An easy protocol for the domino synthesis of diversely functionalized spirocarbocycles and their biological evaluation

Article abstract of DOI:10.1007/s12039-013-0560-1

A base-catalysed domino reaction for the synthesis of spirocarbocycles in excellent yields using chalcones and vinyl malononitriles is reported. This consists of a series of steps including Knoevenagel condensation followed by intermolecular vinylogous Mi

Full text of DOI:10.1007/s12039-013-0560-1

c
J. Chem. Sci. Vol. 126, No. 1, January 2014, pp. 177–185. ꢀ Indian Academy of Sciences.
An easy protocol for the domino synthesis of diversely functionalized
spirocarbocycles and their biological evaluation
SELVARANGAM E KIRUTHIKAa, PARAMASIVAN T PERUMALa,∗, C BALACHANDRANb
and S IGNACIMUTHUb
aOrganic Chemistry Division, CSIR-Central Leather Research Institute, Adyar, Chennai 600 020, India
bDivision of Microbiology, Entomology Research Institute, Loyola College, Chennai 600 034, India
e-mail: ptperumal@gmail.com
MS received 16 June 2013; revised 17 October 2013; accepted 18 October 2013
Abstract. A base-catalysed domino reaction for the synthesis of spirocarbocycles in excellent yields using
chalcones and vinyl malononitriles is reported. This consists of a series of steps including Knoevenagel con-
densation followed by intermolecular vinylogous Michael addition and intramolecular cyclization. The syn-
thesized compounds were evaluated for their antimicrobial activity and the compounds exhibited moderate to
excellent activities.
Keywords. Spirocarbocycles; L-proline; vinyl malononitriles; chalcones; antimicrobial activity.
1. Introduction
have proven to be valuable intermediates for the con-
struction of spirocarbocycles due to their importance in
both synthesis and pharmacology.14,15
Carbon-carbon and carbon-heteroatom bond forming
reactions play a crucial role in organic synthesis. Syn-
thesis of diversely functionalized molecules from sim-
ple starting materials which fulfils both economic and
environmental aspects together is a great challenge to
organic chemists.1a Multicomponent reactions (MCRs)
which involve domino processes assist in achieving this
ideal goal.1 MCRs often do not require the usage of
expensive catalysts, tedious protection and deprotec-
tion processes, purification and isolation procedures.2
Hence, MCRs are of growing importance in the con-
struction of diversely functionalized heterocycles.3
Development of efficient methods to construct spiro
compounds has been a topic of great relevance in
organic synthesis due to their pronounced biological
importance as they are used as anticancer agents,4
antibacterial agents,5 anticonvulsant agents,6 anti-
tuberculosis agents,7a etc.7 Methodologies for the syn-
thesis of spirocyclic compounds range from the classi-
cal approaches involving [3+2] cycloadditions8 to the
recently emerging transition metal-based processes.9
Their synthesis also includes other protocols such as
ring closure methodologies,10 radical cyclizations,11
cleavage of bridged ring systems, etc.12 Such diverse
spirocycles are characterized by a spiro ring fusion with
varied heterocyclic motifs.13 Among the various hete-
rocyclic motifs, isoxazol-5-ones and oxindole moieties
Biological importance of these privileged medicinal
scaffolds possessing a spiro ring fusion led us to the
synthesis of spirocarbocycles. As a continuation of our
research work devoted to the development of multi-
component domino reactions,16 we would herein like to
report the synthesis of spirocarbocycles utilizing base-
catalysed multicomponent domino reactions involving
chalcones and vinyl malononitriles along with biologi-
cal evaluation of the synthesized compounds.
2. Experimental
2.1 Materials, methods and instruments
Melting points were determined on Gallenkamp melt-
ing point apparatus and are uncorrected. Infrared (IR)
spectra were recorded on a Perkin-Elmer FTIR spec-
trophotometer as KBr pellets. 1H and 13C NMR spectra
were obtained in DMSO-d6 on a JEOL spectrometer at
500 and 125 MHz, respectively. Proton chemical shifts
(δ) are relative to tetramethylsilane (TMS, δ = 0.00) as
internal standard and expressed in parts per million. The
number of protons (n) for a given resonance was indi-
cated 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
on a Thermo Finnigan LCQ Advantage MAX 6000
∗For correspondence
177

