Synthesis, antimicrobial and cytotoxic evaluation of spirooxindole[pyrano-bis-2H-l-benzopyrans]

Article abstract of DOI:10.1007/s00044-016-1645-4

Microwave-assisted tandem double condensation between isatins and 4-hydroxycoumarin under zinc triflate catalysis to afford spirooxindoles in excellent yield is reported. The synthesized compounds were screened for their in vitro antibacterial and antifungal activities. The compounds that showed promising antimicrobial activity (3a, 3j and 3m) were studied for their binding affinity towards AmpC-β-lactamase receptor, which revealed that compound 3a is highly stabilized by strong hydrogen bond interactions with in the binding pocket. The synthesized spirooxindoles were also evaluated for their cytotoxic potential against COLO320 adenocarcinoma colorectal cancer cells. Biological assay and in silico studies indicated compound 3n as the most active in terms of low IC₅₀ value (50.7 μM) and least free energy of binding (?8.89 kcal/mol) respectively.

Full text of DOI:10.1007/s00044-016-1645-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1645-4
ORIGINAL RESEARCH
Synthesis, antimicrobial and cytotoxic evaluation of spirooxindole
[pyrano-bis-2H-l-benzopyrans]
3
K. Parthasarathy1,2 Chandrasekar Praveen
K. Saranraj4 C. Balachandran5
●
●
●
●
P. Senthil Kumar6
Received: 30 June 2015 / Accepted: 4 July 2016
© Springer Science+Business Media New York 2016
Abstract Microwave-assisted tandem double condensation
between isatins and 4-hydroxycoumarin under zinc triflate
catalysis to afford spirooxindoles in excellent yield is
reported. The synthesized compounds were screened for
their in vitro antibacterial and antifungal activities. The
compounds that showed promising antimicrobial activity
(3a, 3j and 3m) were studied for their binding affinity
towards AmpC-β-lactamase receptor, which revealed that
compound 3a is highly stabilized by strong hydrogen bond
interactions with in the binding pocket. The synthesized
spirooxindoles were also evaluated for their cytotoxic
potential against COLO320 adenocarcinoma colorectal
cancer cells. Biological assay and in silico studies indicated
compound 3n as the most active in terms of low IC50 value
(50.7 μM) and least free energy of binding (−8.89 kcal/mol)
respectively.
●
●
●
Keywords Spirooxindoles Antibacterial Antifungal
●
Cytotoxicity Molecular docking
Introduction
Spirooxindoles have received paramount importance in
medicinal chemistry, because of their prevalence as sub-
structure in numerous bioactive agents and natural products
(Williams and Cox, 2003; Tang et al., 2001; Tsuda et al.,
2003; Cui et al., 1996; Wang et al., 2005). On the other
hand, 4-hydroxycoumarin is a structural component present
in various pharmaceuticals and clinical drugs like warfarin,
phenprocoumon, acenocoumarol, tioclomarol and brodifa-
coum (Fig. 1; Bo et al., 2010). Based on their unique
pharmacological properties, we speculated that combining
the structural characteristics of both spirooxindole and
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1645-4) contains supplementary material,
which is available to authorized users.
* Chandrasekar Praveen
chandrasekar.praveen@gmail.com
1
Department of Chemistry, Siddha Central Research Institute,
Central Council for Research in Siddha, Arumbakkam, Chennai
600106, Tamil Nadu, India
2
Analytical Research & Development, Orchid Chemicals &
Pharmaceuticals Ltd., Research & Development Centre,
Shozanganallur, Chennai 600119, Tamil Nadu, India
3
Functional Materials Division, CSIR-Central Electrochemical
Research Institute, Karaikudi 630003, Tamil Nadu, India
4
Organic Chemistry Division, CSIR-Central Leather Research
Institute, Adyar, Chennai 600020, Tamil Nadu, India
5
Division of Microbiology, Entomology Research Institute, Loyola
College, Chennai 600034, Tamil Nadu, India
6
Department of Bioinformatics, Orchid Chemicals &
Pharmaceuticals Ltd., Research & Development Centre,
Shozanganallur, Chennai 600119, India
Fig. 1 Pharmaceuticals containing 4-hydroxycoumarin scaffold

Med Chem Res
4-hydroxycoumarin moieties by a hybrid pharmacophore
approach could substantially enhance the biological activity
of the resulting compounds. Consequently, these structu-
rally interesting heterocycles could be utilized in the
synthesis of improved chemical entities and new drug dis-
covery research. However, the synthesis of these types of
compounds has been scarce and a great deal of interest
exists in view of their structural complexity (Bo et al., 2010;
Jain et al., 1996). To the best of our knowledge, only lim-
ited reports were documented for this type of transforma-
tions though with narrow substrate scope. For example, a
substituent at the isatinyl nitrogen was not reported in the
previously reported p-TSA and I2 catalysed protocols
(Almansour et al., 2012a–c). In view of the aforementioned
facts, we intended a simple and straightforward synthesis of
hybrid-spirooxindoles via tandem Knoevenagel/Michael
3f (Chowdhury et al., 2013), 3g (Shi et al., 2012), 3h
(Sharma et al., 2014), 3i (Shakoori et al., 2013), 3j (Rassu
et al., 2012), 3k (Vintonyak et al., 2011), 3o (Wee et al.,
2012) and 3p (Rahmati and Khalesi, 2012) were prepared
by several closely related N-alkylation procedures. The
bacterial strains (Shigella flexneri MTCC 1457, Micro-
coccus luteus MTCC 106, Enterobacter aerogenes MTCC
111, Staphylococcus aureus MTCC 96, Staphylococcus
epidermidis MTCC 3615, Klebsiella pneumoniae MTCC
109, Salmonella typhimurium MTCC 1251, Bacillus sub-
tilis MTCC 441, Staphylococcus aureus (MRSA-methicillin
resistant)) were obtained from the Institute of Microbial
Technology (IMTECH), Chandigarh, India-160 036. Fun-
gal strains (Malassesia pachydermatis and Candida albi-
cans MTCC 227) were obtained from the Department of
Microbiology, Christian Medical College, Vellore, Tamil
Nadu, India. Mueller Hinton agar (MHA) was purchased
from Hi-media Pvt. Ltd, Mumbai, India. COLO320 ade-
nocarcinoma colorectal cancer cell line was obtained from
reaction
between
substituted
isatins
and
4-
hydroxycoumarin. Towards this end, we herein report the
microwave-assisted synthesis of spirooxindole[pyrano-bis-
2H-l-benzopyrans], their antimicrobial and cytotoxic prop-
erties. In addition, molecular docking of the compounds
with appropriate receptors was performed and all these
results were disclosed in this article.
National Institute of Cell Sciences, Pune. Absorbance (λmax
)
was red at 570 nm in an ELISA reader. Cytotoxicity of the
compounds were statistically analysed by Duncan multiple
range test at P = 0.05 with the help of SPSS 11.5 version
software package. All computations for molecular docking
studies were carried out on a Dell Desktop D510 personal
computer (2.4 GHz Pentium 4 processor, Intel, Santa Clara,
CA) running Red Hat Enterprise Linux Client release 5.5.
The time required for each simulation run was on the order
of Real = 1 h 13 min 54.67 s, CPU = 1 h 08 min 54.57 s,
System = 11.75 s on the Pentium machine.
Materials and methods
All commercially available solvents and reagents were used
without further purification. Melting points were determined
in capillary tubes and are uncorrected. Microwave reactions
were performed in CEM-Discover microwave reactor.
Infrared (IR) spectra were recorded on a Perkin-Elmer FTIR
spectrophotometer as KBr pellets. 1H and 13C NMR spectra
were obtained in CDCl3, Acetone-d6, dimethylsulphoxide
(DMSO)-d6 on a Bruker spectrometer at 400 and 100 MHz,
respectively. Proton chemical shifts (δ) are relative to tet-
ramethylsilane (TMS, δ = 0.00) as internal standard and
expressed in parts per million. Spin multiplicities are given
as s (singlet), d (doublet), t (triplet) and m (multiplet).
Coupling constants (J) are given in hertz. Mass spectra were
recorded on a PE-SCIEX API 300 mass spectrometer.
HRMS data were collected on a maxis 10138 mass spec-
trometer. Elemental analyses were recorded using a Thermo
Finnigan FLASH EA 1112CHN analyzer. All the com-
pounds gave C, H and N analysis within 0.5 % of the
theoretical values. Analytical thin-layer chromatography
(TLC) was performed on precoated plastic sheets of silica
gel G/UV-254 of 0.2 mm thickness (Macherey-Nagel,
Duren, Germany) using analytical grade solvents and
visualized with iodine spray (10 % (w/w) I2 in silica gel) or
UV light (λ = 254 and 365 nm). Synthetically good yields of
N-substituted isatins 3b (Shmidt et al., 2012), 3c (Chen
et al., 2010), 3d (Diaz et al., 2008), 3e (Bacchi et al., 2005),
General procedure for the synthesis of compounds 3a-3p
To a pyrex reaction vessel containing a solution of isatin 1
(1.0 mmoL) and Zn(OTf)2 (5 mol%) in DMSO (1 mL) was
added 4-hydroxycoumarin 2 (2.0 mmoL). The resulting
mixture was stirred at 100 °C for 30 min at a power level of
200 W. After completion of the reaction as evidenced by
TLC, the mixture was cooled to room temperature and
poured into ice-cold water (10 mL). The solid obtained was
collected by filtration and washed with copious amount of
cold water (100 mL) and dried under vacuum at 40 °C for
6 h to afford 400 mg of analytically pure product 3a.
1,2-Dihydro-2′,10′,16′-trioxaspiro[indole-3,13′-pentacyclo
[12.8.0.03,12.04,9.017,22]-docosan]-1′(14′),3′(12′),4′
(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-trione (3a)
Off-white powder (MeOH); this compound was prepared
from isatin 1a (147 mg, 1.0 mmoL) and 4-hydroxycoumarin
2 (324 mg, 2.0 mmoL) according to the general procedure to
obtain an off-white powder (400 mg, 92 % yield). mp
178–180 °C (MeOH); FTIR (KBr) νmax: 3411, 1732, 1707,

