'la-Methyl- and 118-Ethoxy-Substitution of [ 1251]-16a-Iodoestradiol

Jun 24, 1990 - Hasrat Mi, Jacques Rousseau, and Johan E. van Lier'. MRC Group in the ...... the Lady Davis Institute for Medical Research, Montreal,...
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J.Med. Chem. 1993,36, 264-271

264

'la-Methyl- and 118-Ethoxy-Substitution of [1251]-16a-Iodoestradiol:Effect on Estrogen Receptor-Mediated Target Tissue Uptake? Hasrat Mi, Jacques Rousseau, and Johan E. van Lier' MRC Group in the Radiation Sciences, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada JlH 5N4 Received August 24, 1992

The 'la-methyl and 118-ethoxy derivatives of 16a-[1251]iodoestradi~l were prepared via halogen exchange with 1251of the corresponding 16&bromoestradiol precursors. The 16a-bromo derivatives were obtained via halogenation of the analogous 17-enol acetate, epimerization to the l6@-isomer, and hydride reduction. Stereochemical assignments were based on high resolution 'H NMR. To evaluate the effect of the nature and stereochemistry of the 16-halo substituent on the relative binding affinity for the estrogen receptor, the analogous 16-chloro derivatives were also prepared. The highest binding affinities were observed with the 7a-methyl-l6a-haloestradiols, particularly the bromo and chloro derivatives while the l6a-iodo derivatives gave somewhat lower values. Both the 11/3-ethoxy and 'la-methyl-16a- [1251]iodoestradiolslocalize in the uteri of immature female rata via a receptor-mediated process. Rapid blood clearance of the I25I-labeled'la-methyl derivative results in lower lZ5Iuptake by the uterus as well as nontarget organs as compared to the 11ssubstituted estradiol analogs. However, uterus to blood and nontarget ratios are more favorable for the 'la-methyl-16a- [12~I]iodoestradiolas compared to the analogous 11s-ethoxy derivatives suggesting that this compound substituted with l23I may be useful for the in vivo imaging of estrogen receptor-rich breast tumors by single photon emission computerized tomography.

Introduction Early detection of mammary tumors is perceived as the best way to reduce the mortality rate from breast cancer in women and also provides the basis for selective therapeutic approachesin the management of this disease. Since a large percentage of breast cancers are rich in estrogen receptor (ER), many attempts to use y-emitting estrogens for ER detection have been made.' Radioisotopes which have been used to label estrogens for receptor imaging include 1311, lZ3I,18F,"Br, and W. Although in vivo images of ER-rich tissues have been reported with some derivatives, none of these products have found routine clinical applications and studies to develop a suitable radiopharmaceutical for ER imaging are in progress in severallaboratories. A radioiodinated estrogen analog with high binding affinity for ER could have important clinical applications through the use of the lZ3Ianalog and single photon emission computerized tomography (SPECT) imaging. Many estrogen analogs have been studied for this purpose' and among them 160iodoestradiol(16a-I&) stands out as a simple derivative which closely resembles the parent molecule, estradiol, and which exhibits high affhity for the estrogen receptor." 16u-lz5IE2has proven to be an excellent ligand for the ER and it is now routinely used for the in vitro quantification of ER in biopsy material. The usefulness of a y-emitting 16a-IEz analog for in vivo ER imaging is however limited due to rapid metabolism of this steroid.5~6Attempts to slow down its metabolism via substitution at the 118position have shown promise.' Addition of a 118-methoxy group decreases metabolism of 16a-IEz while the iodine at C-16 prevents interaction with l7g-hydroxy steroid dehydrogenase: the enzyme which converts estradiol to estrone. Furthermore, substitution at the 118-position increases the stability of the ER-steroid complex and 'Preeented in part at the Eighth International Symposium on Radiopharmaceutical Chemistry, Princeton University, Princeton, NJ, June 24-29, 1990.

reduces nonspecific binding. Likewise, addition of a 7amethyl substituent has ahown to enhance interaction of estradiol with ER.9 Radioiodinated 17a-(iodovinyl)estradiolderivativesloconstitute another classof promising ligands where uptake by ER-rich target tissues has been shown to improve substantially upon lid- or 7a-substitution.lOb In this study we describe the synthesis,characterization, and structure-activity relationships of the 7a-methyl and 11d-ethoxy derivatives of the 16a-halo- (I, Br, C1) and [1251] 16a-iodoestradiolsin relation to the earlier reported 110-methoxy analogs." The lZ5I-labeledproducts were prepared via a rapid halogen exchange reaction. Relative binding affinities (RBA) for the estrogen receptor were measured via an in vitro assay, and tissue distribution and ER-mediated uterus uptake was established in immature female rats.

Chemistry The 16-substituted halo steroids were prepared by an adaptation of the Johnson and Johns procedure'2 (Scheme I). The estrone derivatives (la,b) were converted to the estrone 3-acetate 17-enol acetate (2a,b) and brominated with Brz in acetic acid to give exclusively the 16abromoestrone 3-acetate derivatives (3a,b). The acetate group was then hydrolyzed with concentratedsulfuricacid in absolute ethanol at room temperature to give the 16abromoestrones (4a,b),which were converted to the 16gbromoestrones (Sa,b) via epimerization with LiBr in 2-butanone. The 16g-configurationin the final products was confirmed by the characteristic downfield shift of the C-18 protons in the 'H NMR.13 Whereas the epimeric 7a-methyl derivatives 4a and Sa separated well on a silica gel column, the analogous 116-ethoxyepimers 4b and Sb did not separate under these conditions. The lea-bromo estrones (4a,b) were reduced with lithium aluminum hydride to give a mixture of lea-bromo diols, epimeric at the (2-17 position (6a,7aand 6b,7b). The mass spectra of 6a,7a and 6b,7b showed pairs of molecular ions in about

oO22-2623/93/1836-0264$O4.oO/0 0 1993 American Chemical Society

P~I]-l6a-IodoestradiolDerivatives

Journal of Medicinal Chemistry, 1993, Vol. 36,No. 2 266

Scheme I

2

I 1 4 16a-CI; 17p-OH 1 5 16a-CI; 17a-OH 16: 1wCl; 17p-OH

3: YrOAc 4 Y=OH

6: 17fWl-l 7: 17a4H

5

8

10: YoOAC; 16a-cI 11: Y=OAc; 1Sfl-Cl 12:YrOH; l6a-cI 13: Y =OH; 1Sfl-Cl

a: R=H;

9

X=CH3

b: R=OC&Iq X=H C:

R=OCH3;

d: R=H;

X=H X=H

1:l ratios indicating the presence of bromine in both compounds. The C17-H was shifted downfield towards the C16-H, which is consistent with the cis stereochemistry of the C17a-H and the 16-bromosub~tituent.'~ Reduction of the 16B-bromoderivatives5a,bwith sodium borohydride in ethanol yielded the 16&bromo-17fi-estradiols 8a,b together with the debrominated producta. The 16aiodoestradiol derivatives 9a,b were obtained by halogen exchange of the 16D-bromo in 8a,b using NaI or [12sIlNaI.11414 The halogen exchange reaction (2 h) gave the radioiodinated producta in 60-75 95 yield. The compounds were purified by HPLC on a reverse-phase column in MeOH/H20. The analogous 16-chloroestradiols were obtained by treatment of the diacetates 2a,b with tertbutyl hypOChlOrite,lSwhich afforded a 3:l isomericmixture 10a,lla and 10b,l lb of the 16a/@-c-chloroeatrone3-aceta~ which were converted to the free phenols 12 and 13, respectively, via acid hydrolysis. The 16-chloroepimers 12a,13aand 12b,13bwere separated on a silica gel column. The 16a-chloroestronederivativea 12a,bwere reduced with sodium borohydrideto theC17-epimericalcohols 14a,15a and tab, lbb, whereas reduction of the l6g-chloro analog 13a gave exclusively the 17g-alcoholanalog 16a.

Biological Properties The relative binding affinities (RBA)of 4-9, 12, and 14-16 for the ER were measured via a competitivebinding assay with [3Hlestradio11sand are summarized in Table I. The highest affiiities are observed with the 16ahalogenated steroids containing the natural 178-hydroxy configuration in the order of Br > C1 > I which is in agreement with literature data on related compound^.^^ Between the 7a-methyl and llg-ethoxy derivatives the former gave substantially higher RBA values. Halogenated estrone derivatives showed only a weak binding affinity for the ER.

