Synthesis of new class of spirocarbocycle derivatives by multicomponent domino reaction and their evaluation for antimicrobial, anticancer activity and molecular docking studies

Article abstract of DOI:10.1016/j.ejmech.2014.05.065

A series of 25 new spirocarbocycles were synthesized by a three component reaction that involves few cyclic nucleophiles, vinyl malononitriles and aldehydes with variable substitution patterns. All the synthesized compounds were evaluated for their antimi

Full text of DOI:10.1016/j.ejmech.2014.05.065

Accepted Manuscript
Synthesis of new class of spirocarbocycle derivatives by multicomponent domino
reaction and their evaluation for antimicrobial, anticancer activity and Molecular
docking studies
N. Sudhapriya , P.T. Perumal , C. Balachandran , S. Ignacimuthu , M. Sangeetha ,
Mukesh Doble
PII:
S0223-5234(14)00489-9
10.1016/j.ejmech.2014.05.065
EJMECH 7023
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 March 2014
Revised Date: 9 May 2014
Accepted Date: 26 May 2014
Please cite this article as: N. Sudhapriya, P.T. Perumal, C. Balachandran, S. Ignacimuthu, M.
Sangeetha , M. Doble, Synthesis of new class of spirocarbocycle derivatives by multicomponent domino
reaction and their evaluation for antimicrobial, anticancer activity and Molecular docking studies,
European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.065.
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Highlights
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. One pot synthesis of spirocarbocycles was developed using L-proline.
. Ordinary reaction conditions, wide substrate scope, excellent yield (72-90%).
. Most of the compounds exhibited moderate to good activity against bacteria.
. Docking study of ligands with receptor was further supported by in vitro results.
. Compound 6i showed very good affinity towards the ALK receptor.

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Graphical Abstract
Synthesis of spirocarbocycles was achieved by a three component reaction of cyclic nucleophiles, vinyl malononitriles and aldehydes with
variable substitution patterns.

