- Email: [email protected]
Novel spironolactone-analogs as impurities in spironolactone
Steroids 69 (2004) 647–652
Novel spironolactone-analogs as impurities in spironolactone Hua Chena , Xiao-Yi Wangb , Zhong-Duo Yanga , Yuan-Chao Lia,∗ a
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, PR China b College of Life Science, Beijing University of Technology, Beijing 100022, PR China Received 9 December 2003; received in revised form 13 February 2004; accepted 17 May 2004 Available online 17 September 2004
Abstract Six novel spironolactone-analogs steroids (3–8) were isolated from spironolactone by using various chromatographic methods. Their structures were elucidated by spectrometric analysis. Two of the analogs (3 and 7) were confirmed by X-ray crystallography. The A-ring of compounds 3–7 is opened at C-2 C-3 bond, and compound 7 is an organic polysulfide, which has a rare, nine-membered ring with a five sulfur atom bridge. © 2004 Published by Elsevier Inc. Keywords: Spironolactone; Steroids; Structure elucidation; Spectroscopic methods; X-ray; Organic polysulfide
1. Introduction Spironolactone [1,2] [3-(3-oxo-7␣-acetylthio-17hydroxy-4-androsten-17␣-yl) propionic acid ␥-lactone, 1] has been widely used as an aldosterone-antagonist diuretic. Its production is always accompanied by side reactions leading to unwanted impurities that vary with the different starting materials used. In an attempt to clarify the impurities in spironolactone produced by the process shown in Scheme 1 (4-AD(2) as starting material), six novel compounds (3–8) were isolated and identified (see Fig. 1). In this paper, we report the isolation and identification of these new compounds.
2. Experimental 2.1. General Melting points are uncorrected. Mass spectroscopic measurements: MAT-711 spectrometer, in m/z (rel. %). Op∗
Corresponding author. Tel.: +86 21 50806600x3502; fax: +86 21 50807088. E-mail address: [email protected] (Y.-C. Li). 0039-128X/$ – see front matter © 2004 Published by Elsevier Inc. doi:10.1016/j.steroids.2004.05.018
tical rotation: Jasco-DIP-181 polarimeter, in CHCl3 . IR spectra: Perkin–Elmer-599B IR spectrometer; KBr pellets in cm−1 . 1 H- and 13 C-NMR Spectra: Bruker-AM-400 instrument; δ in ppm relative to SiMe4 as internal standard (=0 ppm), J in Hz. General chromatography: silica gel 60H from Qingdao Haiyang Chemical Group Co., China, HPLC was carried out on HP-1100, with detection at 240 nm on a Lichrosorb RP-18 (10 m, 300 mm × 4 mm) column with methanol–water (75:25) and (70:30) as the mobile phases. 2.2. Raw material The raw material was the mother liquor from repeated crystallization of spironolactone provided by Xianju Zhiyao Erchang, ZheJiang Province, China, in August 2002. 2.3. Extraction and isolation The mother liquor was concentrated in methanol and pyridine at 80 ◦ C for about 8 h, and then, the residue (50 g) was dissolved in dichloromethane. Dichloromethane extracts were chromatographed on silica gel 60H (2 kg)
648
H. Chen et al. / Steroids 69 (2004) 647–652
Scheme 1. The route for synthesizing spironolactone (1).
using a gradient system of cyclohexane–acetone (10:1, 8:1, 6:1, 5:1, 3:1, 2:1, 1:1). Compound 3 (40.6 mg) was isolated from fraction 11–24 in crystalline form, and it was recrystallized from acetone. The residual parts of fractions 11–24 were further fractionated into six parts by silica gel 60H with cyclohexane–acetone (8:1). Among these fractions, the second was repeatedly subjected to silica gel flash chromatography with cyclohexane–acetone (8:1) as solvent to yield mixtures of compounds 4 and 5. Then, the mixtures were rechromatographed by RP-HPLC using methanol–H2 O (70:30) as the mobile phase to obtain compound 4 (15 mg) and compound 5 (20 mg). Compound 7 (23.4 mg) was obtained as colorless crystals in fraction six. Fraction three was rechromatographed in silica gel 60H with cyclohexane–acetone (8:1) and RP-HPLC to yield compound 6 (4.3 mg) and compound 8 (10 mg).
2.3.1. (6S, 7S, 8R, 9S, 10R, 13S, 14S, 17R)-Pregn-[2,3]seco-4-ene-21-carboxylic acid, 6,7-(diacetylthio)-17hydroxy-3-(methoxy)-oxo-, ␥-lactone, (6, 7␣, 17␣)-(3) Colorless crystals; molecular formula: C27 H38 O6 S2 , mp: 274–276 ◦ C, [α]20 D = +11.7 (c = 1.20, CHCl3 ); IR: 1766.5, 1720.2 1693.2, 1608.4, 1 H and 13 C NMR, see Tables 1 and 2. HRESIMS m/z 523.2182 [M + H]+ (calcd. m/z 523.2188, ∆ −1.2 ppm). Anal. (C27 H38 O6 S2 ) C, H, S. Cacl.: 62.07 7.28 12.26 found: 62.37 6.92 12.08. 2.3.2. (6R∗ , 7R∗ , 8R∗ , 9S∗ , 10R∗ , 13S∗ , 14S∗ , 17R∗ )-Pregn-[2,3]-seco-4-ene-21-carboxylic acid, 6,7-(diacetylthio)-17-hydroxy-3-(methoxy)-oxo-, ␥-lactone, (6␣, 7, 17␣)-(4) Amorphous powder; molecular formula: C27 H38 O6 S2 . 1 13 C NMR, see [α]20 D = −44.8 (c = 0.92, CHCl3 ), H and + Tables 1 and 2. ESIMS m/z 523.2 [M + H] and 545.4 [M + Na]+ . 2.3.3. (6S∗ , 7R∗ , 8R∗ , 9S∗ , 10R∗ , 13S∗ , 14S∗ , 17R∗ )-Pregn-[2,3]-seco-4-ene-21- carboxylic acid, 6,7(diacetylthio)-17-hydroxy-3-(methoxy)-oxo-,␥-lactone, (6, 7, 17␣)-(5) Colorless powder; molecular formula: C27 H38 O6 S2 , mp: 106–108 ◦ C, [α]20 D = −1.6 (c = 1.48, CHCl3 ) IR: 1772.3, 1724.1, 1699.0, 1616.1, 1 H and 13 C NMR, see Tables 1 and 2. ESIMS m/z 522.9 [M + H]+ and 545.1 [M + Na]+ .
