Nucl. Med. Biol. Vol. 16, No. 1, pp. 3-9, 1989 Int. J. Radiat. Appl. lnstrum. Part B Printed in Great Britain. All rights reserved

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Synthesis and Evaluation of (17 ,20E)21- [125I ]Iodo- 11-substituted- 19-norpregna-

1,3,5(10),20-tetraene-3,17fl-diols: the Influence of 11-Stereochemistry

on Tissue Distribution of Radioiodinated Estrogens

R O B E R T N. H A N S O N , L O T H A R A. F R A N K E and M I C H A E L L. K A P L A N

Section of Medicinal Chemistry, College of Pharmacy and Allied Health Professions, Northeastern University and Children's Hospital Corporation, Boston, MA 02115, U.S.A.

(Received 11 May 1988)

The 21-tri-n-butylstannyl derivatives of (17~,20E)-ll~t and fl-methoxy-19-norpregna-l,3,5(10),20- tetraene-3,17fl-diol were synthesized and characterized. These compounds, as well as the 1 l-tmsubstituted compound were converted via eleetrophilic ipso radioiododestannylation to the corresponding 21 [~25I]iodo analogs at the no-carrier-added level in 73-90% isolated radiochemical yields. The radiochemical 4(: [IV0tME 2, (l7~2~E)-2l[125I]i~d~-llct-meth~xy-l9-n~rpregna-~3~5(l~)~2~-t~traene-3~7~-di~l] was evalu- ated in immature female rats and the results compared to those previously reported for 4a (IVF~) and 4b (IVfl ME2) to determine the influence of 1 l-substitution on the ability of the compounds to function as estrogen receptor-seeking agents in vivo. The results indicated that the uptake of I 1 ~t-methoxy derivative in the target organ was substantially lower, of shorter duration, with a much smaller specific receptor binding component than the other two radioligands. The distribution profile of the three 17~-iodovinyl estrogens paralleled that previously reported for the corresponding 17~-ethynyl estrogens and this study suggests that the in vivo pharrnacologieal results reported for the 17ct-ethynyl estrogens may be used to predict the in vivo behavior of the corresponding 17~t-iodovinyl analogs.

Introduction

Because the presence of significant levels of specific estrogen receptors in human mammary carcinomas has important ramifications, with respect to both the choice of appropriate therapy and the long term prognosis for the patient, much effort has been expended in developing ),- or positron-emitting trac- ers that could be used for the rapid noninvasive detection of such tumors (McGuire, 1978, 1979; Blarney et al., 1980; Maass and Jonat, 1983; Sledge and McGuire, 1983). The criteria for such tracers, described in numerous reviews, include high affinity for the estrogen receptor, high specific activity, in vivo stability, and low association with nonspecific pro- teins (Gibson et al., 1979; Counsell and Klausmeier, 1979; Eckelman et aL , 1979; Eckelman and Reba, 1981; Katzenellenbogen et aL, 1981b, 1982b; Therain et aL, 1986). The most productive approach has in- volved the preparation of analogs of potent estrogens labeled with one of the radiohalogens (Hochberg, 1979; Katzenellenbogen et aL, 1981 a, 1982; Longcope

et al., 1981; Gibson et al., 1982; Hanson et al. 1982, 1984; Kiesewetter et al., 1984; Mathias et al., 1987) or with carbon-ll (Reiffers et al., 1980; Feenstra et al., 1983). Although agents possessing a nonsteroidal skeleton (Goswami et al., 1980; Landvatter and Katzenellenbogen 1982; Landvatter et al., 1982), e.g. [18F]fluoronorhexestrol, (Landvatter et al., 1983; Kiesewetter et al., 1984) have shown significant po- tential as imaging agents, the bulk of the research has concentrated on derivatives of estradiol. From this effort has emerged 17ct-[125I]iodovinyl-I lfl-methoxy- estradiol (Hanson and Franke, 1984; Franke and Hanson, 1984; Jagoda et al., 1984; Nakatsuka et al., 1984) which was developed independently by two groups and displays the desired properties of high affinity, specific activity, /n vivo stability and tissue selectivity.

