Indi an Jou rnal of Chcmistry Vol. 38B, May 1999, pp. 563-571

Synthesis and biological acti vity of some D-ri I1g modi fi ed estrone deri vati yes

Ranj u Gup ta & Dharalll Paul Jinda l* Univcrsit y In stitutc of PharmacL·u llc.tI Sc icnces, P;lIIjab Un iversit y, ChanJ i ~;, dl. I (,()Il l-i, Inlk

M Bo rras . N Legros & G Lecl ercq Institute Jules 8Q1'det, Centre des TUl11eurs de I' Uni l'e rsite Librc de [3ru xell cs, Rue I k~c r -13orJc t -I- I OOO Brll s,cb. I lcl~i un,

/?ecei\'ed 25 SelJle/llber 1998; accepted (rel'is<,d) 22 March 1999

3-Methoxy- 17-aza-D- hol11 o-1 ,3,5( I O)-estratrienc-16, 17a-d ionc 3. 16, 17a-di oxo- 17-aza-D-homo- 1 ,3,5( IO)-estr;ltric n·J-y i acctate 12 and a serics of rclated compounds havc bcen sy nthcsised fro m 3-methoxy-16-oxil11ino- 1 ,3.5 ( I O)-esu'alriL'n - 17-onc 1 and 3-hydroxy- 16-oximino- I,3,5 ( IO)-estnt trien-17-one 2, respccti ve ly. Thc compounds 3 (DPJ-280). l-t (DPJ-369) . 17 (DPJ-370), 18 (DPJ-354), 19 (DPJ-320), 21 (DPJ- 32 I ), 22 (DPJ-374) and (DPJ-284) ha vc becn cva luatcd for the ir estrogenic/an ti cstrogenic cffccts and the compounds 3-7,8,9,10,12,15,16,18,20,21,23 and 2-t have bcen sc ree ncd for antincoplastic activity at NC I, Bethesda.

D-ring modifi ed steroid, testolactone, a derivative of testosterone is used for the treatmen t of breast carcinoma I .} . I t decreases the circulati ng estrone levels in postmenopausal women with metas tatic breast cancer and behaves as time dependent aromatase inactivator (t ll2=32 min; Ki=35 ~lm)2. A non-steroidal agent, tamox ifen, contallltng 2-dimethylaminoethyl side chain, considered to be important for the antiestrogenic acti ons of tamoxifen and related compounds, is an excell ent drug used for the treatment of hormonal-dependent breast cancer 4,5.

Its principal mode of action has been thought to be as an ant iestrogen 6

.7 in contrast to ami noglutethimide, which is a non-steroidal aromatase inhibitor g.y .

The successful introducti on of steroidal and non­steroidal compounds for the treatment of breast cancer stimulated our interest in the development of new co mpounds containing desired functionalities. We have previously reported 10 the sY:lthesis of heterosteroids which have the functionalities of aminoglutethi mide and 4-hydroxy-4-androstene-3, 17-dione. In thi s study we wanted to assess the es trogenic/antiestrogeni c effects of D-ring modification and fusion involving various groups at the 3-, and 17-position in es trone methyl ether and estrone series.

For thi s purpose, es trone methyl ether was subjected to ox imati on with potassium ferl- butoxide­isoamyl nitrite at room temperature to obtain the keto oxime l it (Scheme I), the structure of which is well

~NH'

o N 0 H

Testolactone Aminoglutethi mide

Tamoxif'en

documented. The ox ime was subjected to Beckmann rearrangement in refluxing acetic anhydride and glac ial acetic acid to obtain the imide derivative 3/cm­I .Characteristic vibrational bands fo r imide system appeared at 1729 and 1673 cm·l. I H NMR spectru m showed singlets at 1.20 (s, J8-CH}), 3.80 (s, -OCH}) and 8.50 (s, NH, exchangeab le) . 3-Methoxy- 17-aza­O-homo-I ,3,5(1 0) -es tratriene-1 6-17a-dione 2 was treated with hydrochlorides of 2-dimethyl­ami noeth y lchloride, 2-diethy lami noeth y lch loride, 1-(2-c hloroethyl) pyrrolidine and 1-(2-chloroethyl )­piperidine, methyl iod ide and Il-butyl iodide, allyl bromide and hydrazine hydrate to obtain the

564 INDIAN J. CHEM., SEC. B, MAY 1999

Scheme I

CH30

Acetic anhydride! pR __________ ~.~ N

Acetic acid 71 0

CH30 ::-...

3 R = H /CH3

4 R =CH2CH~, CH3

C2HS /

5 R = CH~H2N'-... C2Hs

6 R = CH~H2N:J r-'

7 R=CH~H2NJ

10 R=CH~H=CH2

11 R = NH2

Scheme II

-:ompounds 4,5,6,7,8,9,10 and 11, respecti vely (Scheme II). Tertiary amines 4 and 5 were obtained a~ /oily residues, so they were characterised as

. hydrochlorides. All these imide derivatives showed vibrational bands around 1720 and 1670 cm-' in the IR spectrum for imide system and a peak around 4.00 for N-Clh in the '1-1 NMR spectrum.

Similar sequence of reactions was carried out to prepare estrone derivatives. Estrone was converted into 16-oxime 2 and subsequently to imide derivative 12. Treatm~nt of 16,17a-dioxo-17-aza-D-homo-1,3,5(10)-estratrien-3-yl acetate 12 with Il-butyl iodide in presence of anhyd. potass ium carbonate afforded 13, which on alkaline hydrolysi s and treatment with 1-(2-chlorocthyl) pyrrolidine hydrochloride in re tluxing ethyl methyl ketone (MEK) gave 15. ! I J I NMR signals at 0.92 [t, -N (C H~)3CH31 , 1. 1 () (s, 18-CHJ), 3.74 (m, N-CH2) and 4.08 (t, -OCI!:)}. Further treatment of 12 with allyl brdll1ide gave 16 and subsequent hydrolysis yielded 17 . Reaction of 12 with hydrochlorides of 2-dirnethyl­ul'l inoethyJch loride, J -(2-chloroethyl) pyrrolidin.e and J -\ 2-chloroethyl) pipendine and hydrazine hydrate gave the compounds ! 8,1 9.20 and 21. Alkaline

hydrolysis of imide 12 gave 3-hydroxy derivative 22 which on treatment with n-butyl iodide in MEK yielded 3-butoxy-I7 -butyl-I7 -aza-D-homo-l, 3,5(10)­estratriene-I6,17a-dione 23 (Scheme III). In '1-1 NMR spectrum there appeared signals at 8 1.00 [I, 61-1, -N(CI-I2hCH3) and -0 (CH2)3CH3,i.20 (s, 3H, 18-CH3) and 3.60-4.10 (m, 4H, -OCH2 and NCH2) .