Med Chem Res
1662, 1608, 1347, 1101, 751 cm−1; H NMR (400 MHz,
DMSO-d6) δH: 6.82–6.86 (m, 2H, Ar-H), 7.19 (d, J = 7.6
Hz,1H, Ar-H), 7.23 (d, J = 7.7 Hz, 1H, Ar-H), 7.49 (d,
J = 8.2 Hz, 2H, Ar-H), 7.56 (t, J = 7.6 Hz, 2H, Ar-H), 7.79
(t, J = 7.6 Hz, 2H, Ar-H), 8.46 (t, J = 7.7 Hz, 2H, Ar-H),
10.85 (brs, 1H, D2O exchangeable, -NH); 13C NMR (100
MHz, DMSO-d6) δC: 46.5 (C, C-3), 10.33 (C, C-10 and
C-10′), 108.9 (CH, C-5), 112.4 (C, C-17 and C-20), 116.5
(CH, C-13 and C-13′), 121.5 (CH, C-6), 123.6 (CH, C-16
and C-16′), 124.3 (CH, C-7), 125.1 (CH, C-15 and C-15′),
1291 (CH, C4), 131.9 (C, C-9), 133.9 (CH, C14 and C-14′),
144.1 (C, C-8), 151.8 (C, C-12 and C-12′), 154.4 (C, C-18
and C-18′), 157.9 (C=O, C-11 and C-11′), 176.4 (C=O,
C-2); HRMS(ESI): m/z(obs.) = 458.0641 [M+Na]+; Anal.
Calcd for C26H13NO6: C, 71.72; H, 3.01; N, 3.22. Found C,
71.65; H, 2.98; N, 3.20.
NMR (400 MHz, CDCl3) δH: 1.43 (t, J = 7.2 Hz, 3H, -CH3),
3.94 (q, J = 7.2 Hz, 2H, -CH2−), 6.91–6.93 (m, 2H, Ar-H),
6.95–7.01 (d, J = 7.1 Hz, 1H, Ar-H), 7.30–7.34 (m, 3H, Ar-
H), 7.46 (t, J = 7.6 Hz, 2H, Ar-H), 7.66 (t, J = 7.6 Hz, 2H,
Ar-H), 8.11 (d, J = 7.9 Hz, 2H, Ar-H); 13C NMR (100 MHz,
CDCl3) δC: 11.9 (-CH3), 35.8 (-CH2-Me), 46.7 (C, C-3),
104.4 (C, C-10 and C-10′), 108.5 (CH, C-5), 113.1 (C, C-17
and C-20), 117.2 (CH, C-13 and C-13′), 122.4 (CH, C-6),
122.8 (CH, C-16 and C-16′), 123.6 (CH, C-7), 124.9 (CH,
C-4), 129.9 (C, C-9), 131.4 (CH, C-14 and C-14′), 133.6
(C, C-9′), 145.2 (C, C-8), 152.8 (C, C-12 and C-12′), 154.8
(C, C-18), 158.4 (C=O, C-11 and C-11′), 175.1 (C=O, C2);
MS(ESI): m/z = 464 [M+H]+; Anal. Calcd for C26H13NO6:
C, 72.57; H, 3.70; N, 3.02. Found C, 72.67; H, 3.76; N,
3.12.
1
1-Propyl-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-3,13′-
pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′(14′),3′
(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-trione
(3d)
1-Methyl-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-3,13′-
pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′(14′),3′
(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-trione
(3b)
Red powder (MeOH); this compound was prepared from 1-
propyl isatin 1d (189 mg, 1.0 mmoL) and 4-
hydroxycoumarin 2 (324 mg, 2.0 mmoL) according to the
general procedure to obtain a red powder (448 mg, 94 %
yield). mp 179–181 °C (MeOH); FTIR (KBr)νmax: 2950,
2927, 2853, 1738, 1717, 1666, 1609, 1344, 1102, 751 cm−1;
1H NMR (400 MHz, CDCl3) δH: 0.90 (t, J = 6.6 Hz, 3H,
-CH3), 1.61–1.65 (m, J = 7.1 Hz, 2H, -CH2-CH3), 4.17 (t, J
= 7.3 Hz, 2H, -NCH2-), 6.91–6.94 (m, 1H, Ar-H),
7.29–7.35 (m, 3H, Ar-H), 7.44–7.56 (m, 4H, Ar-H), 7.67 (t,
J = 7.5 Hz, 2H, Ar-H), 8.12 (d, J = 7.9 Hz, 2H, Ar-H); 13C
NMR (100 MHz, CDCl3) δC: 11.3 (-CH3), 21.8 (-CH2-Me),
42.4 (-N-CH2-), 47.2 (C, C-3), 102.4 (C, C-10 and C-10′),
107.6 (CH, C-5), 112.7, (C, C-17 and C-20), 117.6 (CH, C-
13 and C-13′), 122.6 (CH, C-6), 122.9(CH, C-16 and C-
16′), 123.8 (CH, C-7), 124.5 (CH, C-4), 129.9 (C, C-9),
131.8 (C, C-9′), 133.7 (C, C-8), 145.9 (C, C-14 and C-14′),
152.4 (C, C-18), 154.3 (C, C-14 and C-14′), 158.1 (C=O,
C-11), 175.3 (C=O, C2); MS(ESI): m/z = 478 [M+H]+;
Anal. Calcd for C29H19NO6: C, 72.95; H, 4.01; N, 2.93.
Found C, 72.90; H, 3.97; N, 2.99.
Off-white powder (MeOH); this compound was prepared
from 1-methyl isatin 1b (161 mg, 1.0 mmoL) and 4-
hydroxycoumarin 2 (324 mg, 2.0 mmoL) according to the
general procedure to obtain an off-white powder (427 mg,
95 %). mp 186–188 °C (MeOH); FTIR (KBr) νmax: 2986,
1
1718, 1662, 1610, 1344, 1097, 754 cm−1; H NMR (400
MHz, DMSO-d6) δH: 3.43 (s, 3H, -NCH3), 6.94–6.96 (m,
2H, Ar-H), 6.99–7.01 (m 1H, Ar-H), 7.31 (t, J = 7.6 Hz,
1H, Ar-H), 7.35 (d, J = 8.3 Hz, 2H, Ar-H), 7.45 (t, J = 7.4
Hz, 2H, Ar-H), 7.65 (t, J = 7.6 Hz, 2H, Ar-H), 8.09 (t, J =
7.7 Hz, 2H, Ar-H); 13C NMR (100 MHz, DMSO-d6) δC:
27.1 (CH3), 51.4 (C, C-3), 104.2 (C, C-10 and C-10′), 108.3
(CH, C-5), 112.9 (C, C-17 and C-20), 117.1 (CH, C-13 and
C-13′), 122.5 (CH, C-6), 123.2 (CH, C-16 and C-16′),
124.8 (CH, C-7), 129.8 (CH, C-4), 130.0 (C, C-9), 133.4
(C, C-9′), 145.8 (C, C-8), 152.7 (C, C-12 and C-12′), 154.7
(C, C-18), 158.3 (C=O, C-11), 175.6 (C=O, C2); MS(ESI):
m/z = 450 [M+H]+; Anal. Calcd for C26H13NO6: C, 72.16;
H, 3.36; N, 3.12. Found C, 72.25; H, 3.28; N, 3.10.
1-Ethyl-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-3,13′-
pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′(14′),3′
(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-trione
(3c)
1-Butyl-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-3,13′-
pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′(14′),3′
(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-trione
(3e)
Off-white powder (MeOH); this compound was prepared
from 1-ethyl isatin 1c (175 mg, 1.0 mmoL) and 4-
hydroxycoumarin 2 (324 mg, 2.0 mmoL) according to the
general procedure to obtain an off-white powder (440 mg,
95 % yield). mp 176–178 °C; FTIR (KBr)νmax: 2983, 2931,
Off-white powder (MeOH); this compound was prepared
from 1-butyl isatin 1e (203 mg, 1.0 mmoL) and 4-
hydroxycoumarin 2 (324 mg, 2.0 mmoL) according to the
general procedure to obtain an off-white powder (466 mg,
1
2868, 1737, 1714, 1663, 1609, 1345, 1100, 757 cm−1; H
95 % yield). mp 185–187 °C (MeOH); FTIR (KBr) νmax:

Med Chem Res
2950, 2923, 2852, 1738, 1712, 1664, 1611, 1341, 1102,
752 cm−1; 1H NMR (400 MHz, CDCl3) δH: 0.90 (t, J = 6.8
Hz, 3H, -CH3), 1.37–1.39 (m, 2H, -CH2-CH3), 1.41–1.44
(m, 2H, -CH2-CH2CH3), 3.99 (t, J = 7.4 Hz, 2H, -NCH2-),
6.91–6.94 (m, 1H, Ar-H), 7.31–7.35 (m, 3H, Ar-H), 7.46 (t,
J = 7.5 Hz, 2H, Ar-H), 7.53–7.57 (m, 2H, Ar-H), 7.66 (t, J
= 7.5 Hz, 2H, Ar-H), 8.10 (d, J = 7.8 Hz, 2H, Ar-H); 13C
NMR (100 MHz, CDCl3) δC: 13.9 (-CH3), 21.8 (-CH2Me),
28.0 (-CH2Pr), 39.8 (-NCH2-), 45.7 (C, C-3), 103.9 (C, C-
10 and C-10′), 108.9 (CH, C-5), 113.1 (C, C-17 and C-20),
117.2 (CH, C-13 and C-13′), 122.1 (CH, C-6), 122.9 (CH,
C-16 and C-16′), 123.8 (CH, C-7), 124.6 (CH, C-4), 129.8
(C, C-9), 131.3 (C, C-9′), 133.6 (C, C-8), 145.6 (C, C-14
and C-14′), 152.2 (C, C-12 and C-12′), 154.5 (C, C-18),
158.3 (C=O, C-11), 175.2 (C=O, C2); MS(ESI): m/z = 492
[M+H]+; Anal. Calcd for C30H21NO6: C, 73.31; H, 4.31; N,
2.85. Found C, 73.25; H, 4.28; N, 2.93.
general procedure to obtain an off-white powder (451 mg,
87 % yield). mp 193–195 °C (MeOH); FTIR (KBr) νmax
:
2952, 2925, 2856, 1739, 1716, 1665, 1609, 1345, 1103,
754; cm−1. 1H NMR (400 MHz, CDCl3) δH: 0.91 (t, J = 6.6
Hz, 3H, -CH3), 1.37–1.38 (m, 4H, -CH2-CH2-CH3),
1.47–1.84 (m, 4H, -NCH2-CH2-CH2-), 3.84 (t, J = 7.8 Hz,
2H, -NCH2-), 6.91–6.96 (m, 2H, Ar-H), 6.98 (d, J = 7.1 Hz,
1H, Ar-H), 7.29–7.44 (m, 3H, Ar-H), 7.46 (t, J = 7.5 Hz,
2H, Ar-H), 7.66 (t, J = 7.5 Hz, 2H, Ar-H), 8.10 (d, J = 7.8
Hz, 2H, Ar-H); 13C NMR (100 MHz, CDCl3) δC 14.3
(-CH3), 22.8 (-CH2Me), 27.0 (-CH2Et), 27.1 (-CH2Pr), 31.8
(-CH2Bu), 41.4 (-NCH2-), 46.7 (C, C-3), 104.4 (C, C-10
and C-10′), 108.6 (CH, C-5), 113.0 (C, C-17 and C-20),
117.1 (CH, C-13 and C-13′), 122.4 (CH, C-6), 122.8, (CH,
C-16 and C-16′), 123.5 (CH, C-7), 124.9 (CH, C-4), 129.8
(C, C-9), 131.4 (C, C-9′), 133.5 (C, C-8), 145.7 (C, C-14
and C-14′), 152.7 (C, C-12 and C-12′), 154.8 (C, C-18),
158.3 (C=O, C-11), 175.4 (C=O, C2); MS(ESI): m/z = 520
[M+H] +, Anal. Calcd for C32H25NO6: C, 73.98; H, 4.85; N,
2.70. Found C, 73.93; H, 4.80; N, 2.66.
1-Pentyl-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-3,13′-
pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′(14′),3′
(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-trione (3f)
1-(Prop-2-en-yl)-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-
3,13′- pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′
(14′),3′(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-
trione (3h)
Off-white powder; this compound was prepared from 1-
pentyl isatin 1f (217 mg, 1.0 mmoL) and 4-hydroxycoumarin
2 (324 mg, 2.0 mmoL) according to the general procedure to
obtain an off-white powder (460 mg, 91 % yield). mp
190–192 °C (MeOH); FTIR (KBr) νmax: 2959, 2905, 2856,
Off-white solid (MeOH); this compound was prepared from
1-allyl isatin 1h (187 mg, 1.0 mmoL) and 4-
hydroxycoumarin 2 (324 mg, 2.0 mmoL) according to the
general procedure to obtain an off-white powder (422 mg,
1
1741, 1710, 1664, 1604, 1349, 1100, 754 cm−1; H NMR
(400 MHz, CDCl3) δΗː 0.91 (t, J = 6.5 Hz, 3H, -CH3),
1.37–1.43 (m, 4H, -CH2-CH2-CH3), 1.47 (q, J = 7.4 Hz, 2H,
-N-CH2-), 4.08 (t, J = 7.8 Hz, 2H, Ar-H), 6.90–6.63 (m 2H,
Ar-H), 6.99 (d, J = 7.3 Hz, 1H, Ar-H), 7.30–7.35 (m 3H, Ar-
H), 7.47 (t, J = 7.6 Hz, 2H, Ar-H), 7.69 (t, J = 7.8 Hz, 2H,
Ar-H), 8.11 (d, J = 7.8 Hz, 2H, Ar-H); 13C NMR (100 MHz,
CDCl3) δC: 14.2 (-CH3), 22.9 (-CH2Me), 27.6 (-CH2Et),
29.1, (-CH2Pr), 41.5 (-NCH2-), 45.7 (C, C-3), 104.0 (C, C-10
and C-10′), 108.9 (CH, C-5), 113.1 (C, C-17 and C-20),
117.1 (CH, C-13 and C-13′), 122.6 (CH, C-6), 122.9 (CH,
C-16 and C-16′), 123.3, (CH, C-7), 124.9 (CH, C-4), 129.7
(C, C-9), 131.6 (C, C-9′), 133.7 (C, C-8), 145.1 (C, C-14 and
C-14′), 152.6 (C, C-12 and C-12′), 154.7 (C, C-18), 158.5
(C=O, C-11), 175.0 (C=O, C2); MS(ESI): m/z = 506 [M
+H]+; Anal. Calcd for C31H23NO6: C, 73.65; H, 4.49; N,
2.77. Found C, 73.59; H, 4.48; N, 2.83.
89 % yield). mp 167–169 °C (MeOH); FTIR (KBr) νmax
:
3065, 1735, 1714, 1664, 1609, 1345,1104, 754 cm−1.1H
NMR (400 MHz, CDCl3) δH: δ 4.54 (d, J = 4.9 Hz, 1H,
=CH2-) 5.31 (d, J = 10.4 Hz, 1H, =CH2-), 5.61 (d, J = 17.2
Hz, 1H, -CH=CH2), 6.02–6.08 (m, 1H, Ar-H), 6.94 (d, J =
7.4 Hz, 2H, -NCH2-), 7.00 (d, J = 7.5 Hz, 1H, Ar-H), 7.29
(t, J = 7.7 Hz, 1H, Ar-H), 7.35 (d, J = 8.3 Hz, 2H, Ar-H),
7.46 (d, J = 7.6 Hz, 2H, Ar-H), 7.65 (d, J = 7.7 Hz, 2H, Ar-
H), 8.11 (d, J = 7.9 Hz, 2H, Ar-H); 13C NMR (100 MHz,
CDCl3) δC: 43.7 (-NCH2-), 46.5 (C, C-3), 104.2 (C, C-10
and C-10′), 109.3 (CH, C-5), 112.9 (C, C-17 and C-20),
117.1 (CH, C-13 and C-13′), 117.8 (allyl=CH2), 122.4
(CH, C-6), 122.6 (CH, C-16 and C-16′), 123.2 (CH, C-7),
124.8 (CH, C-4), 129.6 (C, C-9), 130.9 (C, C-9′), 131.6
(allyl-CH=), 133.4 (C, C-8), 145.2 (C, C-14 and C-14′),
1-Hexyl-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-3,13′-
pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′(14′),3′
(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-trione
(3g)
152.7 (C, C-12 and C-12′), 154.6 (C, C-18), 158.2 (C=O,
+
C-11), 175.2 (C=O, C2); MS(ESI): m/z = 476 [M+H]
,
Anal. Calcd for C29H17NO6: C, 73.26; H, 3.60; N, 2.95.
Found C, 73.31; H, 3.55; N, 2.99.
Off-white solid (MeOH); this compound was prepared
from 1-hexyl isatin 1g (231 mg, 1.0 mmoL) and
4-hydroxycoumarin 2 (324 mg, 2.0 mmoL) according to the
1-(Prop-2-yn-yl)-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-
3,13′- pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′

