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Diterpenoids with the daphnane skeleton from Wikstroemia retusa
Phytochemistry, Vol. 32, No. 1, pp. 141-143, 1993 Printed in Great Britain.
DITERPENOIDS
0031~9422/93 $5.00+0.00 Pergamon Prw Ltd
WITH THE DAPHNANE
SKELETON FROM WZKSTROEMlA
RETUSA SHIRYO
YAGA,KAZUHIKOKINJO,HIROYAHAYASHI,NAOCHIKAMATSUO,FUMIKOABE* and TATSUOYAMAUCHI*
Faculty of Agriculture,University of the Ryukyu, Nishihara-cho,Okinawa 903-01, Japan;*Faculty of PharmaceuticalSciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-01,Japan (Received18 May 1992) Key Word Index-LVikstroemia diterpenoid.
retusa; Thymelaeaceae; daphnane diterpenoid; macrocyclic daphnane
Abstract-Four diterpenoids with the daphnane skeleton were isolated from Wikstroemia retusa, a native plant in the Ryukyu Islands, and the structures established by means of mass and NMR spectroscopy.
INTRODUCTION Wikstroemia retusa A. Gray is indigenous to the Ryukyu Islands of Japan and its bark is used locally for the preparation of paper, as is the bark of other species of Thymelaeaceae in Japan. Since the quality of the paper is fine and it can be stored for long periods of time, the presence of constituents against insect damage was expected. We report the isolation and characterization of four daphnane diterpenoids, of which three are new compounds. RESULTS ANDDISCUSSION The bark was extracted with ethanol and the extract fractionated (see Experimental). This gave four diterpenoids having a daphnane skeleton (l-4). Compound 1 (CJ4Hs,,0s) was identified as huratoxin (‘H NMR and FAB mass spectrometry), previously isolated from Wikstroemia monticola [l] and Hura crepitans [2].
FAB mass spectrometry of 2 gave a [M + Na J ’ peak at m/z 665.3304, suggesting CJBHSOOIO, i.e. a diterpenoid with one acetoxyl group more than 1. In fact, the signal due to one acetyl methyl group was observed at 61.98 in the ‘H NMR spectrum. The ‘H NMR signals, including a terminal methyl at 80.88, olefinic protons due to the E,E-diene system, one proton from an enone group, two exomethylene protons and carbinyl protons at C-7, are in good agreement with those of 1. However, the H-12 signals at 6 1.65 and 2.23 are no longer observed and the H-8 and H-14 signals show downfield shifts (+0.56 ppm and 0.32 ppm, respectively) in 2. Similar downfield shifts were observed between huratoxin and wikstrotoxin C [l], of which H-8 and H-14 were shifted +0.58 ppm and +0.32 ppm, respectively. This suggests the presence of a 12/L~cetoxyl group, and one singlet signal at 84.98 is assignable to H-12oz, retaining 90” of the dihedral angle between H-l lfl.
141
S. YAGA et al.
142
The i3C NMR signal assigments of 2 were confirmed by means of 2D i3C--lH COSY. The signals due to the enone function composed of C-l-C-3 were in good agreement with those of wikstrotoxins Cl]. Three olefinic linkages including one exomethylene group were observed as four doublets, one triplet and one singlet in the range 6 11S- 140. A singlet signal at 6 117.0 can be assigned for an orthoester carbon as described for wikstrotoxin C. The nine signals observed between 660-85 are assignable to the carbons containing oxygen functions at C-4-C-7, C-9, C-12-C-14 and C-20. The structure of 2 is thus determined to be 12/%acetoxy-huratoxin. The ‘H NMR spectrum of 3 is almost superimposable on that of 2. Since the FAB mass spectrum [M+Na]+ peak of 3 (m/z 679.3459, C3,Hs20,,Na) was 14 amu higher than that of 2 