In our ongoing program to better understand the structural requirements for target tissue uptake and selectivity and, therefore, to develop better second generation radioligands, we have undertaken the

4 ROBERT N. HANSON et al.

systematic modification of 17~-[125I]iodovinylestra - diol (Hanson, and Franke, 1984; Franke and Hanson, 1984). In this study we report the influence of the l l-methoxy group and its stereochemistry upon the distribution of radiolabeled estrogens. Although it has been observed that the affinity of estrogens is generally preserved by the introduction of l lft-substituents (Baran, 1967; Raynaud et al., 1979; Gabbard and Segaioff, 1983; Gibson et al., 1984) much less is known concerning the ll~t- isomers. The most significant information concerning this substituent effect is derived from the studies with 17~t-ethynyl-1 l~-methoxyestradiol, RU-16117, which behaves as an impeded estrogen although the in vitro estrogen receptor binding affinity is almost identical to that of the 1 lft-methoxy isomer (Raynaud et al., 1979; Azadian-Boulanger et al., 1978; Raynaud et al., 1984). The objective of the work described herein was to determine whether the localization of the corresponding [nsI]iodovinyl anal- ogs of RU-16117 and moxestrol would parallel either the in vitro or in vivo estrogenic activity of the parent compounds. Based upon the published pharma- cological data for moxestrol and RU-16117 we would predict that the lift-substituted compound would have better uterine localization properties, in terms of uptake and selectivity, than the 11 unsubstituted compound which in turn would have better uterine localization properties, in terms of uptake and selec- tivity, than the l let-substituted ligand. The in vitro receptor binding data would predict the 1 l-unsubsti- tuted ligand to be better than the 11~ and lift methoxy compounds which would be roughly com- parable. As the results indicate, the l lft-methoxy compound, [~25I]4b does exhibit substantially higher target tissue uptake, and the target tissue retention is longer and comparable to the in vivo pharmacological effects observed for the nonradioactive 17~t-ethynyl analog.

Materials

RU-16117 and the starting ketone RU-2656 were graciously supplied by Rouseel-Uclaf (Romaineville, France). All other solvents and reagents were ana- lytical grade and used without purification. The radionuclide Nan~I was purchased from DuPont (Biomedical Products, Billerica, Mass.). The rats were obtained from Charles River.

Instruments

Melting points were taken on a meltemp apparatus in open capillaries and are uncorrected. Infrared spectra were recorded on a Perkin-Elmer 599 spec- trophotometer. IH-NMR spectra were recorded at 300MHz on a Varian NMR spectrometer using Me4Si as an internal standard. ~3C-NMR spectra were recorded on a spectrometer using Me4Si as an internal standard. Mass spectra (El) were obtained at 70 eV on a low resolution mass spectrometer. All

preparative chromatographic separations were per- formed using column chromatography with silica gel (Flash Chromatography grade, J. T. Baker) as the absorbent. Thin layer chromatographic analyses were conducted on 1 x 3 in. precoated TLC plates (silica gel/RP-C18-Whatman). HPLC separations and analyses were carried out on an LDC high performance liquid chromatographic consisting of two Constametric II pumps, a Computer Control Module (CCM), u.v.-detector (fixed wavelength), radiometric detector (Canberra Instruments), and dual pen strip chart recorder (LDC).

( 17~, 20E) - 21 - (tri - n - butyls tannyl) - 19 - norpregna - 1,3,5( lO),20-tetraene-3,17ft-diol 2a

This compound was synthesized from estrone la according to the literature procedure (Hanson et al., 1984) Rf= 0.39 (SiO2), 0.36 (C-18).

