European Journal of Medicinal Chemistry 83 (2014) 190e207

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Synthesis of new class of spirocarbocycle derivatives by

multicomponent domino reaction and their evaluation for
antimicrobial, anticancer activity and molecular docking studies
N. Sudhapriya a, P.T. Perumal a, *, C. Balachandran b, S. Ignacimuthu b, M. Sangeetha c,
Mukesh Doble c
a
Organic Chemistry Division, CSIR-Central Leather Research Institute, Adyar, Chennai 600 020, India
b
Division of Microbiology, Entomology Research Institute, Loyola College, Chennai 600 034, India
c
Department of Biotechnology, Indian Institute of Technology, Chennai 600 036, India

a r t i c l e i n f o a b s t r a c t

Article history: A series of 25 new spirocarbocycles were synthesized by a three component reaction that involves few
Received 5 March 2014 cyclic nucleophiles, vinyl malononitriles and aldehydes with variable substitution patterns. All the
Received in revised form synthesized compounds were evaluated for their antimicrobial activity and the compounds showed
9 May 2014
significant activity. Synthesized compounds 4c, 4i and 6i showed good anticancer activity against A549
Accepted 26 May 2014
Available online 12 June 2014
cancer cell line. Molecular docking studies indicated that compound 4i had the greatest affinity for DNA
gyrase receptor than others and compound 6i had the greatest affinity for anaplastic lymphoma kinase
(ALK) receptor. These compounds can be better therapeutic agents for microbial and cancer cell lines.
Keywords:
Spirocarbocycle
© 2014 Elsevier Masson SAS. All rights reserved.
Multicomponent reaction
Vinylogous Michael addition
Antimicrobial activity
Anticancer activity
Molecular docking

1. Introduction Michael addition reactions with excellent chemo- and stereo

selectivity [9]. The strong electron-withdrawing groups activate the
Spiro compounds exhibit a broad spectrum of important bio- g-position and further it undergoes Michael addition. Vinylogous
activities such as antitumor [1], antidiabetic [2], antibacterial [3], Michael addition was reported as a key step in the preparation of
antitubercular [4], antiinflammatory [5] activities etc [6] due to many spiro compounds [10].
their interesting structures. Spirooxindoles are privileged medici- The methodologies that are accessible for the synthesis of spi-
nal scaffolds that are found in natural and unnatural biologically rocyclic compounds are alkylations [11], rearrangement reactions
active compounds (Fig. 1). The key paradigm of modern drug dis- [12], cycloadditions [13], transition metal catalyzed reactions [14]
covery and the goal of synthetic organic chemistry are being ach- and cleavage of bridged systems [15]. The synthetic and pharma-
ieved by the rapid assembly of structurally different compounds. cological importance of spiro compounds are anchored in their
One of the most potential strategies for the synthesis and library spirocyclic motifs. The captivating framework of spirooxindoles
production of these spiro compounds is achieved through multi- increased the thirst of chemists in preparing these compounds by
component reactions (MCRs) [7]. They have been used widely for numerous methods. Our group has always been interested on the
carbonecarbon bond formation by synthetic chemists. They also synthesis of novel spirooxindole compounds and screening for
offer an eloquent tool for the one pot synthesis of distinct and their biocidal activity [16].
complex molecule as well as small and drug like heterocycles.
Electron-deficient dicyanoalkenes have been reported to behave 2. Result and discussion
as good hydride acceptors in conjugate reduction reactions [8] and
also act as versatile direct vinylogous donors in asymmetric 2.1. Chemistry

* Corresponding author. The biological importance of the spiro compounds directed us to

E-mail address: [email protected] (P.T. Perumal). synthesize spiro-carbocycles. In continuation of our studies in the

http://dx.doi.org/10.1016/j.ejmech.2014.05.065
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207 191

addition of cyclohexylidene malononitrile 3a to provide a precipi-

tate which was filtered off and recrystallized from ethanol to yield
the desired product 4a in 89% yield.
To explain the mechanism of this tandem reaction, a postulated
reaction course is depicted in (Scheme 2). In the first step 1,3-
indandione 1 undergoes condensation reaction with aldehyde by
the removal of water molecule and affords chalcone 1a. In the
second step the proton of vinyl malononitrile 3a is removed by the
mild base to furnish vinylogous carbanion which attacks the acti-
vated double bond of chalcone 1a forming the intermediate 1b.
Among the two paths the first one shows the steric repulsion and
fails to afford a product. The nucleophilic addition to the nitrile
carbon from the least hindered side through path B results in the
formation of intermediate 1c which on isomerisation gives only
one spirocarbocyclic diastereomer.
With these results in hand, we then investigated the substrate
scopes and limitations of the synthesis of spirocarbocycles. First we
started the reaction with 1,3-indandione by varying aldehydes and
alkylidene malononitrile to afford spirocarbocycles (Scheme 3) the
results are listed in Table 2. Aromatic aldehydes having electron
donating groups like 4-methyl and 4-methoxy benzaldehydes
(Table 2, entries 1e5) reacted at faster rates compared with those
that substituted with electron withdrawing groups like 2-fluoro
and 4-chloro benzaldehydes (Table 2, entries 6e10). Various aro-
matic aldehydes such as naphthaldehyde were also examined
Fig. 1. Some biologically important spirooxindole compounds.
(Table 2, entry 11). Besides, our methodology has been used suc-
cessfully in order of acid and base sensitive materials such as het-
eroaromatic aldehydes and corresponding spirocarbocycles were
area of multicomponent domino reaction using vinylogous Michael obtained in excellent yields without the formation of any by
addition methodology [17] we herein report the one pot multi- products (Table 2, entries 12, 13). As it is clear from the obtained
component reaction of vinyl malononitriles with different chal- results, the presented methodology can be used in oxygen and
cones and their antimicrobial, anticancer activity and molecular sulphur containing heteroaromatic aldehydes. Thus the reaction
docking results of ligands to the DNA gyrase and ALK receptor. To was found to be general and has a broad scope due to its applica-
the best of our knowledge, there have been no reports for the bility to a variety of substrates and the products 4ae4m were iso-
synthesis of the spiro moieties derived from oxindole, indanedione lated in excellent yields (79e90%) under milder reaction
and 1,3-cyclohexanedione chalcones. In the present study the vinyl conditions. The results are summarized in Table 2.
malononitriles undergo vinylogous Michael addition with the in To extend the scope of this protocol for this multicomponent
situ generated chalcones followed by intramolecular nucleophilic reaction, studies were continued with other chalcones like oxindole
addition and isomerization respectively to afford spirocarbocycles chalcones using the same protocol which resulted in the formation
in moderate to high yields (Scheme 1). of corresponding spiroxindoles (Scheme 4). The reaction provided
We initiated our study by performing the reaction of cyclo- spiroxindoles in good yields (78e86%) and the results are shown in
hexylidene malononitrile 3a with in situ generated indanedione Table 3. The promising results prompted us to further explore the
chalcone without any base catalyst and using methanol as solvent scope of this protocol. We investigated the reaction of cyclo-
which afforded compound 4a in 40% yield (Table 1, entry 1). Then hexylidene malononitrile with 1,3-cyclohexanedione chalcone. The
the investigatory experiment was performed to improve the yield spirocarbocycles (Scheme 4) were obtained in moderate yields
of the product by varying reaction conditions viz. solvent, tem- (72e74%) and the results are mentioned in Table 3.
perature and base catalyst. All the reaction products are purified by The structure of all prepared compounds were elucidated with
recrystallization using ethanol as a solvent. The observations the aid of IR, 1H and 13C NMR, Mass spectroscopy and elemental
(Table 1) led us to the conclusion that the base has an obvious effect analysis data were discussed for a representative compound 6a. The
on the reaction. Among the selected bases (Table 1, entries 2e6), IR spectrum of 6a exhibited a sharp peak at 3422 cm
1 which
commercially available organocatalyst L-proline was proven to be corresponds to the eNH stretching of eNH2 group and peaks at
the most suitable (Table 1, entries 6e9) as it gives single diaste- 2200 and 1702 cm
1 corresponded to the nitrile and amide
reomer. The reaction of 1,3-indandione 1 with aldehyde 2a and carbonyl respectively. The 1H NMR spectra of 6a showed two sin-
alkylidene malononitrile 3a in the presence of L-proline was glets at d 5.38 and 10.35 for the eNH2 and eNH protons (D2O
completely diastereoselective in affording only the trans diaste- exchangeable) respectively, clearly indicating the incorporation of
reomer whereas the other bases yield mixture of diastereomers. both moieties in the product. The doublet appearing for the Ha
Several solvents were investigated (Table 1, entries 6, 7, 10e12) proton of the product 6a showed a coupling constant value of
and methanol was the best one (Table 1, entries 7e9), although 12.4 Hz indicative of the trans stereochemistry between the Ha and
ethanol also produced comparable results (Table 1, entry 6). The Hb protons which is also clearly evidenced from the single crystal X-
stoichiometry ratio 15 mol% of the catalyst was chosen to be the ray analysis of compound 6a (Fig. 2, Table 4) [18] which further
best for performing the reaction since further increase had no supports the 1H NMR spectroscopy. In 13C NMR spectra, the spiro
impact on the yield of the reaction (Table 1 entries 7e9). Thus the carbon atom displayed a signal at d 82.0 and the amide carbonyl
best result was obtained when 1,3-indandione 1 and aldehyde 2a carbon atom resonated at d 176.6. The mass spectra also exhibited a
were stirred for ten minutes in the presence of 15 mol% of L-proline distinguishing peak at m/z 382 [MþH]þ. This shows that the reac-
as a catalyst for the in situ generation of chalcone followed by the tion of oxindole 5 with aldehyde 2a and alkylidene malononitrile
192 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207

Scheme 1. Synthesis of spirocarbocycle.