Table I. Relative Binding Affiitiea' of Substituted l&Haloeatrogene for Murine Cytoplasmic Estrogen Receptom compd

49 4b 79 7b 69 6b 60

7a

Me

substituents 118 17 keto OEt keto a-OH a-OH OEt @-OH &OH OEt OMe @-OH keto 8-OH OEt &OH 8-OH @-OH OEt keto keto OEt

16 a-Br a-Br a-Br a-Br a-Br a-Br

RBAfSD

**

28.4 3.7 0.8 0.1 Me 13.6 f 0.3 1.1 f 0.1 Me 79.1 f 11.8 16.8 1.0 a-Br 20.e lh Me 8-Br 6.3 0.4 8. Me 8-Br 7.1 f 0.7 8b &Br 0.6 fO.3 9. a-I 66.2 f 3.6 -Me 9b a-I 16.6 f 1.1 12. Me a-C1 33.8 f 3.7 12b a-Cl 0.8 f 0.7 14a Me 8-OH a-C1 71.6 f 7.6 lab a41 14.9 f 0.4 -OEt @-OH a-OH lh Me a-Cl 16.6 0.6 a-OH a-C1 0.8 f 0.0 OEt lSb @-OH 18. Me 8-c1 4.3 0.2 a T h e realtive binding affinity (RBA)is defined aa 100 timcw the ratio between competitor and unlabeled eatradiol concentration required for 60% competition to epecific [aHIeatmdiol binding. Competitive binding between 1 V M [aHIcwtradioland 2 X 109 to 2X M unlabeled ligands wae plotted, and the concentration required for 60% competitionwas used to calculatethe RBA values (mean of three experiments and SD,standard deviation). Value taken from ref llb.

-

*

*

The biodistributionand uterus uptake of the l9-labeled estradiol derivatives 9a and 9b was studied in immature female Fischer rata. The animals were injected via the tail vein with HPLC-purified [laI1-9a or [12sIl-9b(3 pCi, 111KBq) in 200 pL of 9% ethanol-aaline containii 1W Tween-80 and sacrificed at 0.5, 1, 2, 5, and 24 h poetinjection. Receptor-mediateduterine uptake was evaluated in the 1h time group by blocking ER via coinjection with

Ali et al.

266 Journal of Medicinal Chemistry, 1993, Vol. 36, No. 2

Table 11. Tiesue Uptake of Fiecher Rata

1UI after Administration of

l!%Labeled 11B-Ethoxy-and 7a-Methyl-16a-Iodoestradiol to Immature Female % ID/g ( % CV)" 2 h (n = 4) 1 h (+E&)(n= 3)

5 h (n = 4) 24 h (n = 3) [lBI1-9b(7a-methyl) 0.45 (2) 0.38 (19) 0.33 (9) 0.32(21) 0.28 (14) 0.03 (18) blood UterUe 4.32 (25) 5.25 (28) 0.34 (11) 6.35(26) 3.86 (35) 1.64 (13) thyroid 40.33 (31) 45.15 (24) 43.50 (15) 66.19 (40) 320.54 (35) 334.84 (20) muscle 0.54 (1) 0.32 (11) 0.27 (5) 0.15 (32) 0.06 (33) 0.01 (44) fat 2.20 (49) 1.79(28) 1.64 (8) 1.12(25) 0.38 (41) 0.03 (11) kidneys 1.30 (6) 0.83 (3) 0.51 (9) 0.50 (24) 0.25 (22) 0.30 (19) plasma 0.68 (2) 0.58 (19) 0.51 (11) 0.49 (24) 0.41 (12) 0.04 (19) spleen 0.57 (4) 0.43 (6) 0.43 (20) 0.33 (12) 0.22 (29) 0.08 (27) lunge 0.82 (13) 0.54 (7) 0.43 (12) 0.32 (18) 0.19 (15) 0.03 (13) liver 1.94 (10) 1.26 (8) 1.09 (9) 1.09 (10) 0.72 (19) 0.13 (7) [1%1]-9c(11B-ethoxy) blood 1.29 (11) 1.01 (17) 1.09 (9) 0.89 (16) 0.70 (13) 0.17 (18) UterUe 7.85 (17) 11.67 (18) 0.72(9) 13.08 (18) 7.76 (19) 1.64 (36) thyroid 105.65 (19) 110.96 (32) 156.51 (34) 218.87 (21) 703.10(10) 773.37 (45) muscle 1.05 (22) 0.69 (21) 0.72 (20) 0.52 (35) 0.35 (29) 0.13 (52) fat 2.47 (23) 1.51 (24) 1.63 (20) 0.94 (32) 0.52 (14) 0.15 (24) brain 0.68 (16) 0.39 (21) 0.42 (25) 0.25 (33) 0.14 (22) 0.04 (17) kidneys 2.26 (10) 1.83 (25) 1.61 (6) 1.78 (28) 0.91 (17) 0.24 (21) spleen 1.44 (21) 1.49 (19) 1.60(23) 1.76 (19) 1.28 (21) 0.70 (23) lungs 1.72(13) 1.30 (22) 1.18 (14) 1.01 (19) 0.76 (8) 0.23 (11) liver 5.91 (30) 3.48 (26) 4.50 (19) 3.24 (18) 2.31 (9) 0.96 (11) Average % ID/g for 3-5 rata. Each rat was injected iv with 3 pCi (111 kBq) of n.c.a. '2Wabeled steroid in the presence (+E&) or abeence of 60 pg coinjeded estradiol. The incertitude is given in brackets a~ the % CV, e.g. the 5% coefficient of variation corrected for small sample size effect.21

0.5 h (n = 3)

tieeue

1 h (n = 5)

BLOOD CLEARANCE 1.5 T

0 9a

1.0

!I

0 9b v 9c V

1

9d

1I@-ethoxyanalogs 9a and 9b exhibit strong ER-mediated uterus uptake and similar uterus to blood/nontargetratioa (Figure 2).11 The 7a-methyl derivative 9a shows a 2-3 fold lower uterus uptake as compared to the ll@-substituted derivatives however, ita rapid blood and systemic clearanceresults in higher uterus to blood/nontargetratioa at 2 h postinjection (Figure 2).

0

Discussion Among the steroidal estrogens tested bearing different halogens on the D-ring at the 16a-position, the 16a-Br derivatives have the highest affmities for the ER, confirming earlier reports by Heimann et al.,16 Leveaque et 0.0 al.,17 and Longcope et al.18 (Table I). The RBA valuea of 0 5 10 15 20 25 all 116-ethoxy derivatives are much lower than those of HOURS the corresponding 7a-methyl analogs. 16a-Bromo-7aFigure 1. Blood clearance in immature female Fiecher rata after iv injectionwith 3 pCi of l Wabeled 16a-iod~7a-methylestradiol- methylestradiol(6a) has the highest binding affmity for 1,3,5(1O)-triene-3,17~-diol(9a) or 16cr-iodo-ll~-ethoxy-l,3,5(10)- the ER (RBA = 79). The RBA values decrease by triene-3,17/3-diol(9b). The values for the llg-methoxy analog substitution of Br for Cl(14a) or I (9s) ( M A = 71 and 9c and the nonsubstituted l6a-iodoestradiol (9d) were taken 55, respectively). A similar structursactivity relationship from the literature." has been observed in the unsubstituted estradiol s e r i e ~ . ~ 5 The corresponding 16B-epimers exhibited substantially 6Opg of unlabeled estradiol. Tissue uptake of radioiodine diminished binding affinitiesfor the ER. In the llg-eth04 after administrationof [luI1-9a or -9b is presented in Table series, the highest binding affmity was likewise observed I1 as the % of injected dose per gram tissue (%ID/g). for the 17@-hydroxy,lea-bromo derivative 6b (RBA = Blood clearance, uterus uptake, and uptake ratios are 16.8) (Table I). compared to earlier reported data on the C12sIl-16aiodoestradiol (9d) and the [1uIl-11@-methoxy-16a-ioThe radiochemicalpurity of [lsI1-9a and [luI1-9b, after HPLC purification, was in excess of 95%; no other doestradiol ( 9 ~ ) " in Figures 1 and 2. Overall tissue distribution pattern of 9a and 9b resemble those reported radioactive material could be detected on the chromatofor 9c" although some distinct differences are observed gram. The identity of the producte were confiied by in uterine uptake pattern (Figure 2). The uterus uptake their chromatographic mobilities (HPLC) which were of 9s and 9b reaches a maximum between 1-2 h postinidentical to those of the corresponding unlabeled analogs. jection, at which time the 12sIconcentration is the highest Specific activities could not be calculated for these of all organa counted, with the exception of the thyroid compounds since no UV absorption at the moat sensitive (Table 11). Apart from the uterus and thyroid, luIdetector setting was observed in the region of the radioradioactivity level decrease steadily from the first time active HPLC peak. Based on the specific activity of the point measured at 0.5 h, for both [12SIl-9aand -9b. n.c.a. [luI]NaI employed (2125 mCi/mmol) the specific activity of these compounda should exceed 500 Ci/mmol. Compared to the 11@-methoxyderivative 9c and the nonsubstituted 16a-iodoestradio19d,the 7a-methyl and The reverse-phase HPLC system readily separates the Y

