Neomaclafungins AâI: Oligomycin-Class Macrolides from a Marine

Article pubs.acs.org/jnp

Neomaclafungins A−I: Oligomycin-Class Macrolides from a MarineDerived Actinomycete Seizo Sato,* Fumie Iwata, Shoichi Yamada, and Masashi Katayama Central Research Laboratory, Tokyo Innovation Center, Nippon Suisan Kaisha, Ltd. 32-3 Nanakuni 1 chome, Hachioji, Tokyo 192-0991, Japan S Supporting Information *

ABSTRACT: Nine new 26-membered macrolides of the oligomycin subfamily, neomaclafungins A−I, were isolated from the fermentation broth of Actinoalloteichus sp. NPS702, which was isolated from marine sediment collected from Usa Bay, Kochi Prefecture, Japan. Their structures were identified through mass spectrometry and NMR experiments. They belong to the oligomycin class and have several distinct features including the presence of alkane or alkanol branches. Neomaclafungins A−I exhibited significant antifungal activity in vitro against Trichophyton mentagrophytes (ATCC 9533), showing MIC values between 1 and 3 μg/mL.

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M

icroorganisms, including marine-derived microorganisms and particularly actinomycete bacteria and fungi, produce a wide variety of biologically active and structurally unique metabolites.1−6 Hundreds of compounds are isolated from these organisms annually, and they have become a prominent source of natural products.7 Actinomycetes, mainly Streptomyces species, are characterized by their complex secondary metabolism. They have produced more than twothirds of the clinically useful antibiotics of natural origin.8 For example, the oligomycins are macrolides characterized by a 26membered ring fused to a bicyclic spiroketal. The oligomycin antibiotic complex was first isolated from cultures of Streptomyces diastatochromogenes in 1954. Subsequently, many of their analogues have been isolated from Streptomycetaceae. Members of the oligomycin family display a wide variety of bioactivities, including anticancer, antifungal, nematocidal, insecticidal, and immunosuppressive effects.9−13 Furthermore, their use as mitochondrial ATPase inhibitors has aided in the biochemical understanding of the mechanism of oxidative phosphorylation.14−16 These studies suggest that this family of 26-membered macrolide antibiotics has potential to supply drug candidates as well as molecular tools for the examination of biological phenomena. During the course of a screening program to discover new fermentation-derived compounds, antifungal activity was detected in the fermentation extract of strain NPS702. This microorganism was subsequently classified as a strain of Actinoalloteichus sp. based on a 16S rDNA sequence analysis. The chemical investigations reported herein have led to the discovery of the neomaclafungins A (1) to I (9), which possess a macrolide ring similar to that of maclafungin;17 these compounds comprise nine new macrolides of the oligomycin class. Antifungal activities of these new compounds were also investigated. © 2012 American Chemical Society and American Society of Pharmacognosy

RESULTS AND DISCUSSION

Neomaclafungin A (1), the main metabolite from the fermentation broth of Actinoalloteichus sp. NPS702, was isolated as a white, amorphous powder with a molecular formula of C44H76O11 (seven degrees of unsaturation) as determined by high-resolution electrospray-ionization time-of-flight mass spectrometry (HRESITOFMS). This molecular formula was corroborated by 1H and 13C NMR data. The UV spectrum of 1 in methanol exhibited absorption maxima at approximately 224, 231, and 240 nm. The IR spectra indicated the presence of hydroxy (3395 cm−1), carbonyl (1717 cm−1), and conjugated diene (1653 cm−1) moieties. These spectroscopic characteristics and the initial inspections of the 1H and 13C NMR spectra indicated that 1 is a member of the oligomycin class of compounds. On the basis of the 13C chemical shifts, 1 possessed one ester and three double bonds, which accounted for four of the seven total degrees of unsaturation inferred from the molecular formula. This suggested that 1 contained three rings. The 13C NMR chemical shifts of 1 also revealed numerous oxymethine carbon resonances, which supports the likely polyketide origin of 1. When the 1H and 13C NMR data of 1 taken in CDCl3 were compared with those of maclafungin (10), a member of the oligomycin family with five hydroxy groups, the spectra showed significant similarities (Table 1). On the basis of the molecular formula and 13C NMR chemical shifts, 1 has one less methine group adjacent to a heteroatom in the region from 50 to 90 ppm and one less methylene group in the region from 10 to 30 ppm when compared with 10. When the 13C NMR spectrum of 10 was compared with that of 1, it showed a significant upfield shift for C-33 from δC 72.9 to δC Received: October 15, 2012 Published: October 26, 2012 1974