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Synthesis of new class of spirocarbocycle derivatives by multicomponent
domino reaction and their evaluation for antimicrobial, anticancer activity
and Molecular docking studies
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c
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N. Sudhapriya , P.T. Perumal *, C. Balachandran , S. Ignacimuthu , M. Sangeetha , Mukesh
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Doble
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Organic Chemistry Division, CSIR-Central Leather Research Institute, Adyar, Chennai 600
020, India
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Division of Microbiology, Entomology Research Institute, Loyola College, Chennai 600
034, India
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Department of Biotechnology, Indian Institute of Technology, Chennai 600 036, India
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Fax: +91 44 24911589. E-mail: ptperumal@gmail.com
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Abstract:
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A series of 25 new spirocarbocycles were synthesized by a three component reaction that
involves few cyclic nucleophiles, vinyl malononitriles and aldehydes with variable
substitution patterns. All the synthesized compounds were evaluated for their antimicrobial
activity and the compounds showed significant activity. Synthesized compounds 4c, 4i and 6i
showed good anticancer activity against A549 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.
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Keywords: Spirocarbocycle, multicomponent reaction, vinylogous Michael addition,
antimicrobial activity, anticancer activity, Molecular docking.
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*Corresponding author: Tel./fax: +91-44-24913289; email: ptperumal@gmail.com
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1. Introduction
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Spiro compounds exhibit a broad spectrum of important bioactivities such as antitumor [1],
antidiabetic [2], antibacterial [3], antitubercular [4], antiinflammatory [5] activities etc [6]
due to their interesting structures. Spirooxindoles are privileged medicinal scaffolds that are
found in natural and unnatural biologically active compounds [Figure 1]. The key paradigm
of modern drug discovery and the goal of synthetic organic chemistry are being achieved by
the rapid assembly of structurally different compounds. One of the most potential strategies
for the synthesis and library production of these spiro compounds is achieved through
multicomponent reactions (MCRs) [7]. They have been used widely for carbon-carbon bond
formation by synthetic chemists. They also offer an eloquent tool for the one pot synthesis of
distinct and complex molecule as well as small and drug like heterocycles.
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<<Figure 1>>
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Electron-deficient dicyanoalkenes have been reported to behave as good hydride acceptors in
conjugate reduction reactions [8] and also act as versatile direct vinylogous donors in
asymmetric Michael addition reactions with excellent chemo- and stereo selectivity [9]. The
strong electron-withdrawing groups activate the γ-position and further it undergoes Michael
addition. Vinylogous Michael addition was reported as a key step in the preparation of many
spiro compounds [10].
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The methodologies that are accessible for the synthesis of spirocyclic compounds are
alkylations [11], rearrangement reactions [12], cycloadditions [13], transition metal catalyzed
reactions [14] and cleavage of bridged systems [15]. The synthetic and pharmacological
importance of spiro compounds are anchored in their spirocyclic motifs. The captivating
framework of spirooxindoles increased the thirst of chemists in preparing these compounds
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by numerous methods. Our group has always been interested on the synthesis of novel
spirooxindole compounds and screening for their biocidal activity [16].
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2. Result and Discussion
2.1 Chemistry
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The biological importance of the spiro compounds directed us to synthesize spiro-
carbocycles. In continuation of our studies in the area of multicomponent domino reaction
using vinylogous Michael addition methodology [17] we herein report the one pot
multicomponent reaction of vinyl malononitriles with different chalcones and their
antimicrobial, anticancer activity and molecular docking results of ligands to the DNA gyrase
and ALK receptor. To the best of our knowledge, there have been no reports for the synthesis
of the spiro moieties derived from oxindole, indanedione and 1,3-cyclohexanedione
chalcones. In the present study the vinyl malononitriles undergo vinylogous Michael addition
with the in situ generated chalcones followed by intramolecular nucleophilic addition and
isomerization respectively to afford spirocarbocycles in moderate to high yields (Scheme 1).
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<<Scheme 1>>
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We initiated our study by performing the reaction of cyclohexylidene malononitrile
3a with in situ generated indanedione chalcone without any base catalyst and using methanol
as solvent which afforded compound 4a in 40% yield (Table 1, entry 1). Then the
investigatory experiment was performed to improve the yield of the product by varying
reaction conditions viz. solvent, temperature and base catalyst. All the reaction products are
purified by recrystallization using ethanol as a solvent. The observations (Table 1) led us to
the conclusion that the base has an obvious effect on the reaction. Among the selected bases
(Table 1, entries 2-6), commercially available organocatalyst L-proline was proven to be the
most suitable (Table 1, entries 6-9) as it gives single diastereomer. The reaction of 1, 3-
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indandione 1 with aldehyde 2a and alkylidene malononitrile 3a in the presence of L-proline
was completely diastereoselective in affording only the trans diastereomer whereas the other
bases yield mixture of diastereomers.
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Several solvents were investigated (Table 1, entries 6, 7, 10-12) and methanol was the best
one (Table 1, entries 7-9), although ethanol also produced comparable results (Table 1, entry
6). The stoichiometry ratio 15 mol% of the catalyst was chosen to be the best for performing
the reaction since further increase had no impact on the yield of the reaction (Table 1 entries
7-9). Thus the best result was obtained when 1, 3-indandione 1 and aldehyde 2a were stirred
for ten minutes in the presence of 15 mol% of L-proline as a catalyst for the in situ
generation of chalcone followed by the addition of cyclohexylidene malononitrile 3a to
provide a precipitate which was filtered off and recrystallized from ethanol to yield the
desired product 4a in 89% yield.
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<< Table 1>>
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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 activated 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.
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<<Scheme 2>>
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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 1-5) reacted at faster rates compared with those that
substituted with electron withdrawing groups like 2-fluoro and 4-chloro benzaldehydes
(Table 2, entries 6-10). Various aromatic aldehydes such as naphthaldehyde were also
examined (Table 2, entry 11). Besides, our methodology has been used successfully in order
of acid and base sensitive materials such as heteroaromatic aldehydes and corresponding
spirocarbocycles were obtained in excellent yields without the formation of any by products
(Table 2, entries 12, 13). As it is clear from the obtained results, the presented methodology
can be used in oxygen and sulphur containing heteroaromatic aldehydes. Thus the reaction
was found to be general and has a broad scope due to its applicability to a variety of
substrates and the products 4a-n were isolated in excellent yields (79-90%) under milder
reaction conditions. The results are summarized in Table 2.
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<<Scheme 3>>
<<Table 2>>
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To extend the scope of this protocol for this multicomponent reaction, studies were continued
with other chalcones like oxindole chalcones using the same protocol which resulted in the
formation of corresponding spiroxindoles (Scheme 4). The reaction provided spiroxindoles
in good yields (74-86%) and the results are shown in Table 3. The promising results
prompted us to further explore the scope of this protocol. We investigated the reaction of
cyclohexylidene malononitrile with 1, 3-cyclohexanedione chalcone. The spirocarbocycles
(Scheme 5) were obtained in moderate yields (74-76%) and the results are mentioned in
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Table 4.
<<Scheme 4>>
<<Table 3>>
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The structure of all prepared compounds were elucidated with the aid of IR, H and
C
NMR, Mass spectroscopy and elemental analysis data were discussed for a representative
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compound 6a. The IR spectrum of 6a exhibited a sharp peak at 3422 cm which corresponds
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to the -NH stretching of -NH
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group and peaks at 2200 and 1702 cm corresponded to the
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nitrile and amide carbonyls respectively. The H NMR spectra of 6a showed two singlets at δ
5.38 and 10.35 for the -NH and -NH protons (D O exchangeable) respectively, clearly
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indicating the incorporation of both moieties in the product. The doublet appearing for the Ha
proton of the product 6a showed a coupling constant value of 12.4 Hz indicative of the trans
stereochemistry between the H
a
and H
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protons which is also clearly evidenced from the
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single crystal X-ray analysis of compound 6a (Figure 2) [18] which further supports the H
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NMR spectroscopy. In C NMR spectra, the spiro carbon atom displayed a signal at δ 82.0
and the amide carbonyl carbon atom resonated at δ 176.6. The mass spectra also exhibited a
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distinguishing peak at m/z 382 [M+H] . This shows that the reaction of oxindole 5 with
aldehyde 2a and alkylidene malononitrile 3a is completely diastereoselective in affording
only the trans diastereomer. The structures of all spirocarbocycles were consistent with the
above mentioned data.
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<<Figure 2>>
<<Table 4>>
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2.2. Pharmacology
2.2.1 Antimicrobial activity
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In the present work, the antimicrobial activities of 25 synthesized compounds were screened
against nine bacteria and two fungi using in vitro disc diffusion method. The results revealed
that most of the synthesized compounds exhibited antimicrobial activities against E.
aerogens, S. epidermidis, S. aureus (MRSA), S. typhimurium, K. pneumonia and M. luteus.
The results are summarized in Table 5 and Figure 3. Compounds 4b, 4c, 4i and 4k have
shown excellent activities more than the standard drug against both gram-positive and gram-
negative bacteria at 1mg/disc. Moreover the compounds 4a, 4i, 4l, 4m, 6a, 6b, 6i, 6j and 8b
showed good antibacterial activity over the others. All tested compounds showed moderate
antifungal activity against C. albican and M. pachydermatis.
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<<Table 5>>
<<Figure 3>>
The Minimum Inhibitory Concentration (MIC) values of active compounds against bacteria
are given in Table 6 and Figure 4. Significant MIC values were observed against gram
positive and gram negative bacteria. The results revealed that the spirocarbocycles 4c, 4i, 4k
and 6i have shown good antibacterial activity against tested organisms. Among all tested
compounds 4-methyl substituted aromatic ring containing compound 4c has shown
significant MIC values against K. pneumonia, P. vulgaris, S. flexneri, 4-chloro substituted
aromatic ring containing compound 4i is potent against S. aureus (MRSA), P. vulgaris, S.
flexneri and M. luteus. The naphthyl group containing compound 4k is active against S.
aureus (MRSA), S. flexneri and M. luteus. The 2-thiophenyl containing spirooxindole
compound 6i showed significant MIC values against S. aureus (MRSA), P. vulgaris and S.
flexneri.
<<Table 6>>
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<<Figure 4>>
2.2.2. Anticancer activity
Anti cancer activity studies have been performed for the synthesized compounds 4i, 4c and
6i. They showed potent anticancer activity in vitro against A549 lung adenocarcinoma cancer
cell line. Compound 4c showed 59.6% activity at the dose of 50 µg/mL with IC value of 50
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µg/mL. Compound 4i showed 78.8% activity at the dose of 50 µg/mL with IC50 value of 30
µg/mL. Compound 6i showed 83.9% activity at the dose of 50 µg/mL with IC50 value of 20
µg/mL. All concentrations used in the experiment decreased the cell viability significantly
(P<0.05) in a concentration-dependent manner (Table 7).
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<<Table 7>>
2.2.3 Molecular docking studies
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Docking studies were performed to gain insight into the protein inhibitor interactions inside
the enzyme binding sites. Over the past decade DNA gyrase receptor remains one of the most
investigated and validated targets for the development of anti bacterials [19]. Most of the
synthesized spirocarbocyclic compounds have shown significant activity against S. aureus
(MRSA) hence it is thought worthwhile to do docking studies to support the in vitro activity.
All the synthesized new spirocarbocycles were subjected to docking using MOE 2011
software version 7.1. All the prepared compounds were chosen for the docking study of
ligands with the DNA gyrase receptor. To find the potential of these molecules against the
human lung cancer cells the compounds were also docked to the Anaplastic Lymphoma
Kinase (ALK) receptor [20].
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To verify the reproducibility of docking calculations the bound ligand was extracted from the
complexes and submitted for one ligand run calculation. The final docked conformations fall
within 0.5 to 1 Å root-mean-square deviation [21]. Hence it was concluded that this method
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could be used for the docking of other compounds (Figure 5a& b). We have also performed
the docking of the standard ligand Streptomycin with the DNA gyrase receptor for method
validation (Figure 6).
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<<Figure 5>>
<<Figure 6>>
The docked ligand conformations were analyzed in terms of free energy of binding (FEB),
hydrogen bonding and hydrophobic interactions. One hundred (100) docking runs were
performed and the best docked representation of the ligand was selected based on the
conformation with lowest value of FEB.
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Among all compounds docked to the DNA gyrase receptor, compound 4i was the most
active. It had a high binding energy of -11.64 kcal/mol. This compound exactly fits as that of
ligand and shows strong interaction with ARG 1122 aminoacid. (Figure 8)
<<Figure 7>>
The intermediate active compound 4m binds with the DNA gyrase receptor and the
corresponding binding energy is -9.51 kcal/mol. It shows interaction with the aminoacid
GLN 1056 (Figure 8). The least active compound 6h has binding energy value -8.14
kcal/mol, and it shows interaction with ALA 1068 aminoacid (Figure 9).
<<Figure 8>>
<<Figure 9>>
Molecular docking studies of synthesized molecules to the ALK receptor show that, the most
active compound 6i, has a very high binding energy value, -18.43 kcal/mol and it interacts
with the three aminoacids namely, ASP 1249, ASN 1254 and GLY 1272 (Figure10).
Compound 4h, a moderately active compound, has a binding energy value of -12.58 kcal/mol
and it interacts with GLU 1210 as shown in Figure 11. The least active compound 6h has a
binding energy of -11.23 kcal/mol. It interacts with ARG 1209 aminoacid. (Figure 12)
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<<Figure 10>>
<<Figure 11>>
<<Figure 12>>
The preparation of various other spirooxindoles and the detailed study of biological activity
are underway.
3. Conclusion
We have synthesized a new series of 25 spirocarbocycle derivatives through vinylogous
Michael addition. A new, simple, efficient and environmentally benign method involving the
usage of L-proline for the synthesis of spirocarbocycle was developed. By this method, a
diverse spirocarbocycle library has been rapidly constructed with excellent yields without
involving tedious extraction and isolation procedures. All the compounds were evaluated for
their activities against 4 gram positive bacteria, 5 gram negative bacteria and 2 fungi. Most of
the compounds were found to exhibit significant antimicrobial activity. Among them 4c, 4i
and 6i have shown excellent activities and hence are promising candidates as antibacterial
agents. Componds 4c, 4i and 6i also showed good anticancer activity against A549 lung
cancer cell line. The docking scores show that the spirocarbocyclic molecules have good
potential against the human lung cancer cells.
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4. Experimental
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4.1. Chemistry
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 and are uncorrected. Infrared (IR) spectra were recorded on a Perkin Elmer
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FT-IR spectrometer as KBr pellets. H NMR (400 MHz) and C NMR (100 MHz) spectra
were recorded in DMSO-d solutions with TMS as an internal standard on a Brucker Avance
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DPX-400 MHz instrument. Proton chemical shifts (δ) are relative to tetramethylsilane (TMS,
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δ = 0.00) as internal standard and expressed in parts per million. The number of protons (n)
for a given resonance was indicated as nH. Coupling constants (J) are given in hertz. Spin
multiplicities are given as s (singlet), d (doublet), t (triplet) and m (multiplet). Mass spectra
were recorded under HRMS (ESI) using Thermo Scientific Exactive Orbitrap mass
spectrometer and Thermo Finnigan LCQ Advantage MAX 6000 ESI mass spectrometer and
Perkin-Elmer GC-MS. Elemental analysis data were recorded using Thermo Finnigan
FLASH EA 1112 CHN analyzer.
4.1.1. Experimental procedure for the synthesis of (4a-m)
Indanedione 1 (1 mmol), aldehydes 2 (1 mmol) 2a-h were stirred in MeOH in the presence of
L-proline (20 mol%) at room temperature (r.t.) for ten minutes followed by the addition of
alkylidene malononitrile 3a-c (1 mmol) at r.t. for 3 h. The solid precipitated out, and then
was filtered off and purified by recrystallization from ethanol to afford product 4a-m as
yellow crystalline solid.
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4.1.1.1
3'-amino-1,3-dioxo-1'-p-tolyl-1,3,6',7',8',8a'-hexahydro-1'H-spiro[indene-2,2'-
naphthalene]-4'-carbonitrile (4a)
o
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Yellow solid; mp: 224-226 C (Decomposes); IR (cm ): 755, 1246, 1589, 1661, 1704, 1742,
1
2205, 2921 3246, 3345, 3408. H NMR (400 MHz, DMSO-d
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): δ .0.71 (q, 1H, J =12.4Hz),
1.32 – 1.42 (m, 2H), 1.64 – 1.66 (m, 1H), 1.99 (s, 3H), 2.08 – 2.20 (m, 2H), 2.98 (d, 1H, J =
12.8Hz), 3.04 – 3.07 (m, 1H), 5.59 – 5.61 (m, 1H), 6.12 (brs, 2H, -NH , D O exchangeable),
2
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6.61 (d, 1H, J = 7.6 Hz), 6.72 – 6.79 (m, 3H), 7.67 (d, 1H, J = 7.6 Hz), 7.75 - 7.77 (m, 3H).
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C NMR (100MHz, DMSO-d ): δ 20.0, 22.1, 25.4, 27.9, 33.5, 52.2, 63.6, 82.9, 117.4, 118.1,
123.1, 123.7, 126.6, 129.0, 129.2, 129.2, 131.4, 131.8, 133.1, 136.4, 136.6, 142.7, 143.3,
+
151.9, 199.7, 200.2. HRMS (ESI): Mass calculated for C26
H
22
N
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O
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Na [M+Na] 417.1573,
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found, [M+Na] , 417.1573. Anal. Calcd. For: (C H N O ) C, 79.16; H, 5.62; N, 7.10
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Found: C, 79.05; H, 5.73; N, 7.01.
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4.1.1.2.2
6-amino-1',3'-dioxo-4-p-tolyl-1',2,3,3a,3',4-hexahydro-2,5'-spirobi[indene]-7-
carbonitrile (4b)
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Yellow solid; mp 215-216 C (Decomposes); IR (cm ): 790, 1244, 1573, 1664, 1703, 1741,
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2206, 2925, 3246, 3347 3406. H NMR (400 MHz, DMSO-d ): δ 0.98 (q, 1H, J = 10.4), 1.67
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– 1.74 (m, 1H), 1.98 (s, 3H), 2.22 – 2.34 (m, 2H), 3.03 (d, 1H, J = 12.4 Hz), 3.50 – 3.55 (m,
1H), 5.33 – 5.35 (m, 1H), 6.48 (brs, 2H, NH2,
D
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O exchangeable), 6.72 – 6.77 (m, 4H), 7.69
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): δ 20.8, 30.4, 31.3, 42.4, 52.8, 65.2, 78.4,
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– 7.75 (m, 4H); C NMR (100 MHz, DMSO-d
116.0, 117.9, 123.2, 123.7, 129.1, 133.1, 136.4, 136.6, 136.9, 138.5, 142.3, 143.5, 154.6,
+
199.4, 199.9. MS m/z = 381 [M+H] ; Anal. Calcd for C H N O : C, 78.93; H, 5.30; N, 7.36
2
5
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Found: C, 79.94; H, 5.21; N, 7.24.
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4.1.1.3. 3-amino-1',3'-dioxo-1-p-tolyl-1,1',3',6,7,8,9,9a-octahydrospiro[benzo[7]annulene-
2,2'-indene]-4-carbonitrile (4c)
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2
2
2
3
4
5
6
7
8
9
0
1
2
Yellow solid; mp 210-212 C (Decomposes); IR (cm ): 754, 1252, 1590, 1637, 1704, 1741,
1
2200, 2925, 3253, 3366, 3535. H NMR (400 MHz, DMSO-d ): δ 1.14-1.24 (m, 3H), 1.34-
6
1.36 (m, 1H).1.63-1.64 (m, 2H), 1.99 (s, 3H), 2.18-2.31 (m, 2H), 3.13(d, 1H, J = 11.6 Hz),
3.49-3.50 (m, 1H prton merged with solvent peak), 5.76 (t, 1H, J = 6.2 Hz), 6.08(brs, 2H,
NH
2 2
, D O exchangeable)), 6.70 (d, 2H, J = 8.4 Hz), 6.79 (d, 2H, J =8.0Hz), 7.63-7.66 (m,
1
3
1H), 7.74-7.81 (m, 3H); C NMR (100 MHz, DMSO-d
6
): δ 20.7, 25.9, 27.9, 29.8, 31.8, 38.0,
52.2,63.0, 83.3, 118.6, 121.2, 123.1, 124.0, 129.2, 134.6, 136.5, 136.7, 136.9, 137.3, 142.2,
+
143.1, 152.3, 199.0, 199.4. HRMS (ESI): Mass calculated for C H N O [M+H] 409.1911
27
25
2
2
+
found, [M+H] 409.1916; Anal. Calcd for C27
24
H N
2
O
2
: C, 79.39; H, 5.92; N, 6.86;. Found: C,
79.28; H, 6.02; N, 7.94.
2
2
3
4
4.1.1.4.
3'-amino-1'-(4-methoxyphenyl)-1,3-dioxo-1,3,6',7',8',8a'-hexahydro-1'H-
spiro[indene-2,2'-naphthalene]-4'-carbonitrile (4d)
1
2