3a is completely diastereoselective in affording only the trans investigated and validated targets for the development of anti
diastereomer. The structures of all spirocarbocycles were consistent bacterials [19]. Most of the synthesized spirocarbocyclic com-
with the above mentioned data. pounds have shown significant activity against S. aureus (MRSA)
hence it is thought worthwhile to do docking studies to support the
2.2. Pharmacology in vitro activity. All the synthesized new spirocarbocycles were
subjected to docking using MOE 2011 software version 7.1. All the
2.2.1. Antimicrobial activity prepared compounds were chosen for the docking study of ligands
In the present work, the antimicrobial activities of 25 synthe- with the DNA gyrase receptor. To find the potential of these mol-
sized compounds were screened against nine bacteria and two ecules against the human lung cancer cells the compounds were
fungi using in vitro disc diffusion method. The results revealed that also docked to the Anaplastic Lymphoma Kinase (ALK) receptor
most of the synthesized compounds exhibited antimicrobial ac- [20].
tivities against Enterobacter aerogenes, Staphylococcus epidermidis, To verify the reproducibility of docking calculations the bound
Staphylococcus aureus (MRSA), Salmonella typhimurium, Klebsiella ligand was extracted from the complexes and submitted for one
pneumonia and Micrococcus luteus. The results are summarized in ligand run calculation. The final docked conformations fall within
Table 5 and Fig. 3. Compounds 4b, 4c, 4i and 4k have shown 0.5e1 Å root-mean-square deviation [21]. Hence it was concluded
excellent activities more than the standard drug against both gram- that this method could be used for the docking of other compounds
positive and gram-negative bacteria at 1 mg/disc. Moreover the (Fig. 5a and 6). We have also performed the docking of the standard
compounds 4a, 4i, 4l, 4m, 6a, 6b, 6i, 6j and 8b showed good ligand Streptomycin with the DNA gyrase receptor for method
antibacterial activity over the others. All tested compounds showed validation (Fig. 5b).
moderate antifungal activity against Candida albicans and Malas- The docked ligand conformations were analyzed in terms of free
sezia pachydermatis. energy of binding (FEB), hydrogen bonding and hydrophobic in-
The Minimum Inhibitory Concentration (MIC) values of active teractions. One hundred (100) docking runs were performed and
compounds against bacteria are given in Table 6 and Fig. 4. Signif- the best docked representation of the ligand was selected based on
icant MIC values were observed against gram positive and gram the conformation with lowest value of FEB.
negative bacteria. The results revealed that the spirocarbocycles 4c, Among all compounds docked to the DNA gyrase receptor,
4i, 4k and 6i have shown good antibacterial activity against tested compound 4i was the most active. It had a high binding energy
organisms. Among all tested compounds 4-methyl substituted ar- of
11.64 kcal/mol. This compound exactly fits as that of ligand and
omatic ring containing compound 4c has shown significant MIC shows strong interaction with ARG 1122 aminoacid (Fig. 7).
values against K. pneumonia, Proteus vulgaris, Shigella flexneri, 4- The intermediate active compound 4m binds with the DNA
chloro substituted aromatic ring containing compound 4i is gyrase receptor and the corresponding binding energy
potent against S. aureus (MRSA), P. vulgaris, S. flexneri and M. luteus. is
9.51 kcal/mol. It shows interaction with the aminoacid GLN
The naphthyl group containing compound 4k is active against 1056 (Fig. 8). The least active compound 6h has binding energy
S. aureus (MRSA), S. flexneri and M. luteus. The 2-thiophenyl con- value
8.14 kcal/mol, and it shows interaction with ALA 1068
taining spirooxindole compound 6i showed significant MIC values aminoacid (Fig. 9).
against S. aureus (MRSA), P. vulgaris and S. flexneri. Molecular docking studies of synthesized molecules to the ALK
receptor show that, the most active compound 6i, has a very high
2.2.2. Anticancer activity binding energy value,
18.43 kcal/mol and it interacts with the
Anticancer activity studies have been performed for the syn- three aminoacids namely, ASP 1249, ASN 1254 and GLY 1272
thesized compounds 4i, 4c and 6i. They showed potent anticancer (Fig. 10). Compound 4h, a moderately active compound, has a
activity in vitro against A549 lung adenocarcinoma cancer cell line. binding energy value of
12.58 kcal/mol and it interacts with GLU
Compound 4c showed 59.6% activity at the dose of 50 mg/mL with 1210 as shown in Fig. 11. The least active compound 6h has a
IC50 value of 50 mg/mL. Compound 4i showed 78.8% activity at the binding energy of
11.23 kcal/mol. It interacts with ARG 1209
dose of 50 mg/mL with IC50 value of 30 mg/mL. Compound 6i showed aminoacid (Fig. 12).
83.9% activity at the dose of 50 mg/mL with IC50 value of 20 mg/mL. The preparation of various other spirooxindoles and the detailed
All concentrations used in the experiment decreased the cell study of biological activity are underway.
viability significantly (P < 0.05) in a concentration-dependent
manner (Table 7). 3. Conclusion

2.2.3. Molecular docking studies We have synthesized a new series of 25 spirocarbocycle de-
Docking studies were performed to gain insight into the protein rivatives through vinylogous Michael addition. A new, simple,
inhibitor interactions inside the enzyme binding sites. Over the efficient and environmentally benign method involving the usage
past decade DNA gyrase receptor remains one of the most of L-proline for the synthesis of spirocarbocycle was developed. By
 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207 193

Table 1 this method, a diverse spirocarbocycle library has been rapidly

Optimization of reaction conditions for the preparation of spirocarbocycles. constructed with excellent yields without involving tedious
Entry Solvent Catalyst (10 mol%) Conditions Time (h) Yielda (%) extraction and isolation procedures. All the compounds were
1. MeOH e r.t. 48 40
evaluated for their activities against 4 gram positive bacteria, 5
2. EtOH K2CO3 r.t. 6 62 gram negative bacteria and 2 fungi. Most of the compounds were
3. MeOH Et3N r.t. 4 80 found to exhibit significant antimicrobial activity. Among them 4c,
4. EtOH DABCO Reflux 6 54 4i and 6i have shown excellent activities and hence are promising
5. EtOH NaOEt r.t. 5 67
candidates as antibacterial agents. Compounds 4c, 4i and 6i also
6. EtOH L-proline r.t. 3 85
7. MeOH L-proline r.t. 3 86 showed good anticancer activity against A549 lung cancer cell line.
8. MeOH L-proline r.t. 3 89b The docking scores show that the spirocarbocyclic molecules have
9. MeOH L-proline r.t. 3 87c good potential against the human lung cancer cells.
10. CH3CN L-proline r.t. 5 69
11. H2O L-proline r.t. 6 72
12. DMF L-proline r.t. 8 58 4. Experimental
a
Isolated yield of 4a after recrystallization.
b
The reaction was performed using 15 mol% of the catalyst. 4.1. Chemistry
c
The reaction was performed using 20 mol% of the catalyst.

Analytical TLC was performed on precoated aluminium sheets of

silica gel 60F254 of 0.2 mm thickness (Merck, Germany). Melting
points were determined on Gallenkamp melting point apparatus

Scheme 2. Plausible mechanism for the formation of spirocarbocycles.

194 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207

Scheme 3. Synthesis of spirocarbocycle derivatives from 1,3-indandione, substituted aldehydes and alkylidene malononitrile.

