1098
J. Nat. Prod. 2001, 64, 1098-1099
Microbial Transformation of Pyrethrosin Ahmed M. Galal* National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, Mississippi 38677 Received January 12, 2001
Microbial transformation of the germacranolide pyrethrosin (1) using Rhizopus nigricans NRRL 1477 has resulted in the isolation of 6R-acetoxy-1β,4R-dihydroxy-5,7RH,8βH-eudesm-11β,13-dihydro-8,12-olide (5), a new eudesmanolide-type metabolite, in addition to the previously reported eudesmanolides: 2, 3, 4, and 6. The structure elucidation of these metabolites was based primarily on 1D and 2D NMR analyses. The isolated metabolites exhibited cytotoxic, antifungal, and antiprotozoal activities. Pyrethrosin (1), the first recognized germacrane sesquiterpene lactone,1,2 was isolated from the flowering heads of Chrysanthemum cinerariaefolium Visiani3 (Asteraceae). Pyrethrosin exhibits diverse biological activities including cytotoxicity,4 antibacterial,5 antifungal,6 molluscicidal,7 and phytotoxicity.8 Microbial metabolism studies have been used as inexpensive in vitro model systems to predict mammalian metabolism or to increase the efficacy of a drug through metabolic activation.9 Pyrethrosin (1), with its diverse bioactivity range, was subjected to microbial metabolism in an attempt to find more active analogues. Twenty-four growing cultures were screened for their ability to biotransform 1. Of these, Cunninghamella elegans NRRL 1392 and Rhizopus nigricans NRRL 1477 were able to completely deplete and transform 1 into the same five more polar metabolites. R. nigricans (NRRL 1477) was chosen for preparative-scale fermentation due to its higher metabolite yield as compared to C. elegans NRRL. Repeated column chromatography of the crude metabolite mixture furnished five compounds, 2, 3, 4, 5, and 6. Compound 2 was
* To whom correspondence should be addressed. Tel: (662) 915-1019. Fax: (662) 915-7989. E-mail:
[email protected].
10.1021/np0100082 CCC: $20.00
Table 1. 13C NMR Chemical Shift Assignments of Compounds 2, 3, 5, and 7a position
2
3
5
7b
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
78.6, d 31.8, t 34.7, t 143.1, s 54.0, d 69.9, d 56.6, d 76.5, d 40.4, t 43.0, s 40.9, d 178.9, s 14.2, q 14.2, q 109.0, t
76.1, d 33.5, t 123.3, d 133.8, s 54.3, d 71.6, d 59.6, d 76.9, d 39.4, t 41.1, s 41.9, d 179.6, s 15.1, q 13.3, q 24.0, q
78.3, d 27.9, t 40.2, t 72.3, s 57.2, d 72.6, d 57.9, d 76.4, d 43.2, t 41.8, s 41.1, d 178.6, s 14.4, q 16.2, q 24.7, q
79.1, d 24.2, t 40.2, t 71.5, s 56.9, d 71.8, d 57.3, d 75.3, d 42.4, t 40.5, s 40.3, d 177.6, s 13.9, q 16.8, q 24.2, q 21.0, q 169.6, s 21.3, q 170.2, s
21.3, q 171.3, s
21.8, q 170.1, s
a In CDCl at 125 MHz. Carbon multiplicities were determined 3 by DEPT 135° experiments. b At 75 MHz.
previously isolated from the flowers of Chrysanthemum cinerariaefolium Visiani (Pyrethrum flower), while 6 was isolated from Cassinia loevis R.Br. Compounds 3, 4, and 6 were previously synthesized from pyrethrosin (1) using BF3‚OEt2 and p-toluenesulfonic acid to convert the germacrane ring into the eudesmane skeleton.10 The positive ion HRESIFTMS spectrum of 5 displayed a molecular ion peak [M + H]+ at m/z 327.1822, suggesting the molecular formula C17H24O6. The IR spectrum of 5 showed a broad absorption band at 3427 cm-1, suggesting the presence of a hydroxyl group. It also showed a strong absorption band at 1767 cm-1, consistent with the existence of a γ-lactone carbonyl functionality. The 13C and 1H NMR data of 5 (Table 1 and experimental) were in agreement with a eudesmanolide skeleton.12 Inspection of the NMR spectra of 5 revealed close similarity to those of the previously reported compound 6R-acetoxy-1β,4R-dihydroxy5,7RH,8βH-eudes-11-en-8,12-olide (6),12 except for the presence of a methyl group at C-11, resonating at δ 14.4 (d, Me-13) in 5, instead of an exocyclic methylene functionality in 6. The assignment of the relative stereochemistry of the stereocenter C-11 was deduced from NOESY and ROESY data. The β-oriented Me-15 displayed NOESY correlation with the downfield H-11, suggesting a similar stereoorientation. Moreover, the R-oriented acetate methyl at C-6 showed ROESY correlation with Me-13, suggesting R-orientation for Me-13 and supporting the relative stereochemistry assignment of C-13. Therefore, metabolite 5 was
© 2001 American Chemical Society and American Society of Pharmacognosy Published on Web 07/21/2001
Notes
established as 6R-acetoxy-1β,4R-dihydroxy-5,7RH,8βHeudesm-11β,13-dihydro-8,12-olide. Metabolites 2, 3, 4, and 6 were identified by comparing their spectral data with literature.3,10 All isolated new and known compounds were tested for cytotoxicity, antimicrobial, and antimalarial activities using standard methods described elsewhere.11-13 Metabolite 5 and its 1-O-acetate derivative (7) exhibited antiprotozoal activity against Plasmodium falciparum (D6 clone) with IC50 0.88 and 0.32 µg/mL, respectively, without significant toxicity. Compound 7 also displayed a pronounced activity against the chloroquine-resistant strain of P. falciparum (W2 clone) with IC50 0.38 µg/mL. Compound 2 showed considerable in vitro cytotoxic activity against human epidermoid carcinoma (KB) and against human ovary carcinoma (SK-OV-3) with IC50
