Synthesis of 16α-[125I]iodo-5α-dihydrotestosterone and evaluation of its affinity for the androgen receptor

J. srvroid Biochum. Vol. 16. pp. 621 IO 628. 1982 Printed in Great Britain. All rights reserved

0022-4731,82~050621-08503.00/0 Copyright @j 1982 Pergamon Press Ltd

SYNTHESIS OF 16a-[‘251-JIODO-5a-DIHYDROTESTOSTERONE AND EVALUATION OF ITS AFFINITY ANDROGEN RECEPTOR R. M. HonP.

FOR THE

W. ROSNER and R. B. HOCHBERG~

Reproductive Biology Section. Department of Obstetrics and Gynecology and Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine. New Haven. CT 06510 and Department of Medicine, Roosevelt Hospital. Columbia University College of Physicians and Surgeons, New York. NY 10019. U.S.A. (Receired 9 September 198 1) SUMMARY Analogs of 5z-dihydrotestosterone. halogenated at carbon 11. were synthesized as potential gammaemitting ligands for the androgen receptor. These compounds. were chosen for synthesis because estradiol. similarly substituted, is strongly bound to the estrogen receptor, and both androgen and estrogen receptors have generally similar structural requirements for the D-ring. The 16a-halogenated steroids. including 16x-C‘2sl]iodo-5a-dihydrotestosterone were synthesized from 16/I-bromo-Sx-dihydrotestosterone by halogen exchange. The cis-&bromohydrin substrate was synthesized from 5x-androstane-3,17dione by selective ketahzation. dibromination at C-16 and stereoselective reduction. 16e-iodo dihydrotestosterone was devoid of androgen activity in uitw at concentrations at which 5zdihydrotestosterone was fully stimulatory. The 16z-iodo, 16a-bromo. and 16/I-bromo analogs were allowed to compete with [3H]-dihydrotestosterone for binding to the androgen receptor: the 16z-iodo compound had a relative binding affinity l/lOOth and both bromo compounds 1/3Oth that of dihydrotestosterone. In addition, no specific binding was detected when the 16a-[ i2’I]iodo analog was incubated with prostatic cytosol.

lNTRODtJCIlON

The synthesis of gamma-emitting steroids that are capable of binding to appropriate receptors with high affinity and specificity has been the goal of many investigators because of the important practical advantages conferred by gamma-emitters vis-a-vis j-emitting steroids, (labelled with ‘H or r4C). These include very high specific activities, thus enabling receptor analyses to be performed with great sensitivity, and the potential for in ciuo scanning which is made possible by the energetic photon which can be detected externally. Although these properties are of obvious importance for both the evaluation and localization of hormonally dependent tumors, the only successful synthesis of a biologically active, gammaemitting steroid has been 16a-iodoestradiol labelled with either tz51 [l-4] or t3’I [3]. This estrogen binds Visiting Fellow 1980-81. Department of Obstetrics and Gynecology. Yale University School of Medicine, on leave l

from Department of Chemistry, State University of New York, College at Old Westbury. Supported by NIH Fellowship Award Research Service National IF 34 GM076904 from the National Institute of General Medical Sciences. t Recipient of an NIH Research Career Development

Award CA-29591. Address correspondence to: Dr R. B. Hochberg. Department of Obstetrics and Gynecology. Yale University School of Medicine. 333 Cedar Street. New Haven, CT 06510. U.S.A.

