Gallic acid based steroidal phenstatin analogues for selective targeting of breast cancer cells through inhibiting tubulin polymerization

Article abstract of DOI:10.1016/j.steroids.2012.03.012

Phenstatin analogues were synthesized on steroidal framework, for selective targeting of breast cancer cells. These analogues were evaluated for anticancer efficacy against breast cancer cell lines. Analogues 12 and 19 exhibited significant anticancer activity against MCF-7, hormone dependent breast cancer cell line. While analogues 10-14 exhibited significant anticancer activity against MDA-MB-231, hormone independent breast cancer cell line. Compound 10 showed significant oestrogen antagonistic activities with low agonistic activity in in vivo rat model. These analogues also retain tubulin polymerization inhibition activity. The most active analogue 10 was found to be non-toxic in Swiss albino mice up to 300 mg/kg dose. Gallic acid based phenstatin analogues may further be optimized as selective anti-breast cancer agents.

Full text of DOI:10.1016/j.steroids.2012.03.012

Steroids 77 (2012) 878–886
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Gallic acid based steroidal phenstatin analogues for selective targeting
of breast cancer cells through inhibiting tubulin polymerization
Swati Parihar a, Atul Gupta a, Amit K. Chaturvedi a, Jyoti Agarwal a, Suaib Luqman a, Bendangla Changkija b,
Murli Manohar b, Debabrata Chanda a, C.S. Chanotiya a, Karuna Shanker a, Anila Dwivedi b,
Rituraj Konwar b, Arvind S. Negi a,
⇑
a Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Kukrail Picnic Spot Road, Lucknow 226015, UP, India
b Central Drug Research Institute (CSIR-CDRI), Chattar Manzil Palace, Lucknow 226001, UP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Phenstatin analogues were synthesized on steroidal framework, for selective targeting of breast cancer
cells. These analogues were evaluated for anticancer efficacy against breast cancer cell lines. Analogues
12 and 19 exhibited significant anticancer activity against MCF-7, hormone dependent breast cancer cell
line. While analogues 10–14 exhibited significant anticancer activity against MDA-MB-231, hormone
independent breast cancer cell line. Compound 10 showed significant oestrogen antagonistic activities
with low agonistic activity in in vivo rat model. These analogues also retain tubulin polymerization inhi-
bition activity. The most active analogue 10 was found to be non-toxic in Swiss albino mice up to 300 mg/
kg dose. Gallic acid based phenstatin analogues may further be optimized as selective anti-breast cancer
agents.
Received 28 February 2012
Received in revised form 23 March 2012
Accepted 26 March 2012
Available online 5 April 2012
Keywords:
Gallic acid
Phenstatin
Anticancer
Tubulin polymerization inhibition
Acute oral toxicity
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
analogues with potent activity has been developed [8,9]. To
enhance the bioavailability of these lead molecules (CA-1 and CA-
Hormone-dependent breast cancer over-expresses oestrogen
receptor alpha (ER ) in comparison to normal breast tissue [1]
and serves as prognostic indicator of the disease [2]. ER is a well
validated therapeutic target for breast cancer with gold standard
example of tamoxifen, a Selective Estrogen Receptor Modulator
(SERM) [3]. However, several reports suggest tamoxifen to be
potentially associated with risk of endometrial cancers [4]. Tamox-
ifen also exhibits drug resistance in breast cancer [5]. These critical
factors impel to develop newer alternatives of targeted breast can-
cer with improved profile.
4), water soluble prodrugs CA-4P (3) and CA-1P (4) have been pre-
pared as phosphates, which are in the final stage of clinical trials
[10]. Further, structure activity relationship (SAR) based synthesis
of other analogues yielded phenstatin (5) as one of the most active
compounds of this series (Fig. 1) [11,12]. However, like other che-
motherapeutic agents combretastatin often fails to selectively tar-
get cancer cells and also undesired toxicity being a major concern.
a
a
Therefore, we thought to target ER
properties of combretastatin as a new strategy for specifically tar-
geting ER over-expressing hormone dependent breast cancer. In
a with tubulin poisoning
a
Plant molecules for a long have made immense contribution in
enriching repertoire of chemotherapeutic agents. Taxol, vincristine,
vinblastine, podophyllotoxin, camptothecin, indole 3-carbinol and
combretastatin A4 (CA-4) are some of the notable anticancer leads
[6,7]. Combretastatin A4 is an antimitotic agent originally isolated
from the bark of the South African Willow tree Combretum caffrum.
Combretastatin binds to the b-subunit of tubulin at the colchicine
site, thereby strongly inhibiting polymerization of tubulin, causing
cellular disorganization and toxicity. Combretastatin A 4 (1) is an
attractive lead for developing anticancer drugs and several
the present study, we designed several phenstatin analogues on
steroidal framework synthesized through modification of gallic
acid, an abundantly available plant phenolic acid. In our approach
to induce cytotoxicity in the desired molecule, phenstatin (5) was
set as a model molecule, where 3,4,5-trimethoxybenzoyl part was
introduced from gallic acid and the ring A of estradiol unit was
used as second aryl ring to have a phenstatin type arrangement.
The structure activity relationship (SAR) of phenstatin and combre-
tastatin revealed that a diaryl system separated by a linker group
having restricted rotation was essentially required for inducing
anticancer activity of these molecules and one of the rings should
essentially possess 3,4,5-trimethoxyphenyl type arrangement [13].
A series of compounds has been synthesized based on above
prototype. All the compounds were evaluated against breast cancer
⇑
Corresponding author. Tel.: +91 522 2342676; fax: +91 522 2342666.
E-mail address: arvindcimap@rediffmail.com (A.S. Negi).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.03.012

S. Parihar et al. / Steroids 77 (2012) 878–886
879
N
OR2
O
R1
O
O
H3CO
H3CO
R2
H3CO
H3CO
H3CO
OH
OCH3
R1O
OCH3
OCH3
H3CO
OCH3
OCH3
1: R1=H, R2=OH; Combretastatin A4;
2: R1=R2=OH; Combretastatin A1;
Tamoxifen
3: R1=H, R2=OP(O)(OH)2 ; Combretastatin A4 phosphate;
4: R1=R2=OP(O)(OH)2; Combretastatin A1 diphosphate;
Prototype
5: Phenstatin
Fig. 1. Tamoxifen, combretastatins, phenstatin and proposed prototype.
cell lines, ER
a
expressing (MCF-7) and ER-
a
negative (MDA-MB-
anhydrous sodium sulphate. On evaporation, a residue was ob-
tained which on purification over column silica gel yielded pure
benzophenone derivative 10 as light yellow solid (253 mg).