178
Selvarangam E Kiruthika et al.
1473, 1395, 1212, 755, 626 cm−1.1H NMR (DMSO-
d6, 500 MHz): δ 0.96 (q, 1H, J = 12.0 Hz), 1.21 (m,
1H), 1.14 (m, 1H), 1.33 (m, 1H), 1.63 (m, 1H), 2.07 (m,
1H), 3.41 (m, 2H), 4.40 (d, 1H, J = 12.5 Hz), 5.42 (s,
2H, -NH2, D2O exchangeable), 5.56 (m, 1H), 6.71 (m,
2H), 7.03 (t, 1H, J =7.5 Hz), 7.36 (t, 3H, J =7.5 Hz),
7.50 (t, 1H, J =8 Hz), 7.80 (d, 1H, J = 8.5 Hz), 10.57
(s, 1H, -NH, D2O exchangeable). 13C NMR (DMSO-
d6, 125 MHz): δ 14.3, 21.8, 24.9, 27.5, 32.7, 52.0, 54.1,
60.0, 81.0, 110.0, 116.7, 118.2, 121.8, 124.9, 127.3,
128.3, 128.8, 129.6, 131.7, 133.7, 137.7, 143.2, 154.4,
176.0, 200.5. MS (m/z): 396 (M+H)+. Anal. Calcd. for
C25H21N3O2 : C, 75.93; H, 5.35; N, 10.63. Found: C,
75.91; H, 5.36; N, 10.64.
ESI mass spectrometer and Perkin-Elmer GC-MS. Ele-
mental analyses were recorded using a Thermo Finni-
gan FLASH EA 1112CHN analyser. All the compounds
gave C, H and N analysis within 0.5% of the theo-
retical values. Analytical TLC was performed on pre-
coated plastic sheets of silica gel G/UV-254 of 0.2 mm
thickness (Macherey-Nagel, Germany) using analytical
grade solvents and visualizing with UV light (λ = 254
and 365 nm).
2.2 Experimental procedure for the synthesis of 4a–l
3-Phenyl-5-isoxazolone 1 (1 mmol), aldehhydes 2a–k
were stirred in a mixture of EtOH:H2O (2:1) in the pres-
ence of L-proline (15 mol%) for 10 min followed by
the addition of alkylidene malononitrile 3a–c (1 mmol)
at room temperature for 1 h. The solid precipitated out
was filtered off and purified by recrystallization from
methanol to afford product 4a–l as white crystalline
solid.
2.4 Biological evaluation
2.4a Antimicrobial activity: Antimicrobial activities
were carried out using disc diffusion method.17 Petri
plates were prepared with 20 ml of sterile Mueller Hin-
ton agar (MHA) (Hi-media, Mumbai). The test cultures
were swabbed on the top of the solidified media and
allowed to dry for 10 min and a specific amount of syn-
thesized 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 tempera-
ture for compound diffusion. Negative control was pre-
pared using respective solvents. Streptomycin was used
as positive control against bacteria. Ketoconazole was
used as positive control for fungi. The plates were incu-
bated for 24 h at 37◦C for bacteria and for 48 h at 28◦C
for fungi. Zones of inhibition were recorded in mil-
limetres and the experiment was repeated twice. Bacte-
rial inocula were prepared by growing cells in Mueller
Hinton broth (MHB) (Himedia) for 24 h at 37◦C. Fil-
amentous 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 h.
2.2a Spectral data for a representative compound 4a:
White solid. mp: 152–154◦C. νmax (KBr): 3345, 3224,
2926, 2860, 2207, 1790, 1640, 1593, 1514, 1446, 1393,
1151, 908, 881, 764, 691, 640 cm−1.1H NMR (DMSO-
d6, 500 MHz): δ 0.74 (q, 1H, J = 9 Hz), 1.25 (m, 1H),
1.40 (m, 1H), 1.62 (m, 1H), 2.07 (m, 1H), 2.17 (m, 1H),
2.22 (s, 3H), 3.06 (d, 1H, J = 12.5 Hz), 3.18 (m, 1H),
5.76 (m, 1H), 6.14 (d, 1H, J = 8.5 Hz), 6.60 (bs, 2H,
-NH2, D2O exchangeable), 6.86 (d, 1H, J = 7.5 Hz),
7.05 (d, 1H, J = 8 Hz), 7.16 (d, 1H, J = 8.5 Hz),
7.59 (m, 5H). 13C NMR (DMSO-d6, 125 MHz): δ 20.6,
21.5, 25.0, 27.4, 31.3, 50.1, 59.1, 82.1, 117.1, 119.2,
125.6, 126.4, 126.7, 128.6, 129.5, 129.6, 130.3, 130.7,
131.5, 132.3, 137.6, 147.6, 164.3, 176.1 MS (m/z): 410
(M+H)+. Anal. Calcd. for C26H23N3O2 : C, 76.26; H,
5.66; N, 10.26. Found: C, 76.24; H, 5.67; N, 10.27.
2.3 Experimental procedure for the synthesis of 6a–e
3-(2-Oxo-2-phenylethylidene)indolin-2-one1 5(1 mmol)
and alkylidene malononitrile 3a–e (1 mmol) were
stirred in a mixture of EtOH:H2O (2:1) in the presence
of L-proline (15 mol%) at room temperature for 3 h.
The solid precipitated out was filtered off and recrystal-
lized from methanol to afford product 6a–e as a white
solid.
2.4b Minimum inhibitory concentration: Minimum
inhibitory concentration (MIC) studies of the synthe-
sised compounds were performed according to the
standard reference methods for bacteria,18 for fila-
mentous fungi19 and yeasts.20 Required concentrations
(1000, 500, 250, 125, 62.5, 31.25, 15.62 µg/mL and
7.81 µg/mL) of the compound were dissolved in DMSO
(2%), and diluted to give serial two-fold dilutions that
2.3a Spectral data for a representative compound 6b: were added to each medium in 96 well plates. An
White solid. mp: 212–214◦C. νmax (KBr): 3433, 3351, inoculum of 100 µL from each well was inoculated.
3305, 3059, 2930, 2828, 2200, 1708, 1669, 1633, 1575, Antifungal agents such as Ketoconazole for fungi and

