Synthesis of 2-substituted 17β-hydroxy/17-methylene estratrienes and their in vitro cytotoxicity in human cancer cell cultures

Steroids 76 (2011) 1491–1504

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Steroids

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Synthesis of 2-substituted 17b-hydroxy/17-methylene estratrienes

and their in vitro cytotoxicity in human cancer cell cultures

Ganapathy Panchapakesan ^a,b, Vasudevan Dhayalan ^a, Nachiappan Dhatchana Moorthy ^b,

Nidhyanandan Saranya ^b, Arasambattu K. Mohanakrishnan ^a,

⇑

^aDepartment of Organic Chemistry, University of Madras, Guindy Campus, Chennai 600 025, India

^bResearch and Development Centre, Orchid Chemicals and Pharmaceuticals Ltd., 476/14 Old Mahabalipuram Road, Sholinganallur, Chennai 600 119, India

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 4 March 2011

Received in revised form 8 August 2011

Accepted 11 August 2011

Available online 22 August 2011

Synthesis of various types of 2-(alkylaminomethyl) and 2-(aroyl) 17b-estradiol analogs are reported. The

synthesis of similar types of 2-substituted 17-methylene estratriene analogs was also achieved. Synthesis

of chalcone derivatives of 17b-estradiol and 17-methylene estratriene were also realized. All these 2-

substituted estratrienes were tested for their antiproliferative activity by using four different cell lines

from colon, lung, glioma and breast cancers. Among the various 2-substituted estratrienes, the com-

pounds 10d, 14a–h and 17e were found to have in vitro antiproliferative activity comparable to that of

parent analogs 1–4. Comparison of the SAR pattern of these 2-susbtituted estratriene derivatives con-

ﬁrmed that relatively, 17-methylene estratrienes are more active than that of 17b-estradiol analogs.

Keywords:

2-Methoxyestradiol

2-Substituted 17b-estradiols

2-Substituted 17-methylene estratrienes

In vitro cytotoxicity

1. Introduction

tive 2-methoxyestrone by 17b-hydroxy steroid dehydrogenase

type II [7]. To enhance further the oral bio-availability of 2-

Interest in the synthesis of various analogs of 2-methoxyestra-

diol have been stimulated by their cytotoxicity in cancer cell

cultures [1]. During the last ten years, various analogs of 2-meth-

oxyestradiol have been synthesized and studied for their antipro-

liferative activity against human cancer cells. It has been

established that the anticancer activities of 2-substituted estratri-

ene derivatives are independent of the estrogen receptors. These

compounds are indeed thought to bind the colchicine binding-site

of tubulin [2]. Based on the cytotoxicity proﬁle displayed by a

number of 2-substituted estradiol analogs, Cushman and cowork-

ers established that the steric and electronic factors at the 2-posi-

tion of estradiol are prominent contributor to the observed

cytotoxicity, as well as the antitubulin activity [3].

The same group also carried out the synthesis of B-ring

homologated analogs of estradiol, some of which resembled pac-

litaxel (Taxol) in their ability to inhibit tubulin polymerization

[4]. Macdonald et al. synthesized a series of 2-methoxy estradiol

analogs in which the A-ring of the steroid was replaced by

substituted tropone rings [5]. In particular, Rao et al. have car-

ried out extensive work on the synthesis and antimitotic activity

of D-ring modiﬁed 2-methoxyestradiol analogs [6]. It has been

shown that 2-methoxyestradiol is metabolized rapidly into inac-

methoxyestradiol, several 17b-hydroxy modiﬁed derivatives have

been synthesized. The combination of inactivating cellular

metabolism with rapid conjugative inactivation through the

reaction of hydroxyl group emphasizes the need for 2-substi-

tuted estradiol analogs with improved pharmacokinetic proﬁles,

as well as potency. The structure–activity relationship of several

C-17-cyano substituted estratrienes has been reported [8].

Several of these compounds displayed high activity against the

proliferation of cancer cells in vitro. Oral bioavailability of

2-substituted estratriene-3-sulfamate compounds was also con-

ﬁrmed [9]. Treston and coworkers recently reported the synthe-

sis and antiproliferative structure–activity relationship of 2- and

17-substituted estrone analogs [10]. Liou et al. explored a wide

variety of benzophenone derivatives and aroylindoles as poten-

tial antimitotic agents [11]. Recently, Boumendjel and coworkers

reported antimitotic and antiproliferative activities of chalcones

[12]. Hence, the synthesis of similar type of estradiol analogs

possessing aroyl as well as chalcone units at the 2-position

was planned. It is expected that the most useful analogs of 2-

methoxyestradiol should retain its inhibitory effects on tubulin

polymerization and cell growth, while addressing the metabolic

stability of 17b-hydroxy unit. Some of the simplest 2-substituted

estratrienes 1–4 that showed high cytotoxicity against human

cancer cell lines are listed in Fig. 1. It should be noted that al-

most all these 2-substituted estratrienes also exhibited tubulin

polymerase inhibitory activity.

⇑

Corresponding author. Tel.: +91 44 22202813; fax: +91 44 22300488.

E-mail addresses: mohanakrishnan@unom.ac.in, mohan_67@hotmail.com (A.K.

Mohanakrishnan).

doi:10.1016/j.steroids.2011.08.004

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G. Panchapakesan et al. / Steroids 76 (2011) 1491–1504

OH

2.2.1. Synthesis of 2-formylestradiol (6) [15]

Me

Interaction of freshly prepared ethylmagnesium bromide

(2.1 g Mg, 13.1 g bromoethane) with estradiol (4 g, 14.7 mmol)

followed by addition of paraformaldehyde (7 g, 233 mmol) and

subsequent work up of the reaction led to the isolation of crude

2-formyl estradiol as light yellow viscous oil. The crude product

upon trituration with diisopropyl ether furnished 2-formyl estra-

diol 6 (3 g, 70%) as light yellow solid. mp 219–221 °C; IR (KBr,

cm^ꢁ1): 1675 (C@O).

EtO

HO

MeO

HO

2

1

CH₂

Me

EtO

HO

MeO

HO

2.2.2. Representative procedure for preparation of 2-(2⁰-N,N-

3

4

dimethylethylenediamino)methyl estradiol 7a (Procedure A)

Fig. 1. Structures of representative 2-substituted estratrienes 1–4.

To a solution of 2-formyl estradiol 6 (1 g, 3.33 mmol) in DMF

(15 mL) at 5 °C was added sodium sulphate (2 g) followed by

N,N-dimethylethylenediamine (0.59 g, 6.66 mmol) in DMF (5 mL),

and the mixture was stirred for 1 h. To the resulting yellow suspen-

sion was added sodium borohydride (0.5 g, 13.2 mmol) followed

by slow addition of methanol (2 mL) during 30 min. The reaction

mixture was further stirred for 1 h at 5 °C. It was then poured in

to water (40 mL) and washed with ethyl acetate (2 ꢀ 75 mL). The

combined organics were washed with brine (2 ꢀ 40 mL), dried

with sodium sulfate, ﬁltered, and concentrated via rotary evapora-

tion to give light yellowish oil. The crude product was puriﬁed by

silica gel column chromatography using (80:20 ethyl acetate/

methanol) as an eluent to yield 7a as yellow viscous oil (0.84 g,

60%); IR (KBr, cm^ꢁ1): 3402 (OH and NH); ¹H NMR (300 MHz,

DMSO-d₆) d 10.13 (s, 1H), 6.93 (s, 1H), 6.36 (s, 1H), 3.75 (s, 2H),

2.68–2.67 (d, J = 3.6 Hz, 2H), 2.55–2.54 (d, J = 6.2 Hz, 1H), 2.33–

2.30 (t, J = 6.3 Hz, 2H), 2.18–2.17 (d, J = 3.3 Hz, 1H), 2.14 (s, 1H),

2.11 (s, 6H), 1.35 (m, 2H), 1.28–1.04 (m, 13H), 0.65 (s, 3H); m/z (rel-

ative intensity) 373 (MH⁺, 100%); Anal. Calcd. for (C₂₃H₃₆N₂O₂): C,

74.15; H, 9.74; N, 7.52. Found: C, 74.01; H, 9.98; N, 7.30%.

2. Experimental

2.1. Proliferation assays

Human colon carcinoma HCT-116 cells, NCI-H460 non-small

cell lung cancer cells, U251 glioblastoma cells, and MDA MB-435

breast cancer cells were purchased from the American Type Cul-

ture Collection. They were subcultured as per the instructions pro-

vided in the product information sheet. Cell lines were cultured in

Dulbecco’s Modiﬁed Eagle Medium (DMEM) with 10% heat-inacti-

vated fetal bovine serum (FBS, GIBCO), 100 units/ml penicillin and

100 lg/ml streptomycin (GIBCO). Cells were cultured at 37 °C in a

humidiﬁed 5% CO₂atmosphere and were passaged three times

weekly. Cells were passaged by ﬁrst washing with sterile PBS

and then incubating with trypsin-EDTA. The resulting single-cell

suspension was resuspended in fresh DMEM medium (complete),

counted, and seeded at 3 ꢀ 10³cells/well in 96-well plates. Growth

inhibition assays were performed in the presence of different drug

concentrations ranging from 100 lM to 0.01 lM, in a logarithmic

scale. After 48 h, at the end of the incubation, a Sulphorhod-

amine-B (SRB) assay [13,14] was performed to determine the cel-

lular growth and viability. Brieﬂy, cells attached to the bottom of

the plate were ﬁxed by addition of cold trichloroacetic acid (TCA,

4 °C) on the top of the growth medium (ﬁnal TCA 10% w/v). The

plate was placed at 4 °C for 1 h before being gently washed ﬁve

2.2.3. 2-(3⁰-N,N-Dimethylpropylenediamino)methyl estradiol (7b)

Condensation of 2-formyl estradiol 6 (1 g, 3.33 mmol) with N,N-

dimethylpropylenediamine (0.68 g, 6.66 mmol) followed by

sodium borohydride (0.5 g, 13.2 mmol) reduction using the

above-mentioned procedure A and subsequent column chromatog-

raphy puriﬁcation using (60:40 ethyl acetate/methanol) as an elu-

ent afforded compound 7b as a light brown viscous liquid (810 mg,

63%). ¹H NMR (300 MHz, DMSO-d₆) d 10.13 (s, 1H), 7.01 (s, 1H),

6.34 (s, 1H), 3.78 (s, 2H), 2.67 (s, 2 H), 2.22–2.18 (m, 3H), 2.08–

2.03 (m, 8H), 1.82 (m, 4H), 1.56–1.52 (m, 3H), 1.6 (m, 1H), 1.28–

1.19 (m, 9H), 0.65 (s, 3H); m/z (relative intensity) 387 (MH⁺,

100%); Anal. Calcd. for (C₂₄H₃₈N₂O₂): C, 74.57; H, 9.91; N, 7.25.

Found: C, 74.85; H, 9.70; N, 7.06%.

times with tap water. It was allowed to dry in air, then 100 ll of

0.057% w/v SRB dissolved in 1% acetic acid in water was added to

each well for 30 min. At the end of the staining period, unbound

SRB was removed by washing three times with 1% acetic acid.

The plate was air dried again, and 200 ll of 10 mM aqueous Tris

base [tris (hydroxymethyl) aminomethane] was added into each

well to solubilize the cell-bound dye. The plate was shaken for

15 min on a gyratory shaker followed by reading the optical

density (OD) at 530 nm in

a microplate spectrophotometer

(Spectramax Plus, Molecular devices, Softmax Pro Ver. 5.2).

2.2.4. 2-(Cyclopropylamino) methyl estradiol (7c)

Condensation of 2-formyl estradiol 6 (1 g, 3.33 mmol) with

cyclopropylamine (0.38 g, 6.66 mmol) followed by sodium borohy-

dride (0.5 g, 13.2 mmol) reduction using the above-mentioned

procedure A and subsequent column chromatography puriﬁcation

using (30:70 ethyl acetate/hexane) as an eluent afforded com-

pound 7c as a colorless solid (0.95 g, 84%). mp 172–173 °C; ¹H

NMR (300 MHz, DMSO-d₆) d 10.12 (s, 1H), 6.97 (s, 1H), 6.37 (s,

1H), 4.50 (m, 1H), 3.74 (s, 2H), 3.53–3.49 (t, J = 8.4 Hz, 1H), 2.67

(s, 2H), 2.07–2.04 (m, 2H), 1.97–1.75 (m, 3H), 1.6 (m, 1H), 1.28–

1.17 (m, 9H), 0.65 (s, 3H), 0.38–0.36 (t, J = 3.3 Hz, 2H), 0.28–0.27

(d, J = 3.1 Hz, 2H); ¹³C NMR (75 MHz, DMSO-d₆) d 154.3, 135.3,

130.0, 125.3, 122.0, 115.0, 80.0, 50.3, 49.5, 43.5, 42.8, 36.6, 30.0,

29.9, 28.8, 27.0, 26.2, 22.8, 11.2, 5.6; m/z (relative intensity) 342

(MH⁺, 100%); Anal. Calcd. for (C₂₂H₃₁NO₂): C, 77.38; H, 9.15; N,

4.10. Found: C, 77.18; H, 9.39; N, 4.35%.

2.2. General

All melting points were uncorrected. Reagents were purchased

from commercial sources and used as received without puriﬁca-

tion. Solvents were dried by standard procedures. Column chroma-

tography was carried on silica gel (grade 60, mesh size 230–400,

Merck). ¹H and ¹³C NMR spectra were recorded in CDCl₃or

DMSO-d₆using TMS as an internal standard on

a Bruker

300 MHz spectrometer. Chemical shift values were quoted in

ppm and coupling constants were quoted in Hz. Chemical shift

multiplicities were reported as s = singlet, d = doublet, t = triplet,

q = quartet, m = multiplet. Mass spectra were recorded on an Agi-

lent MSD1 SPC spectrometer. Elemental analyses were carried

out on Perkin–Elmer series II 2400 equipment.

G. Panchapakesan et al. / Steroids 76 (2011) 1491–1504

1493

2.2.5. 2-(Ethylglycine) methyl estradiol (7d)

stirred for 10 min, a solution of tert-butyldimethylsilyl chloride

(20 g, 132.7 mmol) in DMF (100 mL) was added slowly. The result-

ing mixture was further stirred for another 3 h at 10 °C, and the

reaction mixture was poured into sodiumbicarbonate solution

(500 mL, 5% w/v) and washed with hexane (2 ꢀ 500 mL). The com-

bined organics were washed with brine solution (2 ꢀ 200 mL),

dried with sodium sulfate, ﬁltered, and concentrated via rotary

evaporation to give light yellow viscous oil. The crude product

was puriﬁed by silica gel column chromatography using (98:2 hex-

ane/ethyl acetate) as an eluent to yield 8 as white solid (14.2 g,

81%). mp 181–182 °C; ¹H NMR (300 MHz, DMSO-d₆) d 10.38 (s,

1H), 7.72 (s, 1H), 7.26 (s, 1H), 6.56 (s, 1H), 3.65–3.61 (t,

J = 8.3 Hz, 1H), 2.85–2.82 (q, J = 4.1 Hz, 1H), 2.47–2.43 (d,

J = 4.2 Hz 2H), 2.3 (m, 1H), 1.90–1.85 (m, 3H), 1.58–1.07 (m, 7H),

1.01 (s, 9H), 0.89 (s, 9H), 0.75 (s, 3H), 0.25 (s, 6H), 0.03–0.02 (d,

J = 6.4 Hz, 6H); m/z (relative intensity) 529 (MH⁺, 100%); Anal.

Calcd. for (C₃₁H₅₂O₃Si₂): C, 70.40; H, 9.91. Found: C, 70.62; H,

9.72%.