ACCEPTED MANUSCRIPT
1
3
o
-1
1
2
3
4
5
6
7
8
9
Yellow solid; mp 194-197 C (Decomposes) ; IR (cm ): 758, 1246, 1588, 1656, 1702, 1739,
1
2202, 2927, 3250, 3343, 3429. H NMR (400 MHz, DMSO-d
6
): δ 0.69 (q, 1H, J = 12.4 Hz),
1.13-1.17 (m, 2H),1.61-1.67 (m, 1H), 2.14-2.22 (m, 2H),2.95 (d, 1H, J = 12.4 Hz), 3.37-3.43
(m, 1H), 3.48 (s, 3H), 5.60-5.62 (m, 1H), 5.94 (brs, 2H, -NH , D O exchangeable), 6.49-6.52
2
2
(m, 2H), 6.64 (d,1H, J = 8 Hz), 6.74 (d,1H, J=8.8 Hz), 7.67 (d, 1H, J = 7.2 Hz), 7.73-7.78 (m,
1
3
6
3H); C NMR (100 MHz, DMSO-d ): δ 22.1, 25.3, 27.7, 30.4, 34.5, 55.3, 63.7, 85.1, 113.6,
114.7, 123.3, 127.5, 131.4, 134.2, 136.8, 140.4, 146.8, 158.3, 172.2, 197.8, 199.0; MS m/z =
+
411 [M+H] ; Anal. Calcd for C26
22 2 3
H N O : C, 76.08; H, 5.40; N, 6.82 Found: C, 76.19; H,
5.30; N, 6.71.
1
1
0
1
4.1.1.5.
6-amino-4-(4-methoxyphenyl)-1',3'-dioxo-1',2,3,3a,3',4-hexahydro-2,5'-
spirobi[indene]-7-carbonitrile (4e)
o
-1
1
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
Yellow solid; mp 206-209 C (Decomposes) ; IR (cm ):1254, 1512, 1573, 1661, 1702, 1739,
1
2204, 2932, 3244, 3345, 3413; H NMR (400 MHz, DMSO-d
6
): δ 0.23-0.28 (m, 1H), 0.98-
1.05 (m, 1H), 1.57-1.63 (m, 2H), 2.34 (d, 1H, J = 7.6 Hz), 2.77 (s, 3H), 2.82-2.90 (m, 1H),
4.65-4.67 (m, 1H), 5.62 (brs, 2H, -NH , D O exchangeable), 5.80 (d , 2H, J = 8 Hz), 6.04-
6.08 (m, 2H),7.00-7.06 (m, 4H); C NMR (100 MHz, DMSO-d ): δ 30.4, 31.3, 42.5, 46.4,
2
2
1
3
6
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 m/z = 397 [M+H] ; Anal. Calcd for C25
H
20
2
N O
3
, C, 75.74; H, 5.08;
N, 7.07 Found: C, 75.83; H, 5.19; N, 7.17.
2
2
0
1
4.1.1.6. 3'-amino-1'-(2-fluorophenyl)-1,3-dioxo-1,3,6',7',8',8a'-hexahydro-1'H-spiro[indene-
2,2'-naphthalene]-4'-carbonitrile (4f)
o
-1
2
2
2
2
3
4
Yellow solid; mp 222-224 C (Decomposes) ; IR (cm ): 759, 1247, 1588, 1662, 1704, 1742,
1
2205, 2919, 3249, 3349, 3410; H NMR (400 MHz, DMSO-d
6
): δ 0.66 ( q, 1H, J = 12.2 Hz),
1.26-1.34 (m, 2H) 1.62-1.65 (m, 1H), 2.05-2.17 (m, 2H), 3.44 (d, 1H, J = 12.5 Hz), 4.48-
1
3