0.5

Journal of Medicinal Chemistry, 1993, Vol. 36, No.2 267

~ ~ I ] - l G a - I o d o e s t r a d i oDerivatives l U T E R U S UPTAKE 20,

I

90 9b 9c 9d

0.5

1

2

5

24

l h (+EZ)

U T E R U S TO BLOOD RATIO 70 60

synthesis could readily be adapted for use with the medicinally important lZ3I(t1p = 13.3 h) isotope. The biodistribution studies with these 1261-labeled steroids revealed persistent retention by the uterus as the most striking property (Table 11). In the case of the 7amethyl derivative 1261-9a,uterus to blood ratios were particularly accentuated due to rapid blood clearance (Figure 1). After 24 h, uterus to blood ratioe for lUI-9a were greater than 4o:l while at 5 h poetinjectionthe % ID/g values still are over 50% of those measured at the maximum concentrations at 1-2 h postinjection. Thus, although the absolute uterus uptake valua in 3'% ID/g are higher for the llg-methoxylethoxy derivatives as compared to the 7a-methyl derivative, the latter gave the best target to nontarget ratios (Figure 2). Accordingly, the could be of par1231-labeled7a-methy1-16a-iodoeetradiol ticular interest for SPECT imaging of estrogen receptors in breast cancer.

Experimental Section

'

50

U T E R U S TO NONTARGET RATIO

I

Figure 2. Uterus uptake in % ID/g (top), uterua to blood ratios (middle),anduterua to nontarget (lung,spleen, and muscle)ratioe (bottom) of 1261-labeled7a-methyl (9a), llg-ethoxy (Sb), 116methoxy (Sc), and nonsubstitutad16a-iodoestradiol(9d).Values for 9c and 9d were taken from the literature.''

16a-iodoproducta from the l6&bromo reactants, as well as possible products from side reactions, such as the 17keto steroids formed by dehydrohalogenation and the 16abromo steroids resulting from epimerization. Sodium thiosulfate was added to the 'Wlabeled solutionto reduce free iodine to I-since commercial [12SI]NaIuponstanding was found to contain a significant amount of 12. In addition, benzoic acid was added to buffer the reaction mixture and to protect the alkali-sensitivebromohydrins. The entire radiochemicalSynthesis,includingpurification, can be completed within 4-6 h to give the radioiodinated estrogens in 6585% radiochemical yield. This rapid

Melting pointa were determined on a Fisher-Johnsapparatus and are u n c o d . Spectral data ('H NMR,BruLer WM 26 epectrometer with M&i aa an integral etandard;maw spectra, HewletbPaclrard Model 5988A quadrupled) were recorded. Microanalyees data were obtained by Guelph Laboratoriea Ltd., Canada Silica gel (60-200 meeh) was used for column chramatography. Silica gel plates coatad with fluorewent indicator (W 264) were uaed for analytical thin-layer chromatography (TLC),and the compoundswere located by their W absorbance and/or color response upon spraying with H&OdEtOH and heating at 120 OC. High performance liquid chromatography (HPLC)waa performed on a reversaphase column (C-18,0DS-2 epherieorb, 5 wm, 25 X 0.94 cm,CSC, Montreal), and the compounda were detected at 280 nm and where appropriate, by their y-radiation which waa registered via a sodium iodide detector. All chemicaleusedare commerciallyavailableand were of the highest chemicalgradeavailable;n.c.a. (non-carrieradded) [lBII NaI waa purchaeed from Amersham Canada Ltd. Steroids were purcbawd fromStmaloidsInc. 17-Ethylenedioxye&a-1,3,5(1O)-triena3,11@-diol waa eyntheeized according to Baranloand converted to llg-ethoxy-3-hydroxyeetra-1,3,5(lO~~en-l7-one with ethyl iodide. 7a-Methylestra- 1,3,5( 10),16-tetraene-3,17-diolDiacetata (2a). A solution of 'la-methyl estrone (la, 375 mg, 1.32 "01) in 3.5 mL of isopropenyl acetate and 0.15 mL of catalyst solution (prepared by mixing 4 mL of isopropenyl acetate + 0.1 mL of HBOd waa refluxed for 2 h. Approximately 1mL of the solvent waa slowly distilled over a period of 1h. An additional 2 mL of isopropenyl acetate and 0.1 mL of catalyst were added, and the solution waa concentrated to one-half of the volume by slow distillation for 1h. "be eolution waa chilled and EtOAc waa added. The EtOAc solution waa washed with icechilled sodium bicarbonate (5%) in water and dried over sodium eulfate (anhydrous)and the eolvent waa removed under reduced preeeure. The reaidue waa purified on a column packed with aluminaoxide. Elution with 10% EtOAc in hexane furniehed 2a (200 mg, 0.64 mmol,M%),which failed to crystallize: 'H NMR (CDCh) 6 0.86 (d, J = 7 Hz, 3 H, 7a-CHa), 0.92 (8, 3 H, 1&CHs), 2.13 (8, 3 H, 17-OCOCHs),2.23 (8, 3 H, 3-OCOCHa), 5.48 (bm,1 H, l&H), 6.86,6.96,7.18 (3 H);MS m / z (re1inten), 368 (M+, 31,326 (lo), 284 (29).270 (361, 255 (151, 159 (11). Further elution gave 7a-methyl~troneacetate (100 mg, 0.3 -01) (23%): 'H NMR (CDCh) 0.82 (d, J 7 Hz,3 H, 7a-C&), 0.92 (e, 3 H, l&CHa), 2.28 (e, 3 H, 3-OCOCHa),6.64,6.92, 7.18 (3 H), MS m / z (re1inten) 326 (M+, 4), 312 (3), 284 (351,270 (34), 185 (ll),145 (13). 1lfi-Lthoxyertra-lf$( lO),l&tetraene-3,17-dol Diacetata (2b). A solution of llg-ethoxyestrone (lb, 970 mg, 3.08 "01) waa treated aa deecribed for 2a to furnish 2b, which failed to crystallize (620 mg, 64% 1: LH NMR (CDCls) 8 1.04 (t,J 7 Hz, 3 H, ll@-OCH&Ha), 1.15 (e, 3 H, l&CHa), 2.18 (E, 3 H, 17-OCOCHs)2.26 (e, 3 H, 3-OCOCHa), 3.28, 3.67 (q, J = 7 Hz,

-

268 Journal of Medicinal Chemistry, 1993, Vol. 36, No.2 2 H, ll@-OCH&Hs),4.26 (m, 1H, lla-H), 5.47 (bra, 1H, 16-H), 6.78 (d, J 2 Hz, 1H, 2-CH), 6.82 (dd, J = 2 and 8Hz, 1H, 2-CH), 7.08 (d, J = 8 Hz, 1H, 1-CH); MS m/z (re1 inten) 389 (M+, 3), 366 (51, 338 (51, 268 (51, 250 (51, 188 (14), 146 (?00). Further elution of the aluminaoxide column wlth 10% EtOAc gave llðoxyestrone acetate (360mg,23%1: mp 120-122 OC; 'H NMR (CDCb) 0.93 (t,3 H, J 7 Hz, 11@-OCH&Hs),1.01 (8, 3 H, l&CHs), 2.15 (8, 3 H, 3-OCOCHs), 3.22, 3.56 (q, J = 7 Hz, 2 H, Il&OCH&&), 4.21 (m, 1 H, lla-H), 6.71 (d, J = 2 Hz, 1 H, 4-CH), 6.76 (dd, J = 2 and 8 Hz, 1H, 2-CH), 7.04 (d, J 8 Hz, 1H, 1-CH);MS m/z (re1inten)356 (M+,281,314 (71,283 (161, 268 (22), 241 (141, 212 (361,188 (17), 170 (31), 146 (100). Bromination of 20 and 2b. A solution of 2a (73.6 mg,0.2 "01) or 2b (550 mg, 1.41 mmol) in ether (4 mL) and acetate buffer (4 mL, 85%acetic acid, 200 mg of potaseium acetate) was cooled to 0 O C . To this solution was dropwiee added 0.68 mL of a molecular bromine solution (preparedfrom 0.08mL of Brz and 6.75 mL of glacialacetic acid) with continuousstirring, whereafter the solutionwas stirred for an additional 10-15 min. The reaction was terminated by the addition of water, whereafter the organic phase was washed with water, 5% sodium thiosulfate, and 6% aqueous sodium bicarbonate, dried over sodium sulfate (anhydrous), fiitered, and evaporated to dryness to afford 3a and 3b. 16a-Bromo-3-acetoxy-7a-met hylestra- 1,3,6(lO)-trien-17one (30). White crystalline solid (55 mg,65%): recrystallized from hexane, mp 158-160 "C; 'H NMR (CDCb), 6 0.92 (d, J

