Synthesis and estrogen activity screening of some new D-secoestrone derivatives Julijana A. Petrovi6, * Vjera M. PejanoviC,? DuSan A. Miljkovi&* and Jovan T. HranisavljeviiS * Institute of Chemistry, Faculty of Sciences, University of Novi Sad, Novi Sad, Yugoslavia; f Pharmaceutical and Chemical Industry Galenika, Belgrade, and f: Institute of General and Physical Chemistry, University of Belgrade,
Yugoslavia; Yugoslavia
Five new D-secoestrone derivatives were synthesized, starting from 3-methoxy-l7-hydroxy-16,17-secoestra-1,3,5(10)-triene-16-nitrile. Newly synthesized compounds showed a complete loss of estrogenic activity.
(Steroids
55276-278,
Keywords: steroids; D-secosteroids;
1990)
16-halogenated secosteroids;
antiestrogens
Introduction
Experimental
Nearly 30 years ago, Stamleri presented evidence that estrogens play a significant role in human female resistance toward atherosclerosis due to their ability to alter serum lipid concentration. However, the effect of estrogens on secondary sex characteristics prevents their therapeutic application. In addition, Baran* demonstrated that in certain D-secoestrone derivatives, estrogenic activity was considerably decreased, while it still significantly affected the blood lipid level. In our earlier reports~5 we established that D ring fragmentation can easily be achieved in some 16-0ximino-17P-hydroxysteroid derivatives under the conditions of Beckmann fragmentation reaction, whereupon the corresponding 16,17-seco-16-cyano-17aldehydes were obtained in high yields. Since then, D-secoestrogens have not been systematically studied. There have been only a few reports in the literature covering this subject: in 1979, Wachter et a1.6 isolated some new D-secosteroids as minor products in estrone 3-methyl ether irradiation, and, in addition, Auchus’ recently reported new data concerning chemistry and pharmacology of 14,15secoestratrienes. The main aim of this work was the synthesis of some new D-secosteroid derivatives, as well as the determination of their residual estrogenic activity, in order to further study their possible hypolipidemic and/or antiestrogenic activity.
Melting points (mp) were determined in open capillary tubes on a Btichi SMP apparatus and are uncorrected. Infrared spectra were recorded in KBr pellets on a Perkin-Elmer M 457 spectrophotometer. Nuclear magnetic resonance (NMR) spectra were determined in CDCl, on a Varian FT-80A spectrometer and are reported in parts per million downfield from a tetramethylsilane internal standard. Mass spectra were recorded on a Finnigan-MAT 8230 (E.I. 70 eV) spectrometer.
Address reprint requests to Dr. Julijana A. PetroviC, Institute of Chemistry, Faculty of Sciences, University of Novi Sad, 2100 Novi Sad, Dr. Ilije DjuriEiCa 4, Yugoslavia. Received July 25, 1989; revised December 8, 1989.
276
Steroids,
1990, vol. 55, June
3-Methoxy-17-p-toluenesulfonyloxy-I6,17secoestra-1,3,5(10)-triene-I6-nitrile (2) 3-Methoxy-17-hydroxy-16,17-secoestra-l,3,5(10)-triene-16-nitrile (1; 1 g, 3.34 mmol) andp-toluenesulfonyl chloride were dissolved in absolute pyridine (30 ml). The reaction mixture was kept at room temperature for 50 hours and was then poured into 6N-HCL (300 ml). The precipitates were collected by filtration, washed with water, and air-dried, giving a crude ptoluenesulfonate ester 2 in a 93% yield. After crystallization from MeOH, 1.06 g (70%) of pure 3-methoxy17-p-toluenesulfonyloxy-16,l7-secoestra-l,3,5(10)-triene-16-nitrile (2), mp 88 to 91°C was obtained. IR spectra (Y,,, , cm-‘): 3,050, 2,225, 1,605, 1,505, 1,375, 1,180; NMR spectra (6, ppm): 0.95 (s, C-18 Me), 2.50 (s, Me from tosyl group), 3.50 (q, C-17 protons, J = 21.25 Hz), 6.60 to 7.80 (7 aromatic protons); mass spectra (m/z): 453 (M+), 241 (M+-CzHdNO), 212 (M+-CH&N-CH20T,-CH3). Anal. calculated for C26H31N04S: C, 68.87; H, 6.84; N, 3.09. Found: C, 68.99; H, 6.80; N, 3.08.
