1532
J. Med. Chem. 1989,32,1532-1538
and the precipitated solid was collected by filtration. Recrystallization from cyclohexane gave 4.3 g (100%) of 32. Diethyl [2-[4-(Benzothiazol-2-yl)phenoxy]ethyl]phosphonate (14). A mixture of chloride 32 (2.0g, 0.0069 mol) and triethyl phosphite (3 mL) was heated a t 190-200 "C for 5 h. After the reaction mixture had cooled, the precipitated solid was collected by filtration. The crude phosphonate was chromatographed on a silica gel column by eluting with a benzene/ EtOAc mixture to give 1.5 g (56%) of 14. Recrystallization from cyclohexane yielded 14 as colorless needles: mp 93.0-94.0 "C. Effect on Coronary Flow in the Isolated Guinea Pig Heart. Male guinea pigs of 400-500-g body weight were killed and exsanguinated and promptly thoracotomized. After cannulation of the ascending aorta, the heart was enucleated. The isolated heart was then perfused with Krebs-Henseleit fluid which was oxygenated with a gaseous mixture of 95% O2 and 5% C02, at 34 h 1 "C under a perfusion pressure of 60 cm of H 2 0 by the methods of Langendorff. The test compound, dissolved in propylene glycol to a concentration of 100 pg/mL, was then infused
at a rate of 0.1 mL/min. The coronary flow was measured with a square wave electromagnetic flow meter (Nihon Kohden, MF-26) with an extracorporal probe (Nihon Kohden, FE) set at the top of the cannula and recorded with a multipurpose polygraph (Nihon Kohden, RM-85). The coronary flows before and after infusion were measured, and the percentage gain in coronary flow was obtained.
Registry No. 2, 41716-26-1;3, 120332-20-9;4, 2682-86-2;5, 41806-42-2;6, 120332-21-0;7, 120332-22-1;8, 120332-23-2;9, 120332-24-3; 10,83524-89-4; 11,120332-25-4; 12,120332-26-5; 13, 120332-27-6;14,120332-28-7; 15,41806-41-1;16,41716-19-2; 17, 67273-40-9;18, 2227-61-4;19, 120332-29-8;20, 23222-36-8;21, 60-23-1;22, 4434-13-3;23, 120332-30-1;24, 120332-31-2;25, 120332-32-3;26, 19654-19-4;27, 2182-80-1;28, 1660-94-2;29, 120332-33-4; 30,6265-55-0; 31,84396-09-8; 32,84396-10-1; triethyl phosphite, 122-52-1;2-(benzimidazo1-2-y1)-5-(bromomethyl)pyridine, 120332-34-5;diethyl phosphite, 762-04-9;fostedil, 75889-62-2.
Diethylstilbestrol-Linked Cytotoxic Agents: Synthesis and Binding Affinity for Estrogen Receptors Karsten Krohn,*-t K o n r a d Kulikowski,t and G u y Leclercqt Institut fur Organische Chemie der Universitat Braunschweig, Hagenring 30, 0-3300Braunschweig, Federal Republic of Germany, and Laboratoire de CancBrologie mammaire, Service de MZdecine, Institut J . Bordet, 1000 Brussels, Belgium. Received August 16, 1988
The syntheses of diethylstilbestrol derivatives with a C4 side chain a t the double bond bearing various functional and potentially alkylating groups (9-25,38-40, 43,44)as well as the coupling product with daunorubicine (41)are described. Derivatives with free phenolic groups show easy isomerization to (2)-stilbenes and styrenes, which could be minimized with silyl protecting groups. Estrogen receptor binding is decreased by polar groups such as carboxylic acids (10)as well as sterically demanding substituents.
