Synthesis of chalcone derivatives on steroidal framework and their anticancer activities

Article abstract of DOI:10.1016/j.steroids.2007.07.012

Chalcone derivatives on estradiol framework have been synthesized. Some of the derivatives showed potent anticancer activity against some human cancer cell lines. Compounds 9 and 19 showed potent activity against MCF-7, a hormone dependent breast cancer cell line. Chalcone 7 was further modified to the corresponding indanone derivative (19) using the Nazarov reaction, which showed better activity than the parent compound against the MCF-7 breast cancer cell line. Active anticancer derivatives were also evaluated for osmotic hemolysis using the erythrocyte as a model system. It was observed that chalcone derivatives showing cytotoxicity against cancer cell lines did not affect the fragility of erythrocytes and hence may be considered as non-toxic to normal cells.

Full text of DOI:10.1016/j.steroids.2007.07.012

steroids 7 2 ( 2 0 0 7 ) 892–900
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Synthesis of chalcone derivatives on steroidal
framework and their anticancer activitiesଝ
Hari Om Saxena, Uzma Faridi, J.K. Kumar, Suaib Luqman, M.P. Darokar,
Karuna Shanker, Chandan S. Chanotiya, M.M. Gupta, Arvind S. Negi∗
Central Institute of Medicinal and Aromatic Plants (CIMAP), P.O. CIMAP,
Kukrail Picnic Spot Road, Lucknow, U.P., India
a r t i c l e i n f o
a b s t r a c t
Article history:
Chalcone derivatives on estradiol framework have been synthesized. Some of the deriva-
tives showed potent anticancer activity against some human cancer cell lines. Compounds
9 and 19 showed potent activity against MCF-7, a hormone dependent breast cancer cell
line. Chalcone 7 was further modified to the corresponding indanone derivative (19) using
the Nazarov reaction, which showed better activity than the parent compound against the
MCF-7 breast cancer cell line. Active anticancer derivatives were also evaluated for osmotic
hemolysis using the erythrocyte as a model system. It was observed that chalcone deriva-
tives showing cytotoxicity against cancer cell lines did not affect the fragility of erythrocytes
and hence may be considered as non-toxic to normal cells.
Received 5 June 2007
Received in revised form
18 July 2007
Accepted 28 July 2007
Published on line 6 August 2007
Keywords:
Chalcone
© 2007 Elsevier Inc. All rights reserved.
Estradiol
Anticancer
Erythrocyte
Osmotic fragility
1.
Introduction
Breast cancer is one of the most common cancers in
women. Due to various reasons, the estrogen receptors are
Chalcone moieties are common substructures in numerous
natural products belonging to the flavonoid family [1–4]. Chal-
cone derivatives are very versatile as physiologically active
compounds and substrates for the evaluation of various
organic syntheses. These compounds have been reported to
possess several biological activities, such as cytotoxic [5–8]
antimalarial [9,10], antileishmanial [11,12], anti-inflammatory
[13,14], anti-HIV [15], antifungal [16] and as tyrosine kinase
inhibitors [17]. Having such varied pharmacological activi-
ties, these molecules have attracted medicinal chemists and
therefore several strategies have been developed to synthesize
them.
over-expressed in these tumour cells and, hence, estrogenicity
is enhanced by many folds leading to excessive proliferation
[18]. Steroids are a fundamental class of biological signaling
molecules with profound chemical, clinical and scientific
significance [19]. Steroids elicit their diverse biological actions
via different functional groups located around the periphery
of their rigid tetracyclic core. A major research target today
is the preparation of novel steroid molecules with reactions
chemically simpler and easier to prepare. This would also
provide a platform to approach the synthesis of new drugs
for tackling important biological problems. In the recent past,
two steroidal molecules, 2-methoxyestradiol and fulvestrant
ଝ
CIMAP Communication No. 2007-24J.
Corresponding author. Tel.: +91 522 2717529x327; fax: +91 522 2342666.
∗
E-mail address: arvindcimap@rediffmail.com (A.S. Negi).
0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2007.07.012

steroids 7 2 ( 2 0 0 7 ) 892–900
893
Fig. 1 – Structures of estradiol (I), 2-methoxyestradiol (II) and fulvestrant (III).
(Fig. 1), have emerged as potent anticancer agents against
breast cancer [20]. 2-Methoxyestradiol has successfully com-
2.
Experimental
pleted Phase II clinical trials [21]. It acts as
a tubulin
2.1.
General
polymerisation inhibitor by interacting at the colchicine site
[22] and also as an antiangiogenic agent by acting at the
tumour vascular system. On the other hand, fulvestrant,
which acts as a pure antiestrogen competing with estradiol
for its receptor binding [23], has already completed phase III
clinical trials [24]. US-FDA approved fulvestrant in 2002 for
the treatment of hormone dependent advanced breast can-
cer, while the European Union approved it in 2004 for the same
purpose.
The anticancer activity of various chalcone molecules is
well studied and chalcone with a trimethoxyphenyl unit
has been reported to be the most cytotoxic (compound 1;
IC50 = 0.21 nM) derivative synthesized so far [25]. Synthesis
of chalcones on steroids has not been studied so far. Thus,
we synthesized various chalcone derivatives on a steroid
frame. Some of them showed potent cytotoxicity against the
MCF-7 hormone dependent breast cancer cells (Fig. 2). The
effect of these chalcones was also studied on the erythro-
cytes osmotic fragility, a model system used to evaluate the
effect of chalcones on membrane stability of a cell at the same
concentration at which anticancer activity was observed.
Estrone was procured from Sigma chemicals, USA. Melting
points were determined on Toshniwal melting point (elec-
trothermal) apparatus and were uncorrected. Dry solvents
were prepared as per reported methods. All the reactions
were performed as per standard procedures and monitored on
Merck aluminium thin layer chromatography (TLC, UV254nm
)
plates. TLC visualization was accomplished by spraying with
a solution of 2% ceric sulphate in 10% aqueous sulphuric acid
and charring at 100–110 ◦C. Column chromatography was car-
ried out on silica gel (60–120 mesh, MERCK chemicals). NMR
experiments were obtained on Bruker Avance-300 MHz instru-
ment with tetramethylsilane (TMS) as an internal standard.
Chemical shifts are given in ı ppm values. All the 1H and nec-
essary 13C spectral data are reported. EI mass spectra were
recorded on Perkin-Elmer TurboMass after dissolving the com-
pounds 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 steroid derivatives has been given as per the
Fig. 2 – Most active chalcone (1) reported earlier and some of the potent steroidal chalcones and indanone against MCF-7
breast cancer cell lines.