0 1990 Butterworth-Heinemann
New D-secoestrone
3-Methoxy-17-fluoro-16,I triene-16-nitrile (3)
7-secoestra-1,3,5(10)-
3-Methoxy-l7-p-toluenesulfonyloxy-16,17-secoestra1,3,5( lO)-triene-16nitrile (2; 1 g, 2.21 mmol) and tetrabutyl ammonium fluoride trihydrate (3.66 g, 11.62 mmol) were dissolved in methyl ethyl ketone (40 ml) and the obtained solution was refluxed for 20 hours. Reaction mixture was then poured into ice-cold water (500 ml) and the crude product was extracted with ether. The extract was washed with water, dried over anhydrous Na#O,, and evaporated to dryness in vacue. The remaining oily product (800 mg) was chromatographed on a silica gel column (80 g, benzene), whereby 340 mg (51%) of 3-methoxy-17-fluoro-16,17secoestra-1,3,5(10)-triene-16-nitrile (3), mp 80 to 81°C was obtained. IR spectra (u-, cm-‘): 2,240, 1,610, 1,500, 1,255; NMR spectra (8, ppm): 0.98 (d, C-18, Me, 4Jur = 3 Hz), 3.75 (s, -OMe), 4.20 (2 AB-quartets, J,b = 9.37 Hz, JH~F = 48.12 HZ, Jnbr = 45.50 HZ, C-17 protons), 6.50 to 7.25 (3 aromatic protons); mass spectrum (m/z): 301 (M+), 300,233,212,41 (CHzC = NH). Anal. calculated for Ci9HZ4NOF: C, 75.79; H, 7.97; N, 4.65. Found: C, 75.14; H, 7.93; N, 4.67.
3-Methoxy-17-chloro-16,17+ecoestra1,3,5(10)-triene-1 Gnitrile (4) 3-Methoxy-17-p-toluenesulfonyloxy-16,17-secoestra1,3,5(10)-triene-16nitrile (2; 1 g, 2.21 mmol) and tetrabutyl ammonium chloride (3.33 g, 11.89 mmol), dissolved in methyl ethyl ketone (40 ml), were refluxed for 42 hours. Reaction mixture was poured into icecold water (300 ml), and precipitates were collected by filtration, washed with water, and air-dried, alfording 0.52 g (74%) of crude 4, mp 101 to 104°C. After repeated crystallization from di-iso-propyl ether-benzene (1 : l), 0.48 g (6%) of pure 3-methoxy-17-chloro16,17-secoestra-1,3,5(10)-triene-16nitrile (4), mp 118 to 119°C was obtained. cm-‘): 2,240, 1,610, 1,500, 1,240; IR spectra (v,, NMR spectra (6, ppm): 1.00 (s, C-18 Me), 3.45 (q, J = 22.50 Hz, C-17 protons), 3.75 (s, -OMe), 6.60 to 7.30 (3 aromatic protons); mass spectra (m/z): 317.5 (M+), 212,41. Anal. calculated for C1sHZ4NOC1:C, 71.81; H, 7.56; N, 4.41. Found: C, 72.30; H, 7.97; N, 4.36.