Structures of the compounds were established on the basis of spectral and elemental analyses. Tertiary amines 18,19 and 20 were characterised as hydrochloride salts. Quaternisation of 15 with methyl iodide gave 24 (Scheme IV).

r

Biological effects

Estrogenidantiestrogenic activity Potential estrogenic/antiestrogenic aCtlvity of the

compounds 3 (DPJ-280), 14 (DPJ-369), 17 (DP]-370), 18 (DPJ-354), 19 (DPJ-320), 21(DPJ-321), 22 (DP]-374) and DPJ-284 (6)'5 was assessed by measuring their binding affinity for the estrogen receptor (ER) as well as their potency to induce the synthesis of the progesterone receptor (PgR) in MCF-7 human breast cancer cells. Before assays, MCF-7 cells maintained in monolayer culture at 37°C [minimal essentinl medium (MEM) supplemented with L-glutamine, antibiotics and 10% heat inactivated fetal calf serum, all from Gibco, Gent, Belgium] were cultured for at least 3 days in 10% serum depleted of unconjugated endogenous steroids by dextrancoated charcoal (DCC) treatment.

Figure 1 shows that most of these compounds were characterized by a weakly binding affinity for ER (note that E,-internal control-binds with as-fold lower binding affinity than E2 in agreement with our previous data'2).

Present data also clearly establish a relationship between the binding affinity of each compound for ER and its potency to induce PgR synthesis (Figure 1 vs Figure 2) .

As expected, the presence in the compounds of a phenolic group (i.e. DPJ-374 vs 280) increases the ER binding affinity.

Establishment of this correlation clearly indicates that present structural modifications of ring D of estrone to imide do not suppress estrogenic activity.

Antineoplastic activity

The compounds (3-7,8,9,10,12,15,16,18,20,21,23 and 24) were tested at National Cancer Institute, Bethesda, Maryland, USA; in ill vitro against the cell panel consisting of 60 lines. A 4R hr continuoLls

GUPTA el al.: SYNTHESIS OF D-RI NG MODIFIED ESTRONE DERI VATI VES 565

2 Ac etic anhydride/ .. Ac etic ac id

12 R = OAc , R I = H

13 R = OAc. R I = C"Hg

14 R = OH. R I = C4Hg

15 R = OCH~H2N~ , R' = C.Hg

16 R=OAc, R' =CH~H=CH2

17 R = OH, R I = CH~H = CH2

CH3 I /

18 R = OH. R = CH2CH2N" I ~CH3

19 R = O"i . R = CH~H2NV

20 R = OH, R'= CH2CH2NJ

21 R = OH. R'= NH2

22 R = OH, n ' = H

23 R = OC.Hg, R' = C.Hg

Schcme II I

15 CH31 ~ ~CH30 N,C4H9

/'1 0

C~\,CH2CH<P ::::-.

CH3 24

Schcme IV

exposure protocol was used and a sulforhodamine B (S RB ) protein assay was used to estimate cell viability or growth. Selec ti vity analysis of the compounds for differen tial ce llular sensiti vity based on the response parameters G [SO, TG I and LC50 indicate that the compounds have stati sti call y insignifi cant an tineopl as tic acti vity for furth er studi es.

Experimental Section

Melting poi nts reported are uncorrected. I H NM R spectra were recorded on Vari an EM-390, 90 MH z and Bruker AC-300 MH z for so lutions in COCl .] using TMS as the intern al reference (chemical shifts in 8, ppm). IR and UV spectra were obtained usin£!. Perki n- Elmer-882 and Lambd a- IS s pec trophoto~ meters, respectively. Mass spectra were recorded on a VG- II-250 J 70S, The pu ri ty of the compounds was exa mined by thin-l ayer chromatography, Elemental ana lyses were carried out on a Perkin- Elmcr-2400. Ultrav iolet spectra were recorded in methanol (A . I'n IllJ X

nm, figures wi thin parentheses refer to log £ va lu es) and IR spectra were recorded in KBr pellets (v , in I 111.1.'

cm- ).

3-Methoxy-17 -aza-0-homo-1,3,5(1 O)-estl'atriene-16,l7a-dione 3. A mi xture of 1 (2.0g), gl. acetic ac id (64 mL) and acetic anh yd ride (90 mL) was reflu xcd

for 20 hI'. Thc rc'> ultant solution was cooled and so lvent removed under reduced pressure. The rcs idue obtained was washed with water, dr ied and crys tal I ised frolll Illcthan ol to afrord 3 ( l.Og, SO C7£;) . Ill p 21R-20 DC; UV: 226.6 (3,63), 277.4 (3. 10), 285 .6 (3.06); IR : 32 10(-NH), 1729, 1673 (imide); IH NMR: 8 1. 20 (3 H,s, 1 8-CH ~ ),3 . 8 0 (3 H,s,-OCH3), 6.66( I H,sA­CfJ ), 6,70( IH ,d,2-CH), 7.26( IH ,d, I-CH), 8.50 ( IH , s. cxehangcable ,-NJ-I): MS :JllIz 3 13. Anal. Fuund: C. 7'2,56 ;H,7.56; 1\ , -UlJ. Ca lc. for CI')H2J N0,1 : C,72.t){J; H,7 .39; N,4A7%.

