A new cytotoxic quinolone alkaloid and a pentacyclic steroidal glycoside from the stem bark of Crataeva nurvala: Study of anti-proliferative and apoptosis inducing property

Article abstract of DOI:10.1016/j.ejmech.2012.12.017

Chemical investigation of stem bark of Crataeva nurvala afforded 5,7-dimethoxy-3-phenyl-1-ethyl-1,4-dihydro-4-quinolone and a steroidal glycoside with unprecedented pentacyclic ring system named crataemine (1a) and crataenoside (2) respectively. The structures of the compounds were determined by spectroscopic analysis. A series of compounds with modification at position 1 of 1a (1a-1c) were prepared. All compounds were screened for cytotoxic activity against HeLa, PC-3 and MCF-7 cells. Only 1a and 2 showed potency against all three cells. Mechanism based study for activity of the compounds demonstrated that it could block the migration of more aggressive HeLa and PC-3 cells and prevent their colony formation ability as well. The compounds potentiated apoptosis in HeLa and PC-3 cells in a significant manner.

Full text of DOI:10.1016/j.ejmech.2012.12.017

European Journal of Medicinal Chemistry 60 (2013) 490e496
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A new cytotoxic quinolone alkaloid and a pentacyclic steroidal
glycoside from the stem bark of Crataeva nurvala: Study of anti-
proliferative and apoptosis inducing property
Sadhna Sinha a, Priyanka Mishra a, Hina Amin b, Bilal Rah b, Debasis Nayak b, Anindya Goswami b,
,c,
Naresh Kumar c, Ram Vishwakarma b, Sabari Ghosal a
*
a Amity Institute of Biotechnology, Amity University, Sector 125, Noida 201303, U.P., India
b Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India
c School of Chemistry, University of New South Wales, Sydney, NSW 2012, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical investigation of stem bark of Crataeva nurvala afforded 5,7-dimethoxy-3-phenyl-1-ethyl-1,4-
dihydro-4-quinolone and a steroidal glycoside with unprecedented pentacyclic ring system named
crataemine (1a) and crataenoside (2) respectively. The structures of the compounds were determined by
spectroscopic analysis. A series of compounds with modification at position 1 of 1a (1ae1c) were
prepared. All compounds were screened for cytotoxic activity against HeLa, PC-3 and MCF-7 cells. Only
1a and 2 showed potency against all three cells. Mechanism based study for activity of the compounds
demonstrated that it could block the migration of more aggressive HeLa and PC-3 cells and prevent their
colony formation ability as well. The compounds potentiated apoptosis in HeLa and PC-3 cells in
a significant manner.
Received 23 September 2012
Received in revised form
5 December 2012
Accepted 8 December 2012
Available online 17 December 2012
Keywords:
4-Quinolones
Steroidal glycoside
Capparaceae
Ó 2012 Elsevier Masson SAS. All rights reserved.
Crataeva nurvala
Cytotoxic activity
1. Introduction
space is to turn to those natural product scaffolds that are
unrepresented.
Plant secondary metabolites and their semi-synthetic deriva-
tives continue to play an important role in cancer chemopreventive
studies. Few anticancer agents amongst a broad arsenal of naturally
occurring compounds currently undergoing clinical trials include
podophyllotoxin, camptothecin, vinblastine and paclitaxel.
Considerable efforts have also been paid to generate promising lead
compounds through high throughput screening (HTS) protocols,
combinatorial chemistry and bioinformatics tools. However,
natural products have an edge as they are evolved for some special
biological purpose and exhibit remarkable HTS hit rates, much
higher than to be expected. Usually these compounds possess
unique built-in chirality which promotes most effective binding to
complex proteins and other three-dimensional biological receptors.
Hence, the best way to find new leads amid unexplored chemical
Crataeva nurvala Buch. Ham. (Capparaceae), an important
medicinal plant of Ayurvedic and Unani system of medicine has
been studied mainly with the stem bark part for obstructive and
nonobstructive uropathies. It is also used for the treatment of
prostate enlargement and bladder sensitivity [1]. Phytochemical
studies showed that it contained lupeol, betulinic acid, b-sitosterol,
stigmasterol [2] and cabadicine [3] as major chemical constituents
of the plant. Lupeol had been reported to reduce the deposition of
calcium oxalate, the most common stone forming constituent in
animals. It also possessed antifartility, anticancer, antitumor, anti-
arthritic, anti-inflammatory, hepatoprotective, and cardioprotec-
tive activities [4]. In our continuous search for new pharmacolog-
ically active agents from Indian medicinal plants, we investigated C.
nurvala for cytotoxic activity. Preliminary screening identified
diethyl ether (Et2O) and n-BuOH fractions of the methanolic extract
(MeOHeH2O; 9:1) of the plant as most active. Hence, subsequent
analysis was carried out with these fractions separately. Herein, we
report i) isolation and characterization of two new compounds viz.,
1a and 2; ii) synthesis of compounds 1ae1c and iii) screening of all
these compounds for cytotoxic activity on human cervical cancer
* Corresponding author. Amity Institute of Biotechnology, Amity University,
Sector 125, Noida 201303, U.P., India. Tel.: þ91 120 4735614; fax: þ91 120 4392947.
