Partial synthesis of some diterpenoids with potential antitumour activity

摘要

J. CHEM. soc. PERKIN TRANS. 1 1991 Partial Synthesis of Some Diterpenoids with Potential Antitumour Activity Mohammed Shaiq Ali, Mark K. Baynham, James R. Hanson and Peter B. Hitchcock The School of Molecular Sciences, University of Sussex, Brighton, Sussex, BNI 9QJ, UK The diterpenoid fungal metabolite, fujenal, has been converted into analogues of the Rabdosia d iterpenoids, ent-7 -hydroxy- 1 5-oxo- 6,7 -secoka ur- 1 6-en-6,19-d ioic acid 6,7-lactone 19-methyl ester and ent-7-acetoxy-I 9-hydroxy-l5-oxo-6,7-secokaur-l6-en-6-oicacid 6,lg-lactone which possess moderate inhibitory activity against HeLa cells. In recent years many ent-kaurenoid diterpenes have been isolated from Chinese and Japanese medicinal plants of the genus Rabdosia (Labiatae). Particular interest has centred on the antitumour activity of these diterpenoids, e.g.trichorabdal A 1.' A number of these compounds have structures in which ring B has been cleaved. Structure-activity relationships have revealed the importance of an a-methyleneketone on ring D and a synergistic increase in activity due to a second oxygen function in the molecule. The fungal metabolite fujenal 2 is a kaurenoid diterpene in which ring B has been ~leaved.~ sometimes in It is f~rrned,~ significant amounts by Gibberella fujikuroi, which is used for the commercial production of gibberellic acid. It was the object of this work to introduce the unsaturated ketone onto ring D of fujenal derivatives and to evaluate the biological activity of the products. The conformation of fujenal has been examined5 in the context of partial syntheses in this area.A well-established route for the introduction of oxygen functions at C- 15 in the tetracyclic diterpenoids involves oxidation with selenium dioxide and hydrogen peroxide or tert- butyl hydroperoxide.6-8 This reaction did not proceed cleanly with fujenal itself, presumably because of the ready oxidation of the aldehyde. However it was successful with a number of derivatives lacking the aldehyde group. Fujenal 2 undergoes an internal aldol condensation with sodium hydride to form an alcohol 3.' Allylic oxidation of this compound with selenium dioxide and hydrogen peroxide gave the 15a-alcohol4. The site of oxidation followed from changes in the I3C NMR spectrum (see Table 1).In particular the signal assigned to C-15 had moved downfield to 6 83.2 whilst those assigned to C-8 and C-16 also showed smaller downfield shifts. The stereochemistry of the hydroxylation was established by 'H NMR spectroscopic studies. Examination of molecular models reveals that the 15a-C-H bond in 3 is approximately 90" to the plane of the C-16=C-17 double bond and thus this proton should exhibit a significant allylic coupling to the 17-H protons.' Decoupling experiments based on irradiating the olefinic proton resonances established their coupling to a signal at 6 2.53 which was assigned to the 15a-H in 3. There was no effect on the 15P-proton resonance which was located by irradiating the 1Sa-signal.There was however a long-range 'W coupling