(17~,20E)- l l f t -me thoxy -21- ( t r i -n -bu ty l s tanny l ) - 19- norpregna- 1,3, 5( l O),20-tetraene- 3,17ft-diol 2b

To the 1 lft-methoxyestrone (0.200 g, 0.67 nmol) in 20mL THF at ambient temperatures was added 0.27mL of n-butyllithium in hexane (2.5 M). The resultant mixture was stirred vigorously to give a homogeneous solution that was maintained under nitrogen. In a separate flask, E-bis(tri-n-butyl- stannyl)ethylene (0.450 g, 0.75 mmol) was dissolved in 10 mL of THF and cooled to 0°C. To this solution was added 0.30mL of n-butylithium in hexane (2.5 M). The resultant solution was stirred for I h and cooled to -78°C for 1 h, then allowed to gradually warm to ambient temperature. The reaction mixture was dried over sodium sulfate (anhyd) filtered and evaporated to dryness. The product was then purified by column chromatography on silica gel using hexane to remove the nonpolar tri-alkylstannyi impurities and hexane-ethyl acetate (5:2) to elute the product which crystallized upon removal of the solvent (0.214g, 0.35retool) 51% yield, m.p. 130-132°C, tH-NMR (CDC13) 0.7-2.3 (m, steroid nucleus plus n-Bu3), 2.65-2.90 (m, 2 H), 3.08 (s, 3H, -OCH3). 4.10 (m, 1H, 11 -CI-H) , 6.07 (d, IH, J - 20 Hz, C21-H ) 6.19 (d, 1H, J = 2 0 H z , C20-H ) 6.49 (d; IH, J = 3.4 Hz, Cu-H), 6.56 (d, d, 1H, J = 3.4, 8.3 Hz, C2-H), 6.95 (d, 1H, J - 8 . 3 H z C : H ) Rf--0.24 (SiO2) , 0.41 (C-18).

(17¢t,20E)- l l c t -me thoxy -21- ( t r i -n-buty ls tannyl) - 19- norpregna- 1,3, 5( l O),20-tetraene- 3,17fl-diol 2c

A solution composed of the 1 lct-methoxy-17~t- ethynylestradiol (RU-16117) (0.120g, 0.37retool), 1 mL tri-n-butylstannane and 5 mg A1BN was heated at 80°C for 8 h. The reaction was cooled and the mixture was purified by column chromatography on silica gel. The excess tri-n-butylstannane was eluted with hexane followed by the product which was eluted with hexane-ethyl acetate (5:2). The product crystallized upon removal of the solvent to give a colorless solid (0.156g, 0.25mmol, 68%). A corn-

Radioiodinated estrogens 5

ponent having a slightly higher Rr was also isolated (0.010g, 0.016mmol, 4%), presumably the 20-Z isomer, m.p. 94-97°C. IH-NMR (CDC13) 0.7-2.2 (m, H, steroid nucleus plus n-Bu3), 2.75 (t, 1H, J = 2H), 3.40 ( , 3H, 11-OCHa), 3.68 (td, IH, J = 3 Hz, 4Hz), 6.10 (d, 1H, J = 2 0 H z , C20-H), 6.21 (d, IH, J = 20Hz, C20-H), 6.54 (d, IH, J = 2.0Hz, C4-H), 6.61 (dd, 1H, J =2.0, 8.3 Hz, C2-H), 7.42 (0, 1H, J = 8.3 Hz, CI-H). Rf= 0.32 (SiO2), 0.42 (C-18).

( 17ct, 20E)- 21- iodo- 19-norpregna- 1, 3, 5( I 0), 20- tetra- ene- 3,17[3-diol 3a

This compound was prepared from 2a according to the literature procedure (Hanson et al., 1984).

(17~t, 20E) - 1 lfl - methoxy - iodo - 19- norpregna - 1, 3, 5- ( l O),20-tetraene- 3,17fl-diol 3b

To the vinylstannane 2b in 5 mL CC14 was added dropwise a 0.1 M solution of 12 in CC14 until the color of iodine persisted. This was followed by the addition of l mL of methanolic KF and l mL 5% sodium bisulfite. The mixture was extracted with ethyl ace- tate. The organic phase was dried over magnesium sulfate (anhyd), filtered and evaporated to dryness. The residue was purified by chromatography on silica gel using chloroform-ethyl acetate (9:1) to give the product as a colorless solid. The yield was virtually quantitative.