E-mail addresses: sabarighosal@gmail.com, sghosal@amity.edu (S. Ghosal).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.017

S. Sinha et al. / European Journal of Medicinal Chemistry 60 (2013) 490e496
491
(HeLa), human prostate cancer (PC-3) and human breast cancer
(MCF-7) cells. The active compounds were also studied for anti-
proliferative and apoptosis inducing property.
reported procedure. Subsequent cyclization of the intermediate in
a solution of biphenyl and diphenyl ether followed by heating at
250 ꢂC produced 5,7-dimethoxy-3-phenyl-1,4-dihydro-4-quino-
lone (1b). Alkylation of 1b with methyl iodide and ethyl iodide in
the presence of potassium carbonate in anhydrous DMF under
nitrogen atmosphere produced 1c and 1a respectively (Scheme 1).
The crude residue was purified further by silica gel column chro-
matography. The structure of 1b and 1c was confirmed by
comparing their physical and spectroscopic data with the reported
literature values.
Crataenoside (2) was obtained as an amorphous solid. The
molecular formula of 2 was established as C33H54O6 by using posi-
tive ion HRESIMS exhibiting an [M þ H]þ peak at m/z 547.3979 and
a quasi-molecular ion peak of [M þ Na]þ at 569.3901. A broad IR
absorption band at 3385 cmꢀ1 suggested the presence of hydroxyl
functionality. The 1H NMR spectrum showed signals for five methyl
2. Results and discussion
2.1. Chemistry
Repeated column chromatography of Et2O fraction of stem bark
of C. nurvala afforded crataemine (1a) while, n-BuOH fraction
afforded crataenoside (2) in minor quantity. The structures of the
compounds were elucidated by 1D and 2D NMR spectroscopy and
mass spectrometry (Fig. 1).
The HRESIMS of 1a gave [M þ H]þ ion peak at m/z 310.1434
indicating the molecular formula of C19H19O3N (calc. 310.1437). The
presence of a conjugated carbonyl and an NR function attached to
an olefinic moiety was revealed by IR absorption bands at 1641 and,
1635 and 1617 cmꢀ1 respectively. The 1H and 13C NMR spectrum
groups including two tertiary methyl groups at
secondary methyl group at 0.93 and two geminal methyl groups at
0.86 and 0.83. Further, it showed one olefinic proton at 5.37,
a broad methine multiplet of a carbinol proton at 3.59 and one
anomeric proton at
d 1.02 and 0.7, one
d
d
d
displayed signals for an NeEt function [
1.43 (t, 3H, J ¼ 6.6 Hz); dC 48.6 and 13.9], a trisubstituted olefinic
moiety [dH 7.44 (s, 1H); dC 139.4], a monosubstituted phenyl ring [
7.57 (d, 2H, J ¼ 8.4 Hz), 7.30 (app t, 2H, J1 ¼ 8.4 Hz, J2 ¼ 1.5 Hz),
7.21 (dd, 1H, J1 ¼ 8.4 Hz and J2 ¼ 1.5 Hz); dC 128.8, 127.8 (each
2 ꢁ CH) and 126.6 (CH)] and a conjugated carbonyl at dC 175.5.
Additionally, two aromatic methoxyl protons at 3.88 and 3.85
(each s, 3H) and two meta coupled aromatic protons at 6.30 and
d
4.04 (q, 2H, J ¼ 7.2 Hz) and
d
d
d
4.40 (d, J ¼ 7.8 Hz). The large coupling constant
d
(7.8 Hz) of the anomeric proton along with five methine and one
methylene carbons at dC 100.6, 76.1, 75.6, 73.1, 69.7 and 61.1 indi-
d
d
cated the presence of a b-D-glucose moiety attached to an aglycon
part. Analysis of 13C and HMQC spectral data of 2 revealed that the
aglycon, bearing 27 carbons contained three quaternary carbons
including one olefinic; nine methine comprising an olefinic; ten
methylenes, and five methyl groups. The long range HMBC corre-
d
d
6.27 (each d, 1H, J ¼ 2.1 Hz) indicated the presence of a 1,2,3,5 tetra
substituted phenyl moiety. It was further supported by the corre-
sponding methoxyl carbons at dC 56.1, 55.3 and two meta coupled
aromatic carbons at 94.1 and 89.8 respectively. These partial
structures were in consistence with an N-Et-4-quinolone moiety
un-substituted at C-2 position. The COSY and HSQC correlations
showed connectivities between five aromatic carbons of a mono-
substituted phenyl ring and between aromatic protons of the qui-
nolone moiety (Fig. 2a). Moreover, the intense 1He1H NOESY cross
peaks between H-2/H2-100; H2-100/H-8 and H2-100/H2-200 confirmed
that C-2 position of the compound was unoccupied while C-3 was
linked to the phenyl ring. Consequently, the structure of 1a was
established as 5,7-dimethoxy-3-phenyl-1-ethyl-1,4-dihydro-4-
quinolone. Comparison of the 1H and 13C NMR data of 1a to those
reported in the literature showed that it possesses striking simi-
larity with the synthetically derived 5,7-dimethoxy-3-phenyl-1-
methyl-1,4-dihydro-4-quinolone (1c). The two compounds differ
essentially only by one methylene group, 1a possessed N-ethyl
functionality whereas 1c contained N-Me group [5].