Yield 62%; mp 177–178 °C; 1H NMR (CDCl3, 300 MHz): d 0.84 (s,
3H, 18-CH3), 1.24–2.26 (m, 13H, rest of the 5XCH2 and 3XCH of ste-
roidal ring), 2.06 (s, 3H, 17-OCOCH3), 2.92 (bs, 2H, 6-CH2), 3.72 (s,
3H, 3-OCH3), 3.88 (s, 6H, 30,50-OCH3), 3.93 (s, 3H, 40-OCH3), 4.69 (t,
1H, 17-CH, J = 8.2 Hz), 6.69 (s, 1H, 4-CH), 7.11 (s, 2H, 20 and 60-CH),
7.27 (s, 1H, 1-CH). 13C NMR (CDCl3, 75 MHz): d 12.38, 21.23, 23.60,
26.56, 27.45, 27.98, 30.31, 37.27, 39.01, 43.34, 44.09, 50.37, 56.19,
56.83, 56.83, 61.10, 82.94, 108.66, 108.66, 112.49, 127.01, 127.19,
133.03, 133.77, 141.27, 143.48, 153.29, 153.29, 155.79, 171.13,
195.34. ESI mass (MeOH): 523 [M+H]+, 545 [M+Na]+, 561 [M+K]+.
IR (KBr, cmꢁ1): 2938, 1736, 1656, 1582, 1501, 1246, 1127.
231) for in vitro cytotoxicity and mode of action studies as tubulin
polymerization inhibition. To estimate bioactivity due to steroidal
framework, in vivo oestrogenicity and anti-oestrogenicity in
female Sprague–Dawley rats were carried out. The most active
analogue 10, was further evaluated for in vivo acute oral toxicity
in Swiss albino mice.
2. Experimental protocols
2.1. General procedures
Melting points were determined on E-Z Melt automated melt-
ing point apparatus, Stanford Research System, USA and were
uncorrected. Gallic acid was procured from S.d. Fine Chemicals, In-
dia and estrone was procured for Sigma, USA. All the reactions
were monitored on Merck aluminium thin layer chromatography
(TLC, UV254 nm) plates. TLC visualization was accomplished by
spraying with a solution of 2% ceric sulphate in 10% aqueous sul-
phuric acid and charring at 100–110 °C. Column chromatography
was carried out on silica gel (60–120 mesh, Thomas Baker). NMR
spectra were obtained on Bruker Avance-300 MHz instrument
with tetramethylsilane (TMS, chemical shifts in d ppm) as an inter-
nal standard. EI mass spectra were recorded on Perkin–Elmer Tur-
boMass GC–MS system after dissolving the compounds in
methanol, while ESI mass spectra were recorded on Shimadzu
LC–MS after dissolving the compounds in methanol. FT-IR spectra
were recorded on Perkin–Elmer SpectrumBX. Nomenclature of ste-
roid derivatives has been given as per the recommendations pub-
lished by the Joint Commission on the Biochemical Nomenclature
(JCBN) of IUPAC [14]. Paclitaxel (Taxol), podophyllotoxin and
tamoxifen were procured from Sigma–Aldrich, USA and centchro-
man was taken from Central Drug Research Institute, India. All
these were used as standard cytotoxic, tubulin polymerization
inhibitor and anti-oestrogenic drugs, respectively.
2.2.1.2. Synthesis of [3-methoxyestra-1,3,5(10)-trien,17b-acetate-
2yl]-(4-methoxyphenyl)-methanone (11). Yield 53%; mp 137–
138 °C; 1H NMR (CDCl3, 300 MHz): d 0.83 (s, 3H, 18-CH3), 1.30–
2.24 (m, 13H, rest of the 5XCH2 and 3XCH of steroidal ring), 2.05
(s, 3H, 17-OCOCH3), 2.92 (bs, 2H, 6-CH2), 3.68 (s, 3H, 3-OCH3),
3.86 (s, 3H, 40-OCH3), 4.68 (t, 1H, 17-CH), 6.67 (s, 1H, 4-CH), 6.89
(d, 2H, 30 and 40-CH), 7.24 (s, 1H, 1-CH), 7.81 (d, 2H, 20 and 60-
CH). 13C NMR (CDCl3, 75 MHz): d 12.43, 21.39, 23.64, 26.50,
27.53, 27.99, 30.36, 37.23, 38.96, 43.32, 44.09, 50.28, 55.76,
56.12, 83.00, 112.28, 113.76, 113.76, 127.01, 127.41, 131.65,
132.54, 132.54, 132.92, 140.89, 155.54, 163.77, 171.32, 195.47.
ESI mass (MeOH): 463 [M+H]+, 485 [M+Na]+, Negative mode: 461
[MꢁH]ꢁ. IR (KBr, cmꢁ1): 2931, 1735, 1655, 1601, 1509, 1251.
2.2.1.3. Synthesis of [3-methoxyestra-1,3,5(10)-trien,17b-acetate-
2yl]-(3,4-dimethoxyphenyl)-methanone (12). Yield 52%; mp 86–
87 °C; 1H NMR (CDCl3, 300 MHz): d 0.83 (s, 3H, 18-CH3), 1.30–
2.24 (m, 13H, rest of the 5XCH2 and 3XCH of steroidal ring), 2.05
(s, 3H, 17-OCOCH3), 2.92 (bs, 2H, 6-CH2), 3.68 (s, 3H, 3-OCH3),
3.86 (s, 6H, 30 and 40-OCH3), 4.68 (t, 1H, 17-CH), 6.67 (s, 1H, 4-
CH), 6.89 (d, 2H, 30 and 40-CH), 7.24 (s, 1H, 1-CH), 7.81 (d, 2H, 20
and 60-CH). 13C NMR (CDCl3, 75 MHz): d 12.37, 21.23, 23.61,
26.52, 27.50, 27.99, 30.29, 37.27, 39.02, 43.35, 44.11, 50.38,
56.21, 56.36, 56.45, 82.96, 110.62, 112.47, 112.47, 125.78, 126.92,
127.50, 131.92, 132.54, 132.97, 140.80, 153.82, 155.65, 171.11,
195.32. ESI mass (MeOH): 493 [M+H]+, 515 [M+Na]+. IR (KBr,
cmꢁ1): 2934, 1736, 1655, 1510, 1270.
