Indian Journal of Chemistry Vol. 42B , January 2003 , pp. 166-172

Synthesis and biological activity of 16-arylidene derivatives of estrone and estrone methyl ether

Maninder Minu* & Dharam Paul lindal

University In 5titutc of Pharmaceutical Sciences, Panjab University , Chandigarh 160014, India

and

G Leclercq & M Borras

institut Jules Bordet , Association Hospita liere de Bruxelles. Centre des Tumeurs de I'ULB, Rue Heger-Bordet 1- 1000, Belgiulll

Received 24 April 200 I .. accepted (revised) 20 March 2002

The synthesis of 16-arylidene derivatives 3, 4, S, 6, 7. 8. 9, 10, 11, 12, 13, 14 and IS is described. These compounds have been tested at NCI, Bethesda, for their antineoplastic acti vity against the cell panel consisting o f 60-ce!l lines and the compounds 3, 4, S, 6 and 7 have also been tested for their ill vitro estrogenic / anti estrogenic activity ; induction o f ERE-dependent luciferase inductio n was measured (MvLN celis).

The formation of active steroids by aromatase has been considered to play an important role in the de-velopment of human breast carcinoma t, at least one-third of all breast cancers establishing that. the estro-gen-dependent carcinoma regresses following estro-gen deprivation2. In the postmenopausal women, the production of estrogen takes place in peripheral tissue from inactive precursors by the action of aromatase3. [t has been documented by several IJboratories that the increased aromatase activity in breast cancer tis-sue is more as compared to the non-malignant paren-chyma3.4 . The enzyme is the rational target for devd-opment of drugs to treat hormonal-dependent breast cancer5-9.

Aminoglutethimide 1 is the pioneer drug of non-steroidal competitive aromatase inhibitors, but it lacks specificit/. In recent years, clinical research of aro-matase inhibitors has accelerated exponentially IO,14. Several oral aromatase inhibitors have been demon-strated to be highly potent and specific inhibitors of estrogen

o

(CrC/" 'I '\ N I -~ ( 1) (2)

h . IS, I 8 V' . d d" h I synt eSlS ' . anous Jl1 anone envatlves ave a so showed good inhibitory effects and more selectivity. The 2-(4-pyridylmethylene)-I-indanone 2 showed good inhibitory effects l9.

It was conceived that selective inhibitors of aroma-tase might be useful as a pharmacological tool to de-vice successful treatment approach for the hormonal dependent breast cancer. Our efforts were focused on designing estrogen and estrogen methyl ether deriva-tives that might inhibit or possess an ti estrogenic activ-ity. From these efforts, several 16-arylidi ne deriva-tives of estrone and estrone methyl ether have emerged which are reported in this paper.

It was planned to synthesise a series of 16-substituted estrone derivatives (Scheme I ). Estrone was subjected to base-catalysed aldol condensation with 3,4,5-trimethoxybenzaldehyde; 4-cyanobenzalde-hyde; 4-nitrobenzaldehyde, 4-isopropylbenzalde-hyde and 4-pyridinecarboxaldehyde to get 3 [IR: 1720 cm' l; IH NMR: () 3.91 (9H: 3,4,5-0CH3) and 7.40 (lH, vinyl-H)]; 4 [UV: Amax 296.0 11m; IR: C = N stretching at 2240 cm' l; IH NMR showed singlet for vinyl-H at 8 7.66]; 5 [IR: 1710 cm' l; IH NMR singlet for vinyl-H at 8 7.48]; 6 CH NMR showed a doublet at 8 1.28] and 7 rIR: 1720 cm' l and the IH NMR showed singlet at 8 7.34 for the vinyl-H].

A series of compounds bearing aryl idine moieties at C-16 position in estrone methyl ether was synthe-sized. The compounds 8- 11 were obtained by aldol condensation using potassium hydroxide as catalyst. For this, estrone methyl ether was condensed with 3,4,5-trimethoxybenzaldehyde; 3,4-dimethoxybenza!de-hyde; cuminaldehyde and vanillin to obtai n 8-11 , re-spectively.