to the estrogen receptor with the same affinity as estradiol and it has been synthesized with t2’1 in carrier-free form (specific activity approximately 2200 Ci/ mmol). From these observations, it was concluded that the 16a halogenation of estradiol leaves its receptor-binding activity intact, and this has been confirmed recently, by several investigators [S, 63. Although several androgen analogs containing iodine, bromine or selenium, (at various positions), have been synthesized, specifically as probes for the androgen receptor [7-10-J, none have been shown to be useful. We chose to synthesize Sr-dihydrotestosterone, substituted with iodine at C-16%, because the similarity in the D-ring requirements for ligands which bind to androgen and estrogen receptors suggests that this C,9 steroid would bind with high affinity to the androgen receptor, just as 16a-iodoestradiol binds to the estrogen receptor. We describe, herein, the synthesis of this compound, 16a-iodo-5xdihydrotestosterone 5, and lZsI-labelled 5, as well as its brominated analogs 4 and 7 (Fig. 1) together with measurement of their affinities for the androgen receptor. EXPERIMENTAL Melting points were obtained in a KofIler Hot Stage or in a Melt-Temp apparatus and are uncorrected. Infrared spectra were recorded in potassium bromide discs using a Nicolet Model 7199 Fourier transform

621

R. M. HOYTEet ul.

622

Fig. 1.

Synthetic scheme for C-16 halogenated kc-dihydrotestosterone.

spectrophotometer. NMR spectra were obtained with a Jeol FX9OQ. 90-MHz Fourier transform instrument. Mass spectra were recorded on a Hewlett Packard Model 5985A spectrometer at 20ev or 70ev. High Performance Liquid Chromatography was performed using a Waters modular system consisting of two Model M6OOOApumps, a U6K injector, and a Model 440 dual wavelength (254 nm. 280 nm) detector. Solvents were HPLC grade and were filtered and deaerated before use. In order to ensure that the halogenated steroids were not contaminated with traces of other C-19-steroids. they were purified by HPLC (described below) before they were used as competitors in the androgen receptor assay. 3,3(&Z-Dierhyl-I.?-propanedioxy)-5a-Androstan-I

7-one

1

Selective ketalization of 5a-androstane-3,17-dione was achieved with 2,2_diethyl-1,3-propanediol according to the equilibrium method of Smith and Newman[l 11. A mixture of 7.8 g (0.027 mol) of Sa-androstane3.1 ‘I-dione. 7 1.5 g (0.542 mol) of 22-diethyl- 1.3propanediol. 3.0 g of ptoluenesulfonic acid monohydrate, and 1.56 liters of 1.2-dimethoxyethane was heated for three days at 5o’C in a flask equipped with a condenser and drying tube. The reaction was quenched by addition of 7.5 ml of triethylamine. Evaporation of the solvent gave a clear oil which was taken up in 100 ml of 1: 1 benzene-ðyl ether and washed three times with 40ml of water. The organic solution was dried over anhydrous magnesium sulfate, filtered, and evaporated to give a slightly yellow oil which was dissolved in 80 ml of acetone and stored overnight at 25’C. The resulting precipitate of the monoketal weighed 7.0 g (65% yield) m.p. 172-176’C. Recrystallization from acetone gave a product with m.p. 175.5-177.O”C (Lit. (12) m.p. 177.0-177.7’C). i.r.:

1732cm-’ (strong, C = 0), 1096 and (strong. Ketal C - 0).

1107 cm-’

16,J6-Dibromo-3.3(2,2-diethyl-I.3-propanediox~)-5zandrostan-17-one

2

A solution of 1.0 g (2.49 mmol) of the above monoketal in 10ml dry tetrahydrofuran (THF) was added, at 25’C under nitrogen, to a suspension of 4.56g of potassium hydride dispersion (22”, in oil. 25.0 mmol) in 10ml dry THF. After 10 min the solution was diluted with 40 ml of dry THF, cooled to - 78’C. and 79.8 g of freshly prepared 30m bromine in methylene chloride (15 mmol Br2) was added drop wise over a period of 80 min. The mixture was stirred at - 78’C for 15 min and then allowed to warm slowly. As the temperature approached O’C. 60 ml of saturated sodium bicarbonate was added. When the temperature reached 2O’C the mixture was transferred to a separatory funnel and shaken with 25 ml IO“,, sodium thiosulfate. The organic phase was separated, dried over anhydrous sodium sulfate, filtered and evaporated. The oily residue was composed mainly of the product and the starting monoketal (TLC); only minor traces of other impurities were present. The oil was chromatographed on 60g of basic alumina (Woelm). activity II, and sequentially eluted with 3OOml isooctane. 300ml 19, ethyl acetate in isooctane. and 600ml F, ethyl acetate in isooctane. The product eluted in the latter solvent mixture (0.5Og. 36”); yield) and was separated cleanly from the unreacted monoketal which was eluted in the same solvent but in the fractions immediately following (0.38 g+ 387; recovery). The product was recrystallited from acetone to give material with m.p. 157-159’C. i.r.: 1762 cm-’ (strong, C = 0). 1105 cm-’ (strong, Ketal C - 0). Mass Spectral Data: 558. 560, 562, (0.6, 1.2, 0.7. Parents); 169 (Base, C,,H,,OZ. A-ring fragmentayion).