Fig. 1. Structures of compounds 3–8.
2.3.4. (7R∗ , 8R∗ , 9S∗ , 10R∗ , 13S∗ , 14S∗ , 17R∗ )-Pregn-[2,3]-seco -4-ene-21-carboxylic acid, 7-(acetylthio)-17-hydroxy-3-(methoxy)-oxo-, ␥-lacton, (7␣, 17␣)-(6) Amorphous powder; molecular formula: C25 H36 O5 S. 1 13 C NMR, see [α]20 D = −36.9 (c = 0.67, CHCl3 ), H and
H. Chen et al. / Steroids 69 (2004) 647–652
649
Table 1 NMR Data on compounds 3–8 [CDCl3 , δ (ppm) (J in Hz)]
1H
Atom 1 H 2 4 6 7 3-OCH3 6-SCOCH3 7-SCOCH3 18 19
1 5.7 brs 3.95 brd (2.63)
2.35 s 0.94 s 1.19 s
3
4
5
6
7
8
0.77 t (7.42) 5.96 s 4.56 d (2.61) 3.82 t (2.61) 3.70 s 2.35 s 2.38 s 0.98 s 1.26 s
0.84 t (7.42) 5.96 d (1.37) 4.88 dd (4.49, 1.37) 3.94 t (4.95) 3.70 s 2.32 s 2.32 s 0.90 s 1.24 s
0.77 t (7.42) 6.02 s 4.32 d (1.38) 3.76 brd (1.38) 3.70 s 2.35 s 2.38 s 0.90 s 1.29 s
0.74 t (7.42) 5.58 s 3.88 dt (6.78, 2.48) 3.65 s
0.84 t (7.28) 4.64 s 5.88 d (6.59) 4.14 dd (6.46,3.98) 3.70 s
3.39 t (8.79) 5.60 brd (2.20) 4.22 brs 3.65 s
2.32 s 0.96 s 1.18 s
0.95 s 1.09 s
2.33 s 0.94 s 0.96 s
Tables 1 and 2. ESIMS m/z 471.4 [M + Na]+ . HRESIMS m/z 449.2359 [M + H]+ (calcd. m/z 449.2362, ∆ −0.6 ppm) and m/z 471.2197 [M + Na]+ (calcd. m/z 471.2181, ∆ 3.4 ppm). 2.3.5. (4S, 7S, 8R, 9S, 10R, 13S, 14S, 17R)-Pregn-[2,3]-seco-5-ene-21-carboxylic acid,4,7-(pentathiopins)-17-hydroxy-3-(methoxy)-oxo-, ␥-lactone, (4␣,7␣,17␣)-(7) Colorless crystals; molecular formula: C23 H32 O4 S5 , mp: 202–204 ◦ C, [α]20 D = −634.0.0 (c = 0.83, CHCl3 ) IR: 1766.5, 1739.5, 1463.7, 1 H and 13 C NMR, see Tables 1 and 2. HREIMS m/z 532.0908 [M + H]+ (calcd. m/z 532.0911, ∆ −0.6 ppm). Anal. (C23 H32 O4 S5 ) C, H, S. Cacl.: 51.86 6.05 30.08. Found: 51.90 6.07 30.17. Table 2 13 C NMR data on compounds 3–8 [CDCl , δ (ppm)] 3 Atom 13 C
1
3
4
5
6
7
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 3-OCH3 6-SCO 6-SCOCH3 7-SCO 7-SCOCH3
35.6 33.9 198.4 126.9 165.5 39.9 45.1 39.0 49.5 38.5 20.5 31.1 45.5 46.0 22.3 35.2 95.4 14.6 17.8 31.1 29.2 176.3
28.3 8.6 168.2 121.3 149.6 53.6 49.2 35.1 40.9 43.1 20.4 31.4 45.3 45.9 22.3 35.1 95.5 14.5 25.2 31.0 29.1 176.5 51.5 192.2 30.3 192.9 31.1
30.4 8.4 166.8 118.4 158.6 50.4 47.1 42.0 40.9 44.7 20.3 31.5 46.5 49.8 22.1 35.5 95.4 14.5 20.3 31.3 29.3 176.7 51.6 193.5 30.3 193.7 30.5
28.4 9.3 166.2 121.4 159.5 52.2 45.6 37.5 41.6 42.1 20.3 31.6 46.6 50.7 22.1 35.4 95.3 14.3 20.8 31.4 29.2 176.6 51.4 191.7 30.3 193.6 30.3
27.5 7.9 168.2 117.2 149.6 42.5 46.3 38.3 41.7 43.5 20.2 31.2 45.2 45.2 22.0 34.9 95.1 14.0 22.2 30.6 28.9 176.1 50.8
28.2 10.6 170.1 50.7 132.0 124.5 52.8 35.6 35.2 42.5 20.3 31.3 45.5 45.5 22.8 35.2 95.6 13.8 23.6 31.1 29.2 176.1 52.9
39.2 24.9 174.5 46.9 146.8 121.8 45.1 35.8 45.2 44.6 21.4 31.0 45.6 45.7 23.0 35.0 95.9 14.4 19.0 30.9 29.2 174.5 52.1
194.1 31.3
194.1 30.4
194.3 31.0
2.3.6. (4S∗ , 7S∗ , 8R∗ , 9S∗ , 10R∗ , 13S∗ , 14S∗ , 17R∗ )-Pregn -[2,3]-seco-5-ene-21- carboxylic acid, 7-(acetylthio)-17-hydroxy-3-(methoxy)-oxo-, ␥-lactone, (4, 7␣, 17␣)-(8) Oil; molecular formula: C25 H34 O5 S. [α]20 D = −118.1 (c = 1.22, CHCl3 ) ESIMS m/z 447.1 [M + H]+ and 469.3 [M + Na]+ , 1 H and 13 C NMR, see Tables 1 and 2. HRESIMS 469.2020 [M + Na]+ (calcd. m/z 469.2025, ∆ −1.0 ppm).
3. Results and discussion The mother liquor was concentrated in methanol and pyridine at 80 ◦ C for about 8 h, and then, the residue was dissolved in dichloromethane. Dichloromethane extracts were further isolated by using various chromatographic methods, including column chromatography and HPLC, to get compounds 3–8 as shown in Fig. 1. Compound 3 was obtained as colorless crystals. Using HRESIMS, its molecular formula was deduced to be C27 H38 O6 S2 . 1 H and 13 C NMR spectra indicated a typical sprinolactone skeleton. The DEPT spectra of 3 indicated that 3 had five methyl, one methoxy, seven methylene, and six methine groups and eight quaternary carbons. Comparison of the NMR data with that of the spironolactone [3] showed that the 17-lactonic ring and 4,5-unsaturated keto were intact, but the A-ring was opened at the C-2 C-3 bond (Fig. 2), as indicated by the methyl signals (δH 0.772-H, t; δC 8.6) and the HMBC cross-peaks of C-10/2-H. The chemical shift of
Fig. 2. Select HMBC (C → H) and ROESY (
) correlations of 3.