and are uncorrected. Infrared (IR) spectra were recorded on a Per- 4.1.1.3. 3-Amino-1 0,3 0 -dioxo-1-p-tolyl-1,1 0,3 0 ,6,7,8,9,9a-octahy-
kin Elmer FT-IR spectrometer as KBr pellets. 1H NMR (400 MHz) and drospiro[benzo[7]annulene-2,20 -indene]-4-carbonitrile (4c).
13
C NMR (100 MHz) spectra were recorded in DMSO-d6 solutions Yellow solid; mp 210e212  C (Decomposes); IR (cm
1): 754, 1252,
with TMS as an internal standard on a Bruker Avance DPX-400 MHz 1590, 1637, 1704, 1741, 2200, 2925, 3253, 3366, 3535. 1H NMR
instrument. Proton chemical shifts (d) are relative to tetrame- (400 MHz, DMSO-d6): d 1.14e1.24 (m, 3H), 1.34e1.36 (m, 1H).
thylsilane (TMS, d ¼ 0.00) as internal standard and expressed in 1.63e1.64 (m, 2H), 1.99 (s, 3H), 2.18e2.31 (m, 2H), 3.13 (d, 1H,
parts per million. The number of protons (n) for a given resonance J ¼ 11.6 Hz), 3.49e3.50 (m, 1H proton merged with solvent peak),
was indicated as nH. Coupling constants (J) are given in hertz. Spin 5.76 (t, 1H, J ¼ 6.2 Hz), 6.08 (brs, 2H, NH2, D2O exchangeable)), 6.70
multiplicities are given as s (singlet), d (doublet), t (triplet) and m (d, 2H, J ¼ 8.4 Hz), 6.79 (d, 2H, J ¼ 8.0 Hz), 7.63e7.66 (m, 1H),
(multiplet). Mass spectra were recorded under HRMS (ESI) using 7.74e7.81 (m, 3H); 13C NMR (100 MHz, DMSO-d6): d 20.7, 25.9, 27.9,
Thermo Scientific Exactive Orbitrap mass spectrometer and 29.8, 31.8, 38.0, 52.2,63.0, 83.3, 118.6, 121.2, 123.1, 124.0, 129.2, 134.6,
Thermo Finnigan LCQ Advantage MAX 6000 ESI mass spectrometer 136.5, 136.7, 136.9, 137.3, 142.2, 143.1, 152.3, 199.0, 199.4. HRMS
and PerkineElmer GCeMS. Elemental analysis data were recorded (ESI): Mass calculated for C27H25N2O2 [MþH]þ 409.1911 found,
using Thermo Finnigan FLASH EA 1112 CHN analyzer. [MþH]þ 409.1916; Anal. Calcd for C27H24N2O2: C, 79.39; H, 5.92; N,
6.86. Found: C, 79.28; H, 6.02; N, 7.94.
4.1.1. Experimental procedure for the synthesis of (4aem)
Indanedione 1 (1 mmol), aldehydes 2 (1 mmol) 2aeh were 4.1.1.4. 3 0 -Amino-1 0 -(4-methoxyphenyl)-1,3-dioxo-1,3,60 ,7 0 ,8 0 ,8a 0 -
stirred in MeOH in the presence of L-proline (20 mol%) at room hexahydro-10 H-spiro[indene-2,20 -naphthalene]-40 -carbonitrile (4d).
temperature (r.t.) for ten minutes followed by the addition of Yellow solid; mp 194e197  C (Decomposes); IR (cm
1): 758, 1246,
alkylidene malononitrile 3aec (1 mmol) at r.t. for 3 h. The solid 1588, 1656, 1702, 1739, 2202, 2927, 3250, 3343, 3429. 1H NMR
precipitated out, and then was filtered off and purified by recrys- (400 MHz, DMSO-d6): d 0.69 (q, 1H, J ¼ 12.4 Hz), 1.13e1.17 (m,
tallization from ethanol to afford product 4aem as yellow crys- 2H),1.61e1.67 (m, 1H), 2.14e2.22 (m, 2H),2.95 (d, 1H, J ¼ 12.4 Hz),
talline solid. 3.37e3.43 (m, 1H), 3.48 (s, 3H), 5.60e5.62 (m, 1H), 5.94 (brs, 2H,
eNH2, D2O exchangeable), 6.49e6.52 (m, 2H), 6.64 (d, 1H, J ¼ 8 Hz),
6.74 (d, 1H, J ¼ 8.8 Hz), 7.67 (d, 1H, J ¼ 7.2 Hz), 7.73e7.78 (m, 3H); 13C
4.1.1.1. 30 -Amino-1,3-dioxo-10 -p-tolyl-1,3,60 ,70 ,80 ,8a0 -hexahydro-10 H- NMR (100 MHz, DMSO-d6): d 22.1, 25.3, 27.7, 30.4, 34.5, 55.3, 63.7,
spiro[indene-2,20 -naphthalene]-40 -carbonitrile (4a). Yellow solid; 85.1, 113.6, 114.7, 123.3, 127.5, 131.4, 134.2, 136.8, 140.4, 146.8, 158.3,
mp: 224e226  C (Decomposes); IR (cm
1): 755, 1246, 1589, 1661, 172.2, 197.8, 199.0; MS m/z ¼ 411 [MþH]þ; Anal. Calcd for
1704, 1742, 2205, 2921, 3246, 3345, 3408. 1H NMR (400 MHz, C26H22N2O3: C, 76.08; H, 5.40; N, 6.82 Found: C, 76.19; H, 5.30; N,
DMSO-d6): d .0.71 (q, 1H, J ¼ 12.4 Hz), 1.32e1.42 (m, 2H), 1.64e1.66 6.71.
(m, 1H), 1.99 (s, 3H), 2.08e2.20 (m, 2H), 2.98 (d, 1H, J ¼ 12.8 Hz),
3.04e3.07 (m, 1H), 5.59e5.61 (m, 1H), 6.12 (brs, 2H, eNH2, D2O 4.1.1.5. 6-Amino-4-(4-methoxyphenyl)-1 0,3 0 -dioxo-1 0,2,3,3a,3 0 ,4-
exchangeable), 6.61 (d, 1H, J ¼ 7.6 Hz), 6.72e6.79 (m, 3H), 7.67 (d, hexahydro-2,50 -spirobi[indene]-7-carbonitrile (4e). Yellow solid; mp
1H, J ¼ 7.6 Hz), 7.75e7.77 (m, 3H). 13C NMR (100 MHz, DMSO-d6): 206e209  C (Decomposes); IR (cm
1):1254, 1512, 1573, 1661, 1702,
d 20.0, 22.1, 25.4, 27.9, 33.5, 52.2, 63.6, 82.9, 117.4, 118.1, 123.1, 123.7, 1739, 2204, 2932, 3244, 3345, 3413; 1H NMR (400 MHz, DMSO-d6):
126.6, 129.0, 129.2, 129.2, 131.4, 131.8, 133.1, 136.4, 136.6, 142.7, d 0.23e0.28 (m, 1H), 0.98e1.05 (m, 1H), 1.57e1.63 (m, 2H), 2.34 (d,
143.3, 151.9, 199.7, 200.2. HRMS (ESI): Mass calculated for 1H, J ¼ 7.6 Hz), 2.77 (s, 3H), 2.82e2.90 (m, 1H), 4.65e4.67 (m, 1H),
C26H22N2O2Na [MþNa]þ 417.1573, found, [MþNa]þ, 417.1573. Anal. 5.62 (brs, 2H, eNH2, D2O exchangeable), 5.80 (d, 2H, J ¼ 8 Hz),
Calcd. For: (C26H22N2O2) C, 79.16; H, 5.62; N, 7.10 Found: C, 79.05; H, 6.04e6.08 (m, 2H),7.00e7.06 (m, 4H); 13C NMR (100 MHz, DMSO-
5.73; N, 7.01. d6): d 30.4, 31.3, 42.5, 46.4, 55.3, 65.3, 78.7, 116.8, 117.9, 123.3, 123.6,
127.8, 136.7, 136.9, 138.0, 142.2, 143.3, 154.4, 158.6, 199.6, 200.3; MS
4.1.1.2. 6-Amino-10,30 -dioxo-4-p-tolyl-10,2,3,3a,30 ,4-hexahydro-2,50 - m/z ¼ 397 [MþH]þ; Anal. Calcd for C25H20N2O3, C, 75.74; H, 5.08; N,
spirobi[indene]-7-carbonitrile (4b). Yellow solid; mp 215e216  C 7.07 Found: C, 75.83; H, 5.19; N, 7.17.
(Decomposes); IR (cm
1): 790, 1244, 1573, 1664, 1703, 1741, 2206,
2925, 3246, 3347, 3406. 1H NMR (400 MHz, DMSO-d6): d 0.98 (q, 1H, 4.1.1.6. 30 -Amino-10 -(2-fluorophenyl)-1,3-dioxo-1,3,60 ,70 ,80 ,8a0 -hex-
J ¼ 10.4), 1.67e1.74 (m, 1H), 1.98 (s, 3H), 2.22e2.34 (m, 2H), 3.03 (d, ahydro-10 H-spiro[indene-2,20 -naphthalene]-40 -carbonitrile (4f).
1H, J ¼ 12.4 Hz), 3.50e3.55 (m, 1H), 5.33e5.35 (m, 1H), 6.48 (brs, Yellow solid; mp 222e224  C (Decomposes); IR (cm
1): 759, 1247,
2H, NH2, D2O exchangeable), 6.72e6.77 (m, 4H), 7.69e7.75 (m, 4H); 1588, 1662, 1704, 1742, 2205, 2919, 3249, 3349, 3410; 1H NMR
13
C NMR (100 MHz, DMSO-d6): d 20.8, 30.4, 31.3, 42.4, 52.8, 65.2, (400 MHz, DMSO-d6): d 0.66 ( q, 1H, J ¼ 12.2 Hz), 1.26e1.34 (m, 2H)
78.4, 116.0, 117.9, 123.2, 123.7, 129.1, 133.1, 136.4, 136.6, 136.9, 138.5, 1.62e1.65 (m, 1H), 2.05e2.17 (m, 2H), 3.44 (d, 1H, J ¼ 12.5 Hz),
142.3, 143.5, 154.6, 199.4, 199.9. MS m/z ¼ 381 [MþH]þ; Anal. Calcd 4.48e4.51 (m, 1H), 5.62e5.64 (m, 1H), 6.06 (brs, 2H, eNH2, D2O
for C25H20N2O2: C, 78.93; H, 5.30; N, 7.36 Found: C, 79.94; H, 5.21; exchangeable), 6.79e6.84 (m, 2H), 6.87e6.90 (m, 1H), 6.93e6.96
N, 7.24. (m, 1H), 7.70 (d, 1H, J ¼ 5 Hz), 7.73e7.76 (m, 3H); 13C NMR
 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207 195

Table 2
Synthesis of spirocarbocycle derivatives from 1, 3-indandione, substituted aldehydes and alkylidene malononitrile.

Entry Aldehyde Vinylogous malononitrile Product Time Yield

1. 3 89

2. 2.5 90

3. 4 86

4. 3.5 87

5. 2.5 88

6. 4 81

(continued on next page)

196 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207

Table 2 (continued )

Entry Aldehyde Vinylogous malononitrile Product Time Yield

7. 3 83

8. 6 82

9. 4.5 84

10. 4 85

11. 5 79

12. 4.5 85

13. 5.5 86
 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207 197

Scheme 4. Synthesis of spirocarbocycle derivatives from oxindole or 1,3-cyclohexanedione, substituted aldehydes and alkylidene malononitrile.

