Synthesis and biological evaluation of 2-(5-substituted-1-((diethylamino) methyl)-2-oxoindolin-3-ylidene)-N-substituted-hydrazinecarbothioamides

Article abstract of DOI:10.1007/s00044-012-0184-x

Various 5-substituted-2-(1-((diethylamino)methyl)-2-oxoindolin-3-ylidene) hydrazinecarbothioamide (4a, b) and 5-substituted-2-(1-((diethylamino)methyl)-2- oxoindolin-3-ylidene)-N-(phenyl-4-substituted)hydrazinecarbothioamide (5a-h) derivatives were synthesized. The compounds were screened for cytotoxicity against human HeLa and CEM T-lymphocytes as well as murine L1210 cells. The compounds were also screened for β-lactamase inhibitory activity, antiviral, antibacterial, and antifungal activity against various strains of microorganisms. Several of these compounds were endowed with low micromolar 50 %-cytostatic concentration (IC₅₀) values, and some were virtually equally potent as melphalan. The most potent inhibitors against the murine leukemia cells (L1210) were also the most inhibitory against human T-lymphocyte (CEM) tumor cells. Derivative 2-(1-((diethylamino)methyl)-2-oxoindolin-3- ylidene)-N-(4-methoxyphenyl)hydrazinecarbothioamide 5c emerged as the most potent cytostatic compound among the tested compounds. Derivatives 4b, 5a, 5b, and 5d showed antiviral activity against HEL cell cultures (IC₅₀ 11-20 μM). Moderate antimicrobial activity was observed for all derivatives. The encouraging cytostatic and antiviral activity data provide an adequate rationale for further modification of these molecular scaffolds. Graphical abstract: Derivative 5c (1.9-4.4 μM) emerged as the most potent cytostatic compound among the tested compounds. Derivatives 4b, 5a, 5b, and 5d showed antiviral activity against HEL cell cultures (IC₅₀ 11-20 μM). [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-012-0184-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2014–2022
DOI 10.1007/s00044-012-0184-x
ORIGINAL RESEARCH
Synthesis and biological evaluation of 2-(5-substituted-
1-((diethylamino)methyl)-2-oxoindolin-3-ylidene)-
N-substituted-hydrazinecarbothioamides
•
Subhas S. Karki Amol A. Kulkarni
•
•
Sujeet Kumar Suresh Kumar Veliyath
•
•
Erik De Clercq Jan Balzarini
Received: 11 April 2012 / Accepted: 25 July 2012 / Published online: 26 August 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Various 5-substituted-2-(1-((diethylamino)methyl)-
2-oxoindolin-3-ylidene)hydrazinecarbothioamide (4a, b) and
5-substituted-2-(1-((diethylamino)methyl)-2-oxoindolin-
3-ylidene)-N-(phenyl-4-substituted)hydrazinecarbothioamide
(5a–h) derivatives were synthesized. The compounds were
screened for cytotoxicity against human HeLa and CEM
T-lymphocytes as well as murine L1210 cells. The com-
pounds were also screened for b-lactamase inhibitory
activity, antiviral, antibacterial, and antifungal activity
against various strains of microorganisms. Several of these
compounds were endowed with low micromolar 50 %-
cytostatic concentration (IC50) values, and some were vir-
tually equally potent as melphalan. The most potent
inhibitors against the murine leukemia cells (L1210) were
also the most inhibitory against human T-lymphocyte
(CEM) tumor cells. Derivative 2-(1-((diethylamino)
methyl)-2-oxoindolin-3-ylidene)-N-(4-methoxyphenyl)hy-
drazinecarbothioamide 5c emerged as the most potent
cytostatic compound among the tested compounds.
Derivatives 4b, 5a, 5b, and 5d showed antiviral activity
against HEL cell cultures (IC50 11–20 lM). Moderate
antimicrobial activity was observed for all derivatives. The
encouraging cytostatic and antiviral activity data provide
an adequate rationale for further modification of these
molecular scaffolds.
Keywords 2,3-Dioxo-2,3-dihydroindole ꢀ
Thiosemicarbazones ꢀ Cytotoxicity assay ꢀ
Antiviral activity ꢀ Antimicrobial activity
Introduction
Isatin has been known for about 150 years and has recently
been found, like 2,3-dioxo-indoles and endogenous poly-
functional heterocyclic compounds, to exhibit biological
activity in mammals (Somogyi, 2001). Schiff bases and
Mannich bases of isatin are known to possess a wide range
of pharmacological properties including antibacterial
(Pandeya et al., 1998; Sarangapani and Reddy, 1994;
Varma and Nobles, 1975), anticonvulsant (Sridhar et al.,
2002; Varma et al., 2004), anti-HIV (Pandeya et al., 1998,
1999a, b, 2000; Sriram et al., 2000), antifungal (Pandeya
et al., 1999a, b), antiviral (Singh et al., 1983), and anti-
cancer activity (Fig. 1) (Karki et al., 2004, 2007, 2009).