Ali et tal. reduced pressure. TLC showed the presence of several compounds, and the mixture was purified by column chromatography over silica gel. Elution with 10% EtOAc in hexane furnished a white c r y h h e compound 7a (8mg; 27%): mp 178-184 OC; HPLC (7030 MeOH/H20) t R = 34 min, lH NMR (CDCb DMSO-&) 6 0.80 (8, 3 H, l&CHs),0.87 (d, J = 7 Hz,3 H, 7a-C&), 3.65 (d, J = 5 Hz, 1H, 17&H), 4.65 (m, 1 H, 16/3-H),6.54 (d, J 3 1 Hz, 1H, 4-CH), 6.64 (dd, J = 2 and 9 Hz, 1 H, 2-CH) 7.17 (d, J = 8 Hz, 1H, 1-CH);MS m/z (re1inten) 366 (38), 364 (M+, 36), 267 (33), 229 (45), 211 (20), 199 (29), 171 (821,167 (67), 147 (100). Anal. (CldIZsBrOz) C, H, Br. Further elution with 15% EtOAc in hexane furnished a crystalline compound 6a (15 y 60%): mp 110-116 OC; HPLC (7030 MeOH/H20) t~ = 31 min, *HNMR (CDCb + DMSO-&) 6 0.80 (a, 3 H, 1&CHa),0.87 (d, J = 7 Hz, 3 H, 7a-C&), 4.0 (d, J = 5.8 Hz, 1H, 17a-H),4.13 (ddd,J1ee,lla= 4.3 Hz; J1as.1~. = 2.4 6.55 (d, J 2 Hz,1H, CCH), Hz, J ~ 7.8~Hz, 1JH, 16&H), ~

+

6.64(dd,J=2and8H~,lH,2-CH),7.14(d,lH,J=8Hz,l-CH); MS m/z (re1inten) 366 (38),364 (M+,38,285 (lo),267 (la), 243 (13), 229 (561,199 (34), 171(591,147 (100). Anal. (CidIyBrOz) C, H, Br. Further elution with 20% EtOAc in hexane afforded

7a-methyl-3,17/3-estradiol. ISa-Bromo-118+thoxyestra-1,3,6( 10)-triene-3,17adio1(7b) and 16a-bromo- 118-ethoxyestra-If&( lO)-triene-3,178-diol (6b). A solution of 16a-bromo-1l~-ethoxy-3-hydroxy-eatra-1,3,6(lO)-trien-l7-0ne(4b,50mg;O.l27mmol) wastreatedasdescribed for 6a. Elution from the silica gel column with 20% EtOAc in ~HZ,~H,~~-C&),O.~~(S,~H,~&CH~),~.~~(S,~H,~-OCOCH~), hexane furnished a white crystalline compound 7b (16 mg, 4.50 (dd,J 1.5 and 4.5 Hz,1H, 16&H), 6.66,6.92,7.18 (3 H); 32%): mp 260-264 O C ; HPLC (7625 MeOH/H20) t R = 16 6, MS m/z (re1inten)406 (M+,3), 364 (38),362 (401,350 (27), 348 'H NMR (CDCb + DMSO-de) S 0.83 ( ~ , H, 3 18-CHs) 0.84 (t,J (29), 284 (a), 214 (12), 172 (11). 7 Hz,3 H, 11/3-OCH2CHa),3.15,3.45 (9, J 7 Hz,2 H, llg160-Bromo-3-acetoxy-11 / 3 4hoxyestra- 1,3,6 (lO)-trien-17OCH&Hs), 3.45 (d, J = 5 Hz,1H, 17&H), 4.11 (m, 1H, lla-H), one (3b). Whitecrystallinesolid(370mg,60.5%1: recrystallized 4.52-4.58 (m, 1 H, 16/3-H),6.37 (d, J = 2 Hz, 1H, 4CH), 6.47 from hexane, mp 214-217 OC; 'H NMR (CDCb) S 1.02 (t,J = 7 (dd, J = 2 and 9 Hz, 1H, 2-CH), 6.80 (d, J = 8 Hz,1H, 1-CH), Hz, 3 H, lI@-OCH&HS), 1.14 (s, 3 H, 1BCHS) 2.27 (8, 3 H, MS m/z (re1inten) 394 (22), 396 (M+,191,350 (13), 348 (131,269 3-OCOCHa),3.32,3.65 (q, J = 7 Hz,2 H, 11fi-OCHzCHa)4.31 (m, (58), 251 (63), 241 (22), 213 (57), 185 (43), 167 (69), 149 (100). 1H, lla-H), 4.63 (dd, J = 1.5 and 4.5 Hz,1H, 16@-H),6.80 (d, Anal. (CdnBrOs) C, H, Br. J =2 Hz,1H, 4CH), 6.86 (dd, J =2 and8 Hz,1H, 2-CH), 7.12 Continuous elution with 20% EtOAc in hexane furnished 6b (d, J = 8 Hz,1H, 1-CH); MS m/z (re1 inten) 436 (6), 434 (M+, as a crystalline compound (25 mg, 50% ): mp 236-237 OC; HPLC 61,355 (281,313 (15), 309 (33), 267 (48),239 (19,167 (50), 157 (7525 MeOH/H20) tR = 16 min; 'H NMR (CDCb + DMSO-&) (28), 147 (100). 6 0.69 (s,3 H, l&CHs), 0.75 (t, J = 7 Hz,3 H, 11&OCH&&), Acid Hydrolysis of 30and 3b. 16a-Bromo-7a-methyleatrone 3.02,3.36 (q, J 7 Hz, 2 H, 11&OCHaCHa),3.62 (d, J 6.6 Hz, acetate (30)(300mg,0.74mmol) or 16a-bromo-llðo~estrone 1H, 17a-H),3.86-3.93 (m, 2 H, lla-H and 16/34),6.26 (d, J acetate (3b) (320 mg,0.74 "01) was hydrolyzed by treatment 2 Hz,1H, 4-CH), 6.34 (dd, J=2 and 8 Hz, 1H, 2-CH), 6.67 (d, with concentrated sulfuric acid (0.1 mL) in anhydrous ethanol J = 8 Hz,1H, 1-CH); MS m/z (re1inten)394 (70), 396 (M+,63), (5 mL) at room temperature. The compound was extracted with 315 (29), 269 (1001,213(831,157 (86).Anal. (CdnBrOa) C, H, CHCb and worked up in the usual manner. Evaporationof the Br. solvent afforded 4a and 4b. Epimerization of 16a-Bromo into 168-BromoKetone. A 16a-Bromo-3-hydroxy-7a-methylestra1,3,6( lO)-trien-17mixture of 16a-bromo-7a-methyleatrone (4a,140mg,0.383mmol) one (4a). White crystalline solid (200 mg, 74.5%): mp 228-233 or 16a-bromo-3-hydroxy-1l~etho.yeetra-1,3,6(lO)-trien-l7-one OC; HPLC (7030 MeOH/HpO) t R = 26 m i q lH NMR (CDCb + (ab,50 mg, 0.127 mmol) and LiBr (140mg) in 1mL of 2-butanone DMSO-&) S 0.917 (d, J = 7 Hz, 3 H, 7a-CHs), 0.946 (e, 3 H, was heated for 1h at 80 OC in a closed 5-mL vial. The reaction l&CHs) 4.57 (dd, 1H, J 1.5 and 5 Hz, 16@-H),6.57 (d, J = 2 mixture was poured into 15 mL of water and extracted with Hz, 1H, 4CH), 6.67 (dd, J = 2 Hz,1H, 2-CH), 7.16 (d, J 8 EtOAc, washed with water, dried over NafiO4 (anhydrous)and Hz, 1 H, 1-CH); MS m/z (re1 inten) 364 (55), 362 (M+,60),322 evaporated to dryness. Column chromatography on silica gel (5), 284 (29),255 (6), 228 (31), 213 (921,171(58), 157 (100). Anal. with 8% EtOAc in hexane gave 6a and 6b. (CldIZJBrOd C, H, Br. 16~-Bromo-9-hydroxy-7a-methylestra-l,3,6( 10)-trien-t7160-Bromo-3-hydroxy-ll~sthoxyertra-1~,6( 10)-trien-17one (a): 80 mg, 67 % ;HPLC (5050THF/HsO) t~ 22 min, mp one (4b). White crystalline solid (270 mg, 93%): mp 250-252 101-106 O C ; 'H NMR (CDCb) 6 0.88 (d, J =7 Hz, 3 H, 7a-C&), OC; HPLC (7625 MeOH/HsO) t R = 17 min; 'H NMR (CDCb + 1.12 (e, 3 H, M-CHS), 4.17 (t, J = 8.6 Hz,1H, 16a-H),6.67 (d, DMSO-&) 6 0.85 (t,J =7 Hz, 3 H, ll@-OCH&Hs),0.96 ( ~ , H, 3 J = 1Hz, 1H, 4-CH), 6.65 (dd, J = 2 and 7 Hz, 1H, 2-CH), 7.14 18-C&), 3.16,3.47 (q, J = 7 Hz, 2 H, Ilg-OCHaCHa), 4.10 (m, (d, J = 8 Hz, 1H, 1-CH); MS m/z (re1inten) 364 (401,362 (M+, 1 H, lla-H), 4.48 (dd, J 1.5 and 5 Hz, 1H, 16&H),6.38 (d, J 44),284 (19), 283 (28), 228 (30),213 (791,206 (791,171 (541,146 = 2 Hz, 1H, 4-CH), 6.46 (dd, J = 2 Hz, and 8 Hz, 1H, 2-CH), (100). Anal. (C1sHd3rO2) C, H, Br. 6.78 (d, 1H, J = 8 Hz,1-CH); MS m/z (re1 inten) 392 (7), 394 16#-Bromo-3-hydro.y- 1l@sthoxyertra-1,3,6(10)-trIen-17(M+,6), 313 (461,267 (52), 239 (161,212 (161,197 (181,167 (341, one (6b). lH NMR analysis indicated that lib c o ~ i s t e dof a 159 (261, 157 (411, 146 (100). Anal. (CpHSsBrOs) C, H, Br. 60:50 mixture of the 16a- and &bromo isomers, which we were 1Ga-Bromo-7a-methylestra-l,3,6( lO)-triene-3,17a-di01(7a) unable to separate. The lH N M R values obtained for the 166and 16a-Bromo-7a-methylertra-1,3,6( lO)-triene-3,178-diol isomer, after subtracting those of the 16a-isomer, include lH (60). A solution of 16a-bromo-7a-methyl-3-hydroxyestra-1,3,& NMR (CDCb) 0.73 (t,J 7 Hz, 3 H, ll@-OCHnC&), 0.98 ( ~ , 3 (lO)-trien-l'l-one (4a, 30 mg,0.082 "01) in THF (5 mL) was H, I&CHa), 3.03,3.34 (9, J 7 Hz,2 H, ll@-OCH&Hs),3.88 (t, added dropwise to a stirred solution of 1M Ld& in THF (0.4 J = 8.7 Hz, 1H, 16a-H), 3.98 (m, 1H, lla-H), 6.23 (d, J =1Hz, mL) at 0 OC. The reaction mixture was stirred for 2 h and then 1H, dCH), 6.31 (dd, J 2 and 7 Hz, 1 H, 2-CH), 6.64 (d, J quenched by the careful addition of water, followed by 10% 8 Hz, 1 H, 1-CH). hydrochloric acid until the solution was strongly acidic. The Redaction of 168-Bromogetones 5.and 6b with N.B&. product was extracted with EtOAc, washed with water, and dried A solution of NaB& (80 mg) in EtOH (5 mL) was added to over NaBO4 (anhydrous)and the solvent was evaporated under