Med Chem Res
(14′),3′(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-
trione (3i)
1-[(2-Fluorophenyl)methyl]-1,2-dihydro-2′,10′,16′-
trioxaspiro[indole-3,13′- pentacyclo
[12.8.0.03,12.04,9.017,22]docosan]-1′(14′),3′(12′),4′
(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-trione (3k)
Off-white solid; this compound was prepared from 1-
propargyl isatin 1i (185 mg, 1.0 mmoL) and 4-
hydroxycoumarin 2 (324 mg, 2.0 mmoL) according to the
general procedure to obtain an off-white powder (445 mg,
Pink solid (MeOH); this compound was prepared from 1-(2-
fluorobenzyl)isatin 1k (255 mg, 1.0 mmoL) and 4-
hydroxycoumarin 2 (324 mg, 2.0 mmoL) according to the
general procedure to obtain a pink solid (500 mg, 92 %
yield). mp 168–170 °C; FTIR (KBr) νmax: 1726, 1654,
91 % yield). mp 177–179 °C (MeOH); FTIR (KBr) νmax
:
3240, 2123, 1734, 1722, 1665, 1609, 1345, 1104, 754 cm−1.
1H NMR (400 MHz, CDCl3) δH: 2.37 (s, 1H, acetylenic-
CH), 4.72 (s, 2H, -NCH2-), 6.97–7.03 (m, 2H, Ar-H), 7.17
(d, J = 7.8 Hz, 1H, Ar-H), 7.35–7.37 (m, 3H, Ar-H), 7.46 (t,
J = 7.5 Hz, 2H, Ar-H), 7.66 (t, J = 7.5 Hz, 2H, Ar-H), 8.10
(d, J = 7.8 Hz, 2H, Ar-H); 13C NMR (100 MHz, CDCl3) δC:
29.6 (-NCH2-), 46.0 (C, C-3), 74.6 (internal propargyl C),
77.5 (terminal propagyl C), 102.8 (C, C-10 and C-10′),
108.6 (CH, C-5), 112.4, (C, C-17 and C-20), 116.6 (CH, C-
13 and C-13′), 122.5 (CH, C-6), 123.7 (CH, C-16 and C-
16′), 124.3 (CH, C-7), 125.2 (CH, C-4), 129.2 (C, C-9),
130.9 (C, C-9′), 134.0 (C, C-8), 143.8 (C, C-14 and C-14′),
151.9 (C, C-12 and C-12′), 154.6 (C, C-18), 157.9 (C=O,
C-11), 174.2 (C=O, C2); MS(ESI): m/z = 474 [M+H]+,
Anal. Calcd for C29H15NO6: C, 73.57; H, 3.19; N, 2.96.
Found C, 73.51; H, 3.23; N, 3.00.
1
1619, 1355, 1106, 750 cm−1. H NMR (400 MHz, CDCl3)
δH: 5.18 (s, 2H, -NCH2-), 6.88 (d, J = 7.9 Hz, 1H, Ar-H),
7.10–7.13 (m, 1H, Ar-H), 7.18 (d, J = 7.2 Hz, 1H, Ar-H),
7.20 (t, J = 7.6 Hz, 1H, Ar-H), 7.29–7.35 (m, 2H, Ar-H),
7.38–7.41 (m, 2H, Ar-H); 7.44 (t, J = 7.5 Hz, 2H, Ar-H),
7.68 (d, J = 7.6 Hz, 2H, Ar-H), 7.73 (t, J = 7.8 Hz, 2H, Ar-
H), 8.15 (d, J = 7.8 Hz, 2H, Ar-H); 13C NMR (100 MHz,
CDCl3) δC: 43.6 (-NCH2-), 49.8 (C, C-3), 104.5 ((C, C-10
and C-10′), 109.3 (CH, C-5), 112.5(C, C-17 and C-20),
115.8 (CH, C-13 and C-13′), 116.1 (CH, C-6), 116.7 (CH,
C-16 and C-16′), 117.2 (CH, C-7), 122.8 (CH, C-4), 123.4
(JC-F = 35.6 Hz), 123.9 (JC-F = 205.1 Hz), 124.1 (C, C-6,
Bn-2F), 124.9 (C, C-5, Bn-F), 127.4 (C, C-4, Bn-F), 127.5,
127.9, 128.8, 129.8 (C, C-9), 130.5 (C, C-3 Bn-2F), 131.0
(ipso-Bn C), 131.1 (C, C-9′), 133.1 (C, C-9′), 135.96 (2-F-
Bn-C), 145.40 (C, C-8), 152.3 (C, C-14 and C-14′), 154.4
(C, C-12 and C-12′), 158.6 (C, C-18), 161.0 (C=O, C-11),
175.8 (C=O, C2); MS (ESI): m/z = 544 [M+H] +, Anal.
Calcd for C33H18FNO6: C, 72.93; H, 3.34; N, 2.58. Found
C, 72.89; H, 3.40; N, 2.62.
1-Benzyl-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-3,13′-
pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′(14′),3′
(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-trione
(3j)
Off-white solid (MeOH); this compound was prepared from
1-benzyl isatin 1j (237 mg, 1.0 mmoL) and 4-
hydroxycoumarin 2 (324 mg, 2.0 mmoL) according to the
general procedure to obtain an off-white powder (493 mg,
94 % yield). mp 154–156 °C; FTIR (KBr) νmax: 1723, 1664,
5-Chloro-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-3,13′-
pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′(14′),3′
(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-trione
(3l)
1
1611, 1345, 1100, 756 cm−1. H NMR (400 MHz, CDCl3)
δH: 5.15 (s, 2H, -NCH2-), 6.68 (d, J = 7.9 Hz, 1H, Ar-H),
6.90 (t, J = 7.4 Hz, 1H, Ar-H), 7.01 (d, J = 7.2 Hz, 1H, Ar-
H), 7.15 (t, J = 7.6 Hz, 1H, Ar-H), 7.29–7.34 (m, 2H, Ar-
H), 7.37–7.42 (m, 3H, Ar-H); 7.47 (t, J = 7.5 Hz, 2H, Ar-
H), 7.63 (d, J = 7.6 Hz, 2H, Ar-H), 7.67 (t, J = 7.8 Hz, 2H,
Ar-H), 8.12 (d, J = 7.8 Hz, 2H, Ar-H); 13C NMR (100 MHz,
CDCl3) δC: 45.6 (C, C-3), 50.2 (-NCH2Bn), 104.3 (C, C-10
and C-10′), 109.7 (CH, C-5), 112.9 (C, C-17 and C-20),
117.2 (CH, C-13 and C-13′), 122.8 (CH, C-6), 123.4 (CH,
C-16 and C-16′), 123.9 (CH, C-7), 124.9 (CH, C-4), 127.4
(benzyl para C), 127.5 (benzyl meta C), 127.9 (benzyl
ortho C), 128.8 (benzyl ipso C), 129.8 (C, C-9), 130.5,
131.0 (C, C-9′), 133.7 (C, C-8), 135.9, 145.4 (C, C-14 and
C-14′), 152.7, (C, C-12 and C-12′), 154.8 (C, C-18), 158.5
(C=O, C-11), 176.0 (C=O, C2); MS(ESI): m/z = 526 [M
+H]+, Anal. Calcd for C33H19NO6: C, 75.42; H, 3.64; N,
2.67%. Found C, 75.45; H, 3.59; N, 2.64 %.
Off-white solid (MeOH); this compound was prepared from
5-chloroisatin 1l (181 mg, 1.0 mmoL) and 4-
hydroxycoumarin 2 (324 mg, 2.0 mmoL) according to the
general procedure to obtain an off-white powder (416 mg,
89 % yield). mp 164–166 °C; FTIR (KBr) νmax: 3395, 1730,
1
1706, 1663, 1610, 1345, 1108, 761 cm−1. H NMR (400
MHz, CDCl3) δH: 6.85 (d, J = 8.3 Hz, 1H, Ar-H), 7.24 (dd,
J = 8.3 and 2.1 Hz, 1H, Ar-H), 7.46 (d, J = 2.1 Hz, 1H, Ar-
H), 7.49 (d, J = 8.3 Hz, 2H, Ar-H), 7.55 (t, J = 7.6 Hz, 2H,
Ar-H), 7.80 (t, J = 7.6 Hz, 2H, Ar-H), 8.45 (d, J = 7.6 Hz,
2H, Ar-H), 10.99 (brs, D2O exchangeable, 1H, -NH); 13C
NMR (100 MHz, CDCl3) δC: 46.6 (C, C-3), 102.7 (C, C-10
and C-10′), 110.2 (C-Cl, C-5), 112.5 (C, C-17 and C-20),
116.6 (CH, C-13 and C-13′), 123.7 (CH, C-6), 124.7 (CH,
C-16 and C-16′), 125.2 (CH, C-7), 125.5 (CH, C-4), 128.9
(C, C-9), 133.7 (C, C-9′), 134.0 (C, C-8), 143.1 (C, C-14
and C-14′), 151.9 (C, C-12 and C-12′), 154.8 (C, C-18),