650
H. Chen et al. / Steroids 69 (2004) 647–652
the 3-keto group shifted from δc 198.4 (in 1) to δc 168.2 (in 3), indicating the methyl ester group on C-3 after the ring opening as shown in Fig. 1. Two acetylthio groups at C-6 and C-7 were deduced from the methyl signals (δH 2.35 s, 2.38 s and δC 30.3, 31.1), the strong 2 JH–H COSY coupling of δH 3.82 (7-H, t, J 2.61 Hz) and δH 4.56 (6-H, d, J 2.61 Hz), and the JC–H couplings between C-6/6-SCOCH3 , 6-H/C-6SCO, and C-7/7-SCOCH3 , 7-H/C-7-SCO in the HMBC spectrum. The relative configurations of the stereogenic centers were studied by means of ROESY experiments. Because the cross peaks between 6-H/8-H and 6-H/19-H were not found, the ␣ orientation of 6-H was determined by the ROESY cross peak between 6-H and 4-H. Comparing the chemical shift and coupling constant of 7-H of 3 with those of 1, the  position of 7-H could be confirmed. The structure and stereochemistry of compound 3 was finally proved by single-crystal X-ray analysis. The perspective view of 3 (Fig. 3) depicts the absolute stereochemistry of the compound, and the atomic numbering is applied. Compound 4, an amorphous powder, had the same molecular weight as 3 as determined by positive ESIMS (m/z 522.9 [M + H]+ and 545.1 [M + Na]+ ). The close similarity of the 1 H and 13 C chemical shifts of 3 and 4 clearly showed that the difference in these compounds might only be the stereochemistry of 6-H and 7-H. It was obvious that the structure of 4 was the same as 3 by interpretation of its HMBC and ROESY. The HMBC correlation of 7-H/C-7-SCO, C-6/7-H,
Fig. 3. Perspective view of compound 3. Crystal structure determination of compound 3. Diffraction data were collected from a single-crystal of 0.35 mm × 0.26 mm × 0.17 mm using a CAD-4 diffractometer with ˚ C27 H38 O6 S2, graphite-monochromated Cu K␣ radiation (λ = 0.71073 A). formula weight M = 522.69, Orthorhombic, space group (from systematic ab˚ b = 12.3441(11) A, ˚ c = 20.4922(19) A, ˚ sences) P21 21 21 , a = 10.6988 (10) A, Dc (Z = 4) = 1.283 mg m−3 . The structure was solved by direct methods with SHELXS-97 [4] and was refined by full-matrix least-squares in F2 mode using SHELXL-97 [5]. Hydrogen positions were calculated from assumed geometries and included SF calculations, but without refinement. Reflections measured were 16632, unique reflections were 6311, R = 0.043, R2w = 0.078. The Absolute structure parameter was determined by refining the Flack [6] parameter to −0.26(9). The molecular plot was drawn with the program ORTEP-III [7]. (The crystal structure has been deposited at the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences (CAS). The deposition number is CCDC-22371).
and 6-H/C-7 suggested that the two acetylthio groups were attached to C-6 and C-7. The interactions of 3-OCH3 -H and C-3 indicated the position of the methyl ester. The stereostructure mainly depended on analyzing the ROESY spectrum. From the ROESY, the  orientation of 6-H was determined by the cross peaks between 6-H and 8-H and the absence of NOE effects between 4-H and 6-H. Because of the observed NOE effects between 7-H and 14-H, the ␣ position of 7-H was evident with regard to the ␣ position of 14-H. Compound 5, a colorless solid, also had the same molecular weight as compound 3 as determined by positive ESIMS. The close similarity of the 1 H and 13 C chemical shifts of 3 and 5 and their HMBC and ROESY spectra also clearly showed that 5 was an isomer of 3. The difference between 5 and 3 may be only the stereochemistry of 7-H. The stereostructure also mainly depended on analyzing the ROESY spectrum. The overhauser effects between 7-H and 9-H showed the ␣ position of 7-H with regard to the ␣ position of 9-H. The ␣ position of 6-H followed from the ROESY cross-peak between 6-H and 7-H. The NOE effects between 6-H and 4-H further demonstrated the ␣ position of 6-H. Compound 6 was isolated in a very minute quantity as an amorphous powder by HPLC. It had the molecular formula C25 H36 O5 S, determined via the quasimolecular ion peak at m/z 449.2359 [M + H]+ (calcd. m/z 449.2362, ∆ −0.6 ppm) and m/z 471.2197 [M + Na]+ (calcd. m/z 471.2181, ∆ 3.4 ppm) in the positive HRESIMS and supported by the proton and carbon counts in the NMR spectra. In the 1 H and 13 C NMR spectra of 6, one acetylthio group at C-6 was missed, and the three proton signals at δH 0.77 (2-H, t) and the methoxy signals (δH 3.65, s; δC 7.9) showed that the other parts of the structure of 6 was the same as 3. By comparing the NMR chemical shift of 7-H in 6 with those in 1 and 3, the ␣orientation of 7-H (δH 3.88, dt, J 6.78 Hz, 2.48 Hz) was determined. Compound 7 was obtained as colorless crystals. The DEPT spectrum showed that it had three methyl, one methoxy, seven methylene, and six methine groups and six quaternary carbons. Comparing the 1 H and 13 C NMR data with that of 3 and 1, it can be seen that there was no acetylthio group, and 7 was determined to be as an A-ring-opened spironolactone-analog with one trisubstituted (δC 132.0 and 124.5, δH 5.88 d) carbon–carbon double bond and two ester keto groups (δC 176.6 and δC 170.1). One keto group (δC 176.6) was in the ␥-lactonic ring, and the other (δC 170.1) was at atom C-3. The same coupling constant was detected at the protons δH 5.88 and 4.14 (6-H/7-H, d, J 6.59 Hz), which indicated that the trisubstituted, unsaturated double bond shifted to C-5 and C-6. Further important evidence came from the correlations signals of C-5/19-H, C-5/7-H, C-5/4-H, C-6/4H, 6-H/C-10, 6-H/C-8, and 6-H/C-4 on the HMBC spectra. The degree of unsaturation of compound 7 was eight, the above evidence proved only seven unsaturation degrees of 7; thus, it was concluded that there must be another ring. Considering the existence of only one proton at C-4 and C-7 and the low chemical shifts of 4-H (δH 4.64) and 7-H (δH 4.14),