A variety of 3-substituted indolin-2-ones have been utilized
as anticancer drugs or drug candidates (Mologni et al.,
2010, Beauchard et al., 2009, Zhang and Go 2009,
Andreani et al., 2010). A representative member of this
class is sunitinib [SU11248, SutentTM; Pfizer, Inc.] which
is currently used in the clinics as a multi-targeting tyrosine
kinase inhibitor with antiangiogenic activity (Fig. 1) (Sun
et al., 2003). 6-Methoxycarbonyl group-substituted indo-
lin-2-ones [BIBF1000, BIBF1120] are potent inhibitors of
VEGFR-1/2/3, PDGFRa, and FGFR-1, with low cross-
reactivity against a panel of other kinases (Fig. 1) (Roth
et al., 2009). While BIBF1120 is currently being evaluated
in phase III clinical trials in the treatment of non-small cell
lung cancer and is in clinical development for other tumor
types. Indirubin was identified as the active ingredient of a
traditional Chinese recipe [Danggui Longhui Wan] that
S. S. Karki (&) ꢀ A. A. Kulkarni ꢀ S. Kumar ꢀ S. K. Veliyath
Department of Pharmaceutical Chemistry, KLE University’s
College of Pharmacy, Rajajinagar, Bangalore 560010,
Karnataka, India
e-mail: subhasskarki@gmail.com
E. De Clercq ꢀ J. Balzarini
Rega Institute for Medical Research, KU Leuven,
Minderbroedersstraat 10, 3000 Leuven, Belgium
123

Med Chem Res (2013) 22:2014–2022
2015
was used for the treatment of chronic myelogenous leu-
kemia (CML) (Fig. 1) (Xiao et al., 2002).
(2) with diethylamine in the presence of formaldehyde. The
synthesis of N-diethylaminomethyl-2-oxo-1,2-dihydroindole
hydrazinecarbothioamides (4a, b, 5a–h) was carried out by
reacting 1-[(diethylamino)methyl]-1H-indole-2,3-dione deriv-
atives (3) with thiosemicarbazide and N4-aryl thiosemicar-
bazides under reflux in ethanol in the presence of catalytic
amounts of glacial acetic acid. 1H-NMR spectroscopy
indicated that the compounds exist as single isomers in
solution.
Thiosemicarbazones of various aldehydes and ketones
occupy a special place among organic ligands, since they
contain various donor atoms and are able to change density
depending on the starting reagents and their reaction con-
ditions. Isatin-3-thiosemicarbazones (1H-indole-2,3-dioxo-
3-thiosemicarbazones) have been studied extensively due
to their important biological activities (Karki et al., 2009),
since 1-methylisatin-3-thiosemicarbazone (Marboran) was
found to be active in the treatment of smallpox. Previous
studies by our group have revealed the promising cyto-
toxic, and anticonvulsant properties of various 2,3-dioxo-
2,3-dihydroindole thiosemicarbazones (Fig. 1) (Karki
et al., 2009). Therefore, we have performed the synthesis
of new N-4-aryl thiosemicarbazone derivatives of substi-
tuted 2,3-dioxo-2,3-dihydroindoles and evaluated them for
cytotoxic, antiviral, and antimicrobial activity.
Biological activity
All of the compounds were assayed for cytotoxicity against
human HeLa and CEM T-lymphocytes as well as murine
L1210 cells. These data are summarized in Table 1.
Compound 5c was the most cytostatic among all com-
pounds evaluated against the tumor cell lines (IC50 in the
low micromolar range (1.9–4.4 lM)).
The most potent inhibitor of murine L1210 cell prolifera-
tion (5c) was also the most inhibitory to human T-lymphocyte
(CEM) and HeLa cell proliferation. The introduction of
R2-benzyl substitution in the thiosemicarbazone derivative of
series 5 often led to more potent inhibitors of tumor cell
proliferation (5a–h). Replacement of the hydrogen by chlo-
rine atom did not improve the potentiation of the cytostatic
activity (compare 5a with 5b) (Table 1). Whereas, replace-
ment of the chlorine atom at R2 by a methoxy group often
resulted in a marked (equal) potentiation of cytostatic activity
(5b with 5c).
Results and discussion
Chemistry
The compounds in series 4 and 5 were prepared by the
methodologies outlined in Scheme 1. The synthesis of
1-[(diethylamino)methyl]-1H-indole-2,3-dione derivatives
(3) was carried out by mannich base reaction of 2,3-dioxy-2,
3-dihydroindoles or 5-chloro-2,3-dioxy-2,3-dihydroindoles
O
Fig. 1 Structures of indolin-2-
ones
H3C
CH3
N
R
CH3
N
O
H3C
NH
O
NH
NH
CH3
N
H
F
O
O
O
O
N
H
N
H
N
H
H3C
O
Indirubin
BIBF 1000 R=dimethylamino
BIBF 1120 R=4-methylpiperazin-1-yl
Sunitinib
R
R
HN
HN
S
S
NH
O
N
NH
O
N
N
N
N
H3C
H3C
Target compounds
R'
Cytotoxic against human & murine cancer cells
123

2016
Med Chem Res (2013) 22:2014–2022
O
O
virus B4 (CV-B4), and respiratory syncytial virus (RSV) in
HeLa cell cultures; influenza A virus H1N1 subtype,
influenza A virus H3N2 subtype, and influenza B virus in
MDCK cell cultures; parainfluenza-3 virus (PI-3V), reo-
virus-1 (RV-1), Sindbis virus (SV), CV-B4, and Punta Toro
virus (PTV) in Vero cell cultures.
R1
R1
R1
ii
i
O
O
N
H
N
NH2
3
1
2
N
iv
iii
Compounds 4b, 5a, 5b, and 5d exhibited moderate
antiviral activity in HEL cells in comparison to standard
compounds. No specific antiviral effects were noted for any
of the compounds in CRFK, MDCK, or Vero cell cultures
(Tables 2, 3, 4, 5, 6).
S
S
NH2
NH
R1
R1
HN
HN
N
N
N
N
O
O
All the synthesized compounds were screened for
b-lactamase inhibitory activity and results are shown in
Table 1. Compounds namely 5a and 5g were capable of
inactivating b-lactamase activity, and for other compounds
activity was moderate in comparing to standard potassium
clavulanate. Titled compounds were also screened
for antibacterial activity against S. aureus, B. subtilis,
K. pneumoniae, E. coli, P. vulgaris, and S. typhi and
antifungal activity against A. niger and C. albicans and
exhibited moderate antimicrobial activity (Table 7).