lations between H-10 (
d
4.40) and C-3 (dC 78.3), and C-10 (dC 100.6) to
3.59) confirmed the position of attachment of the sugar
H-3 (
d
moiety in the aglycon. Additionally, the HMBC connectivities of
methyl protons at CH3-19 to C-10, C-1, C-5 and C-9; CH3-18 to C-13,
C-17, C-14 and C-12; CH3-21 to C-20, C-17 and C-22 showed exactly
the same pattern to that of the partial structure contributed by ABCD
rings of cholesterol [6]. The HMBC correlations also supported the
relative configurations at A to B, B to C and C to D ring junctions,
identical to that of cholesterol types. The 1He1H COSY correlations
between C-6 to C-7, C-3 to C-4 and C-2, and C-20 to C-10 and C-30
further supported the above assumption. However, significant
differences were observed mainly with the carbon signals and their
splitting pattern, arising from the side chain of compound 2 to that
of cholesterol. The comparative study showed that 2 contained ten
methine and eight methylene carbons while cholesterol possessed
nine methine and nine methylene carbons. It revealed the presence
of one more degree of unsaturation. The HMBC spectrum showed
prominent correlations between geminal methyl groups at
d 0.86
In order to understand the significance of naturally occurring N-
ethyl derivative we first prepared N-unsubstituted (1b) derivative
and 0.83 (each d, 3H, J ¼ 6.6 Hz; H-26 and H-27) and corresponding
carbons at dC 18.8 and 18.4. It also showed correlations between C-
25 (dC 27.6) and C-24 (dC 45.3) (Fig. 2b). The COSY cross peaks
by coupling of 3,5-dimethoxyaniline with ethyl
a-formylphenyl
acetate in inert atmosphere producing the intermediate, ethyl (Z)-
3-(3,5-dimethoxyanilino)-2-phenyl-2-propenoate by following the
a
b
Fig. 2. 2D NMR correlations of 1a and 2. (a) Showing HSQC and COSY correlations of 1a
in bold lines and important NOESY correlations by arrows. (b) Showing HSQC and COSY
correlations in bold lines and important HMBC correlations by arrows.
Fig. 1. Chemical structures of crataemine (1a), 1-unsubstituted (1b) and 1-Me (1c)
substituted derivatives of 1a and crataenoside (2).

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S. Sinha et al. / European Journal of Medicinal Chemistry 60 (2013) 490e496
Scheme 1. Synthesis of compounds 1ae1c. Reagents and conditions: (i) reflux, 18 h, N2 atm, toluene, 73%; (ii) biphenyl þ diphenyl ether, 250 ꢂC, 10 min, 60%; (iii) CH3I þ anhydrous
K2CO3, DMF, N2 atm, rt, 18 h, 75%; (iv) C2H5I þ anhydrous K2CO3, DMF, N2 atm, rt, 18 h, 70%.
indicated the presence of sequence C-26, C-27 to C-25 and C-25 to C-
24. Finally, as there was no additional olefinic carbon, the extra one
degree of unsaturation could be devoted only by the formation of an
additional ring. Biogenetically, cholesterol and other phytosterols
can undergo a variety of structural transformation in plants, espe-
cially involving side chains but formation of pentacyclic structures
[7,8] is not common in plant kingdom. A study on the mode of
hydroxylation of steroids at non-activated secondary carbons in
(ECACC). Staurosporine was used as positive control. Compounds
1a and 2 were observed to be active on all three cells whereas 1b
and 1c were completely inactive. Various derivatives of 1a have
been studied widely for remarkable antibacterial and antitumor
activity [14]. Structural modifications in reference to position and
change in functionality and SAR study with respect to these activ-
ities have been examined extensively. Previous studies have shown
that 1b as well as 1c in combination with standard anticancer drugs
is capable of stimulating the recruitment of clonogenic cells in
tumor, making it more sensitive to the conventional treatment with
cytotoxic agents.