Synthesis and biological evaluation of spirocarbocycles
179
Me
O
O
O
O
CHO
NC
CN
NC
H2N
Reaction
conditions
+
+
N
N
Me
1
2a
3a
4a
Scheme 1. Synthesis and screening of reaction conditions for 4a.
Streptomycin for bacteria were included in the assays temperature, base catalysts and adding sequence of
as positive controls. For fungi, the plates were incu- substrates (scheme 1, table 1).
bated for 48 to 72 h at 28◦C and for bacteria the plates
The onset of the uncatalysed model reaction in
were incubated for 24 h at 37◦C. The MIC for fungi ethanol to afford the product 4a in a low yield of 45%
was defined as the lowest extract concentration, show- (table 1, entry 1) prompted us to explore the optimum
ing no visible fungal growth after incubation time. 5 µL base catalyst for the reaction by using a range of base
of tested broth was placed on the sterile MHA plates for catalyst promoters (pyrrolidine, L-proline, DABCO, tri-
bacteria and incubated at respective temperatures. The ethylamine, etc.) (table 1, entries 2–8). The reaction
MIC for bacteria was determined as the lowest concen- afforded comparatively better yields when L-proline
tration of the compound inhibiting visual growth of the was used as a base catalyst (table 1, entry 8). With the
test cultures on the agar plate.
best catalyst L-proline, the reaction was performed in
various solvents which included THF, DMF, MeOH,
water, etc. (table 1, entries 9–15). The reaction was also
found to proceed under neat conditions (table 1, entry
14), but the completion was observed only after pro-
longed hours with a diminished yield of 40%. In case
of L-proline as the base catalyst, a slight increase in
the stoichiometry altered the yield from 85% to 87%
(table 1, entry 16). Further increase had no impact on
the yield of the reaction (table 1, entry 17). The adding
sequence of substrates also influenced the product
3. Results and discussions
3.1 Chemistry
Inourinvestigatoryexperiment, 3-phenyl-5-isoxazolone
1, 4-methyl benzaldehyde 2a and cyclohexylidene
malononitrile 3a were stirred to afford the product
4a under various reaction conditions viz. solvents,
Table 1. Screening of solvents and base catalysts for the synthesis of 4a.
Entry
Solvent
Base Catalysta
Conditions
Time (h)
Yield (%)b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
MeOH
THF
–
rt
rt
10
5
24
10
2.5
4
8
3
3
8
9
1
5
48
6
1
1
45
67
58
59
62
60
66
75
60
65
75
85
68
40
55
87c
86d
Pyrrolidine
DBU
NEt3
K2CO3
NEt3
DABCO
L-Proline
L-Proline
L-Proline
L-Proline
L-Proline
L-Proline
–
Reflux
rt
rt
rt
Reflux
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
H2O
EtOH: H2O
CH3CN
–
DMF
EtOH: H2O
EtOH: H2O
L-Proline
L-Proline
L-Proline
a
All the reactions were performed using 10 mol% of the base catalyst
Isolated yield of 4a after recrystallization
The reaction was performed using 15 mol% of the catalyst
b
c
d
The reaction was performed using 20 mol% of the catalyst