dx.doi.org/10.1021/np300719g | J. Nat. Prod. 2012, 75, 1974−1982

Journal of Natural Products

Article

Neomaclafungin B (2) has a molecular formula of C45H78O11 as established by HRESITOFMS. This formula is one carbon atom and two hydrogen atoms greater than the molecular formula of 1. This result suggests that 2 is a methyl or methylene analogue of 1. A comparison of the 13C NMR and 1 H NMR spectra of neomaclafungins A and B suggests that 2 is the C-45 methyl (δH 1.08, δC 22.4) homologue of 1 (Tables 2 and S1, Supporting Information). The COSY and HMBC signals of the newly identified doublet methyl signal H3-47 correlate to H-45 (δH 3.63, oxymethine proton), C-45 (δC 68.3, oxymethine carbon), and C-44 (δC 40.3). These data and the similarity of other spectroscopic features between 1 and 2 support the former compound as 45-methylneomaclafungin A. Neomaclafungin C (3) was obtained as a white, amorphous powder with a molecular formula of C 45 H 78 O 11 by HRESITOFMS. The molecular composition of 3 indicates a mass of 14 amu less than the mass of 1. The 1H NMR spectrum of neomaclafungin C displayed a high degree of similarity to that of 1, with five oxymethine protons, H-5, H-7, H-9, H-11, and H-13, an oxymethylene proton, H-45, and one ester carbinol proton, H-25. The 13C NMR spectrum showed a significant downfield shift of C-33 from δC 64.6 to δC 69.6 when the NMR spectrum of 1 was compared to that of 3. In addition, COSY correlations of an oxymethine proton (H-33, δH 3.77) to a methylene proton (H2-34, 1.55 and 1.48) and HMBC correlations of H-33 to C-31 (δC 67.4)/C-32 (δC 40.1)/C-34 (δC 31.1)/C-35 (δC 9.7) suggested that the additional methyl group in 3 was located on C-34. The overall NMR data (Tables 2 and S1), including analyses of information from HSQC, COSY, and HSQC experiments, allowed 3 to be assigned as 34-methylneomaclafungin A (neomaclafungin C). Neomaclafungin D (4), a white, amorphous powder, also showed NMR spectra similar to those of 1. The only differences were five resonances in the 1H NMR spectrum at δH 3.78 (1H, m, H-33), 3.65 (1H, m, H-45), 1.53/1.48 (2H, m, H2-34), 1.09 (3H, d, J = 6.3 Hz, H3-47), and 0.97 (3H, t, J = 7.6 Hz, H3-35) instead of at δH 4.04 (1H, m, H-33), 3.44/3.40 (2H, m, H2-45), and 1.23 (3H, d, J = 6.3 Hz, H3-34) in 1. From the HMBC data, a newly observed methyl group (H3-35) correlated with the oxymethine C-33 (δC 69.6) and the geminal methylene C-34 (δC 30.8). Another new methyl geroup (H3-47) correlated with the oxymethine C-45 (δC 70.0) and the geminal methylene C-44 (δC 40.3) (Figure 1). These features are characteristic of 34- and 45-methylneomaclafungin A (2 and 3). Thus, the structure of compound 4 was established as 34,45-dimethylneomaclafungin A, and it was named neomaclafungin D. Neomaclafungin E (5) was isolated as a white, amorphous powder with the molecular formula C43H74O10 by HRESITOFMS analysis. This formula showed seven degrees of unsaturation, the same as for neomaclafungin A (1). A detailed analysis of the 1H and 13C NMR data suggested that 5 had the same 26-membered ring with a bicyclic spiroketal structure as 1, but did not possess a hydroxymethyl group at C-44. This new alkane branch was easily assigned because of the HMBC correlations from δH 1.71 (1H, m, H-24) to both the methylene carbon and the methyl carbon at δC 14.7 (C-43) and 14.9 (C44), respectively. In a manner similar to that used for 1, a comprehensive analysis of the 2D NMR data allowed the full planar structure of neomaclafungin E (5) to be assigned as 44(dehydoroxymethyl)neomaclafungin A (Tables 2 and S1). Neomaclafungin F (6) was also obtained as a white, amorphous powder, and the molecular formula was determined

64.6, a downfield shift for C-32 from δC 37.2 to δC 42.4, a downfield shift for C-3 from δC 148.4 to δC 150.1, an upfield shift for C-4 from δC 49.2 to δC 41.4, and a downfield shift for C-5 from δC 78.6 to δC 80.3. These differences suggested that 1 may be C-34-deoxy-C-34/C-36-didesmethylmaclafungin. Several 2D NMR experiments, including COSY, HSQC, and HMBC, showed that 1 and 10 consist of a common 26membered macrolide core that contains a bicyclic spiroketal. HMBC correlations from the carbinol proton at δH 5.30 (H-25) and the olefinic proton at δH 6.58 (H-3) to the ester carbon at δC 165.2 (C-1) showed the ester and the polyol chain to be connected to the C-25 position of the spiroketal moiety. The terminal moiety, which was different from that of maclafungin, was definitively determined by the key correlations described below and shown in Figure 1. The COSY correlations from the doublet methyl protons H3-34 (δH 1.23) to H-33 (δH 4.04) and HMBC correlations from H3-34 to C-32 (δC 42.4) and C-33 (δC 64.6) established a 2-propanol terminus in place of the dihydroxybutyl group of 10. In addition, the COSY correlations between H3-36 (δH 1.15) and H-4 (δH 2.42) and the HMBC correlations from H3-36 to C-3 (δC 150.1)/C-4 (δC 41.4)/C-5 (δC 80.3) gave important information regarding the substitution of a methyl group for an ethyl residue at the C-4 position in 10. The position of the methyl ether group (δH 3.36, δC 55.8) was confirmed by an HMBC correlation of the singlet methyl protons H3-39 to C-9 (δC 80.9). Consequently, the structure of neomaclafungin A is suggested as 34-deoxy-34,36didesmethylmaclafungin. 1975



