Metabolites ofAspergillus ustus. Part 3. Structure elucidation of austalides G–L

摘要

J. CHEM. SOC. PERKIN TRANS. I 1985 363 Metabolites of Aspergi//us ustus. Part 3. Structure Elucidation of Austalides G-L ' R. Marthinus Horak," Pieter S. Steyn, and Robert Vleggaar National Chemical Research Laboratory, Council for Scientific and Industrial Research, P.0.Box 395, Pretoria 0001, Republic of South Africa Christiaan J. Rabie National Research Institute for Nutritional Diseases, Medical Research Council, P. 0.Box 70, Tygerberg7505, Republic of South Africa The structure elucidation of austalides G-L, chemical derivatization, is described. In an accompanying paper the isolation of 12 meroterpenoid metabolites, austalides A-L, from cultures of Aspergillus ustus (Bainier) Thom. and Church (strain MRC 1163) and the structure elucidation of six of these metabolites, austalides A- F (1(6) are described. We now report the structure elucidation of the austalides G-L (7(12) based on a detailed study of their high-field 'H and n.m.r.spectra, chemical derivatizations, and comparison with the austalides A-F. Austalides G (7),t C2,H3,09, and H (8),C2,H,,0,, are the minor metabolites in the austalide series and probably represent the products of a branch-point in the biosynthetic pathway leading to the highly oxygenated austalides A-F ~(1F-W~I. OCH3 28 33 34 (1) A: R'=COCH3, R2 =H (2) 8: R'=H, ~2 =H 31 32 ( 31 C : R' =COCH3, R2 = OCOCH-J (4)0: R' =H, R2 = OCOCH3 ( 5) E : R' =COCH3, R2 = OH (6) F: R' =H, R2 = OH (7) G: R=COCH3 (8) H: R=H The U.V.maxima (Amax. 221 and 267 nm) of austalides G and H compare well with the corresponding data of austalides A-F. The i.r. absorption band of the C-17 carbonyl groups in the metabolites is masked by that of the phthalide carbonyl group (Amax. 1 740 cm-I). The electron impact mass spectra of these two methyl esters exhibited intense M+ -18 peaks which arise through the facile loss of the elements of water from the based on a study of their H and 3Cn.m.r. spectra and molecular ion. This loss is consistent with the presence of the tertiary hydroxy group at C- 15. The location of the 0-acetyl group at C-13 in austalide G (7) followed from the chemical shift (6,5.391) of the 13-H proton. This proton is part of a four-proton spin system, the first-order analysis of which was confirmed by selective irradiation of the C-13 and C-14 protons in a series of proton-proton decoupling experiments (see Figure 1).The broad singlet at 6, 1.593 which disappeared on addition of deuterium oxide to the sample, is assigned to the proton of the C-15 hydroxy group. The 13Cn.m.r. data of austalide G are in agreement with the proposed structure. The low-field chemical shift of C-14 S, 51.67, 'J(CH) 126.2 Hz is probably due to the inductive effect of the two oxygen atoms which are two bonds removed. The 0' (9) I : R1=OCOCH3, R2=H (10) J : R' = H, R~ = OH (11) K : R1 = R2 =H (12) L : R' = OH, R~=H (18) R' = H, R~=D (19) R' = OD, R2 = D presence of an aliphatic methyl ester is evident from the singlet at 6, 174.47 (C-17) and the quartet at 6, 51.67 'J(CH) 146.6 Hz, C-281 in the single frequency n.0.e.13C~pectrum.~*~ -f The numbering of austalides G, H, K, and L is in accord with the system used for austalides A-F, and 16 is, therefore, omitted. 364 J. CHEM. SOC. PERKIN TRANS. I 1985 Figure 1. The ('H,'H) connectivity pattern of austalide G (7). Values in Hz Alkaline hydrolysis of austalide G (0.1M-potassium hydroxide in methanol) yielded a single product which was identical with austalide H (8). Significantly, the 'H n.m.r. signal of the C-13 methine proton (6,4.629) of austalide H appears as an unresolved multiplet, 0.762 p.p.m. upfield in comparison to the corresponding resonance in the 'H spectrum of austalide G.This signal changed to a well-resolved multiplet (J3.7, 3.1, and 2.5 Hz) upon addition of deuterium oxide to the sample. The location of the secondary hydroxy group at C-13 was demonstrated, using the deuteriated sample, by selective irradiation of the 14-H resonance (6, 1.322, J 2.5 Hz) in a homonuclear decoupling experiment, which changed the 13-H signal to a double doublet (J3.7 and 3.1 Hz). Austalide I (9),m.p. 236-238 "C analysed for C27H3408and had M+ 486. The presence of the phthalide chromophore was evident from the absorption maxima at h,,,, 221 and 266 nm in the U.V. spectrum. The i.r. spectrum of the metabolite had v,,,, 1720-1 750 cm-', assigned to the C-3, C-33, and C-17 carbonyl groups.The presence of the 0-acetyl moiety at C-13 in austalide I was inferred from the chemical shift of the 13-H proton resonance (6, 5.407, m). This resonance changed to a double doublet (J 4.2, 1.9 Hz) on selective irradiation of the doublet signal (6, 1.862, J 3.9 Hz) assigned to 14-H. The single frequency n.0.e. 3Cn.m.r. spectrum of austalide I showed the presence of three doublets at 6,45.94 'J(CH) 125.4 Hz, 55.35 'J(CH) 120.6 Hz, and 69.92 'J(CH) 151.0 Hz. The chemical shift and coupling constant of the last resonance are diagnostic of the oxygen-bearing carbon atom, C-13. The assignment of the doublet signals at 6,45.94 (C-21) and 55.35 (C-14) is based on chemical-shift considerations. The chemical shift of the resonance at 6, 174.40,ascribed to C- 17, compares well with the chemical shift of the carbonyl carbon atom in seven-membered lac tone^.