Indole-based hydrazide-hydrazones and 4-thiazolidinones: Synthesis and evaluation as antitubercular and anticancer agents

Article abstract of DOI:10.3109/14756366.2015.1024673

A new series of indolylhydrazones (6) and indole-based 4-thiazolidinones (7, 8) have been designed, synthesized and screened for in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv. 4-Thiazolidinone derivatives 7g-7j, 8g, 8h and 8j displayed notable antituberculosis (anti-TB) activity showing 99% inhibition at MIC values ranging from 6.25 to 25.0 g/ml. Compounds 7g, 7h, 7i, 8h and 8j demonstrated anti-TB activity at concentrations 10-fold lower than those cytotoxic for the mammalian cell lines. The indolylhydrazone derivative 6b has also been evaluated for antiproliferative activity against human cancer cell lines at the National Cancer Institute (USA). Compound 6b showed an interesting anticancer profile against different human tumor-derived cell lines at sub-micromolar concentrations with obvious selectivity toward colon cancer cell line COLO 205.

Full text of DOI:10.3109/14756366.2015.1024673

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–12
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1024673
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RESEARCH ARTICLE
Indole-based hydrazide-hydrazones and 4-thiazolidinones: synthesis
and evaluation as antitubercular and anticancer agents
1
2
3
Go¨kc¸e Cihan-Ustu¨ndag , Dilek S¸atana , Gu¨l Ozhan , and Gu¨ltaze C¸apan
1
¨
¨
˘
2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Department of Microbiology and Clinical Microbiology, Istanbul Faculty of
3
Medicine, and Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey
Abstract
Keywords
A new series of indolylhydrazones (6) and indole-based 4-thiazolidinones (7, 8) have been
designed, synthesized and screened for in vitro antitubercular activity against Mycobacterium
tuberculosis H37Rv. 4-Thiazolidinone derivatives 7g–7j, 8g, 8h and 8j displayed notable
antituberculosis (anti-TB) activity showing 99% inhibition at MIC values ranging from 6.25 to
25.0 mg/ml. Compounds 7g, 7h, 7i, 8h and 8j demonstrated anti-TB activity at concentrations
10-fold lower than those cytotoxic for the mammalian cell lines. The indolylhydrazone
derivative 6b has also been evaluated for antiproliferative activity against human cancer cell
lines at the National Cancer Institute (USA). Compound 6b showed an interesting anticancer
profile against different human tumor-derived cell lines at sub-micromolar concentrations with
obvious selectivity toward colon cancer cell line COLO 205.
Anticancer activity, antitubercular activity,
hydrazide-hydrazone, indole,
4-thiazolidinone
History
Received 8 January 2015
Revised 18 February 2015
Accepted 19 February 2015
Published online 24 April 2015
Introduction
thiazolidinone acetic acids6, piperidone based 1,3-thiazolidin-4-
ones7, isonicotinyl hydrazide derivatives containing the
4-thiazolidinone nucleus8 and numerous different 2,3-disubsti-
tuted or 2,3,5-trisubstituted 4-thiazolidinones9–11 have been
reported to possess appreciable in vitro antitubercular activity.
In a recent report by Pathak et al., 3-(4-chlorophenyl)-N-[4-oxo-
2-(substitutedphenyl)-1,3-thiazolidin-3-yl]-1H-pyrazole-5-car-
boxamides have been described as promising antitubercular
agents with MIC values in the range of 0.4–4 mg/ml12.
Likewise, spirothiazolidinone featured molecules, N-alkyl sub-
stituted spirothiazolidinones13, spirothiazolidinones bearing the
imidazo[2,1-b]thiazole residue14 and indole-2-carboxamides with
a spirothiazolidinone moiety15, have been found to be effective as
growth inhibitors of MTB. Besides, several small synthetic
molecules with an indole nucleus16–18 or acyl/aroyl hydrazone
moieties19,20 have been reported to possess antitubercular poten-
tial. We have recently reported on the synthesis of novel
spirothiazolidinone derivatives of the 5-fluoro-3-phenyl-1H-
indole scaffold with promising in vitro antitubercular properties
against the Mycobacterium tuberculosis H37Rv strain21. Studies
on the identification of the molecular target for indole-2-
carboxamides suggest that they may inhibit M. tuberculosis
growth by acting on the MmpL3 protein, which belongs to the
family of membrane transporters22.
Tuberculosis (TB) represents an enduring deadly disease and
appears as the second leading cause of infectious disease
mortality worldwide, after the acquired immunodeficiency syn-
drome, caused by the human immunodeficiency virus (HIV). In
2011, WHO reported about 8.7 million new cases of TB (13%
co-infected with HIV) with 1.4 million deaths including almost
1 million HIV-negative and 430 000 HIV-positive individuals1.
The rise in the incidence is partly due to the poverty and inequity
and partly to coinfection with HIV, which greatly increases the
risk of progressing new or latent TB infections to active diseases.
Incomplete drug treatments in third-world countries fuel the
appearance of multi-drug-resistant (MDR) and extensively-
drug-resistant (XDR) strains of the pathogen2. In addition, patient
non-compliance resulting from the long-term therapy of the
infection lasting between 6 and 9 months has contributed to the
emergence of MDR-TB3. Therefore, there is an urgent need to
develop new antitubercular drugs to combat the spread of TB,
particularly its hard-to-kill MDR and latent forms.
The synthetically accessible and pharmacologically active
4-thiazolidinone scaffold has provided the impetus for the
discovery of a number of novel antitubercular agents in recent
years. An early representative actithiazic acid, (ꢀ)-2-(5-carboxy-
pentyl)thiazolidin-4-one (Figure 1), isolated from the culture
broth of a strain of streptomyces shows highly specific in vitro
Several studies have been devoted to the antiproliferative
activity of hydrazide–hydrazone (–CO–NH–N¼CH–) based com-
pounds that have structural similarity to our target indolylhy-
drazones (6). In an early report, Germain et al. identified a series
of acyl hydrazones to be selectively lethal to breast cancer stem
cell enriched populations23. Many acyl/aroyl hydrazones with a
variety of heterocyclic spacers were reported for their antiproli-
ferative properties24–27. More relevant to the present study was the
activity against MTB4,5
. 2-Arylhydrazono-4-oxo-3-phenyl-5-
¨
˘
¨
¨
Address for correspondence: Dr. Gokc¸e Cihan-Ustundag, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul University,
Istanbul 34116, Turkey. Tel: +90 2124400000. Fax: +90 2124400252.
E-mail: gokcechn@istanbul.edu.tr

¨
G. Cihan-Ustundag et al.
¨
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2
J Enzyme Inhib Med Chem, Early Online: 1–12
(a)
(b)
Ethyl 2-benzyl-2-(4-fluorophenylhydrazono)acetate (3)
O
Cl
R
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 0 ꢂC with stirring. The resulting solution of
diazonium salt (2) was poured into a cooled (0 ꢂC) mixture of
ethyl 2-benzyl-3-oxo-butanoate (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.
HN
(CH )
NO
NH
2
N
S
N
H
CH
HOOC
O
2
2 4
R=CH , C H
6 5
3
Figure 1. The structure of actithiazic acid (a) and apoptosis inducers (b).
identification of 5-chloro-3-methyl/phenyl-indole-2-carboxylic
acid benzylidenehydrazides as anticancer agents by Zhang et al.
(Figure 1). These compounds were found to be potent inducers of
apoptosis and inhibited tubulin polimerization in G2/M phase in
breast cancer cell line T47D28. Moreover, some of the
indolyhydrazones synthesized as precursors of 4-thiazolidinone
derivatives in the present study (6a, 6d–h) were previously
patented by Bamaung et al. as angiogenesis inhibitors29.
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.
Encouraged by the above data and in continuation of an
ongoing program aiming at the discovery of new leads with
antitubercular activity, here we report the synthesis, structural
identification and in vitro antitubercular activity of a new series of
5-fluoro-N-[5-(non)substituted-2-(non)substitutedphenyl-4-oxo-
1,3-thiazolidin-3-yl]-3-phenyl-1H-indole-2-carboxamides (7a–7l,
8a–8l) and their precursors 5-fluoro-N2-[(non)substitutedbenzyli-
dene]-3-phenyl-1H-indole-2-carbohydrazides (6a–6l) against
M. tuberculosis H37Rv. In view of the utility of hydrazide–
hydrazone-based compounds for the discovery of novel anti-
proliferative agents, we also report on the preliminary antitumor
screening results of 5-fluoro-N2-(4-methylbenzylidene)-3-phenyl-
1H-indole-2-carbohydrazide (6b), selected by the National Cancer
Institute (NCI) as a prototype.
5-Fluoro-3-phenyl-1H-indole-2-carbohydrazide (5)
A mixture of 4 (0.02 mol), ethanol (20 mL) and H2NNH2ꢃH2O
(98%, 8 mL) was heated under reflux for 6 h. The resulting
brown crystals were filtered off and recrystallized from ethanol–
chloroform. m.p. 222–225 ꢂC; IR(KBr): ꢀmax 3279 (N–H),
1624 (C¼O); 1H NMR (DMSO-d6/200 MHz): d 4.48 (s, 2H,
NH2), 7.10 (td, 1H, J ¼ 9.1, 2.2, H6-ind.), 7.21 (dd, 1H, J ¼ 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-d6/125 MHz): d 104.65 (d, J ¼ 23.7,
C4-ind.), 112.78 (d, J ¼ 26.1, C6-ind.), 114.24 (d, J ¼ 10.0, C7-
ind.), 117.50 (d, J ¼ 4.3, C3-ind.), 127.36 (d, J ¼ 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 ¼ 232.2, C5-ind.),
162.23 (C¼O).
Experimental
Chemistry
General procedure for the synthesis of 5-fluoro-N2-[(non)
substitutedbenzylidene]-3-phenyl-1H-indole-2-carbohydrazides
(6a–6l)
Melting points were determined in open capillary tubes with a
Buchi B-540 melting point apparatus (Bu¨chi Labortechnik AG,
Flawil, Switzerland) and are uncorrected. Microanalyses were
performed on a Thermo Finnigan Flash EA 1112 elemental
analyzer (Thermo Scientific, Waltham, MA). IR spectra were
recorded in KBr discs (ꢀmax in cmꢀ1) on a Perkin-Elmer 1600
FTIR and Shimadzu IRAffinity-1 FTIR (Perkin-Elmer, Waltham,
MA) spectrophotometer (Shimadzu Corporation, Kyoto, Japan).
1H NMR (DMSO-d6),13C NMR (proton decoupled, DEPT-135)
(DMSO-d6) and heteronuclearcorrelation 1H–13C (HSQC,
HMBC) (DMSO-d6) spectra were run on Bruker AC 200
(200 MHz; Bruker BioSpin GmbH, Rheinstetten/Karlsruhe,
Germany) and VarianUNITYINOVA (500 MHz) instruments.
Chemical shifts are reported as d (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 (Thermo Scientific, Waltham, MA).
Analytical HPLC was performed on a Shimadzu LC-10AD VP
Liquid Chromatograph (Shimadzu Corporation, Kyoto, Japan)
equipped with Shimadzu SCL-10A VP System Controller,
Shimadzu DGU-14A Degasser, Shimadzu CTO-10AS VP
Column Oven, Shimadzu SIL-10AD VP Auto Injector and
Shimadzu SPD-M10A VP Diode Array Detector set at 304 nm.
The data were collected and analyzed by using LC Workstation
(Class VP System, Shimadzu). Chromatographic resolutions were
carried out at room temperature on a Kromasil 100-5SIL
microporous silica column (25 cm ꢁ 4.6 mm). Hexane–ethyl acet-
ate (80:20) was used as the mobile phase at a flow rate of 1.2 mL/
min. All solvents were HPLC grade (br.: broad, bnz.: benzylidene,
ind.: indole, thz.: thiazolidinone).