17 -( 2-0imcthylaminoethyl )-3-methoxy-17 -3za-0-homo-1, 3, 5 (10)- estratdene-16,17a-dione (4) hydrochloride. A mi xture of 3 (0.2g), 2-dimethyl ­am inoethyl chloride hydrochloride (0.30 g), anhyd. K2C03 (1.0 g) and KI (0.04 g) in acetonitrile ( 100 mL) was refluxed fo r 20 hI' with continuous st irring. The slurry obtained was filtered and solvent removed under reduced pressure. The oily res idue obtained was purified and characte ri sed by preparing its hydro­chloride salt. The res idue was taken in dry sol ve nt ether and dry hydrochl oric gas was passed through it. The prec ipitated materi al was separated and crystalli sed from dry acetone to givC' hydrochl oride sa lt of 4.(0.14 g, 54. 16%); mp:266-68°C; UV: 235 . 278 ,286; IR: 1723, 1674(imide); IH NMR : 8 1.20 (3 H, s, I8-CH}), 2.66-3.03 [8H, m, CH2N(CH 3h], 3.80 (3H, s, -OCHJ ) , 4.20 (2 H, t , N-CH2), 6.66(1 H, 5,4 -

CH), 6.80 ( I H, d, 2-CH), 7.20 ( I H,d, I-Cf-I) , 12,46 (br, I H.N+H). Anal. Found : C, 65.32; H, 7.69; N, 6.50. Calc. for C2]HJ3 N20 1CI: C, 65.60; H, 7.90; N, 6.66%).

17 - (2-0icthylaminoethyl)-3-methoxy-17 -aza-O­h0I110-1,3,5(10)-cstratrienc-16,17a-dione 5 hydro­chloride. A mi xture of 3 (0.2g) 2-dieth yl aminoelhyl­chl oride hydrochl oride (0.30 g), anh yd. K2C03 ( 1.0 g) and KI (0.04 g) in aceton itrile (300 mL) was reflu xed 1'0 1' 20 hI' with conti nuous stirring. The slurry obta ined was fil tered and solvent removed under red uced pressure. The oily res idue obtai ned was purifi ed and characteri sed by preparing its hydrochl oride salt. The res id ue was taken in dr\' solvent ether and dry hydrochl ori c gas was passcd th rough it. The prec ipi tated lIlaterial was separated and crys talliscd from dry ace tone to give hydrochloride sa lt of 5 (0.1 6 g, 57 .89%); mp 192-94°: UV: 231.2,277.9, 286. 1; IR : 1722. 1674 (im ide) IH NMR: 8 1.23 (3H,s, 18-CH3), 1.50 [6H. t, N­(CI-hCH3h], 2.70-3.53 Im,8 H, CH:N(CJ-I2CHJh and meth ylene protons], 3.80 (3 H, s, -OCHJ) , 4.20 (2 H, t, N-CH2), 6.80 (1 H, s, 4-CH), 6.96 ( I H,d ,2-Cf-I). 7.30 ( I H, d, I-CH), 12.20 (br, I H, N+ H ). Anal. Found : C,

560 Il'\D I.-\ N. !. CII EM .. SEC. 13 . MAY 1999

66.52; H. 7.95: N, 6.29. Ca lc. for C2sH;,·l\cO,CI: C, 66. 86; H. ~ . 31::-.I, 6.24%.

homo-1 , 3, 5(IO)-cstratriene-16, l7a-dionc 6. A mixture of compound 3 (0.5 g) (2-chloroethy l) pyrro ltdine hydrochloride (0.75 g) anhyd. K2C03 (2.5 3-Mcthoxy-17 - (2-pyrrolidinocthyl)- 17 -azu-D-

R BA va l u~s

C E, 10 0 '/.

• E, 20 '/,

OP!

22 X 374 1 . 75'/,

17 • 370 0.47 '/,

I' A 3 69 0 .23'/,

19 0 )2 J 0.07'/, 3 C 26 0 o 07 '/,

21 D 32 1 < 0 .01 '/, I! ... 354 < 0 . 0 1 ' I,

IS 0 284(6)< O.CI'/,

100

'0 ... -c e '0 75 .. C7I

" -C C. v ~ 50 a. .. w

"? ....

- ~~'- --~~~iI];:~~~ " .... , , ' .. , ' .............

'q '~ \

\

\ \ \ \

\ \

- - ------ -------- -\- - --~ --- ---\

~ ...... \ ,

"-

" ~ ' ... -

~

-g 25 \

~ o m

o I I / -Il

10 -10

10 -t

10 -s

10

Molarity of compound

--0-

-7 10

-- ..... -0

-, 10

Figure l- Rcla li \'c billeti ng al"linity of compounds. MCF-7 cells were incubated wilh 10'9 M r 3 H]E~ and increasing amounts of COlll pO Ullds. Bou !1C1 ( 1-I]E2 was subscqucntly measured (100%=-1 .500 Jpm!200p I of ccllular eXlract).

100

75 c .£ ...

50 u :l '0 ,= a: 25 01

0..

'0 0

CIo 01 Cl -2 5 .... r. " v "- -50 0;,

0..

-75

-100

-------

0"1. -- - - Basal 100°'.

..... - - --Optimal (stimulation

I I -II

10 -,

10

-10 at 10 ME,)

-, 10

-7

10 Molarity of tom POUnd

DPJ 22 X 374 17 • 370

14 A 369 19 0 320

3 0 280 21 In 321 18 A 354

<> is

28.qS)

-, 10

Figu 1'\' 2-1 '~ 1~ Induc tioll . 1\1C F, - :('11 ·: \\ '(, IC II1cubalcd ei lher in Iltc absence (Conlrol = O'k ) or lite prcscnce of lO' w M of E (optimal " ll:J, .. 'II = it)0; " 10 r,13111,,11 'I.,' . ,;' h!C o f PgR inducti on. Cell s w(' re L' ult ured in parallel wilh illvcstigated compo~nds at eithcr 10, 10,

10 ' :111d :1' ,1/ . C} IO~ (l I! - PgR 1-:\'c1s we rc ~ l l bscqllcnlly measu red ( P~R In'cls ill fll lO lcs/mg prot.; 2 sets of experimen ts: lSI basal = 176; ('I' I im.1 -= :- ~ 1 , 211 ": basal = 165. opllln:lI = 7(6).