Med Chem Res
158.0 (C=O, C-11), 176.3 (C=O, C2); MS(ESI): m/z = 468
[M−H]−, Anal. Calcd for C26H12ClNO6: C, 66.47; H, 2.57;
N, 2.98. Found C, 66.43; H, 2.51; N, 3.03.
C-11), 177.3 (C=O, C2); MS(ESI): m/z = 479 [M−H]−,
Anal. Calcd for C26H12N2O8: C, 65.01; H, 2.52; N, 5.83.
Found C, 65.06; H, 2.50; N, 5.78.
5-Bromo-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-3,13′-
pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′(14′),3′
(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-trione
(3m)
5-Fluoro-1-methyl-1,2-dihydro-2′,10′,16′-trioxaspiro
[indole-3,13′- pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-
1′(14′),3′(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-
2,11′,15′-trione (3o)
Brown solid (MeOH); mp 187–189 °C; this compound was
prepared from 5-bromoisatin 1m (225 mg, 1.0 mmoL) and
4-hydroxycoumarin 2 (324 mg, 2.0 mmoL) according to the
general procedure to obtain a brown solid (450 mg, 88 %
yield). FTIR (KBr) νmax: 3395, 1730, 1706, 1663, 1610,
Colourless solid (MeOH); this compound was prepared
from 5-fluoro-1-methyl isatin 1o (179 mg, 1.0 mmoL) and
4-hydroxycoumarin 2 (324 mg, 2.0 mmoL) according to the
general procedure to obtain a colourless solid (416 mg, 89
% yield). mp 181–183 °C; FTIR (KBr) νmax: 1734, 1716,
1
1
1345, 1108, 761 cm−1. H NMR (400 MHz, CDCl3) δH:
1669, 1613, 1355, 1103, 760 cm−1. H NMR (400 MHz,
6.80 (d, J = 7.4 Hz, 1H, Ar-H), 7.37 (d, J = 7.6 Hz, 1H, Ar-
H), 7.49 (d, J = 7.5 Hz, 2H, Ar-H), 7.57–7.63 (m, 3H, Ar-
H), 7.82–7.86 (m, 2H, Ar-H), 8.45 (d, J = 7.0 Hz, 2H, Ar-
H), 11.00 (brs, D2O exchangeable, 1H, -NH); 13C NMR
(100 MHz, CDCl3) δC: 46.5 (C, C-3), 102.7 (C, C-10 and C-
10′), 110.7 (C-Br, C-5), 112.5 (C, C-17 and C-20), 113.1
(CH, C-13 and C-13′), 116.5 (CH, C-6), 123.7 (CH, C-16
and C-16′), 125.1 (CH, C-7), 127.3 (CH, C-4), 131.7 (C, C-
9), 133.9 (C, C-9′), 143.5 (C, C-8), 151.9 (C, C-14 and C-
14′), 154.8 (C, C-12 and C-12′), 156.0 (C, C-18), 156.1
(C=O, C-11); 176.1 (C=O, C2); MS(ESI): m/z = 512 [M
−H]−, Anal. Calcd for C26H12BrNO6: C, 60.72; H, 2.35; N,
2.72. Found C, 60.69; H, 2.40; N, 2.68.
CDCl3) δH: 3.38 (s, 3H, -NCH3) 6.88–6.92 (m, 1H, Ar-H),
7.19 (d, J = 8.7 Hz, 1H, Ar-H), 7.33 (d, J = 8.3 Hz, 2H, Ar-
H),7.56 (t, J = 7.7 Hz, 2H, Ar-H), 7.76–7.80 (m, 1H, Ar-H),
7.83 (dd, J = 8.8 and 2.2 Hz, 1H, Ar-H), 8.34 (d, J = 2.2 Hz,
1H, Ar-H), 8.49 (d, J = 8.0 Hz, 2H, Ar-H); 13C NMR (100
MHz, CDCl3) δC: 35.8 (-CH3), 46.7 (C, C-3), 102.9 (C, C-
10 and C-10′), 109.5 (CH, C-5), 112.6 (C, C-17 and C-20),
116.6 (CH, C-13 and C-13′), 116.9 (C, C-9), 119.6 (C, C-
9′), 122.9 (CH, C-6), 123.9 (CH, C-16 and C-16′), 124.1,
(CH, C-7), 126.6, 131.3, 131.4, 133.9, 142.7, 150.6 (C, C-
8), 152.6 (C, C-14 and C-14′), 155.2 (C, C-12 and C-12′),
158.4 (C, C-18), 160.8 (C=O, C-11), 177.3 (C=O, C2); MS
(ESI): m/z = 468 [M+H]+, Anal. Calcd for C27H14FNO6:
C, 69.38; H, 3.02; N, 3.00. Found C, 69.43; H, 3.07;
N, 2.97.
5-Nitro-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-3,13′-
pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′(14′),3′
(12′),4′(9′),5′,7′,17′(22′),18′,20′-octane-2,11′,15′-trione
(3n)
1-(3-{2,11′15′-trioxo-1,2-dihydro-2′,10′,16′-trioxaspiro
[indole-3,13′- pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-
1′(14′),3′(12′),4′(9′),5′,7′,17′(22′),18′,20′-octaen-1-yl}
propyl)-1,2-dihydro-2′,10′,16′-trioxaspiro[indole-3,13′-
pentacyclo[12.8.0.03,12.04,9.017,22]docosan]-1′(14′),3′
(12′),4′(9′),5′,7′,17′(22′),18′,20′-octaene-2,11′,15′-trione
(3p)
Yellow solid (MeOH); this compound was prepared from 5-
nitroisatin 1n (192 mg, 1.0 mmoL) and 4-hydroxycoumarin
2 (324 mg, 2.0 mmoL) according to the general procedure to
obtain a yellow solid (411 mg, 86 % yield). mp 209–211 °C
(MeOH); FTIR (KBr) νmax: 3395, 1730, 1706, 1663, 1610,
1345, 1108, 761 cm−1. 1H NMR (400 MHz, Acetone-d6) δH:
δ 7.17 (d, J = 8.7 Hz, 1H, Ar-H), 7.45 (d, J = 8.3 Hz, 2H,
Ar-H),7.58 (t, J = 7.9 Hz, 2H, Ar-H), 7.81 (t, J = 8.6 Hz,
2H, Ar-H), 8.23 (dd, J = 8.6 and 2.2 Hz, 1H, Ar-H), 8.31 (d,
J = 2.2Hz, 1H, Ar-H), 8.46 (d, J = 8.0 Hz, 2H, Ar-H), 10.59
(brs, D2O exchangeable, 1H, -NH); 13C NMR (100 MHz,
CDCl3) δC: 46.7 (C, C-3), 102.9 (C, C-10 and C-10′), 109.5
(CH, C-5), 112.6 (C, C-17 and C-20), 116.9 (CH, C-13 and
C-13′), 119.6 (CH, C-6), 122.9 (CH, C-16 and C-16′),
125.1 (CH, C-7), 126.6 (CH, C-4), 132.3 (C, C-9), 133.9
(C, C-8), 142.7 (C, C-14 and C-14′), 150.6 (C-NO2, C-5),
152.6 (C, C-12 and C-12′), 155.2 (C, C-18), 158.4 (C=O,
Pale yellow solid (MeOH); this compound was prepared
from bis-isatin 1p (334 mg, 1.0 mmoL) and 4-
hydroxycoumarin 2 (324 mg, 4.0 mmoL) according to the
general procedure to obtain a pale yellow solid (737 mg, 81
% yield). mp > 250 °C (MeOH); FTIR (KBr) νmax: 2928,
2877, 1742, 1734, 1706, 1689, 1612, 1359, 1109, 7601
1
cm−1. H NMR (400 MHz, DMSO-d6) δH: 1.72–1.88 (m,
2H, -CH2-), 3.82–4.02 (q, J = 7.1 Hz, 4H, -NCH2-),
6.84–6.94 (m, 4H), 7.19 (d, J = 7.9 Hz, 2H, Ar-H), 7.26 (d,
J = 7.9 Hz, 2H, Ar-H), 7.49–7.55 (m, 4H, Ar-H), 7.55–7.63
(m, 4H, Ar-H), 7.79–7.90 (m, 4H, Ar-H), 8.46–8.54 (m, 4H,
Ar-H), MS(ESI): m/z = 911 [M+H]+.

Med Chem Res
Experimental procedure for the evaluation of
antimicrobial activity
Absorbance of the converted dye in each well was read on
ELISA reader at 570 nm. From the absorbance, % of inhi-
bition was calculated by using the formula, % of inhibition
= Ac−At/Ac × 100, where Ac is the mean absorbance of
control and At is that of test. From the results, nonlinear
regression graph was plotted between % cell growth inhi-
bition and log10 concentration (μM). The half maximal
inhibitory concentration (IC50 value) was determined aver-
aged from three replicate experiments. Cytotoxicity were
statistically analysed by Duncan multiple range test at P =
0.05 with the help of SPSS 11.5 version software package.
All the synthesized compounds were tested for their anti-
bacterial (against S. epidermidis, S. typhimurium, K. pneu-
monia, B. subtilis, S. flexneri, M. luteus, E. aerogenes, S.
aureus and S. aureus MRSA) and antifungal activities
(against C. albicans, M. pachydermatis) using disc diffusion
method (Bauer et al., 1966). Petri plates were prepared with
20 mL of sterile MHA. The test cultures were swabbed on
the top of the solidified media and allowed to dry for 10 min
and a specific amount of synthesized compound at 1 mg/
disc was added to each disc separately. The loaded discs
were placed on the surface of the medium and left for 30
min at room temperature for compound diffusion. Negative
control was prepared using DMSO. Streptomycin was used
as a positive control against bacterial strains and ketoco-
nazole was used as a positive control for fungal strains. The
plates were incubated for 24 h at 37 °C for bacteria and 48 h
at 28 °C for fungi. The radii of inhibition zones were
recorded in millimeters and the experiment was repeated
twice. Bacterial inoculums were prepared by growing cells
in Mueller Hinton broth for 24 h at 37 °C. The filamentous
fungi were grown on sabouraud dextrose agar slants at 28 °C
for 10 days and the spores were collected using sterile
doubled distilled water and homogenized. Yeast was grown
on sabouraud dextrose broth at 28 °C for 48 h.
Molecular docking studies
The level of antimicrobial activity was studied computa-
tionally by Molegro Virtual Docker (MVD) by automated
docking of the most active compounds (3a, 3j and 3l) to the
binding site of AmpC-β-lactamase. Experimentally, X-ray
crystal structures of AmpC-β-lactamase from Escherichia
coli were retrieved from Protein Data Bank (PDB) database-
1KE4 (Powers and Shoichet, 2002). Computationally,
MVD was used to predict potential binding site on AmpC-
β-lactamase. The MolDock scoring function used by MVD
is derived from the PLP scoring functions (Yang and Chen,
2004). To investigate the potential binding mode of inhi-
bitors, all compounds were subjected to molecular docking
using the AutoDock 1.5.4 docking program. The X-ray
crystal structure of Checkpoint kinase 1 (CHK1) in complex
with pyrazolo[1,5-a]pyrimidine was downloaded from the
protein data bank (PDB ID: 3OT3) and was used for the
docking study. Ligand 2D structures were drawn using
ChemDraw Ultra 7.0. Chem3D Ultra 7.0 was used to con-
vert 2D structure into 3D and the energy minimized using
semi-empirical AM1 method. Minimize energy to minimum
RMS gradient of 0.100 was set in each iteration. All
structures were saved as .pdb file format for input to
AutoDockTools (ADT) version 1.5.4 (http://mgltools.
scripps.edu/). All the ligand structures were then saved in
PDBQT file format, for input into AutoDock version 1.5.4
(http://vina.scripps.edu/). All the ligand structures were then
saved in PDBQT file format, for input into AutoDock
version 1.5.4. For the molecular docking study, protein
structure was obtained from the PDB; the CHK1 structure
PDB ID was 3OT3. The co-crystallized ligand pyrazolo
[1,5-a]pyrimidine in the CHK1 structure was removed. For
the protein structure, all hydrogen atoms were added, lower
occupancy residue structures were deleted, and any
incomplete side chains were replaced using the ADT ver-
sion 1.5.4. Further ADT was used to remove crystal water,
added Gagteiger charges to each atom, and merged the non-
polar hydrogen atoms to the protein structure. The distance
between donor and acceptor atoms that form a hydrogen
bond was defined as 1.9 Å with a tolerance of 0.5 Å, and the
Experimental procedure for the evaluation of
cytotoxicity
The MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-tetra-
zolium bromide] cell proliferation assay (Mossman, 1983)
was used to evaluate the cytotoxic activity of the synthe-
sized compounds against COLO320 adenocarcinoma col-
orectal cancer cell lines. COLO320 cancer cell line was
maintained in complete tissue culture medium RPMI with
10 % fetal bovine serum and 2 mM L-glutamine, along with
antibiotics (about 100 IU/mL of penicillin, 100 µg/mL of
streptomycin) with the pH adjusted to 7.2. Cells (5 × 105)
were seeded in 96 well plates containing medium with
different concentrations such as 500 µg/mL, 250 µg/mL and
100 µg/mL. The cells were cultivated at 37 °C with 5 %
CO2 and 95 % air in 100 % relative humidity. After various
durations of cultivation, the solution in the medium was
removed. An aliquot of 100 µL of medium containing 1 mg/
mL of MTT was loaded to the plate. Incubation at 37 °C for
4 h allowed reduction of MTT by mitochondrial dehy-
drogenase to an insoluble formazan product. Well contents
were removed and the formazan product was solubilized by
the addition of 100 µL of DMSO, which led to the forma-
tion of purple colour. The amount of formazan product is
directly proportional to the number of living cells.