A mixture of 5 (0.005 mol) and an appropriate benzaldehyde
(0.006 mol) was refluxed in 15 mL abs. ethanol for 3 h. The
precipitate obtained was purified either by recrystallization from
ethanol–chloroform or by washing with hot ethanol.
5-Fluoro-N2-(4-methylbenzylidene)-3-phenyl-1H-indole-2-car-
bohydrazide (6b). Brown crystals (91%); m.p. 250–252 ꢂC;
IR(KBr): ꢀmax 3314, 3280 (N–H), 1651 (C¼O); 1H NMR
(DMSO-d6/500 MHz): d 2.32 (s, 3H, 4-CH3), 7.14 (br. t, 1H,
J ¼ 8.3 Hz, H6-ind.), 7.24 (br. d, 2H, J ¼ 4.4 Hz, H3,H5-bnz.),
7.32 (br. d, 2H, J ¼ 9.3 Hz, H4,3-C6H5(H4)-ind.), 7.46–7.56 (m,
7H, H2,H6-bnz. and H7,3-C6H5(H2,H3,H5,H6)-ind.), 8.04 (s, 1H,
N¼CH), 11.33 (s, 1H, CONH), 12.04 (s, 1H, NH); 13C NMR
(HSQC, HMBC) (DMSO-d6/125 MHz): d 21.70 (4-CH3), 104.85
(d, J ¼ 23.5 Hz, C4-ind.), 113.28 (d, J ¼ 25.1 Hz, C6-ind.), 114.38
(d, J ¼ 10.0 Hz, C7-ind.), 118.67 (C3-ind.), 127.08 (d, C3a-ind.),
127.42 (3-C6H5(C4)-ind.), 127.77 (C2, 6-bnz.), 129.15 (3-
C6H5(C3,5)-ind.), 129.95 (C3,5-bnz. and C2-ind.), 130.13 (3-
C6H5(C2,6)-ind.), 131.98 (C1-bnz.), 133.01 (C7a-ind.), 134.01 (3-
C6H5(C1)-ind.), 140.72 (C4-bnz.), 148.13 (C¼N), 158.39 (d,
J ¼ 233.9 Hz, C5-ind.), 158.89 (CONH); MS (ESI-) m/z (%): 370
(MꢀHꢀ, 100). Anal calcd for C23H18FN3O (371.41): C, 74.38; H,
4.88; N, 11.31. Found: C, 74.39; H, 5.09; N, 11.15.
(E)-5-Fluoro-N2-(4-methoxybenzylidene)-3-phenyl-1H-indole-
2-carbohydrazide (6c). See Ref30.
5-Fluoro-3-phenyl-N2-[4-(trifluoromethyl)benzylidene]-1H-
indole-2-carbohydrazide(6i). Yellow needles (92%); m.p. 254–
255 ꢂC; IR(KBr): ꢀmax 3270 (N–H), 1638 (C¼O); 1H NMR

DOI: 10.3109/14756366.2015.1024673
Indole-based hydrazide-hydrazones and 4-thiazolidinones
3
(DMSO-d6/500 MHz): d 7.15 (td, 1H, J ¼ 8.8; 2.0 Hz, H6-ind.),
5-Fluoro-N-[2-(4-methylphenyl)-4-oxo-1,3-thiazolidin-3-yl]-3-
7.32–7.53 (m, 7H, H4, H7,3-C6H5-ind.), 7.78 (br. s, 2H, H2,H6- phenyl-1H-indole-2-carboxamide (7b). White needles (61%); m.p.
bnz.), 7.90 (br. s, 2H, H3,H5-bnz.), 8.16 (s, 1H, N¼CH), 11.61 221–222 ꢂC; IR(KBr): ꢀmax 3357, 3286 (N–H), 1704, 1676
1
(s, 1H, CONH), 12.05 (s, 1H, NH). Anal calcd for C23H15F4N3O (C¼O); H NMR (DMSO-d6/500 MHz): d 2.32 (s, 3H, 4-CH3),
(425.38): C, 64.74; H, 3.55; N, 9.88. Found: C, 64.44; H, 3.47; 3.74 (d, 1H, J ¼ 16.1 Hz, H5-thz.), 3.87 (dd, 1H, J ¼ 16.1; 1.5 Hz,
N, 9.63.
H5-thz.), 5.80 (s, 1H, H2-thz.), 7.12 (td, 1H, J ¼ 9.3; 2.4 Hz, H6-
5-Fluoro-N2-[4-(methoxycarbonyl)benzylidene]-3-phenyl-1H-
ind.), 7.16–7.19 (m, 3H, 2-C6H5(H3,H5)-thz. and H4-ind.), 7.26
indole-2-carbohydrazide (6j). Yellow crystals (94%); m.p. 256– (br. s, 5H, 3-C6H5-ind.), 7.32 (d, 2H, J ¼ 8.3 Hz, 2-C6H5(H2,H6)-
258.5 ꢂC; IR(KBr): ꢀmax 3263 (N–H), 1703 (C¼O), 1649 (C¼O); thz.), 7.47 (dd, 1H, J ¼ 8.8; 4.4 Hz, H7-ind.), 9.92 (s, 1H, CONH),
1H NMR (DMSO-d6/500 MHz): d 3.85 (s, 3H, 4-COOCH3), 7.15 11.83 (s, 1H, NH); 13C NMR (HSQC, HMBC) (DMSO-d6/
(td, 1H, J ¼ 9.0; 2.0 Hz, H6-ind.), 7.33–7.53 (m, 7H, H4,H7,3- 125 MHz): d 21.49 (4-CH3), 30.02 (C5-thz.), 62.44 (C2-thz.),
C6H5-ind.), 7.82 (br. s, 2H, H2,H6-bnz.), 8.00 (br. s, 2H, H3,H5- 104.92 (d, J ¼ 24.0 Hz, C4-ind.), 113.81 (d, J ¼ 26.8 Hz, C6-
bnz.), 8.14 (s, 1H, N¼CH), 11.58 (s, 1H, CONH), 12.07 (s, 1H, ind.), 114.62 (d, J ¼ 9.1 Hz, C7-ind.), 119.79 (d, J ¼ 4.8 Hz,
NH); 13C NMR (HSQC) (DMSO-d6/125 MHz): d 52.91 (4- C3-ind.), 127.08 (d, J ¼ 10.1 Hz, C3a-ind.), 127.26 (C2/3-
COOCH3), 104.91 (d, J ¼ 23.5 Hz, C4-ind.), 113.31 (C6-ind.), C6H5(C4)-ind.), 127.38 (C2/3-C6H5(C4)-ind.), 128.32 (2-C6H5
114.44 (d, J ¼ 8.6 Hz, C7-ind.), 119.13 (C3-ind.), 126.95 (C3a- (C2,6)-thz.), 129.03 (3-C6H5(C3,5)-ind.), 129.83 (2-C6H5(C3,5)-
ind.), 127.48 (3-C6H5(C4)-ind.), 127.90 (C2,6-bnz.), 129.15 thz.), 130.20 (3-C6H5(C2,6)-ind.), 133.19 (C7a-ind.), 133.37 (3-
(3-C6H5(C3,5)-ind.), 129.57 (C4-bnz. and C2-ind.), 130.26 C6H5(C1)-ind.), 135.83 (2-C6H5(C1)-thz.), 139.11 (2-C6H5(C4)-
(C3,5-bnz. and 3-C6H5(C2,6)-ind.), 131.18 (C7a-ind.), 133.18 thz.), 158.36 (d, J ¼ 233.9 Hz, C5-ind.), 160.90 (CONH), 169.65
(3-C6H5(C1)-ind.), 139.10 (C1-bnz.), 146.58 (C¼N), 154.96 (CO-thz.); MS (ESI-) m/z (%): 444 (MꢀHꢀ, 100). Anal calcd for
(CONH), 158.41 (d, J ¼ 233.9 Hz, C5-ind.), 166.50 C25H20FN3O2S (445.51): C, 67.40; H, 4.22; N, 9.43. Found: C,
(4-COOCH3); MS (ESI-) m/z (%): 414 (MꢀHꢀ, 100). Anal 67.24; H, 3.92; N, 9.66.
calcd for C24H18FN3O3 (415.42): C, 69.39; H, 4.37; N, 10.12.
Found: C, 69.34; H, 4.63; N, 9.76.
5-Fluoro-N-[2-(4-methoxyphenyl)-4-oxo-1,3-thiazolidin-3-yl]-
3-phenyl-1H-indole-2-carboxamide (7c). White powder (65%);
m.p. 184–185.5 ꢂC; IR(KBr): ꢀmax 3352, 3292 (N–H), 1697, 1670
N2-(2,6-dichlorobenzylidene)-5-fluoro-3-phenyl-1H-indole-2-
carbohydrazide (6k). Yellow needles (89%); m.p. 205–206 ꢂC; (C¼O); 1H NMR (DMSO-d6/500 MHz):
d 3.74 (d, 1H,
1
IR(KBr): ꢀmax 3309 (N–H), 1673 (C¼O); H NMR (DMSO-d6/ J ¼ 16.1 Hz, H5-thz.), 3.77 (s, 3H, 4-OCH3), 3.86 (dd,
500 MHz): d 7.15 (br. t, 1H, H6-ind.), 7.30–7.51 (m, 10H, H3- 1H, J ¼ 16.1; 1.5 Hz, H5-thz.), 5.80 (s, 1H, H2-thz.), 6.92 (d,
5-bnz. and H4,H7,3-C6H5-ind.), 8.27 (s, 1H, N¼CH), 11.71 2H, J ¼ 8.8 Hz, 2-C6H5(H3,H5)-thz.), 7.12 (td, 1H, J ¼ 9.3;
(s, 1H, CONH), 12.07 (s, 1H, NH). Anal calcd for 2.4 Hz, H6-ind.), 7.18 (dd, 1H, J ¼ 9.8; 2.4 Hz, H4-ind.), 7.26–
C22H14Cl2FN3O (426.27): C, 61.99; H, 3.81; N, 9.86. Found: C, 7.30 (m, 5H, 3-C6H5-ind.), 7.37 (d, 2H, J ¼ 8.8 Hz, 2-
62.12; H, 4.03; N, 9.89.
C6H5(H2,H6)-thz.), 7.48 (dd, 1H, J ¼ 9.0; 4.4 Hz, H7-ind.), 9.88
N2-(2-chloro-6-fluorobenzylidene)-5-fluoro-3-phenyl-1H-
(s, 1H, CONH), 11.83 (s, 1H, NH). Anal calcd for C25H20FN3O3S
indole-2-carbohydrazide (6l). Yellow needles (87%); m.p. 223– (461.51): C, 65.06; H, 4.37; N, 9.10. Found: C, 65.18; H, 4.32; N,
225 ꢂC; IR(KBr): ꢀmax 3324, 3169 (N–H), 1654 (C¼O); 1H NMR 9.13.
(DMSO-d6/500 MHz): d 7.15 (br. t, 1H, J ¼ 8.8 Hz, H6-ind.),
N-[2-(4-chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-5-fluoro-3-
7.30–7.52 (m, 10H, H3-5-bnz. and H4,H7,3-C6H5-ind.), 8.32 phenyl-1H-indole-2-carboxamide (7d). Beige crystals (89%); m.p.
1
(s, 1H, N¼CH), 11.67 (s, 1H, CONH), 12.05 (s, 1H, NH); 234–236 ꢂC; IR(KBr): ꢀmax 3291 (N–H), 1713, 1658 (C¼O); H
MS (ESI-) m/z (%): 410/408 (MꢀHꢀ, 28/100). Anal calcd for NMR (DMSO-d6/500 MHz): d 3.75 (d, 1H, J ¼ 15.6 Hz, H5-thz.),
C22H14ClF2N3O (409.82): C, 64.48; H, 3.84; N, 10.25. Found: C, 3.91 (dd, 1H, J ¼ 16.0; 1.8 Hz, H5-thz.), 5.85 (s, 1H, H2-thz.),
64.43; H, 4.10; N, 10.16.