lo-~~~Sa~ihydrot~tosteronc If@- Bromo- 3.3 - ( 2,2- diethy/ - 1.3 - propunedioxy androstan-

)- 5’a -

17/I-o/ 3

A solution of 110 mg sodium borohydride in 35 ml methanol was added at 0’ C to 405 mg dibromoketone 2 in 8 ml of anhydrous ether. The mixture was stirred overnight at 4-C and then diluted with 80ml of water. The aqueous phase was separated and extracted with three 50 ml portions of ether and these extracts were combined with the organic phase. The organic solution was dried oser anhydrous sodium sulfate. tiltered and evaporated to give a white crystalline residue. Recrystallization from acetone gave 313mg product @I”/, yield) m.p. 90-92°C. i.r.: 3477cm-’ (broad, 17/l-OH). 1105 (strong Ketai C - 0). Mass Spectral Data: 482, 484 (1.8, 1.6. Parents), 403 (2.9, M-W 402 (3.1, M-HBr), 169 (Base, C,OH,,O;, A-ring fragmentation). 1cl/LBromo- I 7/?-hydroxy-5r-androstan-3-one BromoDHT)

4

( 16jb

Sixty mg (0.642 mmol) of the ketal 3 in 10.8 ml of dioxane was mixed with 2.7 ml of 0.1 N hydr~hloric acid and heated in a closed vessel at 37’C for 24 h. The reaction was quenched with 42~1 of triethyiamine, and the solution was evaporated to give a white crystalline residue. The residue was stirred with lOm1 of water and the solid was collected by fiitration. R~rystaiii~tion from aqueous acetone gave 42mg (92”,yieid) of product m.p. 171-172X. i.r.: 3502 cm-’ (broad, 178-OH). 1706cm-’ (strong, C-O). n.m.r. (CDCIs): 54.61 (m. 1, 16a-H), 3.3 (t, 1, J 16.1: = 8.6H1 172-H). 1.04 (5. 3, H-19), 0.92 (S, 3, H-18). Mass Spectral Data: 368. 370 (2.0, 1.8, Parents). 289 (34.6, M-Br). 288 (255, M-HBr), 271 (Base, M-Br-H20). The stereochemistry of 4 was established by its conversion to Sr-androstane-3.17-dione (m.p. and mixed m.p. 129-132.C) when heated for 3 h in methanoiic potassium hydroxide, and by its conversion to 17~-hydroxy-5~-androst~-3-one (5z-dihydrotestosterone) when hydrogenated over 50, palladium on charcoal in the presence of triethyiamine. The identity of both products were confirmed by comparison of their infrared spectra to those of authentic materials. A mixed melting point of the hydrogenation product and authentic Sr-DHT was undepressed (175-177X). whereas a mixture with authentic 17x-hydroxy-5xandrostan-3-one was significantly depressed (152-155 C). 16a-iodo-17fi-hydroxy-5z-androstan-3-one

S

(16r-

IodoDHT) A mixture of 15 mg (0.~~ mmol) bromohyd~n 4, 60mg sodium iodide (O.~m/moie) and 1.5 ml anhydrous acetone was heated at 6O’C for 24 h in a closed (screw cap) test tube. After cooling, the mixture was poured into 15 ml water and extracted with four 10 ml portions of methylene chloride. The extracts were washed with 100, sodium thiosuifate, then water, and