894
steroids 7 2 ( 2 0 0 7 ) 892–900
J = 15.85 Hz). 13C NMR (CDCl3, 75.47 MHz): ␦ 11.4, 23.5, 26.7, 27.5,
30.5, 31.0, 37.1, 39.2, 43.7, 44.2, 50.6, 55.9, 56.8, 56.8, 61.2, 82.1,
107.1, 112.0, 122.0, 122.8, 133.4, 134.5, 141.5, 141.7, 143.0, 153.5,
157.3, 190.9. Electrospray Mass (MeOH): 507.1 [M + H]+, 529.0
[M + Na]+. IR: 3448, 2930, 1656, 1580, 1502, 1251, 1124.
recommendations published by the Joint Commission on the
Biochemical Nomenclature (JCBN) of IUPAC [26]. All the com-
pounds were screened against five human cancer cell lines
by MTT assay for cytotoxic evaluation and compounds show-
ing potent cytotoxicity were further evaluated for erythrocyte
osmotic fragility test to determine their toxicities.
2.2.2.2. Synthesis of 1-(3,4,5-Trimethoxyphenyl)-3-[methox-
2.2.
Chemical syntheses
yestra-1,3,5(10)-trien-17ˇ-acetate,2-yl]-2-propen-1-one
(8).
Substrate 7 (100 mg, 0.19 mmol) in dry pyridine:acetic anhy-
dride (2:1) was left overnight at room temperature. On
completion, the reaction mixture was poured into cold water
to get 8 as a creamish white solid.
2.2.1. Synthesis of 2-formyl,3-methoxyestra-1,3,5(10)-
trien-17ˇ-acetate (6)
Estra 1,3,5(10)-trien-3,17ˇ-diol-3-methyl ether, 17ˇ-acetate (5),
(100 mg, 0.35 mmol) was taken in dry dimethylformamide
(0.2 mL, 2.59 mmol). The reaction mixture was stirred at
5–10 ◦C by using an ice bath. After 10 min, phosphorus oxy-
chloride (0.2 mL, 2.26 mmol) was added dropwise to the
reaction mixture. It was kept at this temperature for 40 min
and then heated at 80 ◦C for 3 h. After completion, the reac-
tion mixture was poured into crushed ice and extracted with
chloroform (3 × 25 mL). The organic layer was washed with
water, dried over anhydrous sodium sulphate and concen-
trated in vacuum. The residue thus obtained was purified
through a silica gel (60–120 mesh) column by eluting with
8–10% hexane–ethyl acetate. Compound 6 was recrystallised
with hexane–chloroform (4:1) to get a creamish white solid
(39 mg).
Yield 36.0%, m.p.170–171 ◦C. 1H NMR (CDCl3, 300 MHz): ␦
0.76 (s, 3H, 18-CH3), 1.99 (s, 3H, OAc), 1.19–2.28 (m, 13H, rest of
the 5XCH2 and 3 XCH of steroidal ring), 2.85 (bs, 2H, 6-CH2), 3.81
(s, 3H, OCH3), 4.61 (t, 1H, 17-CH, J = 8.33 Hz), 6.60 (s, 1H, 4-CH),
7.68(s, 1H, 1-CH), 10.32(s, 1H, CHO). 13C NMR (CDCl3, 75 MHz):
␦ 12.3, 21.2, 23.6, 26.5, 27.3, 28.0, 30.7, 37.2, 38.9, 43.3, 44.0, 50.4,
56.0, 82.9, 112.3, 123.6, 125.9, 133.6, 146.2, 160.3, 171.1, 189.5.
Electrospray mass (MeOH): 357.0 [M + H]+, 378.9 [M + Na]+, 735.1
[2M + Na]+. IR: 2933, 2870, 1733, 1674, 1608, 1495, 1270, 1027.
Yield 86%, m.p.133–35 ◦C. 1H NMR (CDCl3, 300 MHz): ␦ 0.84
(s, 3H, 18-CH3), 1.25–2.32 (m, 13H, rest of the 5XCH2 and 3XCH
of steroidal ring), 2.06 (s, 3H, OAc), 2.90 (bs, 2H, 6-CH2), 3.84 (t,
1H, 18-CH, J = 8.35Hz), 3.87 (s, 3H, 2X OCH3), 3.93 (s, 3H, OCH3),
4.69 (t, 1H, 17-CH, J = 8.92 Hz), 6.65 (s, 1H, 4-CH of steroidal
ring), 7.25 (s, 2H, 2ꢀꢀ & 6ꢀꢀ-CH of benzoyl moiety), 7.48–7.54 (d,
1H, CH–CO, J = 15.75 Hz), 7.50 (s, 1H, 1-CH of steroidal ring),
7.98–8.03 (d, 1H, CH C–CO, J = 15.80 Hz). EI Mass (MeOH): 548
[M]+, 533, 518, 517, 488, 458, 457. IR: 2938, 1732, 1678, 1581,
1502, 1248.
2.2.2.3. Synthesis of 1-(3,4,dimethoxyphenyl)-3-[methoxyestra-
1,3,5(10)-trien-17ˇ-ol,2-yl]-2-propen-1-one (9). Same proce-
dure as for 7. Yield 60.0%, oil, 1H NMR (CDCl3, 300 MHz): ␦
0.79 (s, 3H, 18-CH3), 1.23–2.10 (m, 13H, rest of the 5XCH2
and 3XCH of steroidal ring), 2.90 (bs, 2H, 6-CH2), 3.75 (t, 1H,
17-CH, J = 8.4 Hz), 3.88 (s, 3H, OCH3), 3.97 (s, 6H, 2XOCH3),
6.65 (s, 1H, 4-CH of steroidal ring), 6.94 (d, 1H, 5-CH of ben-
zoyl moiety, J = 8.3 Hz), 7.53 (s, 1H, 1-CH of steroidal ring),
7.59–7.64 (d, 1H, CH–CO, J = 15.74 Hz), 7.62(d, 1H, 2-CH of ben-
zoyl moiety, J = 1.78 Hz), 7.67 (dd, 1H, 6-CH of benzoyl moiety,
J = 8.36, 1.82 Hz), 8.01–8.06 (d, 1H, CH C–CO, J = 15.75 Hz.). EI
Mass (MeOH): 476 [M]+, 458, 428. IR: 3426, 2925, 1652, 1595, 1511,
1263, 1024.
2.2.2. General procedure for the synthesis of chalcones
2.2.2.1. Synthesis of 1-(3,4,5-trimethoxyphenyl)-3-[methox-
yestra-1,3,5(10)-trien-17ˇ-ol,2-yl]-2-propen-1-one (7). Potas-
sium hydroxide solution (7% (w/v), 15 mL) in ethanol–water
(1:2) was cooled to 10 ◦C. Compound 6 (100 mg, 0.28 mmol) and
3,4,5-trimethoxy acetophenone (84 mg, 0.4 mmol) were added
to this stirred solution. The reaction mixture was further
stirred at this temperature for 3 h and then kept in refriger-
ator (5–10 ◦C) overnight. Next day, the reaction mixture was
poured into crushed ice, acidified with diluted HCl (10%, v/v),
extracted with chloroform (3 × 20 mL) and washed with water.
