Regio- and stereoselective synthesis of a library of bioactive dispiro-oxindolo/acenaphthoquino andrographolides via 1,3-dipolar cycloaddition reaction under microwave irradiation

Article abstract of DOI:10.1021/co3001154

Dispiro-pyrrolidino/pyrrolizidino fused oxindoles/acenaphthoquinones have been derived from andrographolide via azomethine ylide cycloaddition to the conjugated double-bond under microwave (MW) irradiation. The reactions are chemo-, stereo-, and regioselective in nature. Change in amino acid from sarcosine/N-benzyl glycine to l-proline changes the regiochemistry. A representative library of 40 compounds along with in vitro anticancer evaluation is reported.

Full text of DOI:10.1021/co3001154

Research Article
pubs.acs.org/acscombsci
Regio- and Stereoselective Synthesis of a Library of Bioactive
Dispiro-Oxindolo/Acenaphthoquino Andrographolides via 1,3-
Dipolar Cycloaddition Reaction Under Microwave Irradiation
Abhijit Hazra,† Yogesh P. Bharitkar,† Debanjana Chakraborty,† Susanta Kumar Mondal,‡ Nupur Singal,‡
Shyamal Mondal,† Arindam Maity,† Rupankar Paira,† Sukdeb Banerjee,† and Nirup B. Mondal*,†
†Department of Chemistry, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4, Raja S. C. Mullick
Road, Jadavpur, Kolkata 700 032, India
‡Chembiotek, TCG Lifesciences Ltd, BIPL, Block-EP & GP, Sec-V, Kolkata 700091, India
S
* Supporting Information
ABSTRACT: Dispiro-pyrrolidino/pyrrolizidino fused oxindoles/acenaphthoquinones have
been derived from andrographolide via azomethine ylide cycloaddition to the conjugated
double-bond under microwave (MW) irradiation. The reactions are chemo-, stereo-, and
regioselective in nature. Change in amino acid from sarcosine/N-benzyl glycine to L-proline
changes the regiochemistry. A representative library of 40 compounds along with in vitro
anticancer evaluation is reported.
KEYWORDS: dispiroheterocycles, pyrrolidino/pyrrolizidino, andrographolide, dipolar cycloaddition, azomethine ylide
numerous alkaloids and pharmacologically important com-
pounds. Spiropyrrolidinyloxindole ring systems are also found
in a number of alkaloids like horsifiline, spirotryprostatine A
and B, elacomine, and so forth.13 Construction of this type of
substituted pyrrolidine or pyrrolizidine rings with spirooxindole
moiety was achieved via cycloaddition reaction of 1,3-dipoles
with olefinic or acetylenic dipolarophiles. Discovered by
Huisgen14a and developed by Woodward and Hoffman14b
and also by Houk et al., this regio- and stereoselective reaction
has since become an important as well as general method for
the construction of five-membered heterocyclic rings.15−17
Reports on the synthesis of derivatives of andrographolide,
the major diterpene constituent of Andrographis paniculata, for
modification of the biological activities are available in the
literature. Bioevaluation of the derivatives has shown
anticancer, α-glucosidase inhibitory18 as also TNF-α and IL-6
expression inhibitory activity.19 Encouraged by these reports,
recently we have synthesized and reported a few dispiro
analogues of andrographolide via azomethine ylide cyclo-
addition in refluxing methanol.20 The results of the biological
evaluation of these compounds encouraged us further to build a
library of dispiro analogues of andrographolides for further
biological evaluation. In this endeavor to access the library of
compounds containing both spiro-oxindole and pyrrolidine/
pyrrolizidine rings attached with the well-known bioactive
INTRODUCTION
■
Exploring novel pharmacological agents with minimum number
of synthetic steps and in less time is a major challenge to the
chemists.1 Besides, chemists are facing another challenge for the
past two decades, namely, that of developing new trans-
formations that are not only efficient, selective, and high
yielding but also environmentally benign. In general, the
conventional approaches involve the use of multistep reaction
sequences. These are typically associated with low yields and
high cost, entail tedious isolation of the resulting products, and
seldom become commercially viable. The use of multi-
component reactions (MCRs) has become a modern approach
to synthesize such novel compounds2 of biological significance.