3-Methoxy-17-bromo-16,17-secoestra1,3,5(10)-triene-16-nitrile (5) 3-Methoxy-17-p-toluenesulfonyloxy-16,17-secoestra1,3,5(10)-triene-16nitrile (2; 1 g, 2.21 mmol) and tetrabutyl ammonium bromide (4.20 g, 12.88 mol) were dissolved in methyl ethyl ketone (30 ml) and the reaction mixture was refluxed for 70 hours. Solution was then poured into ice-cold water (300 ml) and the precipitates were collected by filtration, washed with water, and air-dried, affording 0.80 g of crude 5, mp 122 to 125°C. Repeated crystallization from di-iso-propyl ether-benzene (1 : 1) gave 0.55 g (6%) of analytically
derivatives:
PetroviC et al.
pure 3-methoxy-17-bromo-16,17-secoestra-1,3,5(10)triene-16nitrile (5), mp 129 to 130°C. IR spectra (Y-, cm-i): 2,240, 1,608, 1,510, 1,240; NMR spectra (6, ppm): 1.05 (s, C-18 Me), 3.40 (q, J = 15.0 Hz, C-17 protons), 3.75 (s, -OMe), 6.60 to 7.30 (3 aromatic protons); mass spectra (m/z): 362 (M+), 212, 78,41. Anal. calculated for Ci9H2.+NOBr: C, 62.93; H, 6.62; N, 3.86. Found: C, 63.38; H, 7.06: N, 3.77.
3-Methoxy-17-iodo-16,17-secoestra-1,3,5(10)triene-16~nitrile (6) A solution of compound 2 (1 g, 2.21 mmol) and tetrabutyl ammonium iodide (4.4 g, 11.92 mmol) in methyl ethyl ketone (40 ml) was refluxed for 100 hours. Reaction mixture was then poured into ice-cold water (300 ml) and the precipitates were collected by filtration, washed with water, and air-dried, giving 0.81 g (%) of crude compound 6, mp 124 to 126°C. Repeated crystallization from di-iso-propyl ether-benzene (1: 1) afforded 0.53 g (5%) of analytically pure 3-methoxy-17iodo-16,17-secoestra-1,3,5(10)-triene-16nitrile (6), mp 134 to 135°C. IR spectra (v-, cm-‘): 2,240, 1,605, 1,505, 1,245; NMR spectra (6, ppm): 1.20 (s, C-18 Me), 3.25 (s, C-17 protons), 3.80 (s, -OMe), 6.50 to 7.40 (3 aromatic protons); mass spectra (m/z): 4.09 (M+), 363, 212, 41. Anal. calculated for C19H24NOI: C, 55.70; H, 5.86; N, 3.42. Found: C, 56.23, H, 6.35; N, 3.46.
Biologic test Immature Wistar strain female rats (21 to 23 days old) were randomly divided into groups of seven to 10 animals each. Animals were treated by subcutaneous injection twice a day for 3 days with 0.2 ml of a solution of testing compounds in olive oil. The total administered amounts of compounds 3 through 6 were 200 and 300 pg, while in the case of compound 2 those amounts were 200, 300, 400, and 1,200 pg. The animals were killed on the fourth day. The uteri were removed with the ovaries, dissected free of adhering fat, and blotted dry after expulsion of uterine fluid. The wet weight was recorded.
Results and discussion Five new D-secoestrone derivatives, presented in Scheme 1, were synthesized in this work. The previously synthesized4 secocyanoalcohol 1 with p-toluenesulfonyl chloride in pyridine afforded the corresponding p-toluenesulfonate ester 2. Further treating tosylate 2 with various tetrabutyl ammonium halides in refluxing methyl ethyl ketone afforded 17halogen0 derivatives 3 through 6. All newly synthesized compounds were characterized by usual spectroscopic methods, as well as by satisfactory elemental microanalysis (halogens were only qualitatively detected by Beilstein’s test). The NMR spectra of the newly synthesized compounds showed an interesting behavior for the C-17 protons. Steroids,
1990, vol. 55, June
277
Papers Table 1 Uterotropic Activities of 16,17-Secoestrone 2 Through 6 Compared With E&one
H3CO
Dosage Compound
(&rat)
No. of experimental animals
Derivatives
Uterus weight (mg) (mean + SEMI
2 Control E&one 2
3
1, X= F
s,X=Br
1.