Condensation of 2-formyl estradiol 6 (1 g, 3.33 mmol) with

glycine ethyl ester (0.69 g, 6.66 mmol) in DMF for 3 h followed

by sodium borohydride (0.5 g, 13.2 mmol) reduction using the

above-mentioned procedure A and subsequent column chromatog-

raphy puriﬁcation using (22:78 ethyl acetate/hexane) as an eluent

afforded compound 7d as a pale yellow low melting solid (710 mg,

55%). ¹H NMR (300 MHz, DMSO-d₆) d 9.98 (s, 1H), 6.96 (s, 1H), 6.4

(s, 1H), 4.49 (s, 1H), 4.10–4.08 (d, J = 7.1 Hz, 1H), 3.68 (s, 1H), 3.51

(m, 3H), 2.68 (s, 2H), 2.25–2.15 (m, 2H), 1.95–2.05 (m, 1H), 1.84–

1.81 (m, 3H), 1.5 (m, 1H), 1.32–1.10 (m, 11H), 0.85 (m, 1H), 0.65

(s, 3H); m/z (relative intensity) 388 (MH⁺, 100%); Anal. Calcd. for

(C₂₃H₃₃NO₄): C, 71.29; H, 8.58; N, 3.61. Found: C, 71.04; H, 8.36;

N, 3.85%.

2.2.6. 2-(Ethyl 2-amino butyrate)methyl estradiol (7e)

Condensation of 2-formyl estradiol 6 (1 g, 3.33 mmol) with

ethyl 2-aminobutyrate (0.87 g, 6.66 mmol) followed by sodium

borohydride (0.5 g, 13.2 mmol) reduction using the above-men-

tioned procedure A and subsequent column chromatography puri-

ﬁcation using (12:88 ethyl acetate/hexane) as an eluent afforded

compound 7e as a yellow solid (0.91 g, 66%). IR (KBr, cm^ꢁ1):

3433 (OH and NH), 1734 (C@O); ¹H NMR (300 MHz, DMSO-d₆) d

9.9 (s, 1H), 6.96 (s, 1H), 6.39 (s, 1H), 4.50–4.49 (d, J = 4.0 Hz, 1H),

4.10–4.09 (t, J = 4.2 Hz, 2H), 3.7–3.6 (d, J = 4.0 Hz, 1H), 3.57–3.53

(m, 2H), 3.11 (s, 1H), 2.67 (s, 2H), 2.25–2.15 (m, 1H), 2.05–1.95

(m, 1H), 1.84 (m, 3H), 1.59–1.56 (m, 3H), 1.28–1.17 (m, 11H),

0.87–0.86 (t, J = 7.4 Hz, 3H), 0.65 (s, 3H); ¹³C NMR (75 MHz,

DMSO-d₆) d 173.9, 154.1, 135.6, 130.1, 125.5, 121.5, 115.0, 80.0,

61.0, 60.0, 49.5, 48.2, 43.5, 42.8, 36.6, 29.9, 28.8, 27.0, 26.1, 25.5,

22.7, 14.1, 11.2, 10.0; HRMS Calcd. for C₂₅H₃₇NO₄(M): 415.2723.

Found: 415.2720.

2.2.10. Representative procedure for preparation of 2-benzoyl estradiol

9a (Procedure B)

A freshly prepared solution of phenylmagnesium bromide

(magnesium turnings (0.2 g, 8.23 mmol) and bromobenzene (2 g,

12.7 mmol)) in dry THF (10 mL) was slowly added to the disilylated

aldehyde 8 (1.5 g, 2.84 mmol) in 10 mL of anhydrous THF solution

at 0 °C. After complete addition of Grignard solution, the reaction

mixture was allowed to stir at room temperature for another 1 h.

it was then poured in to 30 mL saturated ammonium chloride solu-

tion and washed with diisopropyl ether (2 ꢀ 50 mL). The combined

organics were washed with brine (2 ꢀ 30 mL), dried (sodium sul-

fate), ﬁltered, and concentrated via rotary evaporation to give

1.8 g of the alcohol as a pale yellow oil [m/z (relative intensity)

607 (MH⁺, 100%)]. The crude product was dissolved in MDC

(50 mL) and stirred with manganese dioxide (2.5 g, 28.7 mmol)

containing powdered molecular sieves 5 Å (2.5 g) for 36 h at room

temperature. The mixture was ﬁltered through a pad of celite, the

ﬁltrate was concentrated in vacuo, to afford 1.7 g of ketone (m/z

(relative intensity) 605 (MH⁺, 100%)). It was then dissolved in

anhydrous THF (25 mL) stirred with tetrabutylammonium ﬂuoride

trihydrate (9 g, 28.5 mmol) and powdered molecular sieves 5 Å

(9 g) for 40 h at room temperature. The mixture was ﬁltered

through a pad of celite, the ﬁltrate was poured in to 40 mL water

and extracted with ethyl acetate (2 ꢀ 50 mL). The combined organ-

ics were washed with brine (2 ꢀ 30 mL), dried with sodium sulfate,

ﬁltered, and concentrated via rotary evaporation to give dark yel-

low viscous oil. The crude product was puriﬁed by silica gel column

chromatography using (10:90 ethyl acetate/hexane) as an eluent to

yield 2-benzoyl estradiol 9a as a light yellow solid (0.67 g, 63%).

mp 120–122 °C; ¹H NMR (300 MHz, DMSO-d₆) d 10.63 (s, 1H),

7.69–7.51 (m, 5H), 7.28 (s, 1H), 6.7 (s, 1H), 4.51–4.50 (d,

J = 4.8 Hz, 1H), 3.52–3.50 (m, 1H), 2.84–2.82 (d, J = 8.0 Hz, 2H),

2.1–2.0 (m, 2H), 1.9–1.8 (m, 3H), 1.7–1.5 (m, 1H), 1.39–1.13 (m,

7H), 0.65 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) d 201.2, 160.9,

147.3, 138.2, 131.8, 131.4, 130.4, 129.1, 128.3, 117.6, 117.1, 81.7,

50.0, 43.6, 43.2, 38.6, 36.5, 30.5, 30.0, 26.8, 26.1, 23.1, 11.1; m/z

(relative intensity) 375 (MH⁺, 100%); Anal. Calcd. for (C₂₅H₂₈O₃):

C, 79.75; H, 7.50. Found: C, 79.98; H, 7.23%.

2.2.7. 2-(2⁰-Hydroxyethylamino)methyl estradiol (7f)

Condensation of 2-formyl estradiol 6 (1 g, 3.33 mmol) with eth-

anolamine (406 mg, 6.66 mmol) followed by sodium borohydride

(0.5 g, 13.2 mmol) reduction using the above-mentioned proce-

dure A and subsequent column chromatography puriﬁcation using

ethyl acetate as an eluent afforded compound 7f as a light brown

low melting solid (0.9 g, 77%). ¹H NMR (300 MHz, DMSO-d₆) d

9.95 (s, 1H), 6.95 (s, 1H), 6.37 (s, 1H), 4.03–4.02 (d, J = 7.1 Hz,

2H), 3.77 (s, 1H), 3.53–3.53 (m, 3H), 3.11 (s, 1H), 2.67 (s, 2H),

2.58–2.57 (t, J = 5.6 Hz, 2H), 2.25 (m, 1H), 1.98 (s, 1H), 1.88–1.75

(m, 3H), 1.7–1.5 (m, 1H), 1.42–1.15 (m, 8H), 0.65 (s, 3H); m/z (rel-

ative intensity) 346 (MH⁺, 100%); Anal. Calcd. for (C₂₁H₃₁NO₃): C,

73.01; H, 9.04; N, 4.05. Found: C, 73.28; H, 9.19; N, 4.25%.

2.2.8. 2-(1⁰-Hydroxybutyl-2⁰-amino) methyl estradiol (7g)

Condensation of 2-formyl estradiol 6 (1 g, 3.33 mmol) with 2-

amino butanol (0.59 g, 6.66 mmol) followed by sodium borohydride

(0.5 g, 13.2 mmol) reduction using the above-mentioned procedure

Aaffordedcompound7gas apaleyellowsolid(0.91 g, 73%). mp171–

172 °C; IR (KBr, cm^ꢁ1): 3432 (OH and NH); ¹H NMR (300 MHz,

DMSO-d₆) d 9.93 (s, 1H), 6.93 (s, 1H), 6.35 (s, 1H), 4.58 (bs, 1H),

3.77 (s, 2H), 3.51–3.33 (m, 3H), 2.67 (s, 2H), 2.4–2.3 (m, 1H), 2.0–

2.1 (m, 1H), 1.98–1.96 (m, 1H), 1.9–1.7 (m, 3H), 1.65–1.50 (m, 1H),

1.32–1.17 (m, 11H), 0.86–0.83 (t, J = 7.4 Hz, 3H), 0.65 (s, 3H); ¹³C

NMR (75 MHz, DMSO-d₆) d 155.0, 135.5, 129.9, 125.2, 121.5, 115.2,

80.1, 61.5, 59.1, 49.5, 48.3, 43.5, 42.8, 38.6, 36.6, 29.9, 28.8, 27.0,

26.2, 23.1, 22.8, 11.2, 10.0; m/z (relative intensity) 374 (MH⁺,

100%); Anal. Calcd. for (C₂₃H₃₅NO₃): C, 73.96; H, 9.44; N, 3.75. Found:

C, 73.74; H, 9.28; N, 3.99%.

2.2.11. 2-(4⁰-Methylbenzoyl)estradiol (9b)

Addition of freshly prepared 4-methylphenylmagnesium bro-

mide (magnesium turnings (0.2 g, 8.52 mmol) and 4-bromotoluene

(2.17 g, 12.7 mmol)) in dry THF (10 mL) to disilylated aldehyde 8

(1.5 g, 2.84 mmol) followed by manganese dioxide (2.5 g,

28.7 mmol) oxidation, tetrabutylammonium ﬂuoride trihydrate

(9 g, 28.5 mmol) mediated desilylation using the procedure B as

mentioned above and subsequent silica gel column chromatography

2.2.9. (3,17-Bis-tert-butyldimethylsilyl)-2-formyl estradiol (8)

To the solution of 2-formyl estradiol 6 (10 g, 33.3 mmol) in DMF

(150 mL) at 10 °C was added triethylamine (18.8 g, 187 mmol) and

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G. Panchapakesan et al. / Steroids 76 (2011) 1491–1504

puriﬁcation (12:88 ethyl acetate/hexane) afforded compound 9b as

light yellow solid (0.66 g, 60%). mp 108–110 °C; ¹H NMR (300 MHz,

DMSO-d₆) d 10.65 (s, 1H), 7.63–7.61 (d, J = 7.8 Hz, 2H), 7.37–7.25 (m,

3H), 6.7 (s, 1H), 4.55–4.54 (d, J = 4.4 Hz, 1H), 3.52 (m, 1H), 2.83 (s,

2H), 2.42 (s, 3H), 2.15–2.0 (m, 2H),1.83–1.8 (m, 3H), 1.7–1.5 (m,

1H), 1.36–1.15 (m, 6H), 0.86 (m, 1H), 0.68 (s, 3H); HRMS Calcd. for

2.2.15. 2-(3⁰,4⁰-Dimethoxybenzoyl)estradiol (9f)

Addition of freshly prepared 3,4-dimethoxyphenylmagnesium

bromide [magnesium turnings (0.2 g, 8.52 mmol) and 1-bromo-

3,4-dimethoxybenzne (2.75 g, 12.7 mmol] in dry THF (10 mL) to

disilylated aldehyde 8 (1.5 g, 2.84 mmol) followed by manganese

dioxide (2.5 g, 28.7 mmol) oxidation, tetrabutylammonium ﬂuo-

ride trihydrate (9 g, 28.5 mmol) mediated desilylation using the

procedure B as mentioned above and subsequent silica gel column

chromatography puriﬁcation (20:80 ethyl acetate/hexane) affor-

ded compound 9f as light yellow solid (0.65 g, 53%). mp 83–

85 °C; IR (KBr, cm^ꢁ1): 1632 (C@O); ¹H NMR (300 MHz, DMSO-d₆)

d 10.51 (s, 1H), 7.33–7.26 (m, 3H), 7.09 (s, 1H), 6.68 (s, 1H), 4.5–

4.49 (d, J = 4.7 Hz, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 3.5 (m, 1H), 2.81

(s, 2H), 2.11–2.08 (m, 2H), 1.82–1.79 (m, 3H), 1.7–1.5 (m, 1H),

1.39–1.11 (m, 6H), 0.85 (m, 1H), 0.68 (s, 3H); m/z (relative inten-

sity) 437 (MH⁺, 100%). Anal. Calcd. for (C₂₇H₃₂O₅): C, 74.29; H,

7.39. Found: C, 74.04; H, 7.64%.

C₂₆H₃₀O₃(M): 390.2195. Found: 390.2199.

2.2.12. 2-(4⁰-Fluorobenzoyl)estradiol (9c)

Addition of freshly prepared 4-ﬂuorophenylmagnesium bro-

mide [magnesium turnings (0.2 g, 8.52 mmol) and 1-bromo-4-

ﬂuorobenzene (2.22 g, 12.7 mmol] in dry THF (10 mL) to disilylated

aldehyde 8 (1.5 g, 2.84 mmol) followed by manganese dioxide

(2.5 g, 28.7 mmol) oxidation, tetrabutylammonium ﬂuoride trihy-

drate (9 g, 28.5 mmol) mediated desilylation using the procedure

B as mentioned above and subsequent silica gel column chroma-

tography puriﬁcation (12:88 ethyl acetate/hexane) gave compound

9c as light yellow solid (0.71 g, 64%). mp 150–152 °C; IR (KBr,

cm^ꢁ1): 3514 (OH), 1631 (CO), 1596 (C@C); ¹H NMR (300 MHz,

DMSO-d₆) d 10.44 (s, 1H), 7.81–7.77 (m, 2H), 7.40–7.35 (m, 2H),

7.28 (s, 1H), 6.71(s, 1H), 4.62 (s, 1H), 3.52 (m, 1H), 2.83 (s, 2H),

2.11 (m, 2H), 1.84–1.81 (m, 3H), 1.70–1.50 (m, 1H), 1.36–1.13

(m, 6H), 0.85 (m, 1H), 0.68 (s, 3H); m/z (relative intensity) 393

(MH⁺, 100%); Anal. Calcd. for (C₂₅H₂₇FO₃): C, 76.12; H, 6.90. Found:

C, 76.36; H, 6.70%.

2.2.16. 2-(3⁰,5⁰-Dimethoxybenzoyl)estradiol (9g)

Addition of freshly prepared 3,5-dimethoxyphenylmagnesium

bromide [magnesium turnings (0.2 g, 8.52 mmol) and 1-bromo-

3,5-dimethoxybenzne (2.75 g, 12.7 mmol] in dry THF (10 mL) to

disilylated aldehyde 8 (1.5 g, 2.84 mmol) followed by manganese

dioxide (2.5 g, 28.7 mmol) oxidation, tetrabutylammonium ﬂuo-

ride trihydrate (9 g, 28.5 mmol) mediated desilylation using the

procedure B as mentioned above and subsequent silica gel column

chromatography puriﬁcation (20:80 ethyl acetate/hexane) gave

compound 9g as bright yellow solid (0.68 g, 55%). mp 68–70 °C;

¹H NMR (300 MHz, DMSO-d₆) d 10.76 (s, 1H), 7.36 (s, 1H), 6.82–

6.8 (m, 3H), 6.74 (s, 1H), 4.56–4.54 (d, J = 6.4 Hz, 1H), 3.82 (s,

6H), 3.55 (m, 1H), 2.86 (s, 2H), 2.11–2.08 (m, 2H), 1.84–1.82 (m,

3H), 1.7–1.5 (m, 1H), 1.39–1.11 (m, 6H), 0.85 (m, 1H), 0.69 (s,

3H); ¹³C NMR (75 MHz, DMSO-d₆) d 198.4, 160.2, 157.6, 145.1,

139.6, 131.1, 128.6, 119.1, 116.7, 106.8, 104.0, 79.9, 55.4, 49.4,

42.9, 42.7, 38.2, 36.3, 29.8, 29.2, 26.4, 25.9, 25.7, 22.7, 11.1; m/z

(relative intensity) 437 (MH⁺, 100%); Anal. Calcd. for (C₂₇H₃₂O₅):

C, 74.29; H, 7.39. Found: C, 74.02; H, 7.60%.

