1974 200113C Nuclear Magnetic Resonance Spectra and Microbiological Hydroxyla-tion of 7a- and 7p-HydroxykaurenolideBy James R. Hanson," Guiseppe Savona, and Michael Siverns, The School of Molecular Sciences, UniversityThe 13C n.m.r. spectra of some kaurenolides have been assigned. Changes in the spectra are used to show thatmicrobiological hydroxylation of 7a-hydroxykaurenolide by Rhizopus arrhizus affords 71x1 1 a-, 7a,l2-, and 7a.13-dihydroxy- and 7a.16a.17-trihydroxykaurenolide. 7P-Hydroxykaurenolide affords 7p.11 a- and 7p.13-dihydroxy-kaurenolide.of Sussex, Brighton BN1 9QJ~~(-HYDROS~-I.;,~URENOLIDE (1) is readily available fromthe fungal metabolite, 7f3-hydroxykaurenolide (2) .lIts derivatives can be converted into gibberellirs2Microbiological hydroxylation of 7a-hydroxykaurenolidewas studied as part of a programme to prepare semi-synthetic gibberellins functionalized at the chemicallyinaccessible sites on ring c.The microbiological trans-formation of tetracyclic diterpenes has been reportedfrom a number of laboratorie~.~ 13C N.m.r. spectro-scopy has a considerable potential as a structural toolin microbiological hydroxylation since it is possibleto assign each I3C resonance in the substrate and metabo-lite and thus from the changes, to locate the site ofhydroxylation. In this paper we report the assignmentof the I3C n.m.r. spectra of some kaurenolides and theiiiicrobiological transformation of 7a- and 7p-hydroxy-kaurenolide by RhizoPz4s arrlzixus, a micro-organismknown to hydroxylate steroids a t C-11.The 13C n.ni.r.spectra were obtained at 25.15 MHzusing a pulsed Fourier transform system with protonabled the C-6 and C-7 resonances to be assigned. Inthis compound the C-7 resonance collapsed and showeda small deuterium isotope shift (0-5 p.p.m.). In the7p- series, C-6 and C-7 were distinguished by the changescaused by acetylation a t C-7. The C-6 resonanceshowed an upfield shift and the C-7 resonance a down-field shift ( c j , ref. 5). The position of the C-6 resonancereflected variations in the nature and stereochemistryof the C-7 substituents. The C-18 and C-20 methylgroups, which gave quartets in the off-resonance spectra,could be distinguished since the 25 p.p.m. re- 3onancewas absent from the spectrum of 7p,l8-diacetoxy-kaurenolide (4).The upfield shift of the C-20 resonancein 7-oxokaurenolide (6) reflects the influence of theshielding cone of the carbonyl group, a feature which isalso seen in the proton spectrum.6The singlets at 34, 42, and 45 p.p.m. were assignedto C-10, C-4, and C-8 respectively on the basis of thevariation of the C-4 resonance on substitution at C-18,the effect of a C-7 carbonyl group on the C-8 resonance,1 N.m.r. spectra of the liaurenolides (p.p.m. from tetramethylsilsne)Carbon atomh v COlll- ~ - __pound 1 2 3 4 5 6 7 8 9 10 11 12 1:; 14 15 16 17 18 19 3038.2 17.3 28 15 42.1 51.2 75.7 71.6 45.4 53.7 33.6 17.8 33.2 S7.3 34-2 50.6 1586 106.8 25.1 181.9 19.137-6 17.35 * 28.1 42.1 51.75 83.7 71.6 45.1 ?5.4 34.2 17.3 * 32.6 37.1.34.3 42.1 159.4 106.9 25.5 183.5 20.5(3) 37.3 17.35 28.15 41.2 51-75 80-15 73.2 44.3 w . 