© Copyright 2003 by the American Chemical Society and the American Society of Pharmacognosy
Volume 66, Number 8
August 2003
Full Papers CT2108A and B: New Fatty Acid Synthase Inhibitors as Antifungal Agents Jodi A. Laakso,†,⊥ Robert Raulli,‡,⊥ Gail E. McElhaney-Feser,§ Paul Actor,‡,⊥ Ted L. Underiner,†,⊥ Brian J. Hotovec,†,⊥ Ursula Mocek,†,⊥ Ronald L. Cihlar,§ and Sheldon E. Broedel, Jr.*,‡,⊥ Dorlin Pharmaceuticals, Inc., 1450 South Rolling Road, Baltimore, Maryland 21227-2349, Panlabs, Incorporated, 11804 North Creek Parkway South, Bothell, Washington 90811-8805, and Department of Microbiology and Immunology, Georgetown University, Washington, D.C. 20007 Received January 31, 2003
A systematic screen for new natural products that displayed antifungal activity by inhibition of fungal fatty acid synthase (FAS) led to the discovery of two new fungal metabolites, designated CT2108A (1) and CT2108B (2). The metabolites were produced by Penicillium solitum (Westling) strain CT2108 and were classified as azaphilones. The structures of these new metabolites were determined using a variety of 1D and 2D NMR experiments, including COSY, HMQC, and HMBC. The chemical conversion of CT2108A to CT2108B was effected using WCl6. The related metabolite, patulodin (3), was also isolated from the fermentation culture of this P. solitum isolate. Both new compounds inhibited fungal FAS, and neither was found to significantly inhibit human FAS activity. The occurrence of fungal infections has escalated significantly in recent years. Increases in the number of patients presenting with such infections has been particularly profound in those immunocompromised by disease or therapies. Currently, only a limited number of antifungal drugs are available for treatment of fungal infections. In addition, use of the most effective antifungal agents, azole derivatives and amphotericin B, suffers from drawbacks including toxicity, fungistatic versus fungicidal activity, and the development of drug resistance. The paucity of effective agents is also due in part to the lack of suitable novel targets for drug screening. * To whom correspondence should be addressed. Tel: 410-455-6319. Fax: 410-455-1155. E-mail:
[email protected]. † Panlabs, Inc. (now part of MDS Pharma Services). ‡ Dorlin Pharmaceuticals, Inc. § Georgetown University. ⊥ Present addresses: Albany Molecular Research, Inc., Bothell Research Center, 18804 North Creek Parkway, Bothell, WA 98011-8805 (J.A.L., U.M.), Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285 (B.J.H.), Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380 (T.L.U.), Athena Environmental Sciences, Inc., 1450 South Rolling Rd., Baltimore, MD 21227 (S.E.B.), Amulet Pharmaceuticals, 1450 South Rolling Rd., Baltimore, MD 21227 (R.R.).
10.1021/np030046g CCC: $25.00
Recent evidence suggests that the enzyme fatty acid synthase (FAS) is a good potential antifungal drug target.1,2 FAS catalyzes the assembly of fatty acids from acetyl, malonyl, and NADPH substrate via seven enzymatic reactions. In bacteria and plants, seven genes encode the seven polypeptides that form an FAS complex.3 In fungi, FAS is a multifunctional enzyme with the seven component activities distributed over two nonidentical polypeptides, R and β, arranged in an R6β6 configuration.4 Conversely, in vertebrates all seven enzymatic reactions are on a single polypeptide in a homodimer configuration and encoded by a single gene.5,6 Despite the diversity of arrangements, all FAS complexes perform the same set of reactions, suggesting that the spatial structure of the reactive sites should be similar. Indeed, comparisons of the deduced polypeptide sequences of FAS from prokaryotic, lower eukaryotic, and higher eukaryotic sources show high degrees of relatedness at catalytic domains and substrate binding sites;7-10 however, major structural differences, including spacing of reactive domains as well as their linear order, are apparent. Because of such structural differences, it is possible that natural products or chemically designed molecules might be identified that differentially affect human and fungal
