J. Nat. Prod. 2009, 72, 679–684
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Semisynthesis and Biological Activity of Stemofoline Alkaloids Morwenna C. Baird,† Stephen G. Pyne,*,† Alison T. Ung,† Wilford Lie,† Thanapat Sastraruji,§ Araya Jatisatienr,§ Chaiwat Jatisatienr,§ Srisulak Dheeranupattana,§ Jaturong Lowlam,§ and Sukanya Boonchalermkit‡ School of Chemistry, UniVersity of Wollongong, Wollongong, NSW, 2522, Australia, Department of Biology, Chiang Mai UniVersity, Chiang Mai, 50202, Thailand, and Department of EnVironmental Quality Promotion, Bangkok, 10400, Thailand ReceiVed December 21, 2008
The semisynthesis of the Stemona alkaloids (3′R)-stemofolenol (1), (3′S)-stemofolenol (2), methylstemofoline (3), and (3′S)-hydroxystemofoline (5) and the unnatural analogues (11E)-methylstemofoline (15) and 3′R-hydroxystemofoline (11) has been achieved starting from (11Z)-1′,2′-didehydrostemofoline (4). This synthesis allowed, for the first time, access to diastereomerically enriched samples of 1 and 2 and the assignment of their absolute configurations at C-3′. These compounds were obtained in sufficient quantities to allow for their biological testing. In a quantitative assay as AChE inhibitors, (11Z)-1′,2′-didehydrostemofoline (4) and (3′S)-hydroxystemofoline (5) were found to be the most active. The Stemona family of alkaloids includes more than 80 different natural products that have been structurally classified into eight groups.1 The pyrrolo[1,2-a]azepine (5,7-bicyclic A,B-ring system) nucleus is common to all compounds in six of these groups, while a pyrido[1,2-a]azepine A,B-ring system (6,7-bicyclic A,B-ring system) is found in the more recently discovered Stemoncurtisine group of Stemona alkaloids.1-5 A miscellaneous group comprising five Stemona alkaloids has also been identified.1 The pure alkaloids derived from the extracts of the leaves and roots of Stemona species have insect toxicity, antifeedant, and insect repellent activities1,3,4,6-8 and antitussive activities.9 In 2005, we reported the structures of six new stemofoline alkaloids, including an inseparable 1:1 mixture of (3′R)-stemofolenol (1) and (3′S)-stemofolenol (2) and the first C19 stemofoline alkaloid, methylstemofoline (3).10 The latter alkaloid was isolated in only 1.9 mg quantity and was of limited supply for further biological screening. Three known stemofoline alkaloids were also isolated, with the major component (11Z)-1′,2′-didehydrostemofoline (4) isolated in 100 mg quantities from the root extract of an unidentified Stemona species (Stemona sp.).10 We now have access to gram quantities of alkaloid 4 to use as a starting material to prepare sufficient quantities of the alkaloids 1, 2, and 3 and their analogues for biological screening. Here we report the semisynthesis of alkaloids 1-3 and some of their analogues and the absolute configuration at C-3′ for compounds 1 and 2. The synthesis of the related alkaloid 5,11 isolated from a different Stemona plant species, is also described. A qualitative study of the inhibitory activities of these compounds on the nicotinic acetylcholine receptor and their antibacterial activities are also reported.
Results and Discussion Dihydroxylation of the 3-butenyl side chain of 4 was first attempted using catalytic K2OsO4 and stoichiometric NMO, since diastereoselectivity was of little significance in this synthesis. Although the desired diol product was obtained, the unwanted dihydroxylated C-11-C-12 product was also observed. The alternative Sharpless asymmetric dihydroxylation reaction conditions using AD-mix-R were then attempted and resulted exclusively in the dihydroxylation of the butenyl side chain, forming the diol product 6, which was readily recrystallized from dichloromethane. It is * To whom correspondence should be addressed. Tel: +61-24221-3511. Fax: +61-24221-4287. E-mail:
[email protected]. † School of Chemistry, University of Wollongong. ‡ Department of Environmental Quality Promotion. § Department of Biology, Chiang Mai University.