5 34-1 17.1 32.5 37.3 34.2 43.2 158.2 307.7 25.7 181.4 20.3(4): 36.3 16.6 23.05 45.8 48-05 81.05 72.9 44.2 55.8 34.2 17.1 32.5 37.4 33.9 4 3 4 1584 107.75 67.95 178.9 21-0 *36.0 16.4 23.0 45.8 47.9 80.05 72.8 42.8 56.5 34.3 15.7 :6.2 42.8 31.7 49.8 821.3 67.6 l i 8 . 4 20.9 * i! 34.4 16.9 28.8 41.4 52.5 76.3 208.9 53.6 57.0 35.3 17.8 al.7 37.3 31.8 47.9 156.65 108.0 26.3 179.8 15.6( 7 ) d 34 9 23.8 68.4 44.7 51.5 80.2 72.8 44.3 55.7 34.0 17.1 32.3 37.2 3 4 4 43.1 157.9 107.8 21.4 179 0 19.1( 8 ) 38.8 17.3 27.8 41.6 50-7 75.6 71.05 45.8 67.1 34.5 65.2 45.0 ?S.0 36.2 50.5 157.6 107.9 25.5 1 S l . Y 21.2(9) 37.8 17.65 28.3 41.4 51.6 75.8 207.1 54.6 68.1 35.3 205.8 49.0 56.8 33.9 51.2 152.6 110.7 26.15 179 5 15.8(16) 37.1 I 5 35 28.3 43.8 51.5 76.1 72.25 43.5 ,)2.2 34.2 19.4 39.6 78.4 41.4 48.05 157.3 106.1 24.2 181.9 20.6(21) 37.4 17.2 27.9 42.8 51.9 83.1 71.5 42.7 55.2 34-2 18.5 39.1 57.7 40.5 42.2 159.7 107.1 25.5 181.8 21.2Acetate 20 9 arid 169 95.b ACetdteS 21.05 * (2), 170.0 and 1704J;. c Acetates 20.95 * (2) and 170.3 (2). d Acetates 20.9 and 21.05, 169.6 and 169 8.* Assignment may be interchanged.noise-decoupling and off-resonance decoupling. Theresults are tabulated. Many of the resonances wereassigned with the aid of their multiplicity in the off-resonance spectra. The C-16 and C-17 olefinic and C-19lactone carbonyl resonances at 158 (s), 107 (t), and179 (s) p.p.m. respectively, were readily recognized.Examination of 7~-2HH-7a-hydroxykaurenolide en-B.E. Cross, It. H. B. Galt, and J. K. Hanson, J . Choi?. SOC.,19G3, 2944.R. 11. 13. Galt and J . 12. Hanson, J . Cheni. Soc., 1965, 1565;B. E. Cross, K. Norton, and J . C. Stewart, J . Chenz. SOC. ( C ) ,1968, 1064; J . R. Haiison and J. Hawker, Phytochenzistry, 1973,12, 1073; J. 13. Bearder, J . MacMillan, and B. 0. Phinney, ibid.,p. 2173.J . R . Hanson and A. I;. White, Tetralaedron, 1968, 24, 6291;A. R. Anderson, R. McCrindle, and J. K. Turnbull, J.G.S. Chem.Comm., 1973, 143; J . P. Beilby, E. L. Ghisalberti, P. R. Jefferies,M. A . Sefton, and 1'. N. Sheppard, Tetvnhedro?z Letters, 1973, 2589;J . R. Bearder, J . MacMillan, C. M. Wels, and B. 0. Phinney,J.C.S.Chew. Cowanz., 1973, 778.and the constancy of the C-10 resonance during thesevariations. C-10 in steroids resonates at 36 p . p . ~ . ~The doublets at 37, 51, and 56 p.p.m. were assignedto C-13, C-5, and C-9 respectively. The variation in theC-13 resonance on converting 7p, 18-diacetoxykaurenolide(4) into its 16-oxo-17-nor-derivative (5) and the variationin the C-5 resonance with the introduction of a C-18substituent (3) --t (4) enabled these centres to bedistinguished. Both the C-12 and C-14 resonances areshifted on the introduction of a C-16 carboiiyl groupD. H. C. Petcrson and H. C. Murray, J . Amer. Chem. SOC.,1952, 74, 1871; P. D. Meister, D. H. Peterson, S. H. Epstein,H. C. Murray, C. M. Keineke, A. Weintraub, and H.M. L.Osborne, ibid., 1954,76, 5679; for a recent ref. see D. S. 13. Smith,N. J. Poole, and W. F. A. Jowett, Phytochemistry, 1973, 12, 561.H. J. Reich, M. Jautelat, M. T. Messe, F. J . W'cigert, andJ . D. Roberts, J . Anzev. Chem. Soc., 1969, 91, 7445; 11. Eggertand C. Djerassi, J . Org. Chem., 1973, 38, 3788.6 J . R. Hanson and A. I;. White, Tetvnhedrori, 1069, 25, 27432002(a) --t (5)J whilst the C-5 and C-9 resonances show ashift on the introduction of a C-7 carbonyl groupr(2) + (6)l.