(100 MHz, DMSO-d6): d 21.7, 25.3, 27.9, 33.4, 42.9, 63.1, 83.6, 118.1, (m, 2H), 2.02e2.05 (m, 1H), 2.36e2.40 (m, 1H), 2.51 (d, 1H,
118.8, 122.9, 123.6, 124.5, 129.2, 130.2, 131.4, 137.3, 142.8, 151.9, J ¼ 11.7 Hz) 2.98e3.00 (m, 1H), 5.07 (t, 1H, J ¼ 5.78 Hz), 5.27 (brs, 2H,
159.2, 198.8, 200.1; MS m/z ¼ 399 [MþH]þ; Anal. Calcd for eNH2, D2O exchangeable), 6.09e6.15 (m, 3H), 6.47 (d, 1H,
C25H19FN2O2: C, 75.36; H, 4.81; N, 7.03 Found: C, 75.47; H, 4.72; N, J ¼ 8.2 Hz), 6.96 (d, 1H, J ¼ 7.4 Hz), 7.07e7.12 (m, 3H); 13C NMR
7.12. (100 MHz, DMSO-d6): d 25.6, 27.8, 29.6, 30.1, 36.3, 52.4, 62.9, 83.4,
118.5, 122.1, 123.3, 123.9, 126.4, 128.4, 129.3, 132.5, 136.5, 137.1, 141.9,
4.1.1.7. 6-Amino-4-(2-fluorophenyl)-1 0,3 0 -dioxo-1 0,2,3,3a,3 0 ,4- 143.0, 151.9, 199.0, 199.1; MS m/z ¼ 429 [MþH]þ; Anal. Calcd for
hexahydro-2,50 -spirobi[indene]-7-carbonitrile (4g). Yellow solid; mp C26H21ClN2O2: C, 72.81; H, 4.94; N, 6.53. Found: C, 72.75; H, 5.15; N,
222e225  C (Decomposes); IR (cm
1): 761, 1245, 1573, 1646, 1708, 6.42.
1742, 2199, 2829, 3258, 3359, 3445; 1H NMR (400 MHz, DMSO-d6):
d 0.25 (q, 1H, J ¼ 11.6 Hz), 1.03e1.04 (m, 1H), 1.57e1.66 (m, 2H), 2.42
4.1.1.10. 30 -Amino-10 -(4-bromophenyl)-1,3-dioxo-1,3,60 ,70 ,80 ,8a0 -hex-
(d, 1H, J ¼ 12.4 Hz), 2.91e2.97 (m, 1H), 4.71e4.72 (m, 1H), 5.78 (brs,
ahydro-10 H-spiro[indene-2,20 -naphthalene]-40 -carbonitrile (4j).
2H, eNH2, D2O exchangeable), 6.09 (t, 1H, J ¼ 9.2 Hz), 6.16e6.20 (m,
Yellow solid; mp 254e256  C (Decomposes); IR (cm
1): 805, 1243,
1H), 6.27e6.34 (m, 2H), 7.00e7.01 (m, 1H), 7.08 (d, 3H, J ¼ 2.9 Hz);
13 1589, 1636, 1696, 1738, 2200, 2948, 3250, 3361, 3452; 1H NMR
C NMR (100 MHz, DMSO-d6): d 35.0, 36.0, 47.2, 48.9, 69.5, 83.4,
(400 MHz, DMSO-d6): d 0.72 (q, 1H, J ¼ 12 Hz), 1.30 (d, 1H,
120.1, 121.8, 122.6, 128.1, 128.3, 129.6, 133.9, 135.1, 141.6, 141.7, 142.2,
J ¼ 11.2 Hz), 1.39e1.42 (m, 1H), 1.65 (d, 1H, J ¼ 9.2 Hz), 2.06e2.07 (m,
147.0, 147.6, 159.2, 163.2, 203.5, 204.0; MS m/z ¼ 385 [MþH]þ; Anal.
1H), 2.15e2.20 (m, 1H), 3.03 (d, 1H, J ¼ 11.2 Hz) 3.34e3.41 (m, 1H),
Calcd for C24H17FN2O2: C, 74.99; H, 4.46; N, 7.29. Found: C, 75.17; H,
5.60e5.62 (m, 1H), 6.18 (brs, 2H, eNH2, D2O exchangeable), 6.70 (d,
4.35; N, 7.38.
1H, J ¼ 7.6 Hz), 7.17 (d, 1H, J ¼ 8.4 Hz), 7.69 (d, 2H, J ¼ 8.4 Hz), 7.69 (d,
1H, J ¼ 7.6 Hz),7.74e7.81 (m, 3H); 13C NMR (100 MHz, DMSO-d6):
4.1.1.8. 30 -Amino-10 -(4-chlorophenyl)-1,3-dioxo-1,3,60 ,70 ,80 ,8a0 -hex-
d 22.0, 25.3, 27.7, 33.3, 51.8, 63.4, 82.8, 117.7, 118.0, 120.9, 123.3,
ahydro-10 H-spiro[indene-2,20 -naphthalene]-40 -carbonitrile (4h).
123.8, 129.0, 131.4, 131.5, 133.5, 135.6, 136.7, 136.8, 142.5, 143.2,
Yellow solid; mp 246e248  C (Decomposes); IR (cm
1): 833, 1093,
151.6, 199.5, 200.0; MS m/z ¼ 459 [MþH]þ; Anal. Calcd for
1244, 1590, 1636, 1697, 1738, 2201, 2923, 3251, 3360, 3452; 1H NMR
C25H19BrN2O2: C, 65.37; H, 4.17; N, 6.10. Found: C, 65.26; H, 4.27; N,
(400 MHz, DMSO-d6): d 0.73 (q, 1H, J ¼ 12.4 Hz), 1.24e1.32 (m, 1H),
6.20.
1.40e1.43 (m, 1H), 1.64e1.67 (m, 1H), 1.97e2.07 (m, 1H), 2.15e2.20
(m, 1H), 3.06 (d, 1H, J ¼ 12.4 Hz) 3.09e3.10 (m, 1H), 5.60e5.62 (m,
1H), 6.19 (brs, 2H, eNH2, D2O exchangeable), 6.76 (d, 1H, J ¼ 7.2 Hz), 4.1.1.11. 30 -Amino-10 -(naphthalen-1-yl)-1,3-dioxo-1,3,60 ,70 ,80 ,8a0 -hex-
6.86 (d, 1H, J ¼ 7.6 Hz), 7.04 (d, 2H, J ¼ 8.4 Hz), 7.69 (d, 1H, ahydro-10 H-spiro[indene-2,20 -naphthalene]-40 -carbonitrile (4k).
J ¼ 7.6 Hz), 7. 78 (q, 3H, J ¼ 7.0 Hz; 13C NMR (100 MHz, DMSO-d6): Yellow solid; mp 213e215  C (Decomposes); IR (cm
1): 774, 1256,
d 22.0, 25.3, 27.7, 33.3, 51.7, 63.5, 82.8, 117.7, 118.0, 123.2, 123.8, 1488, 1585, 1633, 1721, 2195, 2926, 3420, 3466; 1H NMR (400 MHz,
128.5, 128.6, 128.7, 131.4, 132.3, 135.2, 136.7, 136.8, 142.5, 143.2, DMSO-d6): d 0.65 (q, 1H, J ¼ 12.1 Hz), 1.23e1.26 (m, 1H), 1.73e1.79
151.6, 199.5, 200.0; MS m/z ¼ 415 [MþH]þ; Anal. Calcd for (m, 2H), 2.13e2.22 (m, 1H), 2.37 (d, 1H, J ¼ 9.4 Hz), 2.65 (d, 1H,
C25H19ClN2O2: C, 72.37; H, 4.62; N, 6.75 Found: C, 72.28; H, 4.73; N, J ¼ 10.8 Hz) 3.14e3.20 (m, 1H), 5.65e5.67 (m, 1H), 6.10 (brs, 2H,
6.65. eNH2, D2O exchangeable), 7.09e7.11 (m, 2H), 7.23e7.28 (m, 2H),
7.45e7.51 (m, 2H), 7.63e7.70 (m, 4H), 7.79e7.82 (m, 1H); 13C NMR
4.1.1.9. 3-Amino-1-(4-chlorophenyl)-1 0,3 0 -dioxo-1,10,3 0 ,6,7,8,9,9a- (100 MHz, DMSO-d6): d, 27.3, 35.1,36.9, 44.9, 52.5, 63.8, 83.2, 118.0,
octahydrospiro[benzo[7]annulene-2,20 -indene]-4-carbonitrile (4i). 121.8, 123.0, 123.1, 123.7, 125.1, 126.0, 126.7, 128.8, 129.3, 132.1, 133.7,
Yellow solid; mp 180e181  C (Decomposes); IR (cm
1): 833, 1091, 136.1, 142.7, 152.3, 167.6, 199.5, 200.5; MS m/z ¼ 431 [MþH]þ; Anal.
1490, 1590, 1635, 1700, 1741, 2199, 2929, 3366, 3455; 1H NMR Calcd for C29H22N2O2: C, 80.91; H, 5.15; N, 6.51. Found: C, 80.82; H,
(400 MHz, DMSO-d6): d 0.50 (m, 3H), 1.34e1.38 (m, 1H), 1.66e1.69 5.26; N, 6.60.
198 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207

Table 3
Synthesis of spirooxindole derivatives from oxindole or 1,3-cyclohexanedione, substituted aldehydes and alkylidene malononitrile.

Entry Aldehyde/ketone Vinylogous malononitrile Product Time Yield

1. 5 86

2. 6 83

3. 7 84

4. 8 78

5. 10 81

6. 9 80

7. 8 79

8. 7.5 78
 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207 199

Table 3 (continued )

Entry Aldehyde/ketone Vinylogous malononitrile Product Time Yield

9. 9 81

10. 7 82

11. 6 74

12. 6.5 72

4.1.1.12. 30 -Amino-10 -(furan-3-yl)-1,3-dioxo-1,3,60 ,70 ,80 ,8a0 -hexahy- (100 MHz, DMSO-d6): d 22.0, 25.4, 27.7, 35.5, 47.6, 63.3, 82.8, 117.8,
dro-10 H-spiro[indene-2,20 -naphthalene]-40 -carbonitrile (4l). 118.0, 123.4, 124.0, 131.2, 136.6, 136.8, 142.8, 143.3, 151.3, 199.7,
Yellow solid; mp 247e248  C (Decomposes); IR (cm
1): 1243, 1589, 200.0; MS m/z ¼ 387 [MþH]þ; Anal. Calcd for C23H18N2O2S: C,
1660, 1704, 2206, 2923, 3253, 3347, 3422; 1H NMR (400 MHz, 71.48; H, 4.69; N, 7.25. Found: C, 71.36; H, 4.80; N, 7.16.
DMSO-d6): d 0.79 (q, 1H, J ¼ 12 Hz), 1.41e1.43 (m, 2H), 1.69e1.72 (m,
1H), 1.94e2.19 (m, 2H), 2.87e2.92 (m, 1H), 2.98 (d, 1H, J ¼ 12.4 Hz), 4.1.2. Experimental procedure for the synthesis of (6aej)
5.57e5.59 (m, 1H),5.87 (s, 1H) 6.14 (brs, 2H, eNH2, D2O exchange- Oxindole 5 (1 mmol), aldehydes 2 (1 mmol) 2aej were stirred in
able), 7.13 (s, 1H), 7.20 (s, 1H), 7.79e7.80 (m, 1H), 7.84e7.85 (m, 1H); MeOH in the presence of L-proline (20 mol%) at room temperature
13
C NMR (100 MHz, DMSO-d6): d 26.7, 30.1, 32.6, 38.2, 47.6, 67.5, (r.t.) for ten minutes followed by the addition of alkylidene malo-
87.6, 122.2, 122.8, 125.1, 128.1, 128.6, 136.2, 141.5, 146.5, 147.4, 148.1, nonitrile 3aec (1 mmol) at r.t. for 5 h. The solid precipitated out was
148.9, 156.3, 204.7, 204.9; MS m/z ¼ 371 [MþH]þ; Anal. Calcd for filtered off and purified by recrystallization from ethanol to afford
C23H18N2O3: C, 74.58; H, 4.90; N, 7.56. Found: C, 74.48; H, 4.79; N, product 6aej as white crystalline solid.
7.65.
4.1.2.1. 30 -Amino-2-oxo-10 -p-tolyl-60 ,70 ,80 ,8a0 -tetrahydro-10 H-spiro
0 0 0 0 0 0
4.1.1.13. 3 -Amino-1,3-dioxo-1 -(thiophen-2-yl)-1,3,6 ,7 ,8 ,8a -hex- [indoline-3,20 -naphthalene]-40 -carbonitrile (6a). White solid; mp
ahydro-10 H-spiro[indene-2,20 -naphthalene]-40 -carbonitrile (4m). 248e249  C (Decomposes); IR (cm
1):752, 1191, 1388, 1580, 1627,
Yellow solid; mp 248e250  C (Decomposes); IR (cm
1): 1244, 1587, 1702, 2200, 2926. 3057, 3299, 3329, 3422; 1H NMR (400 MHz,
1662, 1704, 1741, 2205, 2923, 3250, 3347, 3411; 1H NMR (400 MHz, DMSO-d6): d 0.72 (q, 1H, J ¼ 12.1 Hz), 1.26e1.29 (m, 1H), 1.37e1.41
DMSO-d6): d 0.83 (q, 1H, J ¼ 12.1 Hz), 1.39e1.48 (m, 2H), 1.68e1.69 (m, 1H), 1.64e1.67 (m, 1H), 2.03e2.07 (m, 1H), 2.12 (s, 3H),
(m, 1H), 2.08e2.21 (m, 2H), 2.93e2.99 (m, 1H), 3.35e3.40 (m, 1H), 2.16e2.21 (m, 1H), 3.02 (d, 1H, J ¼ 12.4 Hz), 3.37e3.41 (m, 1H), 5.38
5.59e5.61 (m, 1H),6.15 (brs, 2H, eNH2, D2O exchangeable), (brs, 2H, eNH2, D2O exchangeable), 5.58e5.60 (m. 1H), 6.37 (d, 1H,
6.55e6.61 (m, 2H), 7.08e7.09 (m, 1H), 7.75e7.82 (m, 4H); 13C NMR J ¼ 8.8 Hz), 6.49 (d, 1H, J ¼ 8 Hz), 6.66 (d, 1H, J ¼ 7.6 Hz), 6.93e7.00
200 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207

1.71e1.74 (m, 1H), 2.10 (s, 3H), 2.24e2.28 (m, 1H), 2.67e2.69 (m,
1H), 3.20 (d, 1H, J ¼ 12.2 Hz), 3.27e3.28 (m, 1H), 5.26 (brs, 2H,
eNH2, D2O exchangeable), 5.70e5.74 (m. 1H), 6.92e6.97 (m, 1H),
6.99e7.03 (m, 2H), 7.05e7.10 (m, 2H), 7.14e7.21 (m, 1H), 7.33e7.40
(m, 2H), 10.42 (brs, 1H, eNH, D2O exchangeable); 13C NMR
(100 MHz, DMSO-d6): d 20.9, 26.0, 28.1, 30.3, 32.3, 37.7, 52.6, 57.6,
82.5, 107.5, 110.0, 119.1, 120.5, 122.2, 125.4, 127.2, 127.6, 128.3, 128.6,
129.0, 129.4, 129.5, 135.9, 136.3, 138.0, 138.3, 142.7, 154.5, 176.0; MS
m/z ¼ 396 [MþH]þ; Anal. Calcd for C26H25N3O: C, 78.96; H, 6.37; N,
10.62. Found: C, 78.87; H, 6.45; N, 10.53.

