Synthesis, antitubercular and anticancer activity of new Baylis-Hillman adduct-derived N-cinnamyl-substituted isatin derivatives

Article abstract of DOI:10.1007/s00044-013-0787-x

Baylis-Hillman adduct-derived N-cinnamyl-substituted isatin derivatives were synthesized and evaluated for their antitubercular activity on Mycobacterium tuberculosis H₃₇Rv strain ATCC 27294 by agar dilution method. Anticancer activity for the

Full text of DOI:10.1007/s00044-013-0787-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1934–1940
DOI 10.1007/s00044-013-0787-x
ORIGINAL RESEARCH
Synthesis, antitubercular and anticancer activity of new Baylis–
Hillman adduct-derived N-cinnamyl-substituted isatin derivatives
•
Sriramoju Bharath Kumar Mettu Ravinder Golla Kishore
•
•
•
Vaidya Jayathirtha Rao Perumal Yogeeswari Darmarajan Sriram
•
Received: 12 June 2013 / Accepted: 10 September 2013 / Published online: 26 September 2013
Ó Springer Science+Business Media New York 2013
Abstract Baylis–Hillman adduct-derived N-cinnamyl-
substituted isatin derivatives were synthesized and evalu-
ated for their antitubercular activity on Mycobacterium
tuberculosis H37Rv strain ATCC 27294 by agar dilution
method. Anticancer activity for the same compounds was
also screened on four different cell lines: Chinese hamster
ovary (CHO cells), Colo 205 (human colon cancer), Sup-
T1 (human lymphoma) and C6 glioma (rat glioma) by
MTT assay method. The compounds (3j–l) have shown
significant activity against Mycobacterium strain and the
compound 3l has shown specific cytotoxic activity.
(Guo and Chen, 1986) and has also been found in humans
as a metabolic derivative of adrenaline (Ischia et al., 1988).
The synthetic flexibility of isatin has led to the synthesis of
an array of substituted derivatives displaying a broad
spectrum of biological properties and the derivatives have
been developed for therapeutic applications. The various
biological properties of isatin include, antibacterial/anti-
fungal (Varma and Nobles 1975), antiviral (Varma and
Nobles 1967), anticancer (Matesic et al., 2008), anti-HIV/
antitubercular (Sriram et al., 2006), antiprotozoal (Raj
et al., 2012, Imam and Varma 1975), antihelminthic (El-
Sawi et al., 1998) and other biological activities (Silva
et al., 2001, Pandeya et al., 2005).
Keywords Baylis–Hillman bromides ꢀ Isatins ꢀ
Antitumor agents ꢀ Cytotoxicity
On the other hand substituted isatin derivatives have
been reported to possess DNA gyrase inhibitor (Oblack
et al., 2005), human rhinovirus 3C protease inhibitor
(Webber et al., 1996), SARS corona virus 3C-like protease
inhibitor (Zhou et al., 2006) and caspase-3 inhibitor
activities (Chu et al., 2005). Investigation of the structure–
activity relationships of isatin derivatives has revealed that
5-halogenated (Sriram et al., 2005) and N-alkylated isatin
(Chen et al., 2005; Singh et al., 2011) derivatives are
proved to be showing marked rise in biological activity.
For example compound 5-fluoro-3-substituted-2-oxindole
(SU11248) has been approved by FDA for the treatment of
gastrointestinal stromal tumours and advanced renal cell
carcinoma (Prenen et al., 2006; Motzer et al., 2006).