The MCR strategy, where three or more simple and flexible
molecules are brought together to rapidly introduce structural
complexity and diversity, offers significant advantages over
conventional linear-type syntheses.3−5 Thus, the choice of an
appropriate reaction condition meeting the aforesaid challenges
is of crucial importance for successful syntheses now-a-days.
Highly substituted pyrrolidines have gained much prom-
inence because they make the central skeleton of many natural
products.6 Some spiropyrrolidines are potential antileukemic
and anticonvulsant agents,7 and possess antiviral and local
anesthetic activities.8 Furthermore, the 3′-spirooxindoles have
been of interest to organic chemists because many such
derivatives are characterized by interesting biological proper-
ties.9−11 Spirooxindoles find many biological applications as
antimicrobial, antitumoral, and inhibitors of human NK-1
receptor, and so forth,12 and constitute the central skeleton for
Received: September 19, 2012
Revised: November 14, 2012
© XXXX American Chemical Society
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natural core andrographolide, we decided to explore the
applicability of microwave (MW) irradiation technique to
develop an efficient, convenient, high yielding, and less time-
consuming green protocol. This technique is being employed in
many reactions in recent years for achieving energy efficiency
and enhancing the rate of reaction as well as product yields.
The reaction has been carried out by mixing the three-
components using 2−3 mL of methanol and irradiating under
MW at 100 °C until completion of the reaction as evidenced by
TLC. The reaction proceeds through decarboxylative con-
densation of isatin with amino acid to generate azomethine
ylide, which subsequently undergoes 1,3-dipolar cycloaddition
with andrographolide to afford novel dispiro cycloadduct
(Scheme 1). Control of the relative stereochemistry at the
spiro center was observed. Anti-ylides are involved in the
reactions which add to dipolarophiles to form the cycloadducts.
Formation of syn-ylide is not observed because of the
unfavorable steric repulsion between the carbonyl group of
oxindole and methyl group of sarcosine or benzyl group of N-
benzyl glycine.21
Characterization of the products was performed by detailed
spectral analysis. In all the cases mass spectral analysis of the
products provided evidence for the success of the cycloaddition
reaction which was further supported by the NMR spectral
data. In the spectra of the products, the signals for the fused six-
membered ring fragments of andrographolide appeared almost
unchanged. From 2D NMR spectroscopic analysis it was
revealed that the reverse regioisomer is predominant in case of
proline and the normal one in case of sarcosine as was reported
in our previous communication.20
RESULTS AND DISCUSSION
■
Initially, the three-component azomethine ylide cycloaddition
reaction of isatin, sarcosine/proline/N-benzyl glycine and
andrographolide was investigated to optimize the green
reaction conditions. Various green reaction protocols, for
example, β-cyclodextrin-water (β-CD), micelle (cetyltrimethy-
lammonium bromide/CTAB-water), water immiscible ionic
liquid 1-butyl-3-methylimidazolium hexafluorophosphate
([bmim] PF6), and MW irradiation were screened for the
optimization of the reaction condition (Table 1). The best
result was obtained under MW irradiation with excellent yield
(80%) in shorter reaction time (15−20 min).
Table 1. Synthesis of 3a/4a/5a under Different Reactions
Conditions
a
entry
condition
temp. (°C)
time (h)
yield (%)
1
2
3
4
5
methanol
reflux
reflux
reflux
80
22
62
35
30
60
82
For the generalization of the method, we extended this
reaction of andrographolide and sarcosine/proline/N-benzyl
glycine with variously substituted isatins and the corresponding
dispiropyrrolidino/pyrrolizidino andrographolide derivatives
were produced in high yields (Tables 2−4).
In case of N-benzyl glycine also the addition follows the
pattern of sarcosine, with the isatin part added from the C13
end and the N-benzyl part from the C12 end. The crucial
water-βCD
water-CTAB
ionic liquid
MW-MeOH
20
22
12
100
0.25
a
Isolated yield.