623

dried over anhydrous magnesium sulfate. Filtration, evaporation, and recrystallization from acetonepetroleum ether (30-WC) gave 14.8 mg (87?;, yield) of impure product. Further recrystallization from absolute ethano1 gave hexagonal plates with m.p. 173.5177.0% (decomp). ir: 3367 (broad. 17/3-OH), 1691 (strong C = O), n.m.r. (CDCI,), 6 3.9-4.2 (m.2.16fl and 17x-H). Jlh.!, = 5.0Hz. 0.99 (S, 3, H-19). 0.73 (S, 3, H-18). Mass Spectral Data: 416 (0.4, Parent), 289 (19.7, M-I), 288 (S, 3. M-HI), 271 (base, M-~-H*O~ The stereochemistry of 5 was established by its conversion to 168. 17/l epoxy-Sz-androstan-3-one 6, m.p. 168-172°C. when heated for 4 h in methanoiic potassium hydroxide. i.r.: (KBr) 1716 cm- ’ (strong C = 0) 834cm-’ and 821 cm- * (medium, epoxy group). n.m.r. (CDCI,): S 3.48 (m 1. 16/3-H), 3.19 (d- 1. Jm7 = 3.1 Hz. 17&H), 1.02 (S,3,H-19) 0.85 (S. 3, H-18). Mass Spectral Data: 288 (36.5. Parent), 273 (15.6, M-CH,), 270 (29.7, M-H,O). 16a- Bromo-17/%hydroxp&-Androsran-3-one

7

( 16a-

Brom~DHT) A mixture of 15mg (0.041 mmof) bromohyd~n 4, 100 mg lithium bromide (1.14 m/mol), and 1.0 ml of N,N-dimethyiformamide was heated at 50 C overnight. The solution was poured into 15 ml of hot water, cooled, and the resulting precipitate collected. Rec~stal~i~tion from a~ton~~troieum ether gave 8.1 mg (54%) of the epimeric bromohydrin m.p. 182.5-184S’C. i.r.: 3386cm-’ (broad- 17/I-OH), 1691 (strong, C = 0). n.m.r. (CDC13): 6 3.8-4.3 (m, 2.168 and 17rH), J16.t7 = 5.5 Hz, 1.0 ($3, Hc19), 0.76 (S, 3, H-18). A mixture of 7 and 4 gave a sig~i~~ntly depressed melting point (161-185X) further confirming their distinctly different identities. 16x-r J25f]lodo-17/?-hydroxyundrostan-3-one [tz51]IodoDHT)

(161-

The halogen exchange reaction with rz31 was performed as previously described [I, 23. In summary, carrier-free [ 1251J sodium iodide in an aqueous soiution (New England Nuclear) was taken to dryness in a microflex reaction vial (Kontes Glass, Co.) by azeotropic evaporation with acetonitriie in a stream of nitrogen. A solution containing 1Ogg of HPtC purified (see below) 16~-bromoDHT in 30 ~1 of dry freshly distilled 2-butanone was added and the reaction vial was tightly closed. The vial was then placed in a heating block held at 66’C and heated overnight, The vial was opened and the contents were dissolved in methylene chloride and transferred to a 16 mm x 100 mm culture tube. The vial was rinsed with three more portions of methytene chloride which were combined (about 3 ml) and then washed with three 1 ml portions of water, The organic phase was evaporated to dryness in a stream of nitrogen and the last traces of water were removed by adding 1 ml of absolute ethano1 which was evaporated to dryness. The residue was

R. M.

624 IS

on1

4 7

HOYTE et PI.

The identity of the radioactive material was confirmed by re-chromatography of 1.14 x lo5 c.p.m. in the presence of 15 pg of carrier. The radioactivity coeluted with the carrier which was detected at 254nm. The eluted material was then mixed with an additional 5 mg carrier. and recrystallized to constant specific activity (Table 1). This was taken as proof of its radiochemical identity and homogeneity.