Inspired with the biological profile of isatins and their
increasing importance in pharmaceutical and biological
fields, and in connection with our research on the design
and synthesis of biologically active and pharmacologically
important new heterocycles (Narender et al., 2006; Rav-
inder et al., 2010, 2012), it was thought worthwhile to
synthesize the title compounds with a view to obtain cer-
tain new chemical entities in order to prepare molecules
Introduction
Isatin (1H-indole-2,3-dione) is a privileged natural product
found in various plants including those of the genus Isatis
S. B. Kumar ꢀ M. Ravinder (&) ꢀ V. Jayathirtha Rao (&)
Crop Protection Chemicals Division, Indian Institute of
Chemical Technology, Uppal Road, Hyderabad 500607, India
e-mail: ravi_iictindia@yahoo.co.in
V. Jayathirtha Rao
e-mail: jrao@iict.res.in
G. Kishore
Department of Biochemistry, School of Life Sciences,
University of Hyderabad, Hyderabad 500046, India
P. Yogeeswari ꢀ D. Sriram (&)
Medicinal Chemistry & Antimycobacterial Research Laboratory
Group, Birla Institute of Technology & Science-Pilani,
Hyderabad 500078, India
e-mail: drdsriram@yahoo.com
123

Med Chem Res (2014) 23:1934–1940
1935
having potentially enhanced biological activities and to
have them evaluated for their bioactivity. Herein, we report
the synthesis of Baylis–Hillman adduct-derived N-cin-
namyl-substituted isatin derivatives and screening of their
antitubercular and anticancer activity.
obtained exclusively rather than SN20-type product. We
reasoned that bulky isatin nucleophile fails to attack at b-
position of the Baylis–Hillman bromide (SN20 path) due to
steric repulsion gained by aromatic group, hence it is
attacking at steric-free allyl carbon having bromide group
via SN2 path and leads to the compound I exclusively
which is confirmed by spectral analysis. A series of com-
pounds (3b–l) were synthesized with this optimized con-
ditions in good yields (80–93 %). All the products
synthesized were well characterized by spectroscopic
techniques. Thus the synthesized products were screened
for their anticancer activity against four different cell lines
by MTT assay method and antituberculosis activity against
Mycobacterium tuberculosis H37Rv strain ATCC 27294 by
agar dilution method.
Results and discussion
Chemistry
The starting substrates, 5-halo-isatin derivatives (2a–d)
were obtained commercially whereas Baylis–Hillman
bromides (1a–c) were prepared by treating Baylis–Hillman
adducts (Basavaiah et al., 2010; Sing and Batra 2008) with
conc. H2SO4/aq. HBr at 0 °C in DCM solvent (Buchholz
and Hoffmann 1991). During the optimization studies, we
focused our efforts on searching suitable reaction condi-
tions for the preparation of target compounds. Initially, the
reaction was carried out between Baylis–Hillman bromide
(1a) and isatin (2a) using different bases such as NaH,
KOH, K2CO3, NEt3, NaOMe and in various solvents such
as DMF, acetonitrile and MeOH (Scheme 1). In our study,
it was observed that K2CO3 in acetonitrile was suitable to
promote and complete the reaction in which the product
(3a) was obtained in good yield (87 %). From mechanistic
view, the nucleophile can attack on allyl bromide of Bay-
lis–Hillman adduct in two fashions (SN2 and SN20) leading
to the formation of two products (I and II) as shown in
Scheme 2, literature also revealed the formation of two
types of products while treating the Baylis–Hillman bro-
mides (Buchholz and Hoffmann 1991) with nucleophile;
but in our study, in this reaction only SN2-type product was
Pharmacology
Antitubercular studies
All the synthesized compounds (3a–l) were screened for their
in vitro antitubercular activity against MTB (M. tuberculosis
H37Rv strain ATCC 27294) by agar dilution method for the
determination of MIC in duplicate. The minimum inhibitory
concentration (MIC; lg/mL) was determined for each com-
pound. The MIC is defined as the minimum concentration of
compoundrequiredtocompletely inhibitthe bacterial growth.
Rifampicin, Isoniazid, Ethambutol and Pyrazinamide were
used as reference drugs. The results of in vitro antitubercular
activities (MIC in lg/mL) along with the standard drugs for
comparison are summarized in Table 1. All the screened
compounds have shown moderate to good in vitro activity
against M. tuberculosis, with MIC in the range of 1.56–25
Scheme 1 Synthesis of N-cinnamyl-substituted isatin derivatives
123

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Med Chem Res (2014) 23:1934–1940
Scheme 2 Nucleophilic
substitution on Baylis–Hillman
bromide
Table 1 In vitro antitubercular
evaluation of 3a–l against M.
tuberculosis H37Rv ATCC
27294
compounds (3a–l) was screened in four cell lines comprising
non-cancerous Chinese hamster ovary cells (CHO) and
cancerous Colo 205 (human colon cancer), Sup-T1 (human
lymphoma) and C6 glioma (rat glioma) cell lines were
determined by MTT assay method. The resulting data show
specific cytotoxicity of compound 3l on cancerous cell lines.