Scheme 1. Synthesis of Novel Di-spiro Pyrrolidino/Pyrrolizidino Andrographolides
B
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Table 2. Yields of 3a−l under Microwave Irradiation
a
b
entry
R1
R2
R3
product (3a−l)
yield (%)
1
H
H
H
H
H
H
H
H
Me
H
H
H
Ph
H
H
H
H
H
H
H
H
Me
F
3a
3b
3c
3d
3e
3f
82
82
81
80
84
82
79
80
82
76
74
70
2
Me
Cl
I
3
4
5
F
6
OMe
Br
H
7
3g
3h
3i
8
9
Me
H
10
11
12
3j
H
Br
H
3k
3l
H
a
b
Reaction was performed at 100 °C for 15 min. Isolated yield.
Table 3. Yields of 4a−n under Microwave Irradiation
a
b
entry
R1
R2
R3
product (4a−n)
yield (%)
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
F
4a
4b
4c
4d
4e
4f
85
82
85
84
88
81
78
80
70
84
83
77
75
73
2
Me
Cl
I
3
4
5
F
6
OMe
Br
H
7
4g
4h
4i
8
Me
Ph
9
H
10
11
12
13
14
H
CH2Ph
H
4j
Me
H
4k
4l
H
H
H
Br
I
4m
4n
H
H
a
b
Reaction was performed at 100 °C for 15 min. Isolated yield.
evidence in support of this addition came from the observed
HMBC correlation in the spectrum of 5c between −CH2N (δ
56.2) and H-11 (δ 2.24) and of C-11 (δ 24.1) with −CH2N
protons (δ 3.18, 2.73) (Figure 1). This was further supported
by the COSY relationship in between −CH2N proton (δ 3.18)
and H-12 (δ 2.53) coupled with the NOESY relationships
between H-9 (δ 1.33) and −CH2N protons (δ 3.18, 2.73) and
between H-11 (δ 2.24) and −CH2N proton (δ 2.73). The
NOESY correlation of β oriented H-9 (δ 1.33) with H-12 (δ
2.53) suggests the latter to be β oriented.
In case of 7-substituted and N-phenyl isatins the reaction rate
was to some extent slow (20 min in MW) with marginally
lower yield of the products as shown in Tables 2−4. The
reaction took place also in water in presence of β-CD or CTAB,
but the rate of the reaction was very slow, and yields were about
30−35% (Table 1). Formation of some insoluble byproduct
may be the reason for the low yield. But under MW irradiation
the reaction occurred within 20 min in a clean manner. The
yields were quite high (up to 80%) with no significant
byproducts. The reaction was also performed in ionic liquid
C
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Table 4. Yields of 5a−k under Microwave Irradiation
a
b
entry
R1
R2
R3
product (5a−k)
yield (%)
1
H
H
H
H
H
H
H
H
H
H
H
Ph
H
H
H
H
H
H
H
Me
F
5a
5b
5c
5d
5e
5f
82
84
82
80
81
78
79
2
Me
Cl
I
3
4
5
F
6
OMe
Br
Me
H
7
5g
5h
5i
c
8
79
c
9
75
c
10
11
H
Br
H
5j
72
c
H
5k
70
a
b
c
Reaction was performed at 100 °C for 15 min. Isolated yield. After 20 min reaction.
Scheme 2. Synthesis of Novel Di-spiro Andrographolide
Derivatives Using Acenaphthoquinone in Place of Isatin
Figure 1. Important correlations of 5c [HMBC (C →blue arrow H),
COSY (green arrow), NOESY (red arrow)].
(IL). Though clean and high yielding, it was comparatively
slower with respect to MW irradiation. However it was faster
than in methanol. The real hurdle faced was the separation of
IL from the product for its recycling, because of the polar
nature of both the product and IL. Considering all the factors,
reaction in methanol assisted by microwave irradiation works
the best.
Using the optimized protocol (for MW) we extended the
synthetic methodology toward another 1,2-diketone acenaph-
thoquinone (6) in place of isatins to synthesize some more
novel dispiro derivatives (7, 8, and 9) with high yield (∼80%)
(Scheme 2).
In case of sarcosine and N-benzyl glycine the acenaph-
thoquinone part got added from the C13 end and the methyl/
N-benzyl part from the C12 end mimicking the case of isatin.