(17ct,2OE)- l lct-methoxy- 19-norpregna- 1,3,5( lO),20- tetraene-3,17fl-diol 4e

The iodovinyl product was obtained from the corresponding vinylstannane 2c using the procedure employed for 4a. The yield was virtually quantitative.

Radioiodination

To 10#L of 0.08N NaOH solution containing 5 mCi (2.27 nmol) Na125I in a sealed conical vial were

. / . added 25 # L of a solution containing 200 nmol of the vinylstannane and 50 #L of a NaOAc-HOAc (pH 4.5) buffer. To this was added 50#L of a solution consisting of H202 (30%): glacial HOAc (2: 1). After stirring at ambient temperature for 5 min the reaction was terminated by the addition of 25/~L of 5% NaHSO 3. The entire contents of the reaction vessel were injected onto a reverse phase HPLC column (C- 18 Whatman PXS/10/25 ODS analytical). Elution with an ethanol-ammonium phosphate (pH 4.5) gradient gave the desired radiochemical free of start- ing vinylstannane and other radioactive species. The identity of the product was confirmed by subsequent injection of an aliquot of the radiochemical mixed with a solution of the authentic iodovinyl material. The radiochemical was stored at 4°C in the dark prior to its in vivo evaluation.

Tissue distribution

Immature female Sprague-Dawley rats (21-25 days, 45-55 g) were injected via a tail vein under light ether anesthesia with 0.1 mL of the solution contain-

ing the radiochemical (10-15/z Ci/animal). Groups o f rats (at least 5 per group) were sacrificed 1-6 h after administration, and samples of blood and tissue of interest were excised, weighed and assayed for radio- activity. Tissue and blood levels were calculated as percent of injected dose x mass (kg) per gram of tissue or blood (% 1D-kg/g). Uterus to blood ratios at the various time points were then calculated. These values are compared to those reported for 4a and 4b. To determine specific binding, groups of immature rats were injected wtih 0.2 mL of a solution contain- ing the radiochemical (10-15pCi) and estradiol (50 ng). The groups were sacrificed at 2 h after injec- tion and the tissue levels determined as previously described. The difference in tissue levels in the pres- ence or absence of estradiol represents the specific estrogen receptor binding component.

Results and Discussion

Chemistry

Our approach involved the synthesis of the 21-tri-n-butylstannyl derivatives which would sub- sequently undergo ipso electrophilic radioiodode- stannylation. Initial studies with the 1 l-unsubstituted compound 2a had demonstrated that protection of the phenolic moeity was unnecessary. The synthesis of the two intermediates was dependent upon the availability of the requisite starting material. The llfl-methoxy isomer was obtained from the 17- ketone lb via addition of the E-tri-n-butylstannyl- vinyllithium (Scheme 1). Column chromatography gave the intermediate 2b in a 51% yield.

For the l lct-methoxy compound, the 17~-alkyne RU-16117 3 underwent free radical initiated hydro- stannation with tri-n-butylin hydride to yield tri-n- buylstannylvinyl derivative (Scheme 1) (Corey, 1976). The reaction upon chromatographic separation gave, in addition to unreacted starting material, a 68% yield of the 21-E-tri-n-butylstannylvinyl derivative plus a 4% yield of a product tentatively identified as the Z-isomer. This observed ratio of E/Z isomers is similar to that reported from the hydrostannation of propargyl alcohols under similar reaction conditions (Corey et al., 1976; Hoyte et aL, 1985; Tolstikov et al., 1986; Hofmeister et al., 1986). The trans (E)-stereochemistry of the 20-double bond for the compounds 2a-c was determined by the ~H-coupling constant; J = 2 0 H z . The E-isomers of the non- radioactive 21-iodo products 4a--c were prepared using the procedure previously reported. The iodination proceeded as evidenced by the virtually instantaneous discharge of iodine color. The reaction gave the desired compounds in a virtually quan- titative yield with no evidence of A-ring iodination. The IH-NMR spectra gave doublets for the protons at C-20 and C-21 with a coupling constant (J = 14 Hz) consistent with the trans stereochemistry.