6,x=
X= CI
4 1
5 Scheme
1 6
Namely, in the NMR spectra of compounds 2 (tosylate), 4 (chloride), and 5 (bromide), these protons appear as AB quartets, having different coupling constants. In the case of fluoro derivative 3, two AB quartets for C-17 protons are present, whereas in the case of iodo derivative 6, the signal for C-17 protons appears as a singlet. An additional explanation for the observed phenomena involves an assumption that in the D-seco derivatives 2 through 5, a quasi ring D can be formed via dipole to dipole interactions between Cf,+ and C,-I-haloge&, thus partially preventing free rotation around the Cl3 to Cl7 bond. To prove or disprove such a hypothesis, NMR spectra of halogen0 derivatives 3 through 6 were taken in a temperature gradient (250 to 330 K). However, no change was noted in the position and shape of signals for C-17 protons. Therefore, the mentioned phenomena can be exclusively assigned to the diastereotopic relationship8 of C-17 protons, being most pronounced in the case of fluoride 3 (with the most polarized and shortest C-halogen bond), while in the case of iodo derivative 6, possessing the least polarized and the longest C-halogen bond, this relationship practically disappears, so that C-17 protons behave equivalently. The newly synthesized derivatives were submitted to screening of their possible residual estrogenic activity using the uterotropic method.9 The results of these tests, in comparison with estrone, are presented in Table 1. Table 1 shows almost complete loss of uterotropic activity for all examined D-secoestrone derivatives (2 through 6) at dosages 22 to 44 times higher than those of estrone. Even a dosage of compound 2 130 times greater (compared with estrone) did not reveal significant uterotropic activity. On the contrary, there is a slight, not yet understood decrease in the average uterus weight in tested
278
Steroids,
1990, vol. 55, June
9 200 300 400 1,200 200 300 200 300 200 300 200 300
30 7 24 8 10 10 10 10 17 16 9 9 9 8
73.2 168.1 72.8 72.4 63.0 87.0 73.0 71.4 67.6 68.3 56.8 59.5 52.6 59.3
+ + 2 + c ? 2 + +? -+ 2 -+ -c
11.3 9.7 14.0 12.4 6.4 12.0 15.4 8.4 11.5 15.0 4.7 10.5 7.8 5.6
animals. This phenomenon will be studied separately. In addition, a possible antiestrogenic, as well as hypocholesterolemic, activity of compounds 2 through 6 will be studied and reported in the near future.
Acknowledgments The authors are grateful to EEC-Yugoslav Common Scientific Fund for financing this work. In addition, the authors are indebted to Zlatica HranisavljeviC and Milica BerkoviC for skillful assistance in performing biological tests.
References 1. 2.
3.
4.
5. 6. 7.
Stamler J (1%3). In: Sandler M, Bourne GH (eds) Atherosclerosis and Its Origin. Academic Press, New York, pp. 231-248. Baran JS (1967). The synthesis, stereochemistry, and biology of 16-hetero and lJ-oxo-o-homo steroids. J Med Chem l&1039-1047. MiijkoviC D, PetroviC J, StajiC M, MiljkoviC M (1973). The
Beckmann fragmentation reaction of some cY-hydroxy ketoximes. J Org Chem 3&3585-3588. MiljkoviC D, PetroviC J (1977). Beckmann fragmentation reaction of 3-methoxy-lJ~-hydroxyestra-l,3,5(10)-trien-16-one oxime. J Org Chem 42:2101-3102. MiljkoviC D, Petrovif J, HadZiC P (1978). Some new ring-D steroids. Tetrahedron 34:3575-3577. Wachten MP, Adams RE, Cotter ML, Settepani JA (1979). Lumi-mestranol and epi-lumi-mestranol. Steroids 33~287-294. Auchus RJ (1988). 14,15-Secoestratrienes as Inactivators of EstradiolDehydronenase From Human Term Placenta: istry, Enzym&logyr Spectroscopy, and Pharmacology.
8.
9.