2.2.13. 2-(4⁰-Methoxybenzoyl)estradiol (9d)

Addition of freshly prepared 4-methoxyphenylmagnesium bro-

mide [magnesium turnings (0.2 g, 8.52 mmol) and 4-bromoanisole

(2.37 g, 12.7 mmol] in dry THF (10 mL) to disilylated aldehyde 8

(1.5 g, 2.84 mmol) followed by manganese dioxide (2.5 g,

28.7 mmol) oxidation, tetrabutylammonium ﬂuoride trihydrate

(9 g, 28.5 mmol) mediated desilylation using the procedure B as

mentioned above and subsequent silica gel column chromatogra-

phy puriﬁcation (15:85 ethyl acetate/hexane) yielded compound

9d as light yellow solid (0.74 g, 65%). mp 90–92 °C; ¹H NMR

(300 MHz, DMSO-d₆) d 10.41 (s, 1H), 7.7–7.68 (d, J = 8.6 Hz, 2H),

7.25 (s, 1H), 7.07–7.05 (d, J = 8.6 Hz, 2H), 6.67 (s, 1H), 4.5–4.49

(d, J = 4.6 Hz, 1H), 3.85 (s, 3H), 3.5 (m, 1H), 2.81 (s, 2H), 2.11–

2.08 (m, 2H), 1.82–1.79 (m, 3H), 1.7–1.5 (m, 1H), 1.39–1.11 (m,

6H), 0.85 (m, 1H), 0.68 (s, 3H); m/z (relative intensity) 405 (MH⁺,

100%); Anal. Calcd. for (C₂₆H₃₀O₄): C, 76.82; H, 7.44. Found: C,

76.60; H, 7.59%.

2.2.17. 2-(3⁰,4⁰,5⁰-Trimethoxybenzoyl)estradiol (9h)

Addition of freshly prepared 3,4,5-trimethoxyphenylmagne-

sium bromide [magnesium turnings (0.20 g, 8.52 mmol) and 1-

bromo-3,4,5-trimethoxybenzene (3.10 g, 12.70 mmol] in dry THF

(10 mL) to disilylated aldehyde 8 (1.50 g, 2.84 mmol) followed by

manganese dioxide (2.50 g, 28.70 mmol) oxidation, tetrabutylam-

monium ﬂuoride trihydrate (9.0 g, 28.50 mmol) mediated desilyla-

tion using the procedure B as mentioned above and subsequent

silica gel column chromatography puriﬁcation (30:70 ethyl ace-

tate/hexane) yielded compound 9h as light yellow solid (0.67 g,

51%). mp 59–61 °C; IR (KBr, cm^ꢁ1): 3312 (OH), 1630 (C@O); ¹H

NMR (300 MHz, DMSO-d₆) d 10.7 (s, 1H), 7.38 (s, 1H), 7.01 (s,

2H), 6.7 (s, 1H), 4.52–4.51 (d, J = 4.6 Hz, 1H), 3.80–3.76 (m, 9H),

3.50 (m, 1H), 2.82 (s, 2H), 2.11 (m, 2H),1.82–1.79 (m, 3H), 1.6–

1.5 (m, 1H), 1.38–1.11 (m, 6H), 0.86 (m, 1H), 0.69 (s, 3H); HRMS

Calcd. for C₂₈H₃₄O₆(M): 466.2355. Found: 466.2352.

2.2.14. 2-(4⁰-Ethoxybenzoyl)estradiol (9e)

Addition of freshly prepared 4-ethoxyphenylmagnesium bro-

mide [magnesium turnings (0.2 g, 8.52 mmol) and 1-bromo-4-eth-

oxybenzene (2.62 g, 12.7 mmol] in dry THF (10 mL) to disilylated

aldehyde 8 (1.5 g, 2.84 mmol) followed by manganese dioxide

(2.5 g, 28.7 mmol) oxidation, tetrabutylammonium ﬂuoride trihy-

drate (9 g, 28.5 mmol) mediated desilylation using the procedure

B as mentioned above and subsequent silica gel column chroma-

tography puriﬁcation (15:85 ethyl acetate/hexane) furnished com-

pound 9e as light yellow solid (0.75 g, 67%). mp 82–84 °C; IR (KBr,

cm^ꢁ1): 1632 (CO), 1598 (C@C); ¹H NMR (300 MHz, DMSO-d₆) d

10.41 (s, 1H), 7.68–7.66 (d, J = 8.5 Hz, 2H), 7.25 (s, 1H), 7.05–7.03

(d, J = 8.5 Hz, 2H), 6.66 (s, 1H), 4.52–4.5 (d, J = 4.6 Hz, 1H), 4.13–

4.11 (d, J = 6.9 Hz, 2H), 3.5 (m, 1H), 2.81 (s, 2H), 2.1–2.08 (m,

2H),1.81–1.78 (m, 3H), 1.7–1.5 (m, 1H), 1.37–1.11 (m, 9H), 0.85

(s, 1H), 0.68 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) d 196.9, 162.0,

156.3, 143.5, 131.6, 130.9, 130.0, 127.8, 120.6, 116.5, 114.0, 79.9,

63.5, 49.5, 43.0, 42.7, 38.3, 36.3, 29.8, 29.2, 26.5, 25.8, 22.7, 14.4,

11.1; m/z (relative intensity) 419 (MH⁺, 100%); Anal. Calcd. for

(C₂₇H₃₂O₄): C, 77.11; H, 7.67. Found: C, 77.38; H, 7.50%.

2.2.18. Representative procedure for preparation of (2E)-3-(3,17b-dihy-

droxy-1,3,5(10)-estratrienyl)-1-(3⁰-methoxyphenyl)prop-2-en-1-one

10a (Procedure C)

To a stirred solution of 2-formyl estradiol 6 (0.75 g, 2.5 mmol),

and 3⁰-methoxy acetophenone (0.5 g, 3.33 mmol) in methanol

(30 mL) was added 6 mL of 50% potassium hydroxide solution,

the reaction mixture was then heated at 70 °C for 6 h. The solvent

was evaporated, and the residue was dissolved in ethyl acetate/

water (100 mL, 3:1), and acidiﬁed with 5 N HCl to pH 6.0. The or-

ganic layer was washed with brine (2 ꢀ 25 mL), dried with sodium

sulfate, ﬁltered, and concentrated via rotary evaporation to give

G. Panchapakesan et al. / Steroids 76 (2011) 1491–1504

1495

dark yellow viscous oil. The crude product was puriﬁed by silica gel

column chromatography using (10:90 ethyl acetate/hexane) as an

eluent to yield trans-isomer 10a as a light yellow solid (0.54 g,

50%). mp 189–190 °C; ¹H NMR (300 MHz, DMSO-d₆) d 9.95 (s,

1H), 8.04–8.00 (d, J = 15.7 Hz, 1H), 7.8–7.68 (m, 3H), 7.54 (s, 1H),

7.5–7.46 (t, J = 8.0 Hz, 1H), 7.23–7.21 (d, J = 8.1 Hz, 1H), 6.61 (s,

1H), 4.52 (s, 1H), 3.84 (s, 3H), 3.56–3.51 (m, 1H), 2.74 (s, 2H),

2.12–2.08 (t, J = 6.9 Hz, 1H), 1.89–1.86 (m, 2H), 1.80–1.77 (m,

1H), 1.60–1.58 (m, 1H), 1.38–1.23 (m, 8H), 0.68 (s, 3H); ¹³C NMR

(75 MHz, DMSO-d₆) d 194.4, 164.7, 160.4, 146.6, 145.6, 144.8,

136.8, 135.0, 130.7, 126.0, 124.8, 124.2, 123.7, 121.0, 118.1, 85.3,

65.0, 60.5, 54.7, 48.6, 48.0, 41.8, 35.1, 34.5, 31.9, 31.1, 28.0, 16.4;

HRMS Calcd. for C₂₈H₃₂O₄(M): 432.2301. Found: 432.2307.

140.3, 133.6, 131.4, 125.7, 119.8, 119.1, 115.8, 106.1, 80.0, 60.1,

56.1, 49.5, 43.4, 42.7, 38.5, 36.5, 29.9, 29.2, 26.6, 25.9, 22.7, 11.2;

m/z (relative intensity) 491 (MH⁺, 100%); Anal. Calcd. for

(C₃₀H₃₆O₆): C, 73.15; H, 7.37. Found: C, 73.01; H, 7.59%.

2.2.22. Preparation of 17-methylene estratriene (11)

The suspension of sodium hydride (7.5 g, (60%) 187 mmol) in

anhydrous DMSO (200 mL) was heated to 60 °C under inert atmo-

sphere for 1 h. To the resulting green coloured solution at RT was

added solution of methyltriphenylphosphonium iodide (75 g,

187 mmol) in DMSO (150 mL) and stirred for 30min. The resulting

red coloured solution was mixed with the solution of estrone (10 g,

37 mmol) in DMSO (150 mL) and stirred for another 20 h at RT. The

resulting mixture was poured in to water (1 L) and extracted with

diisopropyl ether (2 ꢀ 750 mL). The combined organics were

washed with brine (2 ꢀ 250 mL), dried with sodium sulfate, ﬁl-

tered, and concentrated via rotary evaporation to give dark yellow

viscous oil. The crude product was puriﬁed by silica gel column

chromatography using (4:96 ethyl acetate/hexane) as an eluent

to yield the title compound 11 as a dull white solid (9 g, 91%).

mp 135–136 °C ¹H NMR (300 MHz, DMSO-d₆) d 8.99 (s, 1H),

7.06–7.04 (d, J = 8.5 Hz, 1H), 6.65–6.49 (m, 1H), 6.44 (s, 1H),

4.65–4.63 (d, J = 8.7 Hz, 2H), 2.72–2.71 (m, 2H), 2.45 (m, 1H), 2.3

(m, 1H), 1.9–1.84 (m, 4H), 1.39–1.28 (m, 7H), 0.77 (s, 3H); m/z (rel-

ative intensity) 267 (MH⁺, 100%); Anal. Calcd. for (C₁₉H₂₄O): C,

85.03; H, 9.01. Found: C, 85.19; H, 9.26%.

2.2.19. (2E)-3-(3, 17b-Dihydroxy-1,3,5(10)-estratrienyl)-1-(2⁰-triﬂuo-

romethylphenyl)prop-2-en-1-one (10b)

Condensation of 2-formyl estradiol 6 (0.75 g, 2.5 mmol), and 2⁰-

triﬂuoromethylacetophenone (0.62 g, 3.33 mmol) using the

above-mentioned procedure C followed by silica gel column chro-

matographic puriﬁcation (25:75 ethyl acetate/hexane) afforded

10b as pale yellow solid (0.61 g, 52%). mp 221–222 °C; IR (KBr,

cm^ꢁ1): 3463 (OH), 1637 (C@O), 1615 (C@C); ¹H NMR (300 MHz,

DMSO-d₆) d 9.98 (s, 1H), 7.88–7.86 (d, J = 7.8 Hz, 1H), 7.81–7.78

(t, J = 7.4 Hz, 2H), 7.75–7.71 (t, J = 7.6 Hz, 1H), 7.61–7.59 (d,

J = 7.4 Hz, 1H), 7.53 (s, 1H), 7.19 (s, 1H), 6.58 (s, 1H), 4.5–4.49 (d,

J = 4.6 Hz, 1H), 3.54–3.51 (m, 1H), 2.72 (s, 2H), 2.08–2.03 (m, 1H),

1.85–1.75 (m, 3H), 1.58–1.56 (m, 1H), 1.38–1.22 (m, 8H), 0.68 (s,

3H); ¹³C NMR (75 MHz, DMSO-d₆) d 194.5, 155.1, 143.7, 142.1,

139.1, 132.3, 131.7, 130.0, 128.2, 126.5, 125.7, 124.5, 121.9,

118.2, 115.8, 80.0, 67.3, 49.5, 43.2, 42.7, 38.3, 36.4, 29.8, 29.2,

26.5, 25.8, 22.7, 11.1; m/z (relative intensity) 469 (MH^ꢁ, 100%);

Anal. Calcd. for (C₂₈H₂₉F₃O₃): C, 71.47; H, 6.21. Found: C, 71.20;

H, 6.01%.

2.2.23. 2-Formyl 17-methylene estratriene (12)

To the suspension of magnesium turnings (2.1 g, 86.4 mmol) in

THF (20 mL) was added solution of bromoethane (13.1 g,

120 mmol) in THF (10 mL) slowly at RT during 20 min and stirred

for another 30 min at RT. To the resulting clear solution was added

the solution of methylene compound 11 (4.0 g, 14.5 mmol) in THF

(40 mL) at RT and further stirred for 30 min. To the resulting sus-

pension was added hexamethylphosphoramide (7.4 g, 41.3 mmol)

followed by paraformaldehyde (7.0 g, 233 mmol) and heated to

60 °C for 20 h. The resulting yellow reaction mixture was poured

in to water (100 mL) and washed with ethyl acetate (2 ꢀ 100 mL)

at pH 4.0. The combined organics were washed with brine

(2 ꢀ 25 mL) and concentrated via rotary evaporation to give yel-

lowish oil. The crude product was suspended in methanol

(80 mL) at 10 °C and added sodium hydroxide solution (20% w/v,

8 mL) and stirred for 1 h. The resulting yellow clear solution was

poured into the water (160 mL) and washed with ethyl acetate

(2 ꢀ 150 mL) at pH 4.0. The combined organics were washed with

brine (2 ꢀ 50 mL), dried with sodium sulfate, ﬁltered, and concen-

trated via rotary evaporation to give light yellow viscous oil. The

crude product was puriﬁed by silica gel column chromatography

using hexane as an eluent to yield 2-formyl methylene compound

12 as a light yellow solid (3.65 g, 83%). mp 128–130 °C ¹H NMR

(300 MHz, DMSO-d₆) d 10.44 (s, 1H), 10.14 (s, 1H), 7.58 (s, 1H),

6.68 (s, 1H), 4.66–4.64 (d, J = 10.0 Hz, 2H), 3.5 (m, 1H), 2.84–2.82

(s, J = 7.4 Hz, 2H), 2.34–2.3 (d, 1H), 2.29–2.1 (s, 2H), 1.94–1.81

(m, 3H), 1.5–1.13 (m, 6H), 0.77 (s, 3H); m/z (relative intensity)

295 (MH⁺, 100%); Anal. Calcd. for (C₂₀H₂₄O₂): C, 81.04; H, 8.16.

Found: C, 81.18; H, 8.40%.