7.12 (td, 1H, J ¼ 9.0; 2.3 Hz, H6-ind.), 7.18 (dd, 1H, J ¼ 9.8;
2.5 Hz, H4-ind.), 7.24–7.27 (m, 5H, 3-C6H5-ind.), 7.43 (d, 2H,
J ¼ 8.2 Hz, 2-C6H5(H3,H5)-thz.), 7.46–7.49 (m, 3H, 2-
C6H5(H2,H6)-thz. and H7-ind.), 10.01 (s, 1H, CONH), 11.85 (s,
1H, NH); 13C NMR (HSQC) (DMSO-d6/125 MHz): d 29.94 (C5-
thz.), 61.77 (C2-thz.), 104.93 (d, J ¼ 24.0 Hz, C4-ind.), 113.80 (d,
J ¼ 26.4 Hz, C6-ind.), 114.61 (d, J ¼ 9.6 Hz, C7-ind.), 119.72 (d,
J ¼ 5.3 Hz, C3-ind.), 127.06 (d, J ¼ 10.1 Hz, C3a-ind.), 127.33
(C2/3-C6H5(C4)-ind.), 127.35 (C2/3-C6H5(C4)-ind.),128.98 (3-
C6H5(C3,5)-ind.), 129.29 (2-C6H5(C3,5)-thz.), 130.18 (3-
C6H5(C2,6)-ind.), 130.24 (2-C6H5(C2,6)-thz.), 133.22 (C7a-
ind.), 133.37 (3-C6H5(C1)-ind.), 134.20 (2-C6H5(C1/C4)-thz.),
138.20 (2-C6H5(C1/C4)-thz.), 158.37 (d, J ¼ 233.9 Hz, C5-ind.),
161.01 (CONH), 169.51 (CO-thz.). Anal calcd for
C24H17ClFN3O2S (465.93): C, 61.87; H, 3.88; N, 9.02. Found:
C, 62.21; H, 4.21; N, 8.92.
General procedure for the synthesis of 5-fluoro-N-[5-(non)
substituted-2-(non)substitutedphenyl-4-oxo-1,3-thiazolidin-3-yl]-
3-phenyl-1H-indole-2-carboxamides (7a–7l, 8a–8l)
A mixture of 6a–6l (0.0025 mol) and mercaptoacetic acid or 2-
mercaptopropionic acid (0.01 mol) was refluxed in 30 mL dry
benzene for 5–6 h using a Dean-Stark water separator. Excess
benzene was evaporated in vacuo. The resulting residue was
triturated with saturated NaHCO3 solution until CO2 evolution
ceased and was allowed to stand overnight or in some cases
refregerated until solidification. The solid thus obtained was
washed with water, dried and recrystallized from ethanol or
ethanol–chloroform.
5-Fluoro-N-(4-oxo-2-phenyl-1,3-thiazolidin-3-yl)-3-phenyl-
1H-indole-2-carboxamide (7a). White powder (62%); m.p. 219–
220.5 ꢂC; IR(KBr): ꢀmax 3364 (N–H), 1708, 1678 (C¼O); 1H
NMR (DMSO-d6/500 MHz): d 3.75 (d, 1H, J ¼ 15.6 Hz, H5-thz.),
3.89 (dd, 1H, J ¼ 15.9; 1.5 Hz, H5-thz.), 5.84 (s, 1H, H2-thz.),
7.12 (td, 1H, J ¼ 9.3; 2.4 Hz, H6-ind.), 7.18 (dd, 1H, J ¼ 9.8;
2.4 Hz, H4-ind.), 7.25 (br. s, 5H, 3-C6H5-ind.), 7.37–7.39 (m, 3H,
2-C6H5(H3-5)-thz.), 7.44–7.48 (m, 3H, 2-C6H5(H2,H6)-thz. and
H7-ind.), 9.96 (s, 1H, CONH), 11.84 (s, 1H, NH). Anal calcd for
C24H18FN3O2S (431.48): C, 66.81; H, 4.20; N, 9.74. Found: C,
67.15; H, 3.90; N, 9.69.
N-[2-(4-bromophenyl)-4-oxo-1,3-thiazolidin-3-yl]-5-fluoro-3-
phenyl-1H-indole-2-carboxamide (7e). Beige powder (84%); m.p.
1
228–229 ꢂC; IR(KBr): ꢀmax 3295 (N–H), 1713, 1656 (C¼O); H
NMR (DMSO-d6/500 MHz): d 3.75 (d, 1H, J ¼ 16.0 Hz, H5-thz.),
3.91 (dd, 1H, J ¼ 16.0; 1.4 Hz, H5-thz.), 5.84 (s, 1H, H2-thz.),
7.12 (td, 1H, J ¼ 9.2; 2.3 Hz, H6-ind.), 7.18 (dd, 1H, J ¼ 9.6;
2.3 Hz, H4-ind.), 7.26 (br. s, 5H, 3-C6H5-ind.), 7.41 (d, 2H,
J ¼ 8.7 Hz, 2-C6H5(H2,H6)-thz.), 7.47 (dd, 1H, J ¼ 9.2; 4.6 Hz,
H7-ind.), 7.57 (d, 2H, J ¼ 8.7 Hz, 2-C6H5(H3,H5)-thz.), 10.01
(s, 1H, CONH), 11.85 (s, 1H, NH); MS (ESI-) m/z (%): 510/508

¨
G. Cihan-Ustundag et al.
¨
˘
4
J Enzyme Inhib Med Chem, Early Online: 1–12
(M-H-, 100/87). Anal calcd for C24H17BrFN3O2S (510.38): C, (d, 1H, J ¼ 16.0 Hz, H5-thz.), 3.87 (s, 3H, 4-COOCH3), 3.93 (dd,
56.58; H, 3.56; N, 8.23. Found: C, 56.94; H, 3.79; N, 8.12.
5-Fluoro-N-[2-(4-fluorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-3-
1H, J ¼ 15.8; 1.8 Hz, H5-thz.), 5.92 (s, 1H, H2-thz.), 7.12 (td, 1H,
J ¼ 9.2; 2.8 Hz, H6-ind.), 7.18 (dd, 1H, J ¼ 9.8; 2.3 Hz, H4-ind.),
phenyl-1H-indole-2-carboxamide (7f). Beige crystals (89%); m.p. 7.19–7.27 (m, 5H, 3-C6H5-ind.), 7.46 (dd, 1H, J ¼ 9.2; 4.6 Hz,
217–219 ꢂC; IR(KBr): ꢀmax 3284 (N–H), 1704, 1657 (C¼O); H7-ind.), 7.59 (d, 2H, J ¼ 8.7 Hz, 2-C6H5(H2,H6)-thz.), 7.94 (d,
1H NMR (DMSO-d6/500 MHz): d 3.75 (d, 1H, J ¼ 16.0 Hz, H5- 2H, J ¼ 8.2 Hz, 2-C6H5(H3,H5)-thz.), 10.08 (s, 1H, CONH),
thz.), 3.90 (dd, 1H, J ¼ 16.0; 1.8 Hz, H5-thz.), 5.85 (s, 1H, 11.85 (s, 1H, NH); 13C NMR (HSQC) (DMSO-d6/125 MHz): d
H2-thz.), 7.12 (td, 1H, J ¼ 9.2; 2.7 Hz, H6-ind.), 7.17–7.20 (m, 29.91 (C5-thz.), 52.95 (4-COOCH3), 61.87 (C2-thz.), 104.91 (d,
3H, 2-C6H5(H3,H5)-thz. and H4-ind.), 7.27 (br. s, 5H, 3-C6H5- J ¼ 23.5 Hz, C4-ind.), 113.79 (d, J ¼ 26.8 Hz, C6-ind.), 114.60
ind.), 7.46–7.52 (m, 3H, 2-C6H5 (H2,H6)-thz. and H7-ind.), 9.98 (d, J ¼ 9.6 Hz, C7-ind.), 119.74 (d, J ¼ 4.8 Hz, C3-ind.), 127.03
(s, 1H, CONH), 11.84 (s, 1H, NH). Anal calcd for (d, J ¼ 9.6 Hz, C3a-ind.), 127.31 (C2/3-C6H5(C4)-ind.), 127.34
C24H17F2N3O2S (449.47): C, 64.13; H, 3.91; N, 9.35. Found: C, (C2/3-C6H5(C4)-ind.), 128.51 (2-C6H5(C2,6)-thz.), 128.94
64.48; H, 4.17; N, 9.18.
N-[2-(4-cyanophenyl)-4-oxo-1,3-thiazolidin-3-yl]-5-fluoro-3-
(3-C6H5(C2,6)-ind.), 130.14 (2-C6H5(C3,5)-thz./3-C6H5(C3,5)-
ind.), 130.16 (2-C6H5(C3,5)-thz./3-C6H5(C3,5)-ind.), 130.70 (2-
phenyl-1H-indole-2-carboxamide (7g). White crystals (80%); C6H5(C4)-thz.), 133.20 (C7a-ind.), 133.34 (3-C6H5(C1)-ind.),
m.p. 253–255 ꢂC; IR(KBr): ꢀmax 3299 (N–H), 2231 (CꢄN), 144.59 (2-C6H5(C1)-thz.), 158.36 (d, J ¼ 233.9 Hz, C5-ind.),
1
1713, 1655 (C¼O); H NMR (DMSO-d6/500 MHz): d 3.77 (d, 161.09 (CONH), 166.60 (4-COOCH3), 169.60 (CO-thz.); MS
1H, J ¼ 15.6 Hz, H5-thz.), 3.95 (dd, 1H, J ¼ 16.0; 1.5 Hz, H5- (ESI-) m/z (%): 488 (M-H-, 100). Anal calcd for
thz.), 5.93 (s, 1H, H2-thz.), 7.12 (td, 1H, J ¼ 9.3; 2.4 Hz, H6-ind.), C26H20FN3O4S.1/2H2O (498.52): C, 62.64; H, 4.25; N, 8.43.
7.19 (dd, 1H, J ¼ 9.8; 2.4 Hz, H4-ind.), 7.22–7.28 (m, 5H, 3- Found: C, 62.88; H, 4.48; N, 8.26.