Chem-
Washinaton Universitv. St. Louis. .up. l-212, Diss. Abstr. Int. B 1989, 49(10), 4245: Potapov S (1978). The enantiotopic and diastereotopic relationships of atoms and erouos. In: Stereochemistry. Mir, Moskow, pp.-57-62. . Emmens CW (1950). Estrogens. In: Emmens CW (ed) Hormone Assay. Academic Press, New York, pp. 408-410.
Yugoslavia; Yugoslavia
Five new D-secoestrone derivatives were synthesized, starting from 3-methoxy-l7-hydroxy-16,17-secoestra-1,3,5(10)-triene-16-nitrile. Newly synthesized compounds showed a complete loss of estrogenic activity.
(Steroids
55276-278,
Keywords: steroids; D-secosteroids;
1990)
16-halogenated secosteroids;
antiestrogens
Introduction
Experimental
Nearly 30 years ago, Stamleri presented evidence that estrogens play a significant role in human female resistance toward atherosclerosis due to their ability to alter serum lipid concentration. However, the effect of estrogens on secondary sex characteristics prevents their therapeutic application. In addition, Baran* demonstrated that in certain D-secoestrone derivatives, estrogenic activity was considerably decreased, while it still significantly affected the blood lipid level. In our earlier reports~5 we established that D ring fragmentation can easily be achieved in some 16-0ximino-17P-hydroxysteroid derivatives under the conditions of Beckmann fragmentation reaction, whereupon the corresponding 16,17-seco-16-cyano-17aldehydes were obtained in high yields. Since then, D-secoestrogens have not been systematically studied. There have been only a few reports in the literature covering this subject: in 1979, Wachter et a1.6 isolated some new D-secosteroids as minor products in estrone 3-methyl ether irradiation, and, in addition, Auchus’ recently reported new data concerning chemistry and pharmacology of 14,15secoestratrienes. The main aim of this work was the synthesis of some new D-secosteroid derivatives, as well as the determination of their residual estrogenic activity, in order to further study their possible hypolipidemic and/or antiestrogenic activity.
Melting points (mp) were determined in open capillary tubes on a Btichi SMP apparatus and are uncorrected. Infrared spectra were recorded in KBr pellets on a Perkin-Elmer M 457 spectrophotometer. Nuclear magnetic resonance (NMR) spectra were determined in CDCl, on a Varian FT-80A spectrometer and are reported in parts per million downfield from a tetramethylsilane internal standard. Mass spectra were recorded on a Finnigan-MAT 8230 (E.I. 70 eV) spectrometer.
Address reprint requests to Dr. Julijana A. PetroviC, Institute of Chemistry, Faculty of Sciences, University of Novi Sad, 2100 Novi Sad, Dr. Ilije DjuriEiCa 4, Yugoslavia. Received July 25, 1989; revised December 8, 1989.
276
Steroids,
1990, vol. 55, June
3-Methoxy-17-p-toluenesulfonyloxy-I6,17secoestra-1,3,5(10)-triene-I6-nitrile (2) 3-Methoxy-17-hydroxy-16,17-secoestra-l,3,5(10)-triene-16-nitrile (1; 1 g, 3.34 mmol) andp-toluenesulfonyl chloride were dissolved in absolute pyridine (30 ml). The reaction mixture was kept at room temperature for 50 hours and was then poured into 6N-HCL (300 ml). The precipitates were collected by filtration, washed with water, and air-dried, giving a crude ptoluenesulfonate ester 2 in a 93% yield. After crystallization from MeOH, 1.06 g (70%) of pure 3-methoxy17-p-toluenesulfonyloxy-16,l7-secoestra-l,3,5(10)-triene-16-nitrile (2), mp 88 to 91°C was obtained. IR spectra (Y,,, , cm-‘): 3,050, 2,225, 1,605, 1,505, 1,375, 1,180; NMR spectra (6, ppm): 0.95 (s, C-18 Me), 2.50 (s, Me from tosyl group), 3.50 (q, C-17 protons, J = 21.25 Hz), 6.60 to 7.80 (7 aromatic protons); mass spectra (m/z): 453 (M+), 241 (M+-CzHdNO), 212 (M+-CH&N-CH20T,-CH3). Anal. calculated for C26H31N04S: C, 68.87; H, 6.84; N, 3.09. Found: C, 68.99; H, 6.80; N, 3.08.