(21 R ' = H , R 2 = O H( 4 ) R' = R 2 = OAc( 3 ) R ' = H , R ~ = O A C@ OAc, IAcOco-0The two high field triplets at 17 p.p.m. were assignedto C-2 and C-11 as these centres are shielded by major1,3 diaxial interactions.' In steroids these centresare the two highest field triplets (at 20 p.p.m.) andin the pimaranes they resonate a t 18-20 p.p.m.8 Thetriplet at 37 p.p.m. was assigned to C-1, the adjacentaxial methyl group deshielding this centre.' In theandrostane series C-1 resonates at 38 ~ .p . m . ~ whilst inthe pimaranes this resonance is in the range 37-40p.p.m.8 The triplet at 28 p.p.m. shifts to 23 p.p.m.on the introduction of a C-18 substituent (3) + (4)and it was therefore assigned to C-3. The lowest fieldtriplet at 42-50 p.p.m. was assigned to C-15 whichis influenced both by changes a t C-16 and C-7. Theresonances at 32 and 34 p.p.m. were assigned to C-12and C-14 respectively.The microbiological transformation of 7a-hydroxy-kaurenolide (1) was then examined. In order to locatethe metabolites a preliminary transformation wascarried out with "7 p-3H1-7a-hydroxykaurenolide whichrevealed four transformation products.The productswere separated by chromatography. The first meta-bolite (8) (ca. 10 yield) analysed for C,H,O,. Theproton n.m.r. spectrum contained an additional CH-OH' G. C. Levy and G. L. Nelson, ' Carbon-13 Nuclear MagneticResonance for Organic Chemists,' Wiley-Interscience. New York,p. 43.J.C.S. Perkin Iresonance at T 5.9. The C-20 proton resonance wasshifted downfield from T 8.90 in 7or-hydroxykaurenolideto 8.58 in the metabolite. In the 13C n.m.r. spectrum(see the Table) a resonance at 17 p.p.m., which hadbeen assigned to C-11 in 7a-hydroxykaurenolide, wasno longer present and a new doublet appeared at 65.2p.p.m.Furthermore the resonances which had beenassigned to C-12 and C-20 showed downfield shifts.When the metabolite was oxidized with the 8N-chromiumtrioxide reagent, a diketone (9) was obtained whichshowed only a lactone and cyclohexanone carbonylabsorption in the i.r. (vmax. 1792, 1720sh, and 1715cm-l). One carbonyl group in this diketone wassterically hindered since reduction with sodium boro-hydride afforded a 7a-monohydroxyketone (10) v,,3520, 1780, and 1690 cm-l, 7 5.2 (t, J 7 Hz, 6-H) and5.94 (d, J 7 Hz, '?-H). The I3C n.m.r. spectrum ofthe diketone (9), when compared to that of 7-0x0-kaurenolide (6), revealed a marked shift in the C-9resonance (57.0 ---t 68.1 p.p.m.), the C-11 resonance(now at 205.8 p.p.m.), and the C-12 resonance (31.7 --t49.0 p.p.m.).Hence the additional oxygen functionis a t C-11. Further evidence for this came from a studyof the mass spectrum. The hydroxylated metabolite(8), the diketone (9), and the monohydroxy-ketone (10)showed fragments at nz/e 109, 137, and 165 whichare found in the parent kaurenolides and are assignedthe structures ( l l ) , (12), and (13) and arise from ring A.Thus hydroxylation on ring A was excluded. Theco-0( 8 )i(11)c, 0( 1 4 1 (15)mass spectrum of the diketone