4.1.2.3. 30 -Amino-10 -(4-methoxyphenyl)-2-oxo-60 ,70 ,80 ,8a0 -tetrahy-

dro-10 H-spiro[indoline-3,20 -naphthalene]-40 -carbonitrile (6c).
White solid; mp 241e243  C (Decomposes); IR (cm
1): 833, 1185,
1266, 1512, 1573, 1618, 1701, 2195, 2224, 2952, 3276, 3324, 3425; 1H
NMR (400 MHz, DMSO-d6): d 0.72 (q, 1H, J ¼ 12.1 Hz), 1.27e1.30 (m,
1H), 1.37e1.50 (m, 1H), 1.65e1.74 (m, 1H), 2.06e2.08 (m, 1H),
2.16e2.21 (m, 1H), 3.01 (d, 1H, J ¼ 12.8 Hz), 3.31e3.34 (m, 1H),3.60
(s, 3H), 5.43 (brs, 2H, eNH2, D2O exchangeable), 5.57e5.59 (m. 1H),
6.39 (s, 2H), 6.50 (d, 1H, J ¼ 7.6 Hz), 6.76 (d, 1H, J ¼ 8.5 Hz),
6.93e6.97 (m, 1H), 7.05e7.09 (m, 1H), 7.14 (d, 1H, J ¼ 8.6 Hz), 7.28 (d,
Fig. 2. ORTEP diagram of synthesized compound 6a. 1H, J ¼ 7.3 Hz), 10.37 (brs, 1H, eNH, D2O exchangeable); 13C NMR
(100 MHz, DMSO-d6): d 22.3, 25.5, 28.3, 32.8, 52.2, 55.2, 58.0, 81.8,
(m, 2H), 7.06 (t, 1H, J ¼ 7.6 Hz), 7.13 (d, 1H, J ¼ 8 Hz), 7.28 (d, 1H, 110.0, 112.6, 113.9, 114.3, 116.6, 118.5, 122.2, 124.7, 127.6, 129.2, 129.3,
J ¼ 7.6 Hz), 10.35 (brs, 1H, eNH, D2O exchangeable); 13C NMR 129.7, 129.8, 132.6, 133.1, 142.9, 154.5, 158.1, 176.7; MS m/z ¼ 398
(100 MHz, DMSO-d6): d 20.9, 22.3, 25.5 28.3, 32.7, 52.6, 57.9, 82.0, [MþH]þ; Anal. Calcd for C25H23N3O2: C, 75.54; H, 5.83; N, 10.57.
110.0, 116.8, 118.5, 122.2, 124.7, 126.4, 128.1, 128.9, 129.2, 129.7, 132.0, Found: C, 75.45; H, 5.72; N, 10.66.
132.6, 134.5, 135.9, 142.9, 154.5, 176.6; HRMS (ESI): Mass calculated
for C26H22N2O2Na [MþNa]þ: 404.1733, found: 404.1734; Anal. Calcd 4.1.2.4. 30 -Amino-10 -(2-fluorophenyl)-2-oxo-60 ,70 ,80 ,8a0 -tetrahydro-
for C25H23N3O: C, 78.71; H, 6.08; N, 11.02. Found: C, 78.60; H, 6.17; 10 H-spiro[indoline-3,20 -naphthalene]-40 -carbonitrile (6d).
N, 11.12. White solid; mp 265e267  C (Decomposes); IR (cm
1): 753, 1203,
1227, 1489, 1577, 1625, 1701, 2198, 2831, 2939, 3335, 3427; 1H NMR
(400 MHz, DMSO-d6): d 0.74 (q, 1H, J ¼ 12.0 Hz), 1.20e1.23 (m, 1H),
4.1.2.2. 3-Amino-20 -oxo-1-p-tolyl-1,6,7,8,9,9a-hexahydrospiro[benzo 1.36e1.44 (m, 1H), 1.65e1.67 (m, 1H), 2.04e2.21 (m, 2H),3.39e3.43
[7]annulene-2,30 -indoline]-4-carbonitrile (6b). White solid; mp (m, 1H), 3.50 (d, 1H, J ¼ 12.5 Hz), 5.52 (brs, 2H, eNH2, D2O
238e240  C (Decomposes); IR (cm
1):755, 1475, 1577, 1622, 1693, exchangeable), 5.59e5.61 (m. 1H), 6.50 (d, 1H, J ¼ 7.7 Hz), 6.77 (t,
1716, 2200, 2927, 3263, 3350, 3460; 1H NMR (400 MHz, DMSO-d6): 1H, J ¼ 9.0 Hz), 6.92 (t, 1H, J ¼ 7.6 Hz), 7.04e7.10 (m, 3H), 7.21 (d, 1H,
d 1.01 (q, 1H, J ¼ 11.3 Hz), 1.40e1.49 (m, 1H), 1.60e1.67 (m, 3H), J ¼ 7.4 Hz), 7.31 (t, 1H, J ¼ 7.4 Hz), 10.56 (brs, 1H, eNH, D2O
exchangeable); 13C NMR (100 MHz, DMSO-d6): d 22.1, 25.4, 28.0,
Table 4 32.5, 43.5, 57.4, 81.7, 109.8, 115.0, 115.2, 117.0, 118.4, 122.2, 124.6,
Crystal data and structure refinement parameters for compound 6a. 124.8, 128.5, 128.7, 129.1, 129.2, 129.4, 132.2, 142.7, 154.4, 176.6;
HRMS (ESI): Mass calculated for C24H20FN3NaO [MþNa]þ 408.1488,
Empirical formula C25 H23 N3 O
Formula weight 381.46 found, [MþNa]þ 408.1489. Anal. Calcd for C24H20FN3O: C, 74.79; H,
Temperature 293 (2) K 5.23; N, 10.90. Found: C, 74.68; H, 5.14; N, 10.79.
Wavelength 0.71073 Å
Crystal system, space group Monoclinic, P21/n 4.1.2.5. 30 -Amino-10 -(4-chlorophenyl)-2-oxo-60 ,70 ,80 ,8a0 -tetrahydro-
Unit cell dimensions a ¼ 9.3858 (3) Å; b ¼ 21.8349 (6) Å;
c ¼ 9.6525 (2) Å
10 H-spiro[indoline-3,20 -naphthalene]-40 -carbonitrile (6e). White
a ¼ g ¼ 90 solid; mp 258e259  C (Decomposes); IR (cm
1): 747, 1487, 1579,
b ¼ 93.3610 (10) 1629, 1725, 2195, 2923, 3334, 3453; 1H NMR (400 MHz, DMSO-d6):
Volume 1974.76 (9) A3 d 0.76 (q, 1H, J ¼ 12.1 Hz), 1.24e1.27 (m, 1H), 1.38e1.42 (m, 1H),
Z, Calculated density 4, 1.283 Mg m
3
1.66e1.68 (m, 1H), 2.04e2.08 (m, 1H),2.17e2.21 (m, 1H), 3.14 (d, 1H,
Absorption coefficient 0.080 mm
1
F (000) 808 J ¼ 12.4 Hz), 3.37e3.41 (m, 1H) 5.52 (brs, 2H, eNH2, D2O
Crystal size 0.35  0.35  0.30 mm exchangeable), 5.59e5.60 (m. 1H), 6.50 (d, 2H, J ¼ 7.8 Hz),
q range for data collection 2.31e25.00 6.91e6.97 (m, 2H), 7.06e7.08 (m, 1H), 7.22e7.29 (m, 2H), 7.30e7.32
Limiting indices
11  h  11,
25  k  25,
11  l  11 (m, 1H),10.43 (brs, 1H, eNH, D2O exchangeable); 13C NMR
Reflections collected/unique 18019/3486 [R (int) ¼ 0.0335]
Completeness to q ¼ 25.00 100.0%
(100 MHz, DMSO-d6): d 22.2, 25.5, 28.2, 32.4, 52.2, 57.7, 81.7, 110.0,
Absorption correction Semi-empirical from equivalents 116.8, 118.5, 122.3, 124.8, 127.4, 128.3, 128.5, 129.4, 131.7, 132.3,
Max. and min. transmission 0.9865 and 0.9632 133.8, 136.6, 142.8, 154.3, 176.4; MS m/z ¼ 402 [MþH]þ; Anal. Calcd
Refinement method Full-matrix least-squares on F2 for C24H20ClN3O: C, 71.73; H, 5.02; N, 10.46. Found: C, 71.63; H, 5.11;
Data/restraints/parameters 3486/0/275
N, 10.35.
Goodness-of-fit on F2 1.030
Final R indices [I > 2sigma (I)] R1 ¼ 0.0400, wR2 ¼ 0.0965
R indices (all data) R1 ¼ 0.0587, wR2 ¼ 0.1072 4.1.2.6. 30 -Amino-10 -(4-bromophenyl)-2-oxo-60 ,70 ,80 ,8a0 -tetrahydro-
Extinction coefficient 0.0079 (11) 10 H-spiro[indoline-3,20 -naphthalene]-40 -carbonitrile (6f). White
Largest diff. peak and hole 0.240 and
0.158 e A
3 solid; mp 264e266  C (Decomposes); IR (cm
1): 748, 1485, 1577,
CCDC 972722
1631, 1725, 2195, 2922, 3331, 3446; 1H NMR (400 MHz, DMSO-d6):
 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207 201

Table 5
In vitro antimicrobial activity of synthesized compounds.