5

1111

r

In vivo androgen

&,_~~I 0

4

8

I2 I8 20 TIME (min)

24

~1 22

32

Fit 2. High pressure liquid chromatography of 16~ r1 51110doDHT. Reaction oroduct of halogen exchange of kla*“I with 4. Condition; are described-in test. AGows indicate the position in the chromatdgram where the standards migrate.

then dissolved in 5 ml of 9: 1 benzene-ethanol and stored at 4’C for later purification. The benzene-ethanol solution containing the radioiodinated steroid was evaporated to dryness in a stream of nitrogen. and the residue was chromatographed in a reversed phase system consisting of 455; tetrahydrofuran in water on a 4.6 cm x 25 cm uBondapak C-18 column (Waters Associates) at a flow rate of 1 ml/min. The radioiodinated steroid 2.88 x lo9 d.p.m., 53”, yield) eluted in 19.7 minutes (Fig. 2). In this system the 16a-iodo compound is cleanly separated from both 16/?-bromo_5adihydrotestosterone, which migrates in 14.4min and from the 16a-bromo epimer. which migrates in 16.5 min. The molar absorptivity of 16a-iodoDHT at 254nm or 280nm is too low for the lzsI labeled steroid to be quantified’in the U.V.detector when it is purified by HPLC. Consequently. while 16a-iodoDHT could be separated from 16B_bromoDHT, lC-bromoDHT and DHT on the HPLC (Fig. 2) and therefore should be carrier-free, its specific activity could not be verified. However, a minimal specific activity can be calculated if it is assumed that all of the substrate, 16flbromoDHT, used for the synthesis of 16~+[‘~~I]iodoDHT, was converted into an unknown compound with an androgenic potency equal to DHT, and that this compound can not be separated from 16a-[1z51]-iodoDHT by HPLC. In this unlikely circumstance, 2.88 x lo9 d.p.m. of 16a-[‘z’I]-iodoDHT would be contaminated with 27.1 nmol of this hypothetical androgenic product of 16&bromoDHT. and it would yield an apparent specific activity of 48.4 Ci/ mmol. This specific activity is close that of the [‘HI-DHT used in these experiments.

assay

A modification of the method of Hershberger er al. [13], was used to evaluate the androgenic activity of 16a-iodoDHT. Male Sprague-Dawley rats were castrated at the age of 21 days. Beginning on the 28th day of age, the animals were divided randomly into three groups which received daily subcutaneous injections of 0.1 ml sesame oil (control), DHT in oil or 16a-iodoDHT in oil for seven days. On the day after the final dose, the animals were sacrificed, and the seminal vesicles and ventral prostate were removed and weighed. The results are shown in Table 2. In all cases the total indicated weie dividedinto seven qua1 daily doses administered in 0.1 ml of sesame oil. Androgen

receptor

assa)

The interaction of the halogenated steroids with the androgen receptor was measured by a modification of the method described by Zava et al.[l4]. Briefly, 2508 male Sprague-Dawley rats were castrated and 24 h later the prostate glands were removed and homogenized in TEM buffer (10 mM Tris pH 7.4, 1 mM EDTA and 10mM NaMoO,). The homogenate was centrifuged at 105,000 g for 1 h and the resulting cytosol diluted with TEM buffer so that 1 ml contained the equivalent of one prostate. One hundred ~1 of the cytosol preparation was added to 100~1 of TEM buffer containing 1.2 nmol of [‘HI-DHT (5OCi/ mmol) and varying concentrations of either the HPLC purified halogenated steroids or DHT. The mixtures were then incubated at 4% overnight. The receptor bound steroid was precipated with 125 pg of protamine sulfate dissolved in 250~1 of 10mM Tris Table

I.

Crystallization data of iododihydrotestosterone

16~-[‘~~1]-

Fraction

C8

c.p.m.