and the fragment peak at m/z 207 [C,,H,,CO]+ due to the fatty acid side chain in 2 is replaced by the peak at m/z 221 [C,,H,,CO]+, 3 is characterized as a homologue of 2 having a one methylene longer side chain than 2. The FAB mass spectrum of 4 afforded a [M +Na]+ peak at m/z 661.3354, suggesting a molecular formula of C37HS009. Signals arising from rings B and C showed similar chemical shifts and the same coupling patterns as those of 1. Therefore, 4 seemed to be a daphane homologue, although the presence of a benzoyl group was observed. In the 13C NMR spectrum, a singlet signal due
to the orthoester carbon was observed at 6 119.7, showing the presence of a side chain composed of several carbons. Unlike 1-3, the characteristic triplet signal due to the terminal methyl group was replaced by a doublet signal in 4, suggesting that 4 retains a gnidimacrin type macrocyclic structure, in which the second carbon from the terminal position of the side chain is attached to ring A [3]. In fact, no enone system in ring A was observed. The upfield shift of the methyl proton signals at S 1.79 on the enone system in 2 to 6 1.05 in 4 is entirely consistent with the fact that the olefinic linkage of the enone is saturated in 4. Simultaneously, a carbonyl group at C-3 seems to be reduced to a carbinol (682.9). In order to establish the stereochemistry, 2D-NOESY measurements were carried out (Table 1) and the relative configurations at C-l-C-8, C-10, C-i 1 and C-9, were established. Since the carbinol carbon signals of C-5 and C-20 had almost the same chemical shifts as those of 2, and the carbinyl proton signal assignable to H-3 was observed at lower field (65.08), the benzoic acid group is located at the C-3 hydroxyl group as in gnidimacrin [3]. The orientation of H-3 was assigned a, based on NOE to H-5. Compound 4 is thus shown to have the structure represented in formula 4. Daphnane diterpenoids have been isolated from Thymelaeaceae as well as Euphorbiaceae 143. They have interesting biological activities, e.g. antileukaemic [ 1,23,
Table 1. ‘H NMR spectral data of compounds l-3 and 4 CDCI,, TMS as int. stand.; J Hz in parentheses) H
1
2
3
4
1 2 3 5 7 8 10 11 12a
7.63 br s
7.56 br s
7.57 br s
4.25 s 3.44 s 294 d (2) 3.81 d (2) 2.47 dq (9, 7) 1.65 d (14) 2.23 dd (14, 9) 4.42 d(3) 4.91 br s 5.02 br s 1.79 br s 1.18 d(7) 1.79 br s 3.78 d(13) 3.87 d(13) 5.70 d(16) 6.70dd(16,11) 6.06 dd (15,ll) 5.84 dt (l&7)
4.25 s 3.54 s 3.50 d (2) 3.83 d (2) 2.37 q (6) 4.98 s
4.25 s 3.54 s 3.50 d (3) 3.83 d (2) 2.35 q (6) 4.98 s
4.75 d(3) 4.96 br s 5.01 br s 1.83 br s 1.29 d(6) 1.79 br s 3.81 d(13) 3.91 d(13) 5.64 d(16) 6.65 dd (16,lO) 6.04 dd (16,lO) 5.85 dt (16,7)
4.75 d (2) 4.96 br s 5.01 br s 1.83 br s 1.29 d(6) 1.79 br s 3.80 d(13) 3.93 d(13) 5.54d(15) 6.65 dd (15,10) 6.03 dd (15.10) 5.85 dt (16,7)
239 br t (lop”* 1.90 mc 5.08 d(5)” 4.11 P 3.34 sf.g 2.89 d (2)“+’ 3.11 d (11) 2.67 dq (8, 7)‘* 1.62 d (14) 2.13 dd (14.8)” 4.27 d(2)‘+’ 4.85 br sk 4.96 br s’ 1.74 br sJsk 1.45 d(7) 1.05 d(7)b 3.76 d(12r 3.89 d(12)g
2.09 q(7) 0.88 t (6)
2.09 4 (7) 0.88 t (6) (H-15’) 1.98 s (-OAc)
128 14 16 17 18 19 20 2’ 3’ 4 5’ 6 Others
2.09 4 (7) 0.88 t(6) (H-14’)
(H-14’) 1.98 s (-OAc)
Benzoyl moiety 8.04 (2H, dd, 8,l) 7.61 (lH, br t, 8) 7.48 (2H, br t, 8) 0.84 d(7) (H-10’) 2.53 m (H-9’)
a-JOr‘A cross peak was observed between these signals in ZD-NOESY spectrum. *The signal at 62.67 showed a response on irradiation of the proton at 62.39 in NOE difference NMR.