Compounds Zone of inhibition in mm

Gram positive bacteria Gram negative bacteria Fungi

S. epidermidis S. aureus S. aureus (MRSA) M. luteus E. aerogenes S. typhimurium K. pneumonia P. vulgaris S. flexneri C. albicans M. pachydermatis

4a 10 10 11 9 13 14 15 10 8 10 11
4b 14 17 12 12 15 10 15 16 13 10 13
4c 16 13 17 10 18 19 20 22 23 10 10
4d 12 11 NI 11 10 NI 10 NI NI NI 10
4e NI NI 10 8 NI NI 11 NI NI NI NI
4f 14 14 13 15 12 10 16 12 14 10 NI
4g 17 14 16 12 15 12 15 14 10 NI NI
4h 8 9 13 NI 13 10 NI NI 8 NI NI
4i 17 13 21 21 18 18 17 23 22 13 12
4j NI 14 13 NI NI 12 10 NI NI 10 NI
4k 15 12 21 22 17 18 17 19 21 10 9
4l 12 14 13 18 15 10 12 15 14 13 12
4m 12 10 18 10 15 16 14 22 24 10 11
6a 12 11 9 8 13 9 10 12 9 8 10
6b 14 13 15 13 12 16 12 12 11 9 8
6c 9 10 11 8 10 8 9 NI 8 10 13
6d 13 17 12 10 14 11 10 8 9 10 NI
6e 13 NI NI 9 10 12 16 17 10 9 11
6f 10 12 12 8 15 11 10 9 8 10 10
6g 15 NI NI 10 18 14 16 NI 12 NI 9
6h NI NI 10 8 NI NI NI NI NI NI NI
6i 19 13 21 15 17 15 16 23 25 10 12
6j 9 NI NI NI 8 11 NI 10 NI 12 11
8a NI NI NI NI NI NI 10 9 NI NI NI
8b 13 14 15 21 14 11 16 17 19 10 9
Streptomycin 26 14 30 26 22 18 20 30 30 NA NA
Ketoconazole NA NA NA NA NA NA NA NA NA 28 26

NA e not applicable NI e no inhibition.

d 0.75 (q, 1H, J ¼ 12.1 Hz), 1.24e1.26 (m, 1H), 1.38e1.41 (m, 1H), 1702, 2199, 2930, 3279, 3422; 1H NMR (400 MHz, DMSO-d6):
1.65e1.68 (m, 1H), 2.06e2.08 (m, 1H),2.16e2.20 (m, 1H), 3.11 (d, 1H, d 0.83 (q, 1H, J ¼ 12.4 Hz), 1.40e1.48 (m, 2H), 1.70e1.73 (m, 1H),
J ¼ 12.4 Hz), 3.34e3.37 (m, 1H) 5.46 (brs, 2H, eNH2, D2O 2.03e2.21 (m, 2H), 2.97 (d, 1H, J ¼ 12.0 Hz), 3.17e3.24 (m, 1H),
exchangeable), 5.58e5.60 (m. 1H), 6.45 (d, 1H, J ¼ 8.2 Hz), 6.52 (d, 5.44 (brs, 2H, eNH2, D2O exchangeable), 5.55e5.57 (m. 1H), 6.07
1H, J ¼ 7.6 Hz) 6.96 (t, 1H, J ¼ 7.5 Hz)), 7.03e7.10 (m, 2H), 7.17 (d, 1H, (s, 1H), 6.62 (d, 1H, J ¼ 7.7 Hz) 6.81 (s, 1H), 6.97 (t, 1H, J ¼ 7.5 Hz),
J ¼ 8.4 Hz), 7.30 (d, 1H, J ¼ 7.4 Hz), 7.39 (d, 1H, J ¼ 8.4 Hz), 10.42 (brs, 7.12e7.19 (m, 1H), 7.36 (s, 1H), 10.44 (brs, 1H, eNH, D2O
1H, eNH, D2O exchangeable); 13C NMR (100 MHz, DMSO-d6): exchangeable); 13C NMR (100 MHz, DMSO-d6): d 22.2, 25.5, 28.3,
d 22.2, 25.5, 28.2, 32.4, 52.3, 57.7, 81.9, 110.1, 117.0, 118.4, 120.3, 32.6, 44.1, 57.2, 81.8, 110.1, 116.8, 118.5, 121.2, 122.4, 124.2, 129.3,
122.4, 124.8, 128.9, 129.3, 129.4, 130.4, 131.2, 132.2, 134.1, 137.0, 130.2, 132.1, 141.2, 143.1, 143.3, 154.0, 177.1; MS m/z ¼ 358 [MþH]þ;
142.8, 154.2, 176.5; MS m/z ¼ 446 [MþH]þ; Anal. Calcd for Anal. Calcd for C22H19N3O2: C, 73.93; H, 5.36; N, 11.76. Found: C,
C24H20BrN3O: C, 64.58; H, 4.52; N, 9.41. Found: C, 64.69; H, 4.43; N, 73.85; H, 5.45; N, 11.67.
9.52.
4.1.2.9. 30 -Amino-2-oxo-10 -(thiophen-2-yl)-60 ,70 ,80 ,8a0 -tetrahydro-
4.1.2.7. 30 -Amino-10 -(naphthalen-1-yl)-2-oxo-60 ,70 ,80 ,8a0 -tetrahydro- 10 H-spiro[indoline-3,20 -naphthalene]-40 -carbonitrile (6i). White
10 H-spiro[indoline-3,20 -naphthalene]-40 -carbonitrile (6g). White solid; mp 267e269  C (Decomposes); IR (cm
1): 696, 1203, 1476,
solid; mp 245e247  C (Decomposes); IR (cm
1): 783, 1391, 1580, 1578, 1629, 1701, 2200, 2935, 3278, 3423; 1H NMR (400 MHz,
1631, 1703, 2220, 2948, 3333, 3426; 1H NMR (400 MHz, DMSO-d6): DMSO-d6): d 0.85 (q, 1H, J ¼ 12.2 Hz), 1.39e1.42 (m, 2H), 1.69e1.71
d 0.68 (q, 1H, J ¼ 12.1 Hz), 1.00e1.06 (m, 1H), 1.32e1.34 (m, 2H), (m, 1H), 2.07e2.21 (m, 2H), 3.30 (d, 1H, J ¼ 10.8 Hz), 3.46e3.52 (m,
1.97e2.18 (m, 2H), 3.41e3.46 (m, 1H), 4.24 (d, 1H, J ¼ 12.0 Hz), 5.37 1H), 5.44 (brs, 2H, eNH2, D2O exchangeable), 5.57e5.59 (m. 1H),
(brs, 2H, eNH2, D2O exchangeable), 5.61e5.64 (m. 1H), 6.34 (d, 1H, 6.57 (d, 2H, J ¼ 7.6 Hz), 6.72 (s, 1H) 6.96 (t, 1H, J ¼ 7.4 Hz), 7.10 (t, 1H,
J ¼ 7.7 Hz), 6.60e6.64 (m, 1H) 6.78 (t, 1H, J ¼ 7.6 Hz)), 7.27e7.37 (m, J ¼ 7.6 Hz), 7.15e7.16 (m, 1H), 7.27 (d, 1H, J ¼ 7.4 Hz), 10.48 (brs, 1H,
2H), 7.42e7.48 (m, 2H), 7.54e7.56 (m, 1H), 7.59e7.67 (m, 2H), 8.16 eNH, D2O exchangeable); 13C NMR (100 MHz, DMSO-d6): d 22.2,
(d, 1H, J ¼ 8.0 Hz), 10.56 (brs, 1H, eNH, D2O exchangeable); 13C NMR 25.5, 28.1, 34.6, 48.9, 57.9, 81.8, 110.1, 117.0, 118.4, 122.5, 124.6, 125.5,
(100 MHz, DMSO-d6): d 22.2, 25.5, 27.6, 34.5, 44.6, 58.2, 82.1, 109.7, 126.7, 129.4, 129.7, 132.0, 140.3, 143.2, 153.9, 176.6; MS m/z ¼ 374
116.9, 118.6, 121.9, 124.2, 124.3, 125.3, 125.4, 125.5, 125.8, 126.0, [MþH]þ; Anal. Calcd for C22H19N3OS: C, 70.75; H, 5.13; N, 11.25.
126.6, 127.6, 128.6, 128.7, 129.1, 130.9, 132.7, 132.9, 133.3, 133.8, Found: C, 70.64; H, 5.02; N, 11.34.
135.0, 142.4, 155.1, 177.4; MS m/z ¼ 418 [MþH]þ; Anal. Calcd for
C28H23N3O: C, 80.55; H, 5.55; N, 10.06. Found: C, 80.65; H, 5.44; N,
4.1.2.10. 3 0 -Amino-2-oxo-10 -(pyridin-2-yl)-6 0 ,70 ,8 0 ,8a0 -tetrahydro-
10.17.
10 H-spiro[indoline-3,20 -naphthalene]-40 -carbonitrile (6j). White
solid; mp 270e271  C (Decomposes); IR (cm
1): 751, 1199, 1475,
4.1.2.8. 30 -Amino-10 -(furan-3-yl)-2-oxo-60 ,70 ,80 ,8a0 -tetrahydro-10 H- 1574, 1627, 1699, 2199, 2830, 2937, 3325, 3424; 1H NMR (400 MHz,
spiro[indoline-3,20 -naphthalene]-40 -carbonitrile (6h). White solid; DMSO-d6): d 0.83 (q, 1H, J ¼ 12.0 Hz), 1.00e1.03 (m, 1H), 1.35e1.38
mp 236e238  C (Decomposes); IR (cm
1): 754, 1476, 1583, 1633, (m, 1H), 1.63e1.66 (m, 1H), 2.05e2.20 (m, 2H), 3.28 (d, 1H,
202 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207

Fig. 3. Comparison of antimicrobial activity of synthesized compounds and standard drugs.

Table 6
MIC (mg/ml) of compounds tested against bacteria.

Compounds Minimum inhibitory concentration (mg/ml)

Gram positive bacteria Gram negative bacteria

S. epidermidis S. aureus S. aureus (MRSA) M. luteus E. aerogenes S. typhimurium K. pneumonia P. vulgaris S. flexneri

4a 500 250 250 1000 250 250 250 500 1000

4b 250 125 250 250 250 500 250 125 250
4c 250 250 125 250 125 125 62.5 62.5 62.5
4f 250 250 250 250 250 500 125 250 250
4g 125 250 125 250 250 250 250 250 250
4i 125 250 62.5 62.5 125 125 125 62.5 62.5
4k 125 250 62.5 62.5 125 125 125 125 62.5
4l 250 250 250 125 250 500 250 250 250
4m 250 250 125 250 250 125 250 62.5 62.5
6a 250 250 1000 1000 250 1000 500 250 1000
6b 250 250 250 250 250 125 250 250 250
6d 250 125 250 250 250 250 500 1000 1000
6e 250 NI NI 1000 250 250 125 125 250
6f 500 250 250 1000 250 250 500 1000 1000
6g 125 NI NI 250 125 250 125 NI 500
6i 125 250 62.5 250 125 250 125 62.5 62.5
8b 250 250 250 250 250 250 125 125 125
Streptomycin 6.25 6.25 6.25 6.25 25 30 6.25 6.25 6.25

NI e no inhibition.

