The molecular formula of compound 1 was determined to be C33H50O7 by HR electrospray ionization MS. The 1H NMR spectrum showed five methyl signals and a methoxy signal at 3.69 p.p.m. (Supplementary Figure S1). It also showed eight olefinic proton signals and three signals that were attached to oxygenated carbons. Analysis of the 13C NMR and HSQC data confirmed that 1 possessed 33 carbon atoms, including 2 carbonyl carbons and a methoxy carbon, among which 3 sp3 methines were assigned to carbon atoms that bore an oxygen atom (Supplementary Figures S2 and S5). In addition, 1 sp3 acetal carbon signal at 97.2 p.p.m. was observed, indicating that 1 had a 6,6-spiroacetal structure.12 The 13C NMR spectra also showed 10 olefin signals, suggesting the presence of five double bonds. These observations, in conjunction with the UV absorption spectrum and molecular formula, implied that 1 is a reveromycin derivative with an additional methoxy group and the absence of a succinyl group compared with reveromycin A. Its planar structure was elucidated by analysis of the 2D NMR data, as shown in Figure 1. The DQF-COSY and HSQC-TOCSY spectra revealed three spin systems from C-2 to C-7 with a methyl branch at C-4, C-9 to C-14 with a methyl branch at C-12, and C-16 to C-21 with a butyl branch at C-18 (Supplementary Figures S3 and S4). The connections between the spin systems were established by long-range correlations in the HMBC spectrum (Supplementary Figure S6), and the overall structure was established. The methoxy group was assigned to the C-1 position, based on the long-range correlations from the methoxy signal to C-1. The 6,6-spiroacetal core was confirmed, based on the HMBC correlations from H-14, H-17, and H-19 to the acetal carbon and H-14 to C-16. The geometries of the double bonds at Δ2, Δ6 and Δ20 were designated to have E configurations, based on the coupling constants in the 1H NMR spectrum. The Δ9 and Δ22 bonds were E configurations, based on the 13C NMR chemical shift values of C-8-Me and C-22-Me. The absolute configuration at C-5 was determined to be R by the modified Mosher’s method of the dimethyl ester that was converted from 1 by treatment with trimethylsilyl diazomethane.13, 14 The configuration at C-15 was determined to be S* by the NOESY correlations between H-11/H-13, H-16axial/H-18 and H-14equatorial/H-16axial in phase-sensitive NOESY spectrum (Supplementary Figures S7 and S8). The remaining stereochemistry including absolute configuration at C-15 assigning to be S was deduced to be the same as that of reveromycin A, comparing its NMR chemical shifts and optical rotations12, 14 with those of spirofungins A and B.15, 16, 17, 18 Based on these findings, the structure of 1 was determined as shown in Figure 1.

Compound 2 had a similar 1H NMR spectrum as 1, except for a newly observed ethoxy signal that comprised a triplet at 1.26 p.p.m., coupled with a quartet signal at 4.16 p.p.m. and the disappearance of a methoxy signal of 1 (Supplementary Figure S9). The molecular formula was established as C34H52O7 by HR fast atom bombardment MS, which was identical to an ethyl ester form of 1. The planar structure was elucidated in the same manner as that of 1, based on an analysis of 2D NMR data (Supplementary Figures S10–S14). The attachment of the ethoxy group at C-1 was determined, based on the HMBC correlation from the quartet signal to C-1. The stereochemistry was deduced to be the same as 1, based on a comparison of NMR data, and the structure was determined as shown in Figure 1.

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