GUPTA el al.: SYNTHESIS OF D-RING MODIFIED ESTRONE DERIVATIVES 567

g) and KI (0.1 g) in acetonitrile (250 mL) was ref]uxed for 20 hr with continuous stirring. The slurry obtained was filtered and solvent removed under reduced pressure. The residue so obtained was crystallised from dry ether to afford 6 (0.30 g, 45.79%); mp 142-46°C; UV : 229(3.75), 277.4(3.25), 285.4(3.21); IR: 1720,1671 (imide) ; 'H NMR: 81.16

CH (3 H,s,18-CH3), 2.50-2.80 (m,6H,-CH1N< 2, 3.80 (3 H, s, -OCH3), 4.02 (2H,m, N-CH2), 6.80 (2H,m,4-CH and 2-CH), 7.33 (lH, d, I-CH); MS: m/z 410. Anal. Found: C, 72.84; H, 8.73; N, 6.80. Calc. for C2sH34N203: C, 73.13; H, 8.34; N, 6.80%.

3-Methoxy- 17 -(2-pyrrolidinoethyl)-17 -aza-D-homo-l,3,5(10)-estratriene-16,17a-dione(6) hydro­chloride. / Through a solution of compound 6 (0.1 g) in dry acetone (20mL) was passed dry HCI gas. The precipitated material was separated and crystallised from dry acetone to afford hydrocholoride salt of 6 (0.085 g, 77.96%); mp 240A2°C. Anal. Found : C, 66.87; H, 7.65; N, 6.13. Calc. for C2sHJ5N20J CI: C, 67.17; H, 7.89; N,6.27 %.

3-Methoxy-17 -(2-piperidinocthyl)-17 -aza-D-homo-1,3,5(10)-estratriene-16,17a-dione 7. A mixture of compound 3 (0.5 g) 1-(2-chloroethyl )piperidine hydrochloride (0.75 g), anhyd . K2C03 (2 .5 g) and KI (0.1 g) in acetonitrile (250 mL) was ref]uxed for 20 hr wi th continuous stirring. The slurry obtained was fi ltered and solvent removed under reduced pressure. The residue so obtained was crystallised from dry ether to afford 7 (0.30 g, 44.27%); mp 126-28°C; UV: 228.4 (3.74), 277.4 (3. 18), 285.4nm (3.15); lR: 1723, 1667 (imide): 'H NMR: 8 1.13 (3H,s,18-CH3), 2.26-2.63 [6H,t,-CH2N(CH2h], 3. 80 (3H, s, -OCH3), 3.96 (2H,m, N-CH2)' 6.76 (lH,s,4-CH), 6.93 (IH , d, 2-CH), 7.30 (lH, d, I-CH); MS: m/z 424. Anal. Found: C,)3.21; H, 8.71; N, 6.22. Calc. for C26H3(,Nz0 3; C, 73.57; H, 8.54; N, 6.59%.

3-Mcthoxy-17 -( 2-piperidinoethyl )-17 -aza-D-homo-1, 3, 5(10)-estratriene-16, 17a-dione(7) hydro­chloride. Through a solution of 7 (0.1 g) in dry acetone (20 mL) was passed dry HCI gas. The precipitated material was separated and crystallised from dry acetone to afford hydrochloride ~alt of 7. (0.08 g, 73.57%); mp 220-2i'c. Anal. Found: C, 67.39; H, 7.86; N, 5.S5 . Calc. for C26H37Nz03 CI: C, 67 .73; H, 8.09; N,6.07 %.

3-Mclhoxy-17 -methyl -I7 -aza-D-homo- I,3,5(1 0)­cstratrieni~-16,17a-d iol1(.~ 8. Methyl iodide (I mL)

was added to a solution of compound 3 (0.3 g) in dry acetone (ISO mL). Anhyd. K2C03 (1.5 g) was added to the above hot solution with stin·ing. The reaction mixture was refluxed for 3 hr with continuous stirring. The slurry obtained was filtered and solvent removed under reduced pressure. The residue so obtained was washed with water and crystallised from methanol to afford 8 (0.20 g, 63.87%); mp 164-69°C; UV: 226 (3.89),278 (3.33), 285 (3.28); IR: 1721, 1672 (imide); 'H NMR: 8 1.16 (3H,s,18-CH3), 3.16 (3H,s, NCH3), 3.80 (3H, s, OCH3), 6.70 (lH, s, 4-CH), 6.90 (lH, d, 2-CH} 7.26 (I H, d, I-CH); MS: m/z 327. Anal. Found: C, 72.99; H, 7.48; N, 4.20. Calc. for C2oH2SN0 3: C, 73 .36; H, 7.69; N, 4.28%.

17 - Butyl-3-methoxy-17 -aza-D-homo-l,3,5( 10 )­estratriene-16,17a-dione 9. n-Butyl iodide (2 mL) was added to a solution of 3 (0.1 g) in dry MEK (50 mL). To the hot solution was added anhyd. K2C03

(0.5 g) with stirring. The reaction mixture was retluxed for 5 hr with continuous stirring. The slurry obtained was filtered and solvent removed under reduced pressure. The residue so obtained was washed with water and crystallised from n-hexane to afford 9 (0.075 g, 63.69%); mp 83-86°C; UV: 224.27 (3.86),277.4 (3.21); IR : 1721,1666 (imide) 'H NMR: 80.90 [3H,t,N-(CH2hCH3], 1.16 (3H, s, IS-CH3), 3.80 (2H, s, -NCH2), 3.83 (3H, s, OCH3), 6.73 (lH,s,4-CH) 6.90 (lH, d, 2-CH), 7.30 (lH, d, I-CH) . Anal. Found: C, 74.45; H, 7.94; N, 4.06. Calc. for Cn H3,NO j : C, 74.76; H, 8.46; N, 3.79%.