J ¼ 12.1 Hz), 3.53e3.61 (m, 1H), 5.39 (brs, 2H, eNH2, D2O 25.4, 27.8, 32.5, 54.8, 56.8, 81.7, 109.7, 116.4, 118.5, 121.9, 122.2, 124.9,
exchangeable), 5.56e5.58 (m. 1H), 6.46 (d, 1H, J ¼ 7.7 Hz), 6.63e6.74 128.7, 129.2, 133.0, 135.7, 143.1, 148.8, 155.1, 157.9, 175.9; HRMS
(m, 1H) 6.86e6.89 (m, 1H), 6.99e7.03 (m, 2H), 7.24 (d, 1H, (ESI): Mass calculated for C23H21N4O [MþH]þ 369.1710, found,
J ¼ 7.3 Hz), 7.30e7.37 (m, 1H), 8.26e8.33 (m, 1H), 10.26 (brs, 1H, [MþH]þ 369.1717; Anal. Calcd for C23H20N4O: C, 74.98; H, 5.47; N,
eNH, D2O exchangeable); 13C NMR (100 MHz, DMSO-d6): d 22.2, 15.21. Found: C, 74.88; H, 5.36; N, 15.10.

Fig. 4. Comparison of MIC (mg/ml) values of synthesized compounds and standard drugs.
 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207 203

Table 7
Anticancer activity of synthesised compounds against A549 cancer cell line and calculated binding energy with ALK receptor of synthesized spirocarbocycles.

Concentration (mg/mL) Cell inhibition

4c 4i 6i

% Mean ± S.D % Mean ± S.D % Mean ± S.D

5 3.7 0.341 ± 0.00406 12.1 0.311 ± 0.00435 25.7 0.263 ± 0.00491

10 10.5 0.317 ± 0.00296 28.1 0.255 ± 0.00648 45.8 0.192 ± 0.00322
20 18.6 0.288 ± 0.00586 47.5 0.186 ± 0.00435 62.4 0.133 ± 0.00296
30 30.8 0.245 ± 0.00346 56.2 0.155 ± 0.00529 67.8 0.114 ± 0.00291
40 46.6 0.189 ± 0.00462 62.4 0.133 ± 0.00520 75.9 0.085 ± 0.00364
50 59.6 0.143 ± 0.00230 78.8 0.075 ± 0.00491 83.9 0.057 ± 0.00462
Free energy of binding (kcal/mol) 4c 4i 6i

14.55
16.13
18.43

4.1.3. Experimental procedure for the synthesis of (8a & 8b) 4.2.2. Tested microbes
1,3-Cyclohaxanedione 7 (1 mmol), aldehydes 2 (1 mmol) 2aeb The following bacteria and fungi were used for the experiment.
were stirred in MeOH in the presence of L-proline (20 mol%) at Bacteria; S. flexneri MTCC 1457, M. luteus MTCC 106, E. aerogenes
room temperature (r.t.) for ten minutes followed by the addition of MTCC 111, S. aureus MTCC 96, K. pneumonia MTCC 109, S. epi-
alkylidene malononitrile 3a (1 mmol) at r.t. for 3 h. The solid dermidis MTCC 3615, P. vulgaris MTCC 1771, S. typhimurium MTCC
precipitated out, and then filtered off and purified by recrystalli- 1251 and S. aureus (MRSA e methicillin resistant). The reference
zation from ethanol to afford product 8a and 8b as white crystalline cultures were obtained from Institute of Microbial Technology
solid. (IMTECH), Chandigarh, India-160 036; fungi: M. pachydermatis and
C. albicans MTCC 227. All the cultures were obtained from the
Department of Microbiology, Christian Medical College, Vellore,
4.1.3.1. 3 0 -Amino-2,6-dioxo-1 0 -p-tolyl-6 0 ,7 0 ,8 0 ,8a 0 -tetrahydro-1 0 H- Tamil Nadu, India.
spiro[cyclohexane-1,20 -naphthalene]-40 -carbonitrile (8a). White
solid; mp 245e246  C (Decomposes); IR (cm
1):822, 1394, 1588, 4.2.3. Preparation of inoculums
1657, 1688, 1717, 2202, 2923, 3250, 3345, 3422; 1H NMR (400 MHz, Bacterial inoculums were prepared by growing cells in Mueller
DMSO-d6): d 0.11 (q, 1H, J ¼ 11.7 Hz), 0.57e0.65 (m, 1H), 1.29e1.32 Hinton broth (MHB) (Himedia) for 24 h at 37  C. The filamentous
(m, 2H), 1.42e1.48 (m, 1H), 1.59e1.64 (m, 1H), 1.95e2.13 (m, 4H), fungi were grown on sabouraud dextrose agar (SDA) slants at 28  C
2.24 (s, 3H), 2.47e2.52 (m, 1H), 2.73e2.82 (m, 1H), 2.89 (s, 2H), for 10 days and the spores were collected using sterile doubled
5.52e5.54 (m, 1H), 5.91 (brs, 2H, eNH2, D2O exchangeable), 6.71 (d, distilled water and homogenized. Yeast was grown on sabouraud
1H, J ¼ 7.6 Hz), 6.83 (d, 1H, J ¼ 8.0 Hz) 7.08 (d, 1H, J ¼ 7.6 Hz), 7.16 (d, dextrose broth (SDB) at 28  C for 48e72 h.
1H, J ¼ 7.6 Hz); 13C NMR (100 MHz, DMSO-d6): d 14.6, 21.1, 22.1,
25.4, 27.5, 33.3, 54.9, 71.2, 83.1, 117.1, 118.2, 127.5, 129.9, 130.1, 131.3,
4.2.4. Disc diffusion assay
132.0, 133.9, 137.8, 154.3, 209.8, 210.9; HRMS (ESI): Mass calculated
Antimicrobial activities were carried out using disc diffusion
for C23H24N2NaO2 [MþNa]þ 383.1730, found, [MþNa]þ 383.1731
method [22]. Petri plates were prepared with 20 mL of sterile
Anal. Calcd for C23H24N2O2: C, 76.64; H, 6.71; N, 7.77. Found: C,
Mueller Hinton Agar (MHA) (Hi-media, Mumbai). The test cultures
76.75; H, 6.60; N, 7.68.
were swabbed on the top of the solidified media and allowed to dry
for 10 min and a specific amount of synthesised compound at 1 mg/
4.1.3.2. 30 -Amino-10 -(4-methoxyphenyl)-2,6-dioxo-60 ,70 ,80 ,8a0 -tetra- disc was added to each disc separately. The loaded discs were
hydro-10 H-spiro[cyclohexane-1,20 -naphthalene]-40 -carbonitrile (8b). placed on the surface of the medium and left for 30 min at room
White solid; mp 241e243  C (Decomposes); IR (cm
1): 830, 1262, temperature for compound diffusion. Negative control was pre-
1512, 1591, 1659, 1684, 1715, 2204, 2951, 3246, 3346, 3416; 1H NMR pared using respective solvents. Streptomycin was used as positive
(400 MHz, DMSO-d6): d 0.24 (q, 1H, J ¼ 13.3 Hz), 0.62 (q, 1H, control against bacteria. Ketoconazole was used as positive control
J ¼ 10.8 Hz), 1.32e1.38 (m, 2H), 1.49e1.55 (m, 1H), 1.63e1.66 (m, for fungi. The plates were incubated for 24 h at 37  C for bacteria
1H), 1.99e2.16 (m, 4H), 2.53e2.58 (m, 1H), 2.76e2.84 (m, 1H), and for 48 h at 28  C for fungi. Zones of inhibition were recorded in
2.88e2.91 (m, 2H), 3.72 (s, 3H), 5.52e5.54 (m, 1H), 5.93 (brs, 2H, millimetres and the experiment was repeated twice.
eNH2, D2O exchangeable), 6.77 (d, 1H, J ¼ 8.2 Hz), 6.87 (t, 2H,
J ¼ 7.8 Hz) 6.94 (d, 1H, J ¼ 8.6 Hz); 13C NMR (100 MHz, DMSO-d6): 4.2.5. Minimum inhibitory concentration (MIC)
d 14.7, 22.1, 25.4, 27.5, 33.4, 54.4, 55.5, 83.1, 114.2, 115.3, 117.1, 118.2, Minimum inhibitory concentration studies of 17 compounds
128.6, 128.7, 132.0, 132.4, 154.3, 159.3, 209.9, 211.0; HRMS (ESI): were performed according to the standard reference methods for
Mass calculated for C23H25N2O3 [MþH]þ 377.1860, found, [MþH]þ antibacterial activity [23]. The required concentrations (1000 mg/
377.1860; Anal. Calcd for C23H24N2O3: C, 73.38; H, 6.43; N, 7.44. mL, 500 mg/mL, 250 mg/mL, 125 mg/mL, 62.5 mg/mL, 31.25 mg/mL,
Found: C, 73.47; H, 6.34; N, 7.54. 15.62 mg/mL and 7.81 mg/mL) of the compound were dissolved in
DMSO (2%), and diluted to give serial two-fold dilutions that were
added to each medium in 96 well plates. An inoculum of 100 ml
4.2. Biological assays from each well was inoculated. The antifungal agent Ketoconazole
for fungi and Streptomycin for bacteria were included in the assay
4.2.1. Materials and methods for antimicrobial activity as positive controls. For fungi, the plates were incubated for
Streptomycin (Sigma) was used as positive control against 48e72 h at 28  C and for bacteria the plates were incubated for 24 h
bacteria. Ketoconazole (Himedia, Mumbai) was used as positive at 37  C. The MIC for fungi was defined as the lowest extract con-
control against fungi. centration, showing no visible fungal growth after incubation time.
204 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207

Fig. 5. Docking of co-crystallized ligand (5a) and standard drug Streptomycin with the DNA gyrase receptor for method validation (5b)

5 ml of tested broth was placed on the sterile MHA plates for bac-
teria 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 cultures on the agar plate.

4.2.6. Cytotoxic properties

A549 lung adenocarcinoma cancer cell line was obtained from
National Institute of Cell Sciences, Pune. A549 cell line was main-
tained in complete tissue culture medium Dulbecco's Modified
Eagle's Medium with 10% Fetal Bovine Serum and 2 mM L-Gluta-
mine, along with antibiotics (about 100 International Unit/mL of
penicillin, 100 mg/mL of streptomycin) with the pH adjusted to 7.2.
The cytotoxicity was determined according to the method of
Fig. 6. Docking of co-crystallized ligand (crizotinib) with the ALK receptor for method Balachandran et al. [24] with some changes. Cells (5000 cells/well)
validation. were seeded in 96 well plates containing medium with different

Fig. 7. 2D and 3D binding mode of most active compound 4i (FEB ¼
11.64 kcal/mol) with DNA gyrase receptor.

Fig. 8. 2D and 3D binding mode of moderate active compound 4m (FEB ¼
9.51 kcal/mol) with DNA gyrase receptor.
 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207 205

Fig. 9. 2D and 3D binding mode of least active compound 6h (FEB ¼
8.14 kcal/mol) with DNA gyrase receptor.

