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Grindelane derivatives by microbial transformation
Phyrockm~stry, Vol. 30, No. 6. pp. 1847-1848. 1991 Printed in Great Britain.
GRINDELANE
003 I 9422/91 $3.00 t 0.00 Q 1991 Pergamon Press plc
DERIVATIVES
BY MICROBIAL
TRANSFORMATION
ADETUNJI J. ALADESANMI and JOSEPH J. HOFFMANN University of Arizona, Office of Arid Lands Studies, Bioresources Research Facility, 250 E. Valencia Road, Tucson, AZ 85706, U.S.A. (Received
18 September
1990)
Key Word Index-Grindelia comporum; Asteraceae; Aspergih niger; biotransformation; hydroxygrindelic acid; 7z,8z-epoxygrindelic acid; methyl-3-z-hydroxygrindelate.
Abstract-The microbial transformation of grindelic acid by a culture of Aspergihs acid and the known grindelanes: 18hydroxygrindelic acid, 7x&x-epoxygrindelic grindelate.
INTRODUCTION
During the scale-up production of 3b-hydroxygrindelic acid (1) via biotransformation of grindelic acid (2) with Aspergillus niger, several minor components were detected. Four of these yielded significant quantities for isolation and characterization. As the absolute configuration of 2 has been confirmed as a normal labdane Cl], the structures are presented as drawn vs the previously reported ent-labdane configuration [2]. RESULTS AND DISCUSSION
Previously, we reported the facile biotransformation of grindelic, 6,l ‘I-dehydrogrindelic and 7a,8aepoxygrindelic acids into their 3/Ghydroxy derivatives by Aspergillus niger [2, 33. We now report the formation of 3ketogrindelic acid (3) during the large scale production of 1 from 2 by A. niger. Also, the known grindelanes, 7x,8aepoxygrindelic acid and methyl-3/3-hydroxygrindelate were isolated from the broth and identified by comparison with authentic samples [2, 33. Likewise, 18 hydroxygrindelic acid (4) was recovered as its methyl ester and identified by comparing the physical data of 4 and its acetate derivative (5) with previously published data [473.
/
COIR’
R’
R’
&OH,H
Me
H.H
Me
4
Me H Me Me
H,H
CHIOH
!!
Me
H,H
CH,OAc
R’
1
2 3
--
=o
Me
3-ketogrindelic acid; 18-
niger produced 3-ketogrindelic acid and methyl-3+hydroxy-
The EIMS of 3 displayed a [M]’ peak at m/z 348 for C2,HJ204. which was two mass units less than the corresponding methyl ester of 1. The remainder of the mass spectrum displayed the usual diagnostic peaks for grindel-7-enes; m/z 317 [M-OMe], 275 [M-CH,CO,Me], 289 [M-C,H,OH] 210 (base [M - 1381, through a retro-Diels-Alder fragmentation and 109 [B-C5H,02]. The IR spectrum indicated two very strong carbonyl bands at 1730 and 1704 cm- ’ corresponding to an ester and a free ketonic group, respectively. The second carbonyl at 1704 cm- ’ was further confirmed by the NaBH,-methanol reduction of the carbonyl group to a hydroxyl compound that was identical to 1. The identification of 3 as a keto oxidation product of 1 was supported fully by the NMR spectral data. The 13C NMR spectrum displayed two carbonyl signals at 216.55 (C-3) and 171.59(C-15). The ‘H NMR spectrum alsoconfirmed the carbonyl group in 3 to be in the C-3 position because of the downfield shifts of H-l, H-18 and H-19. EXPERIMENTAL