The organic layer was dried over anhydrous sodium sulphate
and evaporated to dryness. The residue thus obtained was
column chromatographed over silica gel (60–120 mesh) and
eluted with 15%ethyl acetate–hexane to obtain the desired
chalcone (7). It was recrystallised with chloroform–hexane
(1:4) to get a light yellowish solid.
Yield 73.0%, m.p.179–182 ◦C. 1H NMR (CDCl3, 300 MHz): ␦
0.74 (s, 3H, 18-CH3), 1.21–1.95 (m, 13H, rest of the 5XCH2 and 3
XCH of steroidal ring), 2.84 (bs, 2H, 6-CH2), 3.69 (t, 1H, 17-CH,
J = 8.35 Hz), 3.83 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 3.89 (s, 6H, 2
X OCH3), 6.60 (s, 1H, 4-CH of steroidal ring), 7.20 (s, 2H, 2ꢀꢀ & 6ꢀꢀ-
CH of benzoyl moiety), 7.43–7.49 (d, 1H, CH–CO, J = 15.80 Hz),
7.46 (s, 1H, 1-CH of steroidal ring), 7.93–7.98 (d, 1H, CH C–CO,
2.2.2.4. Synthesis
of
1-(3-methoxy,4-hydroxyphenyl)-3-
[methoxyestra-1,3,5(10)-trien-17ˇ-ol,2-yl]-2-propen-1-one
(10). Same procedure as for 7. Yield 37%, m.p. 78 ◦C. 1H NMR
(CDCl3, 300 MHz): ␦ 0.80 (s, 3H, 18-CH3), 1.25–2.16 (m, 13H, rest
of the 5XCH2 and 3XCH of steroidal ring), 2.89 (bs, 2H, 6-CH2),
3.78 (t, 1H, 17-CH, J = 8.4 Hz), 3.88 (s, 3H, OCH3), 3.99 (s, 3H,
OCH3), 6.65 (s, 1H, 4-CH of steroidal ring), 6.99 (d, 1H, 5-CH
of benzoyl moiety, J = 8.64 Hz), 7.52 (s, 1H, 2-CH of benzoyl
moiety), 7.58–7.64 (d, 1H, CH–CO, J = 15.78 Hz), 7.65 (s, 1H,
1-CH of steroidal ring), 8.01–8.06 (d, 1H, CH = C–CO, J = 15.78 Hz.
EI Mass (MeOH): 462 [M]+, 400, 399. IR: 3321, 2929, 1661, 1606,
1516, 1290, 1192, 1026.
2.2.2.5. Synthesis of 1-(3,4-methylenedioxyphenyl)-3-[methox-
yestra-1,3,5(10)-trien-17ˇ-ol,2-yl]-2-propen-1-one (11). Same
procedure as for 7. Yield 44.0%, oil, 1H NMR (CDCl3, 300 MHz):
␦ 0.82 (s, 3H, 18-CH3), 1.23–2.37 (m, 13H, rest of the 5XCH2
and 3XCH of steroidal ring), 2.89 (bs, 2H, 6-CH2), 3.75 (t, 1H,
17-CH, J = 7.85 Hz), 3.88 (s, 3H, OCH3), 6.06 (s, 2H, –O–CH2–O–),
6.64 (s, 1H, 4-CH of steroidal ring), 6.89 (d, 1H, 4-CH of ben-
zoyl moiety, J = 8.1 Hz), 7.53–7.58 (d, 1H, CH–CO, J = 15.54 Hz),
7.63(s, 1H, 2-CH of benzoyl moiety), 7.63–7.66 (dd, 1H, 6-CH
of benzoyl moiety), 8.01–8.07 (d, 1H, CH C–CO, J = 15.72 Hz. EI

steroids 7 2 ( 2 0 0 7 ) 892–900
895
Mass (MeOH): 460 [M]+, 445, 427. IR: 3402, 2921, 1656, 1605,
1251, 1034.
2.2.2.10. Synthesis of 1-(2,4-dichlorophenyl)-3-[methoxyestra-
1,3,5(10)-trien-17ˇ-acetate,2-yl]-2-propen-1-one (16). Procee-
ded as per synthesis of substrate 8. Yield 77.0%, oil, 1H NMR
(CDCl3, 300 MHz): ␦ 0.81 (s, 3H, 18-CH3), 2.06 (s, 3H, OAc),
1.28–2.90 (m, 13H, rest of the 5XCH2 and 3XCH of steroidal
ring), 2.88 (bs, 2H, 6-CH2), 4.68 (t, 1H, 17-CH), 3.83 (s, 3H,
OCH3), 6.62 (s, 1H, 4-CH of steroidal ring), 7.15–7.20 (d, 1H,
CH–CO, J = 16.1 Hz), 7.33 (dd, 1H, 5-CH of benzoyl moiety,
J = 8.2 & 1.8 Hz), 7.40 (s, 1H, 6-CH of benzoyl moiety), 7.47 (d, 1H,
3-CH of benzoyl moiety, J = 1.8 Hz), 7.69–7.75 (d, 1H, CH C–CO,
J = 16.3 Hz). EI Mass (MeOH): 528 [M]+, 495, 381. IR: 2931, 1737,
1642, 1603, 1498, 1245, 1102, 1025.
2.2.2.6. Synthesis of 1-(4-methylphenyl)-3-[methoxyestra-
1,3,5(10)-trien-17ˇ-ol,2-yl]-2-propen-1-one (12). Same proce-
dure as for 7. Yield 52.0%, oil, 1H NMR (CDCl3, 300 MHz): ␦
0.80 (s, 3H, 18-CH3), 1.26–2.36 (m, 13H, rest of the 5XCH2
and 3XCH of steroidal ring), 2.89 (bs, 2H, 6-CH2), 3.72 (bt, 1H,
17-CH), 3.88 (s, 3H, OCH3), 6.67 (s, 1H, 4-CH of steroidal ring),
7.29 (d, 2H, 3 & 5-CH of benzoyl moiety, J = 8.0 Hz), 7.52 (s, 1H,
1-CH of steroidal ring), 7.59-7.61 (d, 1H, CH–CO, J = 15.8 Hz),
7.92 (d, 2H, 2 & 6-CH of benzoyl moiety, J = 8.1 Hz), 8.03–8.08
(d, 1H, CH C–CO, J = 15.8 Hz). 13C NMR (CDCl3, 75.47 MHz): ␦
11.4, 21.9, 23.5, 26.7, 27.5, 30.4, 31.0, 37.1, 39.2, 43.7, 44.2, 50.6,
56.0, 82.2, 112.1, 122.1, 122.7, 127.0, 129.3, 129.3, 129.5, 129.5,
133.3, 136.7, 141.0, 141.5, 143.3, 157.3, 191.3. Electrospray mass
(MeOH): 431.1 [M + H]+, 883.2 [2M + Na]+. IR: 3429, 2926, 1656,
1605, 1590, 1499, 1258, 1025.