Med Chem Res
Scheme 1 Proposed synthesis
of target 3a
Table 1 Screening of Lewis acidsa
Entry Catalyst mol % Solvent Temp
acceptor–hydrogen–donor angle was not less than 120◦.
The structures were then saved in PDBQT file format, for
input into AutoDock version 1.5.4. A grid box with
dimension of 40 × 40 × 40 Å3 and was centred on 15.837,
−2.391, 11.647 was created around the binding site of
pyrazolo[1,5-a]pyrimidine on CHK1 protein using auto-
dock tools. The centre of the box was set at pyrazolo[1,5-a]
pyrimidine and grid energy calculations were carried out.
For the Autodock docking calculation, default parameters
were used and 50 docked conformations were generated for
each compound. In order to verify reproducibility of the
docking calculations, the bound ligand (pyrazolo[1,5-a]
pyrimidine) was extracted from the complexes and sub-
mitted for one-ligand run calculation. This reproduced top
scoring conformations of 10 falling within root-mean-
square deviation (rmsd) values of 0.832 to 1.124 Å from
bound X-ray conformation for CHK1, suggesting this
method is valid enough to be used for docking studies of
other compounds. The outputs were exported to VMD and
Pymol for visual inspection of the binding modes and
interactions of the compounds with amino acid residues in
the active sites. Docking of different ligands to protein was
performed using Autodock, the same protocol used in as
that of validation study. All docking were taken into 2.5
million energy evaluations were performed for each of the
test molecules. Docked ligand conformations were analysed
in terms of energy, hydrogen bonding, and hydrophobic
interaction between ligand and receptor protein CHK1.
Detailed analyses of the ligand-receptor interactions were
carried out, and final coordinates of the ligand and receptor
were saved as pdb files. For display of the receptor with the
ligand binding site, VMD software was used.
Time (h) Yield
(%)b
(°C)
1
InCl3
10
10
10
10
10
10
10
10
5
CH3CN 80
12
12
12
12
12
12
12
12
12
48
12
0.5
25
36
32
15
18
28
39
57
59
10
79
92c
2
InCl3
DMSO 80
DMSO 80
CH3CN 80
DMSO 80
DMSO 80
DMSO 80
DMSO 80
DMSO 80
DMSO 25
DMSO 150
DMSO 100
3
In(OTf)3
NiBr2
4
5
NiBr2
6
AgOTf
7
Cu(OTf)2
Zn(OTf)2
Zn(OTf)2
Zn(OTf)2
Zn(OTf)2
Zn(OTf)2
8
9
10
11
12
5
5
5
The bold values are used to highlight the condition which gives
maximum yield of product
a
All reactions were performed using 1.0 mmoL of isatin and
2.0 mmoL of 4-hydroxycoumarin
b
Isolated yield after filtration
c
The reaction was performed under microwave irradiation with a
power level of 200 W
In this effort, we found that the use of indium(III) salts
afforded poor yields of product 3a in both DMSO and
acetonitrile (entries 1–3). Similarly, poor results were
observed, when NiBr2 in acetonitrile and DMSO was
employed (entries 4 and 5). Among the coinage metal cat-
alysts, Cu(OTf)2 gave slightly better yield over AgOTf
(entries 7 and 6 respectively) and Zn(OTf)2 gave a much
improved yield of 57 % (entry 8). Reducing the catalytic
amount of Zn(OTf)2 from 10 to 5 mol% resulted in no
appreciable change in yield (entry 9) and thus evaluated for
subsequent screening. A drastically reduced yield was
observed, when the reaction was performed at room tem-
perature (entry 10). Conversely, by increasing the tem-
perature to 150 °C, the yield was significantly improved to
79 % (entry 11). In order to reduce the reaction time, we
investigated the use of microwave irradiation instead of
conventional heating. To this end, microwave heating of the
model reaction at 100 °C at a power level of 200 W for 30
min afforded an excellent yield of the product 3a (entry 12).
Consequently, this reaction condition was applied to a range
of substituted isatins with 4-hydroxycoumarin (Scheme 2,
Results and discussion
Chemistry
Based on our previous experience with transition metal
catalysis (Praveen and Perumal, 2011a; 2016a, b; Praveen
et al., 2009a–d, 2010a–c, 2011a, 2012a, b, 2013; Praveen
and Ananth, 2016; Jeyaveeran et al., 2016a, b; Parthasar-
athy et al., 2013), we initially screened a few metal catalysts
for the model reaction between 1.0 mmoL of isatin and 2.0
mmoL of 4-hydroxycoumarin at 80 °C (Scheme 1, Table 1).

Med Chem Res
Scheme 2 Zn(OTf)2 catalysed
microwave synthesis of
spirooxindoles 3a-3o
Table 2 Synthesis of targeted spirooxindoles 3a-3o
region from δH 6.84–8.48 ppm and a broad singlet at δH
10.85 ppm corresponds to -NH proton (D2O exchangeable)
of the oxindole moiety. A less intense peak at δC 46.5 ppm
indicates the presence of a quaternary carbon in the aliphatic
region (spiro carbon). Two peaks at δC 157.9 and 176.4
ppm corresponds to the carbonyl carbon of the coumarin
and oxindole, respectively. This observation was supported
by DEPT-135 and 2D chemical shift correlation experi-
ments (see supplementary Data). Finally, HRMS data
showed a peak at m/z(obs.) = 458.0641 corresponds to [M
+Na]+ , where M is the molecular mass of 3a is in well
agreement with the expected m/z(expd.) = 458.0635. All
these experiments and data unambiguously confirmed the
structure of compound 3a. Based on the observed results, a
tentative mechanism was proposed (Scheme 4) for the
formation of representative compound 3a (Boroujeni and
Ghasemi, 2013). According to which, 4-hydroxycoumarin 2
undergoes nucleophilic addition at the C3 carbon of the
zinc-activated isatin to form intermediate I. Dehydration of
intermediate I results in the formation of intermediate II.
The oxophilic zinc again enters the catalytic circle by
interacting with the carbonyl oxygen of II and drives the
addition of another molecule of 4-hydroxycoumarin 2
leading to III. The later upon intramolecular cyclization
results in intermediate IV and subsequent dehydration
affords the product 3a.
Entry
R
R1
Producta
Yield (%)b
1
H
H
3a
3b
3c
3d
3e
3f
92
95
95
94
95
91
87
89
91
94
92
89
88
86
89
2
Methyl
Ethyl
H
3
H
4
Propyl
Butyl
Pentyl
Hexyl
Allyl
H
5
H
6
H
7
H
3g
3h
3i
8
H
9
Propargyl
Benzyl
2-Flurorobenzyl
H
H
10
11
12
13
14
15
a
H
3j
H
3k
3l
Cl
Br
NO2
F
H
3m
3n
3o
H
Methyl
All products were characterized by IR, NMR and mass
b
Isolated yield after filtration
Table 2). The reaction appears to be general and works well
regardless of the electronic nature of the peripheral sub-
stituents (R and R1) on the isatin moiety and offered
excellent yield of products 3a-3o (Table 2). The utility of
this chemistry was further manifested by its applicability to
afford product with propargyl tether 3i without observing
any allene-isomerization (Nandi and Kundu, 2001; Fortes
and Garrote, 1997; Noguchi et al., 1996; Abbiati et al.,
2005). In order to extend the scope of this methodology, we
subjected 1.0 mmoL of bis-isatin 1p with 4.0 mmoL of 4-
hydroxycoumarin under our optimized reaction condition.
The reaction worked well and afforded the corresponding
bis-spiro skeleton 3p in 81 % yield (Scheme 3).
Antimicrobial activity
In the present study, all compounds were tested for their
antibacterial activity against nine bacterial strains (S. epi-
dermidis, S. typhimurium, K. pneumonia, B. subtilis, S.
flexneri, M. luteus, E. aerogenes, S. aureus and S. aureus
MRSA) and antifungal activity against two fungal strains
(C. albicans, M. pachydermatis) using disc diffusion
method (Bauer et al., 1966). Analysis of the screening
results revealed that most of the compounds exhibited
comparable antimicrobial activities against the reference
drugs (Table 3). The most active derivatives of the target
compounds against all bacterial and fungal strains were 3a,
3j and 3m bearing in their structures NH, NBn and C5-Br
moieties, respectively. Compounds 3d, 3e, 3f and 3h pos-
sessing propyl, butyl, pentyl and allyl chains respectively
emerged as the least active as evidenced by their insignif-
icant zones of inhibition. Other compounds 3b, 3c, 3g, 3i,
The structure of all products was confirmed by spectral
1
data (FTIR, H NMR, 13C NMR and MS) and elemental
analyses. Representatively, the IR spectrum of compound
3a showed a broad peak at 3411 cm−1 and a sharp peak at
1708 cm−1 corresponds to the –NH and carbonyl function-
alities of the oxindole core respectively. Sharp stretching
bands at 1732 and 1662 cm−1 suggested the presence of
carbonyl group characteristic of the δ-lactone ring. The H
NMR spectrum of 3a recorded in DMSO-d6 showed 13
protons. Out of which, 12 protons resonated in the aryl
1