See ref. [2] for the description of the microorganism and fermentation procedure, ref. [8] for the analytical procedures and ref. [9] for the GC analysis after methylation with Met-K,CO,-Me,CO. A sample of grindelic acid (2) was afforded by previously published procedures [lo]. The A. niger biotransformation reactions were carried-out in 2.8 I fernbach flasks filled with 1 I of broth. In a typical run the concn of GA was 1 g I-‘. After 10 days the filtrates were recovered by filtration through cheesecloth followed by Whatman no. 3 paper and subsequently passing the combined filtrates and washes (typically, 10 I per run) through a granular charcoal column, which was washed with deionized H,O. Crude 3B-hydroxygrindelic acid was recovered from the charcoal with MeOH followed by CH,Cl,. Compound 1 was purified by combining the organic washes, stripping the solvents in u~cuo and subjecting the Et,0 sol fr. to gravity CC [8] followed by medium pressure LC (Buchi 632a, 40 micron SiO,, 5 x 5Ocm column) with CH,CI,-MeOH (98:2) as the solvent system. After processing 1SOg of substrate, two additional frs were obtained; one (A) with an R, greater than 1 according to TLC (CH,CI,-MeOH-HOAc 196:4: 1) and a second fr. (D) with a
1847
1848
A. J. ALADESANMI and J. J. HOFFMANN
lower R, than 1. After methylation (A) yielded 16.6g and (D) yielded 9OOmg. from which 4 was obtained. Methyl 3-keto-grindelote (3). Fr. A was submited to CC on sthca gel (37Og packed in n-hexane). Elution with n-hexane containing EtOAc (t&90%), EtOAc (lOO%) and MeOH gave 43 frs. Frs 4-7 (2.8g. n-hexane-EtOAc 17:3) was submitted to CC on silica gel. Elution with n-hexane_EtOAc (O-SO%) and EtOAc (100%)gave 41 Its. Compound 3 was isolated from CC frs 17-24 (1.8g) eluted with n-hexane-EtOAc (19~1). A major portion (1.7g) was submitted to CC on sihca gel. Elution with nhexane-EtOAc (49: I-I : I) and EtOAc (100%) gave I1 frs. Fr. 6 (0.66g, n-hexane-EtOAc 9: 1) showed two major spots on TLC and two major peaks on GC and its IR spectrum showed an additional strong carbonyl group. A portion (50 mg) was reduced (NaBH,--McOH) and the product partitioned between Et20 and H,O and washed with 0.1 M NaOH. The major lower spot of the Et,0 extract (22.5mg. n-hexane--EtOAc 7:3) was identical to an authentic sample of I. The unreacted material (upper spot) was identical to methyl 7x,8x-epoxy grindelate. The rest of fr. 6 (0.61 g) was submitted to MPLC (silica gel 6440 pm) and eluted with n-hexane--Me,CO. Fr. 3 (300 mg) was submitted to PTLC on silica gel PF-254 (n-hexane- Me,CO 9: 1. x 2). gave frs A-E. Fr. C (40.24 mg, showed one major spot on TLC), when resubmitted to PTLC (same solvent system) gave 3 (21.17 mg) which was also isolated from various frs for a total of 39.25 mg. The TLC showed one spot and its GC gave a single peak. I R \I::,’ cm _ ‘. I730 (CO,Me), 1704 (C=O), 1667 (C=C). MS mlz (rel. int.): 348 [Ml’ (2.5) Cal.C,,H,,O,, 317 [M-OMe]+ (1.5) 289[M-591’ (1.2),275 [M-C,H,O,]+ (2.5)210[M-1381’ (100). 109 [210-CsH,O,]’ (17.76). ‘HNMR (9OMHq CDCI,): 65.56 (IH, br s. H-7). 3.65 (3H, s, OMe), 2.68 (IH, d, H14eq), 2.64(1H.d, H-14,,). 2.52(2H.d, H-2). 2.41 (2H.d. H-l) 1.80 (3H,d, H-17). 1.29(3H, s. H-16). 1.07(3H.s. H-IX). 1.05 (3H.s. H19, 0.95) (3H, s. H-20). 13CNMR (22.5 MHz CDCI,): 632.4 (C-I). 40.6 (C-2). 216.6 (C-3). 38.3 (C-4). 40.3 (C-5) 24.2 (C-6).