17 -Allyl-3-methoxy-17 -aza-D-homo-l,3,5(1 O)-cs­tratriene-16,17a-dione 10. A mixture of 3 (0.5 g), allyl bromide (2 mL) and dry MEK CWO mL) was heated for 10 min. Anhyd. K2C03 (0.5 g) \'"as added with continuolls stirring. The reaction mixture was refluxed for 4 hr with continuous stirring. The sl urry obtained was filtered and solvent removed under reduced pressure. The residue so obtained wa, crystallised from methanol to afford 10 (0.40 g, 71 %); mp: 138-40°C; UV: 227 (3.77), 277.4 (3.21), 285.6nm (3 .17); IR: 1716,1667 (imide); 'H NMR: 81.20 [3H, s, 18-CH3J, ~ .. 76 (3H, s, O-CH3) , 4.33 (2H, s, N-CH2)' 5.10 (2H, dd, N-CH2CH=CH2),5.70(lH, t, N­CH2CH=CH2), 6.63 (lH, s, 4-CH), 6.76 (lH, d, 2-CH), 7.20 (lH, d, I-CH) . Anal. Found: C, 74.55; H, 8.05; N, 4.30. Calc. for' C22 H27N03: C, 74.75; H, 7.70; N,3 .96%.

17-Amino-3-mcthoxy-17-aza·D-homo-l, 3,5(10)­estratriene-16,17a-dione 11. Tile compound 3 (0.2

56~ INDIAN J. CHEM., SEC. B, MAY 1999

~), was stirred with hydrazine hydrate (20 mL) at room tcmpe raturc under nitrogen for 20 hr. The so lve nt was removed under redu ced pressure and residuc obtaincd was dried overni ght and reflu xed in xy lellt~ fo r 4 hr usi ng Dean-Stark apparatu s. The ~(\ I ve nt was removed ill vacuo. The product obtained \\'as washed wi th pc t. ether, water and crys talli sed from aq. methano l to give 11 (O.l g, 47.07%); Il1p 168-7U:e: UV : 229 (4.03), 277 (3.40); JR : 3250 (-NH stretch ), 17 19, 1656 (im ide); IH NM R : i5 1. 16 (3 H, s, I S-C H1,) , 3.76 (3 H, s, -OCH.1), 4.50 (2 H, br, exchangeab le, N-NH2), 6.53-6.83 (2H, m, 4-CH and 2-CH), 7.33 (l H, d, I-CH); MS: m/z 328. Anal. r ound : C, 69.25; 1-1 , 7.55; N 8. 11 . Ca lc. for C I9 H c~ N ~03 : C, 69.48; H, 7.36 ; N, 8.53%.

3-Hydroxy-16-oximino-I,3,5 (10)-cstratrien-17-one 2. A mi xture of es trone (2.0g) in potass ium l ert­

butox ide rprepared by di sso lving potass ium metal (0.8 g) in lert-bu tanol (20 Il1 L)] was stirred for 2 hr at room temperature under nit rogen atmosphere. Isoamyl nitri te (1.8 mL) was then added and reacti on mi xtu re was kept at room temperature fo r 17 hr. Precipi tated material was disso lved in water (200 mL) anc! so luti on obtained was was hed with ether. Aqueous layer 'vvas separated and acidified with glacia l aceti c acid. Separati on of prod uct was complete in 2 hr. The separated prod uct was filtered, wa <, hed and dri ed . The solid obtained was crys talli sed from metha nol to afford 2 ( 1.2 g, 54%); I11 p 2 18-2() ~ C; UV: 231, 279; JR : 3366 (N-OH), 1733 (C=O); IH NMR: 8 1.06 (3 H, s, 18-CH3), 6.50 ( 11-1 , s, 4-CH), 6.56 ( IH , d, 2-CH), 7.06 ( IH , d, I -CH ), 8.76 ( IH , s, achangeable).

16, 17a -Dioxo-17 -aza-D-homo-l ,3,5(10)-estratri­en-3-yl aceta tc 12. A mixture of compound 2 (0.5 g), a 'el ic an hyd ride (25 mL) and gl. acetic ac id (15 mL) \\ as rcfl uxed for 20 hr under anhyd. condit ions. The solid res idue obtained after removing the so lvent ul ldcr reduced pressure was washed with water. The mate ri al obtained was crys talli sed from acctone to afford 12 (0 .3 g, 52.63 %); mp 196-98°C; UV : 224 (3.64). 266.6 (2.95), 274.2 (2. 89); IR : 1759, (­OCOCrJ.,) , 1720, 1696 (imide); IH NMR: 8 1.22 (3H, s, 18-CHJ), 2.29 (3 H, s, -OCOCH3), 2.88 (3H ,. m, 15-CH:. ), 6.82 (l H, s, 4-CH), 6.88 ( I H, d, 2-CH ), 7.2 8 ( I H, d, I-CH ), 8.50 ( I H, s, exchangeable, -NH):, 13CNMR : 16.36 (CH3), 2 1. 11 (CH]) 11 9.0 (CH), 12 1 ,44 (CH), 126.38 (CI-I) , 136.61 (), 137.48(), 148.68 (C, 3-C), 169.2 (C=O), 172.16 (C, 16-C, C=O), 178.78 (C; 17a-C, C=O). Anal. Found: C, 69.99; H,

6.52; N, 3.93. Calc. for C2oH 2JNO~: C, 70.36; 1-1 , 6.79; ',4. 10%.

17-Butyl-16, 17a-dioxo-17-aza-D-homo-l,3,5 (10)-cstratrien-3-yl acctate 13. A mixture of 12 (O.2g), dry MEK (50 mL) and anhyd . K2CO] (0.5 g) was retlu xed with stirri ng for 10 min. II -Butyl iod ide (2 mL) was added to the stirred so lu tion and mix ture ',\"as relu xed for 5 hI'. The slu rry obtained was fil tered and so lid obtained after reillov ing Ihe so lvcnt was \\ as hed wi th water ~lIld crystal I iscd frolll Illethanol to an'o rc! 13 (0.15 g, 65.2%): mp 174-n GC; UV: 223.6 (3. 79),267 (2.88), 274.2 (2.85); IR: I 754(-OCOCH3),

17 17, 1668 (imide) ; 1 H NM R: 8 0.90 (3 H, t, N­(C H2h -CH}), 1.1 6 (3 H, s, 18-CH3), 2.30 (3 H, S,

-OCOCH3), 3.80 (2 H, t, N-CH2), 6.86 ( 11-1, s, 4-CH), 7.00 (11-1 , d, 2-CH), 7.36 (lH, d, I-CH); MS : mlz 397. Anal. Found : C, 72.29; 1-1, 7.44; , 3.79. Calc. for C24 H31N04: C, 72.5 1; H, 7.86; N, 3.52%.