180
Selvarangam E Kiruthika et al.
Scheme 2. Plausible mechanism for the formation of spirocarbocycle 4a.
formation because the addition of 4-methyl benzalde- 7–12). Varying the ring size of the vinylogous alkyli-
hyde 2a to cyclohexylidene malononitrile 3a resulted dene substrates (table 2, entries 1, 2 and 4) afforded
in the formation of a pyran which reduced the yield the products in good yields. Thus, the reaction was
of 4a. Hence, the best result (table 1, entry 16) was found to be general and has a broad scope due to its
observed when 3-phenyl-5-isoxazolone 1 and 4-methyl applicability to a variety of substrates and the products
benzaldehyde 2a were stirred for 10 min in the presence 4a–l were isolated in excellent yields (75–87%) under
of 15 mol% of L-proline in ethanol: water mixture21 milder reaction conditions. The results are summarized
at room temperature followed by the addition of cyclo- in table 2.
hexylidene malononitrile 3a to afford a precipitate
Structures of compounds 4a–l were consistent with
1
which was filtered off and recrystallized from methanol IR, H and 13C NMR, mass spectroscopy and elemen-
to provide the product 4a in 87% yield.
tal analysis data.The IR spectrum of 4a exhibited peaks
A plausible mechanism to account for the forma- at 2207 and 1790 cm−1 for the nitrile and lactone ester
1
tion of spirocarbocycle 4a is explained in scheme 2. carbonyl groups, respectively. The H NMR spectra of
3-Phenyl-5-isoxazolone 1 adds to 4-methyl benzalde- 4a showed a singlet at δ 6.60 for the –NH2 protons
hyde 2a with concomitant elimination of water leading (D2O exchangeable). The signal at δ 82.1 indicated the
to the formation of chalcone 1a. Alkylidene malono- presence of spiro carbon in the 13C NMR spectra. The
nitrile 3a in the presence of L-proline undergoes inter- mass spectra also exhibited a distinguishing peak at m/z
molecular vinylogous Michael addition onto the acti- 410 [M+H]+. Thus the above data confirms the for-
vated double bond of chalcone 1a forming intermedi- mation of 4a. The structure was further confirmed by
ate 1b. Among the two transition states TS I and TS single crystal X-ray analysis performed for a represen-
II, the former suffers from unwanted steric interac- tative compound 4g (figure 1).22 The stereochemistry
1
tions and congestions. Hence TS II of 1b undergoes was assigned based on H NMR spectroscopy and X-
1
intramolecular addition to the nitrile carbon resulting in ray single crystal analysis. The H NMR spectra for
the formation of a six membered spirocyclic interme- 4a exhibits a doublet for the Ha proton which pos-
diate 1c. The latter undergoes isomerization to provide sesses coupling constant of J = 12.5 Hz characteris-
the spirocarbocyclic diastereomer 4a.
tic of a trans relation between Ha and Hb protons. The
Having optimized the conditions, we expanded the Ha and Hb protons show trans relation with respect to
scope of the reaction by varying the aldehyde and each other which is clearly observed from the single
alkylidene malononitrile substrates. The reaction pro- crystal X-ray analysis of compound 4g which further
1
ceeded smoothly with aldehydes possessing electron- supports the H NMR spectroscopy. This shows that
donating groups. Halogen substituents on the aldehyde the reaction of 3-phenyl-5-isoxazolone 1 with aldehy-
(table 2, entries 2–4) did not have any pronounced effect des 2a-l and alkylidene malononitriles 3a–c is com-
on the yield of the products, but took longer dura- pletely diastereoselective in affording only the trans
tion for the completion of reaction than its counterparts diastereomer.
having electron-rich substituents such as 4-methyl ben-
To extend the utility of this multicomponent reac-
zaldehyde and 3,4-dimethoxy benzaldehyde (table 2, tion, studies were extended to other chalcones such as
entries 1 and 6). Various aldehydes including hetero- isatin chalcones under the same protocol which results
cyclic aldehydes were also explored (table 2, entries in the formation of spiroxindoles. The reaction provided

Synthesis and biological evaluation of spirocarbocycles
Table 2. Synthesis of spirocarbocycles (4a–l).
181
Entry
Aldehyde
Alkylidene
malononitriles
Product
Time (h)
Yield (%) [a]
1
2
3
1
7
3
87
82
81
4
5
10
80
85
6.5
6
7
1
84
82
1.5

182
Selvarangam E Kiruthika et al.
Table 2.
(continued)
Aldehyde
Entry
Alkylidene
malononitriles
Product
Time (h)
Yield (%) [a]
8
9
8
3
83
77
10
11
12
4.5
75
83
80
4
3.5
[a] Isolated yield after recrystallization from methanol
Figure 1. ORTEP diagram of 4g.