higher plants demonstrates that the introduction of the 16
b
hydroxyl group in the biosynthesis of gitoxiginin from pregneno-
lone in Digitalis pupurea proceeds by the direct displacement of the
16
b
proton [9]. A chemical investigation conducted on aerial part of
Initially, MTT assay was performed to define the optimum
concentration at which the compounds were nontoxic to cells. The
cancer cells treated with 50 mM concentration of 1a and 80 mM of 2
Polianthes tuberose reported a bisdesmosidic cholestane glycoside
with an apiofuranoside group at C-16 [10]. On the basis of all these
observations a plausible biogenetic pathway for crataenoside was
proposed as shownin Scheme 2. Compound 2 might be derived from
a steroidal derivative by sequential enzymatic hydroxylation at C-16
followed by cyclization via C-16eC-24 bond formation and subse-
quent reduction of C-24eC-25 bond [11]. Two methine signals at dC
48.6 and dC 45.3 supported the above contention in the favor of C-16
to C-24 linkage. Acid hydrolysis of 2 with 0.2 M HCl/MeOH furnished
(IC50 values) affect the viability of HeLa and PC-3 (data not shown)
cells significantly, whereas less aggressive MCF-7 cells were
affected even at much lower concentration of the compounds. To
test the effect of 1a and 2 on growth kinetics of cancer cells, colony
formation assay were performed in a dose dependent manner as
shown in Figs. 3a and 4a respectively. Sub-lethal doses of the
compounds decreased colony formation ability of HeLa and PC-3
(data not shown) cells in statistically significant manner
(P < 0.05) while MCF-7 cells were unaffected. To examine the effect
of the compounds on cell migration, wound healing assay was
performed on confluent monolayers of HeLa, PC-3 and MCF-7 cells.
After making the wound with a pipette tip, the cells were cultured
in the presence or absence of different concentrations of the
compounds along with vehicle DMSO. The wounded areas were
progressively photographed with Olympus c-7070 with 700M
camera. The vehicle DMSO treated cells (control) were able to
completely fill in the cleared area, whereas treatment with 50 and
the aglycon and one monosaccharide unit which was identified as D-
glucose by co-HPTLC with the authentic sample [12,13].
2.2. Biological evaluation
Compounds 1ae1c and 2 were screened against HeLa, PC-3 and
MCF-7 cell lines purchased from European Collection of Cell Culture
80 mM of 1a and 2 significantly (P < 0.05) inhibited the migration of
HeLa (Figs. 3b and 4b) and PC-3 (data not shown) cells, thereby
reducing their migration capability. The experimental data strongly
demonstrated that both 1a and 2 could block the migration of more
aggressive HeLa and PC-3 cells in a dose dependent manner but not
MCF-7 cells, which exhibited less IC50 values compared to HeLa and
PC-3 cells.
To further elucidate the underlying mechanism for cytotoxicity
of 1a and 2 we performed apoptosis studies on HeLa and PC-3 (data
not shown) cells. After treating the cells with different concentra-
tions of the compounds, the percentage of apoptotic cells was
assessed by propidium iodide staining, followed by flow cytometric
analysis (Figs. 3c and 4c). Interestingly, compounds 1a and 2
observed 76.8% and 63.5% apoptotic cells at 50
mM and 80 mM
Scheme 2. Probable biosynthesis for compound 2.
concentrations compared to 68.6% with 1 M concentration of
m

S. Sinha et al. / European Journal of Medicinal Chemistry 60 (2013) 490e496
493
Fig. 3. Assessment of cytotoxicity and cell motility of 1a at a concentration of 50 mM along with vehicle DMSO and positive control staurosporine (25 nM) or camptothecin (1 mM)
against HeLa cells. (a) Colony formation assay was performed with 1 ꢁ103 cells/well and the number of stained cells per colony was counted randomly and images were captured in
100ꢁ magnifications under inverted microscope. (b) Wound healing assay (0.5 ꢁ 105 cells/well) to assess the degree of wound healing. The scratched areas were quantified in three
random fields in each treatment, and data were calculated from three independent experiments. *P < 0.05 compared with untreated control. (c) Cell cycle analysis to determine cell
cycle distribution. Data were calculated from three independent experiments. (d) HeLa cells (5 ꢁ 104) after fixation were stained with nuclear stain DAPI reagent and were
photographed under fluorescence microscope (100ꢁ magnification) and data were calculated from three independent experiments. Columns mean; bars SD of three independent
experiments. *P < 0.05 compared with untreated control.
positive control camptothecin. By using 4,6-diamidino-2-
phenylindole (DAPI) staining we observed that 1a and 2 induced
apoptosis in HeLa cells within 24 h of incubation as compared to
positive control staurosporine as shown in Figs. 3d and 4d
respectively. The data indicated the occurrence of early apoptosis
with chromatin condensation around the nuclear periphery,
accompanied by nuclear size reduction (white arrow heads).