R2
N
N
5a-h
4a,b
Scheme 1 The reagents used were as follows: i CCl3CH(OH)2/
H2SO4/Na2SO4; R1=H, Cl, ii (C2H5)NH/HCHO, iii NH2C(S)NHNH2,
iv R2C6H4NHC(S)NHNH2. The nature of the R1 and R2 substituent
are presented in Table 1
Replacement of hydrogen atom at R1 by chlorine atom did
not yieldanyimprovement inpotency (compare5a, 5b and 5d
with5e–h). ReplacinghydrogenatomatR1 and R2 bychlorine
resulted in a complete loss of activity (5f). By keeping chlo-
rine atom at R1 and CH3 and OCH3 at R2, resulted in slight
improvement of cytostatic activity (5g and 5h).
Experimental
The antiviral screening of 4a, b and 5a–h was per-
formed using an MTS-based CPE reduction assay (Kumar
et al., 2010) against feline corona virus (FIPV) and feline
herpes virus in CRFK cell culture; herpes simplex virus-1
(KOS) (HSV-1 KOS), herpes simplex virus-2 (G)
(HSV-2G), vaccinia virus (VV), vesicular stomatitis virus
(VSV), and herpes simplex virus-1 TK KOS ACVr (HSV-1
TK KOS ACVr) in HEL cell cultures; VSV, Coxsackie
Chemistry
All reagents were obtained from Sigma-Aldrich, Mumbai, and
Loba Chemie, Mumbai. All the solvents used in these studies
were dried and distilled before use. Melting points (Mp):
Veego VMP-PM digital melting point apparatus, and are
uncorrected. TLC: solvent benzene:ethanol (8:2). UV spectra:
Shimadzu Pharmspec 1700, UV–Vis spectrophotometer. IR
Table 1 Results of cytotoxicity in murine L1210 cells, human HeLa, and CEM T-lymphocytes, and b-lactamase inhibitory activity
a
Compound
R1
R2
IC50 (lM)
Time for
decolorization
of I2 (s)
Activity
(u ml-1
)
Inactivation
(%)
L1210
CEM
HeLa
4a
H
Cl
H
H
H
H
Cl
Cl
Cl
Cl
–
–
121 35
148 15
164 21
123 85
44 22
128.6
121.5
159.5
129.7
123.6
145.8
156.3
142.3
167.9
120.6
–
46.7
49.4
37.6
46.3
48.5
41.2
38.4
42.2
35.7
49.8
–
38.2
34.6
50.2
38.7
35.7
45.5
49.2
44.2
52.6
34.1
–
4b
–
71
11
10
7
0
1
5a
H
13
11
3
1
8.3 0.0
7.6 0.9
4.4 2.4
5b
Cl
5c
OCH3
CH3
H
2.4 0.0
1.9 0.9
5d
29
3
2
12
0
3
12
0
0
5e
49
40
34
5f
Cl
[125
11
[125
6.9 4.3
[125
9.2 0.9
5g
OCH3
CH3
–
2
5h
9.5 0.5
4.6 4.0
8.6 0.3
Melphalan
3.2 0.6
2.2 0.2
2.1 0.02
Control
–
–
–
–
–
–
–
–
79.5
75.5
25.0
–
Potassium clavulanate
–
–
240.50
67.0
a
IC50 concentrations of compounds required to inhibit the growth of the tumor cells by 50 %
123

Med Chem Res (2013) 22:2014–2022
2017
Syntheses of the intermediate 2,3-dioxy-2,3-dihydroindoles
Table 2 Results of anti-FIPV and anti-feline herpes virus activity
and cytotoxicity in CRFK cell cultures
a
b
The synthesis of the intermediate 2,3-dioxo-2,3-dihydro-
indoles was accomplished using a literature methodology
(Marvel and Heirs, 1941) and a previously reported pro-
cedure was used to convert these compounds to the cor-
responding 1-[(diethylamino)methyl]-1H-indole-2,3-dione.
The N4-arylthiosemicarbazides required for the preparation
of 4a, b and 5a–h was prepared by a literature methodol-
ogy (Sen and Sengupta, 1962; Lieber et al., 1957).
Compound
CC50 (lM)
EC50 (lM)
FIPV
Feline herpes virus
4a
73.8
[100
32.3
4.0
[20
[100
[20
[0.8
[0.8
[0.8
[4
[20
[100
[20
[0.8
[0.8
[0.8
[4
4b
5a
5b
5c
3.5
5d
3.8
5e
6.9
General procedure for syntheses of 4a, b
5f
9.1
[4
[4
5g
3.6
[0.8
[4
[0.8
[4
A mixture of the 1-[(diethylamino)methyl]-1H-indole-2,
3-dione or 5-chloro-1-[(diethylamino)methyl]-1H-indole-
2,3-dione (0.005 mol), thiosemicarbazides (0.005 mol),
acetic acid (0.5–1.0 ml), and ethanol (100 ml) was heated
under reflux until the reaction was completed (*4 h).
Approximately half of the ethanol was removed in vacuo
and the solution was left overnight at room temperature.
The precipitated solid was collected, washed with cold
ethanol, and recrystallized from ethanol:chloroform (9:1)
to give the following compounds.