Diterpenoids from Wi~str~~a Table 2. 13C NMR spectral data of com~unds as int. standard) C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
2 160.3 d X36.9 s 209.4 s 83.7 s 71.9 d 60.6 s 64.3 d 35.4 d 72.3 s 47.5 d 44.0 d 78.3 d 78.2 s 80.5 d 143.2 s 113.2 t 18.7 q 18.2 q 9.8 q 65.1 t
4
C
SO.6d 36.3 d 82.9 d 80.1 s* 73.5 d 60.9 s 64.2 d 36.7 d 81.1 s” 47.3 d 35.2 d 36.5 t 83.4 SL 825 d
146.8s
2
1’ 2 3’ 4’ 5
6’ 7’ 8’ 9
.
ICY II’ 12’ 13 14
110.7 t 18.8 q 21.3 q 14.4 q 66.1 t
(CDCl,, TMS
4
117.0s 119.7 s 122.3 d 33.5 t 13S.Od b 27.9 t 128.6 d [ 27.8 t 139.3 d 26.7 t 32.7 t 24.6 t 31.9 t 21.08 29.54 t 37.9 t 29.46 t 29.3 d 29.29 t 12.6q 29.13 t 29.05 t Benzoyl moiety 226 t 167.6 s 14.1 q 129.9 s 129.7 d( x 2) -0Ac 133.4 d 169.6 128.6 d( x 2) 21.1
*Signal assignments were based on 2D ’ 3C-1H COSY spcctra. **bSigntisare inte~~~able.
retusa
143
~xtr~~on and isoiation Air-dried bark and twigs (100 g) were powdered and extracted with hot EtOH. The solvent was evapd in uacuo and the residue extracted with Et@. The Et,0 layer was treated with 1% aq. NaOH. After evapn of the Et,O, the residue was chromatographed on a silica gel column with hexane, hexane-Et&, Et,0 and MeOH. The eluants were further chromatographed on a reversed phase column or subjected to HPLC (ODS, Waters) to afford l(ll.6 mg), 2 (17.1 mg), 3 (10.6 mg), 4 (15.1 mg), Compound 1 was identity as huratoxin by FAB MS and by comparison of the ‘H NMR data with those published [l]. Conzpound 2. Glassy oil, [a]$ + 11.3” (CHCl,; c 0.85), FAB MS m/z 665.3304, C,,H,,OzONa requires 665.3302. m/z 643,583,449,395,207, 185. Compound 3. Glassy oil, [aJ$j (CHCl,; c + 7.9”), FAB MS m/z 679.3459 &H,,O,,,Na requires 679.3458. mfz 657,449,395,221, 185. Compound 4. Glassy oil, [a]Y (CD&; c + 7.6”), FAB MS m/z 661.3354 C3,H,,09Na 661.3353. m/z 639, 449, 185, 105. Acknowledgements-We thank MS Y. Iwase, J. Honda and Mr H. Hanazono of Fukuoka U~versity for NMR and FAB MS measurements. REFERENCES
piscicidal
[2] or antifertility [5]. The macrocyclic daphnane homoiogue was isolated from anon su~orduru during the screening of antilenkaemic agents, and this is its fist isolation from mhtruemia. EXPERI~AL General. ‘H NM& 400 and ‘jC NMR: lOOMHz, CDCIJ, TMS as int. standard. Plant materials. Wikstroemia retusa A. Gray was harvested in Okinawa Island in May 1991. A sampte is &posited in the Herbarium of the University of the Ryukyus (Voucher No. 51353-51359).
1. Jolad, S. D., Hoffmann, J. J., Timmermann, B. N., S&ram, K. H. and Cole, J. R. (1983) J. Nat. prod. 46, 675. 2. Sakata, K., Kawazu, IS., Mitsui, T. and Masaki, N. (1971) Agric. Biol. Chem. 35, 2113. 3. Kupchan, S. M., Shizuri, Y., Murae, T., Sweeny, J. G., Haynes, H. R., Shen, M.-S., Burrick, J. C. and Bryan, R. F. (1976) J. Am. Gem. Sot. 98, 5720. 4. Connolly, J. D. and Hill, R. A. (1991) in ~ie~io~~y of Ter~oids Vol. 2, p. 1033. Chapman & Hall, London. 5. Cu, Z-P., Wang, C., Liang, Z., Lin, R., Lu, R., Zhao, S. and Zhou, B. (1989) Reproduction Contraception 9,48.