The chemotherapy of cancer in its present form suffers from the fact t h a t , i n principle, no difference is m a d e between normal and tumor cells, regardless of whether t h e drug belongs t o the group of alkylants, enzyme inhibitors, or DNA intercalators.' A certain degree of selectivity is mainly due to the higher sensitivity of rapidly growing cells to various kinds of toxic compounds. Numerous efforts have been made to increase the selectivity toward cells and to decrease the systemic toxicity. A possibility for selectivity is offered by hormone-dependent tumors, such as certain breast tumors, which selectively concentrate natural and synthetic estrogens., The idea to induce cytotoxic effects to hormone-dependent t u m o r cells b y covalent linkage of N-mustard groups to t h e steroidal skeleton was tested i n the late six tie^.^ Since that time m a n y compounds have been synthesized and tested in which various ~ - ~(E2), hexcytotoxic groups were linked to e ~ t r a d i o l (1) e s t r o l ~ ' @ ' (2) ~ (HEX), diethylstilbestr01s~J~'~ (3) (DES), or the antiestrogen tamoxifen15 (Chart I). A prerequisite for specificity of these cytotoxic agents is a sufficient binding of the drug to the estrogen receptor, which allows t h e selective u p t a k e into t h e hormone sensitive cells (relative binding affinity compared t o E, = 100%; RBA). Calculations on t h e basis of the number of receptors per cell (about lOOO-lOOOO) and the possible drug concentration show that the R B A value should be at least 1%of that of E,.16 However, chemical modification of estrogens usually produces a dramatic decrease in the binding affinity. Early work on the chemically easy derivatization of the hydroxy groups in E2 (l),HEX (2), arid
Chart I OH
\
Institut fur Organische Chemie der Universitiit Braunschweig. Institut J. Bordet. 0022-2623/89/1832-1532$01.50/0
-OH
HO 3 (diethylstilbestrol. DES)
DES (3) gave products with very low or no RBA, t h u s establishing the essential function of both hydroxy groups (1) Chabner, B. A.; Myers, C. E.; Coleman, C. N.; Johns, D. G. N . Engl. J . Med. 1975, 1107 and 1159. (2) Leclercq, G.; Heuson, J. C. Anticancer Res. 1981,217, 1. (3) Wall, M. E.;Abernethy, G. S.; Carroll, F. I.; Taylor, D. J. J . Med. Chem. 1969, 12, 810. (4) Niculesu-DucHz, I.; Cambanis, A.; THranHuceanu, E. J . Med. Chem. 1967,10, 172. (5) Hamacher, H. Arzmeim.-Forsch./Drug Res. 1979, 29, 463. (6) Hamacher, H. Arzneim-Forsch./Drug Res. 1983, 33, 347. ( 7 ) Schonemann, K. H.; van Vliet, N. P.; Zeelen, F. J. Eur. J . Med. Chem./Chim. Ther. 1980,15, 333. (8) Lam, A.; Begleiter, H. Y. P.; Goldenberg, G. J. J . Med. Chem. 1979, 22,
3
2 (hexestrol, Hex)
1 (estradiol, E p )
200.
(9) Chesne, C.; Leclercq, G.; Pointeau, P.; Patin, H. Eur. J . Med. Chern-Chim. Ther. 1986,21, 3219. (10) Hamacher, H.; Brecht, B. Arch. Pharm. 1977, 310, 662. G 1989 American Chemical Society
Journal of Medicinal Chemistry, 1989, Vol. 32, No. 7 1533
Diethylstilbestrol-Linked Cytotoxic Agents Scheme I
Chart 11
4
11-18 : R1= CH3 19-25: R1=H 11/19 12/20 13/21 14/22 16/23 16/24 17/25 18:
6
7: R ' = O H , R2=Et 8: R ' = E t , R Z = O ~
R 9.:R=CH3 (E) b:R=CH3 ( 2 ) 10:R = H (isomers)
in receptor binding.4J7 On the basis of these results and the fact that DES and HEX are known to have high RBA values18 we worked out a program to investigate the chemistry and receptor binding affinity of DES and HEX derivatives bearing longer chains in the middle part of the molecule remote from the essential hydroxy groups. Of particular interest was the investigation of the influence of side-chain length and polarity of substituents. The present paper on DES and the folowing on HEX report our observations in this regard, including information on the chemical synthesis of such compounds about which little has yet been p u b l i ~ h e d . ' ~ * ~ ~ Chemistry Starting Materials. The starting material for the synthesis of the side-chain DES derivatives was the acid 9 that was previously prepared in connection with a radioimmunoassay for DESz1 The synthesis is outlined in Scheme I and starts with the commercially available deoxyanisoin (4) that was alkylated with iodo ester 5 to yield 622(for improvements on the original procedure, see the Experimental Section). The subsequent Grignard reaction of 6 with ethylmagnesium bromide gave a 92% yield of a 4:l mixture of the threo and erythro esters 7 and 8. Fortunately, the major isomer 7 could be isolated in pure ~~