5h
17.5
[100
[100
[100
HHA (lg ml-1
UDA (lg ml-1
Ganciclovir
a
)
)
19.5
9.1
1.8
2.4
[100
7.3
50 % cytotoxic concentration
b
50 % effective concentration, determined by colorimetric forma-
zan-based MTS assay
2-(1-((diethylamino)methyl)-2-oxoindolin-3-ylidene)hydra-
zinecarbothioamide (4a) % Yield: 83, m.p.: 134–136 °C;
IR (KBr) (cm-1): 1131 (C=S), 1307 (C–N), 1696 (C=O),
3005 (C–H), 3143 (NH), 3238, 3256 (NH2); 1H-NMR
(CDCl3) d (ppm): 1.0 (t, 6H, 2CH3), 2.4 (q, 4H, 2CH2),
4.03 (s, 2H, N–CH2), 7.0–7.7 (m, 4H, Ar–H), 9.53 (s, 2H,
spectra: Shimadzu 8400 S, FT-IR. 1H-NMR spectra:
300 MHz JEOL NMR Spectrophotometer in CDCl3 and
DMSO-d6. Mass spectra: GCMS QP 5050 Shimadzu. All
spectra were obtained from Pune University, Maharashtra,
India.
Table 3 Results of cytotoxicity and antiviral activity of compounds in HEL cell cultures
b
Compound
Minimum cytotoxic
concentrationa (lM)
EC50 (lM)
HSV-1 (KOS)
HSV-2 (G)
VV
VSV
HSV-1 TK- KOS ACVr
4a
[100
[100
[100
100
[100
[100
20
[100
[100
15
[100
20
[100
[100
[100
[20
[100
[100
[100
C20
[20
C20
[20
[20
[20
[20
[250
6
4b
5a
20
5b
15
15
C20
[20
11
5c
100
[20
14
[20
12
[20
5d
100
[20
5e
100
[20
[20
[20
[20
0.08
5
[20
[20
[20
[20
150
1.5
[20
[20
[20
[20
29
[20
5f
100
[20
5g
100
[20
5h
100
[20
Brivudin
Cidofovir
Acyclovir
Ganciclovir
a
[250
[250
[250
[100
[250
[250
[250
[100
10
1.0
0.7
[250
[100
[250
C20
0.09
0.07
Required to cause a microscopically detectable alteration of normal cell morphology
b
Required to reduce virus-induced cytopathogenicity by 50 %
123

2018
Med Chem Res (2013) 22:2014–2022
Table 4 Results of cytotoxicity and antiviral activity of compounds in HeLa cell cultures
b
Compound
Cytotoxicity (lM)
EC50 (lM)
a
CC50
Minimum cytotoxic
concentrationb
VSV
CV-B4
RSV
Visual CPE score
MTS
Visual CPE score
MTS
Visual CPE score
MTS
4a
[100
[100
10.8
[100
[100
C20
C4
[100
[100
[20
[4
[100
[100
[20
[4
[100
[100
[20
[4
[100
[100
[20
[4
[100
[100
[20
[4
[100
[100
[20
[4
4b
5a
5b
9.8
5c
[100
10.7
C4
[4
[4
[4
[4
[4
[4
5d
C20
C20
100
[20
[20
[20
[0.8
[4
[20
[20
[20
[0.8
[4
[20
[20
[20
[0.8
[4
[20
[20
[20
[0.8
[4
[20
[20
[20
[0.8
[4
[20
[20
[20
[0.8
[4
5e
13.5
5f
9.4
5g
13.2
4
5h
[100
[100
[250
[250
C4
DS-5000c
(S)-DHPA
Ribavirin
a
[100
[250
[250
20
14.8
[250
12.1
[100
[250
50
[100
[250
28.5
4
2.8
[250
50
[250
10
[250
4.6
50 % cytotoxic concentration
b
c
Minimum compound concentration that causes a microscopically detectable alteration of normal cell morphology
50 % effective concentration, as determined by a colorimetric formazan-based MTS assay. Data in lg ml-1
Table 5 Results of cytotoxicity and antiviral activity of compounds in Vero cell cultures
b
Compound
Minimum cytotoxic
concentrationa (lM)
EC50 (lM)
PI-3V
RV-1
SV
CV-B4
PTV
4a
[100
[100
100
20
[100
[100
[20
[4
[100
[100
[20
[4
[100
[100
[20
[4
[100
[100
[20
[4
[100
[100
[20
[4
4b
5a
5b
5c
4
[0.8
[20
[20
[20
[0.8
[4
[0.8
[20
[20
[20
[0.8
[4
[0.8
[20
[20
[20
[0.8
[4
[0.8
[20
[20
[20
[0.8
[4
[0.8
[20
[20
[20
[0.8
[4
5d
100
100
100
4
5e
5f
5g
5h
C4
DS-5000c
(S)-DHPA
Ribavirin
a
[100
[250
[250
[100
250
[100
250
20
100
100
[250
[250
[250
[250
[250
112
50
[250
Required to cause a microscopically detectable alteration of normal cell morphology
Required to reduce virus-induced cytopathogenicity by 50 %
Data in lg ml-1
b
c
1
NH2), 11.18 (s, 1H, NH); calc. for C14H19N5OS: C-55.06,
(C=O), 3008 (C–H), 3136 (NH), 3227, 3246 (NH2); H-
NMR (CDCl3) d (ppm): 1.0 (t, 6H, 2CH3), 2.4 (q, 4H,
2CH2), 4.03 (s, 2H, N–CH2), 7.0–7.7 (m, 3H, Ar–H), 9.53
(s, 2H, NH2), 11.18 (s, 1H, NH); calc. for C14H18ClN5OS:
C-49.48, H-5.34 and N-20.61, found C-49.34, H-5.21 and
N-20.69.
H-6.27 and N-22.93, found C-55.18, H-6.15 and N-22.69.
