Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Total Synthesis of Pleofugin A, a Potent Inositol Phosphorylceramide Synthase Inhibitor Toshihiro Kiho,† Mizuka Yokoyama,‡ and Hiroshi Kogen*,§ †
Modality Research Laboratories Daiichi Sankyo Co., Ltd. 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan Medical Science Department Daiichi Sankyo Co., Ltd. 3-5-1, Nihonbashi Honcho, Chuo-ku, Tokyo 103-8426, Japan § Graduate School of Pharmaceutical Sciences, Meiji Pharmaceutical University 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan ‡
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S Supporting Information *
ABSTRACT: X-ray analysis and total synthesis of 1 unambiguously confirmed pleofingin A’s absolute configuration. The total synthesis was achieved by convergent assembly of three fragments (12, 14, and 18). This synthetic approach provides access to derivatives of 1 to search for antifungal agents that will be more effective in clinical use.
F
action of IPC synthase inhibitors is expected to be specific to fungi.4 To date, several natural products have been reported to be IPC synthase inhibitors, including haplofungins,5 khafrefungin,6 aureobasidin A,7 and rustmicin.8 In particular, khafrefungin, aureobasidin A, and rustmicin are potent inhibitors2a,9 and suppress the growth of S. cerevisiae, Candida albicans, and Cryptococcus neoformans. However, all these known inhibitors lack fungicidal activity against A. f umigatus.2a Amphotericin B is used therapeutically as an antifungal agent, but adverse events can occur, making chemotherapy difficult. Pleofungin A has wide-spectrum antifungal activity, with especially strong action against A. fumigatus and C. neoformans.2a Hence, pleofungin A is anticipated as a useful antifungal agent for treating deeply invasive mycoses. Takatsu and co-workers have reported the structural elucidation of pleofungin A (1) by only spectrometric and amino acid analyses.2b The promising biological activity of 1 and our interest in developing effective antifungal agents based on their structure−activity relationships (SARs) prompted us to establish the complete structure of 1 unambiguously. Here, we report the absolute stereochemical structure of 1 determined by X-ray diffraction analysis and the total synthesis of 1. Pleofungin A is a 28-membered cyclic depsipeptide consisting of eight amino acids and two 2-hydroxycarboxylic acids. Interestingly, four components(S)-Hic−OH (2hydroxyisocaproic acid), (S)-Hiv−OH (2-hydroxyisovaleric acid), L-Thr, and N-Me-L-Leuform a moiety with a macrocyclic depsipeptide with three contiguous esters, which is a quite rare structure in cyclic depsipeptides.10 An initial X-
ungal infections are recognized as major diseases that threaten human health.1 The selection of clinically available antifungal drugs is limited, and these drugs also have the problems of adverse effects and the emergence of resistance in several pathogens.1b Pleofungins A−D are antifungal agents isolated from Phoma sp. SANK 13899 by the Sankyo group in 2007.2 They have been shown to inhibit the biosynthesis of inositol phosphorylceramide (IPC). Among them, pleofungin A (1) shows the most potent inhibitory activity against IPC synthase (IC50 = 0.9 nM in Aspergillus f umigatus, 6.4 nM in Saccharomyces cerevisiae) (Scheme 1).2a IPC is an essential structural sphingolipid in the fungal cell membrane.3 Because sphingomyelin is synthesized instead of IPC in mammalian cells, the Scheme 1. Structure of Pleofungin A (1) and Its TES Ether Derivative 1a
Received: June 21, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b01930 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters ray analysis of a crystal of 111 could not successfully solve its structure. Therefore, the natural product 1 was treated with triethylsilyl (TES) chloride to give TES ether 1a as a crystal (mp 189 °C from EtOAc). Treatment of 1a with TBAF completely converted 1a back to 1. Single-crystal X-ray diffraction was then used to elucidate the relative structure of 1a and verify the proposed structure of 1. The crystal structure of 1a suggested that two intramolecular hydrogen bonds between L-Ala and L-Ileu (O47−N23 distance, 3.11 Å; O79−N7 distance, 2.77 Å) caused the 28-memberd ring to form a rectangular shape (Figure 1).
Scheme 2. Retrosynthetic Analysis of Pleofungin A (1)
Scheme 3. Synthesis of Fragment 9
Figure 1. X-ray structure of 1a, the TES ether of 1.