2.2. Chemical synthesis
2.2.1. General procedure for the synthesis of steroidal benzophenones
(10–16)
2.2.1.1. Synthesis of [3-methoxyestra-1,3,5(10)-trien,17b-acetate-
2yl]-(3,4,5-trimethoxyphenyl)-methanone (10). 3,4,5-Trimethoxy-
benzoic acid (250 mg, 1.18 mmol) was taken in thionyl chloride
(2 mL). The reaction mixture was heated at 80 °C for 2 h. On com-
pletion, the excess of thionyl chloride was evaporated in vacuo and
the residue thus obtained was as such processed for next step. The
residue was dissolved in dry dichloromethane (15 mL) and anhy-
drous aluminium chloride (200 mg, 1.5 mmol) was added to it
and stirred at room temperature for 15 min. To the stirred solution,
estradiol 3-methyl ether, 17-acetate (6, 256 mg, 0.78 mmol) was
added and further stirred for 18–20 h. On completion, it was acid-
ified with dil. HCl (10%, 5 mL) and extracted with chloroform
(3 ꢀ 30 mL). Organic layer was washed with water and dried over
2.2.1.4. Synthesis of [3-methoxyestra-1,3,5(10)-trien,17b-acetate-
2yl]-(3,4-methylenedioxyphenyl)-methanone (13). Yield 69%; mp
123–124 °C; 1H NMR (CDCl3, 300 MHz): d 0.78 (s, 3H, 18-CH3),
1.23–2.21 (m, 13H, rest of the 5XCH2 and 3XCH of steroidal ring),
1.98 (s, 3H, 17-OCOCH3), 2.84 (bs, 2H, 6-CH2), 3.62 (s, 3H, 3-
OCH3), 4.61 (t, 1H, 17-CH, J = 8.4 Hz), 5.96 (s, 2H, –O–CH2–O),
6.59 (s, 1H, 4-CH), 6.72 (d, 1H, 50-CH), 7.15 (s, 1H, 1-CH), 7.28 (d,
1H, 60-CH), 7.30 (s, 1H, 20-CH). 13C NMR (CDCl3, 75 MHz): d 12.39,
21.27, 23.62, 26.51, 27.51, 28.00, 30.32, 37.43, 39.01, 43.35,
44.11, 50.37, 56.18, 82.99, 102.01, 107.90, 109.70, 112.41, 126.87,

880
S. Parihar et al. / Steroids 77 (2012) 878–886
126.96, 127.45, 133.04, 133.62, 140.92, 148.27, 151.93, 155.57,
171.20, 194.99. ESI mass (MeOH): 477 [M+H]+, 499 [M+Na]+, 515
[M+K]+. IR (KBr, cmꢁ1): 2934, 1734, 1651, 1605, 1501, 1251.
2.2.2.2. Synthesis of [3-hydroxyestra-1,3,5(10)-trien,17b-acetate-2yl]-
(4-hydroxy,3,5-dimethoxyphenyl)-methanone (17). Yield 76%; mp
133–134 °C. 1H NMR (CDCl3, 300 MHz): d 0.84 (s, 3H, 18-CH3),
1.27–2.26 (m, 13H, rest of the 5XCH2 and 3XCH of steroidal ring),
2.94 (bs, 2H, 6-CH2), 3.95 (s, 6H, 3 and 5-OCH3), 4.70 (dt, 1H, 17-
CH), 6.79 (s, 1H, 4-CH), 7.06 (s, 2H, 20 and 60-CH), 7.61 (s, 1H, 1-
CH), 11.42 (s, 1H, exchangeable, Phenolic OH), 11.72 (s, 1H,
exchangeable, Phenolic OH). 13C NMR (CDCl3, 75 MHz): 12.34,
21.11, 23.55, 26.77, 27.16, 27.94, 30.17, 37.18, 38.92, 43.28,
43.87, 50.37, 56.95, 56.95, 82.87, 108.14, 108.14, 117.76, 118.00,
129.64, 130.25, 131.35, 139.68, 146.83, 147.27, 147.27, 161.15,
171.14, 199.52. ESI mass (MeOH): 495 [M+H]+, 517 [M+Na]+, neg-
ative ion mode: 493 [MꢁH]ꢁ. IR (KBr, cmꢁ1): 3495, 3423, 2937,
1737, 1704, 1587, 1510, 1214.
2.2.1.5. Synthesis of [3-methoxyestra-1,3,5(10)-trien,17b-acetate-
2yl]-(4-methylphenyl)-methanone (14). Yield 58%; mp 81–82 °C;
1H NMR (CDCl3, 300 MHz): d 0.84 (s, 3H, 18-CH3), 0.86–2.26 (m,
13H, rest of the 5XCH2 and 3XCH of steroidal ring), 2.06 (s, 3H,
17-OCOCH3), 2.42 (s, 3H, CH3), 2.93 (bs, 2H, 6-CH2), 3.69 (s, 3H,
3-OCH3), 4.69 (t, 1H, 17-CH, J = 8.4 Hz), 6.68 (s, 1H, 4-CH), 7.21
(d, 2H, 30 and 40-CH, J = 4.1 Hz), 7.27 (s, 1H, 1-CH), 7.72 (d, 2H, 20
and 60-CH). 13C NMR (CDCl3, 75 MHz): d 14.31,21.29, 21.88,
23.63, 26.51, 27.52, 28.01, 30.34, 37.27, 39.01, 43.35, 44.12,
50.36, 56.12, 82.99, 112.43, 127.16, 127.43, 129.12, 129.34,
129.91, 130.30, 133.01, 136.33, 141.12, 143.55, 155.82, 171.18,
196.36. ESI mass (MeOH): 447 [M+H]+, 469 [M+Na]+, 485 [M+K]+,
Negative mode: 445 [MꢁH]ꢁ. IR (KBr, cmꢁ1): 2932, 1734, 1654,
1607, 1496, 1249.
2.2.2.3. Synthesis of [3-hydroxyestra-1,3,5(10)-trien,17b-acetate-2yl]-
(4-methoxyphenyl)-methanone (18). Yield 83%; mp 97–98 °C; 1H
NMR (CDCl3, 300 MHz): d 0.83 (s, 3H, 18-CH3), 1.23–2.23 (m,
13H, rest of the 5XCH2 and 3XCH of steroidal ring), 2.92 (bs, 2H,
6-CH2), 3.90 (s, 3H, 3-OCH3), 4.68 (t, 1H, 17-CH, J = 8.4 Hz), 6.77
(s, 1H, 4-CH), 6.97 (d, 2H, 30 and 50-CH), 7.52 (s, 1H, 1-CH), 7.68
(d, 2H, 20 and 60-CH), 11.92 (s, 1H, exchangeable, Phenolic OH).
