C5-ind.), 159.19 (C=N, C=O), 171.62 (C=O-
thz.);MS (ESI-) m/z (%):471.2 (M-H-, 100).Anal. Calcd for C26H21FN4O2S(472.53): C, 66.09; H,
4.48; N, 11.86. Found: C, 66.32; H, 4.17; N, 11.53.
4.2. Antiviral activity assays
The compounds were evaluated for antiviral activity in cell culture by using cytopathic
effect (CPE) reduction assays with a broad and diverse panel of DNA- and RNA-viruses.27,28
Human cervix carcinoma HeLa cells were used to study vesicular stomatitis virus, Coxsackie B4
virus, and respiratory syncytial virus. African green monkey kidney Vero cells were used to
evaluate para-influenza virus type 3, reovirus type 1, Sindbis virus, Coxsackie B4 virus, and Punta
Toro virus. Viruses evaluated on human embryonic lung fibroblast cells were herpes simplex virus
types 1 and 2, vaccinia virus, and vesicular stomatitis virus. Madin-Darby canine kidney (MDCK)
cells were used for antiviral evaluation against influenza A and B viruses. Feline herpes virus and
feline canine virus were grown in Crandell-Rees feline kidney cells. Finally, HIV-1 and HIV-2
were monitored in human T-lymphoblast MT4 cells.29
To perform the antiviral assays, the viruses were added to subconfluent cultures of the cells
in 96-well plates, and at the same time, the test compounds were added in serial dilutions.
Appropriate reference compounds were included, i.e. the viral entry inhibitor dextran sulfate (MW
5000); the broad antiviral agent ribavirin; the antiherpetic agents ganciclovir and brivudin and the
HIV inhibitor azidothymidine. After 3 to 6 days incubation at 37 °C (or 35 °C in the case of
influenza virus), the compounds’ inhibitory effect on virus-induced cytopathic effect as well as
their cytotoxicity were monitored by light microscopy or by performing the MTS cell viability
assay (CellTiter 96®AQueous One Solution Cell Proliferation Assay from Promega). Antiviral
activity was expressed as the 50% effective concentration (EC50) whereas cytotoxicity was defined
as the minimal cytotoxic concentration (based on microscopy) or CC50 (50% cytotoxic
concentration, assessed by the MTS assay).
Acknowledgements
LN acknowledges excellent technical assistance from L. Persoons, F. De Meyer and K. Erven.

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Graphical abstract
F
C H
6
5
S
C
R
NH
N
H
C
O
NH
NH
6a-g
Compounds with anti-Coxsackie B4 virus activity: 6a (R:CH3), 6b (R:C2H5), 6c (R:C3H7), 6d (R:C3H5)
EC50: 0.4 -2.1 µg/ml
SI: 9-56