C6H5-ind.), 7.47 (dd, 1H, J ¼ 9.0; 4.4 Hz, H7-ind.), 7.64 (d, 2H,
N-[2-(2,6-dichlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-5-
J ¼ 8.3 Hz, 2-C6H5(H2,H6)-thz.), 7.84 (d, 2H, J ¼ 8.3 Hz, 2-C6H5 fluoro-3-phenyl-1H-indole-2-carboxamide (7k). White needles
(H3,H5)-thz.), 10.11 (s, 1H, CONH), 11.87 (s, 1H, NH); MS (67%); m.p. 215–217 ꢂC; IR(KBr): ꢀmax 3298, 3219 (N–H),
(ESI-) m/z (%): 455 (MꢀHꢀ, 100). Anal calcd for C25H17FN4O2S 1720, 1638 (C¼O); H NMR (DMSO-d6/500 MHz): d 3.87 (d,
1
(456.49): C, 65.78; H, 3.75; N, 12.27. Found: C, 65.83; H, 3.70; 1H, J ¼ 16.1 Hz, H5-thz.), 3.91 (dd, 1H, J ¼ 15.9; 2.0 Hz, H5-
N, 12.07.
thz.), 6.73 (d, 1H, J ¼ 1.5 Hz, H2-thz.), 7.12–7.17 (m, 2H, H4,H6-
ind.), 7.26–7.36 (m, 5H, 3-C6H5-ind.), 7.42 (t, 1H, J ¼ 8.3 Hz, 2-
5-Fluoro-N-[2-(4-nitrophenyl)-4-oxo-1,3-thiazolidin-3-yl]-3-
phenyl-1H-indole-2-carboxamide (7h). Yellow crystals (78%); C6H5(H4)-thz.), 7.47–7.53 (m, 3H, 2-C6H5(H3,H5)-thz. and H7-
m.p. 258–260 ꢂC; IR(KBr): ꢀmax 3300 (N–H), 1716, 1654 (C¼O); ind.), 9.97 (s, 1H, CONH), 11.86 (s, 1H, NH); 13C NMR (HSQC)
1H NMR (DMSO-d6/500 MHz): d 3.79 (d, 1H, J ¼ 16.1 Hz, H5- (DMSO-d6/125 MHz): d 30.91 (C5-thz.), 57.01 (C2-thz.), 104.95
thz.), 3.97 (dd, 1H, J ¼ 16.0; 1.5 Hz, H5-thz.), 6.00 (d, 1H, (d, J ¼ 24.0 Hz, C4-ind.), 114.08 (d, J ¼ 26.8 Hz, C6-ind.), 114.72
J ¼ 1.0 Hz, H2-thz.), 7.12 (td, 1H, J ¼ 9.3; 2.4 Hz, H6-ind.), 7.17– (d, J ¼ 9.6 Hz, C7-ind.), 120.46 (d, J ¼ 5.3 Hz, C3-ind.), 126.79
7.23 (m, 4H, H4-ind. and 3-C6H5(H3-5)-ind.), 7.27 (dd, 2H, (C2-ind.), 127.13 (d, J ¼ 10.7 Hz, C3a-ind.), 127.59 (3-C6H5(C4)-
J ¼ 8.1; 1.5 Hz, 3-C6H5(H2,H6)-ind.), 7.47 (dd, 1H, J ¼ 8.8; ind.), 129.08 (3-C6H5(C3,5)-ind.), 129.62 (2-C6H5(C3/5)-thz.),
4.4 Hz, H7-ind.), 7.72 (d, 2H, J ¼ 8.8 Hz, 2-C6H5(H2,H6)-thz.), 130.21 (3-C6H5(C2,6)-ind.), 131.88 (2-C6H5(C3/5)-thz.), 131.92
8.21 (d, 2H, J ¼ 8.8 Hz, 2-C6H5(H3,H5)-thz.), 10.16 (s, 1H, (2-C6H5(C4)-thz.), 133.22 (C7a-ind.), 133.48 (3-C6H5(C1)-ind.),
CONH), 11.88 (s, 1H, NH). Anal calcd for C24H17FN4O4S 135.71 (2-C6H5(C2,6/C1)-thz.), 135.99 (2-C6H5(C2,6/C1)-thz.),
(476.48): C, 60.50; H, 3.60; N, 11.76. Found: C, 60.58; H, 3.62; 158.38 (d, J ¼ 233.9 Hz, C5-ind.), 161.41 (CONH), 169.12 (CO-
N, 11.77.
thz.). Anal calcd for C24H16Cl2FN3O2S (500.37): C, 57.61; H,
5-Fluoro-N-{4-oxo-2-[4-(trifluoromethyl)phenyl]-1,3-thiazoli- 3.22; N, 8.40. Found: C, 57.53; H, 3.51; N, 8.24.
din-3-yl}-3-phenyl-1H-indole-2-carboxamide (7i). Beige crystals
N-[2-(2-chloro-6-fluorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-5-
(73%); m.p. 211–212.5 ꢂC; IR(KBr): ꢀmax 3332, 3176 (N–H), fluoro-3-phenyl-1H-indole-2-carboxamide (7l). White powder
1
1726, 1647 (C¼O); H NMR (DMSO-d6/500 MHz): d 3.78 (d, (67%); m.p. 174–175.5 ꢂC; IR(KBr): ꢀmax 3290, 3209 (N–H),
1
1H, J ¼ 16.0 Hz, H5-thz.), 3.94 (dd, 1H, J ¼ 16.0; 1.8 Hz, H5- 1722, 1641 (C¼O); H NMR (DMSO-d6/500 MHz): d 3.81 (d,
thz.), 5.95 (s, 1H, H2-thz.), 7.12 (td, 1H, J ¼ 9.1; 2.3 Hz, H6-ind.), 1H, J ¼ 15.6 Hz, H5-thz.), 3.87 (br. d, 1H, J ¼ 15.1 Hz; H5-thz.),
7.17–7.20 (m, 4H, H4,3-C6H5(H3-5)-ind.), 7.24–7.27 (m, 2H, 3- 6.40 (s, 1H, H2-thz.), 7.13 (td, 1H, J ¼ 9.3; 2.4 Hz, H6-ind.), 7.18
C6H5(H2,H6)-ind.), 7.47 (dd, 1H, J ¼ 9.2; 4.6 Hz, H7-ind.), 7.66 (br. d, 1H, J ¼ 9.8 Hz, H4-ind.), 7.23–7.38 (m, 7H,
(d, 2H, J ¼ 8.5 Hz, 2-C6H5(H2,H6)-thz.), 7.73 (d, 2H, J ¼ 8.7 Hz, 2-C6H5(H3,H5)-thz. and 3-C6H5-ind.), 7.44–7.50 (m, 2H, 2-
2-C6H5(H3,H5)-thz.), 10.01 (s, 1H, CONH), 11.88 (s, 1H, C6H5(H4)-thz. and H7-ind.), 10.21 (s, 1H, CONH), 11.86 (s, 1H,
NH); 13C NMR (HSQC) (DMSO-d6/125 MHz):
d
29.89 NH); 13C NMR (HSQC, HMBC) (DMSO-d6/125 MHz): d 30.05
(C5-thz.), 61.65 (C2-thz.), 104.92 (d, J ¼ 24.0 Hz, C4-ind.), (C5-thz.), 55.75 (C2-thz.), 104.94 (d, J ¼ 23.5 Hz, C4-ind.),
113.79 (d, J ¼ 26.4 Hz, C6-ind.), 114.60 (d, J ¼ 9.6 Hz, C7-ind.), 113.96 (d, J ¼ 26.4 Hz, C6-ind.), 114.69 (d, J ¼ 9.6 Hz, C7-ind.),
119.68 (d, J ¼ 5.2 Hz, C3-ind.), 124.83 (d, J ¼ 272.2 Hz, 4-CF3), 116.89 (d, J ¼ 23.0 Hz, 2-C6H5(C5)-thz.), 120.17 (br. d, C3-ind.),
126.23 (d, J ¼ 3.8 Hz, 2-C6H5(C3,5)-thz.), 127.02 (d, J ¼ 9.6 Hz, 124.78 (d, J ¼ 11.5 Hz, 2-C6H5(C1)-thz.), 126.37 (2-C6H5(C3)-
C3a-ind.), 127.23 (C2/3-C6H5(C4)-ind.), 127.41 (C2/3-C6H5(C4)- thz.), 127.03 (d, J ¼ 7.2 Hz, C3a-ind.), 127.14 (C2-ind.), 127.49
ind.), 128.88 (2-C6H5 (C2,6)-thz./3-C6H5(C3,5)-ind.), 128.93 (3-C6H5(C4)-ind.),
129.04
(3-C6H5(C3,5)-ind.),
130.18
(2-C6H5(C2,6)-thz./3-C6H5(C3,5)-ind.), 129.92 (d, J ¼ 31.6 Hz, (3-C6H5(C2,6)-ind.), 132.28 (d, J ¼ 10.5 Hz, 2-C6H5(C4)-thz.),
2-C6H5(C4)-thz.), 130.13 (3-C6H5(C2,6)-ind.), 133.24 (C7a-ind.), 133.23 (C7a-ind.), 133.47 (3-C6H5(C1)-ind.), 134.15 (br. d, 2-
133.33 (3-C6H5(C1)-ind.), 144,20 (2-C6H5(C1)-thz.), 158.37 (d, C6H5(C2)-thz.), 158.38 (d, J ¼ 233.9 Hz, C5-ind.), 162.43 (d,
J ¼ 233.9 Hz, C5-ind.), 161.25 (CONH), 169.66 (CO-thz.). Anal J ¼ 256.4 Hz, 2-C6H5(C6)-thz.), 168.77 (CO-thz.); MS (ESI-) m/z
calcd for C25H17F4N3O2S (499.48): C, 60.12; H, 3.43; N, 8.41. (%): 484.5/482.5 (M-H-, 40/100). Anal calcd for
Found: C, 60.34; H, 3.76; N, 8.40.
5-Fluoro-N-{2-[4-(methoxycarbonyl)phenyl]-4-oxo-1,3-thiazo- Found: C, 56.90; H, 3.40; N, 8.19.
lidin-3-yl}-3-phenyl-1H-indole-2-carboxamide (7j). White 5-Fluoro-N-(5-methyl-4-oxo-2-phenyl-1,3-thiazolidin-3-yl)-3-
C24H16ClF2N3O2S.H2O (501.93): C, 57.23; H, 3.22; N, 8.37.
powder (65%); m.p. 141–143 ꢂC; IR(KBr): ꢀmax 3244 (N–H), phenyl-1H-indole-2-carboxamide (8a). White powder (68%);
1718, 1695, 1653 (C¼O); 1H NMR (DMSO-d6/500 MHz): d 3.78 m.p. 233–236 ꢂC; IR(KBr): ꢀmax 3368 (N–H), 1708, 1677

DOI: 10.3109/14756366.2015.1024673
Indole-based hydrazide-hydrazones and 4-thiazolidinones
5
(C¼O); 1H NMR (DMSO-d6/500 MHz): d 1.51 (d, 3H, J ¼ 6.7 Hz, CONH), 11.86 (br. s, 1H, NH); 13C NMR (HSQC) (DMSO-d6/
5-CH3-thz.), 4.05 and 4.14 (q and br. q, 1H, J ¼ 6.8 and 125 MHz): d 20.20, 20.53 (5-CH3-thz.), 38.81, 39.33 (C5-thz.),
J ¼ 6.8 Hz, H5-thz.), 5.82 and 5.85 (2s, 1H, H2-thz.), 7.10–7.19 60.49, 60.82 (C2-thz.), 104.91 (d, J ¼ 23.5 Hz, C4-ind.), 113.81,
(m, 2H, H4,H6-ind.); 7.24 (br. s, 5H, 3-C6H5-ind.), 7.39–7.47 (m, 113.85 (2d, J ¼ 26.4 Hz, C6-ind.), 114.62 (d, J ¼ 9.6 Hz, C7-ind.),
6H, 2-C6H5-thz. and H7-ind.), 10.01 (br. s, 1H, CONH), 11.84 (br. 119.76 (d, J ¼ 4.8 Hz, C3-ind.), 122.70, 122.93 (2-C6H5(C4)-thz.),
s, 1H, NH). Anal calcd for C25H20FN3O2S (445.51): C, 67.40; H, 127.11 (d, J ¼ 9.6 Hz, C3a-ind.), 127.25, 127.29 (3-C6H5(C4)-
4.92; N, 9.43. Found: C, 67.63; H, 5.31; N, 9.51.
ind.), 127.31, 127.49 (C2-ind.), 128.97 (3-C6H5(C3,5)-ind.),
130.16, 130.18 (3-C6H5(C2,6)-ind.), 130.44, 130.77
5-Fluoro-N-[5-methyl-2-(4-methylphenyl)-4-oxo-1,3-thiazoli-
din-3-yl]-3-phenyl-1H-indole-2-carboxamide (8b). Beige needles (2-C6H5(C2,6)-thz.), 132.20, 132.27 (2-C6H5(C3,5)-thz.), 133.22
(60%); m.p. 206–210 ꢂC; IR(KBr): ꢀmax 3234 (N–H), 1700, 1654 (C7a-ind.),
133.34 (3-C6H5(C1)-ind.), 137.93, 138.70
1
(C¼O); H NMR (DMSO-d6/500 MHz): d 1.50–1.53 (m, 3H, 5- (2-C6H5(C1)-thz.), 158.35 (d, J ¼ 233.9 Hz, C5-ind.), 161.10,
CH3-thz.), 2.32 (s, 3H, 4-CH3), 4.04 and 4.12 (q and qd, 1H, 161.14 (CONH), 172.34, 172.46 (CO-thz.); MS (ESI-) m/z (%):
J ¼ 6.8 and J ¼ 6.8; 1.5 Hz, H5-thz.), 5.79 and 5.81 (s and br. s, 524/522 (MꢀHꢀ, 100/92). Anal calcd for C25H19BrFN3O2S
1H, H2-thz.), 7.12 (td, 1H, J ¼ 9.3; 2.4 Hz, H6-ind.), 7.15–7.19 (524.41): C, 57.26; H, 3.95; N, 8.01. Found: C, 57.22; H, 4.22;
(m, 3H, 2-C6H5(H3,H5)-thz. and H4-ind.), 7.24–7.27 (m, 5H, 3- N, 8.30.