ACCEPTED MANUSCRIPT
1
4
1
2
3
4
5
6
4.51 (m, 1H), 5.62-5.64 (m, 1H), 6.06 (brs, 2H, -NH
2H), 6.87-6.90 (m, 1H), 6.93-6.96 (m, 1H), 7.70 (d, 1H, J = 5 Hz), 7.73-7.76 (m, 3H); C
NMR (100 MHz, DMSO-d ): δ 21.7, 25.3, 27.9, 33.4, 42.9, 63.1, 83.6, 118.1, 118.8, 122.9,
2 2
, D O exchangeable), 6.79-6.84 (m,
13
6
123.6, 124.5, 129.2, 130.2, 131.4, 137.3, 142.8, 151.9, 159.2, 198.8, 200.1; MS m/z =399
+
[M+H] ; Anal. Calcd for C H FN O : C, 75.36; H, 4.81; N, 7.03 Found: C, 75.47; H, 4.72;
25
19
2
2
N, 7.12.
7
8
4.1.1.7. 6-amino-4-(2-fluorophenyl)-1',3'-dioxo-1',2,3,3a,3',4-hexahydro-2,5'-spirobi[indene]-
7-carbonitrile (4g)
o
-1
9
0
1
2
3
4
5
6
7
Yellow solid; mp 222-225 C (Decomposes); IR (cm ): 761, 1245, 1573, 1646, 1708, 1742,
1
1
1
1
1
1
1
1
1
2199, 2829, 3258, 3359, 3445; H NMR (400 MHz, DMSO-d
6
): δ 0.25 (q, 1H, J =11.6 Hz),
1.03-1.04 (m, 1H), 1.57-1.66 (m, 2H), 2.42 (d, 1H, J = 12.4 Hz), 2.91-2.97 (m, 1H), 4.71-
4.72 (m, 1H), 5.78 (brs, 2H, -NH , D O exchangeable), 6..09 (t, 1H, J = 9.2 Hz), 6.16-6.20
2
2
1
3
(m, 1H), 6.27-6.34 (m, 2H), 7.00-7.01 (m, 1H), 7.08 (d, 3H, J = 2.9 Hz); C NMR (100
MHz, DMSO-d ): δ 35.0, 36.0, 47.2, 48.9, 69.5, 83.4, 120.1, 121.8, 122.6, 128.1, 128.3,
6
129.6, 133.9, 135.1, 141.6, 141.7, 142.2, 147.0, 147.6, 159.2, 163.2, 203.5, 204.0; MS m/z =
+
385 [M+H] ; Anal. Calcd for C24
2 2
H17FN O : C, 74.99; H, 4.46; N, 7.29. Found: C, 75.17; H,
4.35; N, 7.38.
1
1
8
9
4.1.1.8. 3'-amino-1'-(4-chlorophenyl)-1,3-dioxo-1,3,6',7',8',8a'-hexahydro-1'H-spiro[indene-
2,2'-naphthalene]-4'-carbonitrile (4h)
o
-1
2
2
2
2
2
0
1
2
3
4
Yellow solid; mp 246-248 C (Decomposes); IR (cm ): 833, 1093, 1244, 1590, 1636, 1697,
1
1738, 2201, 2923, 3251, 3360, 3452; H NMR (400 MHz, DMSO-d
6
): δ 0.73 (q, 1H, J = 12.4
Hz), 1.24-1.32 (m, 1H), 1.40-1.43 (m, 1H), 1.64-1.67 (m, 1H), 1.97-2.07 (m, 1H), 2.15-2.20
(m, 1H), 3.06 (d, 1H, J = 12.4 Hz) 3.09-3.10 (m, 1H), 5.60-5.62 (m, 1H), 6.19 (brs, 2H, -
NH , D O exchangeable), 6.76 (d, 1H, J = 7.2 Hz), 6.86(d, 1H, J = 7.6 Hz), 7.04 (d, 2H, J =
2
2
1
4

ACCEPTED MANUSCRIPT
1
5
1
3
1
2
3
4
6
8.4 Hz), 7.69 (d, 1H, J = 7.6 Hz), 7. 78 (q, 3H, J = 7.0 Hz; C NMR (100 MHz, DMSO-d ):
δ 22.0, 25.3, 27.7, 33.3, 51.7, 63.5, 82.8, 117.7, 118.0, 123.2, 123.8, 128.5, 128.6, 128.7,
+
131.4, 132.3, 135.2, 136.7, 136.8, 142.5, 143.2, 151.6, 199.5, 200.0; MS m/z = 415 [M+H] ;
Anal. Calcd for C25
H19ClN
2
O
2
: C, 72.37; H, 4.62; N, 6.75 Found: C, 72.28; H, 4.73; N, 6.65.
5
6
7
4.1.1.9.
3-amino-1-(4-chlorophenyl)-1',3'-dioxo-1,1',3',6,7,8,9,9a-
octahydrospiro[benzo[7]annulene-2,2'-indene]-4-carbonitrile (4i)
o
-1
8
9
0
1
2
3
4
5
6
Yellow solid; mp 180-181 C (Decomposes); IR (cm ): 833, 1091, 1490, 1590, 1635, 1700,
1
1741, 2199, 2929, 3366, 3455; H NMR (400 MHz, DMSO-d ): δ 0.50 (m, 3H), 1.34-1.38
6
1
1
1
1
1
1
1
(m, 1H), 1.66-1.69 (m, 2H), 2.02-2.05 (m, 1H), 2.36-2.40 (m, 1H), 2.51 (d, 1H, J = 11.7 Hz)
2.98-3.00 (m, 1H), 5.07 (t, 1H, J = 5.78 Hz), 5.27 (brs, 2H, -NH
6.15 (m, 3H), 6.47(d, 1H, J = 8.2 Hz), 6.96 (d, 1H, J = 7.4 Hz), 7.07-7.12 (m, 3H); C NMR
(100 MHz, DMSO-d ): δ 25.6, 27.8, 29.6, 30.1, 36.3, 52.4, 62.9, 83.4, 118.5, 122.1, 123.3,
2 2
, D O exchangeable), 6.09-
13
6
123.9, 126.4, 128.4, 129.3, 132.5, 136.5, 137.1, 141.9, 143.0, 151.9, 199.0, 199.1; MS m/z =
+
429 [M+H] ; Anal. Calcd for C H ClN O : C, 72.81; H, 4.94; N, 6.53. Found: C, 72.75; H,
2
6
21
2
2
5.15; N, 6.42.
1
1
7
8
4.1.1.10.
3'-amino-1'-(4-bromophenyl)-1,3-dioxo-1,3,6',7',8',8a'-hexahydro-1'H-
spiro[indene-2,2'-naphthalene]-4'-carbonitrile (4j)
o
-1
1
2
2
2
2
9
0
1
2
3
Yellow solid; mp 254-256 C (Decomposes); IR (cm ): 805, 1243, 1589, 1636, 1696, 1738,
1
2200, 2948, 3250, 3361, 3452; H NMR (400 MHz, DMSO-d
6
): δ 0.72 (q, 1H, J = 12 Hz),
1.30 (d, 1H, J = 11.2 Hz), 1.39-1.42 (m, 1H), 1.65(d, 1H, J = 9.2 Hz), 2.06-2.07 (m, 1H),
2.15-2.20 (m, 1H), 3.03 (d, 1H, J = 11.2 Hz) 3.34-3.41 (m, 1H), 5.60-5.62 (m, 1H), 6.18 (brs,
2 2
2H, -NH , D O exchangeable), 6.70 (d, 1H, J = 7.6 Hz), 7.17 (d, 1H, J = 8.4 Hz), 7.69 (d,
1
5