from the 15P-H to that at 6 2.24 which was assigned to the 14a-proton. Further decoupling studies established the assignments, given in Fig. 1 for the 14P- and 13-protons. Decoupling studies with the hydroxylation product 4 starting from 13-H (6 2.73) led to the identification of the 14P-proton resonance and thence the 14a-proton signal. There was a long- range coupling (J 1.3 Hz) between this signal and the 15-H resonance (6 3.96). Hence this proton is a 15P-H and the hydroxy group has the a-orientation in accordance with oxidations observed previously in similar systems.'.* An alternative way of blocking the aldehyde of fujenal2 is to 12 i7 1 2 3;R=H 4;R=OH 5; R' = H, R2= H2 6; R' = Ac, R2 = H2 7; R' = Ac, R2 = a-OH, P-H 8; R' = Ac, R2= 0 'C02Me 9; R' = CHO, R2= H2 12; R = H2 10; R' = CH20H, R2= H2 13; R = CZ-OH, j3-H 14; R = 011;R' = CH20H, R2= ~L-OH, P-H 15 reduce it with sodium borohydride.' ',12 The anhydride is also reduced and a major product is the 7-hydroxy 6,194actone 5.The alcohol was then converted into its acetate 6 which was oxidized with selenium dioxide and hydrogen peroxide to give the corresponding 15~-alcohol7. Alternatively methanolysis of Table 1 13C NMR spectroscopic data (determined in CDCl,) 6 Carbonatom 3 4 7 8 13 14 15" 1 38.9 39.1 33.5 32.2 34.2 36.3 41.0 2 18.2 18.5 17.6 17.6 19.3 17.5 18.3 3 28.4 28.6 32.5 31.7 36.2 33.8 27.8 4 43.9 44.2 42.8 42.4 44.2 44.2 44.7 5 66.6 66.3 53.4 52.8 58.4 57.0 57.6 6 175.3 175.6 178.5 178.0 174.7 173.7 174.8 7 76.0 73.8 66.0 66.3 72.4 71.3 75.0 8 51.1 55.4 61.6 54.9 51.2 54.9 52.8 9 56.4 54.4 44.0 46.4 56.7 56.2 52.9 10 47.8 47.6 38.4 38.6 40.3 40.3 40.3 11 19.3 19.5 20.8 21.1 17.8 19.6 20.7 12 32.5 32.0 31.6 31.6 31.7 31.5 25.4 13 38.2 36.3 42.9 38.5 42.2 38.2 43.4 14 29.5 25.8 35.6 35.4 40.8 40.1 41.1 15 47.9 83.2 79.6 207.5 80.8 207.8 127.9 16 157.3 162.0 158.6 149.1 159.9 148.3 149.0 17 106.7 111.4 108.1 114.0 108.3 115.7 36.8 18 26.1 26.3 31.2 30.8 29.7 29.3 29.6 19 173.3 172.7 75.7 75.7 175.9 175.5 175.9 20 19.5 19.2 22.7 23.7 18.9 18.6 18.9 OMelOAc 20.9 20.7 51.5 51.4 51.5 171.6 170.7 Determined in C,D,N.14.8 4 4.86 /14 x 0 l2.5 10.5 5 Fig. 1 Coupling constants and assignments for ring D of 3 fujenal 2 with sodium methoxide and methylation of the resultant hemi-methyl ester with diazomethane gave the dimethyl ester 9.12 Reduction of the aldehyde with sodium borohydride gave a separable mixture of the 6,7-lactone 12 and the 7-alcohol 10. Oxidation of the lactone 12 with selenium dioxide and hydrogen peroxide in dioxane gave two products. The first possessed a trisubstituted double bond dH 5.60; 6, 127.93(CH), 149.05(C) and an additional methylene signal (6, 36.76) rather than a methyl signal.An X-ray crystal structure showed that the compound was the dimer 15 (see Fig. 2). The formation of a symmetrical dimer is of interest since oxidations with selenium dioxide have been regarded as being ionic in character.' The second product was assigned the 15-hydroxy structure 13 from its spectral data. Oxidation of the 7- alcohol 10 with selenium dioxide and hydrogen peroxide gave a similar 15-alcohol 11. Oxidation