Further, compound 3l is found to be more toxic (\20 % cell
viability, Fig. 1b, c) towards C6 glioma and Sup-T1 cells.
However, compounds 3a–k are found to be equally toxic to
both cancerous and non-cancerous cells. Interestingly,
compounds 3d–f showed higher cytotoxicity in colon cells
when compared to C6 glioma and Sup-T1cells (Fig. 1b–d).
The results suggest that compound 3l in the series of com-
pounds 3a–l is active on cancer cells, C6 glioma in partic-
ular. More comprehensive activity studies need to be carried
out to establish the anticancer activity of the compound 3l.
Compound
MIC (lg/mL)
agar dilution
method
3a
25
3b
12.5
12.5
12.5
25
3c
3d
3e
3f
6.25
6.25
12.5
6.25
1.56
1.56
1.56
0.1
3g
3h
3i
3j
3k
3l
Bold values indicate the
compounds which are
exhibiting better activity than
other compounds
Rifampicin
Isoniazid
Ethambutol
Pyrazinamide
0.36
7.64
50.77
Experimental
MIC minimum inhibitory
concentration
All chemicals were of research grade and were used as-
obtained commercially from Aldrich. The reactions were
carried out in a round-bottomed flask of 25 mL capacity at
room temperature in an efficient fume hood. The progress of
all the reactions was monitored by TLC, using TLC alu-
minium sheets precoated with silica gel 60 F254 to a
thickness of 0.25 mm (Merck). Flash column chromatog-
raphy was done using silica gel (Merck, 60–120 mesh).
Melting points were determined on a MEL-TEMP II melt-
ing point apparatus and were uncorrected. IR spectra were
lg/mL. All the compounds are more potent than Pyrazina-
mide drug (50.77 lg/mL) and less potent than Rifampicin
(0.1 lg/mL). The compounds 3f, 3g and 3i are showing better
activity than Ethambutol (7.64 lg/mL). The results clearly
indicate that the antitubercular activity of the synthesized
compounds depend on the substituent’s present on both
moieties (isatin, cinnamyl). The compound (3a) that does not
have any substituent on both the moieties as well as the
compounds that are having one halo substituent either on
isatin (3b–d) or cinnamyl moiety (3e) are displaying poor
activity except the compound 3i having nitro group on phenyl
ring. The compounds that are having different halogen sub-
stituents on both the moieties (3f, 3g) are showing moderate
antitubercular activity except the compound 3h, having bro-
mine substituent on both the moieties. Replacement of the
halogen groups on phenyl ring of cinnamyl moiety of com-
pounds (3f–h) with nitro group (3j–l) enhanced the antitu-
bercular activity (1.56 lg/mL), which is close to that of the
standard Isoniazid drug (0.36 lg/mL).
1
recorded on a Perkin-Elmer FT-IR spectrophotometer. H
and 13C NMR spectra were recorded on a Varian Gemini
200 MHz, Bruker Avance 300 MHz spectrometer; TMS
was used as an internal standard in CDCl3/DMSO-d6. Mass
spectra were recorded on VG Micro mass 7070 H (EI),
QSTAR XL High resolution mass spectrometer (HRMS)
and Thermofinnigan ESI ion trap Mass Spectrometer.