17-Butyl-3-hydroxy-17-aza-D-hol11o-1 ,3,5 (10)­cstratriene-16,17a -dionc 14. A mixture of 13 (0.5 g), K2COJ (1.0 g) and 10% aq. methanol (50 mL) was stirred at roo m temperature for 1 hr. The reac tion mi xture was poured in water and the prec ipitated materi al was filtered and dri ed. The solid obtained was crystall ised from methano l to give 14 (0.3 g, 67.35 %), mp 2 10-1 2°C; UV: 223 (3 .72), 280 (3. 13); lR: 17 16, 1650 (i mi de); 1 H NM R: 8 0.92 [31-1 , t, N­(C I-I ~)3CH3], 1.1 6 (3 1-1 , s, 18-Cf-I3), 2.84 (2 1-1 , m, 15-C H2), 3.76 (2 H, t, N-CH 2), 5.49 ( I H, s, exchange­ab le,OlD ; 6.59 (1 1-1 , s, 4-CH) , 6.65 (11-1 , d, 2-CH) , 7. 17 ( 11-1 , d, I-CH); 13C NMR: 13.87 (CH3)" 16.58 (CH.1) 11 3. 14 (CH ), (CH) 115 .14 (CH), 126.49 (CH), 13 1.28 (C), 137.59 (C), 153.94 (C, 3-C), 172.09 (C, 16-C, C=O), 178.9 1 (C, 17a-C, C=O). Anal. 74.61; H, 8.55 ; N, 4.25. Ca lc. for C2~H 2~NOJ: C, 74.33; H, 8.22 ; N, 3.94%.

17-Butyl- 3-(2-pyrrolidinoethoxy)·17 -aza-D-ho-1110-1,3,5 (10) estratricne-16, 17a-diune 15. To a ~olutio n of 14 (0.3g) in dry MEK (150 rnL) was added 1-(2-chloroethyl) py rro li dine hydrochl oride (0.45g) . Reaction mixture was heated for 10 mi n. Anhydrous K2C0 3 and KI (0.0255g) were then added. The mixture was stirred and retluxed for 5 hr. The slurry was filtered and residue obtained after removal of so lve nt was crystalli sed from di stil led water to afford 15 (O.3 Ig, 8 l.57%); mp 74-76°; UV: 224.6 (3.89), 277.8 (3. 18); lR : 172 1, 1667 (imide); IH NMR: 8 0.')2 [3 1-1 . t, N (C H2)3CH3] , 1.1 6 (3 H,s, 18-Clh), 1.80 (6H,I11 , methylene protons), 2.50-3 .03 ( 101-1 , m, pyrro-

GU PTA el al.: SYNTH ESIS OF O-RI NG MO DI FIED ESTRON E DERIV ATIVES 569

lidino and methylene protons) 3;74 (2 H,m N-CH2)' 4.08 (2 H, t, Ct., OCH2), 6.66 ( 1 H,s, 4-CH), 6.75

13 / (l H,d,2-CH), 7.2 1 (lH, d,I-CH) ; C NMR: ' 13 .83 (CH3), 16.54 (CH3) , 67.02 (CH2) 11 2.47 (CH) 114.35 (CH), 126.24 (CH), 131.34 (C), 137 .27(C} 157.04 (C, 3-C), 171.74 (C, 16-C, C=O); 178.69 (C, 17a-C, C=O); MS, mlz 452. Anal. Found: C, 73.87; H, 8.70; N, 5.83. Ca lc. fo r C2sH40N20 f C, 74.29; H, 8.9 1; N, 6.19%.

17 - Butyl-3-(2-pYlTolidinoethoxy) -17 -aza-D-ho­mo-l ,3,5(10)estratriene-16, 17a-dione methiodide 24. Methyl iod ide (0.5mL) was added to a solution of 15 (0.25g) in absolute eth anol (50 mL) at roo m temperatu re and the sol uti on was refl uxed fo r 1 hr. The reaction mixture was cooled, filtered and dried to afford 24 (0.3 g, 90.9%); mp softens at 80°C, melts at 220-22°e. Anal. Found: C, 58 .1 8; H, 7.29; N, 4.39. Calc. fo r C29H43N203l: C, 58 .58; H, 7.28; N, 4.7 1 %.

17 -AlIyl-16-17a-dioxo-17 -aza-D-homo-l,3,5(10)­estratrien-3-yl acetp.tc 16. To a soluti on of 12 (0.5 g) in MEK was added all yl bromide (2 mL) and heated for IO min. Anhydrous K2COJ (1 .5 g) was then added and reaction mi xture was refl uxed wi th stirring fo r 3.5 hr. The slulTY obtained was fil tered and res idue obtained after remova l of solven t was crystallised from meth anol to affo rd 16 (0.47 g, 84 .1 2%) mp 180-82°C; UV: 223 .8 (3.67) , 267 (2.88),274.2 (2.84): IR: 175 I (-OCOCH3) 1720, 1673 (imide); 'H NMR : 8 ] .20 (3 H, s, 18-CH3), 2.26 (3 H, s, -OCOCH3), 4.33 (2H, d, N-CH~), 5. 10 (2H, dd, N-CH ~CI-I=CH2) , 5.60 (1 H, m, N-CH 2CH=CH2), 6.80 ( I H, s, 4-CH), 6.90 C H, d, 2-CH). 7.23 ( IH, d, I-CH ). Anal. Found: C, n. I] ; H, 7.38; N, 3.96. Calc. for C23 Hn N04: C, 72.4 1; H, 7.1 4; N, 3.67 %.