RO

(3) R =H

(4) R =H

(5) R=H

(6) R=H

(7) R =H

(8) R= CH3

(9) R= CH3

(10) R =CH3

(11) R= CH3

(12) R= CH3

(13) R= CH3

(14) R= CH3

(15) R= CH3

MINU el al.: SYNTHESIS OF ESTRONE DERIV ATIVES 167

-eto eH' R1 = '/ _ '\ OCH3 OCH3

R1 = -{}C:N

R1 = -{}N02

R1 = -{} /CH

3 CH - .......... CH3

R1 = 1 ~N -etOCH' R1 = '/ _ '\ OCH3

OCH3

R1 = -6OOH,

'/ _ '\ OCH3

R1 = -{} /CH

3 CH - .......... CH3

R1 = -6oeH,

'/ _ '\ Oft

R1 = -{}N/

CH3

- .......... CH3

R1 = -{}C:N

R1 = -{}N0 2

R1 = -eN :==J Scheme I

The structures of these compounds, 8 ['H NMR singlets at 8 3.90 (9H, 3,4,5-0CH3) and 7.38 (vinyl-H)]; 9 [UV: Amax 334.1 nm; IR: 1720 cm·' and 'H NMR singlets at 8 3.95 (6H, 3,4-0CH3) and 7.41 (vi nyl-If) ; 10 [UV: Amax at 308.8 nm; 'H NMR signal s at 8 1.28 [d, 6H, J=9Hz, -CH(CH3h] and 7.45 (lH, s, vinyl-H) and 11 ['H NMR showed singlets at 8 3.93 (-OCH3), 5.97 (-OH; disappeared Oli 0 20 exchange) and 7.40 (vinyl-H)] were determined with the help of spectral and elemental analyses.

Next, condensation of the aldehydes containing ni -trogen was planned. For thi s, estrone methyl ether was condensed with N,N-dimethylaminobenzalde-hyde; 4-cyanobenzaldehyde; 4-nitrobenzaldehyde and 4-pyridinecarboxaldehyde to get 12-15, respectively. The spectral analyses of these compounds, 12 [UV: Amax at 382.4 nm, IR: 1730 cm-'; 'H NMR: singlets at 8 3.06 {6H, -N(CH3)2 } and 7.41 (lH, vinyl-H)]; 13 [UV: Amax at 294.0 nm; IR showed C == N stretching at 2230 cm·'; and proton resonance singlet for vinyl-H at 8 7.43]; 14 [UV: Amax at 278.2 nm; IR: 1730 cm-'; 'H NMR singlet at 8 7.48 (v inyl-H) and 15 [UV: Amax at 278.6 nm; IR: 1720 cm·' and NMR singlet at 8 7.36 for the vinyl-H] , and CHN analyses confirmed the structures.

The configuration in both estrone methyl ether as well as in estrone series is believed to be Z on the ba-sis of 'H NMR spectroscopy as described earlier20.

Biological activities Antineoplastic activity

The compounds 3 (DPJ-717), 4 (OPJ-716), 5 (OPJ -718), 7 (OPJ-734), 11 OPJ-715), 12 (OPJ-708), 13 (OPJ-720) and 15 (OPJ-714) have been tested fm· antineoplastic activity at National Cancer Institute, Bethesda, Maryland, USA, in vitro against the cell panel consisting of 60 lines at a minimum of five con-centrations at IO-fold di lutions. A 48 hr continuous drug exposure protocol was used, and a sul forhoda-minc B (SRB) protein assay was used to est imate ceil viability or growth . The results obtai ned indicated that the compounds have statistically insignificant anti -neopl astic act ivity for further studies .