2.2.20. (2E)-3-(3, 17b-Dihydroxy-1,3,5(10)-estratrienyl)-1-(2⁰,4⁰,6⁰-

trimethoxyphenyl)prop-2-en-1-one (10c)

Condensation of 2-formyl estradiol 6 (0.75 g, 2.5 mmol), and

2⁰,4⁰,6⁰-trimethoxyphenylacetophenone (0.7 g, 3.33 mmol) using

the above-mentioned procedure C followed by silica gel column

chromatographic puriﬁcation (40:60 ethyl acetate/hexane) fur-

nished 10c as pale yellow solid (0.88 g, 72%). mp 184–186 °C; IR

(KBr, cm^ꢁ1): 3242 (OH), 1630 (C@O), 1603 (C@C); ¹H NMR

(300 MHz, DMSO-d₆) d 9.81 (s, 1H), 7.43–7.38 (d, J = 16.3 Hz, 1H),

7.38 (s, 1H), 6.91–6.87 (d, J = 16.2 Hz, 1H), 6.55 (s, 1H), 6.29 (s,

2H), 4.51–4.50 (d, J = 4.7 Hz, 1H), 4.05 (s, 3H), 3.82 (s, 3H), 3.69

(s, 3H), 2.71 (s, 2H), 2.30 (s, 1H), 2.08 (m, 1H),1.85–1.82 (m, 3H),

1.58–1.56 (m, 1H), 1.38–1.22 (m, 8H), 0.68 (s, 3H); ¹³C NMR

(75 MHz, DMSO-d₆) d 193.8, 161.6, 157.9, 154.5, 141.0, 140.3,

131.5, 127.4, 125.2, 118.6, 115.7, 111.5, 91.0, 80.0, 67.3, 55.7,

55.3, 49.5, 43.3, 42.7, 38.4, 36.5, 29.8, 29.1, 26.6, 25.9, 22.7, 11.2;

m/z (relative intensity) 491 (MH⁺, 100%); Anal. Calcd. for

(C₃₀H₃₆O₆): C, 73.15; H, 7.37. Found: C, 73.43; H, 7.15%.

2.2.21. (2E)-3-(3, 17b-Dihydroxy-1,3,5(10)-estratrienyl)-1-(3⁰,4⁰,5⁰-

trimethoxyphenyl)prop-2-en-1-one (10d)

Condensation of 2-formyl estradiol 6 (0.75 g, 2.5 mmol), and

3⁰,4⁰,5⁰-trimethoxyphenylacetophenone (0.7 g, 3.33 mmol) using

the above-mentioned procedure C followed by silica gel column

chromatographic puriﬁcation (40:60 ethyl acetate/hexane) gave

10d as pale yellow solid (763 mg, 62%). mp 166–168 °C; ¹H NMR

(300 MHz, DMSO-d₆) d 9.96 (s, 1H), 8.03–7.99 (d, J = 15.7 Hz, 1H),

7.79–7.75 (d, J = 15.7 Hz, 1H), 7.66 (s, 1H), 7.35 (s, 2H), 6.61(s,

1H), 4.51–4.49 (d, J = 4.6 Hz, 1H), 3.94 (s, 6H), 3.76 (s, 3H), 3.56

(m, 1H), 2.74 (s, 2H), 2.12–2.08 (m, 1H),1.88–1.85 (m, 2H), 1.80–

1.77 (m, 1H), 1.6–1.58 (m, 1H), 1.38–1.22 (m, 8H), 0.68 (s, 3H);

¹³C NMR (75 MHz, DMSO-d₆) d 188.5, 155.1, 152.8, 141.7, 141.2,

2.2.24. Representative procedure for preparation of 2-(N,N-dimethy-

lhydrazinimino)methyl-17-(1⁰-methylene)estra-1,3,5(10)-triene-3-ol

13a (Procedure D)

To the solution of 2-formyl-17-methylene estratriene 12 (0.2 g,

0.675 mmol) in DMF (5 mL) at 5 °C was added sodium sulphate

(400 mg) and stirred for 5 min. To this, N,N-dimethylhydrazine

(81 mg, 1.35 mmol) in DMF (2 mL) was then added and the mix-

ture was stirred for 1 h. It was then poured in to water (15 mL)

and washed with ethyl acetate (2 ꢀ 25 mL). The combined organics

1496

G. Panchapakesan et al. / Steroids 76 (2011) 1491–1504

were washed with brine (2 ꢀ 15 mL), dried with sodium sulfate,

ﬁltered, and concentrated via rotary evaporation to give light yel-

low semi solid. Triturated of the crude product with hexane

(10 mL), followed by ﬁltration afforded compound 13a as a white

solid (0.19 g, 82%). mp 179–181 °C; ¹H NMR (300 MHz, DMSO-d₆)

d 11.0 (s, 1H), 7.6 (s, 1H), 7.2 (s, 1H), 6.5 (s, 1H), 4.66–4.64 (d,

J = 10.4 Hz, 2H), 2.85 (s, 6H), 2.75 (s, 2H), 2.4–2.30 (m, 1H), 2.25–

2.0 (m, 2H), 1.95–1.7 (m, 3H), 1.39–1.32 (m, 6H), 1.2–1.1 (s, 1H),

0.72 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) d 160.7, 154.1, 138.0,

137.1, 130.6, 125.6, 117.3, 115.3, 100.8, 52.9, 43.8, 43.3, 42.5,

38.2, 35.2, 29.0, 28.9, 27.0, 26.2, 23.4, 18.2; m/z (relative intensity)

337 (MH⁺, 100%); Anal. Calcd. for (C₂₂H₃₀N₂O): C, 78.06; H, 8.93; N,

8.28. Found: C, 78.32; H, 8.80; N, 8.05%.

using the above-mentioned procedure A and subsequent silica

gel column chromatography puriﬁcation (methanol) afforded com-

pound 14a as yellow viscous liquid (0.3 g, 60%). ¹H NMR (300 MHz,

DMSO-d₆) d 12.94 (s, 1H), 6.94 (s, 1H), 6.37 (s, 1H), 4.65–4.63 (d,

J = 9.1 Hz, 2H), 3.75 (s, 2H), 2.7–2.69 (m, 2H), 2.55–2.50 (m, 1H),

2.33–2.29 (t, J = 6.3 Hz, 3H), 2.14–2.13 (m, 1H), 2.11 (s, 6H),

1.93–1.88 (m, 2H), 1.77–1.73 (m, 2H), 1.63 (s, 1H), 1.39–1.31 (m,

8H), 0.75 (s, 3H); m/z (relative intensity) 369 (MH⁺, 100%); Anal.

Calcd. for (C₂₄H₃₆N₂O): C, 78.21; H, 9.85; N, 7.60. Found: C,

78.01; H, 9.71; N, 7.86%.

2.2.29. 2-(2⁰-N,N-Dimethylpropylenediamino)methyl-17-(1⁰-methyle-

ne)estra-1,3,5(10)-triene-3-ol (14b)

2.2.25. 2-(2⁰-(N,N-Dimethylaminoethylimino)methyl-17-(1⁰-methylene)

estra-1,3,5(10)-triene-3-ol (13b)

Condensation of 2-formyl-17-methylene estratriene 12 (0.4 g,

1.35 mmol)

with

N,N-dimethylpropylenediamine

(0.28 g,

Condensation of 2-formyl-17-methylene estratriene 12 (0.2 g,

2.7 mmol) followed by sodium borohydride (0.2 g, 5.26 mmol)

reduction using the above-mentioned procedure A and subsequent

silica gel column chromatography puriﬁcation, methanol as eluent

yielded compound 14b as yellow viscous oil (0.22 g, 43%). ¹H NMR

(300 MHz, DMSO-d₆) d 7.01 (s, 1H), 6.39 (s, 1H), 4.65–4.63 (d,

J = 9.6 Hz, 2H), 3.75 (s, 2H), 2.73–2.70 (m, 2H), 2.51 (m, 1H),

2.33–2.29 (m, 3H), 2.14–2.13 (m, 1H), 2.11 (s, 6H), 1.93–1.88 (m,

2H), 1.77–1.73 (m, 2H), 1.63 (s, 1H), 1.39–1.31 (m, 8H), 1.04–1.02

(m, 2H), 0.98 (s, 1H), 0.75 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) d

161.0, 155.1, 135.6, 135.5, 125.4, 125.3, 115.1, 101.0, 61.9, 57.1,

53.0, 50.7, 45.1, 45.0, 43.8, 43.5, 35.3, 29.0, 28.8, 27.2, 26.3, 23.4,

18.3; m/z (relative intensity) 383 (MH⁺, 100%); Anal. Calcd. for

(C₂₅H₃₈N₂O): C, 78.48; H, 10.01; N, 7.32. Found: C, 78.69; H,

10.26; N, 7.15%.

0.675 mmol)

with

N,N-dimethylethylenediamine

(0.12 g,

1.35 mmol) using the above-mentioned procedure D followed by

trituration of the crude product with hexane afforded compound

13b as a light yellow solid (0.19 g; 79%). mp 101–103 °C; ¹H

NMR (300 MHz, CDCl₃) d 13.1 (s, 1H), 8.32 (s, 1H), 7.26 (s, 1H),

7.15 (s, 1H), 6.67 (s, 1H), 4.68–4.67 (d, J = 6.8 Hz, 2H), 3.7–3.67 (t,

J = 6.8 Hz, 2H), 2.88–2.85 (m, 2H), 2.63–2.6 (m, 2H), 2.53–2.51

(m, 1H), 2.36–2.35 (m, 1H), 2.31 (s, 6H), 2.29–2.20 (m, 1H), 1.98–

1.83 (m, 2H), 1.81–1.8 (m, 1H), 1.4–1.35 (m, 6H), 0.76 (s, 3H);

HRMS Calcd. for C₂₄H₃₄N₂O (M): 366.2671. Found: 366.2665.

2.2.26. 2-(Cyclopropylimino) methyl-17-(1⁰-methylene)estra-1,3,5(10)-

triene-3-ol (¹³C)

Condensation of 2-formyl-17-methylene estratriene 12 (0.2 g,

0.675 mmol) with cyclopropylamine (0.8 g, 1.35 mmol) using the

above-mentioned procedure D followed by trituration of the crude

product with hexane yielded compound ¹³C as a light yellow solid

(0.18 g, 83%). mp 124–126 °C; IR (KBr, cm^ꢁ1): 3448 (OH), 1624

(C@N); ¹H NMR (300 MHz, DMSO-d₆) d 12.3 (s, 1H), 8.6 (s, 1H),

7.3 (s, 1H), 6.5 (s, 1H), 4.67–4.64 (d, J = 10.4 Hz, 2H), 3.1 (s, 1H),

2.78 (s, 2H), 2.4–2.3 (m, 1H), 2.2–2.1 (m, 2H), 1.98–1.7 (m, 3H),

1.4–1.35 (m, 6H), 1.2 (m, 1H), 0.96–0.95 (d, J = 4.8 Hz, 2H), 0.82–

0.81 (s, 2H), 0.76 (s, 3H); m/z (relative intensity) 336 (MH⁺,

100%); Anal. Calcd. for (C₂₃H₂₉NO): C, 82.34; H, 8.71; N, 4.18.

Found: C, 82.20; H, 8.96; N, 4.02%.

2.2.30. 2-(Cyclopropylamino) methyl-17-(1⁰-methylene)estra-1,3,5(10)-

triene-3-ol (14c)

Condensation of 2-formyl-17-methylene estratriene 12 (0.4 g,

1.35 mmol) with cyclopropylamine (0.15 g, 2.7 mmol) followed

by sodium borohydride (0.2 g, 5.26 mmol) reduction using the

above-mentioned procedure A and subsequent silica gel column

chromatography puriﬁcation (10:90 ethyl acetate/hexane) fur-

nished compound 14c as light yellow solid (0.34 g, 76%). mp

114–116 °C; ¹H NMR (300 MHz, DMSO-d₆) d 6.98 (s, 1H), 6.38 (s,

1H), 4.65–4.63 (d, J = 9.2 Hz, 2H), 3.75 (s, 2H), 2.7 (s, 2H), 2.5–

2.47 (m, 1H), 2.34–2.32 (m, 1H), 2.24–2.2 (m, 1H), 2.09–2.05 (m,

2H), 1.91–1.75 (m, 3H), 1.4–1.37 (m, 7H), 1.20–1.10 (m, 1H), 0.75

(s, 3H), 0.38–0.36 (m, 2H), 0.28–0.27 (m, 2H); ¹³C NMR (75 MHz,

DMSO-d₆) d 160.9, 154.4, 135.3, 129.8, 125.3, 122.0, 115.0, 101.0,

52.9, 43.8, 43.5, 38.5, 35.3, 29.0, 28.8, 27.2, 26.3, 23.4, 18.2, 5.6;

HRMS Calcd. for C₂₃H₃₁NO (M): 337.2406. Found: 337.2402.

2.2.27. 2-(2⁰-Hydroxyethylimino)methyl-17-(1⁰-methylene)estra-1,3,5

(10)-triene-3-ol (13d)

Condensation of 2-formyl-17-methylene estratriene 12 (0.2 g,

0.675 mmol) with ethanolamine (0.82 g, 1.35 mmol) using the

above-mentioned procedure D followed by trituration of the crude

product with diisopropyl ether gave compound 13d as light yellow

solid (0.19 g, 85%). mp 151–153 °C; ¹H NMR (300 MHz, CDCl₃) d

12.93 (s, 1H), 8.36 (s, 1H), 7.17 (s, 1H), 6.69 (s, 1H), 4.68–4.67 (d,

J = 4.2 Hz, 2H), 3.92–3.89 (t, J = 5.1 Hz, 2H), 3.75–3.72 (t,

J = 5.1 Hz, 2H), 2.88–2.85 (m, 2H), 2.6–2.45 (m, 1H), 2.4–2.15 (m,

3H), 1.98–1.93 (m, 2H), 1.85–1.75 (m, 1H), 1.6–1.35 (m, 6H),

1.26–1.24 (m, 1H), 0.82 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) d

166.5, 160.9, 158.3, 141.2, 130.3, 128.2, 116.7, 115.7, 101.0, 61.2,

60.7, 52.9, 43.8, 43.1, 38.1, 35.1, 29.2, 29.0, 26.8, 26.1, 23.4, 18.2;

m/z (relative intensity) 340 (MH⁺, 100%); Anal. Calcd. for

(C₂₂H₂₉NO₂): C, 77.84; H, 8.61; N, 4.13. Found: C, 77.60; H, 8.38;

N, 4.41%.