Synthesis and biological evaluation of spirocarbocycles
183
spiroxindoles in good yields (79–83%) and the results –NH2 and -NH protons (D2O exchangeable), respec-
are tabulated in table 3. tively, clearly indicating the incorporation of both the
Structures of compounds 6a–d were consistent with moieties in the product. The doublet appearing for the
IR, 1H and 13C NMR, mass spectroscopy and elemental Ha proton of the product 6b shows a coupling con-
analysis data. The IR spectrum of 6b exhibited peaks stant value of 12.5 Hz indicative of the trans stereo-
at 2200, 1708 and 1669 cm−1 for the nitrile, keto and chemistry between the Ha and Hb protons. In 13C NMR
amide carbonyls, respectively. The 1H NMR spectra of spectra, the spiro carbon atom displayed a signal at δ
6b showed two singlets at δ 5.42 and 10.57 for the 81.0 and the amide and keto carbonyl carbon atoms
Table 3. Synthesis of spirooxindole derivatives (6a–e).
Entry
Vinylogous malononitrile
Product
Time (h)
Yield (%) [a]
1
2
3
83
80
2.5
3
4
2
79
82
3.5
5
4
80
[a] Isolated yield after recrystallization from methanol

184
Selvarangam E Kiruthika et al.
resonated at δ 176.0 and 200.5, respectively. The mass 4. Conclusion
spectra also exhibited a distinguishing peak at m/z 396
[M+H]+. The reaction was also performed using 2-(2,3-
dihydronaphthalen-4(1H)-ylidene)malononitrile 3e as
vinyl malononitrile to afford 6e in 80% yield.
Thus, we have successfully established a new multi-
component domino reaction for the synthesis of poly-
functionalized spirocarbocycles from activated double
bonds such as chalcones and vinylogous malononitriles.
The spirocarbocycles were produced in a single step
in excellent yields without involving tedious extrac-
tion and isolation procedures. The reaction brings forth
a green protocol involving the usage of environmen-
tally benign organocatalyst L-proline in ecofriendly sol-
vent medium such as ethanol–water mixture. It also
eliminates the use of hazardous solvents and expen-
sive catalysts. The compounds were also screened
for antimicrobial activities against four Gram-positive
bacteria and Gram-negative bacteria and also against
two fungi. Most of the compounds have found to exhibit
significant antimicrobial activity of which 4e, 4h and
6b have shown good activity and hence are promising
candidates as antibacterial and antifungal agents.
3.2 Pharmacology
3.2a Antimicrobial activity: In the present study,
antimicrobial activities of synthesized compounds were
screened against bacteria (Bacillus subtilis MTCC 441,
Micrococcus luteus MTCC 106, Enterobacter aero-
genes MTCC 111, Staphylococcus aureus MTCC 96,
Klebsiella pneumoniae MTCC 109, Proteus vulgaris
MTCC 1771, Salmonella typhimurium MTCC 1251
and Staphylococcus aureus (MRSA-methicillin resis-
tant) and fungi (Malassesia pachydermatis and Can-
dida albicans) using in vitro disc diffusion method.
Streptomycin was used as a positive control against bac-
teria and Ketoconazole was used as a positive control
against fungi.
Results revealed that most of the synthesised
compounds exhibited antimicrobial activities against Supplementary information
Bacillus subtilis, Micrococcus luteus, Staphylococcus
The electronic Supplementary information can be seen
at www.ias.ac.in/chemsci.
aureus, Klebsiella pneumoniae, Enterobacter aero-
genes, Proteus vulgaris, Salmonella typhimurium,
Staphylococcus aureus (MRSA-methicillin resistant),
Malassesia pachydermatis and Candida albicans (see
Supplementary information). Compounds 4b, 4c, 4e,
4h, 6a, 6b, 6c and 6d have shown considerable antimi-
crobial activities. Compounds 4d, 4e, 4h, 6c, 6d show
excellent activities more than the standard drug against
the Gram-positive bacteria S. aureus. Among the com-
pounds synthesized and tested for antimicrobial acti-
vity, 4c, 4h and 6b exhibit significant antimicrobial acti-
vities and compound 4h has shown promising activity
against both Gram-positive and Gram-negative bacteria.
Acknowledgements
SEK thanks the Council of Scientific and Industrial
Research (CSIR), New Delhi, India for the research
fellowship. We also thank Department of Chemistry,
Indian Institute of Technology, Madras for X-ray single
crystal analysis.
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have been deposited with the Cambridge Crystallo-
graphic Data centre as supplemental publication no.
CCDC- 905942. Copies of the data can be obtained,
free of charge on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK (fax: +44 01223 336033 or
email: deposit@ccdc.cam.ac.uk)