ESI mass (MeOH): 449 [M+H]+, 471 [M+Na]+. IR (KBr, cmꢁ1):
3448, 3424, 2930, 1718, 1705, 1618, 1510, 1265.
2.2.1.6. Synthesis of [3-methoxyestra-1,3,5(10)-trien,17b-acetate-
2yl]-(4-bromophenyl)-methanone (15). Yield 49%; mp 108–
109 °C; 1H NMR (CDCl3, 300 MHz):
d 0.83 (s, 3H, 18-CH3),
1.25–2.27 (m, 13H, rest of the 5XCH2 and 3XCH of steroidal
ring), 2.05 (s, 3H, 17-OCOCH3), 2.93 (bs, 2H, 6-CH2), 3.66 (s,
3H, 3-OCH3), 4.68 (t, 1H, 17-CH, J = 8.4 Hz), 6.67 (s, 1H, 4-CH),
7.56 (d, 2H, 20 and 60-CH), 7.30 (s, 1H, 1-CH), 7.65 (d, 2H, 30
and 50-CH). ESI mass (MeOH): 511 [M+H]+, 533 [M+Na]+, 549
[M+K]+, Negative mode: 509 [MꢁH]ꢁ. IR (KBr, cmꢁ1): 2931,
1736, 1654, 1607, 1586, 1248.
2.2.2.4. Synthesis of [3-hydroxyestra-1,3,5(10)-trien,17b-acetate-2yl]-
(4-methylphenyl)-methanone (20). Yield 92%; mp 115–116 °C; 1H
NMR (CDCl3, 300 MHz): d 0.84 (s, 3H, 18-CH3), 1.25–2.24 (m,
13H, rest of the 5XCH2 and 3XCH of steroidal ring), 2.09 (s, 3H, –
OCOCH3), 2.43 (s, 3H, 40-CH3), 2. 94 (bs, 2H, 6-CH2), 4.69 (t, 1H,
17-CH, J = 8.4 Hz), 6.79 (s, 1H, 4-CH), 7.31 (d, 2H, 30 and 50-CH),
7.52 (s, 1H, 1-CH), 7.59 (d, 2H, 20 and 60-CH), 11.88 (s, 1H,
exchangeable, Phenolic OH). ESI mass (MeOH): 433 [M+H]+, 455
[M+Na]+, negative ion mode: 431 [MꢁH]ꢁ. IR (KBr, cmꢁ1): 3448,
2926, 1735, 1704, 1602, 1262.
2.2.1.7. Synthesis of [3-methoxyestra-1,3,5(10)-trien,17-one-2yl]-
(3,4,5-trimethoxyphenyl)-methanone (16). Yield 46%; mp 151–
152 °C; 1H NMR (CDCl3, 300 MHz): d 0.88 (s, 3H, 18-CH3), 1.24–
2.51 (m, 13H, rest of the 5XCH2 and 3XCH of steroidal ring), 2.99
(bs, 2H, 6-CH2), 3.73 (s, 3H, 3-OCH3), 3.84 (s, 6H, 30 and 50-OCH3),
3.95 (s, 3H, 40-OCH3), 6.71 (s, 1H, 4-CH), 7.11 (s, 2H, 20 and 60-
CH), 7.28 (s, 1H, 1-CH). 13C NMR (CDCl3, 75 MHz): d 14.23, 21.94,
26.41, 26.77, 30.32, 32.01, 36.06, 38.83, 44.31, 48.23, 51.00,
56.27, 56.86, 56.86, 61.12, 108.71, 108.71, 112.53, 127.15, 128.83,
132.59, 133.72, 141.06, 153.32, 153.32, 153.68, 155.92, 195.28,
221.11. ESI mass (MeOH): 479 [M+H]+, 501 [M+Na]+, 517 [M+K]+.
IR (KBr, cmꢁ1): 2936, 1737, 1657, 1580, 1500, 1227, 1126.
2.2.3. General procedure for synthesizing 2-benzyl analogues (21–23)
2.2.3.1. Synthesis of [3-methoxyestra-1,3,5(10)-trien,17b-acetate-
2yl]-(3,4,5-trimethoxyphenyl)-methane (21). Steroidal benzophe-
none 10 (100 mg, 0.19 mmol) was taken in trifluoroacetic acid
(TFA, 3 mL). The reaction mixture was cooled to 0–10 °C and so-
dium borohydride (80 mg, 2.1 mmol) was added to it in portions
and stirred for 1 h with cooling and then at room temperature
for 2 h. On completion, the reaction mixture was acidified with
dil. HCl (5 mL, 5% v/v) and extracted with chloroform
(3 ꢀ 20 mL). The organic layer was washed with water and evapo-
rated in vacuo and the residue thus obtained was recrystallised
with CHCl3:hexane (1:5) to get compound 21 as creamish white
solid (77 mg).
2.2.2. General procedure for the synthesis of demethylated products
(17–20)
2.2.2.1. Synthesis of [3-hydroxylestra-1,3,5(10)-trien,17b-acetate-
2yl]-(4-hydroxy,3-methoxyphenyl)-methanone (19). Compound 12
(100 mg, 0.20 mmol) was taken in dry dichloromethane (15 mL).
To this stirred solution anhydrous aluminium chloride (200 mg,
1.50 mmol) was added and further stirred at room temperature
for 3 h. On completion, dil. HCl (5% v/v, 2 mL) was added to it
and extracted with chloroform (3 ꢀ 25 mL). Organic layer was
washed with water, dried over anhydrous sodium sulphate and
evaporated to dryness in vacuo. The residue thus obtained was
recrystallised with chloroform-hexane (1:3) to get a creamish
white solid (82 mg).
Yield 79%; mp 139–140 °C; 1H NMR (CDCl3, 300 MHz): d 0.81 (s,
3H, 18-CH3), 1.26–2.20 (m, 13H, rest of the 5XCH2 and 3XCH of ste-
roidal ring), 2.05 (s, 3H, –OCOCH3), 2. 85 (bs, 2H, 6-CH2), 3.81 (s,
12H, 30,40,50-OCH3 and 3-OCH3), 3.86 (s, 2H, –CH2–Ar), 4.67 (t,
1H, 17-CH), 6.45 (s, 2H, 20 and 60-CH), 6.59 (s, 1H, 4-CH), 7.00 (s,
1H, 1-CH). EI mass (MeOH): 508 [M+]. IR (KBr, cmꢁ1): 2935,
1736, 1686, 1592, 1506, 1217, 1166.