ACCEPTED MANUSCRIPT
1
6
1
3
1
2
3
4
5
6
2H, J = 8.4 Hz), 7.69(d, 1H, J = 7.6 Hz),7.74-7.81(m, 3H); C NMR (100 MHz, DMSO-d ):
δ 22.0, 25.3, 27.7, 33.3, 51.8, 63.4, 82.8, 117.7, 118.0, 120.9, 123.3, 123.8, 129.0,
131.4,131.5, 133.5, 135.6, 136.7, 136.8, 142.5, 143.2, 151.6, 199.5, 200.0; MS m/z = 459
+
[M+H] ; Anal. Calcd for C H BrN O : C, 65.37; H, 4.17; N, 6.10. Found: C, 65.26; H,
25
19
2
2
4.27; N, 6.20.
6
7
4.1.1.11.
3'-amino-1'-(naphthalen-1-yl)-1,3-dioxo-1,3,6',7',8',8a'-hexahydro-1'H-
spiro[indene-2,2'-naphthalene]-4'-carbonitrile (4k)
o
-1
8
9
0
1
2
3
4
5
6
Yellow solid; mp 213-215 C (Decomposes); IR (cm ): 774, 1256, 1488, 1585, 1633, 1721,
1
2195, 2926, 3420, 3466; H NMR (400 MHz, DMSO-d
6
): δ 0.65 (q, 1H, J = 12.1 Hz), 1.23-
1
1
1
1
1
1
1
1.26 (m, 1H), 1.73-1.79 (m, 2H), 2.13-2.22 (m, 1H), 2.37 (d, 1H, J = 9.4 Hz), 2.65 (d, 1H, J
= 10.8 Hz) 3.14-3.20 (m, 1H), 5.65-5.67 (m, 1H), 6.10 (brs, 2H, -NH , D O exchangeable),
7.09-7.11 (m, 2H), 7.23-7.28(m, 2H), 7.45-7.51(m, 2H), 7.63-7.70 (m, 4H), 7.79-7.82(m,
2
2
1
3
6
1H); C NMR (100 MHz, DMSO-d ): δ, 27.3, 35.1,36.9, 44.9, 52.5, 63.8, 83.2, 118.0,
121.8,123.0, 123.1, 123.7, 125.1, 126.0, 126.7, 128.8, 129.3, 132.1, 133.7, 136.1, 142.7,
+
152.3, 167.6, 199.5, 200.5; MS m/z = 431 [M+H] ; Anal. Calcd for C29
H
22
N
2
O
2
: C, 80.91; H,
5.15; N, 6.51. Found: C, 80.82; H, 5.26; N, 6.60.
1
1
7
8
4.1.1.12. 3'-amino-1'-(furan-3-yl)-1,3-dioxo-1,3,6',7',8',8a'-hexahydro-1'H-spiro[indene-2,2'-
naphthalene]-4'-carbonitrile (4l)
o
-1
1
2
2
2
2
2
9
0
1
2
3
4
Yellow solid; mp 247-248 C (Decomposes); IR (cm ): 1243, 1589, 1660, 1704, 2206, 2923,
1
3253, 3347, 3422; H NMR (400 MHz, DMSO-d
6
): δ 0.79 (q, 1H, J = 12 Hz), 1.41-1.43 (m,
2H), 1.69-1.72 (m, 1H), 1.94-2.19 (m, 2H), 2.87-2.92 (m, 1H), 2.98 (d, 1H, J = 12.4 Hz),
5.57-5.59 (m, 1H),5.87(s,1H) 6.14 (brs, 2H, -NH , D O exchangeable), 7.13 (s, 1H), 7.20(s,
1H), 7.79-7.80(m, 1H), 7.84-7.85(m,1H); C NMR (100 MHz, DMSO-d ): δ 26.7, 30.1,
32.6, 38.2, 47.6, 67.5, 87.6, 122.2, 122.8, 125.1, 128.1, 128.6, 136.2, 141.5, 146.5, 147.4,
2
2
13
6
1
6

ACCEPTED MANUSCRIPT
1
7
+
1
2
18 2 3
148.1, 148.9, 156.3, 204.7, 204.9; MS m/z = 371 [MH] ; Anal. Calcd for C23H N O : C,
74.58; H, 4.90; N, 7.56. Found: C, 74.48; H, 4.79; N, 7.65
3
4
4.1.1.13. 3'-amino-1,3-dioxo-1'-(thiophen-2-yl)-1,3,6',7',8',8a'-hexahydro-1'H-spiro[indene-
2,2'-naphthalene]-4'-carbonitrile (4m)
o
-1
5
6
7
8
9
0
1
2
Yellow solid; mp 248-250 C (Decomposes); IR (cm ): 1244, 1587, 1662, 1704, 1741, 2205,
1
2923, 3250, 3347, 3411; H NMR (400 MHz, DMSO-d
6
): δ 0.83 (q, 1H, J = 12.1 Hz), 1.39-
1.48 (m, 2H), 1.68-1.69 (m, 1H), 2.08-2.21 (m, 2H), 2.93-2.99 (m, 1H), 3.35-3.40(m, 1H),
5.59-5.61 (m, 1H),6.15 (brs, 2H, -NH , D O exchangeable), 6.55-6.61(m, 2H), 7.08-7.09(m,
1H), 7.75-7.82(m, 4H); C NMR (100 MHz, DMSO-d ): δ 22.0, 25.4, 27.7, 35.5, 47.6, 63.3,
2
2
13
6
1
1
1
82.8, 117.8, 118.0, 123.4, 124.0, 131.2, 136.6, 136.8, 142.8, 143.3, 151.3, 199.7, 200.0; MS
+
m/z = 387 [M+H] ; Anal. Calcd for C23
H
18
N
2
O
2
S: C, 71.48; H, 4.69; N, 7.25. Found: C,
71.36; H, 4.80; N, 7.16
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
0
4.1.2. Experimental procedure for the synthesis of (6a-j)
Oxindole 5 (1 mmol), aldehydes 2 (1 mmol) 2a-j were stirred in MeOH in the presence of L-
proline (20 mol%) at room temperature (r.t.) for ten minutes followed by the addition of
alkylidene malononitrile 3a-c (1 mmol) at r.t. for 5 h. The solid precipitated out was filtered
off and purified by recrystallization from ethanol to afford product 6a-j as white crystalline
solid.
4.1.2.1.
3'-amino-2-oxo-1'-p-tolyl-6',7',8',8a'-tetrahydro-1'H-spiro[indoline-3,2'-
naphthalene]-4'-carbonitrile (6a)
o
-1
2
2
2
2
1
2
3
4
White solid; mp 248-249 C (Decomposes); IR (cm ):752, 1191, 1388, 1580, 1627, 1702,
1
2200, 2926. 3057, 3299, 3329, 3422; H NMR (400 MHz, DMSO-d
6
): δ 0.72 (q, 1H, J = 12.1
Hz), 1.26-1.29 (m, 1H), 1.37-1.41 (m, 1H), 1.64-1.67 (m, 1H), 2.03-2.07(m, 1H), 2.12 (s,
3H), 2.16-2.21 (m, 1H), 3.02 (d, 1H, J = 12.4 Hz), 3.37-3.41 (m, 1H), 5.38 (brs, 2H, -NH2,
1
7

ACCEPTED MANUSCRIPT
1
8
1
2
3
4
5
6
7
D
2
O exchangeable), 5.58-5.60 (m. 1H), 6.37 (d, 1H, J = 8.8 Hz), 6.49 (d, 1H, J = 8Hz), 6.66
(d, 1H, J = 7.6 Hz), 6.93-7.00 (m, 2H), 7.06 (t, 1H, J = 7.6 Hz), 7.13 (d, 1H, J = 8Hz), 7.28
13
2
(d, 1H, J = 7.6 Hz), 10.35 (brs, 1H, -NH, D O exchangeable); C NMR (100 MHz, DMSO-
d6): δ 20.9, 22.3, 25.5 28.3, 32.7, 52.6, 57.9, 82.0, 110.0, 116.8, 118.5, 122.2, 124.7, 126.4,
128.1, 128.9, 129.2, 129.7, 132.0, 132.6, 134.5, 135.9, 142.9, 154.5, 176.6; HRMS (ESI):
+
Mass calculated for C26
H
22
N
2
O
2
Na [M+Na] : 404.1733, found: 404.1734; Anal. Calcd for
C
25
H
23
N
3
O: C, 78.71; H, 6.08; N, 11.02. Found: C, 78.60; H, 6.17; N, 11.12.
8
9
4.1.2.2.
3-amino-2'-oxo-1-p-tolyl-1,6,7,8,9,9a-hexahydrospiro[benzo[7]annulene-2,3'-
indoline]-4-carbonitrile (6b)
o
-1
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
White solid; mp 238-240 C (Decomposes); IR (cm ):755, 1475, 1577, 1622, 1693, 1716,
1
2200, 2927, 3263, 3350, 3460; H NMR (400 MHz, DMSO-d
6
): δ 1.01 (q, 1H, J = 11.3 Hz),
1.40-1.49 (m, 1H), 1.60-1.67 (m, 3H), 1.71-1.74 (m, 1H), 2.10(s, 3H), 2.24-2.28 (m, 1H),
2.67-2.69 (m, 1H), 3.20 (d, 1H, J = 12.2 Hz), 3.27-3.28 (m, 1H), 5.26 (brs, 2H, -NH , D
exchangeable), 5.70-5.74 (m. 1H), 6.92-6.97 (m, 1H), 6.99-7.03 (m, 2H), 7.05-7.10 (m, 2H),
2
2
O
13
7.14-7.21 (m, 1H), 7.33-7.40 (m, 2H), 10.42 (brs, 1H, -NH, D
2
O exchangeable); C NMR
(100 MHz, DMSO-d ): δ 20.9, 26.0, 28.1, 30.3, 32.3, 37.7, 52.6, 57.6, 82.5, 107.5, 110.0,
6
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 = 396 [M+H] ; Anal. Calcd for C26
H
25
N
3
O: C,
78.96; H, 6.37; N, 10.62. Found: C, 78.87; H, 6.45; N, 10.53
2
2
0
1
4.1.2.3. 3'-amino-1'-(4-methoxyphenyl)-2-oxo-6',7',8',8a'-tetrahydro-1'H-spiro[indoline-3,2'-
naphthalene]-4'-carbonitrile (6c)
o
-1
2
2
2
2
3
4
White solid; mp 241-243 C (Decomposes); IR (cm ): 833, 1185, 1266, 1512, 1573,1618,
1
1701, 2195, 2224, 2952, 3276, 3324, 3425; H NMR (400 MHz, DMSO-d
6
): δ 0.72 (q, 1H, J
= 12.1 Hz), 1.27-1.30 (m, 1H), 1.37-1.50 (m, 1H), 1.65-1.74 (m, 1H), 2.06-2.08(m, 1H),
1
8