contains two additionalsignificant peaks at m/e 135 and 163 which are assigned8 E. Wenkert and L. Buckwalter, J . Amer. Chem. SOC., 1972,94, 4367.Q A. J. Kalinovsky, E. P. Serebryakov, A. V. Simolin, V. F.Kucherov, and 0.S. Chizov, Org. iWass Spectrometr-y, 1971, 3, 331974 2003the structures (14) and (15). Their formation requiresthe presence of a correctly oriented y-C-H (e.g. from C-1)for the hydrogen transfer and this limits the site ofoxygentation to C-11. In view of the marked effectof the additional hydroxy-group on the C-20 protonresonance, an effect which was considerably enhancedin iT2H5pyridine, AT 0.47 p.p.m., the C-11 hydroxy-group was assigned the 11 cc-configuration.co-0 CO-0'0 Hco-0I181 (19)The second metabolite (16) to be isolated in 20yield, also gave analytical data for C,H,,O,. Theabsence of an additional CH-OH resonance in the protonn.m.r. spectrum and the presence of a singlet in the13C n.m.r. spectrum at 78.4 p.p.m. replacing the C-13doublet was clearly indicative of a tertiary alcohollocated at C-13.The hydroxy-group showed thecharacteristic effect on the C-17 proton resonances lowhich now appeared at T 5.09 and 4.80, an effect whichwas amplified in 2H,pyridine when the resonancesappeared at 7 4-90 and 4-42. In confirmation of thestructure (Is), the metabolite underwent a Wagner-Meerwein rearrangement l1 on treatment with acidto form a cyclopentanone (vmax. 1745 cm-l) (17) whichpossessed an additional CMe resonance in the protonn.m.r. spectrum T 9-25 (6H) and 8.92 (3H)l.Two minor metabolites were isolated in insufficientquantity for 13C n.m.r. spectroscopy. The first ofthese (18), which was isomeric with the previous metabo-lites, showed the typical kaurenolide ring -4 fragmentsat mje 109, 137, and 165 in its mass spectrum. Hencethe additional oxygen function was located on ringc or D.It possessed an additional CH*OH resonanceat T 6.26 (W, 8 Hz). The corresponding diketonecontained lactone and cyclohexanone absorption in thei.r. (vmZ 1775, 1725, and 1710 cm-I). It did not possesssignificant U.V. absorption. In the n.m.r. spectrumthis diketone showed a doublet at z 6-70 ( J 6 Hz) andlo J. R. Hanson, J . Chem. SOC., 1965, 5036.l1 E. Mosettig, U. Beglinger, F. Dolder, H. Lichti, P. Quitt,and J. A. Waters, J . Amer. Claewz. SOC., 1963, 85, 2306.two more proton signals than 7-oxokaurenolide between7 7.4 and 7.9. The C-17 protons resonated at T 4.68and 4.87 deshielded by an adjacent oxygen function.Reduction of a small sample of the diketone withsodium borohydride regenerated the parent dihydroxy-lactone ca.80 (t.l.c.) together with an epimer (ca.20). Hence this metabolite was tentatively assignedthe structure 701,12( (3)-dihydroxykaurenolide (18), al-though we cannot exclude a 12wconfiguration for thealcohol. The second minor metabolite (19), C~OHamp;~,lacked olefinic proton resonances in the n.m.r. spectrum.It contained a CH2*OH group T (C5D5N) 5-92 ( J 10Hz). When deuterium oxide was added to the solutionthis resonance collapsed to a singlet. Furthermore inthe mass spectrum there was a ready loss of 31 a.m.u.