Estrogenic/ Antiestrogenic activity Compounds 3 (DPJ-717), 4 (DPJ-716), 5 (DPJ-

718), 6 (DPJ-721 ) and 7 DPJ-734) were tested for their in vitro estrogenic / antiestrogeni c activity: in-duction cf ERE-dependent luciferase induction was measured (MVLN cells) , all compounds induced the

168 INDIAN J. CHEM., SEC B, JA NUARY 2003

expression of this reporter gene with an efficiency at least I ,OOO-foid less than EI (optimal induction - 10.9 ); DPl-721 was the less effective compound. Hence, all compounds shared weak estrogenic activ-ity. Binding affinity for ER was also assessed with a human recombinant produced in yeast (Table I). Values were weak ( I % of EI at maximum), in agree-ment with luciferase data (Figures1 and 2).

Experimental Section Melting points reported are uncorrected. IH NMR

spectra were recorded on AC-300F, 300 MHz, Varian EM-390, 60 MHz spectrometers using TMS as an internal standard (chem ical shifts in 8, ppm), IR spec-

Table I - Measurement of binding affinity for ER

Compd Code

4 DPJ - 716

3 DPJ - 717

5 DPJ - 718

6 DPJ - 721

7 DPJ - 734

717, 734 > 716, 718 > 72 I

300

250

200

100

50 I Q w

0+--'--........ -Controls

Binding affinit y for hER (RBA)

EI = 100

0.1

1.0

0.1

< 0.1

1.0

OPJ-716 (4)

tra in KBr (umax in cm-I) on Perkj n-Elmer 882, UV

spectra in methanol (A.max in nm figures within paren-thesis refer to log £ values), on a Lambda 15 spectro-photometer models respectively and mass spectra on V6-11-250 1 70S. The purity of the compounds was established by TLC, which was prepared with silica gel G according to Stahl (E. Merck) using ethyl ace-tate. [odine was used to develop the TLC plates. Ele-mental analyses were calTied out on a Perkin-Elmer 2400 CHN elemental analyzer. Anhydrous sodium sulphate was used as a drying agent.

16-(3, 4, 5-TrimethoxybenzyJidene)-17 -oxo-1 , 3, 5 (10)-estratrien- 3~-ol 3. A solution of estrone (0.25 g), potassium hydroxide (0.25 g) and 3, 4, 5-trimethoxybenzaldehyde (0.3 g) in methanol (SO mL) was refluxed for 15 hr. The completion of reaction was monitored by TLC and the solution was concen-trated in vacuo. Ice-cold water was added, the suspen-sion was acidified with conc. HCI and the precipitate was removed by fi ltration, dried and crystallised from dichloromethane-methanol to afford 3 (0.2 g, 48.78%), m.p. 144-52°; UV: 319.6 (4.22); IR: 3420 (O-H), 1720 (C=O) 1260; I H NMR: 1.00 (3H, s, 18-CHJ), 3.91 (9H, s, 3, 4, 5-0CH3), 5.13 (lH, s, 3-0H; disappeared on 0 20 exchange) 6.6 1 ( I H, d, J=3Hz, 4-CH of estrone), 6.67 ( I H, dd, J=3Hz and 9Hz, 2-CH of estrone), 6.80 (2H, s, 2-CH and 6-CH of 16-(3,4,5-trimethoxybenzylidene)), 7.18 (lH. d, J=3Hz, I-CH

o 10-8 M

OPJ-717 (3) OPJ-718 (5)

COMPOUNDS

Figure I-Expression of Lueiferase in MVLN cells

MINU el al.: SYNTHESIS OF ESTRONE DER IVATIVES 169

300

250

200

=> -' 150 a: i!

100

50

0

I ' , 0 w

Controls DPJ-721 (6)

COMPOUNDS

o 10-8 M

DPJ-734' (7)

*17P OH analogue of DPJ 734 Is also stimulatory

Figure 2-Expression of Luciferase in MVLN cells

of estrone), 7.40 (lH, s, vinyl-H). MS: rnJz 449 (M+) (Found: C, 75.39; H, 7.40. C2sH320S requires C, 74.96; H, 7.19%).