PSI]- 16a-Iodoestradiol Derivatives

Journal of Medicinal Chemistry, 1993, Vol. 33, NO.2 26s

(lO)-trien-17-one (llb), A solution of the enol acetate 2b (500 16@-bromo-7a-methylestrone(Sa, 80 mg, 0.22 "01) or 16gmg, 0.77 m o l ) was treated as deecribed for 120to fiord a white bromo-llg-ethoxyestrone(Sb, 115 mg, 0.292 mmol) in EtOH (5 crystalline solid which according to the 'H NMR ~ 0 ~ h tof. ad mL). The mixture was stirred overnight at room temperature, 3 1 mixture of 16u- and 168-chloroisomera 10band 1lb (260mg, exwas NaBH, was destroyed with acetone, and the reaction 86%):'H NMR (CDCla) 6 1.01 (t,J =7 He,3 H, 11@-OCH&€I& mixture was poured into water, extracted with EtOAc, waehed 1.15 (e, 3 H, ISCHs), 2.26 (E, 3 H, 3-OCOCHs), 3.3, 3.6 (q, J = with water, dried over NafiO4 (anhydrous),and evaporated to 7 Hz, 2 H, 11&OCH2CHs), 4.07 (t, J = 4 Hz,1H, l6u-H), 4.30 dryness. The residue was purified by column chromatography (m, 1H, lla-H), 4.60 (d, J = 7 Hz,1H, 16&H), 6.79 (d, J = 2 on silica gel with 10% EtOAc in hexane to yield 8a or 8b. Hz, 1H, dCH), 6.82 (dd, J = 2 and 8 Hz,1H, 2-CH), 6.86 (d, 16@-Bromo-7a-methylestra-l,3,S( lO)-triene-3,17~-diol J = 8 Hz, 1H, 1-CHI; MS m/z (re1inten)390 (291,392 (M+, ll), (sa): 32 mg, 40%;mp 98-104 "C; HPLC (7030 MeOH/H20) t~ 348 (15), 336 (8), 274 (lo), 267 (13), 258 (321,212 (100). 24 min; 'H NMR (CDCb DMSO-de) 6 0.82 (d, J 6 Hz,3 Acid Hydrolysis of 1 h b and 1la,b. A mixture of 16u- and H, ~ u - C H ~0.93 ) , ( ~ , H, 3 IsCHs), 3.48 (t,J =7 Hz, 1H, 17~-H), 16B-chloro-7a-methylestrone acetate loa and lob (260 mg, 0.64 4.68 (td,J 7 Hz,1H, 16a-H), 6.56 (d, J = 2 Hz, 1H, 4-CH), mmol) and a mixture of 16a- and 16&chloro-ll@-etho~trone 6.65 (dd,J = 2 and 9 Hz, 1H, 2-CH), 7.14 (d, J = 8Hz, 1H, l-CH), acetate lob and l l b (200 mg, 0.51 "01) was hydrolyzed with 8.01 (e, 1H, 17a-OH);MS m/z (re1inten) 366 (33), 364 (M+,36), H&Or (1mL) in absolute EtOH (20 mL) at room temperature 284 (54), 267 (231,229 (331,213 (251,199 (44),185(221,171 (59), for 12 h. The usual workup with EtOAc gave 200 mg of crude 147 (83). Anal. (Cl0H&rO2) C, H, Br. Further elution with product which was purified by column chromatography on silica 15% EtOAc in hexane gave 7~-methylestradiol(20%). gel. Elution with 12-15% EtOAc in hexane furnished 13a, 12a, 16~-Bromo-l1~-ethoxyestra-l,3,5( lO)-triene-3,17B-diol 13b, and 12b. (ab): 50 mg, 43.6%; mp 217-220 OC; HPLC (6535 MeOH/H20) t~ 21 min, 'H NMR (CDCb DMSO-ds) 6 0.78 (t,J = 7 Hz, 16~-Chloro-3--hydro.y-7a-methylest~-l~,S( lO)-trien-ll3 H, 11@-OCH&Hs),0.87 (8, 3 H, l&CHa), 3.04, 3.36 (9, J = 7 one (Ha): 35 mg, 17%; mp 105-112 O C ; HPLC (Kk60 THF/ H20) t~ = 22 miq 'H NMR (CDCb) 6 0.88 (d, J 7 Hz,3 H, Hz, 2 H, llg-OCH2CHs),3.19 (t,J = 8 Hz,1H, 17a-H),3.95 (m, 1H, Ila-H), 4.32 (9,J = 8 Hz, 1H, 16a-H), 6.26 (d, J 2 Hz, 7a-C&), 1.07 (e, 3 H, l&CHa), 4.05 (t, J = 8 Hz,1H, l k - H ) , 1H, 4-CH), 6.36 (dd, 1H, J = 2 and 8 Hz, 2-CH), 6.69 (d, J = 6.57 (d, J = 1Hz,1H, dCH), 6.64 (dd, J = 2 Hz and 6 Hz,1H, 8 Hz, 1H, 1-CH);MS m/z (re1inten) 394 (42), 396 (M+, 41), 350 2-CH), 7.14 (d, J = 8 Hz, 1H, 1-CHI; MS m/z (re1intan) 320 (221, (41), 315 (25),269 (71), 251 (521,241 (241,225 (171,213 (62), 197 318 (M+,61), 254 (30), 228 (47), 213 (100). Anal. (CidI&lOo) (29), 185 (32), 147 (100). Anal. (CaoHnBrOs)C, H, Br. ConC, H, C1. tinuous elution with the same solvent gave a mixture of 6b and 16a-Chloro-3-hydroxy-7a-methy~stra-l~,S( lO)-trien-177b (42 mg, 36951, t R = 26 mh. one (120): 125 mg, 61%; mp 222-225 "C; HPLC (6050THF/ Iodination of 8a and 8b. A mixtureof 16/3-bromo-7a-methylHzO) t~ = 25 min, 'H NMR (CDCb) 6 0.92 (d, J 7 Hz,3 H, 3,17,9-estradiol (8a, 40 mg, 0.109 "01) or 16/9-bramo-llj3~ u - C H ~0.96 ) , (8, 3 H, l&CHa), 4.5 (dd, J = 1 and 8 Hz,1 H, and 7-14 mg of ethoxyeatra-3,17-diol (8b, 19 mg, 0.048 "01) 16&H), 6.57 (d, J = 2 Hz, 1H, 4-CH), 6.66 (dd, J = 2 and 6 Hz, NaI in 1mL of 2-butanone (methylethyl ketone) was heated for 1H, 2-CH), 7.20 (d, J = 8 Hz,1 H, 2-CH); MS m/z (re1 inten) 1h at 105 OC in a closed vial. After cooling, the mixture was 320 (27),318 (M+,76), 283 (5), 254 (20), 228 (45), 213 (100). Anal. poured into 15 mL of water and extracted with EtOAc (15 mL, (CioH~C102)C, H, (21. twice). The extra& were combined, washed with water, dried 1sg-Chloro-3-hydroxy-1lðoxyestra- l,3,S( lO)-trien- 