H. Chen et al. / Steroids 69 (2004) 647–652
651
Table 3 The fragment ion peaks of compound 7 in the HREIMS spectra Mass (m/z)
Relative abundance (%)
∆ (ppm)
Composition
372.2299 404.2011 436.1734 468.1457 532.0908
100.00 9.67 2.72 4.07 0.56
0.2 1.0 0.8 0.6 −0.4
C23 H32 O4 C23 H32 O4 S C23 H32 O4 S2 C23 H32 O4 S3 C23 H32 O4 S5
the last ring must be joined from C-4 to C-7 through a S link bridge. It was important to know the molecular weight to deduce this sulfur-containing ring. The abundance of the two peaks 404 and 468 in ESIMS and EIMS at first misleadingly suggested a five-member ring containing –C-7–S–C-4– or a ring with –C-7–S–O–O–S–C-4– or with –C-7–S–S–S–C-4– fragments. This problem was solved later by HREIMS. The fragment ion peaks are listed in Table 3. The difference in each fragment ion peak was 32, the atomic weight of sulfur, which indicated that the atom S was decomposed from the molecule successively, and the strange big nine-membered ring with five S atoms was constructed by assuring 532 as its molecular weight. The molecular formula was confirmed as C23 H32 O4 S5 . The relative configuration of the stereogenic centers was studied by means of a NOESY experiment. The Overhauser effects between 4-H and 19-H proved the  orientation of 4-H. With comparison of these results to those of 1 and 3, the  position of 7-H was confirmed. Further important evidence came from the cross-peaks of 7-H and 8-H observed in the NOESY spectra. Finally, the structure and stereochemistry of compound 7 were proved by single-crystal X-ray analysis (Fig. 4). Compound 8 was obtained as an oil. Its molecular weight was 446 as determined by the quasimolecular ion peaks at m/z 447.1 [M + H]+ and 469.3 [M + Na]+ by means of the positive ESIMS, and by the quasimolecular ion peak at m/z 469.2020 [M+ Na]+ (calcd. m/z 469.2025, ∆ −1.0 ppm) in the positive HRESIMS. The molecular formula was deduced to be C25 H34 O5 S. The DEPT spectra of 8 indicated that 8 had four methyl, one ester methoxy, eight methylene, and six methine groups and seven quaternary carbons. Comparing the NMR data to those of 3 and 7, the ␥-lactone group and 7acetylthio were shown to be intact. According to the 1 H–1 H COSY and HMQC spectra, two units were deduced as shown in Fig. 5. HMBC correlations between δH 5.96 (4-H) and δC 146.8 (C-5) identified one A/B connection at C-4/C-5, and a correlation between δH 0.94 (18-CH3 ) and δC 39.2 (C-1) established the other A/B linkage at C-1/C-10. On the basis of the ROESY, the Overhauser effects between 6-H and 4-H showed the ␣ position of 4-H relative to the position of 6-H. ROESY correlations were not found between 7-H/9-H and 7-H/14-H, but between 7-H and 8-H, demonstrating the  position of 7-H. In summary, six new compounds were isolated, and their structures were elucidated. Among them, five novel A-
Fig. 4. Perspective view of compound 7. Crystal structure determination of compound 7. Diffraction data were collected from a single-crystal of 0.50 mm × 0.30 mm × 0.11 mm using a CAD-4 diffractometer with ˚ C23 H32 O4 S5, graphite-monochromated Cu K␣ radiation (λ = 0.71073 A). formula weight M = 532.79, Orthorhombic, space group (from systematic ˚ b = 12.3830(10) A, ˚ c = 24.8443(19) A, ˚ absences) P21 21 21 , a = 8.0455(6) A, Dc (Z = 4) = 1.430 mg m−3 . The structure was solved by direct methods with SHELXS-97 and was refined by full-matrix least-squares in F2 mode using SHELXL-97. Hydrogen positions were calculated from assumed geometries and included SF calculations, but without refinement. Reflections measured were 15107, unique reflections were 5677, R = 0.072, R2w = 0.069. The Absolute structure parameter was determined by refining the Flack parameter to 0.06(6). The molecular plot was drawn with the program ORTEP-III. (The crystal structure has been deposited at the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences (CAS). The deposition number is CCDC-23290).
Fig. 5. Two units of compound 8 concluded by COSY and HMQC spectra.
ring-opened spironolactone-analogs (3–7) were obtained and identified. Three of these compounds were stereoisomers. Compound 7 was an organic polysulfide, which had a rare, nine-membered ring with a five sulfur atom bridge. Compound 8 had a five-member ring, which was probably formed via rearrangment of the A-ring. These compounds have similar chemical and biological characteristics to spironolactone, indicating more potent remedies maybe derived from them. Farther biological tests on these compounds are in progress.
Acknowledgements This work was supported by Xianju Zhiyao Erchang, ZheJiang Province, China.
652
H. Chen et al. / Steroids 69 (2004) 647–652
References [1] Spark RF, Melby JC. Hypertension and low plasma renin activity: presumptive evidence for mineralocorticoid excess. Ann Intern Med 1971;75:831–6. [2] Carey RM, Douglas JG, Schweikert JR, Liddle GW. The syndrome of essential hypertension and suppressed plasma renin activity: normalization of blood pressure. Arch Intern Med 1972;130:849–54. [3] Highet RJ, Burke TR, Trager WF, Pohl LR, Menard RH, Taburet AM, et al. Carbon-13 nuclear magnetic resonance studies of spironolactone and several related steroids. Steroids 1980;35(2):119–32.
[4] Sheldriek GM. SHELXS-97. Program for structure solution. G¨ottingen, Germany: Universtiy of G¨ottingen; 1997. [5] Sheldriek GM. SHELXS-97. Program for structure refinement. G¨ottingen, Germany: Universtiy of G¨ottingen; 1997. [6] Flack HD. On enantiomorph-polarity estimation. Acta Crystallogr 1983;A39:876–81. [7] Burqett MN, Johnson CK. ORTEP-III. Oak Ridge Thermal Ellipsoid Plot Program for Crystal Structure illustrations; Report ORNL-6895; Oak Ridge National Laboratory: Oak Ridge, TN, 1996. Windows implementation was by Farrugia, LJ, Department of Chemistry, University of Glasgow, UK, 1998.