DITERPENOIDS
0031~9422/93 $5.00+0.00 Pergamon Prw Ltd
WITH THE DAPHNANE
SKELETON FROM WZKSTROEMlA
RETUSA SHIRYO
YAGA,KAZUHIKOKINJO,HIROYAHAYASHI,NAOCHIKAMATSUO,FUMIKOABE* and TATSUOYAMAUCHI*
Faculty of Agriculture,University of the Ryukyu, Nishihara-cho,Okinawa 903-01, Japan;*Faculty of PharmaceuticalSciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-01,Japan (Received18 May 1992) Key Word Index-LVikstroemia diterpenoid.
retusa; Thymelaeaceae; daphnane diterpenoid; macrocyclic daphnane
Abstract-Four diterpenoids with the daphnane skeleton were isolated from Wikstroemia retusa, a native plant in the Ryukyu Islands, and the structures established by means of mass and NMR spectroscopy.
INTRODUCTION Wikstroemia retusa A. Gray is indigenous to the Ryukyu Islands of Japan and its bark is used locally for the preparation of paper, as is the bark of other species of Thymelaeaceae in Japan. Since the quality of the paper is fine and it can be stored for long periods of time, the presence of constituents against insect damage was expected. We report the isolation and characterization of four daphnane diterpenoids, of which three are new compounds. RESULTS ANDDISCUSSION The bark was extracted with ethanol and the extract fractionated (see Experimental). This gave four diterpenoids having a daphnane skeleton (l-4). Compound 1 (CJ4Hs,,0s) was identified as huratoxin (‘H NMR and FAB mass spectrometry), previously isolated from Wikstroemia monticola [l] and Hura crepitans [2].
FAB mass spectrometry of 2 gave a [M + Na J ’ peak at m/z 665.3304, suggesting CJBHSOOIO, i.e. a diterpenoid with one acetoxyl group more than 1. In fact, the signal due to one acetyl methyl group was observed at 61.98 in the ‘H NMR spectrum. The ‘H NMR signals, including a terminal methyl at 80.88, olefinic protons due to the E,E-diene system, one proton from an enone group, two exomethylene protons and carbinyl protons at C-7, are in good agreement with those of 1. However, the H-12 signals at 6 1.65 and 2.23 are no longer observed and the H-8 and H-14 signals show downfield shifts (+0.56 ppm and 0.32 ppm, respectively) in 2. Similar downfield shifts were observed between huratoxin and wikstrotoxin C [l], of which H-8 and H-14 were shifted +0.58 ppm and +0.32 ppm, respectively. This suggests the presence of a 12/L~cetoxyl group, and one singlet signal at 84.98 is assignable to H-12oz, retaining 90” of the dihedral angle between H-l lfl.
141
S. YAGA et al.
142
The i3C NMR signal assigments of 2 were confirmed by means of 2D i3C--lH COSY. The signals due to the enone function composed of C-l-C-3 were in good agreement with those of wikstrotoxins Cl]. Three olefinic linkages including one exomethylene group were observed as four doublets, one triplet and one singlet in the range 6 11S- 140. A singlet signal at 6 117.0 can be assigned for an orthoester carbon as described for wikstrotoxin C. The nine signals observed between 660-85 are assignable to the carbons containing oxygen functions at C-4-C-7, C-9, C-12-C-14 and C-20. The structure of 2 is thus determined to be 12/%acetoxy-huratoxin. The ‘H NMR spectrum of 3 is almost superimposable on that of 2. Since the FAB mass spectrum [M+Na]+ peak of 3 (m/z 679.3459, C3,Hs20,,Na) was 14 amu higher than that of 2 and the fragment peak at m/z 207 [C,,H,,CO]+ due to the fatty acid side chain in 2 is replaced by the peak at m/z 221 [C,,H,,CO]+, 3 is characterized as a homologue of 2 having a one methylene longer side chain than 2. The FAB mass spectrum of 4 afforded a [M +Na]+ peak at m/z 661.3354, suggesting a molecular formula of C37HS009. Signals arising from rings B and C showed similar chemical shifts and the same coupling patterns as those of 1. Therefore, 4 seemed to be a daphane homologue, although the presence of a benzoyl group was observed. In the 13C NMR spectrum, a singlet signal due