2-(5-chloro-1-((diethylamino)methyl)-2-oxoindolin-3-ylidene)hy-
drazinecarbothioamide (4b) % Yield: 64, m.p.: 214–
216 °C; IR (KBr) (cm-1): 1127 (C=S), 1305 (C–N), 1698
123

Med Chem Res (2013) 22:2014–2022
2019
Table 6 Results of anti-influenza virus activity and cytotoxicity in MDCK cell cultures
c
Compound
Cytotoxicity (lM)
Antiviral EC50 (lM)
a
CC50 Minimum cytotoxic Influenza A
Influenza A
H3N2 subtype
Influenza B
concentrationb
H1N1 subtype
Visual CPE score MTS
Visual CPE score MTS Visual CPE score MTS
4a
4b
5a
5b
5c
5d
5e
5f
85.8
78.3
1.6
100
100
0.8
0.8
4
[20
[20
[0.16
[0.16
[0.8
[0.16
[0.8
[0.8
[0.8
[0.8
45
[20
[20
[20
[20
[20
[20
[20
[20
[20
[20
[0.16 [0.16
[0.16 [0.16
[0.8 [0.8
[0.16 [0.16
[0.8 [0.8
[0.8 [0.8
[0.8 [0.8
[0.8 [0.8
[0.16 [0.16
[0.16 [0.16
[0.8 [0.8
[0.16 [0.16
[0.8 [0.8
[0.8 [0.8
[0.8 [0.8
[0.8 [0.8
[0.16
[0.16
[0.8
[0.16
[0.8
[0.8
[0.8
[0.8
21.8
0.4
9.2
0.2
0.8
C0.8
C0.8
C0.8
4
3.0
2.4
5g
5h
11.1
11.3
Oseltamivir carboxylate [100 [100
39.1
11.5
65.5
24.2
4
5.7
6.8
45
Ribavirin
Amantadine
Rimantadine
a
[100 C100
[500 [500
258.9 500
9
9
9
8.4
45
2
1.5
[500
[100
[500
[100
9
0.1
0.08
50 % cytotoxic concentration, as determined by colorimetric formazan-based MTS assay
b
c
Minimum compound concentration that causes a microscopically detectable alteration of normal cell morphology
50 % effective concentration, as determined by colorimetric formazan-based MTS assay
Table 7 Zone of Inhibition in mm (using 50 lg ml-1 as test solution)
Compound
Antibacterial activity
Antifungal activity
S. aureus
B. subtilis
K. pneumoniae
E. coli
P. vulgaris
S. typhi
A. niger
C. albicans
4a
??
??
??
??
??
??
??
??
??
??
??
??
????
–
??
??
??
??
??
???
??
??
??
??
???
??
??
–
??
4b
???
??
??
??
??
??
??
5a
??
??
???
??
???
??
???
??
5b
??
???
??
???
??
5c
??
???
??
???
???
??
??
5d
???
??
???
??
???
??
??
5e
???
???
???
??
???
???
??
5f
???
??
???
??
???
??
???
???
??
5g
5h
??
??
???
????
–
??
Ofloxacin
Fluconazole
Control
????
–
????
–
????
–
????
–
–
????
?
????
?
?
?
?
?
?
?
???? (31–36), ??? (21–30), ?? (11–20), ? (8–10) in mm
General procedure for syntheses of 5a–h
was heated under reflux until the reaction was completed
(approximately 4 h). Approximately half of the ethanol
was removed in vacuo and the solution was left overnight
at room temperature. The precipitated solid was collected,
washed with cold ethanol, and recrystallized from etha-
nol:chloroform (9:1) to give the following compounds.
A mixture of the 1-[(diethylamino)methyl]-1H-indole-2,
3-dione or 5-chloro-1-[(diethylamino)methyl]-1H-indole-
2,3-dione (0.005 mol), N4-aryl thiosemicarbazides
(0.005 mol), acetic acid (0.5–1.0 ml), and ethanol (100 ml)
123

2020
Med Chem Res (2013) 22:2014–2022
2-(1-((diethylamino)methyl)-2-oxoindolin-3-ylidene)-N-phe-
nylhydrazinecarbothioamide (5a) % Yield: 69, m.p.:
230–232 °C; IR (KBr) (cm-1): 1127 (C=S), 1304 (C–N),
1713 (C=O), 3008 (C–H), 3143, 3217 (NH); 1H-NMR
(CDCl3) d (ppm): 1.0 (t, 6H, 2CH3), 2.4 (q, 4H, 2CH2),
4.03 (s, 2H, N–CH2), 6.9–7.7 (m, 9H, Ar–H), 11.29 (s, 1H,
N–NH), 11.52 (s, 1H, NH); calc. for C20H23N5OS:
C-62.97, H-6.08 and N-18.36, found C-62.53, H-5.87 and
N-18.15.
(C–N), 1713 (C=O), 3007 (C–H), 3123, 3216 (NH);
1H-NMR (CDCl3) d (ppm): 1.0 (t, 6H, 2CH3), 2.4 (q, 4H,
2CH2), 4.03 (s, 2H, N–CH2), 6.4–7.6 (m, 8H, Ar–H), 11.29
(s, 1H, N–NH), 11.52 (s, 1H, NH); calc. for
C20H21Cl2N5OS: C-53.34, H-4.70 and N-15.55, found
C-53.29, H-4.54 and N-15.28.
2-(5-chloro-1-((diethylamino)methyl)-2-oxoindolin-3-yli-
dene)-N-(4-methoxyphenyl)hydrazinecarbothioamide
(5g) % Yield: 73, m.p.: 165–167 °C; IR (KBr) (cm-1):
1123 (C=S), 1329 (C–N), 1716 (C=O), 3008 (C–H), 3127,
N-(4-chlorophenyl)-2-(1-((diethylamino)methyl)-2-oxoin-
dolin-3-ylidene)hydrazine carbothioamide (5b) % Yield:
74, m.p.: 151–153 °C; IR (KBr) (cm-1): 1129 (C=S), 1317
(C–N), 1716 (C=O), 3006 (C–H), 3128, 3209 (NH);
1H-NMR (CDCl3) d (ppm): 1.0 (t, 6H, 2CH3), 2.4 (q, 4H,
2CH2), 4.03 (s, 2H, N–CH2), 6.4–7.7 (m, 8H, Ar–H), 11.29
(s, 1H, N–NH), 11.52 (s, 1H, NH); calc. for
C20H22ClN5OS: C-57.75, H-5.33 and N-16.84, found
C-57.39, H-5.26 and N-16.53.