Yield 87%; mp 105–106 °C; 1H NMR (CDCl3, 300 MHz): d 0.83 (s,
3H, 18-CH3), 1.25–2.23 (m, 13H, rest of the 5XCH2 and 3XCH of ste-
roidal ring), 2.91 (bs, 2H, 6-CH2), 3.95 (s, 3H, 3-OCH3), 4.76 (t, 1H,
17-CH, J = 8.7 Hz), 6.76 (s, 1H, 4-CH), 7.26 (d, 2H, 50 and 60-CH), 7.31
(s, 1H, 20-CH), 7.54 (s, 1H, 1-CH), 11.71 (s, 1H, exchangeable, Phe-
nolic OH), 11.78 (s, 1H, exchangeable, Phenolic OH). ESI mass
(MeOH): 465 [M+H]+, 487 [M+Na]+. IR (KBr, cmꢁ1): 3449, 2931,
1719, 1704, 1636, 1579, 1271.
2.2.3.2. Synthesis of [3-hydroxyestra-1,3,5(10)-trien,17b-acetate-2yl]-
(4-hydroxy,3,5-dimethoxyphenyl)-methane (22). Yield 62%; mp 98–
99 °C; 1H NMR (CDCl3, 300 MHz): d 0.83 (s, 3H, 18-CH3), 1.26–2.24
(m, 13H, rest of the 5XCH2 and 3XCH of steroidal ring), 2.06 (s, 3H,
–OCOCH3), 2.77 (bs, 2H, 6-CH2), 3.78 (s, 6H, 30 and 50-OCH3), 3.93
(s, 2H, –CH2–Ar), 4.68 (t, 1H, 17-CH), 6.47 (s, 2H, 20 and 60-CH),
6.52 (s, 1H, 4-CH), 7.01 (s, 1H, 1-CH), 11.7 (s, 1H, exchangeable,
Phenolic OH). ESI mass (MeOH): 503 [M+Na]+, 519 [M+K]+,

S. Parihar et al. / Steroids 77 (2012) 878–886
881
negative ion mode: 479 [MꢁH]ꢁ. IR (KBr, cmꢁ1): 3424, 2930, 1718,
consecutive days by oral route. A separate group of animals re-
ceived only the vehicle for similar duration served as control. At
autopsy 24 h after the last treatment on day 31 of age, vaginal
smear of each rat was taken and uterus was carefully excised,
gently blotted, and weighed. Premature opening of vagina, cornifi-
cation of vaginal epithelium, and increase in uterine fresh weight
were taken as parameters for evaluation of oestrogen agonistic
activity in comparison to rats of vehicle control group. The objec-
tive was to evaluate oestrogen agonistic effect of the compounds
on the uterus and vagina [18].
1705, 1618, 1510, 1265, 1216.
2.2.3.3. Synthesis of [3-methoxyestra-1,3,5(10)-trien,17b-acetate-
2yl]-(4-methoxyphenyl)-methane (23). Yield 73%; oil; 1H NMR
(CDCl3, 300 MHz): d 0.83 (s, 3H, 18-CH3), 1.25–2.19 (m, 13H, rest
of the 5XCH2 and 3XCH of steroidal ring), 2.07 (s, 3H, –OCOCH3),
2.84 (bs, 2H, 6-CH2), 3.84 (s, 6H, 30 and 40-OCH3), 3.88 (bs, 2H, –
CH2–Ar), 4.70 (t, 1H, 17-CH), 6.57 (s, 1H, 4-CH), 6.81 (s, 2H, 30
and 50-CH), 7.14 (s, 2H, 20 and 60-CH), 7.25 (s, 1H, 1-CH). ESI mass
(MeOH): 517 [M+K]+. IR (neat, cmꢁ1): 2933, 1735, 1509, 1246,
1212.
2.3.5. In vivo anti-oestrogenicity
Twenty-one days old immature female Sprague–Dawley rats
were bilaterally ovariectomized under light ether anaesthesia
and after post-operative rest for 7 days were randomized into dif-
ferent treatment groups. Each group comprised of four or five ani-
mals. Each rat received the compound of the invention and
2.3. Pharmacology
2.3.1. Cell culture
Human breast cancer cell lines MCF-7 and MDA-MB-231 were
American type of cell culture collection (ATCC), USA. Cells were
grown in tissue culture flasks in complete growth medium, DMEM
with 2 mM glutamine, pH 7.4 supplemented with 10% fetal bovine
0.02 mg/kg dose of 17
water once daily for three consecutive days by oral route. A sepa-
rate group of animals receiving only 17 -ethynylestradiol
a-ethynylestradiol in 10% ethanol–distilled
a
serum, 100
l
g/mL streptomycin and 100 units/mL penicillin in a
(0.02 mg/kg) in 10% ethanol–distilled water for similar duration
was used for comparison. At autopsy on day 31 of age, vaginal
smear of each rat was taken and uterus was carefully excised,
gently blotted and weighed. Inhibition in ethynylestradiol induced
cornification of vaginal epithelium and increase in uterine fresh
weight were taken as parameters for evaluation of oestrogen
antagonistic effect of the compounds [18].
CO2 incubator (37 °C, 5% CO2, 90% RH). The cells at subconfluent
stage were harvested from the flask by treatment with trypsin
(0.05% in PBS, pH 7.4, containing 0.02% EDTA) and seeded in tissue
culture plates for assay.
2.3.2. In vitro cytotoxicity evaluation
The cytotoxic activities of the compounds were determined
using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) reduction assay [15]. 1 ꢀ 104 cells/well were seeded in
2.3.6. Oestrogen receptor binding affinity
The relative binding affinity of these compounds for oestrogen
receptor was determined by competitive assay radiometrically as
described earlier [15]. Radiolabeled estradiol 3H[E2] was used as
reference compound.
200 lL DMEM, supplemented with 10% FBS in each well of 96-well
culture plates and incubated for 24 h at 37 °C in a CO2 incubator.
Compounds, diluted to the desired concentrations in culture med-
ium, were added to the wells with respective vehicle control. After
18 h of incubation, 10
lL MTT (5 mg/mL) was added to each well
2.3.7. In vivo acute oral toxicity
and the plates were further incubated for 4 h. Then the supernatant
from each well was carefully removed, formazon crystals were dis-
solved in 100 lL of DMSO and absorbance at 540 nm wavelength
was recorded. IC50 were determined from sigmoidal dose-response
analysis of drug concentration versus percent cell inhibition using
Graphpad Prism software version 3.0. Mean and standard error of
mean (SEM) were calculated using Microsoft Excel and data are ex-
pressed as mean SEM obtained from three independent
experiments.