Specific activity (c.p.m./mg)

X-O X-l ML-1 x-2 ML-2 x-3 ML-3

120.7 141.8 118.2 119.1 82.6 123.9 96.0

2405 2774 2189 2396 1624 23% 1943

19925 19563 18519 20118 19661 19338 20240

Five mg of 16a-iodoDHT were combined with approximately 1OO.OOOc.o.m.of 16a-T*z’IliodoDHT and crvstaliized kom k+&.s ethanol. k-0, &sidue before cry&Ilk ation. X-n. crystalline product from the nlh crystallization. ML-n. residue IeR in the mother liquor from the PI”’crystallization.

16a-lodo-Se-dihydrotestosterone

625

Table 2. Effect of 16a-iodoDHT and DHT on the growth of seminal vesicles and ventral prostate of the castrated rat

~__ Tissue weight (mg f SE) Compound administered

Total dose mg (rmol)

Oil (control) DHT

0 0.3 ( 1.03) 0.6 (2.07) 1.2(4.14) 2.4 (8.28) 0.43 (1.03) 0.86 (2.07) 1.72(4.13) 3.44 (8.27)

I6a-IodoDHT

No. of rats

Seminal vesicle

10.4 f 0.6 19.1 f 1.2 31.6 f 4.2 37.6 & 4.7 70.7 f 7.5 10.4 f 0.5 11.4 f 0.8

Prostate

7.9 f 21.2 f 37.2 + 43.7 f 54.7 k 6.8 f 8.6 f

0.9 2.6 8.2 5.9 6.1 I.0 1.1

- These tissue were not weighed since there was no response at the higher doses. However, the animals were dissected and there was no apparent response upon visual inspection of the organs.

buffer pH 7.4 and after 5 min the precipitate was centrifuged at 1000 g for 10 min. The pellets were washed two times with 0.5 ml of TEM buffer and then extracted twice with 2.5 ml of a toluene based scintillation phosphor. The bound radioactive steroid was determined by counting the combined extracts. When 16z-[‘ZsI]-iodoDHT was tested as a l&and, the conditions for these experiments were exactly as described above with the exception that the izsI labeled steroid was not extracted. The protamine precipitate was counted directly in a gamma counter. RESULTS AND DISCUSSION

The synthesis of [lz51]-iodoDHT is outlined in Fig. 1. The preparation of monoketal 1 by selective ketalization of Sz-androstane-3,17-dione has been extensively studied by Smith and Newman[ll, 123. We chose 2,2-diethyl-1,3-propanediol as the protecting group because of its superior selectivity, hydrolytic stability, and higher yield of the desired monoketal. Bromination of the potassium enolate of 1, generated by the method of Brown[lS], was found to proceed directly to the dibromoketone 2. No monobromo ketone was observed as a product in this reaction. While the overall yield of this step was somewhat low, 36”, it should be noted that 38”/;, of the starting ketone was recovered from the reaction in pure form. Sodium borohydride reduction of dibromoketone 2 gave the 16fi-bromo-17/I-hydrin 3 as the only observed product in accordance with the observation of Shoppee er a/.[163 on a related compound. An alternate route (not described) to 3 by bromination of the enolate of 1 generated from lithium diisopropylamide was found to give a mixture of 162 and 168 bromoketones and therefore requires separation of the epimers or an epimerization step prior to the reduction. Attempted reduction of these epimeric bromoketones gave significant amounts of 16bromo-17~hydrins. In view of these facts, it appears that dibromination of the potassium enolate and subsequent reduc-