16-( 4-Cyanobenzylidene)-17 -oxo-l,3,5(10)-estra -trien-3p-ol 4. Estrone (0.25 g) was dissolved in methanol (50 mL) and potassium hydroxide (0.25 g) was added followed by 4-cyanobenzaldehyde (0.4 g). The reaction mixture was refluxed for 14 hr, proc-essed as usual and the precipitate obtained was fil -tered off, dried and recrystallised from methanol to yield 4 (0.1 g, 28.57%), m.p. 252-56°; UV: 296.0 (4.11); IR: 3400 (O-H), 2240 (C=N), 1730 (C=O) and 1240; 'H NMR: 1.23 (3H, s, 18-CHJ), 6.81 (lH, d, J=3Hz, 4-CH of estrone), 6.93 (lH, dd, J=3Hz and 9Hz, 2-CH of estrone) , 7.37 (lH, d, J=9Hz, l-CH of estrone), 7.66 ( IH, s, vinyl-H), 7.83-7 .97 {4H, m, aromatic protons of 16-(4-cyanobenzylidene)), 8.84 (lH, s, 3-0H; disappeared on 0 20 exchange); MS: rnJz 383 (M+) (Found: C,81.19; H,6.86; N,3 .88. C26H25 N02 requires C, 81.43; H, 6.57; N, 3.65%).

16-(4-Nitrobenzylidene)-17-oxo-l, 3, 5 (10)-estratrien-3p-ol 5. To a solution of estrone (0.25 g) and potass ium hydroxide (0.25 g) in methanol (25 mL), was added 4-nitrobenzaldehyde (0.35 g) and the solution was heated to reflux for 14 hr and the usual processing gave a solid residue which was crystallised from methanol to obtain 5 (0.2 g, 58.82%), m.p. 240-44°; uy 314.4 (4.18); IR: 3460 (O-H), 1700 (C=O), 1250; 'H NMR: 1.02 (3H, s, 18-CHJ), 5.04 (I H, s, 3-OH; disappeared on 0 20 exchange), 6.64 (I H, d,

J=3Hz, 4-CH of estrone), 6.67 (lH, dd, J=3Hz and 9Hz, 2-CH of estrone), 7.18 (lH, d, J=9Hz, 1-CH of estrone), 7.48 (lH, s, vinyl-H), 7.70 {2H, d, J=9Hz, 2-CH and 6-CH of 16-(4-nitrobenzylidene)), 8.28 {2H, d, J=9Hz, 3-CH and 5-CH of 16-(4-nitrobenzyli-dene)}. MS: rnJz 403 (M+) (Found: C, 74.13; H, 6.18; N, 3.48. C25H25N04 requires C, 74.41 ; H, 6.19; 3.47%).

16-(4-Isopropylbenzylidene)-17-oxo-l, 3, 5 (10)-estratrien-3p-ol 6. Cuminaldehyde (0.5 g) was added to a solution of estrone (0.25 g) and potassium hy-droxide (0.25 g) in methanol (35 mL) and the reaction mjxture was refluxed for 14 hr, processed as usual and precipitated solid was filtered, washed with water, dried and crystallised from methanol to yield 6 (0.35 g, 94.59%), m.p. 226-28°; UV : 306.0 (4.32); IR: 3380 (O-H), 1700 (C=O), 1220; 'H NMR: 0.99 (3H, s, 18-CHJ), 1.28 (6H, d, J=9Hz, -CH(CHJh), 5.38 (lH, br, 3-0H; disappeared on 0 20 exchange), 6.61 (lH, d, J=3Hz, 4-CH of estrone), 6.68 (lH, dd, J=3Hz and 9Hz, 2-CH of estrone), 7.17 (lH, d, J=9Hz, loCH of estrone), 7.29 {2H, d, J=6Hz, 3-CH and 5-CH of 16-(4- isopropylbenzylidene)), 7.48 (tH, s, vinyl-H), 7.51 {2H, d; J=9Hz, 2-CH and 6-CH of 16-(4-isopropyl benzylidene) }; MS : rnJz 389 (M+) (Found: C, 83.62; H, 8.26. C2sH320 2 requires C, 83.96; H, 8.05%).