© 2003 American Chemical Society and American Society of Pharmacognosy Published on Web 07/29/2003
1042 Journal of Natural Products, 2003, Vol. 66, No. 8
FAS and that also have a broad spectrum of activity against fungi.11 Previous work with analogues of the FAS inhibitor cerulenin had shown that compounds that inhibit fungal FAS and not human FAS could be identified.11 None of these compounds proved effective as antifungal agents due to irreversible nonspecific protein binding. However, it remained likely that other agents that exploited differences between the respective human and fungal FAS could be discovered from a natural products library. Thus, a mechanism-based screen was employed to identify natural product extracts that (1) inhibited fungal FAS enzyme activity and not human FAS enzyme, (2) showed reversible inhibition of Candida albicans growth in liquid cultures, and (3) exhibited in vitro fungicidal activity. In the course of applying this mechanism-based screen using fungal and actinomycete natural product extracts, two new structurally related azaphilones, designated CT2108A (1) and CT2108B (2), were identified. The producing organism was a strain of Penicillium solitum (CK2108) that was obtained from a duff sample collected in Wyoming. Patulodin (3),12 a related metabolite with antifungal activity, was also isolated from this strain. In this report, the fermentation, isolation, and biological activity of the new compounds are described. The physicochemical properties and details of the structure elucidation and semisynthesis will also be presented. Results and Discussion A total of 10 880 extracts, obtained from 1360 actinomycetes and 1360 fungi, were screened. Eighty-two extracts showed reproducible FAS inhibition and C. albicans growth inhibition. Of these, 30 had no activity against human FAS enzyme. This was a crucial selection criterium, since achieving a good therapeutic index requires that the inhibitor be fungal specific. The organisms yielding the 30 candidate extracts were fermented a second time to identify those cultures that reproducibly yielded active material. Following the re-fermentation, extracts were prepared and again tested for bioactivity; eight strains (six fungi and two actinomycetes) consistently produced an active agent. One of these eight strains was identified as Penicillium solitum Westling (CT2108) and yielded two new compounds, designated CT2108A (1) and CT2108B (2).
The 3 L fermentation of CT2108 yielded 12.4 g of crude ethyl acetate extract. Preparation of an aliquot for coun-
Laakso et al.
tercurrent partition chromatography (CPC) yielded a large amount of insoluble material. CPC fractionation of the soluble material revealed two sets of fractions with biological activity: CPC fractions 17-22 and 26-30. Pooled fraction 17-22 showed good activity against FAS; pooled fraction 26-30 yielded poor FAS activity, yet was equally effective in the susceptibility assay on a per mass basis. UV and 1H NMR analysis suggested that CPC 17-22 and the insoluble material contained the same compound. The two pooled CPC fractions were further analyzed by HPLC, and the fractions were analyzed for biological activity. Fractionation of CPC 17-22 yielded a single HPLC peak (compound 1) that exhibited FAS inhibitory and growth inhibitory activity. HPLC fractionation of CPC 26-30 yielded a single fraction (compound 2) that retained growth inhibitory activity, lacked FAS inhibitory activity, but retained reversible growth inhibition. Examination of the HRFABMS data indicated that the molecular formulas for 1 and 2 differed by only one oxygen atom; the molecular formula of 1 was determined to be C21H22O7, and the molecular formula of 2 was found to contain