4. Experimental
4.1. Chemicals
All solvents used were of analytical grade (CDH Laboratory
Reagents, India). Silica gel 60, 100e200 (Merck, India) and Sepha-
dex LH-20 (Pharmazia) was used for gravity column and silica gel
300e400 (Merck, India) was used for flash column chromatog-
raphy. Pre-coated silica gel 60 GF254 plates were used for analytical
3. Conclusion
TLC. HPTLC (Merck) were used for co-TLC assay. D-Glucose (Merck,
A critical analysis of compounds 1ae1c in reference to cytotoxic
activity revealed that 1a possessing an N-ethyl functionality showed
much higher activity against HeLa and PC-3 cells affording impor-
tant information about structure based drug designing. The study
also afforded a unique pentacyclic steroidal glycoside with anti-
proliferative and apoptosis inducing property. Though few penta-
cyclic polyoxygenated steroids have been reported from marine
microorganisms and plants, however this is the first report of
a cholestane type pentacyclic steroidal glycoside from higher plants.
India) was used as a standard. The spots were visualized under UV
light (254 and 366 nm) and also by spraying with Dragendorff’s
reagent or with anisaldehyde sulfuric acid followed by heating. IR
(KBr) spectra were recorded using a Nicolet model Protégé 460
spectrophotometer. 1H/13C-NMR spectra were recorded on 300/75
and 600/125 MHz Bruker spectrometer using TMS as internal
standard. The chemical shift values are reported in ppm (d) units
and the scalar coupling constants (J) are in Hz. HRESIMS was per-
formed on Agilent-6220 accurate mass LC-TOF system attached

494
S. Sinha et al. / European Journal of Medicinal Chemistry 60 (2013) 490e496
Fig. 4. Cytotoxicity and cell motility assessment of 2 at a concentration of 80 mM along with vehicle DMSO and positive control staurosporine (25 nM) or camptothecin (1 mM)
against HeLa cells. (a) Colony formation assay was performed with 1 ꢁ 103 cells/well and the number of stained cells (crystal violet) per colony was counted randomly and images
were captured in 100ꢁ magnifications under inverted microscope. (b) Wound healing assay (0.5 ꢁ 105 cells/well) to assess the degree of wound healing. The scratched areas were
quantified in three random fields in each treatment, and data were calculated from three independent experiments. *P < 0.05 compared with untreated control. (c) Cell cycle
analysis to determine cell cycle distribution. Data were calculated from three independent experiments. (d) HeLa cells (5 ꢁ 104) after fixation were stained with nuclear stain DAPI
reagent and were photographed under fluorescence microscope (100ꢁ magnification) and data were calculated from three independent experiments. Columns mean; bars SD of
three independent experiments. *P < 0.05 compared with untreated control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
with Agilent 1200 Series HPLC with an eluent of 0.3 mL/min
acetonitrile.
fraction (5.7 ꢁ 85 cm, silica gel 60; 200 mL each) using n-hexane
and stepwise 10e100% gradient of EtOAc followed by solvent
distillation under vacuum, afforded five fractions (F-1 to F-5) by TLC
monitoring under UV light and Dragendorff’s reagent. Further,
chromatography of F-2 (2 ꢁ 70 cm, silica gel 100e200) with n-
hexaneeEtOAc (90:10) eluent afforded previously reported white
precipitate of lupeol in excess (1 g). Additionally, fraction F-3 on
elution with n-hexaneeEtOAc (80:20) produced formerly reported
semisolid precipitate of stigmasterol. Repeated column chroma-
tography of F-5 with n-hexaneeEtOAc (1:1) afforded light yellow
solid of 1a (TLC solvent: n-hexaneeEtOAc, 1:2; 6 mg). The n-BuOH
soluble fraction was purified by flash chromatography with DCM
and 10% stepwise gradient of MeOH affording five fractions
(100 mL; S-1 to S-5) monitored by EtOHeH2SO4 spray reagent (7%
H2S04 in EtOH). Further chromatography of S-2 on Sephadex LH-20
with MeOH as eluent afforded partially purified 2 (9 mg). As the
compound was invisible under PDA detector, it could not be sepa-
rated by HPLC. The final separation was achieved by silica gel
chromatography using gradient elution of DCMeMeOH (99:1 to
95:5). The 5% MeOHeDCM fraction afforded 2 (7 mg) as white solid.
4.2. Plant material
The stem-bark of C. nurvala was procured from a registered
vendor ‘Accolent Dried Herbs’ Parkvale Street, Victoria Point,
Queensland 4165, Australia. A voucher specimen (SG/01/2010) was
authenticated by Dr. M.P. Sharma, Department of Botany, Hamdard
University, New Delhi and has been deposited in the herbarium of
Amity Institute of Biotechnology, Amity University, Noida, India.