126.1 (C-7). 135.2 (C-8). 90.5 (C-9), 47.2 (C-IO), 28.7 (C-l I), 24.9 (C-12) 81.9(C-13),47.9(C-14) 171.6(C-15),27.3(C-16),21.6(C17). 33.6 (C-18). 20.9 (C-19). 16.6 (C-20). 51.3 (CO,-Me). AcknowledgementsWe thank Mr Peter Baker for mass spectral data, Mr Steven J. Dentali for NMR spectral data and conductmg the large-scale fermentations. A.J.A. thanks the Obafemi Awolowo University, Be-lfc. Nigeria for partial sponsorship. Our appreciation is also extended to L. K. Hutter for running the GC and MPLC throughout the course of this project.
REFERENCFS I. Adinolti, M., Greta, M. D. and Mangoni. L. (1988) PItytorhemisfry 27, 1878. 2. Hoffmann, J. J., Punnapayak. H., Jolad, S. D., Bates, R. B. and Camou, F. A. (1988) J. Nat. Prod. 51. 125. 3. Hoffmann, J. J., Jolad, S. D.. Bates. R. B. and Camou. F. A. (1988) Phytochemistry 27, 2125. 4. Rose, A. F. (1980) Phytochemisrry 19, 2689. 5. Rose. A. F.. Jones, K. C., Haddon, W. F. and Dreyer. D. L. (198 1) Phyfochemistry 20, 2249. 6. Guerreiro, E.. Kavka, J.. Saad. J. R.. Orientaly. M. A. and Giordano, 0. S. (1981) Ret:. hinoom. Quim. 12, 77. 7. Bohlmann, F., Ahmed, M., Borthakur, N., Wallmeyer, M., Jakupovic, J., King, R. M. and Robinson. H. (1982) Phyrochemistry 21, 167. 8. Jolad, S. D., Hoffmann, J. J. Schram, K., Cole, J.. Tempesta, M. and Bates. R. (1981) J. Ory. Chem. 46, 4267. 9. Timmermann. B., McLaughlin. S. and Hoffmann, J. J. (1987) Eiochem.
Sysl. Ecol. 15, 401.
10. Timmermann,
Schram, 22, 523.
B.. Luzbetak, D., Hoffmann. J. J., Jolad, S. D.. K.. Klenck. R. and Bates, R. (1983) Phprochemrsrry
GRINDELANE
003 I 9422/91 $3.00 t 0.00 Q 1991 Pergamon Press plc
DERIVATIVES
BY MICROBIAL
TRANSFORMATION
ADETUNJI J. ALADESANMI and JOSEPH J. HOFFMANN University of Arizona, Office of Arid Lands Studies, Bioresources Research Facility, 250 E. Valencia Road, Tucson, AZ 85706, U.S.A. (Received
18 September
1990)
Key Word Index-Grindelia comporum; Asteraceae; Aspergih niger; biotransformation; hydroxygrindelic acid; 7z,8z-epoxygrindelic acid; methyl-3-z-hydroxygrindelate.
Abstract-The microbial transformation of grindelic acid by a culture of Aspergihs acid and the known grindelanes: 18hydroxygrindelic acid, 7x&x-epoxygrindelic grindelate.
INTRODUCTION
During the scale-up production of 3b-hydroxygrindelic acid (1) via biotransformation of grindelic acid (2) with Aspergillus niger, several minor components were detected. Four of these yielded significant quantities for isolation and characterization. As the absolute configuration of 2 has been confirmed as a normal labdane Cl], the structures are presented as drawn vs the previously reported ent-labdane configuration [2]. RESULTS AND DISCUSSION
Previously, we reported the facile biotransformation of grindelic, 6,l ‘I-dehydrogrindelic and 7a,8aepoxygrindelic acids into their 3/Ghydroxy derivatives by Aspergillus niger [2, 33. We now report the formation of 3ketogrindelic acid (3) during the large scale production of 1 from 2 by A. niger. Also, the known grindelanes, 7x,8aepoxygrindelic acid and methyl-3/3-hydroxygrindelate were isolated from the broth and identified by comparison with authentic samples [2, 33. Likewise, 18 hydroxygrindelic acid (4) was recovered as its methyl ester and identified by comparing the physical data of 4 and its acetate derivative (5) with previously published data [473.