ACCEPTED MANUSCRIPT
1
9
1
2
3
4
5
6
7
8
2.16-2.21 (m, 1H), 3.01 (d, 1H, J = 12.8 Hz), 3.31-3.34 (m, 1H),3.60 (s,3H), 5.43 (brs, 2H, -
NH , D O exchangeable), 5.57-5.59 (m. 1H), 6.39 (s, 2H), 6.50 (d, 1H, J = 7.6Hz), 6.76 (d,
1H, J = 8.5 Hz), 6.93-6.97 (m, 1H), 7.05-7.09 (m, 1H), 7.14 (d, 1H, J = 8.6 Hz), 7.28 (d, 1H,
2
2
1
3
J = 7.3 Hz), 10.37 (brs, 1H, -NH, D O exchangeable); C NMR (100 MHz, DMSO-d ): δ
2
6
22.3, 25.5, 28.3, 32.8, 52.2, 55.2, 58.0, 81.8, 110.0, 112.6, 113.9, 114.3, 116.6, 118.5, 122.2,
124.7, 127.6, 129.2, 129.3, 129.7, 129.8, 132.6, 133.1, 142.9, 154.5, 158.1, 176.7; MS m/z =
+
398 [M+H] ; Anal. Calcd for C25
23 3 2
H N O : C, 75.54; H, 5.83; N, 10.57. Found: C, 75.45; H,
5.72; N, 10.66.
9
0
4.1.2.4. 3'-amino-1'-(2-fluorophenyl)-2-oxo-6',7',8',8a'-tetrahydro-1'H-spiro[indoline-3,2'-
1
naphthalene]-4'-carbonitrile (6d)
o
-1
1
1
1
1
1
1
1
1
1
2
2
1
2
3
4
5
6
7
8
9
0
1
White solid; mp 265-267 C (Decomposes); IR (cm ):753, 1203, 1227, 1489, 1577, 1625,
1
1701, 2198, 2831, 2939, 3335, 3427; H NMR (400 MHz, DMSO-d
6
): δ 0.74 (q, 1H, J = 12.0
Hz), 1.20-1.23 (m, 1H), 1.36-1.44 (m, 1H), 1.65-1.67 (m, 1H), 2.04-2.21(m, 2H),3.39-3.43
(m, 1H), 3.50 (d, 1H, J = 12.5 Hz), 5.52 (brs, 2H, -NH , D O exchangeable), 5.59-5.61 (m.
2
2
1H), 6.50 (d, 1H, J = 7.7 Hz)), 6.77 (t, 1H, J = 9.0 Hz), 6.92 (t, 1H, J = 7.6 Hz), 7.04-7.10
(m, 3H), 7.21 (d, 1H, J = 7.4 Hz), 7.31 (t, 1H, J = 7.4 Hz), 10.56 (brs, 1H, -NH, D
2
O
13
6
exchangeable); C NMR (100 MHz, DMSO-d ): δ 22.1, 25.4, 28.0, 32.5, 43.5, 57.4, 81.7,
109.8, 115.0, 115.2, 117.0, 118.4, 122.2, 124.6, 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 C H FN NaO [M+Na]
2
4
20
3
+
408.1488, found, [M+Na] 408.1489. Anal. Calcd for C24
H20FN
3
O: C, 74.79; H, 5.23; N,
10.90. Found: C, 74.68; H, 5.14; N, 10.79.
2
2
2
3
4.1.2.5. 3'-amino-1'-(4-chlorophenyl)-2-oxo-6',7',8',8a'-tetrahydro-1'H-spiro[indoline-3,2'-
naphthalene]-4'-carbonitrile (6e)
o
-1
2
4
White solid; mp 258-259 C (Decomposes); IR (cm ): 747, 1487, 1579, 1629, 1725, 2195,
1
9

ACCEPTED MANUSCRIPT
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0
1
1
2
3
4
5
6
7
8
2923, 3334, 3453; H NMR (400 MHz, DMSO-d
(m, 1H), 1.38-1.42 (m, 1H), 1.66-1.68 (m, 1H), 2.04-2.08(m, 1H),2.17-2.21 (m, 1H), 3.14 (d,
1H, J = 12.4 Hz), 3.37-3.41 (m,1H) 5.52 (brs, 2H, -NH , D O exchangeable), 5.59-5.60 (m.
1H), 6.50 (d, 2H, J = 7.8 Hz)), 6.91-6.97 (m, 2H), 7.06-7.08 (m, 1H), 7.22-7.29 (m, 2H),
6
): δ 0.76 (q, 1H, J = 12.1 Hz), 1.24-1.27
2
2
1
3
7.30-7.32 (m, 1H),10.43 (brs, 1H, -NH, D O exchangeable); C NMR (100 MHz, DMSO-
2
d
6
): δ 22.2, 25.5, 28.2, 32.4, 52.2, 57.7, 81.7, 110.0, 116.8, 118.5, 122.3, 124.8, 127.4, 128.3,
+
128.5, 129.4, 131.7, 132.3, 133.8, 136.6, 142.8, 154.3, 176.4; MS m/z = 402 [M+H] ; Anal.
3
Calcd for C24H20ClN O: C, 71.73; H, 5.02; N, 10.46. Found: C, 71.63; H, 5.11; N, 10.35.
9
0
4.1.2.6. 3'-amino-1'-(4-bromophenyl)-2-oxo-6',7',8',8a'-tetrahydro-1'H-spiro[indoline-3,2'-
1
naphthalene]-4'-carbonitrile (6f)
o
-1
1
1
1
1
1
1
1
1
1
2
1
2
3
4
5
6
7
8
9
0
White solid; mp 264-266 C (Decomposes); IR (cm ): 748, 1485, 1577, 1631, 1725, 2195,
1
2922, 3331, 3446; H NMR (400 MHz, DMSO-d
6
): δ 0.75 (q, 1H, J = 12.1 Hz), 1.24-1.26
(m, 1H), 1.38-1.41 (m, 1H), 1.65-1.68 (m, 1H), 2.06-2.08(m, 1H),2.16-2.20 (m, 1H), 3.11 (d,
1H, J = 12.4 Hz), 3.34-3.37 (m,1H) 5.46 (brs, 2H, -NH , D O exchangeable), 5.58-5.60 (m.
2
2
1H), 6.45 (d, 1H, J = 8.2 Hz)), 6.52 (d, 1H, J = 7.6 Hz) 6.96 (t, 1H, J = 7.5 Hz)), 7.03-7.10
(m, 2H), 7.17 (d, 1H, J = 8.4 Hz), 7.30 (d, 1H, J = 7.4 Hz), 7.39 (d, 1H, J = 8.4 Hz), 10.42
13
2 6
(brs, 1H, -NH, D O exchangeable); C NMR (100 MHz, DMSO-d ): δ 22.2, 25.5, 28.2,
32.4, 52.3, 57.7, 81.9, 110.1, 117.0, 118.4, 120.3, 122.4, 124.8, 128.9, 129.3, 129.4, 130.4,
+
131..2, 132.2, 134.1, 137.0, 142.8, 154.2, 176.5; MS m/z = 446 [M+H] ; Anal. Calcd for
C H BrN O: C, 64.58; H, 4.52; N, 9.41. Found: C, 64.69; H, 4.43; N, 9.52.
24
20
3
2
2
1
2
4.1.2.7 3'-amino-1'-(naphthalen-1-yl)-2-oxo-6',7',8',8a'-tetrahydro-1'H-spiro[indoline-3,2'-
naphthalene]-4'-carbonitrile (6g)
o
-1
2
2
3
4
White solid; mp 245-247 C (Decomposes); IR (cm ): 783, 1391, 1580, 1631, 1703, 2220,
1
2948, 3333, 3426; H NMR (400 MHz, DMSO-d ): δ 0.68 (q, 1H, J = 12.1 Hz), 1.00-1.06
6
2
0