(CH,OH). The metabolite was identified as 7cc,16a,17-trihydroxykaurenolide (19) and was identical with asynthetic sample .6Although 7 (3-hydroxykaurenolide (2) was not apotential substrate for chemical conversion into gib-banes, its hydroxylation was briefly examined.Twomajor metabolites were isolated. The first was assignedthe structure (20), 7p, 11 cc-dihydroxykaurenolide, sinceon oxidation it gave the diketone (9). The llcc-con-figuration was assigned to the hydroxy-group becauseof its marked deshielding effect on both the C-20 andC-7 proton resonances. After this work was completed,this compound was described l2 as a minor metaboliteof Gibberella fujikuroi, strain TP70. The secondmetabolite was the known 7 (3, 13-dihydroxykaurenolide(201 ( 211(21),3J3 which was identified by comparison (i.r. andn.m.r.) with an authentic sample.Its 13C n.m.r.spectrum (see the Table) was in accord with thisstructure .We conclude that microbiological hydroxylation mayprovide a route to kaurenolides which are hydroxylatedon ring c and are suitable for conversion into gibberellins.Furthermore the 13C n.m.r. spectra of this series are acomplementary structural tool to the proton n.m.r.spectra.EXPERIMENTALM.p.s were determined on a Kofler hot-stage apparatus.1.r. spectra were recorded on a Perkin-Elmer 257 spectro-meter for Nujol mulls. lH N.m.r. spectra were determinedfor solutions in 2Hchlor~form on a TTarian AGOA orl2 P. Hedden, J . MacMillan, and M. J. Grinstead, J.C.S.Pevkivz I , 1973, 2773.l 3 E. P. Serebryakov, A. V. Simolin, V. F. Kucherov, andB.V. Rosynov, Tetrahedron, 1970, 23, 5215J.C.S. Perkin IHA 100 spectrometer with tetramethylsilane as an internalstandard. The 13C n.m.r. spectra were determined on aJEOL PFT- 100 Fourier transform spectrometer operatingat 25.15 MHz. The spectral width was 250 p.p.m. using5192 data points and 5--10,000 accumulations. Thepulse length was 7 ps (c. 30") at a pulse interval of 1.0 s.The samples (80-150 mg) were dissolved in 2Hcliloro-form (0.5 ml), and the solvent deuterium provided tlielock signal. Tetramethylsilane was used as an internalstandard. The shifts are estimated t o be accurate to0.1 p.p.m. Mass spectra were determined on an A.E.I.MS9 mass spectrometer operating at 70 eV. Light petrol-eum refers to tlie fraction b.p.60-80".Incubation of 7a-Hydroxykaurenolide will6 Rhizopus ar-rhizus.-Rhizopus arrlzizus was grown on shake culture in 500ml conical flasks a t 25" on a medium l4 (200 ml) comprisingmalt extract (1 g), beef extract (1 g), bacteriological peptone(1 g), corn steep liquor (1 ml), and glucose (5 g) in water(1 1) for 6 days. 7a-Hydroxykaurenolide 1 (2.0 g) inethanol (40 ml) was distributed between 30 flasks and theincubation was continued for a further 4 days. Themycelium was filtered and the broth saturated with sodiumchloride and extracted with ethyl acetate. The extract(2.04 g) was chromatographed on silica (70 g). Elutionwith chloroform gave ent-6P, 7P, 11 P-trihydroxykaur-16-en-19-oic acid 19,6-Zactone (8) (200 nig) as needles (from acetone-light petroleum), m.p.248", a), -31" (c 0.18) (Found:C, 72-5; H, 8.1. C,oH2@, requires C, 72.3; H, 8.5),vmx. 3550, 3520, 1750, 1660, and 896 cni-l, z (in CDCl,)8.78 (3H, s, 18-H3), 8-58 (3H, s, 20-H,), 5.90 (2H, m, 7- and11-H), 5-2 (2H, m, 6- and 17-H), and 4.98 (lH, m, 17-H),(in C,D,N) 8-66 (3H, s, 18-H), 8-11 (3H, s, 20-H,), 5-70(lH, d, J 7 Hz, 7-H), 5.50br (lH, I.T.'