one less oxygen atom. The UV spectra for these compounds were also similar, strengthening the premise that these metabolites were closely related. Because 2 was the most interesting biologically, and because the NMR data obtained for 2 were better than the data obtained for 1, the structure of 2 was pursued first. Identification of two distinct spin systems for 2 was possible based on analysis of the 1D and 2D 1H NMR data (see Table 1). The first spin system, a series of three double bonds, comprised H-9 through H3-15. A large J value was found for each of these vinylic proton signals, indicating that the three double bonds were trans. The only other spin system identified for 2 was a hydroxypropyl group (H-2′ through H3-4′). Four singlet 1H NMR signals (three downfield-shifted and one methyl group) accounted for the remaining signals in the 1H NMR spectrum. The 13C NMR spectrum showed evidence of 12 vinylic/aromatic carbon atoms and three carbonyl groups. Since there were 11 unsaturations present in this molecule, two ring systems were expected. Also present in the 13C NMR/DEPT spectra were signals corresponding to the hydroxypropyl group (a methyl, methylene, and methine group), an oxygenated quaternary carbon, and two methyl groups. The one-bond 1H-13C assignments were made by analyzing the HMQC13 spectrum. These assignments were straightforward and can be found in Table 1. The longrange correlations (obtained from the HMBC14 spectrum) were used to determine the connectivity of 2. HMBC correlations between H-2′a, H-2′b, and H-3′ to the carbon signal at δ 171.1 (C-1′) clearly indicated that this moiety is a hydroxybutanoic acid unit. The spin system comprising the three trans double bonds could be extended to four double bond units (adding C-3 and C-4) on the basis of HMBC data. The methyl group at δ 1.48 was correlated to a quaternary carbon at δ 85.7 and to the two carbonyl carbons at δ 192.6 and 193.5. This methyl group must therefore be attached to C-7, which was in turn flanked by the two carbonyl carbon atoms (C-6 and C-8). Additional HMBC correlations, together with chemical shift information, allowed placement of the two additional double bonds adjacent to the two carbonyl groups at C-6 and C-8. Several key HMBC correlations allowed all the fragments to be connected, thereby forming the requisite two rings. Correlations between H-5 and C-8a and between H-1 and C-4a allowed closure of one ring. A correlation between H-1 and C-3 was key to assigning the pyran ring system. After all
FAS Inhibitors as Antifungal Agents Table 1. 1H and
13C
Journal of Natural Products, 2003, Vol. 66, No. 8 1043
NMR Spectral Data for CT2108A (1) and CT2108B (2)a 1 1H
no. 1 3 4 4a 5 6 7 7-CH3 8 8a 9 10 11 12 13 14 15 1′ 2′a 2′b 3′ 4′
5.89 s 6.11 s 6.28 m 1.62 s 6.19 d (15.2) 6.95 dd (15.1, 11.3) 6.31 dd (14.9, 11.4) 6.56 dd (14.8, 10.8) 6.22 ddd (16,10.9,1) 5.94 dd (14.8, 7.0) 1.79 dd (6.7, 0.6) 2.56 dd (14.3, 7.0) 2.44 dd (14.4, 6.0) 4.11 m 1.20 d (6.2)
2 13C
82.1 154.5 105.4 146.7 120.7 192.5c 85.8 22.2 197.4c 55.5 123.88 136.7 129.9 139.8 132.6 134.5 18.6 171.1 43.2 64.9 23.2
HMBCb
1H
3
8.05 s
3, 5, 8a, 9
6.52 s
4, 7, 8a
5.53 m
6, 7, 8
1.48 s
3, 4, 10, 11 3, 12 9, 10, 13 10, 13, 14 15 12, 15 13, 14
6.30 d (15.0) 7.04 dd (15.3, 11.2) 6.35 m 6.60 dd (15.8, 10.8) 6.22 dd (14.9, 11.3) 5.95 dd (14.8, 7.1) 1.79 br d (6.7)
1′, 3′, 4′ 1′, 3′, 4′
2.52 dd (14.3, 7.0) 2.42 dd (14.4, 6.0) 4.10 m 1.20 d (6.2)
2′, 3′
13C
HMBCb
154.7 156.8 110.5 143.7 108.0 192.6 85.7 22.8 193.5 115.5 121.8 137.1 129.7 140.4 132.6 134.9 18.6 171.1 44.2
3, 4a, 8, 8a
65.1 23.1
3, 5, 8a, 9 4, 7, 8a 6, 7, 8 3, 4, 11 3, 9, 11, 12 9, 13 10, 13, 14 11, 12, 15 12, 15 13, 14 1′, 3′, 4′ 1′, 3′, 4′ 1′ 2′, 3′
a δ in ppm, J in Hz. Data were recorded at 500 and 125 MHz, respectively in acetone-d . bAll correlations represent 2- or 3-bond 6 couplings. nJCH was optimized for 8 Hz. c These carbon atoms may be interchanged.