To develop synthetic routes to various pleofungin analogues to search for more-effective IPC inhibitors and to perform an SAR study of the interesting activity of 1, we set out to complete its total synthesis. We planned to construct the 28-membered macrocyclic ring of 1 by macrolactonization of seco-acid 2 at the positions between (S)-Hiv and (S)-Hic as the key reaction. Compound 2 was divided into two main fragments (ester 4 and hexapeptide 3), as shown in Scheme 2. Then, 3 was further divided into tetrapeptide 5 and dipeptide 6. The convergent assembly of three fragments planned to obtain 1 and its derivatives. The solution-phase coupling of the amino acid or peptide units was conducted using an EDC·HCl (1-ethyl-3-(3(dimethylamino)propyl)carbodiimide) and HOAt (1-hydroxy-7-azabenzotriazol) combination method reported by Carpino.12 It was difficult to synthesize tripeptide 9 via the usual N-terminus elongation method due to easy diketopiperazine formation.13 Thus, L-alanine benzyl ester was used as a starting material and coupled with Boc-N-Me-L-Val 7 followed by deprotection of the benzyl group to give 8 (94%, 2 steps). The condensation of 8 with N-Me-L-Leu benzyl ester afforded compound 9 (92%, 2 steps) as a 2.2:1 mixture of epimers due to partial epimerization. Deprotection of the Boc group gave 10 (Scheme 3). This epimerization at the L-Ala position was sensitive to the coupling conditions, particularly the amount of base, as shown in Table 1. The epimer ratio of 9 (5.2/1) was much improved under the conditions using 3.0 equiv of HOAt and 1.1 equiv of Et3N (entry 2). Furthermore, the conditions without added base gave the best epimer ratio of 9.6/1 (entry 4). Tripeptide 10 was obtained as an inseparable mixture of epimers and was used as-is for further coupling with Boc-L-Thr (TBS) followed by treatment with TBAF to give pure
Table 1. Optimization of the Coupling of Dipeptide 8 with 11
entry 1 2 3 4
additive (equiv) HOAT HOAT HOAT HOAT
(1.2) (3.0) (5.0) (5.0)
base (equiv)
11 (equiv)
yield (%)
ratioa
Et3N (2.6) Et3N (1.1) Et3N (1.0) none
1.0 1.1 1.0 1.0b
92 85 85 84
2.2/1 5.2/1 5.6/1 9.6/1
a
Ratios were determined by 1H NMR analysis in CD3OD. bSalt 11 was neutralized before use.
tetrapeptide 12 as a separable single isomer (59%, 2 steps). Compound 14, which was separately prepared using Boc-L-Ile 13 and a N-Me-L-Leu benzyl ester, was connected to compound 12 to give a hexapeptide 1514 (93%, 2 steps) in the same manner. Removal of the Boc group in compound 15 followed by acylation with (S)-Hic−OH provided acyl hexapeptide 16 in good yield (79%, 2 steps). After hydroB
DOI: 10.1021/acs.orglett.8b01930 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Scheme 4. Synthesis of Pleofungin A (1)
In conclusion, we have unambiguously determined the absolute structure of pleofungin A (1) by X-ray crystallography and the first total synthesis of 1 (6.3% overall yield), a unique natural cyclic depsipeptide possessing three contiguous esters. SAR studies of its derivatives are currently underway to develop new antifungal drugs.
genolysis, the resulting acid was esterified with compound 18, which was prepared by coupling between 17 and (S)-Hiv− OBn. The esterified product was then subjected to the same hydrogenolysis conditions to afford a seco-acid 19 (69%, 3 steps). Partial epimerization also occurred at the N-Me-L-Leu moiety in this esterification step (major/minor = 3.7/1),15 but the minor epimer could be removed at the next purification step after the cyclization. Seco-acid 19 was used in the lactonization reaction by the Yamaguchi method16 to give cyclized product 20 (71%).17 In the final step, the last amino acid, N-Me-L-Ile was introduced. After selective removal of the Boc group in 20, the resulting amine18 was coupled with the side chain Boc-N-Me-L-Ile under neutral conditions to give fully protected 1. Finally, successive deprotection of the Boc and TBS groups provided 1 (70%, 4 steps), which had identical physical and spectral properties to those of natural 1 [1H NMR, 13C NMR spectra, [α]D25 = −135.0 (c = 1.0 in MeOH); natural 1: [α]D25 = −135.7 (c = 1.0 in MeOH)] (Scheme 4).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b01930. Experimental procedures and spectral data for synthesized compounds (PDF) Accession Codes
CCDC 1828023 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing C
DOI: 10.1021/acs.orglett.8b01930 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
1435−1439. (f) Takamatsu, S.; Kim, Y.-P.; Hayashi, M.; Hiraoka, H.; Natori, M.; Komiyama, K.; Omura, S. J. Antibiot. 1996, 49, 95−98. (11) Natural pleofungin A: mp 202−203 °C (n-hexane/i-PrOH), [α]D25 = −135.7 (c = 1.0 in MeOH). Anal. Calcd. For C55H98N8O14; C, 60.31, H, 9.02, N, 10.23. Found: C, 60.30, H, 9.21, N, 10.16. (12) (a) Carpino, L. A.; El-Faham, A. J. Org. Chem. 1994, 59, 695− 698. (b) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397−4398. (c) Carpino, L. A.; El-Faham, A. J. Org. Chem. 1995, 60, 3561−3564. (d) Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997, 97, 2243− 2266. (13) Coin, I.; Beyermann, M.; Bienert, M. Nat. Protoc. 2007, 2, 3247−3256. (14) The coupling of tripeptide 10 with another tripeptide fragment, Boc-L-Ile-N-Me-L-Leu-L-Thr, was unsuccessful for obtaining hexapeptide 15. Only dehydrooxazolone formation at the threonine position was observed under any coupling conditions. (15) This reaction did not optimize for epimerization. (16) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull. Chem. Soc. Jpn. 1979, 52, 1989−1993. (17) Possible macrolactonizations at other positions were also examined, but the present position gave the best result. (18) This amine was so labile that it gradually isomerized to another cyclized product under basic conditions.
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hiroshi Kogen: 0000-0002-5885-5138 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Drs. Tatsuya Yano and Masatoshi Inukai (Daiichi Sankyo) for providing natural pleofungin A and Mr. Youji Furukawa (Daiichi Sankyo) for performing X-ray analysis. This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Sciences (No. 17K08227).
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DEDICATION Dedicated to Professor Paul A. Wender (Stanford University) on the occasion of his 70th birthday. REFERENCES
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DOI: 10.1021/acs.orglett.8b01930 Org. Lett. XXXX, XXX, XXX−XXX