4.3. Chemistry
4.3.1. Extraction, isolation and characterization
The dried stem bark part of C. nurvala (0.25 kg) was extracted
with MeOHeH2O (9:1, 1 L ꢁ 3 ꢁ 24 h) followed by H2O (0.5 L) at
room temperature. The concentrated MeOH extract was suspended
in water and successively partitioned into Et2O (1 L, 8 g) and n-
BuOH (0.5 L, 1.5 g) fractions. Successive chromatography of Et2O

S. Sinha et al. / European Journal of Medicinal Chemistry 60 (2013) 490e496
495
Compound 2 was visualized as bright pink spot on TLC (DCMe
MeOH; 7:3) with the spray reagent.
(d, 1H, J ¼ 2.3 Hz), 3.78 (s, 3H, 5-OMe), 3.73 (s, 3H, 7-OMe), 3.29 (s,
3H, N-CH3); 13C NMR (75 MHz, CDCl3):
d
¼ 175.6 (C-4), 162.8 (C-5),
162.3 (C-7), 142.5 (C-8a), 140.9 (C-2), 135.8 (C-10), 128.6 (C-20, C-60),
127.5 (C-30, C-50), 126.5 (C-40), 123.9 (C-4a), 112.6 (C-3), 94.4 (C-6),
90.2 (C-8), 55.8 (5-OMe), 55.3 (7-OMe), 42.2 (N-Me); HRESI MS: m/
z calc. for C18H18NO3 [M þ H]þ 296.1287; found 296.1282.
4.3.1.1. 5,7-Dimethoxy-3-phenyl-1-ethyl-1,4-dihydro-4-quinolone
(1a). IR (KBr) nmax in cmꢀ1, 1641,1635, 1617, 1560, 1508, 1445, 1340,
1320, 1242, 1215, 1027, 932, 858, 830, 783; 1H NMR (600 MHz,
CDCl3):
d
¼ 7.57 (d, 2H, J ¼ 8.4 Hz, H-20, H-60), 7.44 (s, 1H, H-2), 7.30
(app t, 2H, J1 ¼ 8.4 Hz, J2 ¼ 1.5 Hz, H-30, H-50), 7.21 (dd, 1H,
J1 ¼8.4 Hz, J2 ¼ 1.5 Hz, H-40), 6.30 (d,1H, J ¼ 2.1 Hz, H-6), 6.27 (d,1H,
J ¼ 2.1 Hz, H-8), 4.04 (q, 2H, J ¼ 7.2 Hz, H2-100), 3.88 (s, 3H, 5-OMe),
3.85 (s, 3H, 7-OMe),1.43 (t, 3H, J ¼ 7.2 Hz, H3-200); 13C NMR (75 MHz,
4.3.2.3. Crataenoside (2). 1H NMR (CDCl3eCD3OD; v/v, 2:1,
600 MHz):
d
5.37 (d, 1H, J ¼ 4.9 Hz, H-6), 4.40 (d, 1H, J ¼ 7.8 Hz, H-
10), 3.85 (dd, 1H, J1 ¼ 12 Hz, J2 ¼ 2.4 Hz, H-60
a) 3.71 (dd, 1H,
J1 ¼ 12 Hz, J2 ¼ 5.2 Hz, H-60
b), 3.59 (br m, 1H, H-3), 3.37 (m, 1H, H-
CDCl3):
d
¼ 175.5 (C-4), 163.1 (C-5), 162.5 (C-7), 142.9 (C-8a), 139.4
30), 3.32 (m, 1H, H-40), 3.27 (m, 1H, H-20), 3.20 (t, 1H, J ¼ 6 Hz, H-50),
2.42 (dd, 1H, J1 ¼ 13.1 Hz, J2 ¼ 1.2 Hz, H2-4), 2.27 (t, 1H, H2-4) 1.95
(m, 1H, H-7) 1.66 (m, 1H, H-7), 1.63 (t, 1H, H-25), 1.61 (m, 2H, H2-2),
1.25 (m, 1H, H-24), 1.02 (s, 3H, H-19), 0.93 (d, 3H, J ¼ 6.4 Hz, H-21),
0.86 (d, 3H, J ¼ 6.6 Hz, H-25), 0.83 (d, 3H, J ¼ 6.6 Hz, H-26), 0.7 (s,
3H, H-18).
(C-2), 135.7 (C-10), 128.8 (C-20, C-60), 127.8 (C-30, C-50), 126.6 (C-40),
123.8 (C-4a), 112.9 (C-3), 94.1 (C-6), 89.8 (C-8), 56.1 (5-OMe), 55.3
(7-OMe), 48.6 (C-100),13.9 (C-200); HRESI MS: m/z calc. for C19H19NO3
[M þ H]þ 310.1437, [M þ Na]þ 332.1359; found 310.1434 and
332.1354 respectively.