to the orthoester carbon was observed at 6 119.7, showing the presence of a side chain composed of several carbons. Unlike 1-3, the characteristic triplet signal due to the terminal methyl group was replaced by a doublet signal in 4, suggesting that 4 retains a gnidimacrin type macrocyclic structure, in which the second carbon from the terminal position of the side chain is attached to ring A [3]. In fact, no enone system in ring A was observed. The upfield shift of the methyl proton signals at S 1.79 on the enone system in 2 to 6 1.05 in 4 is entirely consistent with the fact that the olefinic linkage of the enone is saturated in 4. Simultaneously, a carbonyl group at C-3 seems to be reduced to a carbinol (682.9). In order to establish the stereochemistry, 2D-NOESY measurements were carried out (Table 1) and the relative configurations at C-l-C-8, C-10, C-i 1 and C-9, were established. Since the carbinol carbon signals of C-5 and C-20 had almost the same chemical shifts as those of 2, and the carbinyl proton signal assignable to H-3 was observed at lower field (65.08), the benzoic acid group is located at the C-3 hydroxyl group as in gnidimacrin [3]. The orientation of H-3 was assigned a, based on NOE to H-5. Compound 4 is thus shown to have the structure represented in formula 4. Daphnane diterpenoids have been isolated from Thymelaeaceae as well as Euphorbiaceae 143. They have interesting biological activities, e.g. antileukaemic [ 1,23,
Table 1. ‘H NMR spectral data of compounds l-3 and 4 CDCI,, TMS as int. stand.; J Hz in parentheses) H
1
2
3
4
1 2 3 5 7 8 10 11 12a
7.63 br s
7.56 br s
7.57 br s
4.25 s 3.44 s 294 d (2) 3.81 d (2) 2.47 dq (9, 7) 1.65 d (14) 2.23 dd (14, 9) 4.42 d(3) 4.91 br s 5.02 br s 1.79 br s 1.18 d(7) 1.79 br s 3.78 d(13) 3.87 d(13) 5.70 d(16) 6.70dd(16,11) 6.06 dd (15,ll) 5.84 dt (l&7)
4.25 s 3.54 s 3.50 d (2) 3.83 d (2) 2.37 q (6) 4.98 s
4.25 s 3.54 s 3.50 d (3) 3.83 d (2) 2.35 q (6) 4.98 s
4.75 d(3) 4.96 br s 5.01 br s 1.83 br s 1.29 d(6) 1.79 br s 3.81 d(13) 3.91 d(13) 5.64 d(16) 6.65 dd (16,lO) 6.04 dd (16,lO) 5.85 dt (16,7)
4.75 d (2) 4.96 br s 5.01 br s 1.83 br s 1.29 d(6) 1.79 br s 3.80 d(13) 3.93 d(13) 5.54d(15) 6.65 dd (15,10) 6.03 dd (15.10) 5.85 dt (16,7)
239 br t (lop”* 1.90 mc 5.08 d(5)” 4.11 P 3.34 sf.g 2.89 d (2)“+’ 3.11 d (11) 2.67 dq (8, 7)‘* 1.62 d (14) 2.13 dd (14.8)” 4.27 d(2)‘+’ 4.85 br sk 4.96 br s’ 1.74 br sJsk 1.45 d(7) 1.05 d(7)b 3.76 d(12r 3.89 d(12)g
2.09 q(7) 0.88 t (6)
2.09 4 (7) 0.88 t (6) (H-15’) 1.98 s (-OAc)
128 14 16 17 18 19 20 2’ 3’ 4 5’ 6 Others
2.09 4 (7) 0.88 t(6) (H-14’)
(H-14’) 1.98 s (-OAc)
Benzoyl moiety 8.04 (2H, dd, 8,l) 7.61 (lH, br t, 8) 7.48 (2H, br t, 8) 0.84 d(7) (H-10’) 2.53 m (H-9’)
a-JOr‘A cross peak was observed between these signals in ZD-NOESY spectrum. *The signal at 62.67 showed a response on irradiation of the proton at 62.39 in NOE difference NMR.