^ The facile ring-opening of seven- membered lactones on treatment with acid is well documented and can be taken as proof of the presence of such a f~nctionality.~,~For this reason, a solution of austalide 1(9)in a mixture of dichloromethane and methanol was treated with anhydrous hydrogen chloride generated in situ by the addition of thionyl chloride to the reaction mixture.8 After 2 h at room temperature the ester (13), M+ 500, was obtained in 73 yield.Significant features of the 'H n.m.r. spectrum of (13) in comparison with that of austalide I are the unresolved multiplets at 6,4.893 and 4.939 ascribed to the protons of the exocyclic methylene group.A three-proton singlet at 8, 3.683 is indicative of the protons of a methyl ester. It is evident that under these experimental conditions opening of the lactone ring in austalide I leads firstly to the formation of austalide G (7). The facile loss of water from the hydroxy-isopropyl group in (7) then generates the exocyclic methylene moiety. Austalide J (lo), C25H3207, has the characteristic U.V. and i.r. data of austalides A-I, and M+ 444.Although the molecular ions of austalide I (9) and J (10) differ by 42 mass units, austalide J is not simply the deacetyl derivative of austalide I. This was apparent from a comparison of the respective 'H and 13Cn.m.r. data. The resonances at 6, 1.862 and 5.407, assigned respectively to the C-14 and C-13 protons in austalide I were both absent in the 'H spectrum of austalide J.The 13C n.m.r. spectrum of austalide J showed three resonances (6, 75.85, 79.58,and 91.33) due to oxygen-bearing, quaternary, sp3 carbon atoms but none of which could be assigned to an oxygen- bearing methine carbon atom. Austalide J is, therefore, oxygenated at C-14, and as a result it can contain either a six- or a seven-membered lactone ring. This ambiguity was resolved by the chemical shift (6, 173.38) of the lactone carbonyl carbon atom. The corresponding carbon atom in six-membered lactone rings resonates at ca. 6, 167.4 In the preceding paper' it is shown that treatment of the lactone (14) with a methanolic solution of potassium hydroxide followed by acidic work-up leads to the formation of the hemi- ortho ester (15) in high yield (see Figure 2).Austalide J could (14) ii1i OH (15) Figure 2. Transformation of the lactone (14) into the hemi-ortho ester (15). (i) KOH-MeOH, (ii) 0.1~HC1 conceivably exist in solution in equilibrium with its hemi- ortho ester form (17) which could be trapped by methylation. Although the broad-band proton-decoupled '3C n.m.r. spectrum of austalide J in both chloroform and methanol is consistent in each case only with the lactone form (lo), the ortho ester (16) was obtained in 90 yield when austalide J was treated with hydrogen chloride in anhydrous methanol. The same product was also formed by methylation of austalide J J.CHEM. SOC. PERKIN TRANS. I 1985 CHq cH3 OCH3 28 (16) with methyl iodide and potassium carbonate in dry acetone. The efficient conversion of austalide J into its ortho ester form proceeds in each case to completion as methylation of (17) shifts the equilibrium between (10) and (17). CH? ?H3 0Y (10) OH (17) Figure 3. Proposed equilibrium between austalide J (10) and its hemi- ortho ester (17) The 'H n.m.r. spectrum of (16) displayed a three-proton singlet at 6, 3.411, characteristic of the ortho ester methyl protons (28-H) in the austalides A-F (1)-(6). The corresponding methyl carbon atom gives rise to a signal at 6, 48.74 in the '3Cn.m.r. spectrum. The facile transformation of austalide J into the ortho ester (16) indicates that the compound has the same relative configuration as austalide D (14s) and provides chemical proof of the structure. The change in descriptor for this chral centre compared with the corresponding chiral centre in austalide D is merely the result of the sequence rules of the Cahn-Ingold- Prelog system.Austalide K (ll), C2!H?205, and austalide L (12), C2,H3,06, have U.V. data similar to those of austalides A-J. The i.r. spectra of each of these two metabolites showed an absorption band at v,,,. 