17over Na&Od (anhydrous)and evaporated. The reaction mixture one (13b): 4 mg, 2.2%; mp 215-230 OC; HPLC (6050TMF/ was purifed by HPLC. H a ) t~ = 16 min; 'H NMR (CDCb) 6 0.92 (t,J a 7 Hz,3 H, 16a-Jodo-7a-methylestra-l,3,S( lO)-triene3,17~-diol(9a): 11&OCH&Ha), 1.13 ( ~ , H, 3 1&CHs), 3.20,3.53 (9, J 7 Hz,2 35 mg,80%;mp 184-186 OC; HPLC (7030 MeOH/H20) t R = 37 H, 11&OCH2CHs),3.90 (t,J = 8.5 Hz,1H, 16u-H), 4.16 (m, J min; 1H N M R (CDCb DMSO-&) 0.617 (8, 3 H, l&CHs),0.734 2 Hz,1H, lla-H), 6.45 (d, J 2 Hz,1H, dCH), 6.53 (dd, J (d, J = 7 Hz, 3 H, 7a-CHs), 3.93 (td,J = 7 Hz,1H, 16/3-H),4.01 = 2 and 8 Hz,1H, 2-CH), 6.83 (d, J = 8 Hz, 1H, 1-CH);MS m/z (dd, J = 2 and 5 Hz, 1 H, 17a-H), 6.50 (dd, J 2 and 5 Hz, 1 (relinten) 348 (86),350 (M+,301,312 (ll),302 (18,284 (121,275 H, 2-CH), 6.96 (d, J = 8 Hz, 1H, 1-CH); MS m/z (re1inten) 412 (29),267 (37), 258 (271,240 (241,212 (100). Anal. (~nsclos) (M+,100),284 (12), 267 (31),229 (79),173(64),171(60),157 (50). C, H, Cl. Anal. (CipHadO2) C, H, 1. 16~-Chloro-3-hydm~y1lðoxyestra- l a p ( 10)-trien-171 6 d o d o -11g-ethoxyestra-1,3,S( lO)-triene-3,178-diol one (12b): 90 mg, 50%; mp 225-245 OC; HPLC (Kk50 THF/ (9b): 18 mg,84%; mp 245-246 OC; HPLC (7030 MeOWH20) H2O) t~ 17 min; 'H NMF2 (CDCU 6 0.67 (t, J a 7 Hz,3 H, t~ = 21 min; 'H NMR (CDCb + DMSO-ds) 6 0.68 (8, 3 H, 18 llB-OCH&Hs), 0.80 ( ~ , H, 3 18-C&), 2.98,3.29 (9, J 8 Hz,2 CHs), 0.77 (t, J = 7 Hz, 3 H, 11j3-OCH2CHs), 3.04,3.36 (9, J H, 11(?-OCH&Hs), 3.92 (m, 1H, lla-H), 4.22 ( d , J = 8 Hz, 1H, 7 Hz, 2 H, 11@-OCH2CHs), 3.74 (td,J 6 Hz, 1H, 16&H),3.8516&H), 6.17 (d, J 2 Hz,1H, 2-CH), 6.26 (dd, J 2 and 6 Hz, 3.94 (m, 2 H, Ila-H and 17a-H),4.5 (d, J =6 Hz, 1H, 17@-OH), 1H, 2-CH), 6.60 (d, J = 8 Hz,1 H, 1-CH); MS m/z (rel inten) 6.28 (d, J = 2 Hz, 1H, 4-CH), 6.36 (dd, J = 5 and 2 Hz,2-CH), 348 (99),350 (M+,31), 312 (14), 302 (le),276 (341,267 (30),268 6.70 (d, J = 8 Hz, 1 H, 1-CH); MS m/z (re1inten) 442 (M+,48), (29), 240 (l8), 212 (100). Anal. (c&nsClos) c, H ck dd, 315 (8). 269 (49), 251 (41), 225 (31), 213 (67), 157 (100). Anal. 10.16; found, 9.71. (CaoHnIOd C, H, I. Reduction of 12a,12b, and 13a with L i A l a . 16u-Cldom 1~-Chloro-3-acetoxy-7a-met hylestra-1,3,S(10)-trien- 17'la-methylestrone (12a, 50 mg, 0.156 mmol), 16a-chlomll& one (loa) and 16&Chloro-3-acetoxy-7a-methyle~tra-1,3,5ethoxyestrone (12b, 67 mg, 0.19 mmol), or 16~-chlom'la(lO)-trien-l7-one(lla). Asolutionoftheenoldiacetate2a(400 methyleatrone (13a, 31 mg, 0.01 "01) in THF (10 mL) W(UI in acetone (20 mL), buffered with sodium acetate mg, "01) cooled to 0 OC, and a solution of L u in THF (1M, 0.4 mL) (400mg) and glacial acetic acid (0.5 mL) and water (4 mL), was was added dropwise with vigorous stirring. After continuous treated with tert-butyl hypochlorite (0.2mL). The clear solution stirring for 60 min, the reaction mixture was stopped by the was warmed to 50-55 "C, stirred for 90 min, poured into water, additionof EtOAc,followed by water and 109% HC1until strongly extraded with EtOAc, washed with water and aqueous sodium acidic. The mixture was extracted with EtOAc, waehed with bicarbonate (5%), and dried over Na2S01 (anhydrous). Evapwater, dried over Na&Oc (anhydrous) and brought to dryness oration of the sovlent under reduced pressure furnished a white under reduced pressure. The producta were purified by column crystalline solid, which according to the 'H NMR consisted of chromatography on silica gel with 10-20% EtOAc in hexane. a 3 1 mixture of 16u- and 168-chloro isomers 1Oa and 110: mp 16a-Chloro-7a-methyIertra-l,t,S( lO)-triene-3,178-diol 158-160 OC; 'H NMR (CDCb) 6 0.90 (d, J = 7 Hz,3 H, 7a-CH3), (l4a): 25 mg, 50%; mp 129 O C ; HPLC (7030 MeOWHa) t R = 0.98 (8, 3 H, l&CHs), 2.29 (8, 3 H, 3-OCOCHs), 4.18 (dd, J = 2 25 min; 1H N M R (CDCla DMSO-&) 6 0.79 (a, 3 H, l&C&), and 8 Hz, 1H, 168-H), 4.43 (t,J = 8 Hz, 1H, 16a-H),6.60,6.80, 0.83 (d, J = 7 Hz,3 H, 'Ia-CHs), 3.71 (d, J = 4 Hz,1H, 17a-H), 7.2 (3 H); MS m/z (re1 inten) 362 (31,360 (M+, 81,320 (33), 318 4.62 (m, 1H, 168-H), 6.6 (d, J = 2 Hz, 1H, dCH), 6.64 (dd, J (100) 284 (81, 254 (161, 228 (24), 213 (38). = 2 Hz, 1H, 2-CH),7.16 (d, J = 8 Hz, 1H, 1-CHI; MS m/z (re1 16a-Chloro-3-acetoxy-1lðoxyestra-l,3,6( lO)-trien-17one (lob) and 16~-ChIoro-3-acetoxy-1l~athoxyestra-1,3,5- inten), 322 (36), 320 (M+, loo), 278 (171,234 (191,226 (301,213