General experimental procedure for the preparation
of N-cinnamyl-substituted isatin derivative (3a–l)
To a solution of isatin 2a (147 mg, 1.0 mmol) in acetoni-
trile (10 mL), K2CO3 (166 mg, 1.2 mmol) was added fol-
lowed by Baylis–Hillman bromide 1a (305 mg, 1.2 mmol)
at room temperature and the reaction mixture was stirred for
10–12 h. Upon completion of the reaction, the mixture was
filtered and the filtrate was diluted with water (50 mL) and
Cytotoxic evaluation
The in vitro results of antitubercular activity encouraged us
to evaluate their anticancer effects against a panel of four
cell lines. The preliminary cytotoxicity for a set of
123

Med Chem Res (2014) 23:1934–1940
1937
A
B
C6 Glioma
CHO
250
100
80
60
40
20
0
1 mm
1 mm
200
150
100
50
100 µm
100 nm
100 pm
100 µm
100 nm
100 pm
0
3a 3b 3c 3d 3e
3g 3h 3i 3j 3k 3l
3f
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l
Compound
Compound
C
D
COLON
Sup T1
100
80
100
80
1 mm
1 mm
100 µm
100 µm
100 nm
100 pm
100 nm
100 pm
60
60
40
40
20
0
20
0
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l
Compound
Compound
Fig. 1 Analysis of the cytotoxic effects of various derivatives (3a–l)
in non-cancerous cell line (CHO) and in cancerous cell lines (C6
glioma, Suo T1 and Colo 205) was treated with various concentra-
tions (1 mm, 100 lm, 100 nm and 100 pm). A viability assay was
carried out. Experiments were performed in triplicates and data are
expressed as means of the triplicate for determination of % of cell
viability
extracted with EtOAc (50 mL 9 3). The combined organic
layers were dried over Na2SO4, filtered and concentrated
under reduced pressure and the obtained crude product was
purified by silica gel column chromatography (60–120
mesh, eluent: EtOAc/hexane, 3:7) to afford pure cinnamyl-
substituted isatin derivative 3a as solid compound.
1260 cm-1; 1H NMR (300 MHz, CDCl3): d 3.78 (s, 3H, CH3),
4.80 (s, 2H, CH2), 6.54 (d, 1H, J = 8.0 Hz, ArH), 7.28–7.44
(m, 7H, ArH), 7.96 (s, 1H, olefin);13C NMR (75 MHz, CDCl3):
d 37.5, 52.4, 112.3, 118.3, 124.8, 125.2, 128.7, 129.2, 129.3,
133.8, 137.3, 144.4, 148.8, 157.2, 166.6, 181.8; MS (m/z): 356
?
[M?H]?; HRMS (ESI) m/z: C19H15NO4Cl [M?H] calcu-
lated: 356.0689, found: 356.0680.
Methyl(E)-2-[(2,3-dioxo-2,3-dihydro-1H-1-indolyl)methyl]-
3-phenyl-2-propionate (3a) Yield: 87 %; Reddish solid;
m.p. 81–83 °C; IR (KBr) m: 2952 (Ar, C–H str.), 1744
(ester, CO str.), 1700 (CO, str.), 1612 (amide, CO str.),
Methyl(E)-2-[(5-fluoro-2,3-dioxo-2,3-dihydro-1H-1-indolyl)
methyl]-3-phenyl-2-propionate (3c) Yield: 80 %; Reddish
solid; m.p. 114–116 °C; IR (KBr) m: 2956 (Ar, C–H str.),
1746 (ester, CO str.), 1694 (CO, str.), 1621 (amide, CO str.),
1
1469, 1256 cm-1; H NMR (300 MHz, CDCl3): d 3.78 (s,
1
3H, CH3), 4.81(s, 2H, CH2), 6.55 (d, 1H, J = 7.9 Hz,
ArH), 6.98 (t, 1H, J = 7.5 Hz, ArH) 7.26–7.42 (m, 7H,
ArH), 7.96 (s, 1H, olefin); 13C NMR (75 MHz, DMSO-d6):
d 37.6, 51.9, 110.9, 117.3, 122.9, 124.1, 126.1, 128.4, 128.
9, 129.1, 133.9, 137.9, 142.3, 150.7, 157.7, 166.2, 182.8;
MS (m/z): 322 [M?H]?; HRMS (ESI) m/z: C19H16NO4
1482, 1264 cm-1; H NMR (300 MHz, CDCl3): d 3.80 (s,
3H, CH3), 4.82 (s, 2H, CH2), 6.53 (dd, 1H, J = 8.8, 3.7 Hz,
ArH), 7.09–7.38 (m, 7H, ArH), 7.97 (s, 1H, olefin); 13C NMR
(75 MHz, CDCl3, C–F coupling observed): d 37.4, 52.4, 112.
0, 112.1, 118.0, 118.1, 124.1, 124.4, 125.2, 128.6, 129.3, 133.
8, 144.3, 146.5, 157.3, 157.6, 160.6, 166.6, 182.2; MS (m/z):
340 [M?H]?; HRMS (ESI) m/z: C19H15NO4F [M?H]?
calculated: 340.0985, found: 340.0973.