C6H5-ind.), 7.32 (d, 2H, J ¼ 8.0 Hz, 2-C6H5(H2,H6)-thz.), 7.45–
5-Fluoro-N-[2-(4-fluorophenyl)-5-methyl-4-oxo-1,3-thiazoli-
7.49 (m, 1H, H7-ind.), 9.94 and 9.98 (s, 1H, CONH), 11.81 and din-3-yl]-3-phenyl-1H-indole-2-carboxamide (8f). Beige powder
11.88 (s, 1H, NH); MS (ESI-) m/z (%): 458 (MꢀHꢀ, 100). Anal (67%); m.p. 219–220 ꢂC; IR(KBr): ꢀmax 3242 (N–H), 1700, 1661
calcd for C26H22FN3O2S (459.54): C, 67.96; H, 4.83; N, 9.14. (C¼O); 1H NMR (DMSO-d6/500 MHz): d 1.51 and 1.52 (2d, 3H,
Found: C, 67.89; H, 4.94; N, 8.97.
J ¼ 6.8 Hz, 5-CH3-thz.), 4.05 and 4.15 (q and qd, 1H, J ¼ 6.8 and
J ¼ 7.3; 1.5 Hz, H5-thz.), 5.83 and 5.85 (s and d, 1H, J ¼ 1.5 Hz,
5-Fluoro-N-[2-(4-methoxyphenyl)-5-methyl-4-oxo-1,3-thiazo-
(8c).
lidin-3-yl]-3-phenyl-1H-indole-2-carboxamide
White H2-thz.), 7.12 (td, 1H, J ¼ 9.3; 2.4 Hz, H6-ind.), 7.16–7.18 (m,
powder (70%); m.p. 188–189 ꢂC; IR(KBr): ꢀmax 3293 (N–H), 1H, H4-ind.), 7.20 (d, 2H, J ¼ 8.8 Hz, 2-C6H5(H3,H5)-thz.), 7.23–
1
1714, 1639 (C¼O); H NMR (DMSO-d6/500 MHz): d 1.50–1.53 7.26 (m, 5H, 3-C6H5-ind.), 7.48–7.52 (m, 3H, 2-C6H5(H2,H6)-
(m, 3H, 5-CH3-thz.), 3.78 (s, 3H, 4-OCH3), 4.03 and 4.11 (q and thz. and H7-ind.), 9.99 and 10.03 (2s, 1H, CONH), 11.83 and
qd, 1H, J ¼ 6.8 and J ¼ 7.1; 1.5 Hz, H5-thz.), 5.78 and 5.81 (2s, 11.90 (2s, 1H, NH). Anal calcd for C25H19F2N3O2S (463.50): C,
1H, H2-thz.), 6.91–6.94 (m, 2H, 2-C6H5(H3,H5)-thz.), 7.12 (td, 64.88; H, 4.13; N, 9.07. Found: C, 65.05; H, 4.47; N, 8.97.
1H, J ¼ 9.0; 2.4 Hz, H6-ind.), 7.15–7.19 (m, 1H, H4-ind.), 7.23–
N-[2-(4-cyanophenyl)-5-methyl-4-oxo-1,3-thiazolidin-3-yl]-5-
7.29 (m, 5H, 3-C6H5-ind.), 7.36 (d, 2H, J ¼ 8.8 Hz, fluoro-3-phenyl-1H-indole-2-carboxamide (8g). White powder
2-C6H5(H2,H6)-thz.), 7.45–7.49 (m, 1H, H7-ind.), 9.90 and 9.94 (73%); m.p. 199–200 ꢂC; IR(KBr): ꢀmax 3314 (N–H), 2228
(2s, 1H, CONH), 11.81 and 11.88 (2s, 1H, NH); 13C NMR (CꢄN), 1716, 1636 (C¼O); 1H NMR (DMSO-d6/500 MHz): d
(HSQC, HMBC) (DMSO-d6/125 MHz): d 19.97, 20.71 (5-CH3- 1.51 and 1.52 (2d, 3H, J ¼ 6.8 Hz, 5-CH3-thz.), 4.09 and 4.20 (q
thz.), 38.93, 39.25 (C5-thz.), 55.94 (4-OCH3), 60.95, 61.21 (C2- and qd, 1H, J ¼ 6.8 and J ¼ 7.1; 1.5 Hz, H5-thz.), 5.92 and 5.93 (s
thz.), 104.92 (d, J ¼ 24.0 Hz, C4-ind.), 113.77, 113.84 (2d, and d, 1H, J ¼ 1.5 Hz, H2-thz.), 7.12 (td, 1H, J ¼ 9.3; 2.4 Hz, H6-
J ¼ 26.8, J ¼ 26.4 Hz, C6-ind.), 114.55, 114.69 (2-C6H5(C3,5)- ind.), 7.16–7.27 (m, 6H, 3-C6H5,H4-ind.), 7.44–7.48 (m, 1H, H7-
thz.), 114.62 (d, J ¼ 8.1 Hz, C7-ind.), 119.58, 119.82 (2d, C3- ind.), 7.62 (d, 2H, J ¼ 8.3 Hz, 2-C6H5(H2,H6)-thz.), 7.82–7.85 (m,
ind.), 127.05 (d, C3a-ind.), 127.09, 127.19 (3-C6H5(C4)-ind.), 2H, 2-C6H5(H3,H5)-thz.), 10.13 and 10.17 (2s, 1H, CONH),
127.38, 127.41 (C2-ind.), 129.05 (3-C6H5(C3,5)-ind.), 129.66, 11.86 and 11.93 (2s, 1H, NH); 13C NMR (HSQC,DEPT) (DMSO-
129.84 (2-C6H5(C2,6)-thz.), 130.19, 130.22 (3-C6H5(C2,6)-ind.), d6/125 MHz): d 20.34, 20.45 (5-CH3-thz.), 38.69, 39.44 (C5-thz.),
130.47 (2-C6H5(C1)-thz.), 133.17 (C7a-ind.), 133.36 (3- 60.30, 60.68 (C2-thz.), 104.93 (d, J ¼ 24.0 Hz, C4-ind.), 112.04,
C6H5(C1)-ind.), 158.35 (d, J ¼ 233.9 Hz, C5-ind.), 160.46, 112.25 (2-C6H5(C4)-thz.), 113.76 (d, J ¼ 26.8 Hz, C6-ind.),
160.59 (2-C6H5(C4)-thz.), 160.88, 160.94 (CONH), 172.39, 114.56 (d, J ¼ 10.1 Hz, C7-ind.), 119.23 (4-CN), 119.58, 119.75
172.43 (CO-thz.). Anal calcd for C26H22FN3O3S (475.54): C, (2d, J ¼ 5.0 Hz, C3-ind.), 127.04 (d, C3a-ind.), 127.24, 127.30 (3-
65.57; H, 4.66; N, 8.54. Found: C, 65.23; H, 4.83; N, 8.21.
C6H5(C4)-ind.), 127.50 (C2-ind.), 128.91, 128.98 (3-C6H5(C3,5)-
N-[2-(4-chlorophenyl)-5-methyl-4-oxo-1,3-thiazolidin-3-yl]-5- ind.), 129.27 (2-C6H5(C2,6)-thz.), 130.13 (3-C6H5(C2,6)-ind.),
fluoro-3-phenyl-1H-indole-2-carboxamide (8d). White needles 133.24 (C7a-ind./3-C6H5(C1)-ind.), 133.27, 133.33 (2-
(71%); m.p. 211–214 ꢂC; IR(KBr): ꢀmax 3245 (N–H), 1700, C6H5(C3,5)-thz.), 144.41, 145.12 (2-C6H5(C1)-thz.), 158.36 (d,
1655 (C¼O); 1H NMR (DMSO-d6/500 MHz): d 1.51 and 1.52 J ¼ 234.4 Hz, C5-ind.), 161.26 (CONH), 172.37, 172.56 (CO-
(2d, 3H, J ¼ 6.8 Hz, 5-CH3-thz.), 4.06 and 4.16 (q and qd, 1H, thz.); MS (ESI-) m/z (%): 469 (MꢀHꢀ, 100). Anal calcd for
J ¼ 6.8 and J ¼ 7.1; 1.5 Hz, H5-thz.), 5.83 and 5.86 (s and d, 1H, C26H19FN4O2S (470.52): C, 66.37; H, 4.07; N, 11.81. Found: C,
J ¼ 1.5 Hz, H2-thz.), 7.12 (td, 1H, J ¼ 9.0; 2.4 Hz, H6-ind.), 66.35; H, 4.37; N, 11.53.
7.16–7.20 (m, 1H, H4-ind.), 7.22–7.26 (m, 5H, 3-C6H5-ind.),
5-Fluoro-N-[5-methyl-2-(4-nitrophenyl)-4-oxo-1,3-thiazolidin-
7.41–7.49 (m, 5H, 2-C6H5-thz. and H7-ind.), 10.03 (br. s, 1H, 3-yl]-3-phenyl-1H-indole-2-carboxamide (8h). Yellow crystals
CONH), 11.84 and 11.91 (2s, 1H, NH). Anal calcd for (66%); m.p. 193–195 ꢂC; IR(KBr): ꢀmax 3285 (N–H), 1716,
1
C25H19ClFN3O2S (479.95): C, 62.56; H, 3.99; N, 8.76. Found: 1638 (C¼O); H NMR (DMSO-d6/500 MHz): d 1.52 and 1.53
C, 62.75; H, 4.21; N, 8.85.