+

+

+

+

270 Journal of Medicinal Chemistry, 1993, Vol. 36, No.2

Ali et al.

immature femaleFiecherrats, 24 days old, 66-60 g (ChnrlesRiver), were injected with 200 & of 19-labeled 90 or 9b (3 pCi, 111 KBq) via the lateral tail vein. The animals were placed in 16a-Chlor0-7a-methylestra-l,3,S( lO)-triene-3,17a-diol retention cages and thereforenot anesthetizedduringthe injection (158): 10 mg, 20; mp 107-113 OC; HPLC (7030 MeOH/HsO)t~ procedure. The radiopharmaceutical wae dissolved in ethanol = 26 min; IH NMR (CDC& DMSO-de) S 0.80 (e, 3 H, l&CHa), and diluted with sterile physiological saline (0.9% NaCl in HsO) 0.865 (d, J =7 Hz, 3 H, 7a-CHa), 3.85 (d, J 6 Hz, 1H, 17a-H), containing € I Z , 1%Tween-80to give a final ethanol concentration of 4.11 (m,dddJlw,17,,=4.2Hz, Jleg,lb =2.5Hz, J I ~ , I S , S = ~ . ~ 9%. For the receptor saturation studies, 60 pg of unlabeled 1 H, 16/3-H), 6.55 (d, J 2 Hz, 1 H, 4-CH), 6.64 (dd, J 2 and estradiol was coinjected with the radiopharmaceutical. Animaln 7 Hz, 1H, 2-H), 7.14 (d, J = 8 Hz, 1H, 1-HI; MS m / z (re1inten) were sacrificed under deep ether anesthesia by severing the 322 (36), 320 (M+,loo), 242 (lg), 234 (24),227 (25), 213 (241,199 axillary artery,followedby chest ~ p e n i n g Blood . ~ was collected, C, H, C1. (35), 174 (47), 147 (63). Anal. (C&&lO2) tissues of interest were removed, washed with 0.154 M KCl, and 16aChloro- 1 lg-et hoxyestra- 1,3,S(1O)-triene-3,17a-diol blotted dry, and samples were weighed. The radioactivity wae (Ub): 12.2 mg, 18.4%; mp 225-235 OC; HPLC (5050THF/ counted in a Model 1282 Compugamma countar (LKB Wallac, HoO) t~ = 17 min; 'H NMR (CDC&+ DMSO-&) 8 0.65 (8,3 H, Finland) and concentrations were expressed ae % of the iajected LSCHs), 0.68 (t, J = 7 Hz, 3 H, ll&OCH&Ha) 2.95, 3.30 (q, J dose per gram of tissue (%ID/g). Statistical variationa are = 7 Hz, 2 H, ll@-OCHpCHa),3.68 (d, J = 2.5 Hz, 1H, 17&H), presented as the % coefficient of variation corrected for small 3.92 (m, 1H, lla-H), 4.28 (m, 1H, 16/3-H), 6.16 (d, J = 2 Hz, 1 sample size effect (% CV)." H, 4CH), 6.25 (dd, J = 2 and 8 Hz, 1 H, 2-CH), 6.60 (d, J 8 Hz, 1H, 1-CH); MS m / z (re1 inten), 350 (71,352 (M+, 16), 304 Acknowledgment. Generousfmancialsupportfor this (ll),269 (5), 251 (9), 212 (21), 197 (lo), 172 (16), 157 (36),146 work was provided by the Medical Research Council of (100). Anal. (C&I&lOa) C, H C1: cald, 10.10; found, 9.47. 16a-Chloro-1 lg-ethoxyestra-1,3,5(1O)-triene-3,178-diol Canada diid the "Programme d'Actions structurantee" of (14b): 22 mg, 33%;mp 228-240 'C; HPLC (5050 THF/H20) t~ the MESS of QuBbec. We thank Dr. G. Shyamala from = 14 min; lH N M R (CDCh DMSO-de) S 0.71 (8, 3 H, l&C&), the Lady Davis Institute for Medical Research, Montreal, 0.76 (t, J 7 Hz,3 H, 11@-OCH2CHs),3.03,3.35 (q, J 7 Hz, for conducting the receptor binding assays. 2 H,llB-OCH&&), 3.43 (t,J = 6 Hz, 1H, 16&H), 3.86 (dt, J = 1and 6 Hz,1H, 17a-H),3.94 (m, 1H, lla-H), 4.54 (d, J = 5.5 References Hz, 1H, 17@-OH),6.25 (d, J 2 Hz, 1H, 4-CH), 6.34 (dd, J (1) (n) Couneell,R. E.; Klausmeier, W. H. In Principles of Radiop2 and 8 Hz,1H, 2-CH), 6.68 (d, J = 8 Hz, 1H, 1-CH); MS m / z hrmacology; Colombetti,L. G.,Ed.; CRC Prm: Boca Raton, 1979; (re1 inten) 350 (lo), 352 (M+, 26), 304 (71,251 (71, 225 (81, 212 Vol. 11, pp 59-91. (b) Eckelmnn, W. C.; Reba, R. C. In (20), 185 (13), 172 (27), 157 (42), 146 (100). Anal. (c&I&los) Radiopharmaceuticab: Structure-Activity Relatiowhipu; SpenC, H, C1: calcd, 10.10; found, 9.46. cer, R. P., Ed.; G m e and Strntton: New York, 1981;pp 449-458. 16&Chlor0-7a-methylestra-1,3,5(lO)-triene-3,17@-diol (c) Knteenellenbogen,J. A.;Heiman,D.F.; Carlaon,K. E. et al. In Receptor-Binding Radiotracers; Eckeknan, W . C., Ed.; CRC (168): 20 mg, 68%; mp 104-105 OC; HPLC (7030 MeOH/H20) Press: Boca Raton, 198%Vol. I, pp 93-126. tR = 24 min; lH NMR (CDCb DMSO-ds) S 0.83 (d, J = 7 Hz, (2) Hochberg, R. B. Iodine-12blabeled estradiol: a gamma emitting 3 H, 7a-CH.q),0.89 (s,3 H, l&CHS),3.65 (t,J =8 Hz, 1H, 17a-H), analog of estrndiol thnt binds to the estrogen receptor. Science 4.53 (dt, J = 6 Hz, 1 H, 16a-H), 6.57 (d, J 2 Hz, 1H, 4-CH), 1979,205,1138-1140. 6.65 (dd, J = 2 and 6 Hz, 1H, 2-CH), 7.12 (d, 1H, J 8 Hz, 1-CH); (3) Hochberg,R. B.; Rosner, W. Intaractionof16a-[1P61]iodeeatrndiol with estrogen receptor and other etaroid-binding protain. h o c . MS m / z (re1inten) 322 (35), 320 (M+,100),248 (13),227 (351,213 Natl. Acad. Sci. U.S.A. 1980, 77,320-332. (30), 199 (33), 186 (23), 171 (58), 159 (831,145 (92). Further (4) Lippman, M. E.; Do, H. M. T.; Hochberg,R. B. Specirrcestrogen elutionwith 15% EtOAc in hexane gave 7a-methyl-3,17~estradiol receptor binding and biological effecta of Ma-iodoedtradiol on (20%). human breast mcer cells. Cancer Res. 1981,41,3150-3154. Synthesis of 7a-methyl- and llB-Etho~y-l6a-[~~I]io(5) Symea, E. K.; Couleon, W. F.; Ralph, D. N. L. The syntheaia of doestradiola, [lUI]-9a and [lUI]-9b, by Halogen Exchange. 16a-[18111iodeestrndioland evaluation of ita use as n radiotrncer for estrogen receptor positive breast tumors. J.Steroid Biochem. The radioaynthesis of [lmI]-9a and [12611-9b was performed by 1986,22,165-160. a published method.18 Two mCi of Na1=I in 20 pL of water (6) Hochberg, R. B.; MacLueky, N. J.; Chambra, J.; Eisenfield,A. J.; containing 20 mg of Na&Os was added to a reaction vial and Naftolin, F.; Schwartz,P. E. Concentration of [18a-~niodoeatrnevaporated (Np) close to dryness at 60-66 "C; 200 mL of diol in human ovarian tumors in vivo and correlationwith estrogen acetonitrile was added and evaporation was repeated. A solution receptor content. Steroids 1986,419,775-788. (7) Snlmon, J.; C o d i e r e , D.; Couety, C.; Raynaud, J. P. Phnrmaof either 7a-methyl-l6&bromoestradiol (Sa, 1 0 ~or)llg-ethoxycokinetice and metabolism of moxestrol in animals - rat, dog and 16@-astradiol(Sb, 10 pg) in 20 gL of methanol was added to the monkey. J. Steroid Biochem. lSSS,19,1223-1234. reaction vial. The reaction vial was tightly sealed and heated at (8) Grill, H. J.; Moebius, U.; Manz, B.; Pollow, K. 105 OC for 90 min; 100 pL of water was added and the reaction 16a-Iode3,176-estradiok a stable ligand for estrogen receptor mixture was p d i e d by HPLC on a reverse-phase column with determinations in tissue with high [email protected] genaw activity. J. Steroid Biochem. 1983,19,1687-1688. EtOH and water. The compound [lBII-9a eluted at 22 min and (9) (a) Kalvodn, J.; Krahenbuhl, Ch.;Deeaulea, P. A.;Anner, 0. A. [W]-9b at 24 min in 6535 and 7030 MeOH/water, respectively. 7a-Methyl oeetrogene. Helu. Chim. Acta 1967,60,281-288. (b) Estrogen Receptor Binding Assay. Affinity of the estradiol Bucourt, R.; Vignnu, M.; Torelli, V.; Richnrd-Foy,H.; Genet, C.; derivativee for estrogen receptors (ER) was determined by a SecceMillet, C.; Rednvilh, G.; Bnulieu, E. E. New biwpecifc competitive binding assay1' and is expressed as the relative ndeorbenta for thepurificationof estradiolreceptor. J.Biol. Chem. binding affinity (RBA). The RBA is defined as 100 times the 1978,263,8221-8228. (10) (a)Ali,H.;Roueeeau, J.; G M n r i , M.A.; van Lier, J. E. Synthesis, ratio between competitor and the unlabeled estradiol concenreceptor bindq and thue dietribution of (17a,20E)-and (17a,trations required for 50% competition to specific [aHlestradiol 202)-21-[1~~iode19norpregna-l,3,S~lO),~ta~~~3,l7-diol. J. binding. Murine uterine cytoplasmic extracts were incubated at M~d.Chem.l9~,91,1946-1950. (b)Ali,H.;Rouwnu,J.;Ghaffari, 0-4 OC for 18 h with 20 nM of [3Hleatradiol in the absence and M. A.; van Lier, J. E. Synthesis, receptor binding, and tissue presence of competitive steroids ranging from 2 nM to 20 pM. distribution of 7a- and 11/3-substituted(17a,20E)-and (17a,20Z)2 1 - ~ ~ ~ i o d ~ l 9 n o r p ~ - 1 , 3 , 5 ~ 1 0 ) , J. ~ Med. ~~e-3,17~0~ The bound eteroid was separated from free steroid by Sephadex Chem.1991,34,654-860. (c)Napolitano,E.;Fieschi, R.; H m n , LH-20 chromatography. The nonspecific binding (equivalent R. N. S h c h " I & 'vity relntiodp of estrogenicligands a y n h i a to that observed in the presence of a 1Wfold excess of unlabeled and evaluation of (17aJOE)- and ( 1 7 a , 2 0 Z ) - Z l - h a 1 ~ 1 9 - n o ~ ~ estradiol) was 3-496 of the total binding which was subtracted 1,3,6(10),20-tatraene-3,17~-diole.J. Med. Chem. 1991,34, 2764from the total binding to estimate the specific binding. The 2759. (d)J.goda, E. M.; Gibson,R. E.; Goodgold,H.; Ferreirn, N.; Francis, B. E.; Reba, R. C.; Rzeszotareki,W. J.; Eckelman, W. C. specific binding (average of three experiments) in the receptor [I-125117a-iodovinyl116-methoxyestrndiol: in vivo and in vitro preparation was equivalent to 6.3 nM. propertiesof ahigh-affintyestrogen-receptorradiopharmnceutiial. In Vivo Studies. The animal experimenta were conducted J.Nucl. Med. 1984,25,472-477. (e)Nnkntrukn,I.; Ferreirn, N. L.; as previously describedlNb and in accordance with the recomEckelman,W.C.; Francis, B. E.; Rmmotanki, W. J.; G i b n , R. E.; mendations of the Canadian Council on Animal Care and an Jngoda, E. M.; Reba, R. C. Synthesh and evaluation of (17a,20E)in-house Ethica Committee for Animal Experiments. Briefly, 21-[1-1261iodo-19norpr~-1,3,6(10),20-tetrnene-3,17~-diol and (27),199(29),186 (21), 174(50),159(801, 145(71). Anal. (CioHls-