2.2.2.11. Synthesis of 1-(4-fluorophenyl)-3-[methoxyestra-
1,3,5(10)-trien-17ˇ-ol,2-yl]-2-propen-1-one (17). Same proce-
dure as for 7. Yield 43.0%, oil, 1H NMR (CDCl3, 300 MHz): ␦
0.797 (s, 3H, 18-CH3), 1.25–2.12 (m, 13H, rest of the 5XCH2 and
3XCH of steroidal ring), 2.89 (bs, 2H, 6-CH2), 3.73 (distorted t,
1H, 17-CH), 3.88 (s, 3H, OCH3), 6.65 (s, 1H, 4-CH of steroidal
ring), 7.12 (d, 2H, 2 & 6-CH of benzoyl moiety, J = 7.6 Hz), 7.18 (d,
2H, 3 & 5-CH of benzoyl moiety, J = 8.6 Hz), 7.52 (s, 1H, 1-CH of
steroidal ring), 7.54–7.59 (d, 1H, CH–CO, J = 15.8 Hz), 8.02–8.08
(d, 1H, CH C–CO, J = 15.8 Hz). EI Mass (MeOH): 434 [M]+, 416,
403, 385. IR: 3432, 2926, 1678, 1599, 1503, 1227, 1155, 1026.
2.2.2.7. Synthesis of 1-(2,4-dimethylphenyl)-3-[methoxyestra-
1,3,5(10)-trien-17ˇ-ol,2-yl]-2-propen-1-one (13). Same proce-
dure as for 7. Yield 46.0%, m.p.164–166 ◦C. 1H NMR (CDCl3,
300 MHz): ␦ 0.78 (s, 3H, 18-CH3), 1.26–2.21 (m, 13H, rest of the
5XCH2 and 3XCH of steroidal ring), 2.37 (s, 3H, CH3), 2.42 (s,
3H, CH3), 2.87 (bs, 2H, 6-CH2), 3.74 (t, 1H, 17-CH, J = 8.38 Hz),
3.83 (s, 3H, OCH3), 6.62 (s, 1H, 4-CH of steroidal ring), 7.07 (d,
1H, 5-CH of benzoyl moiety, J = 7.3 Hz), 7.22 (d, 1H, CH–CO,
J = 16.1 Hz), 7.26 (s, 1H, 3-CH), 7.41 (s, 1H, 1-CH of steroidal ring),
7.44 (d, 1H, 6-CH of benzoyl moiety), 7.73–7.78 (d, 1H, CH C–CO,
J = 16.2 Hz). 13C NMR (CDCl3, 75.46 MHz): ␦11.4, 20.6, 21.6, 23.5,
26.7, 27.5, 30.4, 31.0, 37.1, 39.2, 43.7, 44.2, 50.6, 55.9, 82.2, 112.1,
121.9, 126.5, 126.7, 127.1, 128.9, 132.4, 133.4, 137.4, 140.7, 141.7,
141.7, 157.2, 197.0. Electrospray Mass (MeOH): 445.1 [M + H]+,
467 [M + Na]+, 911.2 [2M + Na]+. IR: 3492, 2924, 1650, 1608, 1500,
1256, 1026.
2.2.2.12. Synthesis of 1-(4-bromophenyl)-3-[methoxyestra-
1,3,5(10)-trien-17ˇ-ol,2-yl]-2-propen-1-one (18). Same proce-
dure as for 7. Yield 34.0%, oil, 1H NMR (CDCl3, 300 MHz): ␦
0.796 (s, 3H, 18-CH3), 1.28–2.16 (m, 13H, rest of the 5XCH2 and
3XCH of steroidal ring), 2.89 (bs, 2H, 6-CH2), 3.75 (distorted t,
1H, 17-CH), 3.88 (s, 3H, OCH3), 6.65 (s, 1H, 4-CH of steroidal
ring), 7.51–7.56 (d, 1H, CH–CO, J = 15.56 Hz), 7.63 (d, 2H, 2 &
6-CH of benzoyl moiety, J = 8.57 Hz), 7.85–7.88 (d, 2H, 3 & 5-CH
of benzoyl moiety, J = 8.47 Hz), 8.02–8.07 (d, 1H, CH C–CO,
J = 15.78 Hz). EI Mass (MeOH): 494 [M]+. IR: 3428, 2923, 1680,
1586, 1501, 1258, 1069.
2.2.2.8. Synthesis
of
1-(4-chlorophenyl)-3-[methoxyestra-
1,3,5(10)-trien-17ˇ-ol,2-yl]-2-propen-1-one (14). Same proce-
dure as for 7. Yield 41.0%, oil, 1H NMR (CDCl3, 300 MHz): ␦
0.796 (s, 3H, 18-CH3), 1.31–2.37 (m, 13H, rest of the 5XCH2 and
3XCH of steroidal ring), 2.90 (bs, 2H, 6-CH2), 3.73 (distorted t,
1H, 17-CH), 3.88 (s, 3H, OCH3), 6.65 (s, 1H, 4-CH of steroidal
ring), 7.29 (s, 1H, 1-CH of steroidal ring), 7.46 (d, 2H, 3 & 5-CH of
benzoyl moiety, J = 8.4 Hz), 7.52–7.57 (d, 1H, CH–CO, J = 16 Hz),
7.95 (d, 2H, 2 & 6-CH of benzoyl moiety, J = 8.43 Hz), 8.02–8.07
(d, 1H, CH C–CO, J = 15.78 Hz). Electrospray Mass (MeOH):
451.0 [M + H]+. IR: 3506, 2925, 1656, 1591, 1498, 1257, 1092.
2.2.3. Synthesis of 4,5,6-trimethoxy-3-[3-methoxy
estra-1,3,5(10)-trien-17-acetate-2yl]-indan-1-one(19)
In a Borosil test tube, 8 (200 mg, 0.36 mmol) was taken in tri-
fluoroacetic acid (0.5 mL) and the tube was sealed properly.
The reaction mixture was heated at 120 ◦C for 4 h. The reac-
tion was poured into crushed ice and extracted with ethyl
acetate, the organic layer was washed with water, dried over
anhydrous sodium sulphate and evaporated in vacuum. The
crude residue thus obtained was purified through column
chromatography on silica gel using ethyl acetate–hexane as
an eluent. The desired indanone 19 was obtained as an oil.