?
[M?H] calculated: 322.1079, found: 322.1089.
Methyl(E)-2-[(5-chloro-2,3-dioxo-2,3-dihydro-1H-1-indolyl)
methyl]-3-phenyl-2-propionate (3b) Yield: 89 %; Reddish
solid; m.p. 149–151 °C; IR (KBr) m: 2953 (Ar, C–H str.), 1746
(ester, CO str.), 1697 (CO str.), 1608 (amide, CO str.), 1469,
Methyl(E)-2-[(5-bromo-2,3-dioxo-2,3-dihydro-1H-1-indolyl)
methyl]-3-phenyl-2-propionate (3d) Yield: 85 %; Reddish
solid; m.p. 144–146 °C; IR (KBr) m: 2926 (Ar, C–H str.),
123

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Med Chem Res (2014) 23:1934–1940
1742 (ester, CO str.), 1700 (CO, str.), 1605 (amide, CO str.),
1
[M?H]?; HRMS (ESI) m/z: C19H14NO4FBr [M?H]?
1462, 1255 cm-1; H NMR (300 MHz, CDCl3): d 3.80 (s,
calculated: 418.0090, found: 418.0084.
3H, CH3), 4.83 (s, 2H, CH2), 6.49 (d, 1H J = 8.3 Hz,
ArH), 7.28 (d, 2H, J = 7.3 Hz, ArH) 7.35–7.40 (m, 3H,
ArH), 7.52 (dd, 1H, J = 8.3, 2.1 Hz, ArH) 7.58 (d, 1H,
J = 2.1 Hz, ArH), 8.01 (s, 1H, olefin); 13C NMR (75 MHz,
CDCl3): d 37.4, 52.4, 112.7, 116.2, 118.6, 125.1, 127.6,
128.6, 129.3, 133.7, 140.1, 144.4, 149.2, 157.0, 166.5,
181.6; MS (m/z): 400 [M?H]?; HRMS (ESI) m/z:
C19H15NO4Br [M?H]?calculated: 400.0184, found: 400.
0182.
Methyl(E)-3-(4-bromophenyl)-2-[(5-bromo-2,3-dioxo-2,3-
dihydro-1H-1-indolyl)methyl]-2-propionate (3h) Yield:
89 %; Reddish solid; m.p. 169–171 °C; IR (KBr) m: 2923
(Ar, C–H str.), 1744 (ester, CO str.), 1715 (CO, str.), 1604
1
(amide, CO str.), 1464, 1269 cm-1; H NMR (300 MHz,
CDCl3): d 3.78 (s, 3H, CH3), 4.73 (s, 2H, CH2), 6.60 (d,
1H, J = 8.3 Hz, ArH), 7.24 (d, 2H, J = 8.3 Hz, ArH), 7.
52 (d, 2H, J = 8.3 Hz, ArH), 7.56–7.60 (dd, 1H, J = 8.3,
2.3 Hz, ArH), 7.63 (d, 1H, J = 8.3 Hz, ArH), 7.87 (s, 1H,
olefin); 13C NMR(75 MHz, CDCl3): d 37.6, 52.3, 111.7,
118.7, 123.2, 124.7, 125.7, 129.1, 130.1, 131.7, 132.9, 136.
1, 142.3, 149.9, 158.1, 167.4, 181.7; MS (m/z): 478
[M?H]?; HRMS (ESI) m/z: C19H14NO4Br2 [M?H]? cal-
culated: 477.9289, found: 477.9275.
Methyl(E)-3-(4-bromophenyl)-2-[(2,3-dioxo-2,3-dihydro-1H-
1-indolyl)methyl]-2-propionate (3e) Yield: 92 %; Reddish
solid; m.p. 104–106 °C; IR (KBr) m: 2925 (Ar, C–H str.),
1739 (ester, CO str.), 1715 (CO, str.), 1609 (amide, CO str.),
1
1465, 1252 cm-1; H NMR (300 MHz, CDCl3): d 3.78 (s,
3H, CH3), 4.78 (s, 2H, CH2), 6.64 (d, 1H, J = 8.3 Hz,
ArH), 7.05 (t, 1H, J = 7.3 Hz, ArH), 7.22 (d, 2H, J = 8.