10.1021/np800806b CCC: $40.75
suspected that the bulky DHQ2PHAL osmium complex of ADmix-R influenced the regioselectivity such that the more hindered C-11-C-12 double bond could no longer be approached. The diol 6 was then oxidatively cleaved using freshly prepared NaIO4 on silica gel12 to form the aldehyde 7, with an appropriate handle to which a new alkyl side chain can be attached. The classic Wittig reaction was carried out using 1-(triphenylphosphoranyldiene)-2-propanone; however, the triphenylphosphine oxide byproduct proved inseparable from the desired product 8 despite washing with diethyl ether and hexane, column chromatography, and attempted recrystallization. The Horner-Wadsworth-Emmons13 reaction was then utilized and yielded the desired R,β-unsaturated ketone 8 via a clean and simple method (Scheme 1). (R)-(+)- and (S)-(-)-2-Methyl-CBS-oxazaborolidine were used to reduce the ketone 8 diastereoselectively to give the natural product alcohols 1 (dr >95: 0 for an alcohol of R-configuration and δS - δR < 0 for an alcohol of S-configuration. The C-4′ methyl group resonated at δ 1.42 for the (S)-Mosher ester 9 and at δ 1.38 for the (R)-Mosher ester 10. Therefore, the configuration at C-3′ for 1 was assigned as R. According to the literature, this is the expected configuration for the reduction of 8 using the (S)-oxazaborolidine.14-16 The natural product 5 was simply prepared by hydrogenation of 2 over 10% Pd/C in the presence of H2 gas. This compound was obtained as a single diastereomer (dr >98:95% purity based upon 1H NMR analysis. For NMR assignments, all protons and carbons are numbered according to those of (11Z)-1′,2′didehydrostemofoline (4). Physostigmine (esterine) and acetylcholinesterase (906 U/mg, from electric eel) were purchased from Sigma-Aldrich.
682 Journal of Natural Products, 2009, Vol. 72, No. 4 (11Z)-1′,2′-Dihydroxystemofoline (6). To a flask containing ADmix-R (0.182 g) and methanesulfonamide (24.7 mg, 0.260 mmol) in 1:1 tert-butanol/water (1 mL) at 0 °C was added a solution of (11Z)1′,2′-didehydrostemofoline (4) (50.0 mg, 0.130 mmol) in 1:1 ratio of tert-butanol/water (1 mL). The reaction mixture was allowed to warm to rt and left to stir for 3 days. Sodium sulfite (0.2 g) was then added to the reaction and left at rt for 1 h. The mixture was extracted with chloroform (3 × 20 mL), and the combined organic extracts were washed with 2 M KOH, dried over MgSO4, and evaporated in Vacuo. Product 6 was obtained as a white solid (41.0 mg, 0.0979 mmol, 75% yield) of sufficient purity (>95%) to continue to the next step. A small amount was