2.2.31. 2-(2⁰-Hydroxyethylamino) methyl-17-(1⁰-methylene)estra-1,3,

5(10)-triene-3-ol (14d)

Condensation of 2-formyl-17-methylene estratriene 12 (0.4 g,

1.35 mmol) with ethanolamine (0.16 g, 2.7 mmol) followed by so-

dium borohydride (0.2 g, 5.26 mmol) reduction using the above-

mentioned procedure

A and subsequent silica gel column

chromatography puriﬁcation (10:90 methanol/ethyl acetate) gave

compound 14d as yellow solid (0.36 g, 79%). mp 144–146 °C; IR

(KBr, cm^ꢁ1): 3238 (OH & NH), 1623 (C@C); ¹H NMR (300 MHz,

DMSO-d₆) d 7.0 (s, 1H), 6.41 (s, 1H), 4.66–4.63 (d, J = 9.3 Hz,

2H), 3.81 (s, 2H), 3.50–3.48 (t, J = 5.6 Hz, 2H), 2.71–2.7 (m, 2H),

2.62–2.59 (t, J = 5.6 Hz, 2H), 2.45–2.4 (m, 1H), 2.39–2.32 (m,

1H), 2.25–2.05 (m, 2H), 1.91–1.9 (s, 3H), 1.85–1.75 (m, 2H),

1.38–1.17 (m, 7H), 0.77 (s, 3H); m/z (relative intensity) 340

(MH⁺, 100%); Anal. Calcd. for (C₂₂H₃₁NO₂): C, 77.38; H, 9.15; N,

4.10. Found: C, 77.62; H, 9.40; N, 4.38%.

2.2.28. 2-(2⁰-N,N-dimethylthylenediamino)methyl-17-(1⁰-

methylene)estra-1,3,5(10)-triene-3-ol (14a)

Condensation of 2-formyl-17-methylene estratriene 12 (0.4 g,

1.35 mmol) with N,N-dimethylethylenediamine (0.24 g, 2.7 mmol)

followed by sodium borohydride (0.2 g, 5.26 mmol) reduction

G. Panchapakesan et al. / Steroids 76 (2011) 1491–1504

1497

2.2.32. 2-(1⁰-Hydroxy-2⁰-methyl-propyl-2⁰-amino) methyl-17-(1⁰-meth-

ylene)estra-1,3,5(10)-triene-3-ol (14e)

2.25–2.23 (m, 1H), 1.9–1.75 (m, 3H), 1.40–1.32 (m, 6H), 1.27–1.1

(m, 2H), 0.77 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) d 160.9,

154.8, 152.7, 152.6, 136.2, 135.5, 135.4, 129.9, 125.5, 121.3,

115.1, 105.8, 105.2, 100.9, 59.9, 55.8, 55.6, 52.9, 51.9, 49.6, 43.8,

43.5, 38.4, 35.3, 29.0, 28.8, 27.2, 26.3, 23.4, 18.2; m/z (relative

intensity) 476 (MH⁺, 100%); Anal. Calcd. for (C₃₀H₃₉NO₄): C,

75.44; H, 8.23; N, 2.93. Found: C, 75.22; H, 8.05; N, 2.72%.

Condensation of 2-formyl-17-methylene estratriene 12 (0.4 g,

1.35 mmol) with 2-amino-2-methyl-1-propanol (0.24 g, 2.7 mmol)

followed by sodium borohydride (0.2 g, 5.26 mmol) reduction

using the above-mentioned procedure A and subsequent silica

gel column chromatography puriﬁcation (35:65 ethyl acetate/hex-

ane) afforded compound 14e as a white solid (0.33 g, 67%). mp

142–144 °C; ¹H NMR (300 MHz, DMSO-d₆) d 6.93 (s, 1H), 6.34 (s,

1H), 4.65–4.63 (d, J = 9.3 Hz, 2H), 3.74 (s, 2H), 3.24 (s, 2H), 2.73–

2.69 (m, 2H), 2.33–2.31 (m, 2H), 2.24–2.08 (m, 2H), 1.91–1.74

(m, 6H), 1.37–1.34 (m, 6H), 0.98 (s, 6H), 0.75 (s, 3H); ¹³C NMR

(75 MHz, DMSO-d₆) d 160.9, 155.4, 135.3, 129.6, 124.7, 121.9,

115.3, 100.9, 67.6, 53.8, 52.9, 43.8, 43.5, 38.5, 35.3, 29.0, 28.8,

27.2, 26.3, 23.4, 23.1, 23.0, 18.2; m/z (relative intensity) 368

(MH⁺, 100%); Anal. Calcd. for (C₂₄H₃₅NO₂): C, 78.00; H, 9.55; N,

3.79. Found: C, 78.24; H, 9.34; N, 3.99%.

2.2.36. 3-(tert-Butyldimethylsilyl)-2-formyl-17-methylene estratriene

(15)

To the solution of 2-formyl 17-methylene estratriene 12 (10.0 g,

32.9 mmol) in DMF (150 mL) at 10 °C was added triethylamine

(9.4 g, 93.5 mmol) and stirred for 10 min, a solution of tert-butyldi-

methylsilyl chloride (10 g, 66.3 mmol) in DMF (100 mL) was added

slowly. The resulting mixture was further stirred for another 3 h at

10 °C, the reaction mixture was poured into sodium bicarbonate

solution (500 mL, 5% w/v) and washed with hexane (2 ꢀ 500 mL).

The combined organics were washed with brine solution

(2 ꢀ 200 mL), dried with sodium sulfate, ﬁltered, and concentrated

via rotary evaporation to give light yellow viscous oil. The crude

product was puriﬁed by silica gel column chromatography using

hexane as an eluent to yield 15 as white solid (12.1 g, 88%). mp

131–133 °C; IR (KBr, cm^ꢁ1): 1677 (C@O), 1609 (C@C); ¹H NMR

(300 MHz, DMSO-d₆) d 10.44 (s, 1H), 7.58 (s, 1H), 6.68 (s, 1H),

4.66–4.64 (d, J = 8.7 Hz, 2H), 3.5 (m, 1H), 2.84–2.82 (m, 2H),

2.34–2.3 (m, 1H), 2.29–2.1 (s, 2H), 1.94–1.81 (m, 3H), 1.5–1.13

(m, 6H), 1.01 (s, 9H), 0.75 (s, 3H),0.25 (s, 6H); m/z (relative inten-

sity) 411 (MH⁺, 100%); Anal. Calcd. for (C₂₆H₃₈O₂Si): C, 76.04; H,

9.33. Found: C, 76.32; H, 9.13%.

2.2.33. 2-(1⁰-Hydroxy-butyl-2⁰-amino) methyl-17-(1⁰-methylene)estra-

1,3,5(10)-triene-3-ol (14f)

Condensation of 2-formyl-17-methylene estratriene 12 (0.4 g,

1.35 mmol) with 2-amino-1-butanol (0.24 mg, 2.7 mmol) followed

by sodium borohydride (0.2 g, 5.26 mmol) reduction using the

above-mentioned procedure A and subsequent silica gel column

chromatography puriﬁcation (50:50 ethyl acetate/hexane) affor-

ded compound 14f as a white solid (0.35 mg, 71%). mp 83–85 °C;

¹H NMR (300 MHz, DMSO-d₆) d 6.96 (s, 1H), 6.37 (s, 1H), 4.65–

4.63 (d, J = 9.3 Hz, 2H), 3.79 (s, 2H), 3.45–3.41 (m, 2H), 3.35–3.31

(m, 1H), 2.73–2.70 (m, 2H), 2.47–2.41 (m 1H), 2.39–2.34 (m, 1H),

2.31–2.25 (m, 1H), 2.0–2.1 (m, 1H), 1.91–1.75 (m, 3H), 1.47–1.34

(m, 9H), 1.26–1.15 (m, 2H), 0.83–0.8 (t, J = 5.9 Hz, 3H), 0.77 (s,

3H); ¹³C NMR (75 MHz, DMSO-d₆) d 161.0, 155.1, 135.6, 129.7,

125.2, 121.5, 115.1, 101.0, 61.4, 59.1, 52.9, 48.1, 43.8, 43.5, 38.5,

35.3, 29.0, 28.8, 27.2, 26.3, 23.4, 18.3, 10.0; HRMS Calcd. for

2.2.37. 2-Benzoyl-17-(1⁰-methylene)estra-1,3,5(10)-triene-3-ol (16a)

Addition of freshly prepared phenylmagnesium bromide [mag-

nesium turnings (0.2 g, 8.52 mmol) and bromobenzne (4 g,

25.5 mmol] in dry THF (10 mL) to silylated aldehyde 15 (1.5 g,

3.66 mmol) followed by manganese dioxide (3.18 g, 36.6 mmol)

oxidation, tetrabutylammonium ﬂuoride trihydrate (7.5 g,

36.6 mmol) mediated desilylation using the procedure B as men-

tioned above and subsequent silica gel column chromatography

puriﬁcation (4:96 ethyl acetate/hexane) gave compound 16a as

light yellow solid (0.79 g, 58%). mp 145–146 °C; ¹H NMR

(300 MHz, DMSO-d₆) d 10.60 (s, 1H), 7.7–7.51 (m, 5H), 7.3 (s,

1H), 6.7 (s, 1H), 4.63 (s, 2H), 2.85 (s, 2H), 2.18–2.12 (m, 3H),

1.88–1.85 (m, 3H), 1.7–1.5 (m, 1H), 1.38–1.11 (m, 6H), 0.77 (s,

3H); m/z (relative intensity) 371 (MH⁺, 100%); Anal. Calcd. for

(C₂₆H₂₈O₂): C, 83.83; H, 7.58. Found: C, 83.65; H, 7.40%.

C₂₄H₃₅NO₂(M): 369.2668. Found: 369.2660.

2.2.34. 2-(2⁰,4⁰-Dimethoxybenzylamino) methyl-17-(1⁰-methylene)

estra-1,3,5(10)-triene-3-ol (14g)

Condensation of 2-formyl-17-methylene estratriene 12 (0.4 g,

1.35 mmol) with 2,4-dimethoxybenzylamine (0.45 mg, 2.7 mmol)

followed by sodium borohydride (0.2 g, 5.26 mmol) reduction

using the above-mentioned procedure A and subsequent silica

gel column chromatography puriﬁcation (20:80 ethyl acetate/hex-

ane) furnished compound 14g as white solid (0.33 g, 55%). mp 78–

80 °C; IR (KBr, cm^ꢁ1): 3200 (NH & OH), 1612 (C@C); ¹H NMR

(300 MHz, DMSO-d₆) d 7.13–7.11 (d, J = 8.2 Hz, 1H), 6.94 (s, 1H),

6.54–6.53 (d, J = 5.2 Hz, 1H), 6.49–6.47 (m, 1H), 6.37 (s, 1H),

4.65–4.63 (d, J = 9.3 Hz, 2H), 3.77 (s, 2H), 3.75 (s, 6H), 3.74 (s,

1H), 3.57 (s, 1H), 2.71–2.70 (m, 2H), 2.39–2.34 (m, 1H), 2.31–

2.25 (m, 1H), 2.0–2.1 (m, 1H), 1.91–1.75 (m, 4H), 1.39–1.31 (m,

6H), 1.2–1.1 (m, 2H), 0.77 (s, 3H); m/z (relative intensity) 446

(MH⁺, 100%); Anal. Calcd. for (C₂₉H₃₇NO₃): C, 77.82; H, 8.33; N,

3.13. Found: C, 77.65; H, 8.20; N, 3.33%.

2.2.38. 2-(4⁰-Methoxybenzoyl-17-(1⁰-methylene)estra-1,3,5(10)-triene-

3-ol (16b)

Addition of freshly prepared 4-methoxyphenylmagnesium bro-

mide [magnesium turnings (200 mg, 8.52 mmol) and 1-bromo-4-

methoxybenzene (4.74 g, 25.5 mmol] in dry THF (10 mL) to

silylated aldehyde 15 (1.5 g, 3.66 mmol) followed by manganese

dioxide (3.18 g, 36.6 mmol) oxidation, tetrabutylammonium ﬂuo-

ride trihydrate (7.5 g, 36.6 mmol) mediated desilylation using the

procedure B as mentioned above and subsequent silica gel column

chromatography puriﬁcation (5:95 ethyl acetate/hexane) afforded

compound 16b as light yellow solid (0.84 g, 57%). mp 112–

114 °C; ¹H NMR (300 MHz, DMSO-d₆) d 10.40 (s, 1H), 7.71–7.68

(d, J = 8.6 Hz, 2H), 7.27 (s, 1H), 7.07–7.05 (d, J = 8.6 Hz, 2H), 6.68

(s, 1H), 4.63 (s, 2H), 3.85 (s, 3H), 2.83 (s, 2H), 2.27–2.17 (m, 3H),

1.89–1.74 (m, 3H), 1.44–1.33 (m, 7H), 0.8 (s, 3H); ¹³C NMR

(75 MHz, DMSO-d₆) d 196.8, 162.7, 160.7, 156.3, 143.4, 131.6,

130.7, 130.2, 127.8, 120.7, 116.5, 113.6, 101.0, 55.4, 52.8, 43.7,

43.0, 38.0, 35.0, 29.2, 28.9, 26.7, 25.9, 23.4, 18.1; m/z (relative

intensity) 403 (MH⁺, 100%); Anal. Calcd. for (C₂₇H₃₀O₃): C, 80.56;

H, 7.51. Found: C, 80.40; H, 7.74%.

2.2.35. 2-(3⁰,4⁰,5⁰-Trimethoxybenzylamino)methyl-17-(1⁰-methylene)

estra-1,3,5(10)-triene-3-ol (14h)

Condensation of 2-formyl-17-methylene estratriene 12 (0.4 g,

1.35 mmol) with 3,4,5-trimethoxybenzylamine (0.53 g, 2.7 mmol)

followed by sodium borohydride (0.2 g, 5.26 mmol) reduction

using the above-mentioned procedure A and subsequent silica

gel column chromatography puriﬁcation (20:80 ethyl acetate/hex-

ane) afforded compound 14h as white solid (0.33 g, 52%). mp 116–

118 °C; IR (KBr, cm^ꢁ1): 3277 (NH), 1593 (C@C); ¹H NMR (300 MHz,

DMSO-d₆) d 6.98 (s, 1H), 6.66–6.63 (m, 2H), 6.41(s, 1H), 4.65–4.63

(d, J = 9.3 Hz, 2H), 3.93–3.78 (m, 9H), 3.72–3.70 (m, 3H), 3.68–3.57

(m, 2H), 2.71–2.70 (m, 2H), 2.4–2.35 (m, 1H), 2.31–2.27 (m, 1H),

1498

G. Panchapakesan et al. / Steroids 76 (2011) 1491–1504

2.2.39. 2-(4⁰-Ethoxybenzoyl-17-(1⁰-methylene)estra-1,3,5(10)-triene-

3-ol (16c)

2.2.42. (2E)-3-(3-Hydroxy-17(1⁰-methylene)-1,3,5(10)-estratrienyl)-

1-(3⁰-methoxyphenyl)prop-2-en-1-one (17a)

Addition of freshly prepared 4-ethoxyphenylmagnesium bro-

mide [magnesium turnings (0.2 g, 8.52 mmol) and 1-bromo-4-eth-

oxybenzene (5.1 g, 25.5 mmol] in dry THF (10 mL) to silylated

aldehyde 15 (1.5 g, 3.66 mmol) followed by manganese dioxide

(3.18 g, 36.6 mmol) oxidation, tetrabutylammonium ﬂuoride trihy-

drate (7.5 g, 36.6 mmol) mediated desilylation using the procedure

B as mentioned above and subsequent silica gel column chroma-

tography puriﬁcation (5:95 ethyl acetate/hexane) yielded com-

pound 16c as light yellow solid (0.93 g, 61%). mp 140–142 °C; IR

(KBr, cm^ꢁ1): 1633 (C@O), 1596 (C@C); ¹H NMR (300 MHz, DMSO-

d₆) d 10.38 (s, 1H), 7.69–7.67 (d, J = 8.6 Hz, 2H), 7.26 (s, 1H),

7.05–7.02 (d, J = 8.7 Hz, 2H), 6.68 (s, 1H), 4.63 (s, 2H), 4.15–4.1

(q, J = 6.9 Hz, 2H), 2.83 (s, 2H), 2.50 (m 1H), 2.25–2.17 (m, 3H),

1.89–1.76 (m, 3H), 1.44–1.33 (m, 9H), 0.8 (s, 3H); ¹³C NMR

(75 MHz, DMSO-d₆) d 197.4, 161.9, 160.6, 157.4, 144.1, 131.5,

130.7, 129.9, 128.3, 119.4, 116.6, 113.8, 100.9, 63.4, 52.8, 43.7,

43.1, 38.0, 35.0, 29.3, 28.9, 26.7, 25.9, 23.4, 18.1, 14.4; HRMS Calcd.

for C₂₈H₃₂O₃(M): 416.2351. Found: 416.2357.