3 Hz, ArH) 7.45 (t, 1H, J = 7.3 Hz, ArH), 7.51 (m, 3H,
ArH), 7.86 (s, 1H, olefin); 13C NMR (50 MHz, DMSO-
d6): d 37.6, 52.0, 111.0, 117.3, 122.4, 123.0, 124.1, 126.9,
131.2, 131.3, 133.1, 137.9, 141.0, 150.7, 157.8, 166.1,
182.8; MS (m/z): 400 [M?H]?; HRMS (ESI) m/z:
C19H15NO4Br [M?H]?calculated: 400.0184, found: 400.
0185.
Methyl(E)-3-(4-nitrophenyl)-2-[(2,3-dioxo-2,3-dihydro-1H-
1-indolyl)methyl]-2-propionate (3i) Yield: 90 %; Reddish
solid; m.p. 146–148 °C; IR (KBr) m: 2924 (Ar, C–H str.),
1744 (ester, CO str.), 1715 (CO, str.), 1608 (amide, CO str.),
1523, 1464, 1344, 1252 cm-1
;
1H NMR (300 MHz,
CDCl3): d 3.81 (s, 3H, CH3), 4.69 (s, 2H, CH2), 6.81 (d,
1H, J = 8.3 Hz, ArH), 7.07 (t, 1H, J = 7.5 Hz, ArH), 7.
48–7.57 (m, 4H, ArH), 7.94 (s, 1H, olefin), 8.21 (d, 2H,
J = 8.3 Hz, ArH); 13C NMR (75 MHz, CDCl3): d 37.6,
52.1, 111.1, 117.3, 123.0, 123.3, 124.1, 129.0, 130.2, 137.
9, 139.9, 140.9, 147.0, 150.5, 157.9, 165.8, 182.6; MS
(m/z): 389 [M?Na]?; HRMS (ESI) m/z: C19H14N2O6 Na
[M?Na]?calculated: 389.0749, found: 389.0759.
Methyl(E)-3-(4-bromophenyl)-2-[(5-chloro-2,3-dioxo-2,3-
dihydro-1H-1-indolyl)methyl]-2-propionate (3f) Yield:
90 %; Reddish solid; m.p. 154–156 °C; IR (KBr) m: 2949
(Ar, C–H str.), 1745 (ester, CO str.), 1717 (CO, str.), 1608
1
(amide, CO str.), 1467, 1269 cm-1; H NMR (300 MHz,
CDCl3): d 3.77 (s, 3H, CH3), 4.73 (s, 2H, CH2), 6.65 (d,
1H, J = 8.3 Hz, ArH), 7.24 (d, 2H, J = 8.3 Hz, ArH), 7.
41–7.44 (dd, 1H, J = 8.3, 2.3 Hz, ArH) 7.48 (d, 1H, J = 2.
3 Hz, ArH), 7.51 (d, 2H, J = 8.3 Hz, ArH), 7.86 (s, 1H,
olefin); 13C NMR (75 MHz, CDCl3): d 37.6, 52.4, 112.3,
118.3, 123.6, 124.8, 125.9, 129.3, 130.3, 131.8, 132.6, 137.
3, 143.0, 148.8, 157.4, 166.3, 181.6; MS (m/z): 434
[M?H]?; HRMS (ESI) m/z: C19H14NO4ClBr [M?H]?
calculated: 433.9794, found: 433.9779.
Methyl(E)-3-(4-nitrophenyl)-2-[(5-chloro-2,3-dioxo-2,3-
dihydro-1H-1-indolyl)methyl]-2-propionate (3j) Yield:
92 %; Reddish solid; m.p. 164–166 °C; IR (KBr) m: 2924
(Ar, C–H str.), 1744 (ester, CO str.), 1715 (CO, str.), 1604
1
(amide, CO str.), 1514, 1475, 1344, 1262 cm-1; H NMR
(300 MHz, CDCl3): d 3.81 (s, 3H, CH3), 4.66 (s, 2H,
CH2), 6.86 (d, 1H, J = 8.3 Hz, ArH), 7.47–7.51(m, 2H),
7.59 (d, 2H, J = 8.1 Hz, ArH),7.95 (s, 1H, olefin), 8.25
(d, 2H, J = 8.1 Hz, ArH); 13C NMR (50 MHz, DMSO-
d6): d 35.9, 50.4, 111.2, 116.7, 121.5, 125.7, 127.1, 128.5,
130.3, 135.1, 138.4, 139.1, 145.3, 147.3, 155.9, 163.9,
179.8; MS (m/z): 401 [M?H]?; HRMS (ESI) m/z:
C19H14N2O6Cl [M?H]? calculated: 401.0540, found: 401.