ACCEPTED MANUSCRIPT
2
1
1
2
3
4
5
6
7
8
(m, 1H), 1.32-1.34 (m, 2H), 1.97-2.18 (m, 2H), 3.41-3.46 (m, 1H), 4.24 (d, 1H, J = 12.0 Hz),
5.37 (brs, 2H, -NH , D O exchangeable), 5.61-5.64 (m. 1H), 6.34 (d, 1H, J = 7.7 Hz)), 6.60-
2
2
6.64 (m, 1H) 6.78 (t, 1H, J = 7.6 Hz)), 7.27-7.37 (m, 2H), 7.42-7.48 (m, 2H), 7.54-7.56 (m,
1H), 7.59-7.67 (m, 2H), 8.16 (d, 1H, J = 8.0 Hz), 10.56 (brs, 1H, -NH, D O exchangeable);
2
1
3
C NMR (100 MHz, DMSO-d ): δ 22.2, 25.5, 27.6, 34.5, 44.6, 58.2, 82.1, 109.7, 116.9,
6
118.6, 121.9, 124.2, 124.3, 125.3, 125.4, 125.5, 125.8, 126.0, 126.6, 127.6, 128.6, 128.7,
+
129.1, 130.9, 132.7, 132.9, 133.3, 133.8, 135.0, 142.4, 155.1, 177.4; MS m/z = 418 [M+H] ;
23 3
Anal. Calcd for C28H N O: C, 80.55; H, 5.55; N, 10.06. Found: C, 80.65; H, 5.44; N, 10.17.
9
0
4.1.2.8.
3'-amino-1'-(furan-3-yl)-2-oxo-6',7',8',8a'-tetrahydro-1'H-spiro[indoline-3,2'-
1
naphthalene]-4'-carbonitrile (6h)
o
-1
1
1
1
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
White solid; mp 236-238 C. (Decomposes); IR (cm ): 754, 1476, 1583, 1633, 1702, 2199,
1
2930, 3279, 3422; H NMR (400 MHz, DMSO-d
6
): δ 0.83 (q, 1H, J = 12.4 Hz), 1.40-1.48
(m, 2H), 1.70-1.73 (m, 1H), 2.03-2.21 (m, 2H), 2.97 (d, 1H, J = 12.0 Hz), 3.17-3.24 (m, 1H),
5.44 (brs, 2H, -NH , D O exchangeable), 5.55-5.57 (m. 1H), 6.07 (s, 1H), 6.62 (d, 1H, J =
2
2
7.7 Hz) 6.81 (s, 1H), 6.97 (t, 1H, J = 7.5 Hz), 7.12-7.19 (m, 1H), 7.36 (s, 1H), 10.44 (brs, 1H,
13
2 6
-NH, D O exchangeable); C NMR (100 MHz, DMSO-d ): δ 22.2, 25.5, 28.3, 32.6, 44.1,
57.2, 81.8, 110.1, 116.8, 118.5, 121.2, 122.4, 124.2, 129.3, 130.2, 132.1, 141.2, 143.1, 143.3,
+
154.0, 177.1; MS m/z = 358 [M+H] ; Anal. Calcd for C22
H
19
N
3
O
2
: C, 73.93; H, 5.36; N,
11.76. Found: C, 73.85; H, 5.45; N, 11.67.
2
2
0
1
4.1.2.9.
3'-amino-2-oxo-1'-(thiophen-2-yl)-6',7',8',8a'-tetrahydro-1'H-spiro[indoline-3,2'-
naphthalene]-4'-carbonitrile (6i)
o
-1
2
2
2
2
3
4
White solid; mp 267-269 C (Decomposes); IR (cm ): 696, 1203, 1476, 1578, 1629, 1701,
1
2200, 2935, 3278, 3423; H NMR (400 MHz, DMSO-d
6
): δ 0.85 (q, 1H, J = 12.2 Hz), 1.39-
1.42 (m, 2H), 1.69-1.71 (m, 1H), 2.07-2.21 (m, 2H), 3.30 (d, 1H, J = 10.8 Hz), 3.46-3.52 (m,
2
1

ACCEPTED MANUSCRIPT
2
2
1
2
3
4
5
6
1H), 5.44 (brs, 2H, -NH
2 2
, D O exchangeable), 5.57-5.59 (m. 1H), 6.57 (d, 2H, J = 7.6 Hz),
6.72 (s, 1H) 6.96 (t, 1H, J = 7.4 Hz), 7.10 (t, 1H, J = 7.6 Hz), 7.15-7.16 (m, 1H), 7.27 (d, 1H,
13
2 6
J = 7.4 Hz), 10.48 (brs, 1H, -NH, D O exchangeable); C NMR (100 MHz, DMSO-d ): δ
22.2, 25.5, 28.1, 34.6, 48.9, 57.9, 81.8, 110.1, 117.0, 118.4, 122.5, 124.6, 125.5, 126.7, 129.4,
+
129.7, 132.0, 140.3, 143.2, 153.9, 176.6; MS m/z = 374 [M+H] ; Anal. Calcd for
C
22
H
19
N
3
OS: C, 70.75; H, 5.13; N, 11.25. Found: C, 70.64; H, 5.02; N, 11.34.
7
8
4.1.2.10.
3'-amino-2-oxo-1'-(pyridin-2-yl)-6',7',8',8a'-tetrahydro-1'H-spiro[indoline-3,2'-
naphthalene]-4'-carbonitrile (6j)
o
-1
9
0
1
2
3
4
5
6
7
8
9
White solid; mp 270-271 C (Decomposes); IR (cm ): 751, 1199, 1475, 1574, 1627, 1699,
1
1
1
1
1
1
1
1
1
1
1
2199, 2830, 2937, 3325, 3424; H NMR (400 MHz, DMSO-d
6
): δ 0.83 (q, 1H, J = 12.0 Hz),
1.00-1.03 (m, 1H), 1.35-1.38 (m, 1H), 1.63-1.66 (m, 1H), 2.05-2.20 (m, 2H), 3.28 (d, 1H, J =
12.1 Hz), 3.53-3.61 (m, 1H), 5.39 (brs, 2H, -NH , D O exchangeable), 5.56-5.58 (m. 1H),
2
2
6.46 (d, 1H, J = 7.7 Hz), 6.63-6.74 (m, 1H) 6.86-6.89 (m, 1H), 6.99-7.03 (m, 2H), 7.24 (d,
1H, J = 7.3 Hz), 7.30-7.37 (m, 1H), 8.26-8.33 (m, 1H), 10.26 (brs, 1H, -NH, D O
2
13
6
exchangeable); C NMR (100 MHz, DMSO-d ): δ 22.2, 25.4, 27.8, 32.5, 54.8, 56.8, 81.7,
109.7, 116.4, 118.5, 121.9, 122.2, 124.9, 128.7, 129.2, 133.0, 135.7, 143.1, 148.8, 155.1,
+
157.9, 175.9; HRMS (ESI): Mass calculated for C23
H
21
N
4
O [M+H] 369.1710, found,
+
[M+H] 369.1717; Anal. Calcd for C23
20 4
H N O: C, 74.98; H, 5.47; N, 15.21. Found: C, 74.88;
H, 5.36; N, 15.10.
2
2
2
2
0
1
2
3
4.1.3. Experimental procedure for the synthesis of (8a & 8b)
1,3-Cyclohaxanedione 7 (1 mmol), aldehydes 2 (1 mmol) 2a-b were stirred in MeOH in the
presence of L-proline (20 mol%) at room temperature (r.t.) for ten minutes followed by the
addition of alkylidene malononitrile 3a (1 mmol) at r.t. for 3 h. The solid precipitated out,
2
2