(2d, 3H, J ¼ 6.8 Hz, 5-CH3-thz.), 4.11 and 4.21 (q and qd, 1H,
N-[2-(4-bromophenyl)-5-methyl-4-oxo-1,3-thiazolidin-3-yl]-5- J ¼ 6.8 and J ¼ 7.1; 1.5 Hz, H5-thz.), 5.98 and 5.99 (s and d, 1H,
fluoro-3-phenyl-1H-indole-2-carboxamide (8e). Beige powder J ¼ 1.0 Hz, H2-thz.), 7.12 (td, 1H, J ¼ 9.3; 2.4 Hz, H6-ind.), 7.15–
(57%); m.p. 198–200 ꢂC; IR(KBr): ꢀmax 3244 (N–H), 1700, 7.27 (m, 6H, 3-C6H5,H4-ind.), 7.44–7.48 (m, 1H, H7-ind.), 7.71
1654 (C¼O); 1H NMR (DMSO-d6/500 MHz): d 1.51 and 1.52 (d, 2H, J ¼ 8.3 Hz, 2-C6H5(H2,H6)-thz.), 8.18–8.22 (m, 2H,
(2d, 3H, J ¼ 6.8 Hz, 5-CH3-thz.), 4.06 and 4.15 (q and qd, 1H, 2-C6H5(H3,H5)-thz.), 10.16 and 10.20 (2s, 1H, CONH), 11.85
J ¼ 6.8 and J ¼ 6.8; 1.5 Hz, H5-thz.), 5.82 and 5.84 (s and d, 1H, and 11.93 (2s, 1H, NH); 13C NMR (HSQC) (DMSO-d6/
J ¼ 1.5 Hz, H2-thz.), 7.12 (td, 1H, J ¼ 9.3; 2.4 Hz, H6-ind.), 7.15– 125 MHz): d 20.32, 20.44 (5-CH3-thz.), 38.72, 39.48 (C5-thz.),
7.20 (m, 1H, H4-ind.), 7.23–7.26 (m, 5H, 3-C6H5-ind.), 7.39 (d, 60.00, 60.38 (C2-thz.), 104.92 (d, J ¼ 23.5 Hz, C4-ind.), 113.76,
2H, J ¼ 8.8 Hz, 2-C6H5(H2,H6)-thz.), 7.45–7.48 (m, 1H, H7-ind.), 113.83 (2d, J ¼ 26.8, J ¼ 26.4 Hz, C6-ind.), 114.55, 114.60 (2d,
7.54–7.58 (m, 2H, 2-C6H5(H3,H5)-thz.), 10.06 (br. s, 1H, J ¼ 9.6 Hz, C7-ind.), 119.61, 119.74 (2d, J ¼ 4.8 Hz, C3-ind.),

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¨
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6
J Enzyme Inhib Med Chem, Early Online: 1–12
124.42, 124.50 (2-C6H5(C3,5)-thz.), 126.98, 127.08 (2d, 6.41 (s and br. s, 1H, H2-thz.), 7.13 (td, 1H, J ¼ 9.3; 2.4 Hz, H6-
J ¼ 9.6 Hz, C3a-ind.), 127.19, 127.24 (3-C6H5(C4)-ind.), 127.31, ind.), 7.18 (br. d, 1H, J ¼ 9.8 Hz, H4-ind.), 7.24–7.38 (m, 7H,
127.50 (C2-ind.), 128.88, 128.89 (3-C6H5(C3,5)-ind.), 129.37, 2-C6H5(H3,H5)-thz. and 3-C6H5-ind.), 7.44–7.49 (m, 2H,
129.66 (2-C6H5(C2,6)-thz.), 130.15, 130.18 (3-C6H5(C2,6)-ind.), 2-C6H5(H4)-thz. ve H7-ind.), 10.23 and 10.29 (2s, 1H, CONH),
133.23, 133.24 (C7a-ind.), 133.30, 133.33 (3-C6H5(C1)-ind.), 11.87 and 11.90 (2s, 1H, NH); MS (ESI-) m/z (%): 498.5/496.5
146.36, 147.09 (2-C6H5(C1)-thz.), 148,30, 148.43 (2-C6H5(C4)- (MꢀHꢀ, 45/100). Anal calcd for C25H18ClF2N3O2S (497.95): C,
thz.), 158.36 (d, J ¼ 233.9 Hz, C5-ind.), 161.27 (CONH), 172.32, 60.30; H, 3.84; N, 8.44. Found: C, 60.14; H, 4.08; N, 8.50.
172.53 (CO-thz.). Anal calcd for C25H19FN4O4S.1/2H2O
(499.51): C, 60.11; H, 4.08; N, 11.22. Found: C, 60.29; H, Biological assays
4.25; N, 11.25.
5-Fluoro-N-{5-methyl-4-oxo-2-[4-(trifluoromethyl)phenyl]-
BACTEC 460TB assay
1,3-thiazolidin-3-yl}-3-phenyl-1H-indole-2-carboxamide
(8i). The new compounds were evaluated for in vitro antitubercular
White needles (53%); m.p. 217–219 ꢂC; IR(KBr): ꢀmax 3303 activity against M. tuberculosis H37Rv (ATCC 27294) by the
(N–H), 1716, 1636 (C¼O); 1H NMR (DMSO-d6/500 MHz): d BACTEC 460TB (Becton Dickinson, Sparks, MD) system. This
1.51–1.54 (m, 3H, 5-CH3-thz.), 4.09 and 4.18 (q and qd, 1H, rapid and reliable radiometric method is based on the fact that
J ¼ 6.8 and J ¼ 6.8; 1.5 Hz, H5-thz.), 5.94 and 5.96 (s and d, 1H, mycobacteria metabolize fatty acids to CO2. If these fatty acids
J ¼ 1.5 Hz, H2-thz.), 7.12 (td, 1H, J ¼ 9.3; 2.4 Hz, H6-ind.), are radioactively labeled with 14C, the 14CO2 end product can be
7.14–7.20 (m, 4H, 3-C6H5(H3-5),H4-ind.), 7.22–7.25 (m, 2H, quantitatively determined by the Bactec instrument.
3-C6H5(H2,H6)-ind.), 7.44–7.48 (m, 1H, H7-ind.), 7.65 (d, 2H,
2-C6H5(H2,H6)-thz.), 7.71–7.74 (m,
All test compounds were dissolved in dimethyl sulfoxide
2H, (DMSO) at a stock concentration of 4000 mg/ml and kept at
J ¼ 7.8 Hz,
2-C6H5(H3,H5)-thz.), 10.15 (br. s, 1H, CONH), 11.93 (br. s, ꢀ20 ꢂC until used. The final concentrations were prepared with
1H, NH). Anal calcd for C26H19F4N3O2S (513.51): C, 60.81; H, 7H9 broth medium for mycobacteria. The final DMSO concen-
3.93; N, 8.18. Found: C, 60.70; H, 4.25; N, 8.22.
5-Fluoro-N-{2-[4-(methoxycarbonyl)phenyl]-5-methyl-4-oxo-
1,3-thiazolidin-3-yl}-3-phenyl-1H-indole-2-carboxamide
tration was adjusted to 1% and the final concentration of the tested
compounds was adjusted to 25 mg/ml in the BACTEC 12B
(8j). medium. Inoculum for susceptibility testing was used from a
White needles (72%); m.p. 129–131 ꢂC; IR(KBr): ꢀmax 3300 positive BACTEC isolation vial with a growth index (GI) of 500
(N–H), 1713, 1672, 1638 (C¼O); 1H NMR (DMSO-d6/500 MHz): or more. The BACTEC culture was well mixed and 0.1 ml of
d 1.51 and 1.52 (2d, 3H, J ¼ 6.8 Hz, 5-CH3-thz.), 3.87 (s, 3H, culture was added to each of the vials containing the test
4-COOCH3), 4.08 and 4.18 (q and qd, 1H, J ¼ 6.8 and J ¼ 6.8; compounds (25 mg/ml). A control vial was inoculated with a 1:100
1.5 Hz, H5-thz.), 5.91 and 5.92 (s and br. s, 1H, H2-thz.), 7.12 (td, dilution of the culture. The standard vials contained rifampicin
1H, J ¼ 9.3; 2.4 Hz, H6-ind.), 7.15–7.26 (m, 6H, 3-C6H5,H4-ind.), (0.25 mg/ml). Vials were incubated at 37 ꢂC and each vial was
7.44–7.48 (m, 1H, H7-ind.), 7.58 (d, 2H, J ¼ 8.3 Hz, checked daily with a BACTEC 460 instrument. When the control
2-C6H5(H2,H6)-thz.), 7.92–7.96 (m, 2H, 2-C6H5(H3,H5)-thz.), GI reading was at least 30, the results were interpreted by
10.11 (br. s, 1H, CONH), 11.83 and 11.90 (2s, 1H, NH); MS (ESI- calculating the increase in GI from the previous day. If the daily
) m/z (%): 502 (MꢀHꢀ, 100). Anal calcd for C27H22FN3O4S.1/ increase of GI of the control was greater than the daily GI increase
2H2O (512.55): C, 63.27; H, 4.54; N, 8.20. Found: C, 63.15; H, of the test compound vial, the mycobacteria were reported as
4.73; N, 8.08.
N-[2-(2,6-dichlorophenyl)-5-methyl-4-oxo-1,3-thiazolidin-3-
susceptible. If the daily increase of GI of the control was equal to
or less than that of the test compound vial, the organisms were
yl]-5-fluoro-3-phenyl-1H-indole-2-carboxamide (8k). White reported as resistant. The compounds demonstrating antituber-
powder (78%); m.p. 205.5–207 ꢂC; IR(KBr): ꢀmax 3295, 3212 cular activity at 25 mg/ml were further tested at twofold dilutions
(N–H), 1715, 1639 (C¼O); 1H NMR (DMSO-d6/500 MHz): d to determine the actual MIC31–33
1.50 (d, 3H, J ¼ 7.3 Hz, 5-CH3-thz.), 4.17 (qd, 1H, J ¼ 7.1; 2.0 Hz,
.
H5-thz.), 6.69 (d, 1H, J ¼ 2.0 Hz, H2-thz.), 7.14 (td, 1H, J ¼ 9.3;
MTT proliferation assay
2.4 Hz, H6-ind.), 7.17 (dd, 1H, J ¼ 9.2; 2.4 Hz, H4-ind.), 7.23–
The cytotoxicity of the active compounds was determined by the
MTT assay using the rat kidney epithelial cell line (NRK-
52E)34,35. The cell line was obtained from American Type Culture
Collection (ATCC, USA). Cells were incubated in Dulbecco’s
modified Eagle’s medium with 10% fetal bovine serum and
antibiotics (1% penicillin, 1% streptomycin) in 95% O2/5% CO2 at
37 ꢂC. Culture medium was changed every 2 days. The monolayer
cells grown to 75–85% confluency were detached with trypsin-
ethylenediaminetetraacetic acid to make single cell suspensions.
Viable cells were determined using the trypan blue exclusion test
and diluted with medium to give a final density of 105 cells/ml.
The passage number range for the cell line was maintained
between 20 and 25. One hundred microliters of cell suspension
was seeded into 96-well plate at a plating density of 104 cells/well
and incubated to allow for cell attachment at 37 ꢂC in a humidified
5% CO2 atmosphere for 24 h. After 24 h, the cells were treated
with serial concentrations of the test compounds. Test compounds
were initially dissolved in DMSO) and further diluted in distilled
water to produce the end concentrations. Ten microliters of each
concentration was added to plates to obtain final concentrations of
8–250 mg/ml. The final volume in each well was 100 ml and the
plates were incubated at 37 ꢂC in a humidified 5% CO2
7.31 (m, 3H, 3-C6H5(H3-5)-ind.), 7.35 (dd, 2H, J ¼ 8.1; 2.0 Hz,
3-C6H5(H2,H6)-ind.), 7.42 (t, 1H, J ¼ 8.3 Hz, 2-C6H5(H4)-thz.),
7.46–7.50 (m, 2H, 2-C6H5(H3/H5)-thz. and H7-ind.), 7.53 (dd,
1H, J ¼ 8.1; 1.5 Hz; 2-C6H5(H3/H5)-thz.), 10.11 (s, 1H, CONH),
11.91 (s, 1H, NH); 13C NMR (HSQC) (DMSO-d6/125 MHz): d
20.70 (5-CH3-thz.), 39.76 (C5-thz.), 55.79 (C2-thz.), 104.96 (d,
J ¼ 24.0 Hz, C4-ind.), 114.02 (d, J ¼ 26.8 Hz, C6-ind.), 114.67 (d,
J ¼ 9.6 Hz, C7-ind.), 120.36 (d, J ¼ 5.8 Hz, C3-ind.), 126.90 (C2-
ind.), 127.34 (d, J ¼ 9.6 Hz, C3a-ind.), 127.52 (3-C6H5(C4)ind.),
129.02 (3-C6H5(C3,5)-ind.), 129.64 (2-C6H5(C3/5)-thz.), 130.20
(3-C6H5(C2,6)-ind.), 131.88 (2-C6H5(C3/5)-thz.), 131.93
(2-C6H5(C4)-thz.), 133.23 (C7a/3-C6H5(C1)-ind.), 133.50 (C7a/
3-C6H5(C1)-ind.), 135.50 (2-C6H5(C2,6/C1)-thz.), 136.06 (2-
C6H5(C2,6/C1)-thz.), 158.37 (d, J ¼ 233.9 Hz, C5-ind.), 161.50
(CONH), 171.96 (CO-thz.). Anal calcd for C25H18Cl2FN3O2S
(514.40): C, 58.37; H, 3.53; N, 8.17. Found: C, 58.38; H, 3.43;
N, 8.01.