16-(4-Pyridylmethylene)-17-oxo-l, 3,5(10)-estra-trien-3f3- o1 7. A mixture of estrone (0.25 g), potas-si um hydroxide (0.25 g) and 4-pyridine carboxalde-

170 INDIAN J. CHEM., SEC S, JANUARY 2003

hyde ( I mL) was shaken for 10 mi n at room tempera-ture. The reaction mixture was then refluxed for 23 hr and the usual processing gave the precipitate which was separated by filtration , washed thoroughly with water, dried and crystalli sed from methanol to yield 7 (0.1 7 g, 51.52%), m.p. 250-54°; uy 279.3 (4.20); JR: 3260,2930, 1720, 1600, 1490, 1240 and 820 cm"l; IH NMR: 1.00 (3H, S, 18-CH) , 6.59 (lH, d, J=3Hz, 4-CH of estrone), 6.67 (1H, dd, J=3Hz and 9Hz, 2-CH of estrone) 7.15 (1 H, d, J=9Hz, l-CH of estrone), 7.34 (1H, s, vinyl-H), 7.42 (2H, d, J=6Hz, 3-CH and 5-CH of 16-(4-pyridylmethylene)}, 8.60 (1H, s, 3-0H; dis-appeared on DzO exchange) and 8.67 ppm (2H, d, J=6Hz, 2-CH and 6-CH of 16-(4-pyridy lmethylene) }; MS : mlz 359 (M+) (Found : C, 80.48; H,7 .37; N, 4.01. C24H2sN02 requi res C, 80. 19; H, 7.01; N, 3.89%).

3-Methoxy -16-(3,4,5-trimethoxybenzy lidene)-1,3,5(10)-estratrien- 17-one 8. To a solution of es-trone methyl ether (0.5 g) in methanol (35 mL), were added 3,4,5-trimethoxy benzaldehyde (0.75 g) and potassium hydroxide (0.5 g). The reaction mixture was refluxed for 16 hr and excess methanol was re-covered. Ice-cold water was added to the residue and the precipitated product was filtered , dried and crys-tallised from dichloromethane-methanol to obtain 8 (0.45 g, 55.55 %), m.p. 180-82°; UV: 278.8 (4.29); IR: 1730 (C=O), 1250; I HNMR: 0.98 (3H, s, 18-CH)), 3.76 (3 H, s, 3-0CH3), 3.90 (9H, s, 3,4,5-0CHJ), 6.64 (1H, s, J=3Hz, 4-CH of estrone methyl ether), 6.72 (lH, dd~ J=3Hz and 9Hz, 2-CH of estrone methyl ether) , 6.80 (2H, s, 2-CH and 6-CH of 16-(3,4,5-trimethoxybenzy lidene) }, 7.20 (1 H, d, J=9Hz, I-CH of estrone methyl ether) and 7.38 ppm (lH, s, vinyl-H) ; MS : mlz: 462 (M+) (Found: C, 75 .12 ; H,7.37. C29 H340 S requires C, 75 .29; H, 7.4 1 %) .

16-(3,4-Dimethoxybenzylidene)-3-methoxy-1,3,5(10)-estratrien- 17-one 9. Potassium hydrox-ide (0.5 g) was added to a solution of estrone methyl ether (0.5 g) in methanol (35 mL) and then veratral-dehyde (3,4-dimethoxybenzaldehyde) (0.4 g) was added. The reaction mixture was shaken for 10 min at room temperature and refluxed for IS hr. The usual processing gave a solid res idue crystallised from methanol to afford 9 (0.3 g, 39.47%), m.p. 156-58°; UV: 334.1 (4.48); IR: 1720 (C=O), 1220, 1020. IH NMR: 0.98 (3H, s, 18-CH3), 3.85 (3H, s, 3-0CH3) , 3.95 (6H, s, 3,4-0CH3), 6.65 (i H, d, J=3Hz, 4-CH of es trone methyl ether), 6.74 (l H, dd, J=3Hz and 9Hz, 2-CH of estrone methyl ether), 6.93 (l H, d, }:=9Hz, 1-CH of estrone methyl ether), 7.08 (lH, s, 4-CH of estrone methyl ether), 7.25 [2H, m, 2-CH and 6-CH of