C102). C, H, C1.

+

+

+

~gbZ]-16u-Zodoestradiol Derivatives

Journal of Medicinal Chemistry, 1993, Vol. 36, No. 2 271

(15)Heiman,D.F.;Stephen,G.;Senderoff,J.; Katzenellenbogen,J. A,; ~17a,20E~-21-~I-125liodo-ll~-methoxy-~9-norpregna-~,~,~(~~),~~ tetraene3,17,T-diol(17a-[iodovinyl]estradiol derivatives) as high Neeley, R.J. Estrogen recaptorbaeed imagingagenta. 1. S y n t h h specific activity potential radiopharmaceuticale. J. Med. Chem. and receptor binding affinity of nome aromatic and D-ring 1984,27,1287-1291. h . l o g ~ ~ t estrogens. ed J. Med. Chem. 1980,23,994-1002. (11) (a) Zielineki, J.E.; Yabuki, H.; Pahuja, 5.;h e r , J. M.; Hochberg, (16)Schneider,W.; Shyamala,G. Glucocorticoidreaptorn in primary R B. 160-[~~I]Iodo-llg-metholy-l7,T-estradiol: a radiochemical culture of m o w mammary epithelialcells: charaetarization and probe forestrogen-wnsitivetissues. Endocrinology 1986,If 9,130modulation by prolactin and cortbl. Endocrinology 1982,116, 139. (b) Katzenellenbogen,J. A.; McElvany, K. D.; Senderoff, S. 2856-2662. G.; Carlson, K. E.; Landvatter, S. W.; Welch, M. J. (17)Uveaque, C.; MBrand, Y.;Dufour, J. M.; Labrie, C.; Labrie, F. 160-[nBrlBromo-l1~-methoxyestradiol-17~: a gamma-emitting Synthesisandbiologicalactivityof new halo-steroidalantimtrogene. estrogen imaging agent with high uptake and retentionby targetJ. Med. Chem. 1991,34,1624-1630. organs. J. Nuel. Med. 1982,23,411-419. (18)Longcope, C.; Rafkind, I.; A”, T.; Caspi, E.Biological (12)Johneon, W.S.;Johns, W.F. 14-Ieoeetronemethyl ether and ita activitieaof 4-fluoroestrogenanalogues. J.SteroidBiochem. 190, identity with totally syntheticmaterial. J. Am. Chem. SOC. 1967, f9,132&1328. 79,200k2009. (19)Baran, J. 5.A synthesisof lie-hydroxyestrone and related 16-and (13)Arunachalan, T.;Caspi, E.Synthesis of seleno estrogens. J. Org. 17-hydroxyetttratrirnes.J. Med. Chem. 1967,10,118&1190. Chem. 1981,46,3415-3420. (20)Waynforth, H. B. InExperimentol and SurgicalTechniquein the (14) Zielineki,J. E.;h e r , J. M.;Hoffter, P.B.; Hochberg,R. B. The Rot; Academic: London, 1980. syntheaisof 1lfl-methoxy-[ 16a-1~Iliodoentra&ol and itainteraction (21)Sherrer, B. Biostotistique;Morin, G.,Ed.; Chicoutimi, Quebec, with the estrogen receptor in vivo and in vitro. J.Nucl. Med. 1989, Canada, 1964. 30,2W215.