13C NMR (CDCl3eCD3OD; v/v, 2:1, 600 MHz): 139.8 (C-5), 121.3
(C-6), 100.6 (C-10), 78.3 (C-3), 76.1 (C-50), 75.6 (C-30), 73.1 (C-20),
69.7 (C-40), 61.1 (C-60). 56.2 (C-14), 55.5 (C-17), 49.7 (C-9), 48.6 (C-
16), 45.3 (C-24), 41.7 (C-13), 39.2 (C-12), 38.0 (C-4), 36.7 (C-1), 36.1
(C-10), 35.5 (C-20), 33.3 (C-22), 31.3 (C-7), 31.3 (C-8), 28.5 (C-2),
27.6 (C-15), 27.6 (C-25), 25.4 (C-23), 20.4 (C-11), 18.8 (C-27), 18.4 (C-
26), 18.0 (C-19), 17.9 (C-21), 11.0 (C-18); HRESI MS: m/z calc. for
C33H54O6 [M þ H]þ 547.3984, [M þ Na]þ 569.3906; found 547.3979
and 569.3901 respectively.
4.3.2. General procedure for the synthesis of 1b and 1c
1.54 g (10.05 mmol) of 3,5-dimethoxyaniline and 2.12
g
(1.1 equiv) of ethyl- -formylphenyl acetate in 20 mL of toluene was
a
stirred at reflux condition for 18 h under nitrogen atmosphere.
After cooling, the reaction mixture was diluted with toluene
(20 mL), acidified with 10% HCl and then extracted with CHCl3. The
organic layer was dried over MgSO4, filtered and then evaporated
under reduced pressure. The residue was dissolved in methanol
and was stored for crystallization in a freezer at ꢀ80 ꢂC. Repeated
crystallization of the residue afforded 2.2 g (73%) of ethyl (Z)-3-(3,5-
dimethoxyanilino)-2-phenyl-2-propenoate as intermediate. The
intermediate (1.20 g, 3.67 mmol) was added portion wise and
rapidly with stirring, to a solution of biphenyl (1.52 g) and diphenyl
ether (11.60 g) heated to 250 ꢂC for 10 min. The precipitate of 1b
was obtained in excess from the reaction mixture on cooling. The
product was collected by filtration and then rinsed with petroleum
ether. White crystals of 1b (500 mg) were collected after drying.
200 mg (0.7 mmol) of the intermediate was added to 10 mL of
anhydrous N,N-dimethylformamide (DMF) under nitrogen atmo-
sphere. 734 mg of anhydrous K2CO3 (7.5 equiv) and 0.13 mL of
methyl iodide (3 equiv) were added consecutively to the suspen-
sion. The mixture was stirred at room temperature for 18 h. Then
the solvent was evaporated and the residue was extracted with
CH2Cl2 and washed with water. The organic phase was dried over
MgSO4 and the solvent was removed under reduced pressure. The
crude residue was purified on a column of silica (CH2Cl2eEtOAc;
9:1) to produce 150 mg (71%) of 1c.
4.3.3. Acid hydrolysis of 2
Compound 2 (3 mg) was refluxed at 90ꢂC with 2 N HCl in
MeOH (1:4, 1 mL) for 2 h under argon atmosphere. The reaction
mixture was diluted with water and extracted with CHCl3 (10 mL).
The aqueous layer was neutralized with Amberlite MB-3 (Organo
Co.) and then evaporated under reduced pressure to produce the
monosaccharide as residue. The residue was extracted with MeOH
and was analyzed by silica gel HPTLC developed with Me2CO and
2 mM NaOAc (17:3, v/v) and detected by spraying with 0.2%
naphthoresorcinol in Me2CO and 3 N H3PO4 (5:1, v/v) followed by
heating at 105 ꢂC for 5 min (Rf 0.6).
D-Glucose was used as
standard.
4.4. Biology
4.4.1. Cell culture
All cell lines were purchased from European Collection of Cell
Culture (ECACC), Fetal Bovine Serum (FBS), RPMI-1640, Minimum
Essential Medium (MEM), Penicillin G, Streptomycin, Trypsin-EDTA
were obtained from Invitrogen Corp. 5-Diphenyltetrazolium
bromide (MTT), paraformaldehyde, staurosporine, camptothecin,
dimethylsulfoxide (DMSO), Bradford reagent were obtained from
Sigma Chemicals Co. (St. Louis, MO). Propidium iodide and Ultra-
cruz DAPI mounting medium were obtained from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA).
The structure of 1b and 1c was established as 5,7-dimethoxy-3-
phenyl-1,4-dihydro-4-quinolinone and 5,7-dimethoxy-3-phenyl-
1-methyl-1,4-dihydro-4-quinolinone respectively by analysis of
NMR and MS spectral data.
4.3.2.1. 5,7-Dimethoxy-3-phenyl-1,4-dihydro-4-quinolinone
(1b).