N-[2-(2-chloro-6-fluorophenyl)-5-methyl-4-oxo-1,3-thiazoli-
din-3-yl]-5-fluoro-3-phenyl-1H-indole-2-carboxamide (8l). White
powder (74%); m.p. 234–236 ꢂC; IR(KBr): ꢀmax 3287, 3214 (N–
1
H), 1716, 1642 (C¼O); H NMR (DMSO-d6/500 MHz): d 1.51–
1.53 (m, 3H, 5-CH3-thz.), 4.14–4.17 (m, 1H, H5-thz.), 6.37 and

DOI: 10.3109/14756366.2015.1024673
Indole-based hydrazide-hydrazones and 4-thiazolidinones
7
atmosphere for 24 h. Each concentration was tested in triplicate Anticancer screening
and each test was repeated. The medium containing no sample
Primary anticancer assay was performed in accordance with the
protocol of the Drug Evaluation Branch of the National Cancer
Institute (Bethesda, MD). The procedure utilized basicly mea-
sured the cellular protein content of cultures stained with the
protein dye sulforhodamine B spectrophotometrically36.
(growth control) and 0.5% DMSO (solvent control) served as
the controls. There was no difference in the cell count between the
solvent and growth controls.
The growth inhibitory activities of the tested compounds were
determined using the standard colorometric 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Twenty
microliters of MTT solution (5 mg/mL in phosphate buffered
saline) was added to each well and incubated at 37 ꢂC for 4 h. The
medium with MTT was removed, the formed formazan crystals
were solubilized in 100 ml of DMSO and the absorbance was
measured at 570 nm Epoch microplate spectrophotometer system
(BioTek Instruments, Winooski, VT). The percentage cell viabil-
ity was calculated with respect to the solvent control as follows:
Results and discussion
Chemistry
The synthetic approach utilized for the target compounds is
outlined in Scheme 1. The key intermediate 5 and indolylhy-
drazones 6a and 6d–6h were previously synthesized and patented
by Bamaung et al. as angiogenesis inhibitors29.
The structures of the new compounds were characterized by
microanalysis, IR, 1H NMR, 13C NMR (proton decoupled and
DEPT-135), 2D NMR (HSQC and HMBC), electrospray
Abstest
% Cell viability ¼
ꢁ 100:
Abssolvent control
F
F
F
ii
iii
i
C H
CH
C
6
5
2
+
−
NH
N Cl
NHN
COOC H
2
2
2
5
1
2
3
C H
C H
6
5
6
5
F
F
v
iv
O
C H
C
C
O
NH
2
NH
2
5
N
H
N
H
O
4
5
R
1
F
F
R
2
R
vi
2
NH
CH
R
R
N
H
NH
N
C
O
N
N
H
C
O
S
R
1
O
7a-l
6a-l
vii
R
1
F
R
2
R
NH
N
N
H
C
O
S
O
CH
3
8a-l
6-8a: R,R ,R =H
6-8g: R,R =H; R =CN
2 1
1
2
6-8b: R,R =H; R =CH
6-8h: R,R =H; R =NO
2
1
3
2 1 2
6-8i: R,R =H; R =CF
2 1 3
6-8c: R,R =H; R =OCH
2
1
3
6-8d: R,R =H; R =CI
6-8j: R,R =H; R =COOCH
2
1
2 1 3
6-8e: R,R =H; R =Br
6-8k: R,R =Cl; R =H
2 1
2
1
6-8f: R,R =H; R =F
6-8l: R=Cl; R =H; R2=F
2
1
1
* Compounds 6a and 6d-h were previously reported by Ba-Maung et al. (29)
Scheme 1. Synthesis of 6–8. Reagents and conditions: (i) 7% NaNO2, EtOH, conc. HCl, 0 ꢂC; (ii) ethyl 2-benzyl-3-oxo-butanoate, KOH, EtOH, 0 ꢂC;
(iii) conc. HCl, reflux, 4 h; (iv) H2NNH2ꢃH2O, EtOH, reflux, 6 h; (v) (non)substituted benzaldehyde, abs. EtOH, reflux, 5–6 h; (vi) mercaptoacetic acid,
dry benzene, reflux, 5–6 h and (vii) 2-mercaptopropionic acid, dry benzene, reflux, 5–6 h.

¨
G. Cihan-Ustundag et al.
¨
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8
J Enzyme Inhib Med Chem, Early Online: 1–12
Figure 2. Normal-phase HPLC diastereomeric resolution of compounds 8b, 8i, 8k and 8l. Column: Kromasil 100-5SIL microporous silica column
(25 cm ꢁ 4.6 mm); eluent: hexane–ethyl acetate (80:20); flow rate:1.2 mL/min; detection: 304 nm.
ionization mass spectrometry (ESI-MS) and HPLC studies. The 8c enabled definite assignment of the lactam C¼O (d 168.77–
absolute stereochemistry of 6c was determined by an X-ray 172.56 ppm) and the CONH (d 160.88–161.50 ppm) carbons.
The H NMR spectra of 8 displayed two sets of signals for
1
diffraction study30. The absence of the N–H2 resonance of the
intermediate hydrazide (5) at d 4.48 ppm together with the new most of the protons, except for the 2,6-dichloro substituted
1
signals of the N¼CH protons at d 8.00–8.32 ppm in the H NMR derivative, 8k. The 4-thiazolidinone C2-H protons resonated as a
spectra of 6 supported the structure of the new adducts. singlet and a broad singlet/doublet and C5-H protons resonated as
Observation of new lactam C¼O bands (1697–1726 cmꢀ1
)
a quartet and a doublet of quartets. Indole N–H and CONH
besides C¼O amide bands (1636–1678 cmꢀ1) in the IR spectra protons were observed as two separate singlets or one broad
of 7 and 8 proved the aimed cyclization. The 4-thiazolidinone singlet. Most of the proton resonances appeared as duplicate
C2-H of 7 was observed at about d 5.80–6.73 ppm as a singlet or a signals or distorted multiplets (see Experimental section for
doublet (J ¼ 1.0–1.5 Hz) and the C5-H2 resonated at about d 3.74– details). The novel 2,3,5-trisubstituted-4-thiazolidinones (8a–8l)
3.97 ppm as a double doublet with coupling constants in the range bear chiral C-2 and C-5 atoms and were possibly obtained as
of 15.6–16.1 and 1.4–2.0 Hz. Large couplings about 15 Hz mixtures of cis and trans diastereomers. The stereoisomeric
characteristic of geminal interactions supported the chiral nature content and the purity of selected entries 8b, 8i, 8k and 8e were
of the 4-thiazolidinone C2 carbon and small splittings indicated a confirmed by analytical HPLC studies. A simple and rapid
long-range interaction between the cis C2-H and C5-H protons of isocratic HPLC method was developed for the determination of
the rigid 4-thiazolidinone system. These four-bond couplings (W- the diastereomers of compounds 8b, 8i, 8k and 8l chosen as
coupling) across the C2-H and C5-H protons were also observed examples. The resolution of the diastereomers was achieved by
in the 1H NMR spectra of 8 with coupling constants of about 1.0– normal-phase HPLC on a conventional silica column (Kromasil
1.5 Hz. Peaks associated with the indole subunit were observed in 100-5SIL microporous silica column) with hexane–ethyl acetate
the expected regions and were assigned on the basis of 1H–1H and (80:20) as the mobil phase (Figure 2). The retention times and
1H–19F couplings.
peak-area percentages are presented in Table 1. Compound 8k,
13C NMR experiments (proton decoupled and DEPT-135) run which did not display duplicate signals in 1H NMR and 13C NMR
on 5, 8g and 2D NMR experiments (HSQC and HMBC) run on spectra, displayed two peaks on the chromatogram in an
6b, 6j, 7b, 7d, 7i–7l, 8c, 8e, 8g, 8h, 8k allowed unambiguous approximate ratio of 95:5 with a large excess of one of the
assingment of the proton and carbon chemical shifts. The diastereomers. The 5-unsubstituted derivatives 7b, 7i, 7k and 7l
carbocyclic indole carbons which explicitly showed the 13C–19F having a single chiral center at C-2 were also analyzed for
couplings of the 5-fluoro indole core were observed as separate comparison. As expected, the chromatograms showed only single
doublets with characteristic coupling constants related to the ipso, peaks (for representative NMR spectra, see Supplementary
ortho, meta and para positions and allowed definite positional materials).
assignment of the C3, C3a, C4–7 and C7a carbons of 6–8. The
ESI-MS was used to verify the molecular weights of
transformation of 6 to 7 or 8 proceeds via the nucleophilic compounds 6b, 6j, 6l, 7–8b, e, g, j and l. All compounds were
addition of the SH function to the C¼N double bond. Upfield analyzed under negative-ion ESI conditions since none of the
shifts observed in the 13C NMR resonances of the C¼N carbons compounds were responsive to the positive-ion mode.
of 6b and 6g (d 148.13 and 146.58 ppm) supported the expected Deprotonated [MꢀH]ꢀ ions observed in the ESI-MS, confirmed
addition as the resulting typical sp3 hybridized 4-thiazolidinone the calculated molecular weight of compounds 6b, 6j, 6l, 7–8b,
C5 absorbed at d 29.89–39.76 ppm. Resonances assigned to the 4- 8c, 8g and 8l.
thiazolidinone C2 (d 55.75–62.44 ppm) and endocyclic C¼O
Biological activity
(d 168.77–172.56 ppm) further confirmed the aimed conversion.
Cross peaks observed between the 4-thiazolidinone 5-CH3, C5-H
The novel indolylhydrazones (6) and indole-based 4-thiazolidi-
nones (7, 8) were tested for in vitro antitubercular activity against
protons and the lactam C¼O in the HMBC spectrum of 7a, 7l and

DOI: 10.3109/14756366.2015.1024673
Indole-based hydrazide-hydrazones and 4-thiazolidinones
9
Table 1. Retention times and peak area percentages of compounds 7–8b, 8i, 8k, 8l in NP-HPLC.a
7b
7i
7k
7l
8b
8i
8k
8l
Retention time
(min) (Peak-area
percentage)
29.51 (100%) 23.75 (100%) 21.75 (100%) 23.69 (100%) 13.88 (23.49%) 16.63 (66.05%) 8.47 (94.27%) 11.25 (84.82%)
19.30 (76.51%) 27.92 (33.95%) 12.27 (5.73%) 15.28 (15.18%)
aColumn: Kromasil 100-5SIL microporous silica column (25 cm ꢁ 4.6 mm); eluent: hexane–ethyl acetate (80:20); flow rate: 1.2 mL/min; detection:
304 nm.
Table 2. In vitro antitubercular activity and cytotoxicity screening results
of compounds 6–8.
the benzene ring had a profound effect on the activity.
Introduction of electron-withdrawing substituents CN, NO2, CF3
and COOCH3 at the para-position of the phenyl ring, as in 7g–7j,
8g, 8h and 8j, enhanced the biological activity in the majority of
the compounds. The screening results also showed that, substi-
tution at the 5-position of the 4-thiazolidinone system had a
negative effect. The 5-nonsubstituted-4-thiazolidinone derivatives
(7g–7i) were found to be more active in comparison with their
5-methyl substituted congeners (8g–8i), except for compounds 7j
and 8j which displayed similar MIC values (Table 2).