/
COIR’
R’
R’
&OH,H
Me
H.H
Me
4
Me H Me Me
H,H
CHIOH
!!
Me
H,H
CH,OAc
R’
1
2 3
--
=o
Me
3-ketogrindelic acid; 18-
niger produced 3-ketogrindelic acid and methyl-3+hydroxy-
The EIMS of 3 displayed a [M]’ peak at m/z 348 for C2,HJ204. which was two mass units less than the corresponding methyl ester of 1. The remainder of the mass spectrum displayed the usual diagnostic peaks for grindel-7-enes; m/z 317 [M-OMe], 275 [M-CH,CO,Me], 289 [M-C,H,OH] 210 (base [M - 1381, through a retro-Diels-Alder fragmentation and 109 [B-C5H,02]. The IR spectrum indicated two very strong carbonyl bands at 1730 and 1704 cm- ’ corresponding to an ester and a free ketonic group, respectively. The second carbonyl at 1704 cm- ’ was further confirmed by the NaBH,-methanol reduction of the carbonyl group to a hydroxyl compound that was identical to 1. The identification of 3 as a keto oxidation product of 1 was supported fully by the NMR spectral data. The 13C NMR spectrum displayed two carbonyl signals at 216.55 (C-3) and 171.59(C-15). The ‘H NMR spectrum alsoconfirmed the carbonyl group in 3 to be in the C-3 position because of the downfield shifts of H-l, H-18 and H-19. EXPERIMENTAL
See ref. [2] for the description of the microorganism and fermentation procedure, ref. [8] for the analytical procedures and ref. [9] for the GC analysis after methylation with Met-K,CO,-Me,CO. A sample of grindelic acid (2) was afforded by previously published procedures [lo]. The A. niger biotransformation reactions were carried-out in 2.8 I fernbach flasks filled with 1 I of broth. In a typical run the concn of GA was 1 g I-‘. After 10 days the filtrates were recovered by filtration through cheesecloth followed by Whatman no. 3 paper and subsequently passing the combined filtrates and washes (typically, 10 I per run) through a granular charcoal column, which was washed with deionized H,O. Crude 3B-hydroxygrindelic acid was recovered from the charcoal with MeOH followed by CH,Cl,. Compound 1 was purified by combining the organic washes, stripping the solvents in u~cuo and subjecting the Et,0 sol fr. to gravity CC [8] followed by medium pressure LC (Buchi 632a, 40 micron SiO,, 5 x 5Ocm column) with CH,CI,-MeOH (98:2) as the solvent system. After processing 1SOg of substrate, two additional frs were obtained; one (A) with an R, greater than 1 according to TLC (CH,CI,-MeOH-HOAc 196:4: 1) and a second fr. (D) with a
1847
1848
A. J. ALADESANMI and J. J. HOFFMANN
lower R, than 1. After methylation (A) yielded 16.6g and (D) yielded 9OOmg. from which 4 was obtained. Methyl 3-keto-grindelote (3). Fr. A was submited to CC on sthca gel (37Og packed in n-hexane). Elution with n-hexane containing EtOAc (t&90%), EtOAc (lOO%) and MeOH gave 43 frs. Frs 4-7 (2.8g. n-hexane-EtOAc 17:3) was submitted to CC on silica gel. Elution with n-hexane_EtOAc (O-SO%) and EtOAc (100%)gave 41 Its. Compound 3 was isolated from CC frs 17-24 (1.8g) eluted with n-hexane-EtOAc (19~1). A major portion (1.7g) was submitted to CC on sihca gel. Elution with nhexane-EtOAc (49: I-I : I) and EtOAc (100%) gave I1 frs. Fr. 6 (0.66g, n-hexane-EtOAc 9: 1) showed two major spots on TLC and two major peaks on GC and its IR spectrum showed an additional strong carbonyl group. A portion (50 mg) was reduced (NaBH,--McOH) and the product partitioned between Et20 and H,O and washed with 0.1 M NaOH. The major lower spot of the Et,0 extract (22.5mg. n-hexane--EtOAc 7:3) was identical to an authentic sample of I. The unreacted material (upper spot) was identical to methyl 7x,8x-epoxy grindelate. The rest of fr. 6 (0.61 g) was submitted to MPLC (silica gel 6440 pm) and eluted with n-hexane--Me,CO. Fr. 3 (300 mg) was submitted to PTLC on silica gel PF-254 (n-hexane- Me,CO 9: 1. x 2). gave frs A-E. Fr. C (40.24 mg, showed one major spot on TLC), when resubmitted to PTLC (same solvent system) gave 3 (21.17 mg) which was also isolated from various frs for a total of 39.25 mg. The TLC showed one spot and its GC gave a single peak. I R \I::,’ cm _ ‘. I730 (CO,Me), 1704 (C=O), 1667 (C=C). MS mlz (rel. int.): 348 [Ml’ (2.5) Cal.C,,H,,O,, 317 [M-OMe]+ (1.5) 289[M-591’ (1.2),275 [M-C,H,O,]+ (2.5)210[M-1381’ (100). 109 [210-CsH,O,]’ (17.76). ‘HNMR (9OMHq CDCI,): 65.56 (IH, br s. H-7). 3.65 (3H, s, OMe), 2.68 (IH, d, H14eq), 2.64(1H.d, H-14,,). 2.52(2H.d, H-2). 2.41 (2H.d. H-l) 1.80 (3H,d, H-17). 1.29(3H, s. H-16). 1.07(3H.s. H-IX). 1.05 (3H.s. H19, 0.95) (3H, s. H-20). 13CNMR (22.5 MHz CDCI,): 632.4 (C-I). 40.6 (C-2). 216.6 (C-3). 38.3 (C-4). 40.3 (C-5) 24.2 (C-6).
126.1 (C-7). 135.2 (C-8). 90.5 (C-9), 47.2 (C-IO), 28.7 (C-l I), 24.9 (C-12) 81.9(C-13),47.9(C-14) 171.6(C-15),27.3(C-16),21.6(C17). 33.6 (C-18). 20.9 (C-19). 16.6 (C-20). 51.3 (CO,-Me). AcknowledgementsWe thank Mr Peter Baker for mass spectral data, Mr Steven J. Dentali for NMR spectral data and conductmg the large-scale fermentations. A.J.A. thanks the Obafemi Awolowo University, Be-lfc. Nigeria for partial sponsorship. Our appreciation is also extended to L. K. Hutter for running the GC and MPLC throughout the course of this project.
REFERENCFS I. Adinolti, M., Greta, M. D. and Mangoni. L. (1988) PItytorhemisfry 27, 1878. 2. Hoffmann, J. J., Punnapayak. H., Jolad, S. D., Bates, R. B. and Camou, F. A. (1988) J. Nat. Prod. 51. 125. 3. Hoffmann, J. J., Jolad, S. D.. Bates. R. B. and Camou. F. A. (1988) Phytochemistry 27, 2125. 4. Rose, A. F. (1980) Phytochemisrry 19, 2689. 5. Rose. A. F.. Jones, K. C., Haddon, W. F. and Dreyer. D. L. (198 1) Phyfochemistry 20, 2249. 6. Guerreiro, E.. Kavka, J.. Saad. J. R.. Orientaly. M. A. and Giordano, 0. S. (1981) Ret:. hinoom. Quim. 12, 77. 7. Bohlmann, F., Ahmed, M., Borthakur, N., Wallmeyer, M., Jakupovic, J., King, R. M. and Robinson. H. (1982) Phyrochemistry 21, 167. 8. Jolad, S. D., Hoffmann, J. J. Schram, K., Cole, J.. Tempesta, M. and Bates. R. (1981) J. Ory. Chem. 46, 4267. 9. Timmermann. B., McLaughlin. S. and Hoffmann, J. J. (1987) Eiochem.
Sysl. Ecol. 15, 401.
10. Timmermann,
Schram, 22, 523.
B.. Luzbetak, D., Hoffmann. J. J., Jolad, S. D.. K.. Klenck. R. and Bates, R. (1983) Phprochemrsrry