6 ROBERT N. HANSON et al.

HO "~ ~ . . f ~ V

1 ̧ H

HO" " ~ v

la R,: H I~=H

lb Rt: OCl'~ R~H

2a RI:H Re:H

2 b R~= OC~ Rf H

HO

R1 H ~ .¢ CH

R~ OH

( . - . . ) , S. H - - * ~ ''~'~s~""I' AIBN HO" "'7/ v

2C RI:H Rt,=OCl'l=

Scheme 1

Radiochemistry

The stannyl intermediates 2a--c were converted directly to the radioiodinated products [~25I]4a-c us- ing no-carrier-added Na~25I with a substrate to iodide ratio of 200:1, at ambient temperature (Scheme 2). The reaction was terminated after 5 min and the entire mixture was injected onto an HPLC column. Only a single major radioactive peak that eluted after the void volume (inorganic 125I) and well before the unreacted tri-n-butylstannyl precursor was observed. Isolated yields ranged from 73 to 90%. An aliquot of the radioactive fraction was rechromatographed and did not demonstrate either other radioactive com- ponents or a u.v. absorption at the most sensitive setting for the product. A specific activity could not

be determined by u.v. absorption at 254 nm, which is a valley for the estrogen u.v. absorption spectra, but probably exceeded 500 Ci/mmol. The total time for the radioiodination and the isolation of the ]25I-labeled estrogens was less than 1 h to give a product with high specific activity and high radio- chemical and chemical purity. The identity of the labeled material was confirmed by TLC in two sys- tems and by coinjection with the authentic iodinated products on HPLC.

Distribution studies

The in vivo distribution of [~25I]4c in immature female rats were determined at 1, 2 and 6 h following injection. The specific receptor mediated localization

OH

R,. ,,,,,".~"'~ Sn(.-Bu), HO ' ~ , . - J ~ ~ CCI 4

~IM :O2-HOAc Rt . OH

"Sl')-4b I=s1~-4c

Scheme 2

HO / v v

4a RI: H R: :H 4b RI: OCH: R: :H 4c RI: H R::OCH:

Table I. Distribution of 1251

Compound Time (h) Treatment

Radioiodinated estrogens

activity after i.v. administration of estrogenic radioligands in immature female rats

Uterus Liver Lungs Muscle Blood Thyroid ll25I]4a 1

2 2 +E 2 4

[t25|]4b 1 2 2 + E 2 6

[125114¢ 1 1 + E 2 2 2 +E 2 6

0.434 + 0.099 0.144 __. 0.024 0.055 5:0.003 0.044 5:0.010 0.027 + 0.002 0.13 5:0.03 0.465 5:0.085 0.105 + 0.009 0.046 5:0.009 0.043 5:0.005 0.023 5:0.001 0.09 5:0.02 0.129 5:0.010 0.157 5:0.010 0.067 __. 0.004 0.034 5:0.005 0.024 5:0.001 0.10 5:0.02 0.200 5:0.20 0.074 5:0.008 0.025 + 0.002 0.024 5:0.003 0.026 5:0.002 0.33 5:0.14 0.821 + 0.138 0.146 5:0.014 0.079 5:0.009 0.063 5:0.007 0.019 5:0.002 0.15 5:0.04 0.751 5:0.072 0.117 + 0.008 0.050 5:0.004 0.074 5:0.012 0.013 5:0.001 0.13 5:0.04 0.265 5:0.036 0.121 5:0.008 0.048 5:0.010 0.064 5:0.009 0.013 5:0.001 0.14 5:0.04 0.704 5:0.106 0.085 5:0.008 0.027 5:0.003 0.027 5:0.004 0.012 5:0.001 0.43 5:0.16 0.270 + 0.013 0.164 5:0.009 0.084 5:0.004 0.055 + 0.004 0.037 + 0.003 0.13 5:0.02 0.161 5:0.022 0.201 5:0.012 0.093 + 0.004 0.063 5:0.004 0.038 5:0.002 0.10 5:0.02 0.212 5:0.102 0.111 5:0.024 1.036 5:0.004 0.126 5:0.024 0.016 5:0.001 0.10 5:0.03 0.237 + 0.114 0.104 5:0.028 0.037 + 0.002 0.102 5:0.059 0.016 + 0.002 0.10 5:0.03 0.057 5:0.038 0.071 5:0.005 0.024 + 0.002 0.015 5:0.001 0.020 5:0.003 0.42 5:0.05