Yield 23.0%, oil, 1H NMR (CDCl3, 300 MHz): 0.866 (s, 3H, 18-
CH3), 2.83 (bs, 2H, 6-CH2), 4.89 (bs, 1H, 17-CH), 2.57–2.65 (dd,
1H, 2-CH of Indanone ring), 3.09–3.18 (dd, 1H, 2-CH of Indanone
ring), 3.72 (s, 6H, 2 X OCH3), 3.81 (s, 6H, 3 X OCH3), 4.83–4.89 (bs,
1H, H-3 of Indanone ring), 6.58 (s, 1H, 4-CH of steroid ring), 6.81
(s, 1H, 1-CH of steroid ring), 7.11 (s, 1H, 7-CH of Indanone ring).
13C NMR (CDCl3, 75.46 MHz): ␦ 11.4, 14.4, 23.0, 29.7, 31.0, 32.3,
44.0, 44.4, 46.3, 50.0, 55.9, 55.9, 60.4, 60.4, 101.4, 113.1, 125.9,
129.7, 132.0, 133.2, 133.3, 144.5, 148.8, 150.8, 154.9, 155.5, 206.3.
EIectrospray Mass (MeOH): 507.2 [M + H]+. IR: 3447, 2930, 1709,
1601, 1504, 1218, 1162, 1031.
2.2.2.9. Synthesis of 1-(2,4-dichlorophenyl)-3-[methoxyestra-
1,3,5(10)-trien-17ˇ-ol,2-yl]-2-propen-1-one (15). Same proce-
dure as for 7. Yield 35.0%, oil, 1H NMR (CDCl3, 300 MHz): ␦
0.78 (s, 3H, 18-CH3), 1.22–2.14 (m, 13H, rest of the 5XCH2 and
3XCH of steroidal ring), 2.86 (bs, 2H, 6-CH2), 3.69 (distorted t,
1H, 17-CH), 3.83 (s, 3H, OCH3), 6.60 (s, 1H, 4-CH of steroidal
ring), 7.15–7.21 (d, 1H, CH–CO, J = 16.2Hz), 7.33 (dd, 1H, 5-CH
of benzoyl moiety, J = 8.2 & 1.7 Hz), 7.44 (s, 1H, 1-CH of steroidal
ring), 7.40 (d, 1H,6-CH of benzoyl moiety, J = 8.2 Hz), 7.47 (d, 1H,
3-CH of benzoyl moiety, J = 1.7 Hz), 7.67–7.73 (d, 1H, CH C–CO,
J = 16.2 Hz). Electrospray Mass (MeOH): 485.0 [M − 1]+. IR: 3506,
2925, 1656, 1591, 1498, 1257, 1092.

896
steroids 7 2 ( 2 0 0 7 ) 892–900
correspond to 0% hemolysis). The recorded optical density
2.3.
Biological methods
(OD) of the supernatant reflects the degree of hemolysis of the
erythrocytes. The lysis percentage was calculated by dividing
the OD of the supernatant obtained from a particular saline
concentration by the OD of the standard (1 g/L) representing
100% hemolysis. Osmotic fragility curves were constructed
by plotting the lysis percentage against the concentration of
saline solutions. The MEF50 (mean erythrocyte fragility) value,
which is the saline concentration at which 50% of the cells
hemolyse at standard pH and temperature [32], was then
obtained from the curve.
2.3.1. Cell culture
The ATCC (American type of cell culture collection) human
cancer cell lines MCF-7 (hormone dependent breast cancer cell
line), KB (oral and mouth), HepG2 (liver cells), CaCO2 (Colon
cancer) and WRL68 (liver cancer) were obtained from NCCS
Pune, India. Cells were cultured in DMEM with HEPES-25 mM,
0.22% NaHCO3 and 10% FBS.
2.3.2. In vitro anticancer activity using MTT assay
In vitro cytotoxicity testing was performed as described [27].
2 × 103 cells/well were incubated in a 5% CO2 incubator for 24 h
to enable them to adhere properly to the 96 well polystyrene
microplate (Grenier, Germany). The test compound, dissolved
in dimethyl sulphoxide (DMSO, Merck, Germany) in at least
five concentrations, was added into the wells and left for 4 h.
After the incubation, the compound plus media was replaced
with fresh media and the cells were incubated for another 48 h
in the CO2 incubator at 37 ◦C. The concentration of DMSO was
always kept below 1.25%, which was found to be non-toxic
to the cells. Then, 10 ␮L of MTT [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide] was added to each well
and plates were incubated at 37 ◦C for 4 h. 100 ␮L of DMSO
was added to all wells and mixed thoroughly to dissolve the
dark blue crystals. The plates were read on a SpectraMax
190 Microplate reader (Molecular Devices Inc. USA) at 570 nm
within 1 h of DMSO addition.
2.3.4. Data analysis
The experiment was done in triplicate and the inhibitory con-
centration (IC) values were calculated from a dose response
curve. IC50 is the concentration in ␮g/mL required for 50% inhi-
bition of cell growth as compared to that of untreated control.
Complete hemolysis of the erythrocyte suspension
occurred in the 0.1% NaCl solution, for which the hemolysis
was defined as 100%. Hemolysis of the erythrocytes did not
occur in the 0.85% NaCl solution, for which the hemolysis
was defined as 0%. The effective concentration of the NaCl
solution inducing 50% hemolysis (MEF50) of the applied ery-
throcytes was calculated from the hemolysis curve by using
a straight-line equation between the points immediately
adjacent to 50%. All values are expressed as the mean of three
experiments in replicates.
3.
Results and discussion
2.3.3. Determination of osmotic hemolysis of erythrocytes
Blood from healthy human male volunteers (n = 3) with
informed consent was collected for experiments using Hep-
arin (10 units/mL) as the anti-coagulant. The collected blood
was stored at 4 ◦C and used for experiments within four hours
of collection [28].
3.1.
Chemistry
Aldehyde and acetophenone moieties were condensed
together to synthesize these chalcone derivatives (Scheme 1).
The steroidal framework was chosen as the aldehydic sub-
strate and thus, estrone (2) was used as the starting material.
To synthesize the aldehydic substrate (6), the phenolic
hydroxyl of 2 was first protected by methylating with dimethyl
sulphate/anhydrous potassium carbonate in dry acetone
under refluxing conditions to get estrone 3-methyl ether (3)
in a 91% yield. The 17-Keto group of the substrate 3 was
reduced to the corresponding 17-alcohol by using sodium
borohydride in a methanol–chloroform (2:1) mixture to get
estradiol 3-methyl ether (4) in a 94% yield. The 17-hydroxyl
of 4 was acetylated with acetic anhydride in dry pyridine
to get estradiol 3-methyl ether 17-acetate (5). The aldehy-
dic substrate 6 was obtained from estradiol, 3-methyl ether,
17␤-acetate by performing the Vilsmeier formylation reac-
tion, using dimethyl formamide and phosphorus oxychloride
at 80 ◦C [33]. 2-Formyl estradiol 3-methyl ether 17-acetate (6)
was treated with different acetophenone derivatives in 7%
aqueous-alcoholic potassium hydroxide solution to get the
various chalcones on the steroidal moiety with 17-acetate
deprotected. Some of the deprotected chalcones were further
acetylated with acetic anhydride and dry pyridine.