Novel spironolactone-analogs as impurities in spironolactone Hua Chena , Xiao-Yi Wangb , Zhong-Duo Yanga , Yuan-Chao Lia,∗ a
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, PR China b College of Life Science, Beijing University of Technology, Beijing 100022, PR China Received 9 December 2003; received in revised form 13 February 2004; accepted 17 May 2004 Available online 17 September 2004
Abstract Six novel spironolactone-analogs steroids (3–8) were isolated from spironolactone by using various chromatographic methods. Their structures were elucidated by spectrometric analysis. Two of the analogs (3 and 7) were confirmed by X-ray crystallography. The A-ring of compounds 3–7 is opened at C-2 C-3 bond, and compound 7 is an organic polysulfide, which has a rare, nine-membered ring with a five sulfur atom bridge. © 2004 Published by Elsevier Inc. Keywords: Spironolactone; Steroids; Structure elucidation; Spectroscopic methods; X-ray; Organic polysulfide
1. Introduction Spironolactone [1,2] [3-(3-oxo-7␣-acetylthio-17hydroxy-4-androsten-17␣-yl) propionic acid ␥-lactone, 1] has been widely used as an aldosterone-antagonist diuretic. Its production is always accompanied by side reactions leading to unwanted impurities that vary with the different starting materials used. In an attempt to clarify the impurities in spironolactone produced by the process shown in Scheme 1 (4-AD(2) as starting material), six novel compounds (3–8) were isolated and identified (see Fig. 1). In this paper, we report the isolation and identification of these new compounds.
2. Experimental 2.1. General Melting points are uncorrected. Mass spectroscopic measurements: MAT-711 spectrometer, in m/z (rel. %). Op∗
Corresponding author. Tel.: +86 21 50806600x3502; fax: +86 21 50807088. E-mail address: [email protected] (Y.-C. Li). 0039-128X/$ – see front matter © 2004 Published by Elsevier Inc. doi:10.1016/j.steroids.2004.05.018
tical rotation: Jasco-DIP-181 polarimeter, in CHCl3 . IR spectra: Perkin–Elmer-599B IR spectrometer; KBr pellets in cm−1 . 1 H- and 13 C-NMR Spectra: Bruker-AM-400 instrument; δ in ppm relative to SiMe4 as internal standard (=0 ppm), J in Hz. General chromatography: silica gel 60H from Qingdao Haiyang Chemical Group Co., China, HPLC was carried out on HP-1100, with detection at 240 nm on a Lichrosorb RP-18 (10 m, 300 mm × 4 mm) column with methanol–water (75:25) and (70:30) as the mobile phases. 2.2. Raw material The raw material was the mother liquor from repeated crystallization of spironolactone provided by Xianju Zhiyao Erchang, ZheJiang Province, China, in August 2002. 2.3. Extraction and isolation The mother liquor was concentrated in methanol and pyridine at 80 ◦ C for about 8 h, and then, the residue (50 g) was dissolved in dichloromethane. Dichloromethane extracts were chromatographed on silica gel 60H (2 kg)
648
H. Chen et al. / Steroids 69 (2004) 647–652
Scheme 1. The route for synthesizing spironolactone (1).
using a gradient system of cyclohexane–acetone (10:1, 8:1, 6:1, 5:1, 3:1, 2:1, 1:1). Compound 3 (40.6 mg) was isolated from fraction 11–24 in crystalline form, and it was recrystallized from acetone. The residual parts of fractions 11–24 were further fractionated into six parts by silica gel 60H with cyclohexane–acetone (8:1). Among these fractions, the second was repeatedly subjected to silica gel flash chromatography with cyclohexane–acetone (8:1) as solvent to yield mixtures of compounds 4 and 5. Then, the mixtures were rechromatographed by RP-HPLC using methanol–H2 O (70:30) as the mobile phase to obtain compound 4 (15 mg) and compound 5 (20 mg). Compound 7 (23.4 mg) was obtained as colorless crystals in fraction six. Fraction three was rechromatographed in silica gel 60H with cyclohexane–acetone (8:1) and RP-HPLC to yield compound 6 (4.3 mg) and compound 8 (10 mg).
2.3.1. (6S, 7S, 8R, 9S, 10R, 13S, 14S, 17R)-Pregn-[2,3]seco-4-ene-21-carboxylic acid, 6,7-(diacetylthio)-17hydroxy-3-(methoxy)-oxo-, ␥-lactone, (6, 7␣, 17␣)-(3) Colorless crystals; molecular formula: C27 H38 O6 S2 , mp: 274–276 ◦ C, [α]20 D = +11.7 (c = 1.20, CHCl3 ); IR: 1766.5, 1720.2 1693.2, 1608.4, 1 H and 13 C NMR, see Tables 1 and 2. HRESIMS m/z 523.2182 [M + H]+ (calcd. m/z 523.2188, ∆ −1.2 ppm). Anal. (C27 H38 O6 S2 ) C, H, S. Cacl.: 62.07 7.28 12.26 found: 62.37 6.92 12.08. 2.3.2. (6R∗ , 7R∗ , 8R∗ , 9S∗ , 10R∗ , 13S∗ , 14S∗ , 17R∗ )-Pregn-[2,3]-seco-4-ene-21-carboxylic acid, 6,7-(diacetylthio)-17-hydroxy-3-(methoxy)-oxo-, ␥-lactone, (6␣, 7, 17␣)-(4) Amorphous powder; molecular formula: C27 H38 O6 S2 . 1 13 C NMR, see [α]20 D = −44.8 (c = 0.92, CHCl3 ), H and + Tables 1 and 2. ESIMS m/z 523.2 [M + H] and 545.4 [M + Na]+ . 2.3.3. (6S∗ , 7R∗ , 8R∗ , 9S∗ , 10R∗ , 13S∗ , 14S∗ , 17R∗ )-Pregn-[2,3]-seco-4-ene-21- carboxylic acid, 6,7(diacetylthio)-17-hydroxy-3-(methoxy)-oxo-,␥-lactone, (6, 7, 17␣)-(5) Colorless powder; molecular formula: C27 H38 O6 S2 , mp: 106–108 ◦ C, [α]20 D = −1.6 (c = 1.48, CHCl3 ) IR: 1772.3, 1724.1, 1699.0, 1616.1, 1 H and 13 C NMR, see Tables 1 and 2. ESIMS m/z 522.9 [M + H]+ and 545.1 [M + Na]+ .
Fig. 1. Structures of compounds 3–8.