0544.
Methyl(E)-3-(4-bromophenyl)-2-[(5-fluoro-2,3-dioxo-2,3-
dihydro-1H-1-indolyl)methyl]-2-propionate (3g) Yield:
93 %; Reddish solid; m.p. 89–91 °C; IR (KBr) m: 2925 (Ar,
C–H str.), 1740 (ester, CO str.), 1713 (CO, str.), 1622
(amide, CO str.), 1484, 1263 cm-1
;
1H NMR
Methyl(E)-3-(4-nitrophenyl)-2-[(5-fluoro-2,3-dioxo-2,3-
(300 MHz,CDCl3): d 3.78 (s, 3H, CH3), 4.74 (s, 2H, CH2),
6.65 (dd, 1H, J = 8.5, 3.6 Hz, ArH), 7.15–7.25 (m, 4H,
ArH), 7.50 (d, 2H, J = 8.5 Hz, ArH) 7.86 (s, 1H, olefin);
13C NMR (75 MHz, CDCl3): d 37.6, 52.5, 111.8, 112.1,
118.1, 123.6, 124.2, 124.5, 126.0, 130.4, 131.9, 132.7, 142.
9, 146.6, 157.4, 157.7, 160.6, 166.5, 182.0; MS (m/z): 418
dihydro-1H-1-indolyl)methyl]-2-propionate
(3k) Yield:
82 %; Reddish solid; m.p. 169–171 °C; IR (KBr) m: 2924
(Ar, C–H str.), 1739 (ester, CO str.), 1713 (CO, str.), 1616
1
(amide, CO str.), 1514, 1484, 1342, 1265 cm-1; H NMR
(300 MHz, CDCl3): d 3.81 (s, 3H, CH3), 4.72 (s, 2H, CH2),
123

Med Chem Res (2014) 23:1934–1940
1939
Cytotoxicity assay procedure
6.81 (dd, 1H, J = 8.3, 3.4 Hz, ArH), 7.23-7.29 (m, 2H,
ArH), 7.56 (d, 2H, J = 8.5 Hz, ArH), 7.99 (s, 1H, olefin),
8.24 (d, 2H, J = 8.5 Hz, ArH); 13C NMR (75 MHz,
CDCl3): d 37.9, 52.7, 112.2, 112.4, 118.1, 123.3, 123.7,
124.3, 124.6, 128.3, 129.6, 140.5, 141.5, 146.5, 147.7, 157.
9, 160.7, 166.0, 181.8; MS (m/z): 407 [M?Na]?; HRMS
(ESI) m/z: C19H13N2O6FNa [M?Na]?calculated: 407.
0655, found: 407.0661.
The potential effects on cell viability were investigated by
using the MTT assay (Sigma, USA) [3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide] as an indicator of
metabolically active cells (Van de Loosdrecht et al., 1994;
Alley et al., 1988). The four different cell lines used are
CHO, Colo 205, Sup-T1 and C6 glioma were seeded into
96-well plates in a volume of 200 mL of culture medium and
incubated overnight at 37 °C in a CO2 incubator before
addition of test compound. Cells were then exposed to
known concentrations of the compound to be tested (1 mM,
100 lM, 100 nM and 100 pM expressed as final concen-
tration) for 16 h at 37 °C in a CO2 incubator with 5 % CO2.