Hamacher, H. Arch. Pharm. 1978, 311, 184. Hamacher, H.; Mangold, C. Arch. Pharm. 1983, 316, 271. Katzenellenbogen, J. A.; McGorrin, R. J.; Tatee, T.;Kempton, R. J.; Carlson, K. E.; Kinder, D. H. J . Med. Chem. 1981, 24, 435. Vilkas, M.; Epsztein, R.; Sainton, J.; Leclercq, G. Eur. J.Med. Chem.-Chim. Ther. 1982, 17, 191. Wei, L. L.; Katzenellenbogen, B.; Robertson, D. W.; Sompson, D. M.; Katzenellenbogen, J. A. Breast Cancer Res. Treat. 1986, 7, 77. Katzenellenbogen, J. A,; Katzenellenbogen, B. S. Breast Cancer Res. Treat. 1982, 2, 347. Leclercq, G.; Heuson, J.-C. Cancer Treatment Rep. 1978,62, 1255. PBteri, E. J. Chem. SOC.1940, 833. Pons, M.; Michel, F.; de Paulet, A. C.; Gilbert, J.; Miquel, J.-F.; PrBcigoux, G.; Hospital, M.; Ojasoo, T.; Raunaud, J.-P. J. Steroid. Biochem. 1984, 20, 137. Zablocki, J. A.; Katzenellenbogen, J. A.; Carlson, K. E.; Norman, M. J. J . Med. Chem. 1987,30, 829. Krohn, K. Hoppe-Seyler's 2.Physiol. Chem. 1977, 358, 1551. Krohn, K.; Hemme, C. Liebigs Ann. Chem. 1978, 726.
R2 CH20H CHzCI CHzI CH20Et CH3 (CH2)4CH3 CONEt2 COz-t-BuPh
form by crystallization. The stereocenters are lost on dehydration and both isomers 7 and 8 can be transformed to the corresponding stilbenes. On treatment with p toluenesulfonic acid at 110 OC both dehydration and ester cleavage take place to afford a mixture of at least five compounds, the major components being the (E)- and (2)-stilbenes 9a and 9b (65%) together with 35% of isomeric styrenes as shown by analysis of the 400-MHz 'H NMR spectrum. A chromatographic purification of the most important (E)-stilbene methyl ether was possible. Styrene formation does normally not occur in the formation of DES, but it is known to take place with unsymmetrically substituted derivative^.^^ A simultaneous dehydration/ester and ether cleavage is effected by treatment with boron tribromide in dichloromethanez2 or with potassium hydroxide in ethylene glycol at 200 OCZ4to afford phenolic acid 10 (90%) as a mixture of isomers as shown in Scheme I. Our first goal was the synthesis of the DES alkyl halides which are potential alkylants. To this end acids 9a/9b were reduced to alcohol 11 (mixture of isomers) with lithium aluminum hydride in 94% yield and converted to chloride 12 with triphenylphosphine/carbontetrachloride% (Chart 11). It was possible to purify pure (E)-stilbene 12 and also the other derivatives 13-18 from the reaction mixtures containing (2)-stilbenes and styrenes by using a special TLC technique (see the Experimental Section). (Structural assignment was based on the chemical shift of the methyl group in the 'H NMR spectrum at 0.77 ppm, which is characteristic for the E configurationz6). A one-step conversion of alcohol 11 to the corresponding iodide 13 was possible by using the method of Scheffold by treatment of 11 with N,N-dicyclohexyl-N-methyldicyclohexylcarbodiimide.27 For binding studies it was of interest to include long-chain ethers, which were obtained by treatment of 11 with sodium hydride and ethyl iodide to afford the ethyl ether 14. Longer aliphatic chains could be attached by alkylation of deoxyanisoin (4) with n-butyl bromide and n-octyl bromide followed by Grignardation with ethylmagnesium bromide and dehydration to provide intermediates 15 and 16, respectively. The final stage of the reaction sequence was the liberation of the phenolic groups. Crystalline pure (E)-stilbenes 23 and 24 were isolated after methyl ether cleavage with BBr3 or KOH. (23) Hamacher, H.; Bormann, B. Arch. Pharm. 1981, 314, 257. (24) Corse, J. U.S. Patent 2,325,307, July 27, 1943. (25) Appel, R. Angew. Chem. 1975,87,863;Angew. Chem., Int. Ed. Engl. 1975, 14, 801. (26) Winkler, V. W.; Nyman, M. A.; Egan, R. S. Steroids 1971,17, 197. (27) Scheffold, R.; Saladin, E. Angew. Chem. 1972,84, 158; Angew. Chem., Int. Ed. Engl. 1972, 11, 229.