In view of anti-cancer activity of compound 10 in in vitro mod-
el, acute oral toxicity of the same was carried out in Swiss albino
mice. Experiment was conducted in accordance with the Organiza-
tion for Economic Co-operation and Development (OECD) test
Guideline No. 423.
For the study, 16 mice (eight male and eight female) were taken
and divided into four groups comprising two male and two female
mice in each group weighing between 20 and 25 g. The animals
were maintained at 22 5 °C with humidity control and also on
an automatic dark and light cycle of 12 h. The animals were fed
with the standard rat feed and provided ad libitum drinking water.
Mice of group 1 were kept as control and animals of groups 2–4
were kept as experimental. The animals were acclimatized for
7 days in the experimental environment prior to the actual exper-
imentation. The test compound was suspended in distilled water
using traces of ethanol as a co-solvent and was given at 5, 50
and 300 mg/kg body weight to animals of groups 2–4, respectively.
Control animals received only vehicle.
2.3.3. Tubulin polymerization inhibition assay
Tubulin polymerization experiment was done as per reported
method [16,17] using ‘assay kit’ from Cytoskeleton, USA. In brief,
tubulin protein (3 mg/mL) in tubulin polymerization buffer
(80 mM PIPES, pH 6.9, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP
and 15% glycerol) was placed in pre-warmed 96-well microtiter
plates at 37 °C in the presence of test compounds with variable
concentrations. All samples were mixed well and polymerization
was monitored kinetically at 340 nm every min for 1 h using Spec-
tramax plate reader. Podophyllotoxin was used as standard inhib-
itor of tubulin polymerase and DMSO as negative control. The IC50
value was determined from dose-dependent analysis and is de-
fined as the concentration that inhibits the rate of polymerization
by 50%.
2.3.7.1. Observational, haematological, biochemical and gross patho-
logical study. The animals were checked for mortality and any signs
of ill health at hourly interval on the day of administration of drug
and there after a daily general case side clinical examination was
carried out including changes in skin, mucous membrane, eyes,
occurrence of secretion and excretion and also responses like lach-
rymation, pilo-erection respiratory patterns etc. Also changes in
gait, posture and response to handling were also recorded. In
addition to observational study, body weights were recorded and
blood and serum samples were collected from all the animals on
7th day after experiment and were analysed for total RBC, WBC, dif-
ferential leucocyte count, haemoglobin percentage and biochemical
2.3.4. In vivo oestrogenicity
Twenty-one-days-old immature female Sprague–Dawley rats
were bilaterally ovariectomized under light ether anaesthesia
and after post-operative rest for 7 days were randomized into dif-
ferent treatment groups. Each group comprised of four or five ani-
mals. Each rat received the compound once daily for three

882
S. Parihar et al. / Steroids 77 (2012) 878–886
parameters like total cholesterol, triglycerides, creatinine, SGPT and
SGOT activity. The animals were then sacrificed and were necrop-
sed for any gross pathological changes. Weights of vital organs like
liver, heart, kidney etc. were recorded.
MCF-7 and MDA-MB-231 (Table 1), while rest of the compounds
were either poorly active or inactive (IC50 > 15 M) Out of these,
l
compounds 12 and 19 showed significantly higher activity in
ER-positive MCF-7 cells indicating that their action may involve
oestrogen receptor (ER). Whereas compounds 10, 11, 13 and 14
showed higher activity in ER-negative MDA-MB-231 cells possibly
indicating lack of role of ER involvement. Most selectively active
analogues in ER-bearing cell MCF-7 i.e., 12 and 19 possess a 3,4-
dimethoxybenzoyl or 3-methoxy,4-hydroxybenzoyl unit attached
to estradiol unit.
Analogue 10 with highest activity in MDA-MB-231 possessed
3,4,5-trimethoxybenzoyl arrangement. On removing methoxy
groups from the benzoyl ring cytotoxicity was reduced. p-Methyl
substitution at benzoyl group (compound 14) exhibited good activ-
ity and comparable to 3,4,5-trimethoxy group. p-Bromo substitu-
3. Results and discussion
3.1. Chemistry
The synthetic strategy adopted was as depicted in Scheme 1. Es-
trone and gallic acid were modified suitably to get the compounds
of desired prototype. Compound 6 was synthesized from estrone as
reported earlier [19]. Gallic acid was methylated with dimethyl
sulphate in 20% aqueous alkali to produce 3,4,5-trimethoxybenzoic
acid (8) [20]. Compound 8 was transformed to its acid chloride i.e.,
3,4,5-trimethoxybenzoyl chloride (9) by heating it with thionyl
chloride [21]. To get benzophenone 10, Friedel-Craft’s acylation
reaction was done on 6 and 9 in presence of anhydrous aluminium
chloride in dry dichloromethane. Similarly, various other acid chlo-
rides were used to get other analogues 11–15.
These benzophenone moieties on treatment with excess of alu-
minium chloride in DCM yielded o and p-demethylated analogues
17–20. Initially, these demethylated products were synthesized in
the separate steps but when the molar ratio of aluminium chloride
was enhanced by several folds in the Friedel–Craft’s reaction step,
we got both the methylated and demethylated products in the
same reaction. Some of the benzophenone analogues were treated
with sodium borohydride in TFA to yield corresponding benzyl
derivatives (21–23).
tion showed very poor cytotoxicity.
o and p-Demethylated
compounds (compounds 17–20) showed comparable cytotoxicity
to methylated products but on reducing keto group of benzophe-
none to methylene group, the cytotoxicity was reduced drastically.
It might be due to loss of restricted rotation of the phenyl rings,
which was pre-requisite for inducing cytotoxicity. We also evalu-
ated non-specific cytotoxicity of the molecules. For this purpose,
we evaluated their cytotoxicity against human cell lines of normal
tissue origin, HEK-293 (human embryonic kidney cells). Only com-
pound 12 and 17 showed moderate toxicity against normal human
cell line, whereas other compounds are significantly safe with IC50
higher than 50 lM. This further supports that our hybrid analogues
are acting on cancer cells in selective fashion.
The Table 2 shows the tubulin polymerization inhibition activ-
ity of these analogues [16,17]. Analogue compound 10 possessed
antitubulin activity comparable to podophyllotoxin, a standard
antitubulin drug. Rest of the compounds were considered inactive
3.2. Biological results and discussion
due to low inhibition (% inhibition < 30% at 20 lg/mL) concentration.