0 1990 Butterworth-Heinemann
New D-secoestrone
3-Methoxy-17-fluoro-16,I triene-16-nitrile (3)
7-secoestra-1,3,5(10)-
3-Methoxy-l7-p-toluenesulfonyloxy-16,17-secoestra1,3,5( lO)-triene-16nitrile (2; 1 g, 2.21 mmol) and tetrabutyl ammonium fluoride trihydrate (3.66 g, 11.62 mmol) were dissolved in methyl ethyl ketone (40 ml) and the obtained solution was refluxed for 20 hours. Reaction mixture was then poured into ice-cold water (500 ml) and the crude product was extracted with ether. The extract was washed with water, dried over anhydrous Na#O,, and evaporated to dryness in vacue. The remaining oily product (800 mg) was chromatographed on a silica gel column (80 g, benzene), whereby 340 mg (51%) of 3-methoxy-17-fluoro-16,17secoestra-1,3,5(10)-triene-16-nitrile (3), mp 80 to 81°C was obtained. IR spectra (u-, cm-‘): 2,240, 1,610, 1,500, 1,255; NMR spectra (8, ppm): 0.98 (d, C-18, Me, 4Jur = 3 Hz), 3.75 (s, -OMe), 4.20 (2 AB-quartets, J,b = 9.37 Hz, JH~F = 48.12 HZ, Jnbr = 45.50 HZ, C-17 protons), 6.50 to 7.25 (3 aromatic protons); mass spectrum (m/z): 301 (M+), 300,233,212,41 (CHzC = NH). Anal. calculated for Ci9HZ4NOF: C, 75.79; H, 7.97; N, 4.65. Found: C, 75.14; H, 7.93; N, 4.67.
3-Methoxy-17-chloro-16,17+ecoestra1,3,5(10)-triene-1 Gnitrile (4) 3-Methoxy-17-p-toluenesulfonyloxy-16,17-secoestra1,3,5(10)-triene-16nitrile (2; 1 g, 2.21 mmol) and tetrabutyl ammonium chloride (3.33 g, 11.89 mmol), dissolved in methyl ethyl ketone (40 ml), were refluxed for 42 hours. Reaction mixture was poured into icecold water (300 ml), and precipitates were collected by filtration, washed with water, and air-dried, alfording 0.52 g (74%) of crude 4, mp 101 to 104°C. After repeated crystallization from di-iso-propyl ether-benzene (1 : l), 0.48 g (6%) of pure 3-methoxy-17-chloro16,17-secoestra-1,3,5(10)-triene-16nitrile (4), mp 118 to 119°C was obtained. cm-‘): 2,240, 1,610, 1,500, 1,240; IR spectra (v,, NMR spectra (6, ppm): 1.00 (s, C-18 Me), 3.45 (q, J = 22.50 Hz, C-17 protons), 3.75 (s, -OMe), 6.60 to 7.30 (3 aromatic protons); mass spectra (m/z): 317.5 (M+), 212,41. Anal. calculated for C1sHZ4NOC1:C, 71.81; H, 7.56; N, 4.41. Found: C, 72.30; H, 7.97; N, 4.36.
3-Methoxy-17-bromo-16,17-secoestra1,3,5(10)-triene-16-nitrile (5) 3-Methoxy-17-p-toluenesulfonyloxy-16,17-secoestra1,3,5(10)-triene-16nitrile (2; 1 g, 2.21 mmol) and tetrabutyl ammonium bromide (4.20 g, 12.88 mol) were dissolved in methyl ethyl ketone (30 ml) and the reaction mixture was refluxed for 70 hours. Solution was then poured into ice-cold water (300 ml) and the precipitates were collected by filtration, washed with water, and air-dried, affording 0.80 g of crude 5, mp 122 to 125°C. Repeated crystallization from di-iso-propyl ether-benzene (1 : 1) gave 0.55 g (6%) of analytically
derivatives:
PetroviC et al.