1
3224 (NH); H-NMR (CDCl3) d (ppm): 1.0 (t, 6H, 2CH3),
2.4 (q, 4H, 2CH2), 3.75 (s, 3H, O–CH3), 4.03 (s, 2H,
N–CH2), 6.3–7.7 (m, 7H, Ar–H), 11.29 (s, 1H, N–NH),
11.52 (s, 1H, NH); calc. for C21H24ClN5O2S: C-56.56,
H-5.42 and N-15.70, found C-56.26, H-5.32 and N-15.47.
2-(5-chloro-1-((diethylamino)methyl)-2-oxoindolin-3-yli-
dene)-N-p-tolylhydrazinecarbothioamide (5h) % Yield:
72, m.p.: 178–180 °C; IR (KBr) (cm-1): 1128 (C=S), 1309
(C–N), 1711 (C=O), 3008 (C–H), 3125, 3228 (NH);
1H-NMR (CDCl3) d (ppm): 1.0 (t, 6H, 2CH3), 2.32 (s, 3H,
CH3), 2.4 (q, 4H, 2CH2), 4.03 (s, 2H, N–CH2), 6.3–7.7 (m,
8H, Ar–H), 11.29 (s, 1H, N–NH), 11.52 (s, 1H, NH); calc.
for C21H24ClN5OS: C-58.66, H-5.63 and N-16.29, found
C-58.43, H-5.38 and N-16.31.
2-(1-((diethylamino)methyl)-2-oxoindolin-3-ylidene)-
N-(4-methoxyphenyl)hydrazinecarbothioamide (5c) % Yield:
77, m.p.: 177–179 °C; IR (KBr) (cm-1): 1119 (C=S), 1325
1
(C–N), 1706 (C=O), 3008 (C–H), 3120, 3202 (NH); H-
NMR (CDCl3) d (ppm): 1.0 (t, 6H, 2CH3), 2.4 (q, 4H,
2CH2), 3.75 (s, 3H, O–CH3), 4.03 (s, 2H, N–CH2), 6.3–7.7
(m, 8H, Ar–H), 11.29 (s, 1H, N–NH), 11.52 (s, 1H, NH);
calc. for C21H25N5O2S: C-61.29, H-6.12 and N-17.02,
found C-61.09, H-6.09 and N-16.80.
Cytotoxicity assays
The methodology for undertaking the HeLa, CEM, and
L1210 assays has been published previously (Baraldi et al.,
2004). In brief, varying concentrations of the compounds
were incubated at 37 °C for 72 h (HeLa and CEM
T-lymphocytes) or 48 h (L1210 cells) in 200-ll 96-well-
microtiter plates, and the viable tumor cell number was
counted at the end of the incubation period using a Coulter
Counter (Coulter Electronics, Harpenden Hertz, U.K.).
2-(1-((diethylamino)methyl)-2-oxoindolin-3-ylidene)-
N-p-tolylhydrazinecarbothioamide (5d) % Yield: 67,
m.p.: 191–193 °C; IR (KBr) (cm-1): 1125 (C=S), 1297
1
(C–N), 1706 (C=O), 3008 (C–H), 3135, 3219 (NH); H-
NMR (CDCl3) d (ppm): 1.0 (t, 6H, 2CH3), 2.32 (s, 3H,
CH3), 2.4 (q, 4H, 2CH2), 4.03 (s, 2H, N–CH2), 6.3–7.7
(m, 9H, Ar–H), 11.29 (s, 1H, N–NH), 11.52 (s, 1H, NH);
calc. for C21H25N5OS: C-63.77, H-6.37 and N-17.71, found
C-63.42, H-6.14 and N-17.39.
Antiviral assay
2-(5-chloro-1-((diethylamino)methyl)-2-oxoindolin-3-yli-
dene)-N-phenylhydrazinecarbothioamide (5e) % Yield:
72, m.p.: 181–183 °C; IR (KBr) (cm-1): 1125 (C=S), 1315
(C–N), 1718 (C=O), 3008 (C–H), 3123, 3219 (NH);
1H-NMR (CDCl3) d (ppm): 1.0 (t, 6H, 2CH3), 2.4 (q, 4H,
2CH2), 4.03 (s, 2H, N–CH2), 6.9–7.7 (m, 8H, Ar–H), 11.29
(s, 1H, N–NH), 11.52 (s, 1H, NH); calc. for C20H22
ClN5OS: C-57.75, H-5.33 and N-16.84, found C-57.51,
H-5.37 and N-16.27.
The antiviral screening of 4a, b and 5a–h was performed
against FIPV and feline herpes virus in CRFK cell culture;
HSV-1 KOS, HSV-2G, VV, VSV, and HSV-1 TK KOS
ACVr in HEL cell cultures; VSV, CVB4, and RSV in HeLa
cell cultures; PI-3V, RV-1, SV, CV-B4, and PTV in Vero
cell cultures; influenza A virus H1N1 subtype, influenza A
virus H3N2 subtype, and influenza B virus in MDCK cell
cultures and the results are expressed as the 50 % effective
concentration (EC50). Cells, grown to confluency in
96-well plates, were infected with 100 CCID50 of virus,
one CCID50 being the 50 % cell culture infective dose. At
the time of virus infection, serial dilutions of the com-
pounds were added. The cultures were incubated at 37 °C
2-(5-chloro-1-((diethylamino)methyl)-2-oxoindolin-3-ylidene)-
N-(4-chlorophenyl)hydrazinecarbothioamide (5f) % Yield:
79, m.p.: 219–221 °C; IR (KBr) (cm-1): 1114 (C=S), 1310
123

Med Chem Res (2013) 22:2014–2022
2021
for 3 days, until complete CPE was observed in the
infected and untreated virus control.