The crucial evidence in support of this addition came from the
observed HMBC correlation in the spectrum of 7 between
signals of −CH2N (δ 60.3) and H-11 (δ 2.38, 2.26) as also C-
11 (δ 24.9) and −CH2N protons (δ 3.56, 3.15). Further, COSY
relationship between peaks for −CH2N proton (δ 3.56) and H-
12 (δ 3.11) together with the NOESY relationships among
peaks for H-9 (δ 1.61) and −CH2N protons (δ 3.15) as also
between exomethylene protons (δ 4.73) and a −CH2N proton
(δ 3.11) strongly supported the proposed structure (Figure 2).
Similar relationship was also observed in case of 8 [HMBC
between −CH2N (δ 57.1) and H-11 (δ 2.49, 2.18), C-11 (δ
24.5) and −CH2N protons (δ 3.57, 2.97); COSY between one
−CH2N proton (δ 3.57) and H-12 (δ 2.97) as also H-11 (δ
D
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Figure 4. ORTEP representation of 8.
Figure 2. Important correlations of 7 [HMBC (C →blue arrow H),
COSY (green arrow), NOESY (red arrow)].
2.49); NOESY relationships between H-9 (δ 1.51) and −CH2N
protons (δ 3.57, 2.97) and also between exomethylene protons
(δ 4.80) and −CH2N proton (δ 2.97)], strongly supporting the
proposed structure (Figure 3). The NOESY relationship of the
Figure 5. Important correlations of 9 [HMBC (C →blue arrow H),
COSY (green arrow), NOESY (red arrow)].
Inspired by the positive results from the biological screening
of the previous 15 compounds against some cancer cell lines
[unpublished data], anticancer activity of these 40 compounds
compared to andrographolide was evaluated against seven
cancer cell lines (Table 5).
The enhancement of activity in the N-benzyl glycine series
seems to be very promising, as indicated from their IC50 values,
and demand further evaluation for SAR analysis, toxicity
profiles, and mechanistic pathways.
Figure 3. Important correlations of 8 [HMBC (C →blue arrow H),
COSY (green arrow), NOESY (red arrow)].
acenaphthoquinone aromatic proton (δ 7.78) (2′) with the N-
benzylic proton (δ 3.31) also helped in the confirmation of
structure 8 (Figure 3). The NOESY correlation of β oriented
H-9 (δ 1.61 or 1.51) with H-12 (δ 3.11 or 2.97) confirms the
latter also to be β oriented.
This was ultimately confirmed by single crystal X-ray
crystallography of 8 (Figure 4) where the β-orientation of C7
and C9 protons was clearly visible, and the chirality deduced at
the C7, C16, and C20 center are respectively R, R, and R
(according to X-ray crystallographic numbering). This is
applied to all the figures presented.
CONCLUSION
■
In conclusion, a facile, atom-economic synthesis of novel
dispiro compounds of andrographolide has been achieved via
1,3-dipolar cycloaddition of azomethine ylides (derived from
isatins or acenaphthoquinone and few aminoacid derivatives)
under MW irradiation to Δ12 of andrographolide. When the
amino acid was changed from sarcosine or N-benzyl glycine to
proline, the opposite regioisomer was produced, as determined
by 2D NMR and confirmed by X-ray crystallographic analysis.
All the reactions are chemoselective in nature as only one of the
two double bonds takes part in the reaction. Biological
evaluation indicates the promise held out by N-benzyl glycine
derivatives that need to be further explored.
As noted in reactions with isatin, the acenaphthoquinone
part added from the C12 end when proline was introduced in
the reaction (in case of 9) and the proline part from the C13
end (Figure 5) as evidenced from detailed NMR spectra.