17-Allyl-3-hydroxy-17-aza-D-homo-l , 3, 5 (1 0)­estratricnc-16,17a-dione 17. A mi xture of 16 (0.4 g) K2COJ (1.5 g) and aqueous meth anol (50 mL) was sti rred at room temperature fo r I hr. The slurry obtaincd was poured into water. The prccipit:ltcd materi al was crysta l I ised fro m methano l to afford 17 (0.325 g, 9 1.54%); Il1p 238-40°C; UV: 226 (3 .72), L80 (3 .26); IR : 3454, (-OH) 171 9,166 1 (imide); IH

MR . 0 I :'! I '.-"i .~ , I ~-C I U , 4.30 (2Jl, \:. ~ Cff.I , 5.03 (2H, dd , N-CH~CH=CH2 ) 5. 56, ( I H, Ill, N­CH 1CH=CH c). 6.50 ( I H, s, 4-Cff), 6.60 ( I H , d, 2-CIf). 7.00 ( I! I, d, loCH) , 8.M (! H, ~ ,c xchangcaj,k ,

O /-f ): An al. Found: C, 74. 10; II , 7.73: N, 4.41 Calc . forC 211i . .. N0. ,· C. 74.30; l1. 7.42; N, 4. l 3%.

17 - ( 2-Dimethylaminoethyl )-3-hydroxy-17 -aza­D-homo-1,3,5 (10) estratrielle-16,17a-dione(18) hydrochloride. To a solution of 12 (0.3 g) in dry acetonitril e (200 mL) was added 2- dimethylamino­ethylchloride hydrochloride (0.45 g) and heated for 10 mi n. To the hot solution, was added anhydrous K2C0 3 (l .0 g) and KI (0.60 g). The mixture was reflu xed fo r 6 hr with continuous stirring. The slurry obtained was filtered and the residue obtained after removal of solvent was sti rred with K2C03 (0.5 g) in ag. methanol for 3 hr. The resultant mixture was poured into distilled water and extracted with dichloro­methane. The oil y residue obtained after removal of solvent was puri fied and characterised by preparing its hydrochloride salt. The residue was taken in dry solvent ether and HCI gas was passed th rough it . The preci pitated mate rial was separated and crystall ised from dry acetone to give hydrochloride salt of 18 (0.15 g, 47.9%); mp 280-82°C; UV: 230.9, 280.5; l R : 340 1 (-OH), 1723 ,167 1 (imide) ; IH NMR : 81. 14 (3 H, s, I S-CH 3), 2.5 1 (6H, m, methylene protons), 2. 80 [8 H, s, -CH2N(CH3hl, 3.95 (2H, m, N-CH2) , 6"+7 (1 H, s, 4-CH), 6.56 (l H, d, 2-CH) , 7.07 (l H, d, 1-CH), Anal. Found : C, 64.65; H, 7.80; N, 6.80. Calc. fo r C22 H3IN20 )CI : C, 64.92; H, 7.67; N, 6.88%.

3-Hydroxy-17 -(2-pyrrolidinocthyl)-17 -aza-D-ho­mo-l,3,5(10) cstratriene-16, 17a-dione (19) hydro­chloride. To a sol ution of 12 (0.5 g) in dry acetoni tri le (150 mL) was added 1-(2-chloroethyl) pyrroli dine hydrochl oride (0.75 g) and hcated for 10 min . To the hot soluti on was added anhyd . K2COJ

(0.1 g) and KI (0.06 g). The mixturc was rc rluxed for 6 hr with conti nuous st irring. The slurry obtained was fil tcred and the residue obtained aft er remo\ :11 of solvent was stirred wi th K}C03 (0.5 g) in aq. meth anol for 3 hr. The resultant mi xture was poured into di stilled water and extracted with di chloro­methane. The oily res idue obtained 3fter removal of solve nt was purified and characterised by preparing its hydroch loride salt. The residue was taken in dry solvent ether and dry hydroch loric gas was passed through it. The preci pitated material was separated and cryst;l llised from dry acetone to g.i ve hydrochlOI';de salt of 19 (0. ] 0 g, 15.8%): mp j 76-~'I~ C; Uv . 2:1: . :~n . -': 1 , "1,1 ' 1 ( ,\1 1 \ 1"7 " " - .

(imide) ; I H NMR : 8 1. 18 (3 H, s, I ~ -C f-ll ) ' 4.04 (2 ]1 .

111, N-CH2)' 6.48 ( IH , s, -CH). 6.56 ( I H, d, .2 C1I " 7.0 1 ( I H, d, loCH): 8.92 (br, IH). J\n,ti. F(~ lind . C 66.23 ; H, 8.08 ; N, 6.30. Calc. for C21 H n '· n ,(" C' 66.57 ; I-J, 7.68; 1, 6.4 7%.

570 INDIAN J. CHEM ., SEC. B, MA Y 1999

3-Hydroxy-17 -(2-piperidinoethyl)-17 -aza-D-homo-1,3,5(10) estratriene-16, 17a-dionc (20) hydro­chloride. To a solution of 12 (0.2 g) in dry acetonitrile (l00 mL) was added 1-(2-chloroethyl) piperidine hydrochloride (0.3 g) and heatcd for 10 min . To the hot solution was added anhyd. K2C03 (0.75 g) and KI (0.04 g). The mixture was retluxed for 6 hr with continuous stirring. The slurry obtained was filtered and the residue obtained after removal of solvent was stirred with K2C03 (0.5 g) in ag. methanol for 3 hr. The resultant mixture was poured into distilled water and extracted with dichloro­methane. The oi ly residue obtained after removal of solvent was purified and characterised by preparing its hydrochloride salt. The residue was taken in dry solvent ether and dry hydrochloric gas was passed through it. The precipitatcd material was separated and crystallised from dry acetone to give hydrochloride salt of 20 (0.07 g, 27%); mp 198-200°C; UV: 224, 280; IR: 3461 (-OH) 1723,1674 (imide); IH NMR : 8 1.14 (3H, s, 18-CH3), 4.02 (2H, m, N-Cfl2)' 6.47 (lH, s, 4-CH), 6.56 (IH, d, 2-CH), 7.10 (I H, d, l-CH), 9.64 (br, IH). Anal. Found: C, 67.35; H, 8.15; N, 5.92. Calc. for C2s HJsN20 JCI: C, 67.60; H, 7.89; N, 6.27%.