Med Chem Res
Scheme 3 Synthesis of bis-spiro framework 3p
Scheme 4 Plausible mechanism for the formation of compound 3a
3k and 3l exhibited moderate activities against the tested
strains. Compound possessing the bis-spiro skeleton 3p
exhibited very poor activity against all the strains.
each well was inoculated. The references, ketoconazole for
fungi and streptomycin for bacteria, were included in the
assays as positive controls. For fungi, the plates were
incubated for 48-72 h at 28 °C and for bacteria the plates
were incubated for 24 h at 37 °C. The MIC for fungi was
defined as the lowest concentration showing no visible
fungal growth after the incubation time. Five microlitres of
tested broth was placed on the sterile MHA plates for
bacteria and incubated at respective temperature. The MIC
for bacteria was determined as the lowest concentration of
the compound inhibiting the visual growth of the test cul-
tures on the agar plate. The MIC values of active com-
pounds against bacteria and fungi are given in Table 4,
which revealed that compound 3a devoid of any substitu-
tion showed activity against E. aerogenes (62.5 µM) and
S. aureus MRSA (62.5 µM). Compound 3j possessing
N-benzyl substituent showed activity against S. epidermidis
(62.5 µM), S. aureus (62.5 µM) and C. albicans (62.5 µM).
Minimum inhibitory concentration
The activity of the test compounds which gave positive
results (3a-3c, 3g and 3i-3m) was assessed in terms of
minimum inhibitory concentration (MIC) by standard
reference methods (Duraipandiyan and Ignacimuthi, 2009;
Raj et al., 2013). MIC is defined as the maximum dilution of
the test compound that inhibits the growth of the micro-
organism. The selected compounds were dissolved in
DMSO to prepare a series of descending concentration
(1000 µM, 500 µM, 250 µM, 125 µM, 62.5 µM, 31.25 µM,
15.62 µM and 7.81 µM). These solutions were then diluted
to give serial two-fold dilutions and were added to each
medium in 96 well plates. An inoculum of 100 µL from

Med Chem Res
Table 3 Zone of inhibition
(mm) of compounds 3a-3o
(1 mg/disc)
Organism
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n 3o control
Bacteria
S. epidermidis
S. typhimurium
K. pneumoniae
B. subtilis
21
16
8
9
8
2
8
8
8
8
9
9
9
2
1
3
2
1
1
3
2
4
4
2
3
3
1
2
2
2
3
3
5
2
6
1
1
5
4
2
11
8
3
1
5
2
2
2
3
4
6
8
22
9
8
10 21
14
7
8
2
1
3
1
1
2
2
10 25
18
10 20
10 15
8
9
18 10
22 11
9
8
8
9
8
8
17 11 10 16
10
8
23
26
22
20
9
9
8
9
9
8
9
21
25
20
9
6
2
25
30
26
S. flexneri
25
21
8
9
M. luteus
10
11
11
10
E. aerogenes
S. aureus
18 10
11 12
26 12
10 19
11 22
10 13 13
9
9
14
28
4
14
30
S.aureus MRSA
Fungi
8
25 10
7
C. albicans
M. pachydermatis
24 10
8
2
2
2
2
3
2
3
3
2
4
3
2
2
23
12
8
8
3
8
26
13
2
3
8
4
28
26
12
2
The bold values are used to highlight those compounds which gives maximum zone of inhibition values
Control: Streptomycin (standard antibacterial agent) and Ketoconazole (standard antifungal agent)
Table 4 MIC of selected
Organism
3a
3b
3c
3g
3i
3j
3k
3l
3m
Control
compounds
Bacteria
S. epidermidis
S. typhimurium
K. pneumoniae
B. subtilis
500
500
500
250
250
250
62.5
500
62.5
500
500
500
250
500
500
250
250
250
500
-
250
500
500
500
500
250
250
250
250
500
500
500
500
500
500
500
250
500
62.5
125
250
125
125
125
125
62.5
250
500
500
250
500
500
500
500
125
250
500
500
500
500
500
500
250
500
500
62.5
500
250
250
250
125
250
62.5
125
25
30
500
500
500
500
500
500
500
6.25
25
S. flexneri
6.25
6.25
25
M. luteus
E. aerogenes
S. aureus
6.25
6.25
S. aureus MRSA
Fungi
C. albicans
500
250
500
250
250
62.5
500
500
500
25
The bold values are used to highlight the minimum MIC values
Control: Streptomycin (standard antibacterial agent) and Ketoconazole (standard antifungal agent)
Compound 3m possessing bromo substituent at the C5-
carbon of the oxindole core exhibited activity against
S. epidermidis (62.5 µM) and S. aureus (62.5 µM).
cell viability significantly (P<0.05) in a concentration-
dependent manner. Cytotoxicity of each sample was
expressed as IC50 value. The IC50 value is the concentration
of test sample that causes 50 % inhibition of cell growth
averaged from three replicate experiments (Table 5).
Attempts to understand the SAR of the spirooxindoles
started with the substituent at the C5 position, which
revealed that replacement of C5-Cl (3l, IC50 > 100 µM) by a
C5-Br (3m, IC50 = 52.7 µM) did increase the potency to a
great extent. Similar trend was observed, when C5-Br (3m,
IC50 = 52.7 µM) was replaced by C5-NO2 (3n, IC50 = 50.7
µM) and resulted in slight escalation of activity. These
observations could be attributed to the increase in size and
electronegativity (Cl < Br < NO2) of the respective sub-
stituent. It is apparent fro.m the data presented in Table 5
that with every increase in the number of –CH2 spacer, the
inhibitory potency also increased. This drift was observed in
Cytotoxicity
In continuation of our ongoing project on bio-active het-
erocycles (Praveen et al., 2010c, 2011a–c, 2012a, b, 2013;
Parthasarathy et al., 2013), an attempt has been made to
evaluate the cytotoxicity of all compounds (3a-3o) against
colorectal adenocarcinoma cell lines (COLO320) by MTT
assay (Mossman, 1983). The cytotoxic results were com-
pared with reference drug cyclophosphamide which showed
90 % inhibition at a concentration 150 μM. The tested
compounds exhibited maximum cytotoxicity against
COLO320 cells at a concentration of 200 to 50 µM. All
concentrations used in the experiment could decrease the

Med Chem Res
Table 5 IC50 and FEB values of compounds 3a-o
compound. Compounds possessing butyl (3e, IC50 = 51.8
µM) and 2-fluorobenzyl groups (3k, IC50 = 51.8 µM)
respectively showed same but significant inhibitory poten-
cies. Compounds 3f, 3g, 3i and 3l emerged as the least
active among the tested series as evident from their poor
cytotoxocity (IC50 values of >100 µM).
Entry
Compounds
IC50 (µM)
FEB (kcal/moL)a
1
3a
57.2
54.5
53.9
51.1
51.8
>100
>200
54.2
>100
53.1
51.8
>100
52.7
50.7
55.3
–
−8.24
−8.44
−8.34
−8.63
−8.87
−7.58
−6.71
−8.55
−7.74
−8.31
−8.44
−7.73
−8.77
−8.89
−8.33
−9.53
2
3b
3
3c
4
3d
5
3e
Molecular docking
6
3f
7
3g
The precise prediction of ligand-protein complexes is of
fundamental importance in modern structure-based drug
design (Taylor et al., 2002). The ligand matching and the
corresponding docking score could confirm the binding
efficiency of each molecule at the binding site. To this end,
selected ligands were docked into AmpC-β-lactamase and
CHK receptors to evaluate the antimicrobial and cytotoxic
activities, respectively. Docking of ligands to the receptor
was assessed using Moldock scoring function for anti-
microbial and AUTODOCK for cytotoxicity and all the
results were discussed in the following sections.
8
3h
9
3i
10
11
12
13
14
15
16
a
3j
3k
3l
3m
3n
3o
Standardb,c
FEB Free Energy of Binding
b
Cyclophosphamide was used as the standard for cytotoxic studies,
Binding towards AmpC-β-lactamase
which showed 90 % inhibition (156 μM).
Pyrazolo[1,5-a]pyrimidine was used as the standard for docking
c
The level of antimicrobial activity was studied computa-
tionally by MVD by automated docking of the most active
compounds 3a, 3j and 3m to the binding site of AmpC-β-
lactamase. The MolDock scoring function used by MVD is
derived from the PLP scoring functions. The MolDock
further improves these scoring functions with new hydrogen
bonding term and new charge schemes. The ligand matching
and the corresponding scoring could confirm the binding
efficiency of each molecule at the binding site which are
evaluated and ranked according to Moldock scoring function
(Vanderpool et al., 2009). Experimentally, X-ray crystal
structures of AmpC-β-lactamase from E. coli were retrieved
from PDB database-1KE4 (Powers and Shoichet, 2002).
Computationally, MVD was used to predict the potential
binding site on AmpC-β-lactamase. Based on the cavities
detected, the ligands 3a, 3j and 3m were docked with the
active site of amino acids (Figs. 2, 3 and 4). The active site
consisted of amino acid residue that bound to the corre-
sponding amino acids such as ASN346, THR316, ALA318
and SER64. However, ASN346 was found to be conserved
in the active site. The binding energy of ligand 3a with
protein active site was found to be −117.819 kcal mol−1. The
best poses for molecule 3j shows two interactions with
lowest energy of −113.067 kcal mol−1 which binds to active
site amino acids ASN346 and ARG148 respectively. The
best poses for molecule 3m shows three interactions with
lowest energy of −107.056 kcal mol−1 which binds to active
site amino acids ASN346, GLU272 and ARG148 respec-
tively. The docking study revealed that compound 3a
exhibited the minimum binding energy which indicates its
analogues containing NH (3a, IC50 = 57.2 μM), NMe (3b,
IC50 = 54.5 μM), NEt (3c, IC50 = 53.9 μM) and NPr (3d,
IC50 = 51.1 μM), which showed the activity increased in the
order NPr > NEt > NMe > NH. Conversely, a completely
opposite trend was observed when the carbon chain goes
beyond the three methylene spacer 3d. For example, a
slightly reduced potency was observed for the NBu analo-
gue (3e, IC50 = 51.8 μM) when compared to the NPr ana-
logue (3d, IC50 = 51.1 μM). Higher congeners such as the
N-pentyl (3f, IC50 > 100 μM), and N-hexyl (3g, IC50 > 200
μM) exhibited a drastically decreased cytotoxicity. Among
the unsaturated systems, compound with a propargyl group
(3i, IC50 > 100 μM) demonstrated significant cytotoxicity
against its allyl counterpart (3h, IC50 > 100 μM). Assess-
ment of the aryl groups revealed that compound with 2-
fluorobenzyl substituent (3k, IC50 = 51.8 μM) exhibited an
elevated cytotoxicity than those containing the benzyl (3j,
IC50 = 53.1 μM). This enhanced activity is mainly attributed
to the differences in size and electronegativity offered by
the fluorine atom. However, when the fluorine atom was
located on the C5-position (3o, IC50 = 55.3 μM), it renders
the molecule less active than its unsubstituted counterpart
(3b, IC50 = 54.5 μM). Analysis of the combined results as
depicted in Table 5 indicates that compound with a -NO2
group (3n, IC50 = 50.7 µM) positioned at the C5 of the
oxindole core emerged as the most active. Compound with
N-propyl substituent (3d, IC50 = 51.1 µM) was found to be
almost active as 3n and appeared as the second most active