E
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Table 5. IC50 Values of Compounds 3a−3l, 4a−4n, 5a−5k, and 7−9
IC50 (μM)
Comp ID
CHO
HepG-2
HeLa
A431
MCF-7
Caco-2
MDCK
a
3a
3b
3c
3d
3e
3f
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
31.2
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
18.4
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
49.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
49.2
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
33.4
>50.0
>50.0
>50.0
>50.0
40.9
>50.0
>50.0
ND
ND
ND
ND
ND
ND
ND
3g
3h
3i
ND
>50.0
>50.0
>50.0
>50.0
>50.0
ND
ND
3j
ND
3k
3l
ND
ND
4a
4b
4c
4d
4e
4f
>50.0
>50.0
31.8
>50.0
>50.0
36.2
>50.0
28.9
>50.0
>50.0
>50.0
>50.0
30.6
>50.0
ND
ND
9.8
14.3
ND
12.9
18.5
18.8
21.5
31.6
16.4
ND
32.8
46.0
>50.0
>50.0
28.8
>50.0
>50.0
36.5
>50.0
>50.0
26.4
>50.0
13.1
>50.0
22.2
ND
4g
4h
4i
ND
27.0
>50.0
25.0
35.5
>50.0
26.7
>50.0
36.4
ND
32.4
24.8
17.7
>50.0
27.1
>50.0
>50.0
26.4
ND
4j
26.9
22.0
23.7
>50.0
25.4
38.9
39.0
ND
4k
4l
20.0
26.2
28.5
23.1
>50.0
24.9
>50.0
39.9
>50.0
36.3
>50.0
37.1
>50.0
33.7
ND
4m
4n
5a
5b
5c
5d
5e
5f
ND
24.2
26.8
26.1
26.2
30.9
ND
22.0
20.9
14.0
9.5
18.9
18.8
12.4
12.5
43.5
12.0
13.3
ND
19.4
13.9
13.7
12.1
10.9
ND
10.2
9.7
9.8
9.0
12.7
10.0
37.8
ND
48.7
43.9
40.4
33.1
43.2
16.2
16.5
14.6
9.0
14.4
14.4
12.3
12.7
16.6
15.7
12.7
10.6
7.3
5g
5h
5i
13.1
10.0
8.9
10.0
12.8
8.1
7.5
7.5
8.1
15.4
ND
10.8
12.9
12.2
13.9
13.7
ND
13.8
13.0
13.4
>50.0
10.8
39.3
20.9
5j
11.5
10.6
10.4
12.5
13.9
ND
5k
7
13.8
9.3
8.9
8.2
16.0
ND
>50.0
9.6
>50.0
8.1
>50.0
7.4
>50.0
10.5
>50.0
8.9
ND
8
ND
9
30.9
30.3
29.6
39.2
>50.0
29.4
>50.0
14.1
1
46.0
33.4
27.1
15.0
a
ND: Not done.
chromatographic absorbents were procured from E. Merck
(Germany) and SRL (India) Ltd. unless otherwise indicated.
Biology. Cell lines (CHO, HepG2, HeLa, A-431, MCF-7,
MDCK and Caco-2) were obtained from National Centre for
Cell Sciences, Pune, India. MEM-alpha, FBS (fetal bovine
serum), penicillin−streptomycin were purchased from Gibco,
India. Cell culture flasks, plates, and tips were obtained from
Tarsons, Kolkata, India. DMEM, other components of media,
and all other chemicals were purchased from Sigma, St. Louis,
U.S.A.
Typical Experimental Procedure for Synthesis of Dispiro
Products. A mixture of 1 (0.58 mmol, 200 mg), isatin (0.6
mmol, 90 mg) or acenapthoquinone (0.6 mmol, 110 mg), and
proline (0.61 mmol, 70 mg) or sarcosine (0.61 mmol, 54 mg)
or N-benzyl glycine (0.61 mmol, 101 mg) was taken in a
conical flask. The contents of the flask were mixed thoroughly
EXPERIMENTAL PROCEDURES
■
General Information. Chemistry. All the compounds
evaluated in this work were synthesized in one-pot sequences
using a mono mode Discover microwave reactor (CEM Corp.,
Matthews, NC, U.S.A.). Melting points were determined in
capillaries and are uncorrected. IR spectra were recorded as KBr
pellets using a JASCO 410 FTIR spectrometer. The NMR
spectra were recorded using a Bruker 600 DPX spectrometer
operating at 600 MHz for 1H and 150 MHz for 13C respectively
in DMSO-d6/CDCl3 with TMS as internal standard. Mass
spectra (positive mode) were obtained on a Q-TOF micro TM
mass spectrometer in the electrospray ionization mode.