1H NMR (300 MHz, CDCl3):
d
¼ 7.79 (s, 1H, H-2), 7.56 (d, 2H,
J ¼ 7.5 Hz, H-20, H-60), 7.3 (app t, 2H, J ¼ 7.5 Hz, H-30, H-50), 7.19 (app
t,1H, J ¼ 7.8 Hz, H-40), 6.50 (d,1H, J ¼ 2.3 Hz), 6.30 (d,1H, J ¼ 2.3 Hz),
3.80 (s, 3H, 5-OMe), 3.76 (s, 3H, 7-OMe); 13C NMR (75 MHz, CDCl3):
4.4.2. Cell proliferation assay
The cell viability was determined by standard MTT dye uptake
method. Briefly, HeLa, PC-3 and MCF-7 cells (3 ꢁ 103 cells/well)
were plated into 96 well plate and were treated with different
concentrations of compounds 1ae1c and 2 in triplicate so that
the final concentration of DMSO was 0.2 %. After 48 h incubation,
MTT (0.5 mg/mL) solution was added and was further incubated
at 37 ꢂC for 4 h. The amount of colored formazan derivatives was
determined by measuring optical density (OD) using a micro-
plate reader (Infinite M200 PRO) at 570 nm. The percentage
viability was determined according to the protocol described in
Ref. [15].
d
¼ 175.3 (C-4), 162.9 (C-5), 162.5 (C-7), 142.8 (C-8a), 139.4 (C-2),
135.6 (C-10), 128.8 (C-20, C-60), 127.6 (C-30, C-50), 126.5 (C-40), 123.8
(C-4a), 112.7 (C-3), 94.1 (C-6), 89.8 (C-8), 55.9 (5-OMe), 55.4 (7-
OMe); HRESI MS: m/z calc. for C17H16NO3 [M þ H]þ 282.1130;
found 282.1125.
4.3.2.2. 5,7-Dimethoxy-3-phenyl-1-methyl-1,4-dihydro-4-
quinolinone (1c). 1H NMR (300 MHz, CDCl3):
d
¼ 7.77 (s, 1H, H-2),
7.53 (d, 2H, J1 ¼ 7.6 Hz, H-20, H-60), 7.26 (app t, 2H, J ¼ 7.6 Hz, H-30,
H-50), 7.15 (app t, 1H, J ¼ 7.6 Hz, H-40), 6.46 (d, 1H, J ¼ 2.3 Hz), 6.27

496
S. Sinha et al. / European Journal of Medicinal Chemistry 60 (2013) 490e496
4.4.3. Clonogenic assay
Conflict of interest
The assay was performed according to the previously described
method [16]. Briefly, HeLa, PC-3 (data not shown) and MCF-7 cells
were plated at a seeding density of (1 ꢁ 103 cells/well) in 6 well
tissue culture grade plates. After 24 h the culture medium was
changed and new medium was added and cells were exposed to
various concentration of compounds 1a and 2 along with vehicle
DMSO for 5 days in 5% CO2 incubator at 37 ꢂC. Later on, the obtained
colonies were fixed with 4% paraformaldehyde and were stained
with 0.5% crystal violet solution. The colonies from the plates were
counted and averaged from the observed fields randomly (n ¼ 3)
and photographed with Olympus c-7070 wide 700M inverted
microscope camera.
None of the authors of the above manuscript have declared any
conflict of interest within the last three years which may arise from
being named as an author on the manuscript.
Acknowledgments
Authors acknowledge Endeavour Research Award Committee,
Australian Government for financial support, Dr. Donald Thomas,
University of New South Wales for NMR support, Dr. Sally Duck,
Monash University for providing mass spectroscopic data, Dr. N.G.
Ramesh, IIT Delhi for valuable inputs and Dr. A.K. Chauhan, Founder
President, Amity University, for his continuous motivation and
encouragement.
4.4.4. Wound healing assay
Briefly, HeLa, PC-3 (data not shown) and MCF-7 cells were
plated in 6 well plates at a seeding density of 5.5 ꢁ 105 cells/well
and grown overnight to form a confluent monolayer. An artificial
Appendix A. Supplementary data
wound was made by a sterile pipette tip (200 mL) after 24 h of
serum starvation and washed with serum free medium to remove
floated and detached cells. A photograph was recorded (time 0 h).
Thereafter, the cells were successively treated in a medium con-
taining low serum (1%), in the presence of different concentrations
of compounds 1a and 2 along with vehicle DMSO for 24 h. The
wounded areas were progressively photographed with Olympus c-
7070 with 700M camera (100ꢁ magnification). The percentage of
wound closure was estimated by the following equation: wound
closure % ¼ [1 ꢀ (wound area at t1/wound area at t0) ꢁ 100%],
where t1 is the time after wounding and t0 is the time immediately
after wounding [17].
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.12.017.
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