Both the compounds 10 and 21 possessed a 3,4,5-trimethoxyphenyl
In MTT assay [15] compounds 10, 11, 12, 13, 14 and 19 exhib-
ited significant cytotoxicity against human breast cancer cell lines
R1
R1
R2
R2
A
O
C
O
H3CO
H3CO
H3CO
Cl
vi
H3CO
H3CO
+
H3CO
H3CO
OCH3
6: R1=H, R2=OAc;
7: R1-R2=O;
10: R1= H, R2=OAc;
16: R1-R2=O
9
OAc
OAc
B
O
O
C
R1
vi
Cl
+ R1
H3CO
H3CO
11: R1=4-methoxy-;
6
viii
12: R1=3,4-dimethoxy-;
13: R1=3,4-methylenedioxy-;
14: R1=4-methyl-;
vii
15: R1=4-bromo-;
OAc
OAc
O
R2
R1
R1
HO
21: R1=OCH3, R2=3,4.5-trimethoxy-;
22: R1=OH, R2=4-hydroxy, 3,5-dimethoxy-;
23: R1=OCH3, R2=4-OCH3
viii
17: R1=4-hydroxy, 3,5-dimethoxy-
18: R1=4-methoxy-;
19: R1=3-methoxy, 4-hydroxy-;
20: R1=4-methyl-;
Scheme 1. Reagents and conditions: (i) Me2SO4, K2CO3, dry acetone-DCM (1:1), reflux, 87%; (ii) NaBH4, MeOH:DCM (2:1), RT, 2 h, 91%; (iii) Ac2O, dry pyridine, dry DCM, RT,
3 h, 94%; (iv) Me2SO4, 20% aqueous KOH, 0–10 °C for 30 min, then reflux, 2 h, 68%; (v) SOCl2, 80 °C, 1 h; (vi) Crude mass of (v), dry DCM, anhydrous AlCl3, RT, 3–4 h, 46–69%;
(vii) DCM, anhydrous AlCl3, RT, 2–3 h, 76–92%; (viii) NaBH4, TFA, 0 °C, 1 h then RT, 3–4 h, 62–79%.

S. Parihar et al. / Steroids 77 (2012) 878–886
883
Table 1
Table 1 (continued)
In vitro cytotoxicity of phenstatin analogues by MTT Assay.
Compound
Cytotoxicity against human cancer cell lines
IC50 M)
Compound
Cytotoxicity against human cancer cell lines
IC50 M)
(l
(l
MCF-7
0.02
MDA-MB-231
7 0.02
HEK-293
46 0.03
MCF-7
>50
MDA-MB-231
0.03
HEK-293
4
N
O
OAc
OAc
5
>50
O
MeO
MeO
MeO
OMe
10
11
Tamoxifen
>50
14 0.26
>50
O
aValues represent means SEM of a minimum of three observations.
MeO
MeO
OAc
8
0.10
20 0.001
10 0.18
42 0.06
Table 2
O
In vitro tubulin polymerization inhibition activity of selected analogues.
MeO
MeO
Tubulin polymerization inhibitionb IC50
0.99
(lM)
MeO
Compound
12
OAc
OAc
OAc
>50
>50
O
O
MeO
O
O
MeO
MeO
OMe
MeO
10
21
13
14
OAc
1.60
7.18
>50
>50
>50
9.17 0.32
35.23 0.06
>50
>50
>50
>50
O
MeO
MeO
O
MeO
OMe
H3C
MeO
OAc
OAc
O
O
MeO
MeO
N
Br
MeO
23
15
0.74
O
O
O
MeO
MeO
MeO
OMe
16
OAc
Tamoxifen
>50
>50
48 0.06
48 0.04
1.2a
1.0a
O
OH
MeO
MeO
HO
OMe
MeO
HO
OMe
OMe
17
Combretastatin A4
OAc
>50
>50
O
O
MeO
MeO
OH
OMe
OMe
MeO
HO
18
Phenstatin
OAc
12 0.01
30 0.03
25 0.02
32 0.01
>50
>50
a
O
Literature values from Ref. [11].
Rest of the compounds were found inactive at 20 lg/mL concentration.
MeO
HO
b
HO
19
OAc
system, while on demethylating some of the methoxy groups re-
sulted in loss of the activity.
O
In vivo oestrogenicity and anti-oestrogenicity were determined
as per reported method [18]. Among these analogues, compounds
13, 16, 18, 21 and 22 exhibited high level of oestrogenicity (Ta-
ble 3). These compounds exhibited low level of cytotoxicity against
MCF-7 cell lines. It might be due to their oestrogenic nature which
activates proliferation of tumour cells. While the compounds 10,
11, 12, 15, and 19 exhibited very low oestrogenicity. Rest of the
compounds possessed moderate agonistic activity. On the other
hand compounds 10, 13, 15, 17, 18 and 19 exhibited high level of
anti-oestrogenic activity. Ideally, a low level of oestrogenicity
and high level of anti-oestrogenicity is desirable in these com-
pounds. These analogues showed a low level of binding affinity
to oestrogen receptors. Only compound 16 showed RBA (5.1%) bet-
ter than that of tamoxifen (1.05%)’. It might be due to interference
of bulky phenyl ring close to 3-hydroxy of ring A of steroidal
framework, which takes part in oestrogen receptor binding.
H3C
HO
O
20
OAc
>50
>50
>50
>50
>50
MeO
MeO
MeO
OMe
21
OAc
47 0.04
O
MeO
HO
HO
OMe
22
OAc
24 0.06
17.16 0.02
>50
O
MeO
MeO
23

884
S. Parihar et al. / Steroids 77 (2012) 878–886
Table 3
Oestrogenicity and anti-oestrogenicity of synthesized analogues.
S. No.
1.
Compd. code
Dose (mg/kg)
E Uterine weight (mg)* Mean S.E.
AE Uterine weight (mg)* Mean S.E.
%Gain**
5.88
%Inhibition***
41.10
%RBA
10
Control
EE
11
Control
EE
12
Control
EE
13
Control
EE
14
Control
EE
15
Control
EE
16
Control
EE
17
Control
EE
18
Control
EE
19
Control
EE
20
Control
EE
21
Control
EE
10
–
0.02
10
–
0.02
10
–
0.02
10
–
0.02
10
–
0.02
10
–
0.02
10
–
0.02
10
–
0.02
10
–
0.02
10
–
0.02
10
–
0.02
10
–
0.02
10
–
0.02
n.d.