Experiments were carried in vitro by adding heparinized
blood to hypotonic solutions of varying concentrations of
phosphate buffered saline (0.85–0.10%). Phosphate buffered
saline stock (10%) was prepared by dissolving 5 g of sodium
chloride, 1.3655 g of disodium hydrogen orthophosphate and
0.243 g of sodium dihydrogen orthophosphate in 100 mL of
autoclaved double distilled water. From this stock, working
standards of 0.85–0.10% were prepared. The tubes were incu-
bated at 37 ◦C for 60 min with mild shaking and the extent
of hemolysis was measured colorimetrically at 540 nm [29,30].
Results are expressed in terms of mean erythrocyte fragility
(MEF50), which is the level of hemolysis of the erythrocytes at
50% saline concentrations. Similarly, prior to the experiment,
heparinized blood was incubated with effective concentration
of chalcones and indanone (19.7–182.4 ␮M) at 37 ◦C for 60 min.
Only those concentrations at which the compounds showed
anticancer activity (or higher) were chosen.
Aliquots of saline solutions, with concentrations ranging
from 10 to 1 g/L were prepared as described by Papart et al.
[31]. The chalcone treated erythrocytes were then transferred
to the tubes containing decreasing concentrations of saline
solutions. After careful mixing, the cell suspensions were left
to equilibrate for 30 min and then centrifuged at 3000 rpm for
5 min. The absorbance of supernatants was read at 540 nm,
standardized against a blank assay (10 g/L saline supernatant
One of the chalcone derivatives was further modified
to the corresponding 1-indanone derivative (19) using the
trifluoroacetic acid catalyzed Nazarov cyclisation reaction
(Scheme 2). Chalcone 8 was heated with trifluoroacetic acid
for 4 h in a sealed tube yielding indanone 19 in a 23% yield [34].

steroids 7 2 ( 2 0 0 7 ) 892–900
897
Scheme 1 – Synthesis of chalcone derivatives on estradiol framework.
Chalcones represent an important class of anticancer
molecules. Hence, various types of chalcones have been syn-
thesized as anticancer agents. In most cases, the anticancer
activities reported were found in ␮M concentrations. Chalcone
1 has been reported to be the most active synthesized chal-
cone so far, having IC50 value at 0.21 nM concentration against
K562 human leukemia cells [25]. We synthesized various estra-
diol chalcone derivatives, which may act as anticancer agents.
One of the chalcone derivatives (8) has been modified to an
indanone derivative (19). All these chalcones were obtained in
good yields after chromatographic separations (Isolated yields
34–86%) and showed high levels of cytotoxicities (Table 1).
lines MCF-7, KB, HepG2, CaCO2 and WRL68 using the MTT
assay. Tamoxifen, Paclitaxel (Taxol) and Podophyllotoxin were
used as reference compounds.
Although these compounds were screened for various
human cancer cell lines, our main interest was in their activ-
ity against MCF-7 breast cancer cells. Among these chalcones,
9 was found to be the most active (IC50 = 7.3 ␮M) followed by
indanone derivative 19 (IC50 = 9.8 ␮M) against the MCF-7 hor-
mone dependent breast cancer cell line where tamoxifen is
the only drug of choice. Chalcones 8, 10 and 13 also possessed
moderate level of cytotoxicity against MCF-7 cancer cells hav-
ing IC50 values of 72.9 ␮M, 64.9 ␮M and 56.3 ␮M, respectively.
The remaining chalcones exhibited low cytotoxicity levels
(IC50 values higher than 100 ␮M), except 12 (IC50 = 697 ␮M, inac-
tive). Among these, 7 also possessed high levels of cytotoxicity
against WRL68, HepG2, and CaCO2 cancer cells, having IC50
values of 49.5 ␮M, 49.5 ␮M, and 16.7 ␮M, respectively.
3.2.
Bioactivity
The anticancer activity of all the synthesized chalcones and
indanone was evaluated against various human cancer cell
Scheme 2 – Conversion of steroidal chalcone to a steroidal indanone derivative.

898
steroids 7 2 ( 2 0 0 7 ) 892–900
Table 1 – Cytotoxicity of different compounds against various human cancer cell lines by the MTT assay
S. no.
Compound no.
Human cancer cells
HepG2 IC50 ␮M
49.4
MCF-7 IC50 ␮M
KB IC50 ␮M
CaCO2 IC50 ␮M
WRL68 IC50 ␮M
a
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
7
8
9
10
11
12
13
14
15
16
17
18
177.86
72.9
7.3
–
16.7
100.3
189.0
140.7
141.3
116.2
168.9
200.0
185.9
142.0
49.4
145.9
–
127.7
a
–
189.0
a
a
64.9
a
194.8
206.5
a
184.7
197.6
a
174.4
162.7
a
56.3
200.0
180.1
191.4
a
a
a
a
a
a
154.9
144.6
a
a
160.9
a
a
138.2
151.8
9.88
0.027
0.006
8.45
161.2
126.7
172.8
a
a
a
19
138.3
0.013
0.001
20.5
3.9
167.9
0.40
0.008
0.002
128.4
0.027
0.005
4.83
Tamoxifen
Paclitaxel (Taxol)
Podophyllotoxin
2.28
0.009
4.83
a
Means inactive (IC50 values > 200 ␮M); IC50 values mean of three experiments in replicate.
The structure and activity relationship (SAR) of these
steroidal chalcones against the MCF-7 cancer cells line
was studied. Earlier reports [25] on SAR of these class of
compounds showed that the chalcone (1) with the 3,4,5-
trimethoxy benzoyl unit was the most cytotoxic one whereas,
in the case of steroidal chalcones, compound 9, containing
the 3,4-dimethoxy benzoyl unit, was the most active (nearly
24fold higher activity than 7, which has a 3,4,5-trimethoxy
benzoyl unit). Using the Nazarov reaction, compound 8 yielded
indanone derivative 19, which showed a better cytotoxic activ-
ity than the parent compound and comparable to 9. The
cytotoxicity was reduced when one of the methoxy group
of 9 was replaced with a phenolic hydroxyl (10). When the
3,4-dimethoxy benzoyl unit of 9 was replaced with the 3,4-
methylenedioxy benzoyl unit in chalcone 11 the anticancer
activity was lost. 4-Halogented (F/Cl/Br) chalcone derivatives
(14, 15, 16, 17 & 18) were either inactive or poorly active against
the MCF-7 human cancer cell line.