Andrographolide was isolated from A. paniculata in the usual
way. Isatins, acenaphthoquinone and α-amino acids were
purchased from Alfa-Aesar Company. All other solvents and
F
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Funding
with 2−3 mL methanol. Then the crude mixture was irradiated
under MW at 100 °C. The progress of the reaction was
monitored by TLC in chloroform−methanol (9:1) and
visualization was accomplished in UV or by LB spray. After
completion of the reaction, the contents were directly dissolved
in chloroform−methanol (1−2%) and subjected to column
chromatography using chloroform−methanol (0.5% methanol
in chloroform) as eluant. The product was crystallized from an
acetonitrile-benzene mixture.
This work received financial assistance from the Council of
Scientific and Industrial Research (CSIR, Net Work Project-
0009), Government of India and Department of Science and
Technology (West Bengal-India). A.H., B.Y., A.M., S.M., and
R.P. are recipients of Research Fellowships from CSIR.
Notes
The authors declare no competing financial interest.
Cell Culture and MTT Assay. MDCK cells were
maintained in growth media containing MEM-alpha (Gibco)
supplemented with 10% FBS (Gibco) and penicillin−
streptomycin (100U/mL for each). Other cell lines were
maintained in DMEM media containing 10% FBS and 40 μg/
mL gentamycin.
ACKNOWLEDGMENTS
■
Our thanks are due to Dr. B. Achari (Emeritus Scientist, CSIR)
for helpful suggestions and Mr. K. Sarkar, E. Padmanabhan, and
S. Samaddar for recording HRMS, NMR, and IR spectra,
respectively. Special thanks go to Prof. B.C. Ranu of Indian
Association for the Cultivation of Science for giving permission
to use the microwave reactor.
HeLa cells were plated (in 96 well plates) at 6000 cells/well/
180 μL media. For other cell lines it was 4000 cells/well/180
μL media. Cell seeding density was optimized so that the wells
without any inhibitor can make up to 90% confluency at the
end of the incubation period/experiment. The plates containing
cells were placed in 37 °C incubator with 5% CO2 and 95%
relative humidity for 24 h. Media was aspirated off and replaced
with 180 μL of fresh media. Test compounds were dissolved at
a concentration of 5 mM (DMSO), and further serial half
dilutions were made in DMSO to reach to a concentration of
0.078 mM. These substocks were further 10-fold diluted in
respective growth media. Then 20 μL of growth media
containing test compounds was added (n = 2) in 96-well test
plate to produce final working conc of 50 to 0.78 μM (DMSO:
1%). Each plate had cell control, vehicle control, media control,
and standard inhibitor Doxorubicin at 10 μM.
The plates were placed back into the incubator for 72 h. 20
μL of MTT (5 mg/mL of PBS pH7.2) was added in each well,
and the plates further incubated for 4 h. The plates were
centrifuged (2500 rpm, 10 min), media flicked off and formazan
crystals dissolved in 150 μL of DMSO. Absorbance was
measured at 510 nm using Spectramax M5 (Molecular Devices,
U.S.A.). Cell death at each conc was determined based on OD
difference of the test well from that of wells of vehicle control.
If the highest test concentration (50 μM) shows less than 50%
cell death then IC50 (concentration that causes 50% cell death)
is reported as >50 μM or else it was calculated using GraphPad
Prism 5.0 software. The MTT assay experiment was performed
in duplicate wells each day and in three separate days.22,23
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C NMR, and HRMS data along with spectral copy of all
compounds associated with this Article. This material is
available free of charge via the Internet at http://pubs.acs.org.
Crystallographic data in CIF format are available free of charge
via the Internet from the Cambridge Crystallographic Data
Centre under deposition no. CCDC 893411. These data can be
obtained free of charge via www.ccdc.cam.ac.uk/conts/
retrieving.html (or from the Cambridge Crystallographic Data
Centre, 12, Union Road, Cambridge CB2 1EZ, U.K.; fax: +44
1223 336033; or deposit@ccdc.cam.ac.uk).
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AUTHOR INFORMATION
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Corresponding Author
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*Fax: +91-33-2473-5197. E-mail: nirup@iicb.res.in.
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