3
29.5 1.65g
27.86 2.43
–
91.25 5.96a,b
27.86 2.43
135.6 2.60a
106.75 2.12a,b
34.5 0.86
121.66 2.04
100.35 1.77a,b
34.5 0.86
Inactive
2.
35.02 2.00g
34.5 0.86
–
1.5
0.72
184
17.89
25.17
38.97
30.5
0.08
3.
34.75 0.90g
34.5 0.86
–
0.19
121.66 2.04
88.2 4.14a,b
34.5 0.86
4.
98.25 2.49a
34.5 0.86
–
0.20
121.66 2.04a
95.62 0.64a,b
34.5 0.86
5.
52.07 2.45a
34.5 0.86
–
50.9
1.22
118
0.14
121.66 2.04
83.22 3.03a,b
26.84 0.68
139.33 3.48a
102 0.70a,b
27.86 2.43
135.6 2.60
70 0.70a,b
27.86 2.43
135.6 2.60
64.72 1.86a,b
26.57
6.
27.17 0.42g
26.84 0.68
–
49.87
31.12
60.85
53.43
45.5
Inactive
5.1
7.
60.75 1.25a
27.86 2.43
–
8.
35 2.01g
27.86 2.43
–
25.62
145
Inactive
Inactive
0.23
9.
65.3 2.25a
26.57 1.27
–
108.33 1.22a
88.12 1.69a,b
26.84 0.68
139.33 3.48
99.44 1.63a,b
26.84 0.68
139.33 3.48
88.03 2.95a,b
26.57 1.27
108.33 1.22
80.52 2.89a,b
26.57 1.27
108.33 1.22
n.d.
10.
11.
12.
13.
30.07 0.71g
26.84 0.68
–
12.03
28.16
145
34.4 1.21e
26.84 0.68
–
35.4
0.13
65.15 3.77a
26.57 1.27
–
24.98
34.15
Inactive
Inactive
22
Control
EE
78.22 2.94a
26.57 1.27
–
194
14.
15.
23
n.d.
n.d.
248.66
n.d.
27.36
n.d.
1.07
Tamoxifen
Control
EE
Tamoxifen
Control
EE
Centchroman
Control
EE
52.3 5.3a
15.0 0.57
–
46.32 2.92a,d
15.0 0.57
–
0.02
10
–
0.02
10
–
66.35 0.44
76.55 2.30a,b
26.57 1.27
108.33 1.22
46.0 2.9a,b
17.4 0.56
16.
17.
61.17 3.64a
26.57 1.27
–
130
106
38.99
65.9
–
–
36.0 1.5c
17.4 0.56
–
0.02
101.23 2.1
n.d. = not determined; EE = 17a-ethynylestradiol.
*
Values are mean S.E. (n = 4–5).
Percent of vehicle control group.
**
***
Percent of 17 a-ethynylestradiol per se treated group. Statistical analysis was done by ANOVA and Newman Keul’s test. p values >0.05 were considered insignificant; p
values a < 0.001, c < 0.01, e < 0.05, and g > 0.05 Vs control; p values b < 0.001, d < 0.01, f < 0.05 and h > 0.05 Vs E2. %Gain = oestrogenicity and %Inhibition = anti-oestrogenicity.
In acute oral toxicity, no morbidity and mortality was observed
throughout the experimental period. However, group of animals
treated with 10 at 300 mg/kg showed slight depression and inac-
tivity for a period of 20 min and were then recovered very shortly
which could be due to the bulkiness of the dosage volume. Blood
and serum samples upon analysis showed non-significant changes
in all the parameters studied like total RBC, WBC count, differential
leucocyte count, haemoglobin, serum total cholesterol, triglycer-
ides, creatinine level, SGPT and SGOT activity (Table 4 and
Fig. 3). Similarly, animals on gross pathological study showed no
changes in any of the organs studied including their absolute and
relative weights (Fig. 2A and B). Therefore, the experiment showed
that 10 is well tolerated by the Swiss albino mice up to the dose
level of 300 mg/kg body weight as a single acute oral dose. How-
ever, sub-acute and/or chronic experiment with the test drug
needs to be carried out to look for any adverse effect on repeated
exposure to the test drug compound 10 for its future development
[22].
A 3,4,5-trimethoxyphenyl unit is considered to interact with
tubulin and exerts tubulin polymerization inhibition. During mito-
sis, microtubule polymerization and depolymerization are tightly
controlled. Disruption of this process can cause cell cycle arrest
and subsequently cell death [23]. The anticancer effect of the com-
pounds having this type arrangement is due to antiproliferation of
cancer cells through disturbance of mitotic spindle function which
leads to cell apoptosis [24] and also disrupting cell signalling path-
ways of endothelial cells in tumour vasculature leading to selective
shutdown of blood flow through tumours [25,26]. Several combre-
tastatin analogues have already been reported to act like this
[27,28].

S. Parihar et al. / Steroids 77 (2012) 878–886
885
Table 4
Effect of compound 10 on body weight, haemogram and serum biochemical parameters in Swiss albino mice (n = 4).
Parameters
Dose of compound 10 at mg/kg body weight
Control
5 mg/kg
50 mg/kg
300 mg/kg
Body weight (g)
21.94 0.85
7.85 0.45
5.05 0.88
16.23 1.07
148.39 20.63
137.95 9.05
0.55 0.06
23.84 0.59
8.62 0.71
5.08 0.75
14.51 0.33
165.38 11.02
157.91 16.31
0.32 0.06
12.63 0.67
25.02 1.38
22.01 1.16
7.82 0.86
5.03 0.20
18.16 1.81
163.56 20.04
146.91 5.42
0.43 0.03
22.67 1.06
10.24 .60
7.80 0.67
17.05 0.66
146.63 6.87
155.38 17.35
0.40 0.09
12.20 2.52
32.53 2.30
Total RBC count (millions/mm3)
Total WBC count (thousands/mm3)
Haemoglobin (g/dL)
Serum total cholesterol (mg/dL)
Serum triglycerides (mg/dL)
Serum creatinine (mg/dL)
SGPT (U/L)
7.32 0.88
26.61 1.68
9.09 0.90
26.79 2.22
SGOT (U/L)
Fig. 2. (A and B): Effect of compound 10 on absolute and relative organ weights in Swiss albino mice (n = 4).
Acknowledgements
The authors thank to Director CIMAP for constant support and
encouragement. This work was supported by a Grant from Depart-
ment of Science and Technology (DST), Govt. of India.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2012.
03.012.
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