Krohn et al.
1534 Journal of Medicinal Chemistry, 1989, Vol. 32, No. 7 Chart I11
Table I. Estradiol Receptor Binding Affinities of DES Derivatives
R'O 26-37: R'=TBDMS 3 9 - 4 4 : R'= H
OMe 0
OH
0
I
R2
26/10 27/38 28/39 29/40
COOH C02CH3 C02C6Hg COoCfiHiCl
no. HO
I
42 (dad
35/19 CH2OH 3 8 / 4 3 CH2S03CH3 3 7 / 4 4 CH20CONHCH2CH2CI
However, treatment of the alkali-labile halides 12 and 13 with BBr3 and alcohol 11 and ether 14 with KOH at 200 OC resulted again in mixtures of (E)-and Wstilbenes 19-22 as well as styrenes in a similar ratio previously observed for 10. In order to study the chemical stability in comparison to similar N-mustards some amides and esters were prepared from acids 9a/9b. The coupling of an activated p-nitrophenyl ester28with diethylamine afforded amide 17. The corresponding 4-tert-butylphenyl ester 18 was synthesized in a similar manner. Chromatographic separation of the pure @)-stilbene was possible, but BBr3 treatment of 17 again gave a mixture of phenolic isomers. Careful analysis of the 400-MHz lH NMR spectrum showed the presence of about 60% of (E)-stilbene 17% of (Z)-stilbene, and 23% of styrenes. The easy E-2 isomerization of nonsteroidal estrogens is known,29but the isomerization to styrenes had no precedent. The extreme tendency of the unsymmetrically substituted DES derivatives toward double-bond isomerization made it necessary to liberate the phenols under much milder conditions. The phenolic acid 10 (as a mixture of isomers) was silylated with tert-butyldimethylsilyl chloride and subsequent selective ester cleavage of a persilated intermediate gave acid 26 that was the starting material for a number of acyl derivatives listed in Chart 111. Methyl ether 27 was obtained on treatment of 26 with diazomethane, and the substituted phenyl esters 28-31 were prepared with DCCI in the coupling reaction of acid 26 with the corresponding phenols. The pentachlorophenyl ester 31 proved to be an excellent precursor for the further preparation of acyl amides. Treatment with diethanolamine afforded the bis(hydroxyethy1)amide 33 accompanied by some mono(hydroxyethy1) amide 32 from impurities of the diethanolamine. All of these phenyl esters and amides could be chromatographically purified to the pure (E)-stilbenes. In order to investigate the biological activity and RBA of DNA intercalators linked to estrogens, the antitumor antibiotic daunorubicin was reacted with the activated ester 31 to afford the N-coupled adduct 34. A number of DES derivatives were prepared from alcohol 35, which is readily available by lithium aluminum hydride reduction of pure (E)-stilbene ester 27. Mesylation of 35 gave mesylate 36. A carbamidic 6-chloroethylamino function was introduced by using the method of Staab30 ~
R'
41 10 24 39 40 25 44 22 19 21 43 20 23
R
CH20SOZCHs CH4.21 CH,
RBA no binding 0.1 0.2 0.3 0.3 1.0 1.0 1.2 1.5 1.5 1.8 3.0 10.0
by reaction of protected alcohol 35 with carbonyldiimidazole followed by treatment with P-chloroethylamine to afford 37. Fluoride deprotection of the isomericallypure (E)-stilbenes 26-29 and 34-37 gave the bisphenols 10, 38-40, and 41-44 containing less than 10% of isomeric (2)-stilbenes and styrenes as shown by 'H NMR. However, the compounds smoothly isomerized in aqueous solution to equilibrium of (E)-and (2)-stilbenes and styrenes. Estrogen Receptor Binding Table I gives the relative binding affinity (E, = 100)for several of the synthetic DES derivatives. With the exception of the daunomycin derivative 41, which is totally devoid of binding affinity (RBA