B. subtilis, K. pneumoniae, E. coli, P. vulgaris, and S. typhi
and antifungal activity against A. niger and C. albicans.
This activity was expressed in terms of diameters of zone
of inhibition in mm.
Cytotoxicity assay
The cytotoxicity of the compounds was evaluated in par-
allel with their antiviral activity in uninfected cells, and is
expressed as the minimum cytotoxic concentration to cause
a microscopically detectable alteration of normal cell
morphology (HEL, HeLa, CRFK, MDCK, and Vero cells).
Conclusion
In summary, a series of indolin-2-ones with N-diethyl
amino and various thiosemicarbazide moiety were
designed and synthesized. The cytotoxicity of all synthe-
sized compounds was evaluated against two human cancer
cell lines (CEM and HeLa) and murine leukemia cell line
(L1210). Compound 5c displayed an excellent cytotoxicity
against all three cell lines tested; in particular, it showed
potent cytotoxicity against CEM and L1210 cancer cell
lines. The preliminary structure–activity relationship
(SAR) studies revealed that combination of indolin-2-one
core structure and 4-methoxy phenyl thiosemicarbazide
moiety at the 3-position was more favorable. All the syn-
thesized compounds were screened for cytotoxicity,
b-lactamase inhibitory activity, antiviral, antibacterial, and
antifungal activity against various strains of microorgan-
isms. Derivative 4b, 5a, 5b, and 5d showed antiviral
activity against HEL cell cultures in the range of
11–20 lM, in comparison with IC50 values of 29, 10,
[250, and [100 lM for standard brivudin, cidofovir,
acyclovir, and ganciclovir, respectively. Mild to moderate
antimicrobial activity was observed. The encouraging
biological data provide an adequate rationale for further
modification of these molecular scaffolds.
Antiproliferative assays
The cytostatic effects of the test compounds on murine
leukemia cells (L1210), human T-lymphocyte cells (CEM),
and human cervix carcinoma cells (HeLa) were evaluated
as follows: an appropriate number of cells suspended in
growth medium were allowed to proliferate in 200-ll wells
of 96-well-microtiter plates in the presence of variable
amounts of test compounds at 37 °C in a humidified CO2-
controlled atmosphere. After 48 h (L1210), 72 h (CEM), or
96 h (HeLa), the number of cells was counted in a Coulter
counter. The IC50 value was defined as the compound
concentration required to inhibit cell proliferation by 50 %.
b-lactamase inhibitory assay
All reagents were equilibrated to 30 °C in a water bath
before adding them to the reaction tubes (20 9 150 mm.
Pyrex test tubes) in the following order: first, 1 ml of
gelatin solution (1 % in 0.1 M phosphate buffer, pH 7.0),
50 ll of enzyme, one drop of starch solution (1 % soluble
starch), 1 ml of Penicillin solution (Crystalline Sodium
Penicillin
G (Benzyl penicillin) IP, Alembic Ltd.)
1,660 lg mg-1, dissolved in 0.1 M phosphate buffer, pH
7.0, to contain not less than 5,000 lg ml-1), 3 ml of
sample solution and finally, 2 ml of iodine (0.01 N iodine
in 0.1 M potassium iodide) was added. Then the time of
decolorization of iodine was recorded with a stopwatch;
after addition of substrate (synthesized compounds), stan-
dard (Potassium Clavulanate) and blank was determined
using water in place of sample solution.
References
Andreani A, Bellini S, Burnelli S, Granaiola M, Leoni A, Locatelli A,
Morigi R, Rambaldi M, Varoli L, Calonghi N, Cappadone C,
Zini M, Stefanelli C, Masotti L, Shoemaker RH (2010)
Substituted E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-
2-ones with antitumor activity. Effect on the cell cycle and
apoptosis. J Med Chem 53:5567–5575
Baraldi PB, Nunez MDelC, Tabrizo MA, De Clercq E, Balzarini J,
Bermejo J, Estevez F, Romagnoli R (2004) Design, syntheses
and biological evaluation of hybrid molecules containing
a-methylene-c-butyrolactones and polypyrrole minor groove
binders. J Med Chem 47:2877–2886
Unit Penicillinase activity is expressed in Pollock and
Torriani unit. One unit is the amount of enzyme which will
hydrolyze 1 lM Sodium Penicillin G in 1 h at pH 7.0 at
30 °C.
´
Beauchard A, Laborie H, Rouillard H, Lozach O, Ferandin Y, Guevel
RL, Guguen-Guillouzo C, Meijer L, Besson T, Thiery V (2009)
´
Synthesis and kinase inhibitory activity of novel substituted
indigoids. Bioorg Med Chem 17:6257–6263
Antimicrobial screening
Karki SS, Mazumder UK, Gupta M, Bhattacharya S, Rathinasamy S,
Thangavel S (2004) Synthesis, anticancer and antibacterial
activity of some novel mononuclear Ru(II) complexes. Chem
Pharm Bull 52:178–185
Karki SS, Shrikanth T, Satyanarayana YD, Balzarini J, De Clercq E
(2007) Synthesis, anticancer and cytotoxic activities of some
The antimicrobial assays were performed for synthesized
compounds by cup-plate method of all the synthesized
compounds, as antibacterial activity against S. aureus,
123

2022
Med Chem Res (2013) 22:2014–2022
mononuclear Ru(II) compounds. Bioorg Med Chem 15:6632–
6641
Sarangapani M, Reddy VM (1994) Synthesis and antimicrobial
activity of 1-[(N, N-disubstituted amino)methyl]-3-[(2-phenyl-
3,4-dihydro-4-oxoquinazoline-3-yl]indole-2-one. Indian J Het-
erocycl Chem 3:257–260
Sen AB, Sengupta SK (1962) Possible antiamoebic agents. Part XIX.