$ 11 Hz, 11-H), and 4.95(3H, 111, 6-H and 17-H,). Furthcr elution with chloroformgave ent-6P,7P, 12~-tril~ydroxykaur-l6-en-19-oic acid 19,6-lactone (18) (30 mg) which crystallized from acetone-light petroleum as needles, m.p. 212-220deg;, a, -15"( c 0-2) (Found: C, 72.3; H, 8.0. C2,H2,04 requires C,72.3; H, 8-5), v, 3400br, 1755, 1660, and 900 cm-l,'c 8-86 (3H, s, 2O-H,), 8.68 (3H, s, 18-H3), 6-24 (lH, m TV,8 Hz, 12-H), 5.85 (lH, d, J 7 Hz, 7-H), 5.2 (2H, m, 6- a n i17-33), and 4-80 (lH, in, 17-H).Further elution withchloroform gave ent-GP, 7P, 13-tvi3zydroxykaur- 16-en- 1 9-oicacid 19,6-Zactone (16) (400 nig) which crystallized fromethyl acetate as plates, m.p. 220-222", a, - 131" (c 0.2 inpyridine) (Found: C, 72.4; H, 8.05. C,0H2s04 requiresC, 72.2; H, a), vmx. 3440br, 1750, 1670, and 910 cm-l,z 8-87 (3H, s, 20-H,), 8.68 (3H, s, 18-H,), 6.05 (lH, d, J7 Hz, 7-H), 5.15 (lH, ni, 6-H), 5.09br (lH, s, 17-H), and4-80br (lH, s, 17-H), (in C,D,N) 8.75 (3H, s, 2O-H,), 8-63(3H, s, 18-H3), 5.90 (lH, d, J 7 Hz, 7-H), 5.0 (lH, m, 6-H),4-90 (lH, m, 17-H), and 4-42 (IH, m, 17-H). Elutionwith cthyl acetate gave eizt-6P,7P, 16P, 17-tetrahydroxy-kauran- 19-oic acid 19,g-lactone u-liicli crystallized fromacetone-light petroleum as prisms, m.p.228-230" (lit.,6223--226"), a, -130" (c 0-2 in pyridine) (Found: C,68.3; H, 8.5. Calc. for C,,H,,O,: C, 68-5; H, 8.6?4),identical (n.ni.r. and i.r.) with tlie saniple describedpreviously.Oxidation of 7a, 1 1a-DiJLydroxyJzauvenolide (8) .-The lac-tone (100 nig) in acetone (10 nil) was treated with 8 ~ -chromium trioxide reagent l5 (0.3 nil) at room temperaturefor 1 11. Methanol was added, and the solution concen-J. W. Blunt, I. M. Clark, J. amp;I. Evans, Sir E. R. H. Jones,G. D. Mcskins, and J. T. Pinhey, J . Clicm. SOC. (C), 1071, 1136.trated, diluted with water, and tlie product recovered inethyl acetate. ent-6P-Hydroxy-7,l l-diosokaur-16-en-l9-oic acid (9) crystallized from acetone-light petroleum asneedles, m.p.260-265" (1it.,l2 247-250"), a, -Go (c0.3) (Found: C, 73.5; H, 7-45. Calc. for C20H2404: C,73-1; H, 7*4), v,,, 1790, 1720, 1715, 1660, and 890 cm-1,T 9.12 (3H, s, 20-H,), 8.69 (3H, s, 18-H3), 7.80 (lH, d, J7 Hz, 5-H), 7.00 (lH, m, 9-H), 5-15 (lH, d, J 7 Hz, 6-H),and 4.86 and 4.80 (2H, m, 17-H,).Reduction of the Diketone (9).-The diketone (40 mg)in methanol (2 nil) was treated with sodium borohydride(50 mg) for 2 h a t room temperature. Dilute hydrochloricacid was added and the product (20 mg) was recoveredin chloroform. ent-6P, 7P-Dilzydvoxy- 1 2-oxohaur- 16-en-1 Y-oic acid 19,6-lactone (10) crystallized from acetone-lightpetroleum as needles, m.p.190-192", u, -40" (c 0-2)(Found: C, 72-3; H, 8.3. C2,H2,04 requires C, 72.7;H, 7.9y0), v,,,~. 3510, 1780, 1690, 1650sh, and 880 cm-1,'c 8.71 (3H, s, 18-H3), 8-65 (3H, s, 2O-H,), 7-04br (lH, s,and 4-97 and 4.91 (2H, m, 17-H,).Renrvnngement of 7u, 13-~ihydron.yizaztrenolid~ (1 6) .