Diterpenoids from Wi~str~~a Table 2. 13C NMR spectral data of com~unds as int. standard) C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
2 160.3 d X36.9 s 209.4 s 83.7 s 71.9 d 60.6 s 64.3 d 35.4 d 72.3 s 47.5 d 44.0 d 78.3 d 78.2 s 80.5 d 143.2 s 113.2 t 18.7 q 18.2 q 9.8 q 65.1 t
4
C
SO.6d 36.3 d 82.9 d 80.1 s* 73.5 d 60.9 s 64.2 d 36.7 d 81.1 s” 47.3 d 35.2 d 36.5 t 83.4 SL 825 d
146.8s
2
1’ 2 3’ 4’ 5
6’ 7’ 8’ 9
.
ICY II’ 12’ 13 14
110.7 t 18.8 q 21.3 q 14.4 q 66.1 t
(CDCl,, TMS
4
117.0s 119.7 s 122.3 d 33.5 t 13S.Od b 27.9 t 128.6 d [ 27.8 t 139.3 d 26.7 t 32.7 t 24.6 t 31.9 t 21.08 29.54 t 37.9 t 29.46 t 29.3 d 29.29 t 12.6q 29.13 t 29.05 t Benzoyl moiety 226 t 167.6 s 14.1 q 129.9 s 129.7 d( x 2) -0Ac 133.4 d 169.6 128.6 d( x 2) 21.1
*Signal assignments were based on 2D ’ 3C-1H COSY spcctra. **bSigntisare inte~~~able.
retusa
143
~xtr~~on and isoiation Air-dried bark and twigs (100 g) were powdered and extracted with hot EtOH. The solvent was evapd in uacuo and the residue extracted with Et@. The Et,0 layer was treated with 1% aq. NaOH. After evapn of the Et,O, the residue was chromatographed on a silica gel column with hexane, hexane-Et&, Et,0 and MeOH. The eluants were further chromatographed on a reversed phase column or subjected to HPLC (ODS, Waters) to afford l(ll.6 mg), 2 (17.1 mg), 3 (10.6 mg), 4 (15.1 mg), Compound 1 was identity as huratoxin by FAB MS and by comparison of the ‘H NMR data with those published [l]. Conzpound 2. Glassy oil, [a]$ + 11.3” (CHCl,; c 0.85), FAB MS m/z 665.3304, C,,H,,OzONa requires 665.3302. m/z 643,583,449,395,207, 185. Compound 3. Glassy oil, [aJ$j (CHCl,; c + 7.9”), FAB MS m/z 679.3459 &H,,O,,,Na requires 679.3458. mfz 657,449,395,221, 185. Compound 4. Glassy oil, [a]Y (CD&; c + 7.6”), FAB MS m/z 661.3354 C3,H,,09Na 661.3353. m/z 639, 449, 185, 105. Acknowledgements-We thank MS Y. Iwase, J. Honda and Mr H. Hanazono of Fukuoka U~versity for NMR and FAB MS measurements. REFERENCES
piscicidal
[2] or antifertility [5]. The macrocyclic daphnane homoiogue was isolated from anon su~orduru during the screening of antilenkaemic agents, and this is its fist isolation from mhtruemia. EXPERI~AL General. ‘H NM& 400 and ‘jC NMR: lOOMHz, CDCIJ, TMS as int. standard. Plant materials. Wikstroemia retusa A. Gray was harvested in Okinawa Island in May 1991. A sampte is &posited in the Herbarium of the University of the Ryukyus (Voucher No. 51353-51359).
1. Jolad, S. D., Hoffmann, J. J., Timmermann, B. N., S&ram, K. H. and Cole, J. R. (1983) J. Nat. prod. 46, 675. 2. Sakata, K., Kawazu, IS., Mitsui, T. and Masaki, N. (1971) Agric. Biol. Chem. 35, 2113. 3. Kupchan, S. M., Shizuri, Y., Murae, T., Sweeny, J. G., Haynes, H. R., Shen, M.-S., Burrick, J. C. and Bryan, R. F. (1976) J. Am. Gem. Sot. 98, 5720. 4. Connolly, J. D. and Hill, R. A. (1991) in ~ie~io~~y of Ter~oids Vol. 2, p. 1033. Chapman & Hall, London. 5. Cu, Z-P., Wang, C., Liang, Z., Lin, R., Lu, R., Zhao, S. and Zhou, B. (1989) Reproduction Contraception 9,48.