{3-methoxy-16-(3,4-dimethoxybenzylidene) }] 7.41 (l H, s, vinyl-H); MS : mlz 432 (rvn (Found: C, 77 .58 ; H,7 .26. C28H320 4 requires C, 77 .74; H, 7.46%) .

16-(4-Isopropylbenzylidene )-3-methoxy-1,3,5 (10)-estratrien-17-one 10. A mixture of estrone methyl ether (0.5 g) , methanol (40 mL), cuminaldehyde (0.4 g) and potass: um hydroxide (0.5 g) was refl uxed for 14 hr, processed as usual and the prec ipitate obtained was crystalli sed from dichloromethane-methanol to afford 10 (0.65 g, 89 .04%); m.p. 188-90°; UV : 308.8 (4.88); IR: 1735 (C=O), 1220, 1020; IH NMR : 0.97 (3H, s, 18-CHJ), 1.28 (6H, d, J=9Hz, -CH(CHJ)2) , 3.78 (3H, s, 3-0CH3), 6.66 ( I H, d, J=3Hz, 4-CH of estrone methyl ether), 6.74 (1 H, dd, J=3Hz and 9Hz, 2-CH of estrone methyl ether) , 7.23 (l H, d, J=9Hz, 1-CI! of estrone methyl ether), 7.29 (2H , d, J=9Hz, 2-CH and 6-CH of 16-(4-isopropy lbenzy lidene)}, 7.45 (2H, s, vinyl-H), 7.51 (2H, d, J=9Hz, 3-CH and 5-CH of l6-(4-i sopropy lbenzy lidene)}; MS : mlz: 414 (M '-) (Found: C, 84.26; H,8 .44. C29H340 2 requires C, 84.0 I; H,8.27%) .

3-Methoxy-16-( 4-hydroxy-3-methoxybenzylidene)-1,3,5(1O)-estratrien-17-one 11. Estrone methyl ether (0.5 g) was dissolved in methanol (40 mL). Vanillin (4-hydroxy-3-methoxybenzaldehyde) (0.75 g) and potassium hydroxide pellets (0.5 g) 'Nere added to thi s solution . The reaction mixtu re was refluxed for 23 hr. Methanol was recovered under reduced pressure and ice-cold water was added. The suspension so obtained was acidified to yield the precipitated product, wh ich was filtered , dried and crystalli sed from methanol to afford 11 (0.4 g, 54.79%), m.p. 222-24°. UV (MeOH): 337.6 (4.32); IR: 3480 (O- H), 1720 (C=O), 1240; IH NMR: 0.9 1 (3H, s, 18-CHJ) , 3.78 (3H, s, 3-OCH) , 3.93 (3H, S, -OCHJ of 16-(4-hydroxy-3-methoxybenzy lidene function)}, 5.97 (lH, s, -OH; disappeared on D20 exchange), 6.66 ( I H, d, }=3Hz, 4-CH of estrone methyl ether), 6.75 (1 H, dd, J=3Hz and 9Hz, 2-CH of estrone methyl ether), 6.98 (l H, d, J=9Hz, I -CH of estrone methyl ether), 7.21 (3H, m, aromatic protons of 16-(4-hydroxy-3-methoxy-benzylidene)}, 7.40 (I H, s, vi nyl-H) ; MS : mlz 418 (M+) (Found : C,77.36; H,7.46. C27H300 4 requires C, 77.48; H, 7.23%).