*Mean + SD for N = 5-6 rats. +E 2 ffi 50 #g estradiol coinjected with radiotracer. Thyroid values are percent of injected dose (% ID).

was evaluated by the coadministration of the radio- chemical with 50 ng of estradiol and performing the tissue distribution at 2 h, the difference in tissue levels at 2 h representing the degree of receptor mediated localization. The percent injected dose and mass (kg) per gram of tissue or plasma and the tissue to plasma ratios were calculated and compared to that pre- viously obtained with 4a and 4b (Tables 1 and 2).

The biological distribution study in rats revealed a market effect of the ll-stereochemistry on estrogen responsive tissue uptake, retention and selectivity. Compared to the 1 l-unsubstituted compound ([125I]4a), the 1 lfl-methoxy radiochemical possessed almost twice the uterine uptake (0.75-0.82vs 0.43--0.47% 1D-kg/g at 1-2h) and much greater retention. The ll~-methoxy derivative did not localize as well in the uterus and its 6 h retention was poor. The degree of receptor mediated uptake, as represented by suppression in the presence of exogenous estradiol, displayed the same pattern. The l lfl-methoxy derivative had the greatest specific binding followed by the unsubstituted and 1 l~-methoxy compounds. The tissue to blood ratios (Table 2) also present the effect of stereochemistry where the uterus to blood ratios for [~25I]4a and [t25I]4b are 10-20 times that of the nontarget tissue while the uterus to blood ratio of [~25I]4c is only 2-5 times greater. Therefore the localization of the radio- labeled estrogens on the largest tissue very closely parallels the pharmacological effects of the corre- sponding 17~-ethynyl analogs rather than the in vitro binding affinities. As has been described (Raynaud et

Table 2. Tissue to blood ratios following i.v. administration of estrogenic radioligands in immature female rats

Compound Time (h) Uterus L i v e r Lungs Muscle

[J25I~a 1 16.1 5.3 2.0 1.6 2 20.2 4.6 2.0 1.9 4 7.7 2.8 1.0 1.0

['25I]4b 1 43.0 7.7 4.2 3.3 2 56.1 8.8 3.8 3.6 6 ~.7 7.1 2.3 2.3

[125I]4c 1 7.3 4.4 2.3 1.5 2 16.7 8.1 2.3 6.4 6 2.9 3.6 1.2 0.8

al., 1979) moxestrol is a highly potent, slowly dis- sociating estrogen; ethynyl estradiol is less potent and dissociates somewhat more rapidly, and RU-16117, the l l~-methoxy 17~-ethynyl estradiol, is a weak estrogen that rapidly dissociates from the receptor. The receptor binding affinities of these estrogens rank the 17~-ethynyl estradiol higher than the 1 lfl-methoxy derivative which is only slightly higher than the l l~-methoxy isomer. Therefore it is the pharmacological effects which take into account re- ceptor dissociation, recirculation and metabolism that must also be considered in predicting the properties of the radioligands.

In summary, we have described an improved method to prepare the radiolabeled (17~,20E)-21- iodo - 19- norpregna- 1,3, 5(10),20 = tetraene- 3,178 - diols (17~-iodovinyl estrogens) using electrophilic ipso radioiododestannylation. The evaluation of the three radiochemicals reported in this study indicates that the 11-stereochemistry exerts a marked effect on the localization of the agent in vivo and supports the development of the 1 lfl-methoxy or similarly substi- tuted estrogens as potential estrogen receptor binding radiopharmaceuticals.

Acknowledgements--This work has been supported in part by PHS grants 5R01-CA31624 and 5R01-CA41399 and DOE contract DE-AC01-76EV4115.

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