Synthesis of 1,3,4-thiadiazolines and di-1,3,4-thiadiazolines.
J Indian Chem Soc 39:628–634
Karki SS, Bahaduria VS, Rana V, Kumar S, Prasanna GS, Das U,
Balzarini J, De Clercq E, Dimmock JR (2009) 1-Arylmethyl-2,3-
dioxo-2,3-dihydroindole thiosemicarbazones as leads for devel-
oping cytotoxins and anticonvulsants. J Enzym Inhib Med Chem
24:537–544
Kumar D, Judge V, Narang R, Sangwan S, De Clercq E, Balzarini J,
Narasimhan B (2010) Benzylidene/2-chlorobenzylidene hydra-
zides: synthesis, antimicrobial activity, QSAR studies and
antiviral evaluation. Eur J Med Chem 45:2806–2816
Singh SP, Shukla SK, Awasthi LP (1983) Synthesis of some
3-(40-nitrobenzoylhydrazono)-2-indolinones as a potential anti-
viral agents. Curr Sci 52:766–769
Somogyi L (2001) Transformation of isatin 3-acylhydrazones under
acetylating conditions: synthesis and structure elucidation of
1,50-disubstituted 30-acetylspiro[oxindole-3,20-[1,3,4]oxadiazo-
lines]. Bull Chem Soc Jpn 74:873–881
Sridhar SK, Pandeya SN, Stables JP, Ramesh A (2002) The wide
pharmacological versatility of semicarbazones, thiosemicarba-
zones and their metal complexes. Eur J Med Chem 16:129–132
Sriram D, Pandeya SN, Nath G, De Clercq E (2000) Synthesis,
antibacterial, antifungal and anti-HIV evaluation of Schiff and
Mannich bases of isatin and its derivatives with triazole.
Arzneimittelforschung 50:55–59
Sun L, Liang C, Shirazian S, Zhou Y, Miller T, Cui J, Fukuda JY, Chu
JY, Nematalla A, Wang XY, Chen H, Sistla A, Luu TC, Tang F,
Wei J, Tang C (2003) Discovery of 5-[5-fluoro-2-oxo-1,2-
dihydroindol-(3Z)-ylidenemethyl]-2, 4-dimethyl-1H-pyrrole-3-
carboxylic acid (2-diethylaminoethyl)amide, a novel tyrosine
kinase inhibitor targeting vascular endothelial and platelet-
derived growth factor receptor tyrosine kinase. J Med Chem
46:1116–1119
Lieber E, Pillai ECN, Hites RD (1957) Reactions of nitrous acid with
4-substituted thiosemicarbazides. Can J Chem 35:832–842
Marvel CS, Heirs GS (1941) In isatin. In: Blatt AH (ed) Organic
syntheses collection, vol 1. Wiley, New York, pp 327–330
Mologni L, Rostagno R, Brussolo S, Knowles PP, Kjaer S, Murray-
Rust J, Rosso E, Zambon A, Scapozza L, McDonald NQ,
Lucchini V, Gambacorti-Passerini C (2010) Synthesis, structure–
activity relationship and crystallographic studies of 3-substituted
indolin-2-one RET inhibitors. Bioorg Med Chem 18:1482–1496
Pandeya SN, Yogeeswari P, Sriram D, Nath G (1998) Synthesis and
antimicrobial activity of N-Mannich bases of 3-[N0-sulphadoox-
imino] isatin and its methyl derivatives. Bull Chim Farm
137:321–324
Pandeya SN, Yogeeswari P, Sriram D, De Clercq E, Pannecouque C,
Witvrouw M (1999a) Synthesis and screening for anti-HIV
activity of some N-Mannich bases of isatin derivatives. J Che-
mother 45:192–196
Pandeya SN, Sriram D, Nath G, De Clercq E (1999b) Synthesis,
antibacterial, antifungal and anti-HIV evaluation of Schiff and
Mannich bases of isatin derivatives with 3-amino-2-methylmer-
capto quinazolin-4(3H)-one. Pharm Acta Helv 74:11–17
Pandeya SN, Sriram D, Nath G, De Clercq E (2000) Synthesis,
antibacterial, antifungal and anti-HIV activities of Norfloxacin
Mannich bases. Eur J Med Chem 35:249–255
Varma RS, Nobles WL (1975) Antiviral, antibacterial, and antifungal
activities of isatin N-Mannich bases. J Pharm Sci 64:881–882
Varma M, Pandeya SN, Singh KN, Stables JP (2004) Anticonvulsant
activity of Schiff bases of isatin derivatives. Acta Pharm
54:49–56
Xiao Z, Hao Y, Liu B, Qian L (2002) Indirubin and meisoindigo in
the treatment of chronic myelogenous leukemia in China. J Leuk
Lymph 43:1763–1768
Roth GJ, Heckel A, Colbatzky F, Handschuh S, Kley J, Lehmann-
Lintz T, Lotz R, Tontsch-Grunt U, Walter R, Hilberg F (2009)
Design, synthesis, and evaluation of indolinones as triple
angiokinase inhibitors and the discovery of a highly specific
6-methoxycarbonyl-substituted indolinone (BIBF 1120). J Med
Chem 52:4466–4480
Zhang W, Go ML (2009) Functionalized 3-benzylidene-indolin-
2-ones: inducers of NAD(P)H-quinone oxidoreductase 1 (NQO1)
with antiproliferative activity. Bioorg Med Chem 17:2077–2090
123