8-Methyl-Pyridoxatin: A Novel N-Hydroxy Pyridone from Fungus OS-F61800 That Induces Erythropoietin in Human Cells Ping Cai, †,| David Smith, ‡ Bernadette Cunningham, ‡ Sheryl Brown-Shimer, ⊥ Barry Katz, † Cedric Pearce, † Debra Venables, § and David Houck* ,† MYCOsearch, a subsidiary of OSI Pharmaceuticals, 4727 University Drive, Suite 400, Durham, North Carolina 27707, OSI Pharmaceuticals, 106 Charles Lindbergh Blvd, Uniondale, New York 11553, Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, and Oncogene Science Diagnostics, 80 Rogers Street, Cambridge, Massachusetts 02142 Received October 9, 1998 In the course of screening for small-molecule modulators of erythropoietin gene expression, a novel N-hydroxy pyridone was isolated from a culture of OS-F61800. Its structure was elucidated by extensive 1 H and 13 C NMR spectroscopic and chemical studies. This compound induced erythropoietin gene expression fivefold at a concentration of 0.3 μM, which is about threefold greater potency than our previously identified erythropoietin inducers. As described in previous communications, 1,2 we have screened for inducers of erythropoietin (EPO) gene expres- sion using luciferase-reporter technology. 3,4 Continued screening resulted in the isolation of a novel pyridone (1) from the fermentation extract of fungus OS-F61800, a sterile-hyaline culture, collected from Six Mile Cypress Slough in Florida. In this note, we report the isolation and structure elucidation of this cyclic hydroxamic acid. In addition, we provide data on the ability of 1 and related compounds to induce human EPO in cell-based assays. The dried methanol extract from a 3-L fermentation culture of OS-F61800 was partitioned into hexane, chlo- roform, and methanol-water. The CHCl 3 fraction was subjected to isolation by LH-20 flash chromatography, followed by reversed-phase HPLC to yield 1 as a white powder. Elemental analysis established a molecular for- mula of C 16 H 23 NO 3 , which was confirmed by HRFABMS. The IR spectrum of 1 had a carbonyl absorption at 1625 cm -1 . The presence of a pyridone ring was supported by both 1 H and 13 C NMR data. In the 1 H NMR spectrum, 1 displayed a pair of coupled doublets at δ 7.53 (d, J ) 8.5, H-6) and 5.91 (d, J ) 8.5, H-5), which correlated to the carbons at δ 132.7 and 98.8, respectively, in the HMQC spectrum. The proton at δ 5.91 displayed two- and three- bond correlations to the quaternary carbons at δ 164.6 (C- 4) and 113.4 (C-3), respectively, in the HMBC spectrum; the proton resonance at δ 7.53 had three-bond correlations to carbons corresponding to δ 164.6 (C-4) and 161.1 (C-2), suggesting a 3-alkyl-4-hydroxy-2-pyridone substructure. In addition to the pyridone protons, the DQCOSY spectrum of 1 also demonstrated the presence of two isolated proton- spin systems. One of these was a monosubstituted double bond, in which an olefinic doublet of doublets (δ 4.87, dd, J ) 17, 10 Hz, H-13) coupled to two olefinic protons at δ 4.69 (dd, J ) 17, 1.5 Hz, H-14 a ) and 4.67 (dd, J ) 10, 1.5 Hz, H-14 b ), respectively. The second proton-spin system consisted of four methylene protons, three methine protons, and two groups of methyl protons; the most downfield methine-proton doublet at δ 2.61 (H-7) coupled to the methine-proton multiplet at δ 2.89 (H-12), which in turn coupled to a methyl-proton doublet at δ 0.63 (H-17) and one methylene proton at δ 0.66 (H-11′). This methylene proton was also coupled to its geminal partner at δ 1.75 (H-11) and to the most upfield methine proton at δ 1.78 (H-10), which in turn coupled to the methyl doublets at δ 0.88 (H-16) and a pair of methylene protons at δ 1.12 (H- 9′) and 1.32 (H-9), respectively. A cyclohexane substructure was suggested by those correlations and by the observed HMBC correlations of a quaternary carbon at δ 45.3 (C-8) with both H-7 and H-9. The monosubstituted double bond and a methyl group were attached to a common quaternary carbon, as indicated by HMBC correlations of C-8 (δ 45.3) to the double-bond proton at δ 4.68 (H-14) and the methyl- proton singlet at δ 1.07 (H-15). Clear HMBC correlations of the methine proton at δ 2.61 (H-7) with the carbons at δ 113.4 (C-3), 164.6 (C-4), and 161.1 (CdO, C-2) revealed the connection of the cyclohexane ring to the pyridone C-3 position. The complete 1 H and 13 C assignments agreed with a molecular formula of C 16 H 22 NO 2 , one oxygen less than that obtained from elemental analysis and HRMS. Because 1 H and 13 C chemical shift analyses eliminated the possibility of another carbon-bonded oxygen, we expected that the oxygen must be attached to the pyridone nitrogen, and this hypothesis was supported by the results of diazomethane methylation. Treatment of 1 with freshly prepared CH 2 N 2 produced two methylated derivatives (2, 3). Both com- pounds had a molecular ion of m/z 305 in the positive ESMS spectrum, suggesting that methylation occurred at both the C-hydroxyl and the N-hydroxyl groups. The UV absorption spectra of the two products were characteristic of the structures; the major product (2) had an absorption spectrum similar to 1 (λ max 290 nm), whereas the maximal UV absorption of the minor product shifted to 272 nm, indicative of the tautomer, a 4-pyridone (3). * To whom all correspondence should be addressed. Tel.: 919-489-4700. Fax: 919-490-3745. E-mail dhouck@osip.com. † MYCOsearch, a subsidiary of OSI Pharmaceuticals. ‡ OSI Pharmaceuticals. § Department of Medicinal Chemistry, Utah University. ⊥ Oncogene Science Diagnostics. | Present Address: Wyeth-Ayerst Research; 401 N. Middletown Road, Building 96A, Pearl River, NY 10965. 397 J. Nat. Prod. 1999, 62, 397-399 10.1021/np980450t CCC: $18.00 © 1999 American Chemical Society and American Society of Pharmacognosy Published on Web 01/28/1999