1700 cm-l, characteristic of a carbonyl group (C- 17) in a six-membered ring.g A number of signals in the 'H n.m.r. spectrum of austalide K (11) exhibited extensive fine structure as a result of multiple geminal and vicinal proton-proton coupling (Figure 4).The resonances of the C-12, C-13, and C-14 protons appear as deceptively simple multiplets in the 'H n.m.r. spectrum since one vicinal and both geminal proton-proton coupling constants have the same magnitude. Extensive 'H-('H) homonuclear decoupling experiments were, therefore, necessary to analyse the various multiplets and assign them to specific protons. The resultant proton-proton connectivity pattern is illustrated in 365 1 0 0 OCH3 'I (6.9)' Figure 4. The ('H,'H) connectivity pattern of austalide K (11). Values in Hz Figure 4. The 13-Ha proton is assigned to the eight-line multiplet at 6,1.785 (J 13.9, 13.9, 13.9, and 3.0 Hz) since it is the only proton with an antiperiplanar orientation with two different neighbouring protons (12-H, and 14-H).The magnitude of the geminal coupling constant of the C-18 protons can be explained by the presence of a carbonyl group at C-17 (see Figure 4). A singlet resonance at 6,216.23 in the '3Cn.m.r. spectrum of austalide K was assigned to the carbonyl carbon atom, C-17. The corresponding carbon aton in 4,4-dimethyl-3-ketosteroids resonates at ca. Sc 216." The chemical-shift values of the C-14 S, 54.07 D, 'J(CH) 120.9 Hz and C-15 (6,47.09 S) resonances confirmed the lack of oxygenation at these centres. The structural assignment of austalide K (11) is corroborated by the results obtained from an n.m.r. study of its deuteriated derivative (18), obtained by refluxing the compound in a solution of sodium methoxide in O-2Hmethanol.'2 As a result of the base-catalysed ring-opening of the phthalide moiety in the compound, the reaction mixture was acidified during work- up with deuteriated acid obtained from a 1:l mixture of deuterium oxide and acetyl chloride.A comparison of the 'H n.m.r. spectrum of (18) with that of austalide K shows that the signals assigned to the C-18 protons are absent. In addition, the C-19 protons give rise to an AX spin system (J 13.3 Hz). The broad-band proton-decoupled 3C n.m.r. spectrum of the deuteriated derivative (18) showed 24 singlet signals plus an ill-resolved multiplet (6, 33.50) of very low relative intensity. This multiplet, assigned to C-18, is centred upfield (A6 -0.41 p.p.m.) from the resonance position of C-18 in the spectrum of austalide K (ll).13 The relatively low intensity of the C-18 signal in the spectrum of (18) is due to the loss of n.0.e.and the fact that it is split into five lines as a result of spin coupling with the two deuterium atorns.l4 Furthermore, the C-19 signal exhibits an upfield shift (AS -0.08 p.p.m.) whereas the C-17 resonance is shifted downfield (A6 0.13 p.p.m.).'2*13 These isotope shifts support the structural assignment of austalide K( 11). The structural assignment of austalide L(12) is based on the same approach as described for austalide K. First-order analysis of the multiplets in the 'H n.m.r. spectrum of austalide L yielded the chemical shifts and coupling constants.The values of the coupling constants, as corroborated by 'H-{'H} decoupling experiments, indicated the geminal and vicinal proton-proton coupling patterns in the molecule, as outlined in Figure 5. The deuteriated derivative (19) of austalide L was obtained as described above for austalide K. The presence of three deuterium atoms in the molecule is evident from the mass Figure 5. The ('H,'H) connectivity pattern of austalide L (12). Values in Hz spectral data. The H n.m.r. spectrum lacks the signals assigned to the C-14 hydroxy group and the C-18 methylene protons, and the signal assigned to the C-19 protons of (19) appears as a pair of doublets (J 12.9 Hz). A comparison of the 13Cn.m.r. data of the trideuterioketone (19) and of austalide L (12) indicated an upfield shift (A6 -0.10 p.p.m.) for the C-19 resonance whereas the (2-14 signal is shifted downfield (A6 0.11 p.p.m.).Deuteriation at C-18 in (19) causes an upfield shift (A6 -0.15 p.p.m.) instead of a downfield shift ' see austalide K (ll) in the resonance position of the C-17 carbonyl carbon atom due to the presence of the C-14 hydroxy group. The austalides, e.g. austalide D (4), are biosynthetically derived from 6-farnesyl-5,7-dihydroxy-4-methylphthalide,a known intermediate in the biosynthesis of mycophenolic acid,' by stereospecific ring closure to give austalide K (ll).3 Hydroxylation at C-14 of austalide K (11) proceeds with retention of configuration to give austalide L (12). An enzymatic Baeyer-Villiger oxidation (retention of configura- tion) produces the seven-membered hydroxy-lactone ring of austalide J (10) with the correct stereochemical orientation of the substituents for in vivo cyclisation to an ortho ester.This cyclisation was demonstrated in vitro for austalide J (10) (see above). The detailed biosynthetic studies on the austalides will be described in a subsequent publication. ExperimentalFor general directions see reference 1. Alkaline Hydrolysis of Austalide G (7).-Austalide G (10 mg) was hydrolysed according to the procedure described in reference 1 to give the diol (8) (8 mg, 87), identical with an authentic sample of austalide H (8). Acid-catal