The osmotic fragility profiles of the control and in vitro
chalcone(s)-treated erythrocytes are shown in Fig. 3. Vari-
ous reference compounds were also used, namely, curcumin
as a positive control, hydrogen peroxide as a negative con-
trol, tamoxifen and podophyllotoxin as standard anticancer
molecules. In vitro treatment of erythrocytes with chalcone(s)
resulted in an altered osmotic fragility profile, which is evi-
dent by the shift of the curve to the left and a decrease in
MEF values, representing decreased cell lysis. The MEF50 val-
ues of the control, the reference compounds and the in vitro
chalcone(s)-treated erythrocytes are tabulated in Table 2. Ery-
throcyte osmotic fragility is used as a marker of erythrocyte
tensile strength and is related to cellular deformability, useful
in the clinical detection of many diseases including anemia
[36–40]. The significance of this parameter lies in the fact that
it gives information about the metabolism and membrane sta-
Chalcones have been reported to act as tubulin polymer-
ization inhibitors. Having structural similarity with colchicine,
these are believed to bind to the same site of tubulin [35]. Tubu-
lin exists as a heterodimer of alpha and beta-subunits. This
dimmer can couple together to make profilaments consisting
of alternating alpha and beta subunits. Several profilaments
(12 or more) can bind together to form pipe like structures
known as microtubulin (polymer of tubulin). The structure
plays a vital role in various biochemical processes of cell sur-
vival and growth. One of these is the formation of the mitotic
spindle, without which mitosis would not take place.
Hsu et al. [7] studied the molecular mechanism of some of
the chalcones against the MCF-7 cancer cells and found that
chalcones were involved in the; (1) induction of apoptosis, (2)
blockage of cell cycle progression by cell cycle-related factor
regulation, (3) initiation of Fas/Fas ligand pathway, (4) trigger
of mitochondrial pathway, and (5) modulation of Bcl-2 fam-
ily protein. Thus, the authors demonstrated that chalcones
may be a promising chemopreventive agent for breast cancer
treatment.
Fig. 3 – Osmotic hemolysis curve of erythrocyte.

steroids 7 2 ( 2 0 0 7 ) 892–900
899
[4] Zhang Y, Shi S, Zhao M, Jiang Y, Tu P. A novel chalcone from
Coreopsis tinctoria nutt. Biochem Syst Ecol 2006;34:766–9.
[5] Modzelewska A, Pettit C, Achanta G, Davidson NE, Huang P,
Khan SR. Anticancer activities of novel chalcone and
bis-chalcone derivatives. Bioorg Med Chem 2006;14:3491–5.
[6] Anto RJ, Sukumaran K, Kuttana G, Rao MNA, Subbaraju V,
Kuttana R. Anticancer and antioxidant activity of synthetic
chalcones and related compounds. Cancer Lett 1995;97:33–7.
[7] Hsu YL, Kuo PL, Tzeng WS, Lin CC. Chalcone inhibits the
proliferation of human breast cancer cell by blocking cell
cycle progression and inducing apoptosis. Food Chem
Toxicol 2006;44:704–13.
[8] Lawrence NJ, Patterson RP, Ooi LL, Cook D, Ducki S. Effects of
␣-substitutions on structure and biological activity of
anticancer chalcones. Bioorg Med Chem Lett 2006;16:5844–8.
[9] Dominguez JN, Leon C, Rodrigues J, Gamboa de Dominguez
N, Gut J, Rosenthal PJ. Synthesis and evaluation of new
antimalarial phenylurenyl chalcone derivatives. J Med Chem
2005;48:3654–8.
Table 2 – Evaluation of the effect of chalcones on MEF
values of erythrocyte osmotic fragility
S. no.
Condition
Mean erythrocyte
fragilitya
Concentration
(␮M)
1.
2.
3.
4.
5.
6.
7.
8.
9.
Control
7
8
9
10
13
19
Curcumin
Podophyllotoxin
Tamoxifen
Hydrogen peroxide
0.69
0.685
0.71
0.52
0.70
0.69
0.55
0.51
0.69
0.65
0.77
–
177.8
182.4
7.35
64.9
56.3
19.7
25.8
241.5
1.34
2000
10.
11.
a
Values are mean of three experiments in replicate.
[10] Alain Valla A, Valla B, Cartier D, Guillou RL, Labia R, Florent L,
et al. New syntheses and potential antimalarial activities of
new ‘retinoid-like chalcones’. Eur J Med Chem 2006;41:142–6.
[11] Nielsen SF, Christensen SB, Cruciani G, Kharazmi A, Liljefors
T. Antileishmanial chalcones: statistical design, synthesis,
and three-dimensional quantitative structure-activity
relationship analysis. J Med Chem 1998;41:4819–32.
[12] Boeck P, Falca CAB, Leal PC, Yunes PA, Filho VC,
Torres-Santos EC, et al. Synthesis of chalcone analogues
with increased antileishmanial activity. Bioorg Med Chem
2006;14:1538–45.
[13] Lee SH, Seo GS, Kim JY, Jin XY, Kim HD, Sohn DH. Heme
oxygenase 1 mediates anti-inflammatory effects of
2ꢀ,4ꢀ,6ꢀ-tris(methoxymethoxy)chalcone. Eur J Pharmacol
2006;532:178–86.
[14] Yang HM, Shin HR, Cho SH, Bang SC, Song GY, Ju JH, et al.
Structural requirement of chalcones for the inhibitory
activity of interleukin-5. Bioorg Med Chem 2007;15:104–11.
[15] Cheenpracha S, Karalai C, Ponglimanont C, Subhadhirasakul
S, Tewtrakul S. Anti-HIV-1 protease activity of compounds
from Boesenbergia pandurata. Bioorg Med Chem
bility of the erythrocytes. The present study was undertaken to
study the effect of chalcone(s) on the osmotic hemolysis pro-
file of erythrocytes. The decrease in the erythrocytes’ osmotic
hemolysis observed suggests that the chalcone derivatives,
which showed cytotoxicity against a cancer cell line, are non-
toxic to erythrocytes since they did not affect the fragility of
these cells. However, further work is needed to understand
how these chalcones interact and affect the fluidity and per-
meability of the erythrocyte membrane.
In conclusion, the chalcone derivatives synthesized on a
steroidal framework showed good anticancer activity against
hormone dependent breast cancer cells and were found to
be non-toxic to erythrocytes. Thus, steroidal chalcones may
be developed as potent anticancer agents. These derivatives
may be further studied for antiestrogenic activity and recep-
tor binding affinity in order to better understand their mode
of action. The present study already gives some insight about
its further optimization for better activity.
2006;14:1710–4.