Condensation of 2-formyl-17-methylene estratriene 12

(750 mg, 2.53 mmol) and 3⁰-methoxyacetophenone (500 mg,

3.33 mmol) using the above-mentioned procedure C followed by

silica gel column chromatographic puriﬁcation (10:90 ethyl ace-

tate/hexane) afforded 17a as light yellow solid (542 mg, 50%). mp

220–219 °C; IR (KBr, cm^ꢁ1): 3272 (OH), 1636 (C@O); ¹H NMR

(300 MHz, DMSO-d₆) d 9.98 (s, 1H), 8.04 (d, J = 15.7 Hz, 1H), 7.8–

7.71 (d, J = 15.7 Hz, 1H), 7.73–7.70 (d, J = 9.8 Hz, 2H), 7.55 (s, 1H),

7.5–7.46 (m, 1H), 7.24–7.21 (m, 1H), 6.62 (s, 1H), 4.68–4.65 (d,

J = 12.2 Hz, 2H), 3.84 (s, 3H), 3.56–3.51 (m, 1H), 2.77 (s, 2H),

2.65–2.55 (m, 1H), 2.3–2.15 (m, 2 H), 1.95–1.7 (m, 3H), 1.43–1.1

(m, 6H), 0.8 (s, 3H); m/z (relative intensity) 427 (MH^ꢁ, 100%); Anal.

Calcd. for (C₂₉H₃₂O₃): C, 81.27; H, 7.53. Found: C, 81.06; H, 7.37%.

2.2.43. (2E)-3-(3-Hydroxy-17(1⁰-methylene)-1,3,5(10)-estratrienyl)-

1-(4⁰-ethoxyphenyl)prop-2-en-1-one (17b)

Condensation of 2-formyl-17-methylene estratriene 12 (0.75 g,

2.53 mmol) and 4⁰-ethoxyacetophenone (0.55 g, 3.33 mmol) using

the above-mentioned procedure C followed by silica gel column

chromatographic puriﬁcation (10:90 ethyl acetate/hexane) and

subsequent trituration with diisopropyl ether afforded 17b as pale

yellow solid (0.62 g, 55%). mp 221–222 °C; ¹H NMR (300 MHz,

DMSO-d₆) d 9.91 (s, 1H), 8.10–8.09 (d, J = 8.6 Hz, 2H), 7.97 (m,

1H), 7.84 (m, 1H), 7.7 (s, 1H), 7.07–7.05 (d, J = 8.6 Hz, 2H), 6.62

(s, 1H), 4.68–4.65 (d, J = 13.0 Hz, 2H), 4.15–4.13 (m, 2H), 2.77 (s,

2H), 2.7–2.55 (m, 2H), 2.35–2.15 (m, 2H), 1.95–1.7 (m, 3H), 1.41–

1.1 (m, 9H), 0.8 (s, 3H); m/z (relative intensity) 441 (MH⁺, 100%);

Anal. Calcd. for (C₃₀H₃₄O₃): C, 81.41; H, 7.74. Found: C, 81.23; H,

7.93%.

2.2.40. 2-(3⁰,4⁰-Dimethoxybenzoyl-17-(1⁰-methylene)estra-1,3,5(10)-

triene-3-ol (16d)

Addition of freshly prepared 3,4-dimethoxyphenylmagnesium

bromide [magnesium turnings (0.2 g, 8.52 mmol) and 4-bromo-

1,2-dimethoxybenzene (5.5 g, 25.5 mmol] in dry THF (10 mL) to

silylated aldehyde 15 (1.5 g, 3.66 mmol) followed by manganese

dioxide (3.18 g, 36.6 mmol) oxidation, tetrabutylammonium ﬂuo-

ride trihydrate (7.5 g, 36.6 mmol) mediated desilylation using the

procedure B as mentioned above and subsequent silica gel column

chromatography puriﬁcation (20:80 ethyl acetate/hexane) affor-

ded compound 16d as light yellow solid (0.99 g, 63%). mp 137–

138 °C; IR (KBr, cm^ꢁ1): 3158 (OH), 1654 (C@O), 1630 (C@C); ¹H

NMR (300 MHz, DMSO-d₆) d 10.45 (s, 1H), 7.34–7.27 (m, 3H),

7.08–7.06 (d, J = 8.3 Hz, 1H), 6.69 (s, 1H), 4.63 (s, 2H), 3.85 (s,

3H), 3.80 (s, 3H), 2.84 (s, 2H), 2.5 (m, 1H), 2.21–2.17 (m, 3H),

1.89–1.86 (m, 3H), 1.41–1.33 (m, 6H), 0.78 (s, 3H); ¹³C NMR

(75 MHz, DMSO-d₆) d 196.8, 160.7, 156.5, 152.6, 148.4, 143.5,

130.7, 130.1, 127.9, 124.4, 120.4, 116.5, 111.6, 110.5, 101.0, 55.6,

55.4, 52.8, 43.7, 43.0, 38.0, 35.0, 29.2, 28.9, 26.7, 26.0, 23.4, 18.1;

m/z (relative intensity) 431 (MH⁺, 100%); Anal. Calcd. for

(C₂₈H₃₂O₄): C, 77.75; H, 7.46. Found: C, 77.51; H, 7.30%.

2.2.44. (2E)-3-(3-Hydroxy-17(1⁰-methylene)-1,3,5(10)-estratrienyl)-

1-(2⁰-triﬂuoromethylphenyl) prop-2-en-1-one (17c)

Condensation of 2-formyl-17-methylene estratriene 12

(0.75 g, 2.53 mmol) and 2⁰-triﬂuoromethylacetophenone (0.62 g,

3.33 mmol) using the above-mentioned procedure C followed by sil-

ica gel column chromatographic puriﬁcation (6:94 ethyl acetate/

hexane) and subsequent trituration with diisopropyl ether afforded

17c as light yellow solid (0.79 g, 67%). mp 208–210 °C; IR (KBr,

cm^ꢁ1): 3321 (OH), 1655 (C@O), 1612 (C@C); ¹H NMR (300 MHz,

DMSO-d₆) d 10.00 (s, 1H), 7.88–7.86 (d, J = 7.7 Hz, 1H), 7.82–7.75

(m, 2H), 7.73–7.71 (m, 1H), 7.61–7.59 (d, J = 7.7 Hz, 1H), 7.54–7.48

(m, 2H), 7.24–7.20 (m, 1H), 6.59 (s, 1H), 4.66–4.63 (d, J = 10.5 Hz,

2H), 2.75 (s, 2H), 2.47 (s, 1H), 2.27–2.22 (m, 1H), 2.18–2.1 (m,

1H),1.91–1.72 (m, 3H), 1.42–1.12 (m, 6H), 0.77 (s, 3H); ¹³C NMR

(75 MHz, DMSO-d₆) d 194.5, 160.8, 155.1, 143.7, 142.0, 132.3,

131.5, 130.0, 128.2, 126.6, 126.5, 125.7, 124.6, 118.2, 115.9, 101.0,

52.9, 43.7, 43.2, 38.1, 35.1, 29.2, 29.0, 26.8, 25.9, 23.4, 18.2; HRMS

Calcd. for C₂₉H₂₉F₃O₂(M): 466.2120. Found: 466.2129.

2.2.41. 2-(3⁰,4⁰,5-Trimethoxybenzoyl-17-(1⁰-methylene)estra-1,3,5(10)-

triene-3-ol (16e)

Addition of freshly prepared 3,4,5-trimethoxyphenylmagne-

sium bromide [magnesium turnings (0.2 g, 8.52 mmol) and 5-bro-

mo-1,2,3-trimethoxybenzene (6.3 g, 25.5 mmol] in dry THF

(10 mL) to silylated aldehyde 15 (1.5 g, 3.66 mmol) followed by

manganese dioxide (3.18 g, 36.6 mmol) oxidation, tetrabutylam-

monium ﬂuoride trihydrate (7.5 g, 36.6 mmol) mediated desilyla-

tion using the procedure B as mentioned above and subsequent

silica gel column chromatography puriﬁcation (20:80 ethyl ace-

tate/hexane) furnished compound 16e as light yellow solid

(0.81 g, 48%). mp 65–68 °C; IR (KBr, cm^ꢁ1): 1630 (C@O), 1569

(C@C); ¹H NMR (300 MHz, DMSO-d₆) d 10.72 (s, 1H), 7.4 (s, 1H),

7.02 (s, 2H), 6.72 (s, 1H), 4.63 (s, 2H), 3.8 (s, 6H), 3.77 (s, 3H),

2.87–2.85 (d, J = 7.4 Hz, 2H), 2.5 (m, 1H), 2.23–2.18 (m, 3H),

1.89–1.87 (m, 3H), 1.43–1.33 (m, 6H), 0.77 (s, 3H); ¹³C NMR

(75 MHz, DMSO-d₆) d 197.3, 160.7, 157.2, 152.5, 144.5, 141.2,

132.7, 130.9, 128.5, 119.4, 116.7, 107.0, 101.0, 60.1, 56.0, 52.8,

43.7, 43.0, 38.0, 35.0, 29.2, 29.0, 26.6, 26.1, 23.4, 18.1; m/z (relative

intensity) 461 (MH^ꢁ, 100%); Anal. Calcd. for (C₂₉H₃₄O₅): C, 75.30;

H, 7.41. Found: C, 75.04; H, 7.70%.

2.2.45. (2E)-3-(3-Hydroxy-17-(1⁰-methylene)-1,3,5(10)-estratrienyl)-

1-(2⁰,4⁰,6⁰-trimethoxyphenyl) prop-2-en-1-one (17d)

Condensation of 2-formyl-17-methylene estratriene 12

(0.75 g, 2.53 mmol) and 2⁰,4⁰,6⁰-trimethoxyacetophenone (0.7 g,

3.33 mmol) using the above-mentioned procedure C followed by

silica gel column chromatographic puriﬁcation (14:86 ethyl ace-

tate/hexane) and subsequent trituration with diisopropyl ether

afforded 17d as a pale yellow solid (0.56 g, 45%). mp 201–203 °C;

IR (KBr, cm^ꢁ1): 3366 (OH), 1605 (C@O); ¹H NMR (300 MHz,

DMSO-d₆) d 9.98 (s, 1H), 7.41 (s, 2H), 6.91–6.87 (m, 1H), 6.66 (s,

1H), 6.30 (s, 2H), 4.66–4.65 (d, J = 7.5 Hz, 2H), 3.85 (s, 6H), 3.70

(s, 3H), 2.75 (s, 2H), 2.47–2.42 (m, 1H), 2.25- 2.11 (m, 2H), 1.92–

1.76 (m, 3H), 1.58–1.56 (m, 1H), 1.36–1.23 (m, 6H), 0.77 (s, 3H);

m/z (relative intensity) 487 (MH⁺, 100%); Anal. Calcd. for

(C₃₁H₃₆O₅): C, 76.20; H, 7.43. Found: C, 76.44; H, 7.20%.

G. Panchapakesan et al. / Steroids 76 (2011) 1491–1504

1499

2.2.46. (2E)-3-(3-Hydroxy-17(1-methylene)-1,3,5(10)-estratrienyl)-

1-(3⁰,4⁰,5⁰-trimethoxyphenyl) prop-2-en-1-one (17e)

(50 mL) was heated at reﬂux for 10 h. Removal of solvent followed

by silica gel column chromatography puriﬁcation (5:95 ethyl ace-

tate/hexane) yielded 18c as a colorless solid (0.77 g, 83%). mp

227–229 °C; ¹H NMR (300 MHz, DMSO-d₆) d 9.94 (s, 1H), 7.85–

7.81 (d, J = 16.1 Hz, 1H), 7.47 (s, 1H), 6.62–6.58 (m, 1H), 6.47 (s,

1H), 4.69–4.66 (d, J = 12.0 Hz, 2H), 4.19–4.15 (m, 2H), 2.77–2.74

(m, 2H), 2.35–1.76 (m, 7H), 1.48–1.17 (m, 9H), 0.78 (s, 3H); ¹³C

NMR (75 MHz, DMSO-d₆) d 166.9, 160.9, 154.6, 140.7, 140.6,

131.2, 125.6, 118.3, 115.9, 115.7, 101.0, 59.6, 52.9, 43.8, 43.3,

38.2, 35.2, 29.0, 26.9, 25.9, 23.4, 18.2, 14.2; m/z (relative intensity)

365 (MH⁺, 100%); Anal. Calcd. for (C₂₄H₃₀O₃): C, 78.65; H, 8.25.

Found: C, 78.68; H, 8.02%.

Condensation of 2-formyl-17-methylene estratriene 12 (0.75 g,

2.53 mmol) and 3⁰,4⁰, 5⁰-trimethoxyacetophenone (0.7 mg,

3.33 mmol) using the above-mentioned procedure C followed by

silica gel column chromatographic puriﬁcation (40:60 ethyl ace-

tate/hexane) and subsequent trituration with diisopropyl ether

yielded 17e as a pale yellow solid (0.76 g, 52%). mp 204–206 °C;

¹H NMR (300 MHz, DMSO-d₆)

d 9.96 (s, 1H), 8.04–8.00 (d,

J = 15.6 Hz, 1H), 7.81–7.77 (d, J = 15.7 Hz, 1H), 7.69 (s, 1H), 7.36

(s, 2H), 6.62 (s, 1H), 4.68–4.64 (d, J = 14.7 Hz, 2H), 3.89 (s, 6H),

3.76 (s, 3H), 3.17–3.19 (d, J = 5.2 Hz, 1H), 2.46 (m, 1H), 2.77 (s,

2H), 2.26–2.15 (m, 2H), 1.95–177 (m, 3H), 1.46–1.21 (m, 6H),

0.77 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) d 188.4, 160.9, 155.1,

152.8, 141.7, 141.2, 140.2, 133.6, 131.3, 125.6, 119.8, 119.1,

115.8, 106.0, 101.0, 60.1, 56.1, 52.9, 43.8, 43.4, 35.2, 29.2, 29.0,

26.9, 26.0, 23.4, 18.3; m/z (relative intensity) 487 (MH⁺, 100%);

Anal. Calcd. for (C₃₁H₃₆O₅): C, 76.20; H, 7.43. Found: C, 76.01; H,

7.59%.

2.2.50. 2-(E)-3⁰-Hydroxy-1⁰-propenyl-17-(1⁰-methylene)estra-1,3,

5(10)-triene-3-ol (18d)

A 1 M solution of DIBAL in toluene (10 mL, 10 mmol) was slowly

added to a solution of ester 18c (0.75 g, 2.05 mmol) in dry THF

(10 mL) at 10 °C under nitrogen. After the addition was completed,

the reaction mixture was stirred at the same temperature for 1 h. It

was then poured into the mixture of ethyl acetate/water (100 mL,

3:1). The organic layer was washed with brine (2 ꢀ 25 mL), dried

with sodium sulfate, ﬁltered, and concentrated via rotary evapora-

tion to give yellow viscous oil. The crude product was puriﬁed by

silica gel column chromatography (30:70 ethyl acetate/hexane)

to yield compound 18d as yellow solid (0.52 g, 78%). mp 171–

173 °C; IR (KBr, cm^ꢁ1): 3386 (OH), 1616 (C@C); ¹H NMR

(300 MHz, DMSO-d₆) d 9.27 (s, 1H), 7.29 (s, 1H), 6.76–6.72 (d,

J = 16.0 Hz, 1H), 6.55 (s, 1H), 6.34–6.28 (m, 1H), 4.80–4.77 (m,

1H), 4.71–4.69 (d, J = 10.3 Hz, 2H), 4.13–4.07 (m, 2H), 2.79–2.76

(m, 2H), 2.35–1.76 (m, 7H), 1.48–1.17 (m, 6H), 0.83 (s, 3H); ¹³C

NMR (75 MHz, DMSO-d₆) d 160.9, 152.3, 136.1, 130.5, 128.7,

124.5, 123.1, 121.0, 115.3, 101.0, 62.2, 52.9, 43.8, 43.4, 38.4, 35.2,

29.0, 28.8, 27.2, 26.2, 23.4, 18.2; HRMS Calcd. for C₂₂H₂₈O₂(M):

324.2089. Found: 324.2086.

2.2.47. Representative procedure for preparation 2-ethenyl-17-(1⁰-

methylene)estra-1,3,5(10)-triene-3-ol 18a (Procedure E)

To a suspension of methyltriphenylphosphonium iodide (3.41 g,

8.44 mmol) in anhydrous THF (25 mL) at 0 °C under nitrogen was

added 1 M solution of Li[N(SiMe₃)₂] (11 mL, 11 mmol) in THF and

stirred for 10 min at 0 °C to get yellow clear solution. To this, com-

pound 12 (0.5 g 1.69 mmol) in anhydrous THF (5 mL) was added

and the turbid mixture was stirred for 4 h at room temperature.