tion as described above is a superior route to the desired 16/I-bromo-17B-hydrin. Hydrolysis of 3 with dilute hydrochloric acid in aqueous dioxane [I l] gave 16/I-bromoDHT 4. Reaction of 4 with sodium iodide in acetone and with lithium bromide in N,N-dimethylformamide gave 16a-iodoDHT 5 and 16a-bromoDHT 7, respectively. The stereochemistry of the groups at C-16 and C-17 of 4 and 5 were proven by the method of Fieser et a/.[ 173. Catalytic hydrogenation of 4 gave DHT, demonstrating that the hydroxyl group at C-17 has the 178 configuration. Dehydrohalogenation of 4 with potassium hydroxide in methanol gave Sa-androstane3,17dione showing the cis geometry of the bromine and hydroxyl group. Similar dehydrohalogenation of 5 gave the 16/I, 178 epoxide 6 demonstrating the tram geometry of the iodine and hydroxyl group. In addition, the stereochemical assignments for 4, 5. and 7 are supported by the NMR coupling constants. Ji6., ,, and the chemical shifts for H-16, H-17, and H-18 which are consistent with those reported by other workers for 16, 17-disubstituted steroids [18,19]. The immature rat model was used to test 16a-iodoDHT, 5, for its adrogenicity in cico. The iodinated steroid, 5. was also tested for its ability to bind to the androgen receptor in citro using rat prostatic cytosol. It is obvious from the data in Table 2 that this steroid did not cause an increase in the weight of the seminal vesicles or the prostate. The highest dose of the iodinated steroid tested was almost ten times greater than that of the lowest dose of DHT that caused appreciable growth of these organs. Since binding to the receptor is a prerequisite for androgen action, this in rice experiment would seem to indicate that 5 was not a ligand for the androgen receptor. However, other factors also could explain these negative results. For example. this steroid could have been an anti-androgen. It would have bound to the androgen receptor and have no effect in this assay. Alternatively. this lack of androge-

R. M. HOYTE er ul.

626

nit activity could have been due to metabolic factors. causing a rapid clearance. rather than lo a low affinity for the androgen receptor. Consequently. the binding of 16a-[‘251]-iodoDHT to the androgen receptor was measured in a series of experiments. Prostatic cytosol prepared in the usual way was incubated with 8000 c.p.m. 16a-[’ 251]-iodoDHT It radioinert DHT (1. 5. 10, 25. 50. 100 nm) or f radioinert S(1. 5, 10. 25.50.100.500. 1000 nMl. The bound steroid was quantified by protamine precipitation of the receptor as described above. In the assays conducted with [ “‘I]-DHT alone. 26.3”” of the counts were bound. This percentage did not change at any concentration of either radioinert DHT or 5, thus indicating that there was no specific binding of C’251]-DHT. Further. prostatic cytosol incubated with either 20.000 cpm 16~-[‘~~I]-iodoDHT +- I PM 5 or 50.000 c.p.m. 16a-[‘r”I]-iodoDHT + 1 FM 5 bound respectively. 25.6.24.3.25.6 and 27.8”” of the added radioactivity. again demonstrating the lack of specific binding. Positive controls. using the same cytosol with I.2nM [“HI-DHT. bound 39.89, of the [‘HIDHT in the absence of carrier DHT and 17.1”, and 6.29, in the presence of 10 and 1OOnm radioinert DHT. respectively. Since. as explained above. the specific activity of the lz51 labelled steroid could not be ascertained. its use as a ligand in the above experiments might be suspect. To eliminate this uncertainty. another experiment was performed to determine whether non-radioactive 5 could displace [‘HI-DHT from the prostatic androgen receptor. In addition 16a-bromoDHT, 7, and 16/l-bromoDHT. 4. were also tested as inhibitors of the binding of DHT to its receptor. The binding of [‘HI-DHT in the presence of increasing concentration of these halogenatcd steroids is compared lo non-labelled DHT. It is obvious. from the results