[16] Svetaz L, Tapia A, Lopez SN, Furlan RLE, Petenatti E, Pioli R,
et al. Antifungal chalcones and new caffeic acid esters from
Zuccagnia punctata acting against soybean infecting fungi. J
Agric Food Chem 2004;52:3297–300.
Acknowledgements
[17] Nerya O, Musa R, Khatib S, Tamir S, Vaya J. Chalcones as
potent tyrosinase inhibitors: the effect of hydroxyl positions
and numbers. Phytochemistry 2004;65:1384–95.
[18] Jordan VC. Antiestrogens and selective estrogen receptor
modulators as multifunctional medicines. 1. Receptor
interactions. J Med Chem 2003;46:883–907.
Authors are thankful to the Director, CIMAP for constant
encouragement and providing necessary facilities. The award
of Junior Research Fellow to one of the authors (HOS) from Uni-
versity Grants Commission (UGC) is duly acknowledged. The
project work was supported from Department of Science and
Technology (DST), Government of India.
[19] (a) O’Malley BW, editor. Hormones and signalling. San Diego:
Academic Press; 1998;
(b) Parker MG, editor. Steroid hormone action. Oxford: IRL
Press; 1993.
r e f e r e n c e s
[20] Seeger H, Diesing D, Guckel B, Wallwiener D, Mueck AO,
Huober J. Effect of tamoxifen and 2-methoxyestradiol alone
and in combination on human breast cancer cell
proliferation. J Steroid Biochem Mol Biol 2003;84:255–7.
[21] Sweeney C, Liu G, Yiannoutsos C, Kolesar J, Horvath D, Staab
MJ, et al. A phase II multicenter, randomized, double-blind,
safety trial assessing the pharmacokinetics,
[1] Nowakowska Z. A review of antiinfective and
anti-inflammatory chalcones. Eur J Med Chem
2007;42:125–37.
[2] Akihisa T, Tokuda H, Hasegawa D, Ukiya M, Kimura Y, Enjo F,
et al. Chalcones and other compounds from the exudates of
Angelica keiskei and their cancer chemopreventive effects. J
Nat Prod 2006;69:38–42.
pharmacodynamics, and efficacy of oral 2-methoxyestradiol
capsules in hormone-refractory prostate cancer. Clin Cancer
Res 2005;11:6625–33.
[3] Narender T, Shweta, Tanvir K, Srinivasa Rao M, Srivastava K,
Puri SK. Prenylated chalcones isolated from Crotalaria genus
inhibits in vitro growth of the human malaria parasite
Plasmodium falciparum. Bioorg Med Chem Lett 2005;15:2453–5.
[22] D’Amato RJ, Lin CM, Flynn E, Folkman J, Hamel E.
2-Methoxyestradiol, an endogenous mammalian metabolite,
inhibits tubulin polymarisation by interacting at the
colchicine site. Proc Natl Acad Sci 1994;91:3964–8.

900
steroids 7 2 ( 2 0 0 7 ) 892–900
[23] Dauvois S, White R, Parker MG. The antiestrogen ICI 182780
disrupts estrogen receptor nucleocytoplasmic shuttling. J
Cell Sci 1993;106:1377–88.
[24] Howell A. Fulvestrant (Faslodex): current and future role in
breast cancer management. Crit Rev Oncol-Hematol
2005;57:265–73.
[25] Lawrence NJ, Rennison D, McGown AT, Hadfield JA. The total
synthesis of an aurone isolated from Uvaria hamiltonii:
aurones and flavones as anticancer agents. Bioorg Med
Chem Lett 2003;13:3759–63.
[26] IUPAC. Joint Commission on Biochemical Nomenclature
(JCBN). Nomenclature of steroids. Pure Appl Chem
1989;61:1783–822.
and normal red cell membranes. Biochim Biophys Acta
1992;1103:296–302.
[33] Vilsmeier A, Haack A. Uber die Einwirkung von
halogenphosphor auf alkyl-formanilide. Eine neue methode
zur Dalstellung SekundOler und tertiaue
p-alkylamino-benzaldehyde. Chem Ber 1927;60:119–22.
[34] Lawrence NJ, Simon E, Armitage M, Greedy B, Cook D, Ducki
S, et al. The synthesis of indanones related to
combretastatin A-4 via microwave-assisted Nazarov
cyclisation of chalcones. Tetrahedron Lett 2006;47:
1637–40.
[35] Lowrence N, McGown A, Ducki S, Hadfield J. The interaction
of chalcones with tubulin. Anticancer Drug Design
2000;15(2):135–41.
[27] Woerdenbag HJ, Moskal TA, Pras N, Malingre´ TM, Farouk S,
El-Feraly H, et al. Cytotoxicity of Artemisinin-related
endoperoxides to ehrlich ascites tumor cells. J Nat Prod
1993;56(6):849–56.
[36] Burke BE, Shotton DM. Erythrocyte membrane skeleton
abnormalities in hereditary spherocytosis. Br J Hematol
1983;54:173–87.
[28] Luqman S, Rizvi SI. Effect of amiloride on the osmotic
fragility and sodium/potassium adenosine triphosphatase
activity of erythrocytes. Asia Pac J Pharmacol 2004;16:
53–5.
[37] Sen G, Mukopadhaya R, Ghosal J, Biswas T. Combination of
ascorbate and ␣ tocopherol as a preventive therapy against
structural and functional defects of erythrocytes in visceral
leishmaniasis. Free Rad Res 2004;38:527–34.
[29] Luqman S, Obli Prabu KV, Pal A, Saikia D, Darokar MP,
Khanuja SPS. Antibiotic-induced alterations in the osmotic
resistance of erythrocytes is modulated by ␤-carotene and
l-ascorbic acid. Nat Prod Commun 2006;1:481–6.
[30] Dacie JV, Lewis SM. Practical hematology. Orient Longman;
1984. pp. 152–156.
[38] Sirichotiyakul S. Erythrocyte osmotic fragility test for
screening of ␣-thallassemia and ␤-thallassemia trait in
pregnancy. Int J Gynecol Obst 2004;86:347–56.
[39] Drabko K, Kuwalczyk J. Imbalance between pro-oxidative
and anti-oxidative processes in children with neoplastic
disease. Med wieku rozwoj 2004;8:217–23.
[31] Papart AK, Lorenz PB, Papart ER, Gregg JR, Chase AM. The
osmotic resistance (fragility) of human red cells. J Clin Invest
1947;26:636–40.
[40] Vijayamalini M, Manoharan S. Lipid peroxidation, vitamin C,
E and reduced glutathione levels in patients with pulmonary
tuberculosis. Cell Biochem Funct 2004;22:19–22.
[32] Kucuk O, Lis LJ, Dey T, Mata R, Westerman MP, Yachnin S, et
al. The effects of cholesterol oxidation products in sickle