The resulting mixture was poured into the mixture of ethyl ace-

tate/water (150 mL, 3:1), and acidiﬁed with 5 N HCl to pH 5.0.

The organic layer was washed with brine (2 ꢀ 25 mL), dried with

sodium sulfate, ﬁltered, and concentrated via rotary evaporation

to give yellow viscous oil. The crude product was puriﬁed by silica

gel column chromatography using (4:96 ethyl acetate/hexane) as

an eluent to yield compound 18a as colorless liquid (0.4 g, 81%).

¹H NMR (300 MHz, DMSO-d₆) d 9.27 (s, 1H), 7.29 (s, 1H), 6.9–

6.83 (m, 1H), 6.51 (s, 1H), 5.73–5.68 (m, 1H), 5.11–5.08 (d,

J = 11.3 Hz,1H), 4.66–4.63 (d, J = 9.9 Hz, 2H), 2.74–2.7 (m, 2H),

2.25–2.12 (m, 4H), 1.98–1.73 (m, 3H), 1.38–1.23 (m, 6H), 0.78 (s,

3H); ¹³C NMR (75 MHz, DMSO-d₆) d 161.0, 152.5, 137.0, 132.2,

130.5, 122.9, 121.4, 115.4, 112.2, 101.0, 59.7, 52.9, 43.8, 38.4,

35.2, 29.0, 27.1, 26.1, 23.4, 20,7, 18.3, 14.0; m/z (relative intensity)

293 (MH⁺, 100%); Anal. Calcd. for (C₂₁H₂₆O): C, 85.67; H, 8.90.

Found: C, 85.95; H, 8.71%.

2.2.51. 2-(E)-3⁰4⁰,5⁰-Trimethoxyphenyl-1⁰-vinly1-17-(1⁰-methylene)

estra-1,3,5(10)-triene-3-ol (18e)

Wittig reaction of aldehyde 12 (0.5 g, 1.69 mmol) with 3,4,5-tri-

methoxybenzyl

triphenylphosphonium

bromide

(4.41 g,

8.45 mmol) was performed using the above-mentioned procedure

E except that the reaction time was extended up to 12 h. The usual

workup followed by column chromatographic puriﬁcation (15:85

ethyl acetate/hexane) afforded pure trans-isomer 18e as a white

solid (0.61 g, 79%). mp 215–217 °C; ¹H NMR (300 MHz, DMSO-d₆)

d 9.39 (s, 1H), 7.44 (s, 1H), 7.30 (d, 1H), 7.26 (s, 1H), 6.84 (s, 2H),

6.55 (s, 1H), 4.67–4.64 (d, J = 12.0 Hz, 2H), 3.82 (s, 6H), 3.66 (s,

3H), 2.74–2.7 (m, 2H), 2.26–2.15 (m, 3H), 198–1.78 (m, 4H),

1.48–1.2 (m, 6H), 0.78 (s, 3H); m/z (relative intensity) 459 (MH⁺,

100%); Anal. Calcd. for (C₃₀H₃₆O₄): C, 78.23; H, 7.88. Found: C,

78.06; H, 7.63%.

2.2.48. 2-(E)-Propenyl-17-(1⁰-methylene)estra-1,3,5(10)-triene-3-ol

(18b)

Wittig reaction of aldehyde 12 (0.5 g, 1.69 mmol) with ethyltri-

phenylphosphonium iodide (3.53 g, 8.45 mmol) using the above-

mentioned procedure E followed by column chromatographic puri-

ﬁcation (3:97 ethyl acetate/hexane) afforded pure trans-isomer

18b as a colorless liquid (0.45 g, 86%). IR (KBr, cm^ꢁ1): 3343 (OH),

1615 (C@C); ¹H NMR (300 MHz, DMSO-d₆) d 9.11 (s, 1H), 7.19 (s,

1H), 6.56–6.52 (d, J = 15.9 Hz, 1H), 6.47 (s, 1H), 6.17–6.12 (m,

1H), 4.65–4.63 (d, J = 9.4 Hz, 2H), 2.72–2.67 (m, 2H), 2.26–2.12

(m, 4H), 198–1.72 (m, 6H), 1.42–1.11 (m, 6H), 0.77 (s, 3H); ¹³C

NMR (75 MHz, DMSO-d₆) d 160.9, 151.9, 135.5, 130.3, 126.6,

123.0, 122.7, 121.6, 115.2, 100.8, 52.9, 43.8, 43.4, 38.4, 35.2, 29.0,

28.8, 27.2, 26.2, 23.4, 18.7, 18.2; m/z (relative intensity) 307

(MH⁺, 100%); Anal. Calcd. for (C₂₂H₂₈O): C, 85.66; H, 9.15. Found:

C, 85.87; H, 9.23%.

2.2.52. Preparation of 3-hydroxy-17a-methyl estratriene (19)

The solution of 17-methylene estratriene 11 (5.0 g,

18.6 mmol) in methanol (200 mL) was hydrogenated under 5 kg

hydrogen pressure for 3 h at RT. The mixture was ﬁltered

through celite powder and concentrated via rotary evaporation

to yield the title compound 19 as white solid (5 g, 99%). mp

133–134 °C ¹H NMR (300 MHz, DMSO-d₆) d 8.99 (s, 1H), 7.06–

7.04 (d, J = 8.5 Hz, 1H), 6.65–6.49 (m, 1H), 6.44 (s, 1H), 2.80 (s,

2H), 2.28–2.25 (m, 2H), 2.1 (m, 1H), 1.81–1.76 (m, 4H), 1.45–

1.24 (m, 7H), 0.85 (s, 3H), 0.6 (s, 3H); m/z (relative intensity)

271 (MH⁺, 100%); Anal. Calcd. for (C₁₉H₂₆O): C, 84.39; H, 9.69.

Found: C, 84.20; H, 9.93%.

2.2.49. Ethyl-2-(E)-2⁰⁰-acryl-17-(1⁰-methylene)estra-1,3,5(10)-triene-

3-ol (18c)

A solution of aldehyde 12 (0.75 g, 2.53 mmol) and ethyl(triphe-

nylphosphoranylidene)acetate (1.32 g, 3.8 mmol) in dry THF

1500

G. Panchapakesan et al. / Steroids 76 (2011) 1491–1504

2.2.53. Preparation of 2-formyl-3-hydroxy-17

(20)

a

-methyl estratriene

diisopropyl ether afforded compound 21c as a white solid

(0.19 g, 84%). mp 152.5–153 °C; IR (KBr, cm^ꢁ1): 3432 (OH), 1623

(C@N); ¹H NMR (300 MHz, DMSO-d₆) d 12.4 (s, 1H), 8.43 (s, 1H),

7.25 (s, 1H), 7.11 (s, 1H), 6.64 (s, 1H), 2.94–2.91 (m, 1H), 2.85–

2.82 (m, 2H), 2.29–2.17 (m, 3H), 1.89–1.17 (m, 4H), 1.55–1.37

(m, 10H), 0.88 (s, 3H), 0.6 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) d

162.6, 157.2, 140.6, 130.8, 127.4, 116.7, 115.5, 54.2, 44.5, 43.3,

41.8, 38.4, 36.8, 29.7, 29.3, 27.2, 26.0, 23.9, 13,7, 11.7, 8.8; m/z (rel-

ative intensity) 338 (MH⁺, 100%); Anal. Calcd. for (C₂₃H₃₁NO): C,

81.85; H, 9.26; N, 4.15. Found: C, 81.70; H, 9.03; N, 4.39%.

To the suspension of magnesium turnings (2.1 g, 86.4 mmol) in

THF (20 mL) was added solution of bromoethane (13.1 g,

120 mmol) in THF (10 mL) slowly at RT during 20 min and stirred

for another 30 min at RT. To the resulting clear solution was added

the solution of methyl compound 19 (4 g, 14.5 mmol) in THF

(40 mL) at RT and further stirred for 30 min. To the resulting sus-

pension was added hexamethylphosphoramide (7.4 g, 41.3 mmol)

followed by paraformaldedhyde (7 g, 233 mmol) and heated to

60 °C for 20 h. The resulting yellow reaction mixture was poured

in to water (100 mL) and washed with ethyl acetate (2 ꢀ 100 mL)

at pH 4.0. The combined organics were washed with brine

(2 ꢀ 25 mL) and concentrated via rotary evaporation to give yel-

lowish oil. The crude product was suspended in methanol

(80 mL) at 10 °C and added sodium hydroxide solution (20% w/v,

8 mL) and stirred for 1 h. The resulting yellow clear solution was

poured into the water (160 mL) and washed with ethyl acetate

(2 ꢀ 150 mL) at pH 4.0. The combined organics were washed with

brine (2 ꢀ 50 mL), dried with sodium sulfate, ﬁltered, and concen-

trated via rotary evaporation to give light yellow viscous oil. The

crude product was puriﬁed by silica gel column chromatography

using hexane as an eluent to yield 2-formyl methyl compound

20 as off white solid (3.65 g, 83%). mp 194–196 °C ¹H NMR

(300 MHz, DMSO-d₆) d 10.42 (s, 1H), 10.13 (s, 1H), 7.56 (s, 1H),

6.67 (s, 1H), 2.8 (s, 2H), 2.28–2.25 (m, 1H), 2.1 (m, 1H), 1.81–1.76

(m, 4H), 1.45–1.24 (m, 8H), 0.85 (s, 3H), 0.60 (s, 3H); m/z (relative

intensity) 297 (MH⁺, 100%); Anal. Calcd. for (C₂₀H₂₆O₂): C, 80.50; H,

8.78. Found: C, 80.75; H, 8.59%.

2.2.57. 2-(Hydroxyethylimino)methyl-17-(1⁰-methyl)estra-1,3,5(10)-

triene-3-ol (21d)

Condensation of 2-formyl compound 20 (0.2 g, 0.67 mmol) with

ethanolamine (53 mg, 0.87 mmol) using the above-mentioned pro-

cedure D followed by trituration of the crude product with diiso-

propyl ether afforded compound 21d as a white solid (0.18 g,

79%). mp 195–197 °C; ¹H NMR (300 MHz, CDCl₃) d 12.86 (s, 1H),

8.36 (s, 1H), 7.26 (s, 1H), 7.16 (s, 1H), 6.68 (s, 1H), 3.92–3.89 (t,

J = 5.1 Hz, 2H), 3.74–3.72 (t, J = 5.1 Hz, 2H), 2.87 (s, 2H), 2.28–

2.18 (m, 2H), 1.90–1.17 (m, 10H), 0.82 (s, 3H), 0.6 (s, 3H); ¹³C

NMR (75 MHz, DMSO-d₆) d 166.5, 158.2, 141.3, 130.6, 128.1,

116.7, 115.7, 61.2, 60.7, 54.2, 44.5, 43.2, 41.8, 38.5, 36.8, 29.7,

29.2, 27.2, 26.0, 24.0, 13.8, 11.8; m/z (relative intensity) 342

(MH⁺, 100%); Anal. Calcd. for (C₂₂H₃₁NO₂): C, 77.38; H, 9.15; N,

4.10. Found: C, 77.18; H, 9.39; N, 4.32%.

3. Results and discussion

3.1. Chemical synthesis

2.2.54. 2-(N,N-Dimethylhydrazinimino)methyl-17-(1⁰-methyl)estra-1,

3,5(10)-triene-3-ol (21a)

Commercially available estrone 5 upon NaBH₄reduction fol-

lowed by selective 2-formylation of resulting 17b-estradiol using

non-lithiation protocol [15] led to aldehyde 6, which on condensa-

tion with various amines/amino acid ethyl esters followed by

reduction afforded amino derivatives 7a–g, Scheme 1.

As per the objective, the synthesis of estradiol derivatives con-

taining aroyl units at the 2-position was initiated. Accordingly,

interaction of the aldehyde 8 with freshly prepared aryl Grignards

Condensation of 2-formyl compound 20 (0.2 g, 0.67 mmol) with

N,N-dimethylhydrazine (52 mg, 0.87 mmol) using the above-men-

tioned procedure D followed by trituration of the crude product

with diisopropyl ether afforded compound 21a as a white solid

(0.2 g, 87%). mp 208–209 °C; IR (KBr, cm^ꢁ1): 3360 (OH), 1590

(C@N); ¹H NMR (300 MHz, DMSO-d₆) d 11.0 (s, 1H), 7.6 (s, 1H),

7.24 (s, 1H), 6.5 (s, 1H), 2.85 (s, 6H), 2.74 (s, 2H), 2.30–2.27 (m,

1H), 2.12 (m, 1H), 1.80–1.68 (m, 6H), 1.47–1.23 (m, 6H), 0.8 (s,

3H), 0.55 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) d 154.1, 137.9,

137.4, 131.0, 125.5, 116.9, 115.3, 54.2, 44.5, 43.3, 42.4, 41.7, 38.5,

36.9, 29.7, 29.1, 27.3, 26.0, 23.9, 13.5, 11.6; m/z (relative intensity)

341 (MH⁺, 100%); Anal. Calcd. for (C₂₂H₃₂N₂O): C, 77.60; H, 9.47; N,

8.23. Found: C, 77.79; H, 9.20; N, 8.02%.

OH

Me

O

Me

i) NaBH₄

OHC

THF-MeOH

ii) EtMgBr/(CH₂O)n

THF-HMPA

61% (2 steps)

2.2.55. 2-(2⁰-(N,N-Dimethylaminoethyl)imino)methyl-17-(1⁰-methyl)

estra-1,3,5(10)-triene-3-ol (21b)

HO

5

6

Condensation of 2-formyl compound 20 (0.2 g, 0.67 mmol) with

N,N-dimethylethylenediamine (76 mg, 0.87 mmol) using the

above-mentioned procedure D followed by trituration of the crude

product with diisopropyl ether afforded compound 21b as a white

solid (0.2 g, 81%). mp 130–132 °C; ¹H NMR (300 MHz, CDCl₃) d 13.1

(s, 1H), 8.31 (s, 1H), 7.26 (s, 1H), 7.14 (s, 1H), 6.68 (s, 1H), 3.7 (m,

2H), 2.86–2.85 (m, 2H), 2.63–2.59 (m, 2H), 2.29 (s, 6H), 2.17–

2.15 (m, 1H), 1.89–1.69 (m, 6H), 1.5–1.1 (m, 6H), 0.88 (s, 3H),

0.58 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) d 165.7, 158.1, 141.2,

130.6, 127.9, 116.4, 115.7, 59.5, 56.7, 54.2, 45.2, 44.5, 43.2, 41.8,

38.4, 36.8, 29.7, 29.3, 27.2, 26.0, 23.9, 13.7, 11.7; HRMS Calcd. for

OH

Me

i) R¹NH₂/Na₂SO₄

DMF

R¹NH

ii) NaBH₄/MeOH

55-84% (2 steps)

HO

7a-g

Compound R¹

Yield (%)

7a

7b

7c

(CH₂)₂NMe₂

(CH₂)₃NMe₂

Cyclopropyl

60

63

84

C₂₄H₃₆N₂O (M): 368.2828. Found: 368.2821.