MIC
(mg/ml)a
IC50
(mg/ml)b
Compound
R
R1
R2
R3
6b
6c
6i
6j
6k
6l
7a
7b
7c
7d
7e
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
Cl
Cl
–
CH3
OCH3
CF3
COOCH3
H
H
H
CH3
OCH3
Cl
H
H
H
H
Cl
F
H
H
H
H
H
H
H
H
H
H
Cl
F
H
H
H
H
H
H
H
H
H
H
Cl
F
–
–
–
–
–
–
H
H
H
H
H
H
H
H
H
H
H
425
425
425
425
425
425
425
425
425
425
425
425
6.25
6.25
12.5
25.0
425
425
425
425
425
425
425
425
12.5
12.5
425
25.0
425
425
0.25
NTc
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
113.4
4125
4250
4125
NT
NT
NT
NT
NT
NT
NT
NT
60.0
125.0
NT
4250
NT
NT
The active compounds, 7g–7j, 8g, 8h and 8j, were further
examined for cytotoxicity using the rat kidney epithelial cell line
(NRK-52E) up to 125.0 or 250.0 mg/ml. Higher concentrations
could not tested due to solubility problems. The cytotoxicity
results presented in Table 2 are expressed as the concentrations
inhibiting 50% of the cell growth (IC50). The tested compounds
were found not to be cytotoxic at concentrations up to 125.0 or
250.0 mg/ml, except for 4-CN substituted derivatives 7g and 8g
which exhibited low cytotoxicity at 113.4 and 60.0 mg/ml,
respectively. All the tested compounds showed a differential
between active and cytotoxic doses. Compounds 7g, 7h, 7i, 8h
and 8j may be considered for further development as potential
anti-TB agents, as they demonstrated anti-TB activity at concen-
trations 10-fold lower than those cytotoxic for the mammalian
cell lines37.
5-Fluoro-N2-(4-methylbenzylidene)-3-phenyl-1H-indole-2-car-
bohydrazide (6b, NSC 752358) was selected by the NCI (USA) as
a prototype for evaluation in the in vitro preclinical antitumor
screening program against 60 human tumor cell lines derived
from nine different types of cancer, namely, leukemia, melanoma
and cancers of the lung, colon, brain, ovary, breast, prostate and
kidney36. This in vitro screen was subdivided into a pretest and a
main test. Within the one-dose pretest consisting of approximately
60 tumor cell lines, compound 6b was added at a single dose of
10 mM to each cell line. The results were reported as the
percentage of growth of the treated cells in comparison with that
of the untreated control cells. Compound 6b which satisfied
predetermined threshold inhibition criteria was passed on for
evaluation in the main test, consisting of 60 cell lines over a five-
dose range (0.01–100 mM).
Br
F
CN
7f
7g
7h
7i
7j
7k
7l
8a
8b
8c
8d
8e
NO2
CF3
COOCH3
H
H
H
CH3
OCH3
Cl
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
–
Br
F
CN
8f
8g
8h
8i
8j
8k
8l
NO2
CF3
COOCH3
H
H
–
Rifampicin
–
4250
aMIC, minimum inhibitory concentration required to inhibit the growth of
99% of M. tuberculosis H37Rv (ATCC 27294).
bIC50, 50% cytotoxic concentration against NRK-52E cells in vitro.
cNT, not tested.
Values in bold indicate the MIC and IC50 values of the most active
compounds 7g–j, 8g, 8h and 8j.
M. tuberculosis H37Rv using the BACTEC 460TB system31–33
.
The antituberculosis (anti-TB) drug, rifampicin, was used as the
positive control. The minimum concentration of compound
required to hinder 99% of bacterial growth in the culture was
referred as the MIC. Compounds 7g–7j, 8g, 8h and 8j were
assayed using twofold dilutions starting at 25.0 mg/ml. As reported
in Table 2, compounds 7g–7j, 8g, 8h and 8j exhibited significant
anti-TB activity showing 99% inhibition at MIC values ranging
from 25.0 to 6.25 mg/ml. The most potent entries were the CN
an NO2 substituted derivatives (7g and 7h) with MIC values of
6.25 mg/ml.
Table 3 shows the anticancer data for compound 6b. The NCI
standard anticancer agent 5-fluorouracil (NSC 19893) was used as
the reference compound38. As can be seen from Table 3, 6b
exhibited significant anticancer activity at sub-micromolar con-
centrations against most of the tested cell lines. The GI50 values
were between 0.01– and 0.1 mM with a full panel mean value of
0.039 mM, except for SF-268 (CNS Cancer), SK-MEL-28 (mel-
anoma) and T-47D (breast cancer) cell lines. Compound 6b
demostrated higher 50% growth inhibition activity than the
standard 5-fluorouracil (NSC 19893) did against all of the cell
lines tested except for SR (leukemia), MALME 3M (melanoma),
OVCAR-3 (ovarian cancer) and T-47D (breast cancer). It is
especially noteable that 6b had an obvious selectivity toward
colon cancer COLO 205 cell line at both the GI50 (0.018 mM),
The screening results showed that the 4-thiazolidinone scaffold
had an essential role in anti-TB activity since precursors (6) were
inactive at the tested concentrations. When the structures of the
active molecules were examined, it seemed that substitutions on

¨
10 G. Cihan-Ustundag et al.
¨
˘
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 3. In vitro anticancer activity of compound 6b against 60 human tumor cell lines at five-dose levels in comparison with data of
NCI’s standard 5-fluorouracil.a
NSC 752358 (6b)
NSC 19893 (5-fluorouracil)
TGI
b
d
Panel/cell line
GI50
TGIc
LC50
GI50
LC50
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
0.033
0.026
0.038
0.038
0.033
0.028
4100
0.072
15.5
15.5
13.8
4100
4100
4100
70.8
4100
4100
10.0
2.51
4.00
0.32
0.050
0.025
100.0
100.0
100.0
50.1
50.1
10.0
100.0
100.0
100.0
100.0
100.0
100.0
40.7
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
0.034
0.074
0.033
0.036
0.031
0.039
0.036
0.013
4100
4100
0.81
30.2
19.1
26.9
14.5
0.040
4100
4100
4100
4100
4100
4100
4100
4100
0.20
63.1
100.0
100.0
100.0
39.8
7.94
50.1
63.1
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
63.1
0.40
79.4
0.32
0.20
0.063
7.94
0.018
0.030
0.023
0.025
0.025
0.025
0.039
0.034
21.4
0.069
0.083
NTe
0.066
4100
0.063
4100
4100
4100
4100
17.4
0.16
0.050
0.25
0.10
0.16
0.20
1.00
63.1
39.8
3.98
50.1
63.1
39.8
100.0
100.0
100.0
25.1
100.0
100.0
100.0
100.0
HT29
KM12
SW-620
4100
CNS cancer
SF-268
SF-295
SF-539
SNB-19
1.32
34.7
4100
50.1
38.0
4100
83.2
1.58
0.25
0.063
4.00
79.4
1.00
100.0
3.98
79.4
79.4
100.0
79.4
100.0
100.0
100.0
100.0
100.0
100.0
0.019
0.025
0.050
0.032
0.036
0.060
0.070
4100
11.7
SNB-75
U251
34.7
4100
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
0.028
0.067
0.022
0.021
0.031
0.25
0.020
0.061
0.042
4100
66.1
0.061
0.048
NT
4100
0.045
4100
34.7
4100
4100
4100
2.75
4100
4100
0.29
0.25
50.1
2.51
100.0
79.4
100.0
63.1
39.8
79.4
39.8
79.4
100.0
100.0
100.0
100.0
100.0
79.4
0.050
1.00
0.079
63.1
1.00
0.50
4.00
0.50
4100
4100
100.0
100.0
0.032
0.025
0.046
0.050
0.037
0.022
0.027
1.82
0.048
4100
4100
4100
0.053
0.077
4100
NT
1.26
0.015
4.00
10.0
1.58
0.32
31.6
0.32
79.4
50.1
31.6
12.6
63.1
100.0
50.1
4100
4100
4100
4100
4100
100.0
100.0
100.0
100.0
100.0
19.9
0.031
0.038
0.048
0.033
0.025
0.045
0.044
0.038
12.3
24.5
4100
3.63
0.085
4100
4100
21.4
4100
4100
4100
4100
63.1
4100
4100
4100
0.79
0.40
0.32
0.079
2.51
0.50
1.26
1.58
50.1
100.0
31.6
2.00
31.6
25.1
79.4
50.1
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
DU-145
0.034
0.023
20.4
0.052
4100
0.29
2.51
0.40
100.0
100.0
100.0
100.0
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
MG_MIDf
0.029
0.033
0.027
0.032
24.0
0.023
0.039
4100
0.23
1.78
4100
4100
4100
4100
4100
4100
63.1
0.079
6.31
10.0
10.0
7.94
50.1
39.8
100.0
100.0
50.1
NT
100.0
100.0
100.0
100.0
100.0
NT
2.34
4100
0.067
4.37
NT
0.85
42.7
95.5
aData obtained from the NCI’s in vitro disease-oriented human tumor cell screen. All values were given in mM. Highest concentration was
100 mM unless otherwise reported.
bMolar concentration that inhibited 50% net cell growth.
cMolar concentration leading to total growth inhibition.
dMolar concentration leading to 50% cell death.
eNot tested.
fAverage activity parameter over all cell lines.

DOI: 10.3109/14756366.2015.1024673
Indole-based hydrazide-hydrazones and 4-thiazolidinones 11
4-phenyl-3-thiosemicarbazones and 2-[(phenylmethylene)hydra-
zono]-4-oxo-3-phenyl-5-thiazolidineacetic acids. Bioorg Med
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752358) was retested using the five-dose screen and the
reproducibility of its cellular actions was confirmed.
7. Aridoss G, Amirthaganesan S, Kim MS, et al. Synthesis, spectral
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Conclusion
In the search for effective and selective antitubercular agents, we
achieved the synthesis of novel hydrazone (6) and 4-thiazolidi-
none (7, 8) derivatives of the 5-fluoro-3-phenyl-1H-indole
scaffold. All the newly synthesized compounds (6–8) were
evaluated for in vitro anti-TB activity against M. tuberculosis
H37Rv. The 4-thiazolidinone derivatives 7g–7j, 8g, 8h and 8j
displayed appreciable anti-TB activity showing 99% inhibition at
MIC values ranging from 25.0 to 6.25 mg/ml along with rather low
cytotoxicity against mammalian cell lines. Compounds 7g, 7h, 7i,
8h and 8j demonstrated anti-TB activity at concentrations 10-fold
lower than those cytotoxic for the mammalian cell lines. The
screening results warrant further investigation to evaluate in vivo
anti-TB activity of promising analogs and elucidate the mechan-
ism of the anti-TB potential of this class of compounds.
The antitumor potential of compound 6b, which was selected
by the NCI as a prototype, has been also investigated. Although
the biochemical targets of the molecule have not been identified
yet, it showed an interesting anticancer profile against different
human tumor-derived cell lines at sub-micromolar concentrations
with GI50 values between 0.01 and 0.1 mM with a full panel mean
value of 0.039 mM, except for SF-268 (CNS cancer), SK-MEL-28
(melanoma) and T-47D (breast cancer) cell lines. Compound 6b
demonstrated an obvious selectivity toward colon cancer COLO
205 cell line at both the GI50 (0.018 mM), TGI (0.034 mM) and
LC50 (0.063 mM) levels. These encouraging preliminary results
make relevant structures an interesting avenue toward the
discovery of a new class of anticancer agents worthy of further
examination and scientific scrutiny.
˘
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Investigations directed toward the modification of the indole
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antitubercular and anticancer activity.
Acknowledgements
We thank the National Cancer Institute (NCI) (Bethesda, MD, USA) for
anticancer screening.
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Declaration of interest
This work was supported by The Research Fund of Istanbul University
(Project Number T-2827) and The Scientific and Technological Research
¨
_
_
Council of Turkey (TUBITAK, BIDEB-2211). The authors declare no
conflict of interest.
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