pure 3-methoxy-17-bromo-16,17-secoestra-1,3,5(10)triene-16nitrile (5), mp 129 to 130°C. IR spectra (Y-, cm-i): 2,240, 1,608, 1,510, 1,240; NMR spectra (6, ppm): 1.05 (s, C-18 Me), 3.40 (q, J = 15.0 Hz, C-17 protons), 3.75 (s, -OMe), 6.60 to 7.30 (3 aromatic protons); mass spectra (m/z): 362 (M+), 212, 78,41. Anal. calculated for Ci9H2.+NOBr: C, 62.93; H, 6.62; N, 3.86. Found: C, 63.38; H, 7.06: N, 3.77.
3-Methoxy-17-iodo-16,17-secoestra-1,3,5(10)triene-16~nitrile (6) A solution of compound 2 (1 g, 2.21 mmol) and tetrabutyl ammonium iodide (4.4 g, 11.92 mmol) in methyl ethyl ketone (40 ml) was refluxed for 100 hours. Reaction mixture was then poured into ice-cold water (300 ml) and the precipitates were collected by filtration, washed with water, and air-dried, giving 0.81 g (%) of crude compound 6, mp 124 to 126°C. Repeated crystallization from di-iso-propyl ether-benzene (1: 1) afforded 0.53 g (5%) of analytically pure 3-methoxy-17iodo-16,17-secoestra-1,3,5(10)-triene-16nitrile (6), mp 134 to 135°C. IR spectra (v-, cm-‘): 2,240, 1,605, 1,505, 1,245; NMR spectra (6, ppm): 1.20 (s, C-18 Me), 3.25 (s, C-17 protons), 3.80 (s, -OMe), 6.50 to 7.40 (3 aromatic protons); mass spectra (m/z): 4.09 (M+), 363, 212, 41. Anal. calculated for C19H24NOI: C, 55.70; H, 5.86; N, 3.42. Found: C, 56.23, H, 6.35; N, 3.46.
Biologic test Immature Wistar strain female rats (21 to 23 days old) were randomly divided into groups of seven to 10 animals each. Animals were treated by subcutaneous injection twice a day for 3 days with 0.2 ml of a solution of testing compounds in olive oil. The total administered amounts of compounds 3 through 6 were 200 and 300 pg, while in the case of compound 2 those amounts were 200, 300, 400, and 1,200 pg. The animals were killed on the fourth day. The uteri were removed with the ovaries, dissected free of adhering fat, and blotted dry after expulsion of uterine fluid. The wet weight was recorded.
Results and discussion Five new D-secoestrone derivatives, presented in Scheme 1, were synthesized in this work. The previously synthesized4 secocyanoalcohol 1 with p-toluenesulfonyl chloride in pyridine afforded the corresponding p-toluenesulfonate ester 2. Further treating tosylate 2 with various tetrabutyl ammonium halides in refluxing methyl ethyl ketone afforded 17halogen0 derivatives 3 through 6. All newly synthesized compounds were characterized by usual spectroscopic methods, as well as by satisfactory elemental microanalysis (halogens were only qualitatively detected by Beilstein’s test). The NMR spectra of the newly synthesized compounds showed an interesting behavior for the C-17 protons. Steroids,
1990, vol. 55, June
277
Papers Table 1 Uterotropic Activities of 16,17-Secoestrone 2 Through 6 Compared With E&one
H3CO
Dosage Compound
(&rat)
No. of experimental animals
Derivatives
Uterus weight (mg) (mean + SEMI
2 Control E&one 2
3
1, X= F
s,X=Br
1.