7d

7e

7f

CH₂CO₂Et

55

66

77

73

2.2.56. 2-(Cyclopropylimino) methyl-17-(1⁰-methyl)estra-1,3,5(10)-

triene-3-ol (21c)

Condensation of 2-formyl compound 20 (0.2 g, 0.67 mmol) with

cyclopropylamine (50 mg, 0.87 mmol) using the above-mentioned

procedure D followed by trituration of the crude product with

CH(Et)CO₂Et

CH₂CH₂OH

CH(Et)CH₂OH

7g

Scheme 1. Synthesis of 2-alkylaminomethyl 17b-estradiols 7a–g.

G. Panchapakesan et al. / Steroids 76 (2011) 1491–1504

1501

followed by MnO₂oxidation and subsequent desilylation gave 2-

CH₂

Me

aroyl estradiols 9a–h, Scheme 2. Next, the synthesis of vinyl teth-

ered 2-aroyl estradiols was planned. As expected, condensation of

17b-estradiol-2-carboxaldehyde 6 with substituted acetophenones

[16] using methanolic KOH as a base, furnished trans-chalcones

10a–d.

CH₃PPh₃I

5

NaH/DMSO

91%

HO

CH₂

11

Since, recent reports identiﬁed that the replacement of 17b-OH

group with a methylene unit exhibited maximum cytotoxicity

[3d,10], a synthesis of 2-substituted estratriene derivatives con-

taining an exocyclic methylene at C-17 was planned. Accordingly,

the Wittig reaction of estrone 5 with methyltriphenylphospho-

nium iodide using NaH as a base in dry DMSO, afforded 11, which

on regio-selective formylation as mentioned in the case of 17b-

estradiol [15] gave 17-methylene aldehyde 12. The condensation

of aldehyde 12 with amines led to the isolation of 17-methylene

imine analogs 13a–d. Alternatively, condensation of 12 with

amines followed by NaBH₄reduction afforded 2-alkylaminomethyl

estradiol analogs 14a–h, Scheme 3.

The preparation of additional analogs of 17-methylene estratri-

enes is depicted in Scheme 4. TBDMS protection of phenolic OH

group of 12 furnished 15. Addition of arylmagnesium bromides

to aldehyde 15, followed by oxidation and subsequent de-silyla-

tion, produced the 17-methylene 2-aroyls estratrienes 16a–e. Con-

densation of 12 with substituted acetophenones [16] using

methanolic KOH furnished vinyl tethered 17-methylene 2-aroyl

estratrienes 17a–e. The Wittig reaction of aldehyde 12 with alkyl

phosphonium salts using LiHMDS as base yielded 2-substituted

17-methylene estratrienes 18a–e, Scheme 4.

OHC

HO

EtMgBr/(CH₂O)_n

THF-HMPA

11

12

83%

12

CH₂

Me

R¹NH₂/Na₂SO₄

DMF

R¹

N

79-85%

HO

13a-d

Compound R¹

Yield (%)

(CH₃)₂N

(CH₂)₂NMe₂

13a

13b

13c

82

79

83

cyclopropyl

CH₂CH₂OH

13d

85

CH₂

Me

i) R²NH₂/Na₂SO₄

DMF

R²NH

12

ii) NaBH₄/MeOH

43-79% (2 steps)

HO

14a-h

Compound R²

Yield (%)

14a

14b

14c

60

43

76

(CH₂)₂NMe₂

(CH₂)₃NMe₂

cyclopropyl

OTBDMS

Me

OHC

TBDMSCl/Et₃N

14d

14e

14f

79

67

71

CH₂CH₂OH

CH(Me)₂CH₂OH

CH(Et)CH₂OH

6

DMF

81%

TBDMSO

8

OH

Me

14g

14h

2,4-dimethoxybenzyl

55

52

i) ArMgBr/THF

ii) MnO₂/DCM

O

3,4,5-trimethoxybenzyl

Ar

8

Scheme 3. Synthesis of 2-imino and 2-alkylaminomethyl 17-methylene

iii) TBAF/THF

51-65% (3 steps)

estratrienes.

HO

9a-h

Compound Ar

Yield (%)

Finally, 17a-methyl-2-iminoestratrienes 21a–d were prepared

9a

9b

9c

phenyl

p-tolyl

63

60

64

using the routine procedure as mentioned in Scheme 5. As ex-

pected, heterogenous reduction of 17-methylene unit of 11 pro-

duced compound 19. A selective 2-formylation of 19 using non-

lithiation protocol [15] led to aldehyde 20, which on condensation

with various amines provided 17a-methyl 2-iminoestratriene

derivatives 21a–d, Scheme 5.

4-fluorophenyl

9d

p-anisyl

65

63

53

9e

9f

4-ethoxyphenyl

3,4-dimethoxyphenyl

9g

3,5-dimethoxyphenyl

55

9h

3,4,5-trimethoxyphenyl

51

3.2. Antiproliferative activity

OH

Me

The synthesized 2-substituted estratriene analogs were evalu-

ated for their antiproliferative activities against four different hu-

man cell lines of diverse tumour origin. The in vitro GI₅₀(growth

inhibition) values of estratrienes against individual cell lines are

presented in Table 1. The MGM values were calculated by averag-

ing the GI₅₀values of the four cell lines tested. It should be noted

that in those cases wherein growth inhibition values for one or

O

ArCOCH₃

Ar

6

MeOH-KOH

50-72%

HO

10a-d

Compound Ar

Yield (%)

10a

10b

10c

more cell lines are >100

calculated.

The Table 1 also includes antiproliferation values of the most

active reference compounds, 2-methoxyestradiol 1 [3a], 2-ethoxy-

estradiol 2 [3a], 2-ethoxy-17-methylene estratriene-3-ol 3 [3d],

lM, the respective MGM values are not

m-anisyl

50

52

2-trifluoromethyl

2,4,6-trimethoxyphenyl

72

10d

3,4,5-trimethoxyphenyl 69

Scheme 2. Synthesis of 2-aroyl and 2-aroylvinyl 17b-estradiols.

and 2-methoxy-17-methylene estratriene-3-ol

4

[10]. The

1502

G. Panchapakesan et al. / Steroids 76 (2011) 1491–1504

CH₂

Me

OHC

H₂/Pd-C

TBDMSCl/Et₃N

DMF

11

12

EtOAc-MeOH

99%

HO

TBDMSO

19

Me

88%

15

CH₂

Me

EtMgBr/(CH₂O)_n

THF-HMPA

OHC

HO

i) ArMgBr/THF

ii) MnO₂/DCM

O

19

Ar

83%

15

iii) TBAF/THF

20

Me

HO

48-63% (3 steps)

16a-e

R¹NH₂/Na₂SO₄

DMF

R¹

Compound Ar

Yield (%)

N

20

16a

16b

16c

phenyl

58

57

61

79-87%

HO

p-anisyl

21a-d

4-ethoxyphenyl

Compound R¹

Yield (%)

16d

16e

3,4-dimethoxyphenyl

3,4,5-trimethoxyphenyl

63

48

21a

87

81

84

Me₂N

21b

21c

21d

(CH₂)₂NMe₂

cyclopropyl

CH₂

Me

O

79

CH₂CH₂OH

ArCOCH₃

Ar

12

Scheme 5. Synthesis of 2-imino 17b-methyl estratrienes 21a–d.

MeOH-KOH

45-67%

HO

17a-e

the range of 3.8–40 lM. Indeed, electronic withdrawing effect of

Compound Ar

Yield (%)

cyano group imposed on A-ring is responsible for completely inac-

tive nature of 2-cyanoestradiol [3c]. However, moderate activity

portrayed by compounds 9a–h containing an electron withdraw-

ing carbonyl function seems to indicate that the electronic factor

alone may not be a sole criteria for the observed activity of 2-

substituted estratrienes. The SAR of 9a–h against the four cell lines

indicated that the presence of either varying number of methoxy

groups or its absence have only negligible inﬂuence on their anti-

proliferation potential. Among the four chalcone derivatives 10a–d

tested, only 10c and 10d containing three methoxy groups dis-

played antiproliferative activities comparable to that of reference

compounds 1–4. The compound 10d wherein three methoxy

groups are far away from the carbonyl function showed maximum

17a

17b

17c

3-methoxyphenyl

4-ethoxyphenyl

50

55

3-trifluoromethylphenyl

67

17d

17e

2,4,6-trimethoxyphenyl

3,4,5-trimethoxyphenyl

45

52

CH₂

Me

R

RCH₂PPh₃I

12

LiHMDS/THF

HO

18a-e

cytotoxicity with an MGM value of 1.8

chalcone derivatives 10a/10b containing methoxy/triﬂuoromethyl

unit showed moderate activity (MGM value 6–9 M). Comparison

of SAR of 9h and 10d clearly indicated the separation of estradiol

and 3,4,5-trimethoxybenzoyl unit using a vinyl tether has signiﬁ-

cantly enhanced the antiproliferative potential.

Among the four imine derivatives 13a–d tested for their anti-

proliferative potential, three of them showed comparable activity.

All the eight 2-alkylamino side chain compounds 14a–h containing

lM. The remaining two

Compound

R

H

Yield (%)

18a

18b

81

86

l

Me

18c

18d

18e

CO₂Et

83

78

79

CH₂OH

3,4,5-trimethoxyphenyl

Scheme 4. Synthesis of 2-aroyl, 2-aroylvinyl and 2-vinyl 17-methylene

estratrienes.

17-methylene displayed growth inhibition values (MGM ꢂ2

lM)

comparable to that of the reference compounds 1–4. Contrary to

the 17b-OH containing 2-alkylamino compounds 7a–g, in the case

of 17-methylene system 13a–d reduction of imine-double bond

led to a signiﬁcant increase in the antiproliferative potential. In

particular, saturation of imine unit of 13a led to dramatic increase

compounds containing 2-alkylamino side chain 7a–g were found

to have moderate inhibitory effects (MGM 23–50 lM) on cell pro-

liferation. The compound 7d/7e containing an electron withdraw-

in the cytotoxicity value (from MGM 53.7 to 2.3 lM). Most of the

ing ester unit in the 2-alkylamino side chain was found to be

17-methylene compounds 16a–c and 16e possessing an electron

withdrawing benzoyl unit at the 2-position were found to be inac-

tive. Surprisingly, the transformation of the 17b-OH of 9f into the

exocyclic methylene compound 16d has signiﬁcantly enhanced

its activity in HCT-116 and U-251 human cancer cell lines. To reach

any further conclusions, however, the observed selectivity pattern

needs to be further veriﬁed with additional estratriene analogs.

The 17-methylene chalcone derivatives 17a–d did not show any

signiﬁcant cytotoxicity in all the four cancer lines tested. However,

a 17-methylene chalcone derivative 17e containing 3,4,5-trime-

completely inactive (>100 lM). Although, it has been conﬁrmed

that the electron releasing substituents at 2-position of estratri-

enes are favorable for growth inhibition [3], the moderate activity

displayed by the estratrienes 7a–c, 7f and 7g are surprising. It

should be noted that the corresponding unsaturated imine analogs

of 7a–c exhibited comparatively better antiproliferative activity

with MGM value of ꢂ16

lM [3c,3d]. The steric-bulkiness of the

2-substituents of 7a–g did not have any signiﬁcant inﬂuence on

their activity. The compounds 9a–h possessing an electron with-

drawing benzoyl unit at the 2-position exhibited GI₅₀values in

G. Panchapakesan et al. / Steroids 76 (2011) 1491–1504

1503

Table 1

1.1

l

M and 3.4

l

M, respectively [3a]. Hence, it is reasonable to

Antiproliferative assays of 2-substituted estradiol analogs.

conclude that presence of simultaneous double bonds at the C-2

and C-17 positions of estratrienes 18a–e are not favorable for

growth inhibition. The antiproliferative potential of imino com-

Compound Colon

HCT-116

Lung

NCIH-460

Glioma

U-251

Breast MDA

MB-435

MGM^b

M)^a

0.7

0.18

0.56

0.6

30

38

14

41

23

14.5

10

20

17

15

19

17

19

28

pounds 21a–d containing 17a-methyl unit was found to be moder-

Growth inhibition (GI₅₀in

l

1

2

3

4

7a

7b

7c

7d

0.47

0.26

0.6

0.65

25

52.5

38

>100

52

46.5

40

0.36

0.016

0.54

0.62

25

60

20

>100

21

39

15

17

13

19

17

13

40

15

7

18

4.8

2.1

50

2.9

38

0.08

<0.01

0.08

0.15

10

ND

20

ND

25

21

20

21

29

ND

26

18

ND

20

3

0.4

ate and the activity was comparatively better compared to the

corresponding imino derivatives containing 17-b-OH and 17-

methylene units.

0.058

0.44

0.54

22.5

50.2

23

4. Conclusions

In conclusion, different types of 2-substituted estratrienes con-

taining 17b-OH, 17-methylene, 17a-methyl units were synthe-

7e

7f

7g

26.7

27.5

17

17.2

17.7

18

13.4

27

15.7

6.2

9.1

4

1.8

53.7

2.5

5.7

3.6

2.3

1.6

1.9

2

sized and tested for their antiproliferative activity by using four

different cell lines from colon, lung, glioma and breast cancers.

Among the various 2-substituted estratrienes, ten compounds

10d, 14a–h and 17e were found to have in vitro antiproliferative

activity comparable to that of parent compounds 1–4. Comparison

of the SAR pattern of these 2-susbtituted estratriene derivatives

conﬁrmed that relatively, 17-methylene estratrienes are more ac-

tive. Work is in progress to synthesize additional analogs of 17-

9a

9b

9c

9d

9e

9f

9g

9h

13

14

16

12

3.8

13

12

10

16

5

7

10a

10b

10c

10d

13a

13b

13c

13d

14a

14b

14c

14d

14e

14f

14g

14h

16a

16b

16c

16d

16e

17a

17b

17c

17d

17e

18a

18b

18c

18d

18e

21a

21b

21c

21d

methylene and 17a-methyl estratriene analogs to further under-

stand their antiproliferative potential.

9

15

8

2

30

1.8

1.9

35

2.5

3.2

3

2.5

2

1.8

2

1.8

2

1.7

2

70

50

45

0.45

>100

34

62

15

8

1.6

18

19

>100

25

60

4.5

2.2

100

2.5

6.8

3.6

2.8

0.9

2.4

1.8

1.7

2.2

2.3

2.2

>100

50

70

14

3.2

18

6

2.6

17

>100

22

>100

40

Acknowledgements

2.1

8.9

4.8

1.8

1.6

1.5

2

1.6

2.8

2

2.2

>100

30

3

The authors thank Mr. J. Venkatesan and Mr. K. Parthasarathy,

Analytical Division, Orchid Pharma, for providing HPLC, NMR and

mass spectral data. The authors thank the Department of Science

and Technology (DST-FIST) for a 300-MHz NMR spectrometer.

2.2

1.9

1.8

2.3

2.8

1.9

2.5

>100

0.6

30

>100

19

15

1.4

21

2.4

2

2.2

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Article Doi

Synthesis of 2-substituted 17β-hydroxy/17-methylene estratrienes and their in vitro cytotoxicity in human cancer cell cultures

DOI: 10.1016/j.steroids.2011.08.004

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Full text of DOI:10.1016/j.steroids.2011.08.004

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