2.3.4. (7R∗ , 8R∗ , 9S∗ , 10R∗ , 13S∗ , 14S∗ , 17R∗ )-Pregn-[2,3]-seco -4-ene-21-carboxylic acid, 7-(acetylthio)-17-hydroxy-3-(methoxy)-oxo-, ␥-lacton, (7␣, 17␣)-(6) Amorphous powder; molecular formula: C25 H36 O5 S. 1 13 C NMR, see [α]20 D = −36.9 (c = 0.67, CHCl3 ), H and
H. Chen et al. / Steroids 69 (2004) 647–652
649
Table 1 NMR Data on compounds 3–8 [CDCl3 , δ (ppm) (J in Hz)]
1H
Atom 1 H 2 4 6 7 3-OCH3 6-SCOCH3 7-SCOCH3 18 19
1 5.7 brs 3.95 brd (2.63)
2.35 s 0.94 s 1.19 s
3
4
5
6
7
8
0.77 t (7.42) 5.96 s 4.56 d (2.61) 3.82 t (2.61) 3.70 s 2.35 s 2.38 s 0.98 s 1.26 s
0.84 t (7.42) 5.96 d (1.37) 4.88 dd (4.49, 1.37) 3.94 t (4.95) 3.70 s 2.32 s 2.32 s 0.90 s 1.24 s
0.77 t (7.42) 6.02 s 4.32 d (1.38) 3.76 brd (1.38) 3.70 s 2.35 s 2.38 s 0.90 s 1.29 s
0.74 t (7.42) 5.58 s 3.88 dt (6.78, 2.48) 3.65 s
0.84 t (7.28) 4.64 s 5.88 d (6.59) 4.14 dd (6.46,3.98) 3.70 s
3.39 t (8.79) 5.60 brd (2.20) 4.22 brs 3.65 s
2.32 s 0.96 s 1.18 s
0.95 s 1.09 s
2.33 s 0.94 s 0.96 s
Tables 1 and 2. ESIMS m/z 471.4 [M + Na]+ . HRESIMS m/z 449.2359 [M + H]+ (calcd. m/z 449.2362, ∆ −0.6 ppm) and m/z 471.2197 [M + Na]+ (calcd. m/z 471.2181, ∆ 3.4 ppm). 2.3.5. (4S, 7S, 8R, 9S, 10R, 13S, 14S, 17R)-Pregn-[2,3]-seco-5-ene-21-carboxylic acid,4,7-(pentathiopins)-17-hydroxy-3-(methoxy)-oxo-, ␥-lactone, (4␣,7␣,17␣)-(7) Colorless crystals; molecular formula: C23 H32 O4 S5 , mp: 202–204 ◦ C, [α]20 D = −634.0.0 (c = 0.83, CHCl3 ) IR: 1766.5, 1739.5, 1463.7, 1 H and 13 C NMR, see Tables 1 and 2. HREIMS m/z 532.0908 [M + H]+ (calcd. m/z 532.0911, ∆ −0.6 ppm). Anal. (C23 H32 O4 S5 ) C, H, S. Cacl.: 51.86 6.05 30.08. Found: 51.90 6.07 30.17. Table 2 13 C NMR data on compounds 3–8 [CDCl , δ (ppm)] 3 Atom 13 C
1
3
4
5
6
7
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 3-OCH3 6-SCO 6-SCOCH3 7-SCO 7-SCOCH3
35.6 33.9 198.4 126.9 165.5 39.9 45.1 39.0 49.5 38.5 20.5 31.1 45.5 46.0 22.3 35.2 95.4 14.6 17.8 31.1 29.2 176.3
28.3 8.6 168.2 121.3 149.6 53.6 49.2 35.1 40.9 43.1 20.4 31.4 45.3 45.9 22.3 35.1 95.5 14.5 25.2 31.0 29.1 176.5 51.5 192.2 30.3 192.9 31.1
30.4 8.4 166.8 118.4 158.6 50.4 47.1 42.0 40.9 44.7 20.3 31.5 46.5 49.8 22.1 35.5 95.4 14.5 20.3 31.3 29.3 176.7 51.6 193.5 30.3 193.7 30.5
28.4 9.3 166.2 121.4 159.5 52.2 45.6 37.5 41.6 42.1 20.3 31.6 46.6 50.7 22.1 35.4 95.3 14.3 20.8 31.4 29.2 176.6 51.4 191.7 30.3 193.6 30.3
27.5 7.9 168.2 117.2 149.6 42.5 46.3 38.3 41.7 43.5 20.2 31.2 45.2 45.2 22.0 34.9 95.1 14.0 22.2 30.6 28.9 176.1 50.8
28.2 10.6 170.1 50.7 132.0 124.5 52.8 35.6 35.2 42.5 20.3 31.3 45.5 45.5 22.8 35.2 95.6 13.8 23.6 31.1 29.2 176.1 52.9
39.2 24.9 174.5 46.9 146.8 121.8 45.1 35.8 45.2 44.6 21.4 31.0 45.6 45.7 23.0 35.0 95.9 14.4 19.0 30.9 29.2 174.5 52.1
194.1 31.3
194.1 30.4
194.3 31.0
2.3.6. (4S∗ , 7S∗ , 8R∗ , 9S∗ , 10R∗ , 13S∗ , 14S∗ , 17R∗ )-Pregn -[2,3]-seco-5-ene-21- carboxylic acid, 7-(acetylthio)-17-hydroxy-3-(methoxy)-oxo-, ␥-lactone, (4, 7␣, 17␣)-(8) Oil; molecular formula: C25 H34 O5 S. [α]20 D = −118.1 (c = 1.22, CHCl3 ) ESIMS m/z 447.1 [M + H]+ and 469.3 [M + Na]+ , 1 H and 13 C NMR, see Tables 1 and 2. HRESIMS 469.2020 [M + Na]+ (calcd. m/z 469.2025, ∆ −1.0 ppm).
3. Results and discussion The mother liquor was concentrated in methanol and pyridine at 80 ◦ C for about 8 h, and then, the residue was dissolved in dichloromethane. Dichloromethane extracts were further isolated by using various chromatographic methods, including column chromatography and HPLC, to get compounds 3–8 as shown in Fig. 1. Compound 3 was obtained as colorless crystals. Using HRESIMS, its molecular formula was deduced to be C27 H38 O6 S2 . 1 H and 13 C NMR spectra indicated a typical sprinolactone skeleton. The DEPT spectra of 3 indicated that 3 had five methyl, one methoxy, seven methylene, and six methine groups and eight quaternary carbons. Comparison of the NMR data with that of the spironolactone [3] showed that the 17-lactonic ring and 4,5-unsaturated keto were intact, but the A-ring was opened at the C-2 C-3 bond (Fig. 2), as indicated by the methyl signals (δH 0.772-H, t; δC 8.6) and the HMBC cross-peaks of C-10/2-H. The chemical shift of
Fig. 2. Select HMBC (C → H) and ROESY (
) correlations of 3.