16-( 4-N, N -Dimethylaminobenzylidene )-3-metho-xy-l,3,5 (10)-estratrien-17-one 12. 4-N,N-Dimethyl-ami nobenzaldehyde (0.4 g) and potass ium hydrox ide pellets (0.5 g) were added to the solution of es trone methyl ether (0.5 g) in methanol (40 mL). The reac-tion mixture was refluxed for 14 hr and the usual process the so lid residue which was crystalli sed from

MINU et at.: SYNTHESIS OF ESTRONE DERiVATIVES 171

methanol to yield 12 (0.25 g, 34.30%); m.p. 196-200°; UV: 38204 (4048); IR: 1730 (C=O), 1220, 1010; IH NMR: 0.97 (3H, s, 18-CHJ), 3.06 (6H, s, -N(CHJh), 3.78 (3 H, S, 3-0CHJ), 6.65-6.74 (3H, m, aromatic protons of estrone methyl ether), 7.21 {2H, d, 2-CH and 6-CH of 16-(4-N,N-dimethylaminobenzylidene)}, 7041 (lH, s, vinyl-H), 7.47 {2H, d, J=9Hz, 3-CH and 5-CH of 16-(4-N,N-dimethylaminobenzylidene)} ; MS: mlz 415 (M+) (Found: C, 81.23; H,7.87; N,3.45. C28H33N02 requires C, 80.92; H, 8.00; N, 3.37%).

16-(4·Cyanobenzylidene)-3-methoxy-l, 3, 5( 10)-estratrien-17-nne 13. Potassium hydroxide (0.5 g) was added to a mixture of estrone methyl ether (0.5 g), methanol (40 mL) and 4-cyanobenzaldehyde (0.75 g). The reaction mjxture was refluxed for 14 hr and processed as usual. The precipitate obtained was crys-tallised from methanol to give 13 (0.82 g, 98.63%), m.p. 232-36°. UV: 294.0 (4.24); IR: 2230, (C=N), 1730 (C=O), 1220, 1010; I H NMR: 1.01 (3H, s, 18-CHJ), 3.78 (3H, S, 3-0CHJ), 6.66 (l H, d, J=3Hz, 4-CH of estrone methyl ether), 6.75 (lH, dd, J=3Hz and 9Hz, 2-CH of estrone methyl ether) , 7.23 (lH, d, J=9Hz, I-CH of estrone methyl ether), 7043 (1 H, s, vinyl-H), 7.64 {2H, d, J=9Hz, 2-CH and 6-CH of 16-(4-cya nobenzylidene)} 7.71 {2H, d, J=9Hz, 3-CH and 5-CH of 16-(4-cyanobenzylidene)}; MS: mlz 397 (M+) (Found : C,81.83; H,6.99; N,3.32. C27 H27N02 requires C, 81.57; H, 6.85; N, 3.52%).

3-Methoxy-16-(4-nitrobenzylidene)-I, 3, 5 (10)-estratrien-17-one 14. Estrone methyl ether (0.5 g) was taken in methanol (35 mL) to which 4-nitrobenzaldehyde (004 g) and potassium hydroxide pellets (0.5 g) were added. The reaction mixture was shaken for 30 min at room temperature and then re-fluxed for 15 hr. Usual processing gave solid residue which was crystallised from dichloromethane- metha-nol to afford 14 (0.72 g, 98.63%), m.p. 216-22°; UV: 278.2 (4.25); IR: 1730 (C=O), 1250; IH NMR: 1.02 (3H, s, 18-CHJ), 3.78 (3H, s, 3-0CHJ), 6.66 (I H, d, J=3Hz, 4-CH of estrone methyl ether), 6.75 (lH, dd, J=3Hz and 9Hz, 2-CH of estrone methyl ether) , 7.23 (lH, d, J=9Hz, l-CH of estrone methyl ether), 7048 (I H, s, vinyl-H), 7.71 {2H, d, J=9Hz, 2-CH and 6-CH of 16-( 4-nitrobenzylidene)} and 8.28 {2H, d, J=9.Hz, 3-CH and 5-CH of 16-(4-nitrobenzylidene)} MS: rnJz 417 (M+) (Found: C,75.13; H,6.77; N,3.11. C26H27 N04 requires C, 74.79; H, 6.52; N, 3.23 %).