purified by column chromatography for characterization. Diol 6 can also be recrystallized from CH2Cl2 to yield pure compound. Rf ) 0.28 in MeOH/EtOAc (1:4); mp 188-190 °C (color changed to brown); [R]22 D +252.9 (c 0.89, CHCl3); IR νmax 3201, 2340, 1735, 1613, 1144, 1001 cm-1; 1H NMR (CDCl3) δ 4.24 (s, 1H, H-2), 4.14 (s, 3H, OCH3), 3.74 (s, 1H, H-1′β), 3.56 (t, J ) J 6.5 Hz, 1H, H-2′β), 3.53 (bs, 1H, H-9a), 3.24 (m, 1H, H-5a), 3.09 (m, 1H, H-10), 3.02 (m, 1H, H-5b), 2.98 (d, J ) 6.0 Hz, 1H, H-7), 2.32 (m, 1H, H-6a), 2.08 (s, 3H, H-16), 2.02 (d, J ) 12.5 Hz, 1H, H-1a), 1.84 (m, 2H, H-6b, H-9), 1.78 (m, 1H, H-1b), 1.64 (sextet, J ) 7.0 Hz, 1H, H-3′a), 1.54 (quintet, J ) 7.0 Hz, 1H, 3′b), 1.38 (d, J ) 6.5 Hz, 3H, H-17), 0.97 (t, J ) 7.0 Hz, 3H, H-4′); 13C NMR (CDCl3) δ 169.6 (C-15), 162.7 (C-13), 147.9 (C-11), 128.0 (C-12), 112.5 (C-8), 98.7 (C-14), 86.3 (C-3), 77.5 (C-2), 73.6 (C-2′), 73.0 (C-1′), 61.7 (C-9a), 58.8 (O-CH3), 50.6 (C-7), 50.5 (C-5), 47.6 (C-9), 34.4 (C-10), 32.8 (C-1), 28.5 (C-6), 26.1 (C-3′), 18.2 (C17), 10.0 (C-4′), 9.1 (C-16); EIMS m/z 419 (28%) [M]+; HREIMS m/z 419.1949 [M]+, calcd for C22H29NO7 419.1940. (5Z)-5-[(2S,2aR,6S,7aS,7bS,8R,9S)-7b-(1E)-1-Formylhexahydro9-methyl-4H-2,2,6-(epoxy[1]propanyl[3]ylidene)furo[2,3,4-gh]pyrrolizin-10-ylidene]-4-methoxy-3-methyl-2(5H)-furanone (7). To a vigorously stirred suspension of silica gel (1.83 g) and diethyl ether (9.2 mL) was added a warm (ca. 40 °C) solution of NaIO4 (177 mg, 0.826 mmol) in water (1.5 mL). Diol 6 (266.0 mg, 0.635 mmol) was dissolved in chloroform (1 mL) and added to the silica gel/NaIO4 mixture, which was left to stir at rt for 1 h. The mixture was filtered and the silica gel was washed with chloroform. Impure product 7 was obtained as a yellow oil and taken immediately through to the next step to avoid decomposition due to the instability of the aldehyde. Rf ) 0.15 in MeOH/EtOAc (1:4); IR νmax 2919, 2848, 1726, 1619, 1071, 1020 cm-1; 1H NMR (CDCl3) δ 9.73 (s, 1H, CHO), 4.61 (s, 1H, H-2), 4.15 (s, 3H, O-CH3), 3.62 (s, 1H, H-9a), 3.26 (s, 1H, H-7), 3.15 (dd, J ) 7.5 Hz, 6.5 Hz, 2H, 2 × H-5), 3.10 (m, 1H, H-10), 2.08 (s, 3H, H-16), 2.05 (s, 1H, H-1a), 1.89 (m, 3H, 2 × H-6, H-9), 1.86 (m, 1H, H-1b), 1.40 (d, J ) 6.0 Hz, 3H, H-17); 13C NMR (CDCl3) δ 197.6 (CHO), 169.6 (C-15), 162.6 (C-13), 147.5 (C-11), 128.1 (C-12), 112.6 (C-8), 98.9 (C-14), 89.4 (C-3), 76.5 (C-2), 61.2 (C-9a), 58.9 (O-CH3), 49.4 (C-7), 49.2 (C-5), 47.9 (C-9), 34.4 (C-10), 33.5 (C-1), 26.9 (C6), 18.2 (C-17), 9.1 (C-16); EIMS m/z 359 (27%) [M]+; HREIMS m/z 359.1363 [M]+, calcd for C19H21NO6 