Med Chem Res
Fig. 2 Putative binding pose of
compound 3a in AmpC-β-
lactamase (left panel). Docking
of compound 3a in AmpC-β-
lactamase, where hydrogen
bonding interactions are shown
in dotted lines (right panel)
Fig. 3 Putative binding pose of
compound 3j in AmpC-β-
lactamase (left panel). Docking
of compound 3j in AmpC-β-
lactamase, where hydrogen
bonding interactions are shown
in dotted lines (right panel)
strong affinity with the protein and has been stabilized by
strong hydrogen bond interaction in the binding pocket.
program. As a reference to other compounds, pyrazolo[1,5-
a]pyrimidine was used as a model drug to show the binding
modes of CHK1 inhibitors. From the docking scores, the
free energy of binding (FEB) of all compounds were cal-
culated (Table 5). The overlaid pose of bound and docked
pyrazolo[1,5-a]pyrimidine ligand in the active site of CHK1
is shown in Figure 5. The reader will be reminded that the
binding interactions of all the docked compounds (3a-3o)
were shown in supplementary Fig. 6–21 (see supplementary
Material). The docking results revealed that compound 3n
as the most active with a calculated binding energy of
−8.89 kcal/mol. The least binding energy was exhibited by
compound 3g with a binding energy of –6.71 kcal/mol. To
elucidate the interaction mechanism, the most potent inhi-
bitors among the data set, were selected for more detailed
analysis. The ligand-enzyme interaction analysis shows that
Leu137, Glu91, Cys87, Asp148, Ser88, Gly90 and Tyr86
are the important residues present in the active site. As
shown in supplementary Fig. 6, pyrazolo[1,5-a]pyrimidine
binds to the kinase through key H-bond interactions: (i)
Between the -NH of cyclohexyl ring and the -COO of
Glu91 (−O···HN, 1.90 Å, 160.9°); (ii) Between the back-
bone carbonyl oxygen of Cys87 and the amino of
Binding towards CHK1
CHK1 is a protein kinase from the CAMKL kinase family,
is a key element in the DNA damage response pathway and
plays a crucial role in the S-G2-phase checkpoint (Labroli
et al., 2011). Each phase of the cell cycle is regulated by
several checkpoint mechanisms, such as DNA damage or
spindle assembly checkpoints. First-line therapies for cancer
include radiation and chemotherapy and are designed to
induce cell death by damaging the DNA material of can-
cerous cells. Because cancer cells utilize the checkpoints to
facilitate repair of their DNA, the inhibition of CHK1
represents an attractive strategy for sensitizing cancer cells
to cytotoxic chemotherapeutics. To this end, all compounds
were docked into the relative binding site of CHK1 crystal
structures and the optimal conformations of these com-
pounds were determined. To investigate the potential
binding mode of inhibitors, all the compounds were sub-
jected to molecular docking using the AutoDock 1.5.4

Med Chem Res
Fig. 4 Putative binding pose of
compound 3m in AmpC-β-
lactamase (left panel). Docking
of compound 3m in AmpC-β-
lactamase, where hydrogen
bonding interactions are shown
in dotted lines (right panel)
Fig. 5 Overlaid pose of bound
and docked co-crystal
pyrimidine ring (−O···HN, 2.46 Å, 129.8°; -O···HN, 3.47 Å,
150.2°); (iii) Between the nitrogen atom of pyrazole ring
and the -NH group of Cys87 residue (−N···HN, 2.37 Å,
157.6°); (iv) The interaction between the nitrogen atom of
another pyrazole ring and the side chain of Asp148 is
bridged by a structural molecule W502; (v) Pyrazolo[1,5-a]
pyrimidine ring all form arene-cation interaction with
Leu137 that further enhance the binding activity. The
docking results were well consistent with the in vitro CHK1
inhibitory biological activity (IC50). The binding mode of
compounds 3a-3o is shown in supplementary Figs. 6–21.
The specific cleft in which the compounds bind in the active
site contains polar residues (Glu85, Tyr86, Cys87, Ser88,
Gly89, Gly90, Glu91, Ser147 and Asp148) and non-polar
residues (Ala36, Leu84 and Val23). The compounds 3a-3e,
3h and 3m-3o are well placed inside the active site and
demonstrate the following interactions. (i) The oxygen atom
of spirooxindole (supplementary Figs. 6–21) forms H-bond
with –NH group of Cys87 (-O…HN, 2.15 Å). (ii) The –NH
group of indole ring forms H-bond with the backbone –CO
of Glu85 (-O…HN, 1.95 Å). It is remarkable to note that the
large spiro-moiety has sufficient room to get introduced into
the hydrophobic pocket composed by Ala36, Leu84 and
Val23. The docking score was improved, when the R sub-
stituents were H, methyl, ethyl, propyl and butyl (3a-3e).
Interestingly, the CHK1 inhibitory activity also improved in
the same way. The similar binding mode and docking score
as well as CHK1 inhibitory activity was also observed in
compounds 3h and 3m-3o. The pentyl (3f) and hexyl (3g)
groups were not able to accommodate into the active site;
hence the docking score and CHK1 inhibitory activity also
reduced. Ligands with bulky substituents like benzyl (3j)
and 2-fluorobenzyl (3k) showed different binding mode on
the same active site. The oxygen atom of indole ring (sup-
plementary Figs. 15 and 16) forms H-bond with –NH group
of Glu84 (-O…HN, 2.76 Å). The –NH group of indole ring
forms H-bond with the backbone –CO of Glu85 (-O...HN,
2.15 Å). Based on the above observations, we conclude that
the network of H-bonds formed between CHK1 and the
compounds are crucial for molecular recognition.

Med Chem Res
N-alkylisatin bisthiocarbonohydrazones.
99:397–408
J
Inorg Biochem
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In conclusion, we have disclosed Zn(OTf)2 catalysed effi-
cient synthesis of spirooxindole[pyrano-bis-2H-l-benzo-
pyrans] via tandem double condensation reaction between
4-hydroxycoumarin and isatin. The beneficial advantages of
this methodology are simple reaction conditions, no chro-
matographic techniques and excellent chemical yields.
Antimicrobial evaluation of all compounds against a wide
panel of antimicrobial strains revealed that three compounds
(3a, 3j and 3m) showed comparable inhibitory ability
against the reference drugs. Molecular docking studies of
these compounds with AmpC-β-lactamase receptor revealed
that compound 3a which exhibited minimum FEB
(−117.819 kcal/moL) indicates its strong affinity towards
the targeted protein and has been stabilized by strong
hydrogen bond interaction in the binding pocket. All com-
pounds were also screened for their in vitro cytotoxicity
against COLO320 cancer cells and computationally studied
for their binding affinity towards CHKl receptor. Our
overall results from biological assay and molecular in silico
studies demonstrated that 3n is the most active compound in
terms of its low IC50 value (50.7 μM) and least FEB (−8.89
kcal/mol). We are currently engaged in understanding the
effect of compounds on the host cell and their mechanism of
action and will be reported in the due course.
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Acknowledgments One of the authors K.P. thanks the management
of M/s Orchid Chemicals and Pharmaceuticals Ltd., Chennai, India. C.
P. acknowledges the Department of Science and Technology (DST),
New Delhi, India for providing DST-INSPIRE faculty award (IFA-
MS-27).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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