I

shown in Fig. 3. that these halogenated steroids compete with C3H]-DHT for receptor sites only poorly when compared to DHT. The relative binding affinity of the iodinated steroid was lOO-fold less than DHT. and the brominated compounds, 4 and 7, were both 33-fold less. When the data for DHT was analyzed as described by Scatchard[ZO], an association constant of 4.6 x 10s M-’ was calculated. The unexpected failure of the compounds in this study to show evidence of high affinity for the androRecent gen receptor requires an explanation. studies [21] have indicated an important relationship between the conformation of the D-ring of androgens and their affinity for their receptor protein. It has been proposed that the optimal position of an oxygen atom at C-17 is one in which it is very close to the mean plane of the B- and C-rings of the steroid nucleus. Small departures from this position towards either the a-side or b-side are shown to be associated with decreased affinity. This is ostensibly related to the effectiveness of hydrogen bonding with the receptor protein. The conformations of the D-ring of steroids have been extensively investigated using X-ray and theoretical methods [22-241. and spectroscopic techniques [ 19.25,26]. These studies. which have recently been reviewed [27] indicate that most steroids, with the exception of 17-keto steroids, have D-ring conformations which are intermediate between the halfchair (Fig. 4a) and the 13/?-envelope (Fig. 4b). Most androgenic steroids thus far investigated appear to have D-ring conformations which can be described as distorted 13fi-envelopes [21]. In considering the effect of substituents at C-16 it can be seen from molecular models that the limiting 13/&envelope conformation would have substituents at C-16 eclipsed with hydrogens at C-15 leading to an increase in energy (Pitzer’s

I

8

I

1

0

f

2

3

LOGCoNCENTRATIOW tnM) Fig. 3. Competition of C-16 halogenated analogs of dihydrotestosterone for sites on the androgen receptor. The androgen receptor assay was performed with rat prostate cytosol incubated with 1.2 nM [‘HI-DHT. Details are provided in the text. & = [‘HI-DHT specifically bound in the absence of any other steroid (in this experiment 6.P0 of the added radioactivity). B p [‘HI-DHT speciftcally bound in the presence of the indicated steroid. Ail data were corrected for nonspecik binding which was determined with 100 nm DHT and constituted 0.P; of the added radioactivity. All points represent the mean of duplicate experiments.

I~-I~o-5a~ihydrot~tosterone

627 REFERENCES

1. Hochberg

a

b

Fig. 4. D-Ring Conformations of I7~-Hydroxy-C-l9-Steraids. (at represents the half chair and (b) represents the 138 envelope conformation.

This torsional eclipsing energy can be relieved if the conformation assumed by the D-ring changes toward the half-chair. As a consequence. the 178 hydroxyl group will tilt upward toward the &side of the molecules, and away from the optimal position for strong receptor binding described above. Thus, in accordance with these effects, substituents at C-16 should have the effect of decreasing affinity for the receptor. In support of this hypothesis, it is noted that GOFOef a/.[283 have reported considerable reductions in androgenic activity upon introduction of alkyl substituents at C-16 in testosterone and 19-nortestosterone. While conformational deformations may be an important factor in accounting for decreased receptor affinity they may not be Ihe only influence. Both the size and electronegativity of a large halogen atom at C-16 could have important effects on the steroidreceptor interaction. It is possible that such large atoms are sterically incompatible with the preferred orientation of the receptor protein in its complex with the steroid. or that the electron rich halogen atoms lead to electrostatic repulsive forces which decrease the binding energy. Unfortunately. too little is known about the nature of these effects to allow a determination of their relative importance. The differences between the ~6-halodihydrotestosFerones. 4, 5, 7, on the one hand. and l&-iodoestradiol on the other, with respect to their affinities for their respective receptors is at first surprising. IF has. in fact. been proposed that estrogens and androgens bind similarly to their receptors through hydrogen bonds to the 178 hydroxyl group [29]. The results of this study suggest that the two receptors bind differently in that the androgen receptor is more sensitive to the effects of substituents at C-16 than is the estrogen receptor.

Strain).

.&~~~o~sl~~~e~ stirs-This work was supported by Grants AM28562. CA-29591 and RR08180 from the United States Public Health Service. National institutes of Health and a grant from the Orentreich Foundation for the Advancement of Science. We gratefully acknowledge Wayne Douglas for excellent technical assistance and Gale Iannone for the preparation of the manuscript.

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