3-Methoxy-16-(4-pyridylmethylene)-1, 3, 5 (10)-estratrien-17-one 15. A mixture of estrone methyl ether (0.5 g), methanol (40.0 mL), 4-pyridine carbox-aldehyde (2 mL) and potassium hydroxide (0.5 g) was

shaken for 30 min at room temperature and then re-fluxed for 23 hr. The usual processing gave the pre-cipitate which was crystallised from methanol to af-ford 15 (0.3 g, 96.77%), m.p. 188-90°; UV: 278.6 (4.33); IR: 1720 (C=O), 1260, 1050; IH NMR: 1.01 (3H, s, 18-CH3), 3.78 (3H, S, 3-0CHJ), 6.66 (lH, d, J=3Hz, 4-CH of estrone methyl ether), 6.75 (lH, dd, J=3Hz and 9Hz, 2-CH of estrone methyl ether), 7.26 (lH, d, J=9Hz, 1-CH of estrone methyl ether), 7.36 (lH, s, vinyl-H), 7040 {2H, d, J=-=6Hz, 3-CH and 5-CH of 16-(4-pyridylmethylene)}, 8.68 {2H, d, J=6Hz, 2-CH and 6-CH of 16-(4-pyridylmethylene)}; MS: rnJz 373 (M+) (Found: C,80.92; H,7A6; N,3A2. C2sH27N02 requires C, 80043; H, 7.24; N, 3.75%).

Assessment of estrogenic / antiestrogenic po-tency of a compound

Experimental Protocol A. Measurement of binding affinity for ER

Human recombinant produced in yeast was over-night incubated at OAoC with 5nM eH]E2 in the ab-sence or presence of increasing amounts of either unlabelled E2 (control) or investigated compound (l nM to 10 1lM). Unbound ligands were then removed by a DCC treatment and the residual radioactivity measured by sci ntillation counting.

B. ERE-regulated luciferase expression MVLN cells (tk-vit ERE stably transfected MCF-7

cells) were cultured for 3 to 4 days in 0 35 mm Falcon dishes (planting density 80.000/dish) in 10% serum depleted of endogenous steroid (DCC treatment. Inves-tigated compound (with or without 10 nM E2) was sub-sequently added to the medium and the culture pursued until Firefly luciferase assay (24 hr). For that purpose, medium was removed and cells washed twice with PBS. A minimal volume (250 ilL) of a 5-fold diluted lysis solution (Promega E 153A) was then added to the dishes and the latter maintained under mild agitation for 20 min to extract luciferace. Lysed cells were sub-sequently detached with scraper (Coster 3010) and cen-trifuged for 5 sec at 12,000 g to clarify their extracts. Twenty ilL of such extracts were finally mixed at roOI11 temperature with 100 ilL of a luciferase reactant me-dium (Promega E151A / E152A) prepared according to the manufacturer's protocol. Induced light was meas-ured with a Berthold luminometer with a blank (Rela-tive Light Unit, RLU). To compare RLU data, protein content of each extract was measured by the

172 INDIAN J. CHEM., SEC B, JANUARY 2003

Coomassie method (PIERCE) and the data expressed per mg of protein.

Acknowledgement We gratefully acknowledge the financial support

from the Council of Scientific and Industrial Re-search, New Delhi and Panacea Biotech, New Delhi. We also acknowledge Mr R A Marks, Searle, Illinoi s for the generous supply of estrone. We also wish to thank Dr V L Narayanan for carry ing out the antineo-plastic activity of the compounds.

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