connections were made (based on available NMR data), there remained only two open valences. The only option was to attach C-1′ to C-7 via an ester linkage. All other HMBC correlations are consistent with the proposed structure. The stereochemistry of 2 was deduced using two methods. The absolute configuration at C-7 of the azaphilones has been clearly established by optical methods.15,16 The sign of the Cotton effect at the longest wavelength in the CD spectrum depends on the stereochemistry at C-7. The CD curve for 2 (∆390 -1.7) provided clear evidence for the S-configuration at C-7. The only other asymmetric carbon atom in 2 was C-3′. Application of Horeau’s method17 established the absolute stereochemistry at C-3′ to be S. The gross structure of 1 was determined on the basis of comparison to the structure determined for 2 and the NMR data obtained for 1. The molecular formula for 1 contained one additional oxygen atom compared to 2. In the 13C NMR spectrum, two vinylic carbon signals were replaced by two downfield-shifted aliphatic signals. This suggested that a double bond had been replaced by an epoxide functionality. Based on examination of 1D NMR data alone, it appeared that it was the C-1/C-8a double bond that had been replaced by the epoxide. Data from 1H-1H COSY, HMQC, and HMBC experiments confirmed our assignment of the structure of CT2108A as 1. Further corroboration for this structure was obtained when the 13C NMR data for 1 were compared to the published NMR data for the related metabolite patulodin (3).12 The chemical shifts for the carbon atoms in 1 differed by less than 2 ppm from the published values for 3 (excluding those carbon atoms in the divergent side chain at C-7). The assignments for C-6 and C-8, unfortunately, could not be made unequivocally. On the basis of chemical shift values, it seemed likely that the carbon atom at δ 197.4 be assigned to C-8. The carbonyl at C-6 was conjugated and therefore would probably resonate further upfield (δ 192.5). Biosynthetic considerations suggested that 1 and 2 would likely have the same stereochemistry. Because 1 was synthetically converted to 2 (see below), and the semisynthetic 2 gave the same CD curve as the natural product, the stereochemistry at C-7 could be assigned as S for 1 as
well. Likewise, the stereochemistry at C-3′ for 1 was assumed to be the same as was determined for 2 because the epoxide could be converted to the alkene with retention of optical properties. The stereochemistry of the epoxide was not determined. Further support for the stereochemical relationship between 1 and 2 was obtained by chemically converting 1 to 2. This conversion was also sought because the isolated yield of 2 from the fermentation culture was substantially less than the yield for 1, and additional material was desired for further biological testing. A variety of methods to chemically convert epoxide 1 to alkene 2 were evaluated by analyzing the resulting crude reaction product mixtures by LC-MS. These trials included treating 1 with a variety of phosphorus reagents (Ph3P,18 (EtO)3P,19 Ph3PS-Ph3P,20 and [(PhO)3PMe]I-BF3OEt221), zinc reagents (Zn-HOAc,22Zn-NaI-HOAc-NaOAc,23 and Zn-Cu24), and other reducing metal/Lewis acid combinations (TiCl3-LiAlH425 and WCl6-2 n-BuLi26). At the end, utilization of the WCl6-2 n-BuLi reagent proved most efficacious. This reagent allowed for the preparation and isolation of gram quantities of pure 2, whose physicochemical properties (1H NMR, MS, CD, and UV) were identical to those of the natural product. The biological activity of CT2108A and B is listed in Table 2. Compound CT2108A inhibited fungal FAS activity in vitro with an IC50 of 160 µg/mL. Most important, neither compound inhibited human FAS. Both compounds exhibited reversible growth inhibition of C. albicans. The MIC90 values were 6.25 and 3.124 µg/mL using a microdilution susceptibility test for 1 and 2, respectively. The minimal fungal concentrations (MFC) were 25 and 6.25 µg/mL, respectively. In the disk diffusion assay, both compounds produced a clear zone of inhibition. Addition of long-chain fatty acids (Tween-40 and mysteric acid) to the culture medium partially reversed the growth inhibition. This fatty acid mixture had no effect on enzyme inhibition of either compound, implying that an interaction of the compounds with the fatty acids was not the likely cause of reversal. Rescue from growth inhibition by exogenous fatty acids suggests that the intracellular target of the compounds is FAS; however, a secondary target cannot be definitively
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Laakso et al.
Table 2. Effect of CT2108A (1) and CT2108B (2) on FAS Activity and Growth of C. albicans CT2108A FAS inhibitiona IC50 (Candida FAS) IC50 (human FAS) growth inhibition MIC MIC + fatty acidsb MFC DDA
CT2108B
cerulenin
160 >1000
250 >1000
0.5 0.5
6.25 50 25 8 clear
3.13 50 6.25 10-13 clear
5 >1000 5 20-30 hazy
a IC , minimum inhibitory concentration (MIC) and minimum 50 fungicidal concentration (MFC) values are expressed in µg/mL. Disk diffusion assay (DDA) zone of inhibition is expressed in mm. b Medium was supplemented with 1.0% Tween 40 and 0.03% mysteric acid. The addition of Tween 40 or mysteric acid to an enzyme assay has no effect on inhibition.
Table 3. Minimum Inhibitory Concentration of CT2108A (1) and CT2108B (2) against Clinical Isolatesa microorganism
no. tested
Candida albicans C. tropicalis C. lusitaniae C. guillurmondii C. krusei C. glabrata C. parapsilosis C. dublinensis S. cerevisiae Cryptococcus neoformansb
4 1 3 1 3 3 4 2 4 2
Cryptococcus laurinticb
2
CT2108A 24 h 48 h
CT2108B 24 h 48 h
3.9 NT 8.3