650
H. Chen et al. / Steroids 69 (2004) 647–652
the 3-keto group shifted from δc 198.4 (in 1) to δc 168.2 (in 3), indicating the methyl ester group on C-3 after the ring opening as shown in Fig. 1. Two acetylthio groups at C-6 and C-7 were deduced from the methyl signals (δH 2.35 s, 2.38 s and δC 30.3, 31.1), the strong 2 JH–H COSY coupling of δH 3.82 (7-H, t, J 2.61 Hz) and δH 4.56 (6-H, d, J 2.61 Hz), and the JC–H couplings between C-6/6-SCOCH3 , 6-H/C-6SCO, and C-7/7-SCOCH3 , 7-H/C-7-SCO in the HMBC spectrum. The relative configurations of the stereogenic centers were studied by means of ROESY experiments. Because the cross peaks between 6-H/8-H and 6-H/19-H were not found, the ␣ orientation of 6-H was determined by the ROESY cross peak between 6-H and 4-H. Comparing the chemical shift and coupling constant of 7-H of 3 with those of 1, the  position of 7-H could be confirmed. The structure and stereochemistry of compound 3 was finally proved by single-crystal X-ray analysis. The perspective view of 3 (Fig. 3) depicts the absolute stereochemistry of the compound, and the atomic numbering is applied. Compound 4, an amorphous powder, had the same molecular weight as 3 as determined by positive ESIMS (m/z 522.9 [M + H]+ and 545.1 [M + Na]+ ). The close similarity of the 1 H and 13 C chemical shifts of 3 and 4 clearly showed that the difference in these compounds might only be the stereochemistry of 6-H and 7-H. It was obvious that the structure of 4 was the same as 3 by interpretation of its HMBC and ROESY. The HMBC correlation of 7-H/C-7-SCO, C-6/7-H,
Fig. 3. Perspective view of compound 3. Crystal structure determination of compound 3. Diffraction data were collected from a single-crystal of 0.35 mm × 0.26 mm × 0.17 mm using a CAD-4 diffractometer with ˚ C27 H38 O6 S2, graphite-monochromated Cu K␣ radiation (λ = 0.71073 A). formula weight M = 522.69, Orthorhombic, space group (from systematic ab˚ b = 12.3441(11) A, ˚ c = 20.4922(19) A, ˚ sences) P21 21 21 , a = 10.6988 (10) A, Dc (Z = 4) = 1.283 mg m−3 . The structure was solved by direct methods with SHELXS-97 [4] and was refined by full-matrix least-squares in F2 mode using SHELXL-97 [5]. Hydrogen positions were calculated from assumed geometries and included SF calculations, but without refinement. Reflections measured were 16632, unique reflections were 6311, R = 0.043, R2w = 0.078. The Absolute structure parameter was determined by refining the Flack [6] parameter to −0.26(9). The molecular plot was drawn with the program ORTEP-III [7]. (The crystal structure has been deposited at the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences (CAS). The deposition number is CCDC-22371).
and 6-H/C-7 suggested that the two acetylthio groups were attached to C-6 and C-7. The interactions of 3-OCH3 -H and C-3 indicated the position of the methyl ester. The stereostructure mainly depended on analyzing the ROESY spectrum. From the ROESY, the  orientation of 6-H was determined by the cross peaks between 6-H and 8-H and the absence of NOE effects between 4-H and 6-H. Because of the observed NOE effects between 7-H and 14-H, the ␣ position of 7-H was evident with regard to the ␣ position of 14-H. Compound 5, a colorless solid, also had the same molecular weight as compound 3 as determined by positive ESIMS. The close similarity of the 1 H and 13 C chemical shifts of 3 and 5 and their HMBC and ROESY spectra also clearly showed that 5 was an isomer of 3. The difference between 5 and 3 may be only the stereochemistry of 7-H. The stereostructure also mainly depended on analyzing the ROESY spectrum. The overhauser effects between 7-H and 9-H showed the ␣ position of 7-H with regard to the ␣ position of 9-H. The ␣ position of 6-H followed from the ROESY cross-peak between 6-H and 7-H. The NOE effects between 6-H and 4-H further demonstrated the ␣ position of 6-H. Compound 6 was isolated in a very minute quantity as an amorphous powder by HPLC. It had the molecular formula C25 H36 O5 S, determined via the quasimolecular ion peak at m/z 449.2359 [M + H]+ (calcd. m/z 449.2362, ∆ −0.6 ppm) and m/z 471.2197 [M + Na]+ (calcd. m/z 471.2181, ∆ 3.4 ppm) in the positive HRESIMS and supported by the proton and carbon counts in the NMR spectra. In the 1 H and 13 C NMR spectra of 6, one acetylthio group at C-6 was missed, and the three proton signals at δH 0.77 (2-H, t) and the methoxy signals (δH 3.65, s; δC 7.9) showed that the other parts of the structure of 6 was the same as 3. By comparing the NMR chemical shift of 7-H in 6 with those in 1 and 3, the ␣orientation of 7-H (δH 3.88, dt, J 6.78 Hz, 2.48 Hz) was determined. Compound 7 was obtained as colorless crystals. The DEPT spectrum showed that it had three methyl, one methoxy, seven methylene, and six methine groups and six quaternary carbons. Comparing the 1 H and 13 C NMR data with that of 3 and 1, it can be seen that there was no acetylthio group, and 7 was determined to be as an A-ring-opened spironolactone-analog with one trisubstituted (δC 132.0 and 124.5, δH 5.88 d) carbon–carbon double bond and two ester keto groups (δC 176.6 and δC 170.1). One keto group (δC 176.6) was in the ␥-lactonic ring, and the other (δC 170.1) was at atom C-3. The same coupling constant was detected at the protons δH 5.88 and 4.14 (6-H/7-H, d, J 6.59 Hz), which indicated that the trisubstituted, unsaturated double bond shifted to C-5 and C-6. Further important evidence came from the correlations signals of C-5/19-H, C-5/7-H, C-5/4-H, C-6/4H, 6-H/C-10, 6-H/C-8, and 6-H/C-4 on the HMBC spectra. The degree of unsaturation of compound 7 was eight, the above evidence proved only seven unsaturation degrees of 7; thus, it was concluded that there must be another ring. Considering the existence of only one proton at C-4 and C-7 and the low chemical shifts of 4-H (δH 4.64) and 7-H (δH 4.14),