of the 15-alcohols 7 and 13 to the corresponding 15-ketones 8 and 14 was achieked using chromium trioxide in pyridine, a procedure which has been successful in previous work. The unsaturated ketones possessed the anticipated spectral characteristics (eg. 6, 207.82, 148.30, 115.72 for C-15, C-16 and C-17 in 14).J. CHEM. SOC. PERKIN TRANS. I 1991 When tested* at concentrations of 3 and 7.5 ~m-~,the compounds 8 and 14 reduced the growth of HeLa cells by about 50 over a period of 4 days. They are thus fairly potent inhibitors of cell growth. Thus fujenal 2, a by-product of the gibberellin fermentation, may be transformed on the one-hand to compounds with potential plant-growth regulatory acti- vity9.15 and on the other to compounds with potential tumour inhibitory activity. Experimental General Ekperimental Details.--'H and '3CNMR spectra were determined at 360 and 90.56 MHz respectively on a Bruker WM 360 spectrometer for solutions in deuteriochloroform except where otherwise stated; J values are given in Hz. TR spectra were determined as Nujol mulls.Solutions were dried over sodium sulphate. Light petroleum refers to the fraction b.p. 60-80 "C. Silica for chromatography was Merck 9385. Hydroxylation of the Anhydride 3.-ent-6P-Hydroxy-7-nor-5P-gibberell- 16-ene-5@, 19-dioic acid anhydride 3' (500 mg) and selenium dioxide (200 mg) in dioxane (8 cm3) were treated with 30 hydrogen peroxide (4 cm3) dropwise at room temperature for 1 h. The mixture was cooled in ice and poured into aqueous sodium hydrogen carbonate. The solution was extracted with dichloromethane. The extract was dried, the solvent was evaporated and the residue was chromatographed on silica. Elution with ethyl acetate-light petroleum (1 :4) gave the starting material (290 mg) followed by ent-6P,15P-dihydroxy-7- nor-5P-gibberell- 16-ene-5P, 19-dioic acid anhydride 4 (145 mg) which crystallized from ethyl acetate-light petroleum as prisms, m.p.20201 "C (Found: c,69.2; H, 7.3. C2,H2,05 requires C, 69.3; H, 7.6"/d), v,,,/cm-' 3440br, 1840, 1780 and 1660; S 0.95 (3 H, s, 20-H), 1.49 (3 H, s, 18-H), 1.85 (1 H, dd, J 5.1 and 11.8, 14- H), 2.33 (1 H,dd, J 11.8 and 1.3, 14-H), 2.73 (1 H, dd, J5.1 and 8.8, 13-H), 3.96 (1 H, d, J 1.3, 15-H), 4.55 (1 H, s, 7-H) and 5.19 and 5.24 (each 1 H, s, 17-H). Hydroxylation ojthe Ester 10.-ent-7-Hydroxy-6,7-secokaur-16-ene-6,19-dioic acid 6,19-dimethyl ester' 10 (1.5 g) in dioxane (24 cm3) was treated with selenium dioxide (600 mg) and hydrogen peroxide (30, 12 cm3) at room temperature for 1 h. The products were recovered as above to give a gum which was chromatographed on silica. Elution with ethyl acetate-light petroleum (2:3) gave ent-7,15P-dihydro.xy-6,7-secokaur-16-ene-6,19-dioic acid 6,19-dirnethyl ester 11 (530 mg) as a gum, (Found: C, 67.1; H, 8.6.C2,H3,06 requires C, 67.0; H, 8.7), v,,,/cm-' 3481, 1733, 1719 and 1654; 6 1.16 (3 H, s, 20-H), 1.49 (3 H, s, 18-H), 3.61 and 3.62 (each 3 H, s, OMe), 3.76 and 4.28 (each 1 H, d, J 11,7-H), 4.20 (1 H, br s, 15-H) and 5.08 and 5.20 (each 1 H, br s, 17-H). On one occasion a 7,16,17-triol was isolated as a gum, (Found: C, 64.4; H, 8.6. C22H,,O7 requires C, 64.1; H, 8.873, v,,,/cm-' 3400, 1730 and 1710; 6 1.30 (3 H, s, 20-H), 1.37 (3 H, s, 18-H), 3.53 (1 H, d, J 11.7,7-H) 3.63 and 3.69 (each 3 H, s, OMe), 3.77 (1 H, d, J 12.8, 17-H), 4.09 (1 H, d, J 11.7, 7-H) and 4.80 (1 H, d, J 12.8, 17-H).Hj,dro.xylation of' the Ester 12.-ent-7-Hydroxy-6,7-seco-kaur-l6-ene-6,19-dioic acid 6,7-lactone 19-methyl ester' 12 (1 g) in dioxane (30 cm3) was treated with selenium dioxide (450 mg) and hydrogen peroxide (30; 8 cm3) at room temperature for 1 h. The products were recovered as above to give a gum which was chromatographed on silica. Elution with ethyl acetate-light petroleum (3 :7) gave the 17,17'-dimer of ent-7- * We thank Dr. E. A. Hamilton (ICI Pharmaceuticals) for carrying out these determinations. J. CHEM. SOC. PERKIN TRANS. 1 1991 268 1 Fig. 2 X-Ray molecular structure of the 17,17'-dimer (15) Table 2 Crystal data and structure refinement details for the X-ray Table 3 Fractional atomic co-ordinates ( x SO4) structure 15 X Y Formula C42H5208 M 684.9 4 233(2) 5 099(5) 3 673( 1) Crystal size (mm) 0.20 x 0.15 x 0.10 5 556(2) 5 128(5) 4 632( 1) Crystal system monoclinic 5 851(2) 2 085(4) 3 680( I) Space group P2,(no. 4) 6 834(2) 1442 2 989( 1) a, b, c', (A), S 11.759(9), 7.618(2), 21.409(7), 5 371(3) 6 977(6) 2 350(2) 105.83(4) 5 149(4) 7 925(6) 2 927(2) v (A)3 1845.1 6 145(3) 7 605(6) 3 526(2) F(OO0) 2, 1.23, 736 6 295(3) 5 661(5) 3 719(2) Z, D,(g.~m-~), p Mo-Ka (cm-') 0.8 6 354(3) 4 539(5) 3 114(2) Total unique reflections 3497 6 316(3) 2 635(5) 3 288(2) Significant reflections I > a(1) 2319 7 493(3) 1971(6) 2 541(2) R 0.039 6 693(3) 2 544(6) 1 892(2) R' 0.048 6 159(3) 4 414(5) 1 887(1) 5 522(3) 4 952(5) 2 415(2) 5 409(3) 4 852(6) 1 178(2) 4 776(3) 3 318(7) 758(2)hydroxy-6,7-secokaur- 15-ene-6,19-dioic acid 6,7-lactone 19-5 524(3) 1654(6) 860( 2) methyl ester 15 which crystallized from pyridine as prisms, m.p.5 785(3) 1 158(6) 1579(2) 250 "C (Found: C, 72.7; H, 8.5. C42H5808 requires C, 73.0; H, 7 404(3) 2 608(6) I386(2) 8.5), v,,,/cm-' 1729; G(C,D,N) 1.34 (3 H, s, 20-H), 1.47 (3 6 766(3) 2 098(6) 817(2) H, s, 18-H), 2.97 (1 H, s, 5-H), 3.69 (3 H, s, OMe), 3.80 and 4.68 7 052(3) 2 142(7) 168(2) 7 469(3) 5 412(7) 4 232(2) (each 1 H, d, J 12, 7-H) and 5.60 (1 H, br s, 15-H). Further 5 236(3) 5 202(6) 3 979(2) elution with ethyl acetate-light petroleum (2: 3) gave ent-7,lSP- 4 309(3) 4 099(6) 2 310(2) dihydro.xy-6,7-secokaur-16-ene-6,19-dioic acid 6,7-lactone 19- 4 612(3) 4 873(8) 4 935(2) methyl ester 13 (560 mg) which crystallized from ethyl acetate as 10 134(3) 5 63 l(5) -3 599(1) needles, m.p.248-250°C (Found: C, 69.6; H, 8.4. C2,H3,05 9 991(2) 3 510(5) -4 323 I) requires C, 69.6; H, 8.3), v,,,/cm-' 3459 and 1724; 6 1.24 10 974(2) 2 457(5) -2 751(1) (3 H, S, 20-H), 1.34 (3 H, S, 18-H), 2.86 (1 H, S, 5-H), 3.69 (3 H, S, 10 273(2) 1351(4) -2 OW(I) 7 568(3) 5 736(6) -2 924(2) OMe), 4.03 (1 H, br s, 15-H), 4.37 and 4.43 (each 1 H, d, J 13, 7- 7 528(4) 5 924(7) -3 634(2)H) and 5.12 and 5.23 (each 1 H, s, 17-H). 7 607(4) 4 139( 7) -3 929(2) 8 769(3) 3 199(6) -3 616(2) Hydroxylution of the Lactone 6.-ent- 7-Ace toxy- 19- hydroxy- 8 938(3) 3 107(6) -2 860(2) 6,7-secokaur-l6-en-6-oic acid 6,194actone 612 (1.03 g) in 10 123(3) 2 301(6) -2 546(2) 9 304(4) 1085(6) -1 71 l(2)dioxane (30 cm3) was treated with selenium dioxide (400 mg) 8 998(3) 2 716(6) -1 399(2)and hydrogen peroxide (30; 8 cm3) for 1 h.The products were 8 223(3) 4 087(5) -1 889(2)recovered as above to give a gum which was chromatographed 8 653(3) 4 736(5) -2 482(2) on silica. Elution with ethyl acetate-light petroleum (3 :7) gave 7 877(3) 5 615(6) -1 495(2) 15p, 19-dihydroxy-6,7-secokuur-ent-7-aceto.u~-16-en-6-oic acid 8 798(4) 6 168(6) -866(2) 6,19-luctonc~7 (9 13 mg) which crystallized from light petroleum 9 460(3) 4 590(6) -482( 2) as needles, m.p. 162 "C (Found: C, 70.1; H, 8.6.C22H3205 10 058(3) 3 610(6) -925(2) 8 279(3) 2 230(6) -922(2)requires C, 70.2; H, 8.679, v,,,/cm-' 3472, 1765 and 1741; 6 8 557(3) 3 239(6) -407(2)1.12 (3 H, S, 20-H), 1.27 (3 H, S, 18-H), 2.16 (3 H, S, OAC), 3.75 8 085(4) 3 335(7) 182(2) and 4.04 (each 1 H, d, J9, 19-H), 4.25 (1 H, br s, 15-H), 4.28 and 8 686(4) 1 306(8) -3 865(2) 4.41 (each 1 H,d, J 12,7-H)and 5.1 1 and 5.23 (each 1 H, s, 17-H). 9 719(3) 4 243(7) -3 817(2) 9 676(3) 6 034(6) -2 298(2) 0.uidrtion of the Hydro.uj1 Lactone 13 with Chromium 10 745(4) 4 558(9) -4 609( 2) Trio.uirle--The above lactone 13 (520 mg) was treated with a solution of chromium trioxide (5 g) in a mixture of pyridine (9 cm3) and dichloromethane (125 cm3) for 30 min at room solvent was evaporated to give a gum which was chromato- temperature.Aqueous sodium hydroxide was added and the graphed on silica. Elution with ethyl acetate-light petroleum product was isolated with dichloromethane. The extract was (3:7) gave ent-7-hydroxy- 15-oxo-6,7-secokuur- 16-ene-6,19-dioic washed with aqueous copper sulphate and water and dried. The acid 6,7-lactone 19-methyl ester 14 (450 mg) which crystallized from ethyl acetate as plates, m.p. 160 "C (Found: C, 69.7; H, 7.9. C21H2805 requires C, 67.0; H, 7.8), v,,,/cm-' 1723 and 1646; S 1.24 (3 H, S, 20-H), 1.40 (3 H, S, 18-H), 2.85 (1 H, S, 5-H), 3.16 (1 H, br s, 13-H), 3.69 (3 H, s, OMe), 3.80 and 4.60 (each 1 H, d, J 13,7-H) and 5.37 and 5.97 (each 1 H, s, 17-H). Oxidation of the Hydroxy Lactone 7 with Chromium Trioxide.-The above hydroxy lactone 7(600 mg) was treated with a solution of chromium trioxide (6 g) in pyridine (10 cm3) and dichloromethane (200 cm3) for 30 min at room temperature.Aqueous sodium hydroxide was added and the product was recovered in dichloromethane. The organic layer was washed with aqueous copper sulphate, water and dried. The solvent was evaporated to give a residue which was chromatographed on silica. Elution with ethyl acetate-light petroleum (1 :3) gave ent-7-acetoxy-19-hydroxy-15-oxo-6,7-secokaur-16-en-6-oic acid 6,19-lactone 8 (475 mg) as a foam (Found: C, 67.7; H, 8.2. C2,H3,0S*H20 requires C, 67.3; H, 8.2), vmax/cm-' 1734br and 1652; 6 1.13 (3 H, s, 20-H), 1.20 (3H,s, 18-H),3.11 (1 H, brs, 13-H),3.77and4.03(each 1 H,d, J 9, 19-H), 4.23 and 4.58 (each 1 H, d, J 11,7-H) and 5.27 and 5.92 (each 1 H, br s, 17-H).Crystal Structure Determination.-A summary of the crystal data and structure refinement details are given in Table 2. The data were collected from a crystal mounted on an Enraf-Nonius CAD4 diffractometer operating in the 8-28 mode with A8 = (0.8 + 0.35 tan 0)' and a maximum scan time of one minute and with monochromated Mo-Ka radiation (A = 0.710 69 A). Unique reflections were measured for 2 c 0 30(F2) were used in the refinement where o(F2) = (02(1)+ (0.041)2)*/L,. The structure was solved by direct methods using SHELXS-86.I6 Refinement was by full matrix least squares with non-hydrogen atoms anisotropic and weights of w = l/02(F).Hydrogen atoms were held fixed at calculated positions with Uiso= 1.3Ue, for the parent carbon atom. Programs from the Enraf-Nonius SDP-Plus package were run on a Micro-Vax computer. Fractional atomic co- ordinates are shown in Table 3. The remaining crystallographic data has been deposited at the Cambridge Crystallographic Data Centre.* * For full details of the CCDC deposition scheme see 'Instructions for Authors,'J. Chem. SOC.,Perkin Trans. I, 1991, Issue 1. J. CHEM. SOC. PERKIN TRANS. I 1991 Acknowledgements We thank the AFRC and the British Council for financial support and ICI Pharmaceuticals for a gift of fujenal. Part of this work was carried out under the HEJ Institute, University of Karachi-University of Sussex Link Scheme.We thank Professor Atta-ur-Rahman and Dr. D. R. M. Walton for establishing this link. We thank Dr. N. F. Elmore (ICI Pharmaceuticals) for arranging the bio-assay. References 1 E. Fujita and M. Node in Progress in the Chemistry of Organic Natural Products, 1984,46,78. 2 K. Fuji, M. Node, M. Sai, E. Fujita, S. Takeda and N. Unemi, Chem. Pharm. Bull., 1989,37, 1472 and refs. therein. 3 B. E. Cross, R. H. B. Galt and J. R. Hanson, J. Chem. Soc., 1963, 5052. 4 B. E. Cross, R. H. B. Galt, J. R. Hanson, P. J. Curtis, J. F. Grove and A. Morrison, J, Chem. SOC.,1963, 2937. 5 A. G. Avent, C. Chamberlain, J. R. Hanson and P. B. Hitchcock, J. Chem. Soc., Perkin Trans. I, 1985,2493. 6 A. Carcia-Granados, A. Parra-Sanchez and A. Y. Pena Carrillo, Anales de Quim., 1980, 76, 85. 7 S. C. Dolan and J. MacMillan, J. Chem. SOC.Perkin Trans. I, 1985, 2741. 8 A. G. Avent, J. R. Hanson, P. B. Hitchcock and B. H. de Oliveira, J. Chem. Soc., Perkin Trans. I, 1990,2661. 9 J. R. Hanson, C. L. Willis and K. P. Parry, J. Chem. Soc., Perkin Trans. 1, 198 1, 3020. 10 S. Sternhell, Pure Appl. Chem., 1964, 14, 15. 11 R. H. B. Galt and J. R. Hanson, J. Chem. Soc., 1965,1565. 12 M. K. Baynham, J. M. Dickinson, J. R. Hanson and P. B. Hitchcock, J. Chem. Soc., Perkin Trans. 1, 1987, 1987. 13 E. N. Trachtenberg in Oxidation,ed. R. L. Augustine, Marcel Decker, New York, 1970, vol. 1, ch. 3, p. 119. 14 J. R. Cannon, P. W. Chow, P. R. Jefferies and G. V. Meehan, Aust. J. Chem., 1966,19,861. 15 M. K. Baynham, J. M. Dickinson and J. R. Hanson, Phytochemistry, 1988,27,761 and refs. therein. 16 G. M. Sheldrick in Crystallographic Computing 3, eds. G. M. Sheldrick, C. Kruger and R. Goddard, Oxford University Press, 1985, pp. 175-1 89. Paper 1/03258E Received 1st July 199 1 Accepted 15th July 199 1