After drug exposure, the culture medium was removed and
20 lL of MTT reagent (diluted in culture medium, 1 mg/mL)
was added. After incubating for 5 h in a humidified atmo-
sphere, the MTT/medium was removed and DMSO (200 lL)
was added to dissolve the formazan crystals. Absorbance of
the coloured solution was measured by an ELISA using a NJ-
2300 microplate spectrophotometer with a test wavelength
of 570 nm. Results were evaluated by comparing the
absorbance of the wells containing compound-treated cells
with the absorbance of wells containing 0.1 % DMSO alone
(solvent control). Conventionally, cell viability was esti-
mated to be 100 % in the solvent control. All assays were
performed in triplicate and mean standard deviation values
were used to estimate cell viability.
Methyl(E)-3-(4-nitrophenyl)-2-[(5-bromo-2,3-dioxo-2,3-
dihydro-1H-1-indolyl)methyl]-2-propionate (3l) Yield:
85 %; Reddish solid; m.p. 159–161 °C; IR (KBr) m: 2955
(Ar, C–H str.), 1742 (ester, CO str.), 1713 (CO, str.), 1599
1
(amide, CO str.), 1514, 1471, 1343, 1260 cm-1; H NMR
(300 MHz, CDCl3): d 3.81 (s, 3H, CH3), 4.67 (s, 2H, CH2),
6.85 (dd, 1H, J = 9.4, 3.8 Hz, ArH), 7.22-7.28 (m, 2H,
ArH), 7.58 (d, 2H, J = 8.5 Hz, ArH), 7.94 (s, 1H, olefin),
8.24 (d, 2H, J = 8.5 Hz, ArH); 13C NMR (75 MHz,
CDCl3): d 37.8, 52.7, 112.8, 116.6, 123.3, 123.7, 127.8,
129.1, 129.6, 136.6, 140.5, 141.7, 147.7, 149.2, 157.4, 166.
0, 181.2; MS (m/z): 445 [M?H]?; HRMS (ESI) m/z:
C19H14N2O6Br [M?H]? calculated: 445.0035, found: 445.
0046.
Antitubercular studies procedure
Twelve compounds (3a–l) were tested in vitro against M.
tuberculosis H37RV strain ATCC 27294 which is suscep-
tible to control drugs (Rifampicin, Isoniazid, Ethambutol
and Pyrazinamide). MIC was determined using agar dilu-
tion method in Middlebrook 7H11 medium with oleic
acid–albumin–dextrose (OADC) growth supplement. The
compounds and control drugs were dissolved in DMSO and
diluted twofold to obtain ten serial dilutions of each com-
pound. Appropriate volumes of compounds were incorpo-
rated into duplicate plates of Middlebrook 7H11 agar
medium supplemented with 10 % Middlebrook supplement
OADC. Inoculums of M. tuberculosis H37Rv (Mycobacte-
rium strains) were grown in Middlebrook 7H11 agar slants
with OADC growth supplement adjusted to 1 mg/mL (wet
weight) in Tween 80 (0.05 %) saline diluted to 10-2 to
give a concentration of approximately 107 cfu/mL. A 5 lL
amount of bacterial suspension was spotted into 7H11 agar
tubes containing 10-fold serial dilutions of compounds and
control drugs per mL. MIC values were determined after
incubation at 37 °C and final readings were recorded after
28 days. The MIC (in lg/mL) was recorded as the lowest
concentration/highest dilution of the compounds/control
drugs that completely inhibited the growth of Mycobacte-
rium cultures. This method is similar to that recommended
by the National Committee for Clinical Laboratory Stan-
dards (NCCLS) for the determination of MIC in duplicate.
Conclusion
In conclusion, a series of new Baylis–Hillman adduct-derived
N-cinnamyl-substituted isatin derivatives was synthesized in a
simple and efficient manner. All the synthesized compounds
were evaluated for their antitubercular and anticancer activi-
ties. These compounds have inhibited Mycobacterium strains,
especially compounds 3j–l have shown overall good activity
onM. tuberculosisH37Rvstrain ATCC27294. Thecompound
3l showed a specific cytotoxicity on cancerous cell lines Colo
205, Sup-T1 and C6-glioma, whereas it is non-cytotoxic to
non-cancerous cell line (CHO).
Acknowledgments The authors thank Director, IICT for encour-
agement of this Main Lab Project work. We are grateful to Prof.
Anand Kumar Kondapi, Department of Biochemistry, University of
Hyderabad for helping in bio-evaluation of anticancer activity. SBK
and MR thank CSIR, New Delhi for fellowships.
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