6,x=
X= CI
4 1
5 Scheme
1 6
Namely, in the NMR spectra of compounds 2 (tosylate), 4 (chloride), and 5 (bromide), these protons appear as AB quartets, having different coupling constants. In the case of fluoro derivative 3, two AB quartets for C-17 protons are present, whereas in the case of iodo derivative 6, the signal for C-17 protons appears as a singlet. An additional explanation for the observed phenomena involves an assumption that in the D-seco derivatives 2 through 5, a quasi ring D can be formed via dipole to dipole interactions between Cf,+ and C,-I-haloge&, thus partially preventing free rotation around the Cl3 to Cl7 bond. To prove or disprove such a hypothesis, NMR spectra of halogen0 derivatives 3 through 6 were taken in a temperature gradient (250 to 330 K). However, no change was noted in the position and shape of signals for C-17 protons. Therefore, the mentioned phenomena can be exclusively assigned to the diastereotopic relationship8 of C-17 protons, being most pronounced in the case of fluoride 3 (with the most polarized and shortest C-halogen bond), while in the case of iodo derivative 6, possessing the least polarized and the longest C-halogen bond, this relationship practically disappears, so that C-17 protons behave equivalently. The newly synthesized derivatives were submitted to screening of their possible residual estrogenic activity using the uterotropic method.9 The results of these tests, in comparison with estrone, are presented in Table 1. Table 1 shows almost complete loss of uterotropic activity for all examined D-secoestrone derivatives (2 through 6) at dosages 22 to 44 times higher than those of estrone. Even a dosage of compound 2 130 times greater (compared with estrone) did not reveal significant uterotropic activity. On the contrary, there is a slight, not yet understood decrease in the average uterus weight in tested
278
Steroids,
1990, vol. 55, June
9 200 300 400 1,200 200 300 200 300 200 300 200 300
30 7 24 8 10 10 10 10 17 16 9 9 9 8
73.2 168.1 72.8 72.4 63.0 87.0 73.0 71.4 67.6 68.3 56.8 59.5 52.6 59.3
+ + 2 + c ? 2 + +? -+ 2 -+ -c
11.3 9.7 14.0 12.4 6.4 12.0 15.4 8.4 11.5 15.0 4.7 10.5 7.8 5.6
animals. This phenomenon will be studied separately. In addition, a possible antiestrogenic, as well as hypocholesterolemic, activity of compounds 2 through 6 will be studied and reported in the near future.
Acknowledgments The authors are grateful to EEC-Yugoslav Common Scientific Fund for financing this work. In addition, the authors are indebted to Zlatica HranisavljeviC and Milica BerkoviC for skillful assistance in performing biological tests.
References 1. 2.
3.
4.
5. 6. 7.
Stamler J (1%3). In: Sandler M, Bourne GH (eds) Atherosclerosis and Its Origin. Academic Press, New York, pp. 231-248. Baran JS (1967). The synthesis, stereochemistry, and biology of 16-hetero and lJ-oxo-o-homo steroids. J Med Chem l&1039-1047. MiijkoviC D, PetroviC J, StajiC M, MiljkoviC M (1973). The
Beckmann fragmentation reaction of some cY-hydroxy ketoximes. J Org Chem 3&3585-3588. MiljkoviC D, PetroviC J (1977). Beckmann fragmentation reaction of 3-methoxy-lJ~-hydroxyestra-l,3,5(10)-trien-16-one oxime. J Org Chem 42:2101-3102. MiljkoviC D, Petrovif J, HadZiC P (1978). Some new ring-D steroids. Tetrahedron 34:3575-3577. Wachten MP, Adams RE, Cotter ML, Settepani JA (1979). Lumi-mestranol and epi-lumi-mestranol. Steroids 33~287-294. Auchus RJ (1988). 14,15-Secoestratrienes as Inactivators of EstradiolDehydronenase From Human Term Placenta: istry, Enzym&logyr Spectroscopy, and Pharmacology.
8.
9.
Chem-
Washinaton Universitv. St. Louis. .up. l-212, Diss. Abstr. Int. B 1989, 49(10), 4245: Potapov S (1978). The enantiotopic and diastereotopic relationships of atoms and erouos. In: Stereochemistry. Mir, Moskow, pp.-57-62. . Emmens CW (1950). Estrogens. In: Emmens CW (ed) Hormone Assay. Academic Press, New York, pp. 408-410.