--Thekaurenolide (40 mg) was hcated under reflux in aqueousetlianolic hydrochloric acid (4 nil) for 1 11, The product(30 nig) was recovered in ethyl acetate. ent-6P,7P-Di-hydvoxy- 13-inethyl- 180x0- 17-nov- 13i3-ltauran- 19-oic acid19,Wactone ( 17) crystallized Prom acetone-light petroleumas prisms, imp. 241--243", a, - 119" (c 0.1) (Found:C, 71.8; H, 8.9. CzoH2804 requires C, 72.3; H, 8.5),v,,,,. 3540, 1778, and 1745 cni-l, 7 8.98 (GH, s, 2O-H, antl13-Me), 8.64 (3H, s, 18-H:J, G-17 ( L H , q, J 7 and 10 Hz,collapses to a d, J 7 Hz, on addition of D,O, 7-H), and 5.08,(lH, q, J 4 and 7 Hz, 6-H).Oxidation of 7a, 12-dihydr0,~~llrauvelzolide ( 18) .--The kau-renolide (10 mg) in acetone (2 nil) was treated with 8 ~ -chromium trioxide reagent (0.1 ml) for 1 h.Methanolwas added and the solution was concentrated, poured intowater, and the product recovered in ethyl acetate. ent-G~-Hyd~oxy-7,12-dioxokaur-16-en-l9-oi~ acid II),G-lacto?zecrystallized from acetone-light petroleum as needles,m.p. 289-290" (Found: amp;I+, 335. C20H2404 requires 17.1,328), v,,, 1775, 1725~11, 1710, 1655, aiid 895 cm-l, T 9.22(3H, s, 2O-H,), 8.66 (3H, s, 18-H3), 670 (lH, d, J G Hz),5.10 (lH, d, J 7 Hz, 6-H), and 4.87 and 4-68 (1H each, s,Incubation of 7P-Hydr0,~yFzauvenoZicle with Rliizopusarrhizus.-The kaurenolide (2.3 g) in ethanol (40 ml) wasdistributed between 28 flasks of Rhizopus awhizus culturedas described previously.After a further 4 days themycelium was filtered and tlie broth extracted with ethylacetate. Unchanged 7P-hydroxy1;aureiiolide ( 1.4 g) wasrecovercd from the mycelium. Cliromatography of thebroth extract on silica gave, in the fractions eluted withchloroform, ent-6P,7a, 1 lP-trihydrosykaur-16-en-19-oic acid(75 mg) which crystallized Prom acetone-light petroleunias needles, n1.p. 254", a,, -34" (c 0-2 in pyridine) lit.,l?1n.p. 251-253", a, -23" (in CHCl,) (Found: C, 72.6;H, 8.0. Calc. for C20H2S04: C, 72.3; H, 8.6), 7(CD,),CO 8.82 (3H, s, 20-H3), 8-75 (3H, s, 18-H3), 5-78(lH, m, Wp 11 Hz, 11-H), 5.45 (2H, in, G- and 7-H), antl5-21 and 5-08 (2H, 17-H,). Oxidation of the kaurenolidewith 8x-chromium trioxidc reagent gave ent-6fLhydroxy-7 , l l-diosokaur-16-en-19-oic acid,12 identical (i.r. and15 R. G. Curtis, E. R. H. Jones, 1. 31. Heilbron, and G. I?.Woods, J . Cltem. SOC., 1953, 457.H), 5.90 (lH, d, J 7 Hz, 7-H), 5.13 (lH, t, J 7 Hz, G-H),1 7-H,)1974 2005t.1.c.) with the sample described previously. Furtherelution with chloroform gave eut-69,7a, 13-trihydroxy-ksur-16-en- 19-oic acid 19,0-lactone (2 1) (100 nig) whichcrystallized from acetone-light petroleum as needles,m.p. 260--261', a, - 11" (c 0.18 in pyridine) (lit.,3 m.p.261-263", 11t.,13 m.p. 259-262") (Found: C, '72-1; H,84. Calc. for C,,H,,O,: C, 72.3; H, 8-5), vmas. 3520,1740, 1660, and 900 ciW1, 7 (CD,),CO 9.18 (3H, s, 20-H,),8.75 (3H, s, 18-H3), 5.76 (lH, cl, J 7 Hz, 7-H), 5.40 (IH, t ,J 7 Hz, 6-H), 5-30 (lH, s, 17-H), ancl 4.80 (lH, s, 17-H).We thank Dr. M. G. Combe for helpful advice and for agift of the culture of RRhizofius awJzzzus, Mrs. A. Wardfor growing the fermentations, ancl the S.R.C. for financialsupport.4/444 Received, 7th Muamp;, 1974
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