Fig. 10. 2D and 3D binding mode of most active compound 6i (FEB ¼
18.43 kcal/mol) with ALK receptor.

Fig. 11. 2D and 3D binding mode of intermediate active compound 4h (FEB ¼
12.58 kcal/mol) with ALK receptor.

concentrations such as 50, 40, 30, 20, 10 and 5 mg/mL. The cells were 540 nm in an Enzyme linked immune sorbant assay reader. Cyto-
cultivated at 37  C with 5% CO2 and 95% air in 100% relative hu- toxicity of each sample was expressed as the half maximal inhibi-
midity. After various durations of cultivation, the solution in the tory concentration (IC50) value. The IC50 value is the concentration
medium was removed. An aliquot of 100 mL of medium containing of test sample that causes 50% inhibition of cell growth, averaged
1 mg/mL of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazo- from three replicate experiments.
lium bromide was loaded in the plate. The cells were cultured for
4 h and then the solution in the medium was removed. An aliquot of 4.3. Molecular docking studies
100 mL of DMSO was added to the plate, which was shaken until the
crystals were dissolved. The cytotoxicity against cancer cells was Protein and ligand preparation were carried out using MOE
determined by measuring the absorbance of the converted dye at (Molecular Operating Environment) 2011 software tool version 7.1.
206 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207

Fig. 12. 2D and 3D binding mode of least active compound 6h (FEB ¼
11.23 kcal/mol) with ALK receptor.

A PDB entry 2XCS was selected for antibacterial docking study and (b) S.V. Karthikeyan, B.D. Bala, V.P.A. Raja, S. Perumal, P. Yogeeswari, D. Sriram,
Bioorg. Med. Chem. Lett. 20 (2010) 350e353.
was processed with MOE software. The structural issues such as
[5] K.M. Amin, M.M. Kamel, M.M. Anwar, M. Khedr, Y.M. Syam, Eur. J. Med. Chem.
capping, completing residues with missing atoms and selecting 45 (2010) 2117e2131.
appropriate alternate locations were corrected automatically using [6] (a) M.J. Pawar, A.B. Burungale, B.K. Karale, Arkivoc Part XIII (2009) 97e107;
structure preparation application available with the MOE software. (b) B. Thadhaney, D. Sain, G. Pernawat, G.L. Talesara, Indian J. Chem. 49B
(2010) 368e373.
An additional step was carried out to delete extraneous cofactors or [7] (a) M. Li, F.M. Gong, L.R. Wen, Z.R. Li, Eur. J. Org. Chem. (2011) 3482e3490;
unbound water and the co-crystallized ligand. The active site of (b) S. Bruch, S.S.V. Berkel, B. Westermann, Chem. Soc. Rev. 42 (2013)
protein was identified by using MOE's site finder application. Ligand 4948e4962.
[8] (a) Y.C. Chen, D. Xue, J.G. Deng, X. Cui, J. Zhu, Y.Z. Jiang, Tetrahedron Lett. 45
2D structures were drawn using ChemBioDraw Ultra 11.0 (Che- (2004) 1555e1558;
mOffice 2008). Ligand 2D structures were converted to mol2 (b) D. Xue, Y.C. Chen, X. Cui, Q.W. Wang, J. Zhu, J.G. Deng, J. Org. Chem. 70
format using the file convertor Open Babel GUI. Energy minimiza- (2005) 3584e3591.
[9] (a) D. Xue, Y.C. Chen, L.F. Cun, Q.W. Wang, J. Zhu, J.G. Deng, Org. Lett. 7 (2005)
tion was performed to adjust hydrogen and lone pairs and to 5293e5296;
calculate partial charges. The Protein and ligand structures were (b) J.W. Xie, L. Yue, D. Xue, X.L. Ma, Y.C. Chen, Y. Wu, J. Zhu, J.G. Deng, Chem.
energy minimized using of MMFF94x force field implemented in Commun. (2006) 1563e1565;
(c) J.W. Xie, W. Chen, R. Li, M. Zeng, W. Du, L. Yue, Y.C. Chen, Y. Wu, J. Zhu,
MOE. The energy was minimized to the minimum gradient of 0.05. J.G. Deng, Angew. Chem. Int. Ed. 46 (2007) 389e392;
The final refined poses were ranked by the MM/GBVI binding free (d) T.B. Poulsen, C. Alemparte, K.A. Jørgensen, J. Am. Chem. Soc. 127 (2005)
energy estimation. The best docked representation of the ligand 11614e11615;
(e) T.B. Poulsen, M. Bell, K.A. Jørgensen, Org. Biomol. Chem. 4 (2006)
was chosen based on the conformation with lowest binding free
2001e2011.
energy out of 100 docking runs. The final docked conformations [10] (a) M.N. Elinson, A.I. Ilovaisky, V.M. Merkulova, F. Barba, B. Batanero, Tetra-
were within the range of 0.5e1 Å root-mean-square deviation. hedron 69 (2013) 7125e7130;
For the docking of synthesized molecules to the ALK structure (b) Naoto Chatani, Terukiyo Hanafusa, Bull. Chem. Soc. Jpn. 63 (1990)
2134e2135;
with the PDB id 2XP2, the same procedure followed as for the DNA (c) A.K. El-Shafei, A.A. Sultan, A.M. Soliman, E.A. Ahmed, Synth. Commun. 25
gyrase receptor. (1995) 3211e3217.
[11] ^t, Tetrahedron Lett. 22 (1981) 629e632;
(a) J. Ficini, G. Revial, J.P. Gene
(b) R. Kaiser, P. Naegeli, Tetrahedron Lett. 13 (1972) 2009e2012.
Acknowledgements [12] B.M. Trost, D.C. Lee, J. Am. Chem. Soc. 110 (1988) 6556e6558.
[13] B. List, R.A. Lerner, C.F. Barbas, J. Am. Chem. Soc. 122 (2000) 2395e2396.
[14] (a) T.L. Liu, Z.L. He, H.Y. Tao, Y.P. Cai, C.J. Wang, Chem. Commun. 47 (2011)
N.S.P thanks the Council of Scientific and Industrial Research 2616e2618;
(CSIR), New Delhi, India for the research fellowship and SAIF, IITM, (b) B.M. Trost, N. Cramer, S.M. Silverman, J. Am. Chem. Soc. 129 (2007)
12396e12397.
Chennai for single crystal XRD analysis.
[15] (a) S. Chikaoka, A. Toyao, M. Ogasawara, O. Tamura, H. Ishibashi, J. Org. Chem.
68 (2003) 312e318;
(b) M. Inui, A. Nakazaki, S. Kobayashi, Org. Lett. 9 (2007) 469e472;
Appendix A. Supplementary data (c) F. Gonzalez-Lopez de Turiso, D.P. Curran, Org. Lett. 7 (2005) 151e154;
(d) T. Lanza, R. Leardini, M. Minozzi, D. Nanni, P. Spagnolo, G. Zanardi, Angew.
Supplementary data related to this article can be found at http:// Chem. Int. Ed. 47 (2008) 9439e9442.
[16] (a) A. Nandakumar, P. Thirumurugan, P.T. Perumal, P. Vembu,
dx.doi.org/10.1016/j.ejmech.2014.05.065. M.N. Ponnuswamy, P. Ramesh, Bioorg. Med. Chem. Lett. 20 (2010)
4252e4258;
(b) K. Karthikeyan, P.M. Sivakumar, M. Doble, P.T. Perumal, Eur. J. Med. Chem.
References 45 (2010) 3446e3452;
(c) G. Bhaskar, Y. Arun, C. Balachandran, C. Saikumar, P.T. Perumal, Eur. J. Med.
[1] (a) A.S. Girgis, Eur. J. Med. Chem. 44 (2009) 91e100; Chem. 51 (2012) 79e91.
(b) C. Young-Won, S. Angela, S. Bao-Ning, M. Qiuwen, C. Hee-Byung, [17] (a) T.H. Babu, P. Sujeet, D. Muralidharan, P.T. Perumal, Synlett 14 (2010)
R. Soedarsono, K. Leonardus, R. Agus, F. Norman, S. Steven, K. Douglas, J. Nat. 2125e2129;
Prod. 71 (2008) 390e395. (b) T.H. Babu, A.J. Abragam, D. Muralidharan, P.T. Perumal, Tetrahedron Lett.
[2] R. Murugan, S. Anbazhagan, S.S. Narayanan, Eur. J. Med. Chem. 44 (2009) 51 (2010) 994e996;
3272e3279. (c) T.H. Babu, P.T. Perumal, Synlett 3 (2010) 341e344.
[3] S.A. Van der Sar, J.W. Blunt, M.H. Munro, Org. Lett. 8 (2006) 2059e2061. [18] Crystallographic Data for Compound 6a in this Paper have been Deposited
[4] (a) P. Prasanna, K. Balamurugan, S. Perumal, P. Yogeeswari, D. Sriram, Eur. J. with the Cambridge Crystallographic Data Centre as Supplemental Publication
Med. Chem. 45 (2010) 5653e5661; No. CCDC-972722. Copies of the data can be obtained, free of charge on
 N. Sudhapriya et al. / European Journal of Medicinal Chemistry 83 (2014) 190e207 207

application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: þ44 01223 (b) R. Ottana, R. Maccari, J. Mortier, A. Caselli, S. Amuso, G. Camici, A. Rotondo,
336033 or email: [email protected] G. Wolber, P. Paoli, Eur. J. Med. Chem. 71 (2014) 112e127.
[19] B. Matjaz, P. Andrej, R. Miha, A. Gregor, T. Dusan, J. Med. Chem. 55 (2012) [22] C. Balachandran, V. Duraipandiyan, N.A. Al-Dhabi, K. Balakrishna, N.P. Kalia,
6413e6426. V.S. Rajput, I.A. Khan, S. Ignacimuthu, Indian J. Microbiol. 52 (2012) 676e681.
[20] J.J. Cui, M. Tran-Dube, H. Shen, M. Nambu, P.P. Kung, M. Parish, L. Jia, J. Meng, [23] C. Balachandran, V. Duraipandiyan, K. Balakrishna, R. Lakshmi Sundaram,
L. Funk, I. Botrous, M. McTigue, N. Grodsky, K. Ryan, E. Padrique, G. Alton, A. Vijayakumar, S. Ignacimuthu, N.A. Al-Dhabi, Environ. Chem. Lett. 11 (2013)
S. Timofeevski, S. Yamazaki, Q. Li, H. Zou, J. Christensen, B. Mroczhowski, 303e308.
S. Bender, R.S. Kania, M.P. Edwards, J. Med. Chem. 54 (2011) 6342e6363. [24] C. Balachandran, V. Duraipandiyan, S. Ignacimuthu, Asian Pac. J. Trop. Biomed.
[21] (a) C.R. Corbeil, C.I. Williams, P. Labute, J. Comput. Aided Mol. Des. 26 (2012) (2012) 712e716.
775e786;