ACCEPTED MANUSCRIPT
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3
1
2
3
4
and then filtered off and purified by recrystallization from ethanol to afford product 8a and
8b as white crystalline solid.
4.1.3.1.
3'-amino-2,6-dioxo-1'-p-tolyl-6',7',8',8a'-tetrahydro-1'H-spiro[cyclohexane-1,2'-
naphthalene]-4'-carbonitrile (8a)
o
-1
5
6
7
8
9
0
1
2
3
4
White solid; mp 245-246 C (Decomposes); IR (cm ):822 1394, 1588, 1657, 1688, 1717,
1
2202, 2923, 3250, 3345, 3422; H NMR (400 MHz, DMSO-d
6
): δ 0.11 (q, 1H, J = 11.7 Hz),
0.57-0.65 (m, 1H), 1.29-1.32 (m, 2H), 1.42-1.48 (m, 1H), 1.59-1.64 (m, 1H), 1.95-2.13 (m,
4H), 2.24 (s, 3H), 2.47-2.52 (m, 1H), 2.73-2.82 (m, 1H), 2.89 (s,2H), 5.52-5.54 (m, 1H), 5.91
(brs, 2H, -NH , D O exchangeable), 6.71 (d, 1H, J = 7.6 Hz), 6.83 (d, 1H, J = 8.0 Hz)) 7.08
2
2
1
3
1
1
1
1
1
6
(d, 1H, J = 7.6 Hz), 7.16 (d, 1H, J = 7.6 Hz); C NMR (100 MHz, DMSO-d ): δ 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, 132.0, 133.9,
+
24 2 2
137.8, 154.3, 209.8, 210.9; HRMS (ESI): Mass calculated for C23H N NaO [M+Na]
+
383.1730, found, [M+Na] 383.1731 Anal. Calcd for C23
24 2 2
H N O : C, 76.64; H, 6.71; N,
7.77. Found: C, 76.75; H, 6.60; N, 7.68.
1
1
5
6
4.1.3.2.
3'-amino-1'-(4-methoxyphenyl)-2,6-dioxo-6',7',8',8a'-tetrahydro-1'H-
spiro[cyclohexane-1,2'-naphthalene]-4'-carbonitrile (8b)
o
-1
1
1
1
2
2
2
2
2
7
8
9
0
1
2
3
4
White solid; mp 241-243 C (Decomposes); IR (cm ):830 1262, 1512, 1591, 1659, 1684,
1
1715, 2204, 2951, 3246, 3346, 3416; H NMR (400 MHz, DMSO-d
6
): δ 0.24 (q, 1H, J = 13.3
Hz), 0.62 (q, 1H, J =10.8 Hz ), 1.32-1.38 (m, 2H), 1.49-1.55 (m, 1H), 1.63-1.66 (m, 1H),
1.99-2.16 (m, 4H), 2.53-2.58 (m, 1H), 2.76-2.84 (m, 1H), 2.88-2.91 (m, 2H), 3.72 (s, 3H),
5.52-5.54 (m, 1H), 5.93 (brs, 2H, -NH
2 2
, D O exchangeable), 6.77 (d, 1H, J = 8.2 Hz), 6.87 (t,
1
3
2H, J = 7.8 Hz)) 6.94 (d, 1H, J = 8.6 Hz); C NMR (100 MHz, DMSO-d
6
): δ 14.7, 22.1,
25.4, 27.5, 33.4, 54.4, 55.5, 83.1, 114.2, 115.3, 117.1, 118.2, 128.6, 128.7, 132.0, 132.4,
+
154.3, 159.3, 209.9, 211.0; HRMS (ESI): Mass calculated for C H N O [M+H] 377.1860,
2
3
25
2
3
2
3

ACCEPTED MANUSCRIPT
2
4
+
1
2
found, [M+H] 377.1860; Anal. Calcd for C23
24 2 3
H N O : C, 73.38; H, 6.43; N, 7.44. Found:
C, 73.47; H, 6.34; N, 7.54.
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
4.2. Biological assays
4.2.1. Materials and methods for antimicrobial activity
Streptomycin (Sigma) was used as positive control against bacteria. Ketoconazole (Himedia,
Mumbai) was used as positive control against fungi.
4.2.2. Tested microbes
The following bacteria and fungi were used for the experiment. Bacteria; Shigella flexneri
MTCC 1457, Micrococcus luteus MTCC 106, Enterobacter aerogenes MTCC 111,
Staphylococcus aureus MTCC 96, Klebsiella pneumoniae MTCC 109, Staphylococcus
epidermidis MTCC 3615, Proteus vulgaris MTCC 1771, Salmonella typhimurium MTCC
1251 and Staphylococcus aureus (MRSA- methicillin resistant). The reference cultures were
obtained from Institute of Microbial Technology (IMTECH), Chandigarh, India-160 036;
fungi: Malassesia pachydermatis and Candida albicans MTCC 227. All the cultures were
obtained from the Department of Microbiology, Christian Medical College, Vellore, Tamil
Nadu, India.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
4.2.3. Preparation of inoculums
Bacterial inoculums were prepared by growing cells in Mueller Hinton broth (MHB)
(Himedia) for 24 h at 37°C. The filamentous fungi were grown on sabouraud dextrose agar
(SDA) 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 (SDB) at 28°C for
48-72 h.
4.2.4. Disc diffusion assay
Antimicrobial activities were carried out using disc diffusion method [22]. Petri plates were
prepared with 20 ml of sterile Mueller Hinton Agar (MHA) (Hi-media, Mumbai). The test
2
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4
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7
8
9
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
cultures were swabbed on the top of the solidified media and allowed to dry for 10 min and a
specific amount of synthesised compound at 1mg/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 respective
solvents. Streptomycin was used as positive control against bacteria. Ketoconazole was used
as positive control for fungi. The plates were incubated for 24 h at 37°C for bacteria and for
48 h at 28°C for fungi. Zones of inhibition were recorded in millimetres and the experiment
was repeated twice.
4.2.5. Minimum inhibitory concentration (MIC)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
Minimum inhibitory concentration studies of 17 compounds were performed according to
the standard reference methods for antibacterial activity [23]. The required concentrations
(1000 µg/mL, 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL, 15.62 µg/mL
and 7.81µg/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 µl
from each well was inoculated. The antifungal agent Ketoconazole for fungi and
Streptomycin for bacteria were included in the assay as positive controls. For fungi, the
plates were incubated for 48 to 72 hours 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 extract concentration, showing
no visible fungal growth after incubation time. 5 µl 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 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 maintained in complete tissue culture medium
2
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Dulbecco's Modified Eagle's Medium with 10 % Fetal Bovine Serum and 2mM L-Glutamine,
along with antibiotics (about 100 International Unit/mL of penicillin, 100 µg/mL of
streptomycin) with the pH adjusted to 7.2. The cytotoxicity was determined according to the
method of Balachandran et al. [24] with some changes. Cells (5000 cells/well) were seeded
in 96 well plates containing medium with different concentrations such as 50, 40, 30, 20, 10
and 5 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 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyl-tetrazolium 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 100 µL of DMSO was added to
the plate, which was shaken until the crystals were dissolved. The cytotoxicity against cancer
cells was determined by measuring the absorbance of the converted dye at 540 nm in an
Enzyme linked immune sorbant assay reader. Cytotoxicity of each sample was expressed as
the half maximal inhibitory concentration (IC50) value. The IC50 value is the concentration
of test sample that causes 50% inhibition of cell growth, averaged from three replicate
experiments.
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4.3. Molecular docking studies
Protein and ligand preparation were carried out using MOE (Molecular Operating
Environment) 2011 software tool version 7.1. A PDB entry 2XCS was selected for
antibacterial docking study and was processed with MOE software. The structural issues
such as capping, completing residues with missing atoms and selecting appropriate alternate
locations were corrected automatically using structure preparation application available with
the MOE software. An additional step was carried out to delete extraneous cofactors or
unbound water and the co-crystallized ligand. The active site of protein was identified by
using MOE’s site finder application. Ligand 2D structures were drawn using ChemBioDraw
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Ultra 11.0 (ChemOffice 2008). Ligand 2D structures were converted to mol2 format using
the file convertor Open Babel GUI. Energy minimization was performed to adjust hydrogen
and lone pairs and to calculate partial charges. The Protein and ligand structures were energy
minimized using of MMFF94x force field implemented in MOE. The energy was minimized
to the minimum gradient of 0.05. The final refined poses were ranked by the MM/GBVI
binding free energy estimation. The best docked representation of the ligand was chosen
based on the conformation with lowest binding free energy out of 100 docking runs. The
final docked conformations were within the range of 0.5 to 1 Å root-mean-square deviation.
For the docking of synthesized molecules to the ALK structure with the PDB id 2XP2, the
same procedure followed as for the DNA gyrase receptor.
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Acknowledgements
N.S.P thanks the Council of Scientific and Industrial Research (CSIR), New Delhi, India for
the research fellowship and SAIF, IITM, Chennai for single crystal XRD analysis.
References
[1] (a) A.S. Girgis, Eur. J. Med. Chem. 44 (2009) 91-100.
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(b) C. Young-Won, S. Angela, S. Bao-Ning, M. Qiuwen, C. Hee-Byung, R. Soedarsono, K.
Leonardus, R. Agus, F. Norman, S. Steven, K. Douglas, J. Nat. Prod. 71 (2008) 390-395.
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[2] R. Murugan, S. Anbazhagan, S. S. Narayanan, Eur. J. Med. Chem. 44 (2009) 3272-3279.
[3] S.A. Van der Sar, J.W. Blunt, M.H. Munro, Org. Lett. 8 (2006) 2059-2061.
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[4] (a) P. Prasanna, K. Balamurugan, S. Perumal, P. Yogeeswari, D. Sriram, Eur. J. Med.
Chem. 45 (2010) 5653-5661.
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(b) S.V. Karthikeyan, B. D. Bala, V.P.A. Raja, S. Perumal, P. Yogeeswari, D. Sriram,
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