359.1369. (5Z)-5-[(2S,2aR,6S,7aS,7bS,8R,9S)-7b-(1E)-1-(3′-Oxo-1′-butenyl)hexahydro-9-methyl-4H-2,2,6-(epoxy[1]propanyl[3]ylidene)furo[2,3,4gh]pyrrolizin-10-ylidene]-4-methoxy-3-methyl-2(5H)-furanone (8). To a flask containing LiCl (29.6 mg, 0.699 mmol) in acetonitrile (7.1 mL) under a nitrogen atmosphere was added diethyl (2-oxopropyl)phosphonate (135.7 mg, 0.699 mmol, 0.13 mL), N,N-diisopropylethylamine (75.3 mg, 0.582 mmol, 0.10 mL), and aldehyde 7 (209.1 mg of crude from previous reaction, 0.582 mmol) dissolved in acetonitrile (1 mL). The reaction mixture was allowed to stir at rt for 2 days. The mixture was diluted with water (20 mL) and extracted with CHCl3 (3 × 20 mL), and the combined organic extracts were washed with brine (20 mL), dried over MgSO4, and evaporated in Vacuo. Purification by column chromatography using EtOAc to MeOH/ EtOAc (15:85) as eluent gave ketone 8 as a semisolid in 60% yield from 6 (151.9 mg, 0.381 mmol). Rf ) 0.33 in MeOH/EtOAc (1:4); [R]D21 +327.4 (c 0.39, CHCl3); IR νmax 2955, 1742, 1675, 1619, 968 cm-1; 1H NMR (300 MHz, CDCl3) δ 6.81 (d, J ) 15.8 Hz, 1H, H-2′), 6.38 (d, J ) 15.8 Hz, 1H, H-1′), 4.32 (s, 1H, H-2), 4.15 (s, 3H, O-CH3), 3.56 (bs, 1H, H-9a), 3.13-3.02 (m, 3H, 2 × H-5, H-10), 2.97 (t, J ) 3.3 Hz, 1H, H-7), 2.29 (s, 3H, H-4′), 2.08 (s, 3H, H-16), 1.99 (d, J ) 12.3 Hz, 1H, H-1a), 1.88-1.82 (m, 3H, 2 × H-6, H-9), 1.77 (dt, J ) 12.3, 3.3 Hz, 1H, H-1b), 1.40 (d, J ) 6.6 Hz, 3H, H-17); 13C NMR (75 MHz, CDCl3) δ 197.9 (CO), 169.6 (C-15), 162.7 (C-13), 147.9 (C11), 143.8 (C-2′), 130.6 (C-1′), 128.1 (C-12), 112.7 (C-8), 98.8 (C-
Baird et al. 14), 83.2 (C-3), 80.0 (C-2), 61.0 (C-9a), 58.9 (O-CH3), 52.4 (C-7), 48.2 (C-5), 47.6 (C-9), 34.5 (C-10), 32.7 (C-1), 27.7 (C-4′), 26.8 (C6), 18.3 (C-17), 9.2 (C-16); EIMS m/z 399 (23%) [M]+; HREIMS m/z 399.1687 [M]+, calcd for C22H25NO6 399.1682. (3′R)-Stemofolenol (1). To a solution of (S)-(-)-2-methyl-CBSoxazaborolidine (4.46 mg, 0.0161 mmol) and 1 M borane-methyl sulfide complex in CH2Cl2 (0.01 mL) at 0 °C was added ketone 8 (32.1 mg, 0.0805 mmol) in CH2Cl2 (1 mL). The mixture was allowed to stir at 0 °C for 1.5 h, after which time ethanolamine (1 mL) was added and the mixture was allowed to stir at rt for 18 h. The reaction mixture was quenched with methanol (2 mL), diluted with saturated sodium bicarbonate solution (20 mL), and extracted with chloroform (3 × 20 mL). The combined organic extracts were washed with brine (40 mL), dried over MgSO4, and evaporated in Vacuo. Purification by column chromatography using EtOAc to MeOH/EtOAc (1:9) as eluent gave 1 as a white solid in 66% yield (21.4 mg, 0.0534 mmol, dr ) >95:
