J. CHEM. SOC. PERKIN TRANS. 1 1992 Oxidation of Aphidicolin and Its Conversion into 19-Noraphidicolan-I 6p-01 John F. Gordon? James R. Hanson,*na Andrew G. Jarvisa and Arnold H. Ratcliffeb a School of Molecular Sciences, University of Sussex, Brighton, Sussex, BNI 9QJ, UK ICI Pharmaceuticals, Mereside, Alderle y Park, Macclesfield, Cheshire, SK I0 4JG,UK The preparation of the 3a,l8-monoacetonide of aphidicolin and its selective oxidation at C-17, is described. Catalytic oxidation of aphidicolin affords 16~-hydroxy-3-oxo-l9-noraphidicolan-l7-0ic acid. The conversion of this into 19-noraphidicolan-I 6p-01 and its biotransformation by the fungus, Cephalosporium aphidicola, to a 1 9- noraphidicolin, is reported. The diterpenoid fungal metabolite, aphidicolin 1, 'is a specific inhibitor2 of DNA polymerase a and, consequently, it has 1 R'-d=H 2 R' = R3= H, F? = R4= BuhepSi 3 R'=R2=R3=H,R4=Ts 4 R'=R3=H,f$=R4=Ts 5 R', R2= 0,R3 = R5 = CHzOH, R4= OH 6 R' = HI R2= OH, R3= CHO, R' = OH, R5= C02H 7 R1,R2= 0,R3 = CHO, R4= OH, R5= C02H 8 R',$ = 0, R3= CHzOH, R4= OH, R5 = C02H 9 R' = H, R2= OH, R3= CHzOH, R4, R5= 0 10 R' = HI R2= OH, R3= CHzOH, R4= OH, R5= CHO 11 R' = H, R2= OH, R3= R5 = CHO, R4 = OH 12 R' = H, R2 = OH, R3 = CHzOH, k= OH, = Me 13 R' = H, R2= OH, R3= CHO, R4= OH, R5 = Me 14 R' = H, R2= OH, R3= CHO, R4= OH, R5= C02Me 15 R' = H, R2=OH, R3= CHzOH, R'=OH, F? = C02Me 16 develop some selective oxidations of these hydroxy groups.These studies form the subject of this paper. Prior work5 had shown that the primary alcohols of aphidicolin could be protected as the 17,18-bis(tert-butyl-dimethylsilyloxy) derivative 2.Oxidation with pyridinium chlorochromate and removal of the protecting groups afforded the 3-ketone 5. The same compound has been obtained6 by the microbial oxidation of aphidicolin with Chaetomium funiculurn and Streptomyces griseus. On the other hand oxidation 'of the unprotected aphidicolin 1 with pyridinium dichromate gave a mixture of 17-carboxylic acids 6 8 in which oxidation had also taken place at C-3 and/or C-18. More vigorous oxidation with chromic acid ' afforded the diketo acid 16 in which not only had the C(16)-euro;(17) bond been cleaved but ring A had also been opened. The selective cleavage of the C(16F(17) bond can be achieved ' by oxidation with periodic acid which affords the 17-nor-16-ketone 9.The hydroxy groups of aphidicolin differ in the ease with which they form derivatives. Thus, at low temperatures aphidi- Colin forms a 17-mono-toluene-p-sulfonate 3 whilst on more prolonged treatment with toluene-p-sulfonyl chloride in pyri- dine at room temperature, it gives a 17,18-ditoluene-p-sulfonate 4. However the relatively mild Pfitzner-Moffatt oxidation with dicyclohexylcarbodiimide(DCC) in dimethyl sulfoxide (DMS0)-trifluoroacetic acid, which also relies on esterification for the oxidation, showed only a limited regiospecificity and gave a mixture of the 17-aldehyde 10 and the 17,18-dialdehyde 11. The location of the aldehyde group at C(17) in 10 followed from its oxidation with periodic acid which gave the known ' 3a,l8-dihydroxy-17-noraphidicolan-16-one9.Since the 17-aldehyde was required for further study, attempts were made to obtain a better yield. The selective protection of the glycols using acetonides was attempted. Although it was possible to prepare and separate the 3a,l8- and 16p, 17-monoacetonides, 17 and 21, the yields were not satisfactory. The mono-acetonides R' 21received attention as an antiviral and tumour-inhibitory agent.3 17 R' =OH, R2= CH2OH Aphidicolin possesses two primary, a secondary and a tertiary 18 R', R*= o alcohol disposed as a 1,2- and a 1,3-glycol. In the course of 19 R' = OH, R2= CH~OTS biosynthetic studies4 on this molecule, it has been necessary to 20 R' = OH, R2 = CHO were distinguished by oxidation of the 3a,l8-monoacetonide 17 with chromium trioxide in pyridine which gave the known' 3a, 18-monoacetonide of 3a, 18-dihydroxy- 17-noraphidicolan- 16-one 18.A simple method for preparing the 3a,18-mono- acetonide involved firstly preparing the 17-monotoluene-p-sulfonate 3and thence the 3a,l8-acetonide 19. Hydrolysis of the toluene-p-sulfonate with potassium hydroxide in DMSO then gave the 3a,18-monoacetonide of aphidicolin 17. This, in turn, was oxidized under the Pfitzner-Moffatt conditions to give the 17-aldehyde 20. Oxidation of aphidicolane-3a,l6P,18-triol12 under Pfitzner-Moffatt conditions gave the 18-aldehyde 13 in rather poor yield.Catalytic oxidation may show contrasting regiospecifi- city.'.' Catalytic oxidation of aphidicolin 1 with oxygen and finely divided platinum gave a mixture of 17-carboxylic acids which were separated as their methyl esters. The major product was the 19-nor compound 22. Its IR spectrum contained 22 R1, R2= 0,R3= Me, R4 = C02Me 23 R' = OH,R2= H,R3 = Me, R4 = CH20H 24 R1 = H,R2= OH,R3= R4= CH2OH carbonyl(l699 and 1720 cm-') and hydroxy absorptions (3475 cm-'). In the 'H NMR spectrum the 18-H3 signal (6 0.99) now appeared as a doublet (J 6.5 Hz). The minor products were assigned the structures 14 and 15. The location of the primary hydroxy group at C(18) in 15 followed from the position of the CH20H resonances (6 3.38 and 3.47, J 11 Hz) which fall within the range found for the 18-CH20H in the 17-nor-ketone 9, the 16-deoxy and 16-met hylene analogues. Reduction of the 19-nor compound 22 with lithium alum- inium hydride gave a triol 23, the stereochemistry of which was assigned on the basis of its 'H NMR spectrum.A selective population transfer decoupling experiment based on irradiating the methyl signal at 6 1.21 (d, J 6.5 Hz) led to the identification of the 4-H resonance at 6 1.52. Analysis of this resonance and that of the secondary alcohol S 3.24 (J 10 Hz) d (J 4.8 Hz), revealed J3,410 and J4,511.5 Hz. Thus there are diaxial relationships between the protons at C-3, C-4 and C-5. An NOE experiment based on irradiating the 20-H signal (6 0.89) led to an 8 enhancement of the 4-H signal (6 1.52).Hence this hydrogen atom is p and the stereochemistry of the triol 23 is as shown. Epimerization of the C-4 methyl group has taken place presumably during the loss of C-18. Hence these compounds are regarded as 19-noraphidicolanes. This also confirms the assignment made by Rozassa '' to a metabolite of aphidicolin produced by Trichothecium roseurn. The triol was converted into its ditoluene-p-sulfonate which was then reduced with lithium aluminium hydride to afford 19- noraphidicolan-16-0125. This compound is the 19-nor analogue of a precursor 26" of aphidicolin 1 in Cephalosporium aphidicola. Its biotransformation by C. aphidicola was therefore examined. We have shown that chlorocholine chloride (CCC) can inhibit the biosynthesis of aphidicolin by C.aphidicola. Although the inhibition is not complete at concentrations that are not also lethal to the organism, nevertheless, the reduction in the amount of aphidicolin that is formed, is sufficient to facilitate the detection of the bio-transformation products of J. CHEM. SOC. PERKIN TRANS. 1 1992 bsol;, 25 R=H 27 R=H26R=Me 28 R=Me exogenous substrates. 19-Nor-aphidicolan- 16p-01 25 was in- cubated with C. aphidicola for 29 days. The metabolites were isolated and chromatographed. The aphidicolin fractions were carefully monitored by conversion into their bis-acetonides. H NMR and mass spectroscopy showed that the 'last' aphidicolin fraction comprised the bis-acetonides 27 and 28 of aphidicolin 1 and 19-noraphidicolin 24 in a 1: 1 ratio.In the mass spectrum of the bisacetonide of aphidicolin 28 the most prominent peak is M -CH, (C26H4204 -CH,, m/z 403). There was a cor-responding peak at m/z 389 (C25H4004 -CH,). In the 'H NMR spectrum there were distinct resonances e.g. 6 0.87 (20-H), 2.37 (5-H), 3.87 (2 H, m, 18-H,) which could be assigned to a 19-noraphidicolin bisacetonide. In conclusion, we have shown that although there is only limited selectivity in the oxidation of the C-17 primary alcohol over that at C-18, this may be enhanced by the careful choice of protecting groups. Experimental General Procedure.--'H NMR spectra were determined at 80 and 360 MHz on Bruker WP 80 and WM 360 spectrometers for solutions in deuteriochloroform except where otherwise stated; J-values in Hz. IR spectra were determined as Nujol mulls.Solutions were dried over sodium sulfate. Light petroleum refers to the fraction, b.p. 60-80 "C. Silica for chromatography was Merck 9385. P$tzner-Mojfatt Oxidation of Aphidico1in.-Aphidicolin (1 g) was dissolved in dimethyl sulfoxide (DMSO) (4 cm3) and benzene (10 cm3). Pyridine (0.36 cm3), trifluoroacetic acid (0.1 1 cm3) and DCC (1.8 g) were added to this solution and the mixture was then stirred overnight. The solvents were removed under reduced pressure and the residue was dissolved in ethyl acetate (50 cm3). The solution was stirred for 15 min and then filtered through Celite. The filtrate was washed with dil. hydrochloric acid, aqueous sodium hydrogen carbonate and brine and dried.The solvent was evaporated to give a gum which was chromatographed on silica. Elution with ethyl acetate-light petroleum (1 :1) gave 3a, 16P-dihydroxyaphidi- colane- 17,18-dial 11 (300 mg) as a gum (M +,305.2 14. Calc. for C20H3004-CHO, 305.212); v,,,/cm-' 3350 and 1720; 6-(90 MHz) 0.92 and 0.97 (each 3 H, s, 19-, 20-H), 3.7 (1 H, m, 3-H) and 9.49 and 9.54 (each 1 H, s, CHO). Further elution gave 3a,l6~,18-trihydroxyaphidicolan-17-al 10 (170 mg) as a gum (M', 307.228. Calc. for C,oH3204 -CHO 307.227); v,,,/cm-' 3350 and 1720; S(60 MHz) 0.86 and 1.02 (each 3 H, s, 19-, 20-H), 3.6 (2 H, m, 18-H), 3.8 (1 H, m, 3-H) and 9.6 (I H, s, CHO). Periodate Oxidation of the Aldehyde 10.-A solution of the aldehyde 10 (110 mg) in pyridine (5 cm3) and water (1.5 cm3) was treated with aqueous periodic acid (50; 0.4 cm3) for 15 min at room temperature. The solvents were evaporated and the J.CHEM. SOC. PERKIN TRANS. 1 1992 residue taken up in ethyl acetate. The extract was washed with dil. hydrochloric acid, aqueous sodium hydrogen carbonate and brine and dried. The solvent was evaporated and the residue was chromatographed on silica. Elution with ethyl acetate gave 3a,18-dihydroxy-17-noraphidicolan-16-one9 (63 mg), m.p. 155 "C (lit., 155-156 "C) identified by its IR spectrum. Partial Acetonide Formation with Aphidico1in.-Conc. sulfuric acid (1 drop) and acetone (0.1 cm3) were added to a solution of aphidicolin (200 mg) in tetrahydrofuran (THF) (25 cm3) at room temperature.The reaction was monitored by TLC. After 30 min two products were observed and further acetone (0.06 cm3) was added. After 3 h the starting material had disappeared and a third product was becoming apparent. The acid was quenched by the addition of aqueous sodium hydrogen car- bonate and the solvents were evaporated. The residue was extracted with ethyl acetate and the extract was washed with brine, dried, and evaporated. The resultant gum was chromato- graphed on silica with toluene+!thyl acetate (9:l) as eluent to give the bisacetonide of aphidicolin 28 (108 mg), m.p. 144- 145 "C (lit.,' 145-146 "C) identified by its 'H NMR spectrum. Elution with toluene-ethyl acetate (1 :1) gave 16P,17-iso- propylidenedioxyaphidicolane-3a,18-di0l21 (1 2 mg), m.p.197- 201 "C; m/z 378 (l, M+) and 363 (45, M -CH,); 6 0.70 (3 H, s, 19-H), 0.98 (3 H, s, 20-H), 1.34 and 1.40 (each 3 H, s, 02CMe2), 3.37 (2 H, s, 18-H), 3.51 and 3.74 (each 1 H, d, J 8, 17-H) and 3.66 (1 H, m, 3-H). Further elution gave 3a, 1 8-isopropylidenedioxyaphidicolane-16p,17-diol 17 (49 mg), m.p. 161-163 "C (Found: C, 73.4; H, 10.3. C23H3804 requires C, 73.0; H, 10.1); v,,,/cm-' 3390 d(80 MHz) 0.72 (3 H, s, 19-H), 0.99 (3 H, s, 20-H), 3.03 and 3.80 (2 H, d, J 12, 18-H), 3.41 (2 H, s, 17-H) and 3.60 (1 H, m, 3-H). Oxidation of the Acetonide 17.-Dry chromium trioxide (160 mg) was added to a solution of dry pyridine (0.24 cm3) in dry dichloromethane (4 cm3) under argon.The deep red solution was stirred at room temperature for 15 min and then the acetonide 17 (100 mg) in dichloromethane (0.5 cm3) was added. After 10 min (TLC control) the reaction mixture was filtered through Celite which was repeatedly washed with ether. The combined filtrates were washed with aqueous sodium hydrogen carbonate and brine and dried. The solvents were evaporated to give a gum which was chromatographed on silica. Elution with toluene-ethyl acetate (9 :I) gave 3a, 18-isopropylidenedioxy- 17- noraphidicolan-16-one 18 (80 mg), m.p. 147-149 "C (lit.,' 143- 146 "C), identified by its IR and NMR spectra. 3a,18-Isopr op y lidenedioxy -1 7 -(p-toly lsulfon y1oxy)aph idicol- an- 16p-0119-The 17-monotoluene-p-sulfonate 3 of aphidicolin (500 mg) was heated under reflux in acetone (25 cm3) containing toluene-p-sulfonic acid (5 mg) for 30 min.The reaction mixture was neutralized with aqueous sodium hydrogen carbonate and then evaporated. The residue was taken up in ethyl acetate, washed with brine, dried and evaporated to give a residue. This was chromatographed on silica with toluenethyl acetate (6: 1) as eluent to give the title aphidicolane 19 (540 mg), m.p. 145- 148 "C (Found: C, 67.4; H, 8.7. C,0H440,S requires C, 67.6; H, 8.3); v,,,/cm-' 3450 and 1600; 6(80 MHz) 0.82 (3 H, s, 19-H), 0.96 (3 H, s, 20-H), 1.39 (6 H, s, 02CMe2), 2.45 (3 H, s, ArMe), 3.03 and 3.80 (each 1 H, d, J 12, 18-H), 3.60 (1 H, m, 3-H) and 3.66 and 3.92 (each 1 H, d, J 9, 17-H). Hydrolysis of the Toluene-p-sulfonate 19.-The above tolu- ene-p-sulfonate 19 (500 mg) in aqueous DMSO (15; 30 cm3) was treated with potassium hydroxide (1.2 g) at 80 "C for 3 h after which the mixture was cooled and poured into water (150 cm3).The products were recovered in ether and the extract was 3021 washed with brine, dried and evaporated to give a gum which was chromatographed on silica with toluene-ethyl acetate (1 :1) as eluent to give the aphidicolane 17 (390 mg) as needles, m.p. 161-163 "C, identical with the sample described above. Pjtzner-Moffatt Oxidation of Aphidicolane 17.-The above acetonide 17 (380 mg) in dry benzene (5 cm3) and dry DMSO (5 cm3) was treated with pyridine (0.08 cm'), trifluoroacetic acid (0.04 cm3) and dicyclohexylcarbodiimide (0.65 g) and the mixture stirred overnight in a tightly stoppered flask.The mixture was filtered through Celite which was then repeatedly washed with ether, The filtrates were washed with brine, dried, and evaporated and the residue was chromatographed on silica with ethyl acetatelight petroleum (1:l) as eluent to give 16P- hydroxy-3a,18-isopropylidenedioxyaphidicolan-17-a1 20 (295 mg), m.p. 197-201 "C (Found: C, 72.5; H, 9.8. C23H3@4 requires C, 73.3; H, 9.6); vmax/cm-l 3510 and 1725; 6(80 MHz) 0.74 (3 H, s, 19-H), 1.0 (3 H, s, 20-H), 3.24 and 3.64 (each 1 H, d, J 10,18-H), 3.62 (1 H, m, 3-H) and 9.60 (1 H, s, 17-H). Pfitzner- Moflat t Oxidation of Aphidicolane-3a, 1 6p, 1 8-trio1 12.-Pyridine (120 mg), trifluoroacetic acid (85 mg) and DCC (0.93 g) were added to a solution of the triol 12 (480 mg) in anhydrous DMSO (3 cm3) and benzene (5 cm3) and the mixture stirred at room temperature for 30 h.Although TLC indicated that most of the starting material was unchanged, the solvents were evaporated and the residue was taken up in ethyl acetate (30 cm3). Water (5 an3)was added and the mixture was stirred for 30 min before being filtered through Celite. The filtrate was washed with dil. hydrochloric acid, aqueous sodium hydrogen carbonate and brine, dried and evaporated. The residue was chromatographed on silica with ethyl acetate-light petroleum (2:3) as eluent to give 3a,16P-dihydroxyaphidicolan-18-a113 (55 mg) which crystallized from ethyl acetate-light petroleum as needles, m.p.172 "C (Found C, 75.1; H, 10.2. C20H3203 requires C, 75.0; H, 10.0); vma,/cm-' 3495, 3440 and 1710; 6 1.00 and 1.05 (each 3 H, s, 19- and 20-H), 1.15 (3 H, s, 17-H), 3.78 (1 H, t, J 2.7, 3-H) and 9.57 (1 H, s, 18-H). Further elution gave the unchanged triol (180 mg). Aerial Oxidation of Aphidicolin 1.-Adams catalyst (3.0 g) in methanol (50 cm3) was reduced under hydrogen and then washed with water. The black solid was suspended in water (50 cm3) and added to a suspension of aphidicolin (3.5 g, finely powdered) in water (500 cm3). A stream of air was bubbled through the vigorously stirred mixture at 90deg;C for 24 h. The mixture was filtered through Celite whilst hot and then allowed to cool. It was acidified to pH 2 with dil.hydrochloric acid and then extracted with ethyl acetate. This extract was separated into acidic and neutral fractions with aqueous sodium hydrogen carbonate. The acidic fraction contained most of the material (3.2 8). This was dissolved in methanol (200 cm3) and treated with an excess of diazomethane in ether. The excess of diazomethane was destroyed with acetic acid and the solvents were evaporated to give a residue which was chromatographed on silica. Elution with ethyl acetate-toluene (3 :2) gave methyl 16P-hydroxy-3-0x0- 19-noraphidicolan- 17-oate 22 (1.4 g) which crystallized from ethyl acetate as needles, m.p. 135-137 "C (Found: C, 72.2; H, 9.4. C20H30O4 requires C, 71.9; H, 9.0); vmax/cm-' 3475, 1720 and 1699; 6 0.99 (3 H, d, J 6.5, 18-H), 1.16 (3 H, s, 20-H), 3.76 (3 H, s, OMe).Further elution gave methyl 3a,l6P-dihydroxy-18-oxoaphi-dicolan-17-oate 14 (450 mg) as a gum; m/z (CI, NH,) 382 (M + NH4)+, 364 (M), 347 (M -OH) and 329 (347 -H20); vmax/cm-' 3430 and 1723; 6 1.00 and 1.10 (each 3 H, s, 19-and 20-H), 3.72 (1 H, s, W, 4 Hz, 3-H), 3.75 (3 H, s, OMe) and 9.38 (1 H, S, 18-H). Further elution gave methyl 3a,16P,18-trihydroxyaphidicolan-17-oats 15 (405 mg) which crystallized from ethyl acetate as prisms, m.p. 189-191 "C (Found: C, 68.8; H, 9.4. C21HS4O.j requires C, 68.8; H, 9.4); v,,/cm-' 3520, 3395, 3275 and 1736; 6 0.71 (3 H, s, 19-H), 0.95 (3 H, s, 20-H), 3.38 and 3.47 (each 1 H, d, J 11, 18-H), 3.68 (1 H, br s, 3-H) and 3.75 (3 H, s, OMe).Reduction of the Keto Ester 22.-The above keto ester 22 (1.35 g) in THF (10 cm3) was added to a stirred suspension of lithium aluminium hydride (0.8 g) in dry THF (100 cm3) under nitrogen and heated under reflux for 2.5 h. The mixture was cooled to 0 "C and the excess of reagent was destroyed by the addition of ethyl acetate followed by the dropwise addition of water. The mixture was acidified with dil. hydrochloric acid and stirred at room temperature for 30 min. The THF was removed under reduced pressure and the residue was extracted with ethyl acetate. The extract was washed with aqueous sodium hydrogen carbonate and brine, dried and evaporated to give 19-nor- uphidicolune-3/3,16~,17-trioZ23 (1.01 g) which crystallized from ethyl acetate-methanol as cubes, m.p.159-161 "C (Found: C, 70.4; H, 10.6. C,9H,203~H,0 requires C, 69.9; H, 10.4); v,,,/cm-' 3335 and 1030; G(C,D,N) 0.89 (3 H, s, 20-H), 1.21 (3 H, d, J 6.3, 18-H), 3.24 (1 H, t of d, J 10 and 4.8, 3-H) and 3.72 and 3.80 (each 1 H, d, J 12, 17-H). 19-Noruphidicolun- 16p-01 25.-Toluene-p-sulfonyl chloride (1.61 g) was added in portions over a period of 3 d to a solution of 19-noraphidicolane-3~,16~,17-triol23(870 mg). After 4 d the mixture was poured into dil. hydrochloric acid and the product was extracted with ethyl acetate. The extract was washed with aqueous sodium hydrogen carbonate and brine, dried and evaporated. The residue was chromatographed on silica with ethyl acetate-light petroleum (3 :7) as eluent to give 3/3,17-di(p- tolylsulfonyloxy)- 19-noraphidicolan- 16p-01 (790 mg) as a gum; mjz (FAB) 599 (M -OH) 427 (599 -TsOH) and 255 (427 -TsOH); v,,,/cm-' 3400, 1600 and 1175; 6 0.70 (3 H, d, J 6.3, 18-H), 0.83 (3 H, s, 20-H), 2.40 and 2.41 (each 3 H, s, ArMe), 3.78 and 3.84 (each 1 H, d, J 10, 17-H), 4.04 (1 H, t, d, J 10, 5.6,3-H) and 7.29, 7.32,7.75 and 7.76 (each 2 H, d, J 10, ArH).The above toluene-p-sulfonate (665 mg) in THF (5 cm3) was added to a stirred suspension of lithium aluminium hydride (0.8 g) in dry THF (100 cm3) under nitrogen and heated under reflux for 2 h. The mixture was cooled to 0 "C and the excess of reagent was destroyed by the addition of ethyl acetate followed by the dropwise addition of water. The mixture was acidified with dil.hydrochloric acid and stirred for 30 min. The THF was removed under reduced pressure and the residue was extracted with ethyl acetate. The extract was washed with aqueous sodium hydrogen carbonate and brine, dried and evaporated. The residue was chromatographed on silica with ethyl acetate- light petroleum (3: 17) as eluent to give 19-noraphidicolan-16P- 01 25 (245 mg) which crystallized from ethyl acetate as needles, m.p. 128-130 "C (Found: C, 82.3; H, 11.6. C19H3,O requires C, 82.6; H, 11.7); v,,,/cm-' 3370 and 1115; 6, 0.79 (3 H, d, J 6.3, 18-H), 0.88 (3 H, s, 20-H) and 1.13 (3 H, s, 17-H); 6, 13.4 (C-20), 21.2 (C-18), 22.0 (C-2), 24.6 (C-14), 26.7 (C-6), 27.0 (C-7), 28.1 (C-17), 31.7 (C-4), 32.2 (C-1), 32.8, 33.3 and 34.2 (C-15, -13, -ll), 36.6 (C-3), 39.6 (C-lo), 39.8 (C-8), 45.5 (C-5), 46.7 (C-12), 46.8 (C-9) and 73.0 (C-16).J. CHEM. SOC. PERKIN TRANS. 1 1992 Incubation of 19-Noruphidicolun- 16p-01 25 with Cephalo- sporium aphidico1a.-The fungus was grown on a medium as described previou~ly.'~ The substrate 25 (145 mg) and CCC (110 mg) were dissolved in ethanol (6 cm3) and DMSO (15 cm3) containing Tween 80 (2 drops). This solution was evenly distributed between three Thompson bottles (2.25 dm3) of C. uphidicolu on the sixth day after inoculation. On the 35th day, the culture was filtered and the broth was extracted with ethyl acetate. The metabolites were chromatographed on silica. Elution with ethyl acetatelight petroleum (1 :4) afforded the untransformed substrate (31 mg).Further elution with ethyl acetate gave three 'aphidicolin' fractions: A (78 mg), 5 (52 mg) and C (12 mg). Each fraction was separately treated with acetone and toluene-p-sulfonic acid to afford the bis-acetonides. Analysis by 'H NMR showed that the products from fractions A and B were pure aphidicolin bis-acetonide 28. Fraction C acetonides were examined by MS and 'H NMR; m/z 403 (C,,H42O, -CH,; compound 28) and 389 (C25H4004 -CH,; compound 27); dH 0.87 (3 H, s, 20-H), 1.25, 1.34, 1.40 and 1.43 (each 3 H, s, 02CMe,), 2.37 (1 H, m, 5-H), 3.54 and 3.76 (each 1 H, d, J 8.3, 17-H), 3.87 (2 H, m, 18-H) and 4.05 (1 H, m, 3-H) for compound 27 together with resonances attributable to aphidicolin bis-acetonide 28.' The signals were in the ratio 1 :1 suggesting the presence of 3a, 18; 16p,17-bisisopropylidenedioxy-19-noraphidicolane 27 (cu.6 mg). Acknowledgements We thank the SERC and ICI Pharmaceuticals for a CASE studentship for A. G. J. References 1 W. Dalziel, B. Hesp, K. M. Stevenson and J. A. J. Jarvis, J. Chem. Soc., Perkin Trans. I, 1973,2841. 2 S. Ikegami, T. Taguchi, M. Ohashi, M. Oguro, H. Nagano and Y. Mano, Nature (London), 1978,275,458. 3 For a review, see S. Spadari, F. Focher, F. Sala, G. Ciarrocchi, G. Koch, A. Falaschi and G. Pedrali-Noy, Arzneim.-Forsch., 1985, 35, 1108. 4 M. J. Ackland, J. Gordon, J. R. Hanson, B. L. Yeoh and A. H. Ratcliffe, J. Chem. SOC.,Perkin Trans. I, 1988, 1477 and refs. therein. 5 A. H. Ratcliffe, unpublished work. 6 J. Ipsen, J. Fuska, A. Fuskova and J. P. Rosazza, J. Org. Chem., 1982, 47, 3278. 7 S. Hiranuma, T. Shimizu, H. Yoshioka, K. Ono, H. Nakone and T. Takahashi, Chem. Pharm. Bull., 1987,35,1641. 8 K. E. Pfitzner and J. G. Moffatt, J. Am. Chem. Soc., 1965,87,5661. 9 K. Heyns and L. Blazajewicz, Tefrahedron,1960,9,67. 10 R. P. A. Sneeden and R. B. Turner, J. Am. Chem. Soc., 1954,77, 190. 11 J. Ipsen and J. P. Rosazza, J. Nat. Prod., 1984,47,497. 12 M. J. Ackland, J. R. Hanson, B. L. Yeoh and A. H. Ratcliffe, J. Chem. Soc., Perkin Trans. I, 1985,2705. 13 M. J. Ackland, J. R. Hanson and A. H. Ratcliffe, J. Chem. SOC.,Perkin Trans. I, 1984,275 1. Paper 2/04290H Received 10th August 1992 Accepted 21st August 1992
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