H. Chen et al. / Steroids 69 (2004) 647–652
651
Table 3 The fragment ion peaks of compound 7 in the HREIMS spectra Mass (m/z)
Relative abundance (%)
∆ (ppm)
Composition
372.2299 404.2011 436.1734 468.1457 532.0908
100.00 9.67 2.72 4.07 0.56
0.2 1.0 0.8 0.6 −0.4
C23 H32 O4 C23 H32 O4 S C23 H32 O4 S2 C23 H32 O4 S3 C23 H32 O4 S5
the last ring must be joined from C-4 to C-7 through a S link bridge. It was important to know the molecular weight to deduce this sulfur-containing ring. The abundance of the two peaks 404 and 468 in ESIMS and EIMS at first misleadingly suggested a five-member ring containing –C-7–S–C-4– or a ring with –C-7–S–O–O–S–C-4– or with –C-7–S–S–S–C-4– fragments. This problem was solved later by HREIMS. The fragment ion peaks are listed in Table 3. The difference in each fragment ion peak was 32, the atomic weight of sulfur, which indicated that the atom S was decomposed from the molecule successively, and the strange big nine-membered ring with five S atoms was constructed by assuring 532 as its molecular weight. The molecular formula was confirmed as C23 H32 O4 S5 . The relative configuration of the stereogenic centers was studied by means of a NOESY experiment. The Overhauser effects between 4-H and 19-H proved the  orientation of 4-H. With comparison of these results to those of 1 and 3, the  position of 7-H was confirmed. Further important evidence came from the cross-peaks of 7-H and 8-H observed in the NOESY spectra. Finally, the structure and stereochemistry of compound 7 were proved by single-crystal X-ray analysis (Fig. 4). Compound 8 was obtained as an oil. Its molecular weight was 446 as determined by the quasimolecular ion peaks at m/z 447.1 [M + H]+ and 469.3 [M + Na]+ by means of the positive ESIMS, and by the quasimolecular ion peak at m/z 469.2020 [M+ Na]+ (calcd. m/z 469.2025, ∆ −1.0 ppm) in the positive HRESIMS. The molecular formula was deduced to be C25 H34 O5 S. The DEPT spectra of 8 indicated that 8 had four methyl, one ester methoxy, eight methylene, and six methine groups and seven quaternary carbons. Comparing the NMR data to those of 3 and 7, the ␥-lactone group and 7acetylthio were shown to be intact. According to the 1 H–1 H COSY and HMQC spectra, two units were deduced as shown in Fig. 5. HMBC correlations between δH 5.96 (4-H) and δC 146.8 (C-5) identified one A/B connection at C-4/C-5, and a correlation between δH 0.94 (18-CH3 ) and δC 39.2 (C-1) established the other A/B linkage at C-1/C-10. On the basis of the ROESY, the Overhauser effects between 6-H and 4-H showed the ␣ position of 4-H relative to the position of 6-H. ROESY correlations were not found between 7-H/9-H and 7-H/14-H, but between 7-H and 8-H, demonstrating the  position of 7-H. In summary, six new compounds were isolated, and their structures were elucidated. Among them, five novel A-
Fig. 4. Perspective view of compound 7. Crystal structure determination of compound 7. Diffraction data were collected from a single-crystal of 0.50 mm × 0.30 mm × 0.11 mm using a CAD-4 diffractometer with ˚ C23 H32 O4 S5, graphite-monochromated Cu K␣ radiation (λ = 0.71073 A). formula weight M = 532.79, Orthorhombic, space group (from systematic ˚ b = 12.3830(10) A, ˚ c = 24.8443(19) A, ˚ absences) P21 21 21 , a = 8.0455(6) A, Dc (Z = 4) = 1.430 mg m−3 . The structure was solved by direct methods with SHELXS-97 and was refined by full-matrix least-squares in F2 mode using SHELXL-97. Hydrogen positions were calculated from assumed geometries and included SF calculations, but without refinement. Reflections measured were 15107, unique reflections were 5677, R = 0.072, R2w = 0.069. The Absolute structure parameter was determined by refining the Flack parameter to 0.06(6). The molecular plot was drawn with the program ORTEP-III. (The crystal structure has been deposited at the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences (CAS). The deposition number is CCDC-23290).
Fig. 5. Two units of compound 8 concluded by COSY and HMQC spectra.
ring-opened spironolactone-analogs (3–7) were obtained and identified. Three of these compounds were stereoisomers. Compound 7 was an organic polysulfide, which had a rare, nine-membered ring with a five sulfur atom bridge. Compound 8 had a five-member ring, which was probably formed via rearrangment of the A-ring. These compounds have similar chemical and biological characteristics to spironolactone, indicating more potent remedies maybe derived from them. Farther biological tests on these compounds are in progress.
Acknowledgements This work was supported by Xianju Zhiyao Erchang, ZheJiang Province, China.
652
H. Chen et al. / Steroids 69 (2004) 647–652
References [1] Spark RF, Melby JC. Hypertension and low plasma renin activity: presumptive evidence for mineralocorticoid excess. Ann Intern Med 1971;75:831–6. [2] Carey RM, Douglas JG, Schweikert JR, Liddle GW. The syndrome of essential hypertension and suppressed plasma renin activity: normalization of blood pressure. Arch Intern Med 1972;130:849–54. [3] Highet RJ, Burke TR, Trager WF, Pohl LR, Menard RH, Taburet AM, et al. Carbon-13 nuclear magnetic resonance studies of spironolactone and several related steroids. Steroids 1980;35(2):119–32.
[4] Sheldriek GM. SHELXS-97. Program for structure solution. G¨ottingen, Germany: Universtiy of G¨ottingen; 1997. [5] Sheldriek GM. SHELXS-97. Program for structure refinement. G¨ottingen, Germany: Universtiy of G¨ottingen; 1997. [6] Flack HD. On enantiomorph-polarity estimation. Acta Crystallogr 1983;A39:876–81. [7] Burqett MN, Johnson CK. ORTEP-III. Oak Ridge Thermal Ellipsoid Plot Program for Crystal Structure illustrations; Report ORNL-6895; Oak Ridge National Laboratory: Oak Ridge, TN, 1996. Windows implementation was by Farrugia, LJ, Department of Chemistry, University of Glasgow, UK, 1998.