17 - Amino-3-hydroxy-17 -aza-D-homo-l,3,5(10)­estratriene-16, 17a-dione 21. A mixture of 12 (1.0 g) and hydrazine hydrate (80%, 25 mL) was stirred overnight under nitrogen atmosphere at room temperature. The solvent was removed under reduced pressure. The solid residue obtained was dried overnight and was retluxed with xylene using Dean­Stark apparatus. Solvent was removed ill vaclIo and product obtained was washed with pet. ether and water. Resultant product was crystallised from methanol to afford 21 (0.6 g, 68.1 %); mp 300-02°C; UV: 225.2,(3.73); IR : 3421 (-OH) 1719,1665 (imide) ; I H NMR : (5 1.20 (3H, s, 18-CH3), 5.10 (2H, br, N-NH2)' 6.50 (lH, s, 4-CH), 6.60 (IH, d, 2-CH), 7.00 (lH, d, I-CH), 8.70 (lH , s, -OH) . Anal. Found: C, 68 .53 ; H, 7.14; N, 8.77. Calc . for ClsHn N20 3: C, 68.76; H, 7.05; N, 8.91 %.

3-Hydroxy-17 -aza-D-homo-l,3,5 (10)-estratri-enc-16,17a-dione 22. A mixture of 12 (0.2 g), K2C03 (0.5 g) and ag . methanol (50 mL) was stirred at room temperature for I hr. The reaction mixture was poured in water and extracted with dichloromethane. The solid obtained after removing the solvent was crystallised from methanol to afford 22 (0.15 g, 65.77%); mp 294-96°C; UV: 224.8,(3 .66); 280nm

(3.22); IR: 3428 (-OH) 1716,1671 (imide); 'H NMR (5 1.16 (3H, s, 18-CH3) , 6.43 (I H, s, 4-CH), 6.46 (l H, d, 2-CH) , 7.03 (I H, d, I-CH), 8.80 (l H, s, exchange­able). Anal. Found: C, 71.98; H, 7.06; N, 5.07.Cale. for Cl sH2IN03: C, 72.21; H, 7.07; N,4.67%.

3- l3utoxy-17 -butyl-17 -aza-D-homo-l,3,5(10)-es­tratricne-16,17a-dione 23. To a stirred solution of 12 (0.4 g) in MEK (150 mL) was added K2C03 (2.0g). The reaction mixture was heated and stirred for 10 min . n-Butyl iodide was then added and the mixture retluxed for 6 hr. The slurry was filtered and solid obtained after removal of solvent was crystallised from ag. methanol to afford 23 (0.38 g, 69.2%); mp 98-100°; UV: 229.4 (3 .81 ); 278.5 (3.23); 286.6 (3.18); IR: 1719,1669 (imide); IH NMR: (5 1.00 [6H, t, N-(CH2)3CH3 and -O-(CH2hCH3], 1.20 (3H, s, 18-CHJ) , 3.60-4.10 (4H, m, -OCH2 and N-CH2), 6.66 (lH, s, 4-CH), 6.70 (lH, d,2-CH), 7.20 (lH, d, 1-CH). Anal. Found: C, 75.40; H, 8.81; N, 3.22. Cale. for C26H37N03: C, 75.87; H, 9.06; N, 3.40%.

Pharmacology

Material and Methods

I3inding affinity to estrogen receptorl2. Relative binding affinity (RBA) of the compounds 3 (DPJ-280), 14 (DPJ-369), 17 (DPJ-370), 18 (DPJ-354), 19 (DPl-320), 21 (DPl-321), 22 (DPJ-374) and DPl-284 to estradiol (E2) and estrone (E I) was performed by who le cell assay, a method which provides an index of the abi lity of the compounds to acti vate ERI2. In practice cells were plated in 24-mul tiwell dishes (1.5x104 cells I well in mew without phenol red and 10% DCC treated serum. After 4 days of culture, thi s medium was removed and replaced by a serum free medium containing 10.9 M eH]Ez (-- 80 Ci/mole, Amersham, UK) with or without increasing amounts of investigating compounds. After 1 hr of incubation, this medium was removed and monolayer washed three times with an ice cold buffered saline solution (PBS). Bound eH]E2 was then extracted from the monolayer by a final incubation with (250 ilL) ethanol at room temperature for 20 min . The radioactivity of these extracts 200 ilL was then measured by scintil lation counting. Specifically incorporated radioactivity in the presence of unlabeled E2 and a given compound gave the RBA values of the latter for ER ; RBA= [l so]E21 [150] ([150]E2/(l50)][Iso]compound x l 00. All measurements were performed in triplicate.

GUPTA etal.: SYNTHESIS OF D-RING MODIFIED ESTRONE DERIVATIVES 571

Progesterone receptor induction 13, Cells were incubated for 3 days in 10% DCC-treated serum in the presence of a given compound at either 10. 10

, 10.8

or 10.6 M (T-175 flasks); control cells were maintainted in culture with or without E2 at 10-10 to establish the optimal level in PgR induction (ratio of receptor levels respectively in the absence or presence of E2). Treated and untreated cells were subsequently detached from the flasks with 1 mM EDTA in Hank's balanced salt solution (HBSS) without Ca2+ and Mg2+. Cellular suspensions were centrifuged at 800g and the pellets were washed once with (HBBS) and twice with the homogenization buffer (i.e., lOmm phosphate buffer, pH 7.4, containing 1.5 mM EDTA and I' mM monothioglycerol). Cytosol was obtained by homo­genization of the final pellet by means of a teflon­glass homogenizer and centrifugation at 130.000 g for 1 hr (cytosol protein content 1 to 3.5 mg/mL). Cytosol PgR (cytosol PgR concentrations were measured by Scatchard plot analysis according to the procedure established by the EORTC Receptor groupl 4 using eH]ORG-2058 (50Cilmmole; Amersham U .K ) as labelled ligand).

Acknowledgement

The authors are thankful to Dr V L Narayanan , NCI, Bethesda, USA, for carrying out the antineo-

plastic activity. We are grateful to Panjab University for providing financial support, and "The MEDIC Foundation" for sponsoring the financial cost of biological studies. We extend our thanks to Mis Schering Aktien-gesell Schaft. Berlin, Germany, for the generous supply of estrone methyl ether. We thank Mr S.K. Sethi for his technical assistance.

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