Article

Table 1. Complete NMR Data for Neomaclafungin A (1) and Maclafungin (10) in CDCl3 maclafungin (10) literature17

neomaclafungin A (1) experimental no. 1 2 3 4 5 6 7 8

δC,a multb 165.2, 121.9, 150.1, 41.4, 80.3, 40.4, 78.6, 37.9,

C CH CH CH CH CH CH CH2

9 10

80.9, CH 37.8, CH2

11 12 13 14

73.9, 45.0, 75.0, 32.7,

15

28.3, CH2

16 17 18 19 20

131.0, 132.0, 130.2, 137.5, 45.4,

CH CH CH CH2

CH CH CH CH CH

21 22

30.8, CH2 29.7, CH2

23 24 25 26

70.3, 40.0, 71.0, 36.3,

27 28

97.4, C 29.6, CH2

29

26.4, CH2

30 31 32

30.5, CH 67.4, CH 42.4, CH2

33 34 35 36

CH CH CH CH2

δH,c mult (J in Hz) 5.78, d (15.6) 6.58, d (15.6, 10.4) 2.42, ddq (10.4, 9.6, 6.3) 3.64, d (9.6) 1.32, m 4.06, d (10.0) 1.572,f m 1.504,f m 3.75, tt (10.1, 10.1) 1.86, dd (13.7, 11.6) 1.434,f m 3.43, dd (11.6, 8.9) 1.503f, m 3.54, dd (11.2, 8.8) 1.70, m 1.46, m 2.27, m 5.42, ddd (14.7, 9.2, 5.6) 6.09, dd (14.7, 10.5) 5.93, dd (14.7, 10.5) 5.34, dd (14.7, 9.7) 1.91, m 1.49, m 1.47, m 1.624,f m 1.15, m 3.80, m 1.87, m 5.30, dt (12.2, 4.8) 1.78, dd (12.9, 4.8) 1.67, dd (12.9, 12.2)

COSY 3 2, 3, 4, 5, 6, 7,

4 5, 36 6 7, 38 8 9

HMBC 1, 1, 2, 3, 5, 9, 7,

4 2, 4, 5, 36 3, 5, 36 4, 6, 7, 36, 38 38 38 9

δCd 164.8, 123.5, 148.4, 49.2, 78.6, 40.7, 78.8, 38.0,

C CH CH CH CH CH CH CH2

8, 10 9, 11

8, 9, 11

81.0, CH 38.0, CH2

10, 11, 12, 13,

13 11, 40 12 15

73.8, 45.3, 75.2, 32.9,

12 13, 40 14 15

14, 16 15, 16, 17, 18, 19, 20,

17 18 19 20 21, 41 22

CH CH CH CH2

28.4, CH2 14, 15, 16, 17, 22, 20,

15, 18, 17, 20, 42 22,

18 19 20 41 23, 41

21, 23

131.0, 132.1, 130.0, 137.9, 45.4, 30.8,

CH CH CH CH CH CH2

29.4, CH2

22, 24 23, 25, 43 24, 26 25

21, 25, 27, 43 23, 26, 44 1, 26, 43 24, 25, 27, 28

70.3, 40.0, 70.9, 36.5,

29

26, 27, 29

97.5, C 29.9, CH2

28, 30

27, 28, 30, 31, 46

26.5, CH2

29, 31, 46 30, 32 31, 33

29, 31 27, 29, 32, 33, 46 31, 34

30.8, CH 67.3, CH 37.2, CH2

64.6, CH 24.7, CH3

1.622,f m 1.432,f m 2.10, tt (13.5, 4.1) 1.38, d (13.5) 1.573,f m 3.99, d (10.4) 1.61, m 1.26, m 4.04, m 1.23, d (6.3)

32, 34 33

31, 32 32, 33

17.74,f CH3

1.15, d (6.3)

4

3, 4, 5

72.9, 71.6, 19.8, 23.8,

CH CH CH3 CH2

37 38 39 40 41

4.1, 55.8, 12.9, 27.4,

6 12 20, 42

5, 6, 7 9 11, 12, 13 19, 20, 42

11.5, 4.1, 55.9, 12.9, 27.7,

CH3 CH3 CH3 CH3 CH2

42 43

12.1, CH3 17.71,f CH2

41 24

20, 41 24

12.2, CH3 17.7, CH2

44

32.8, CH2

45

24, 43, 45

32.9, CH2

45

62.6, CH2

0.86, d (6.9) 3.36, s 0.75, d (6.8) 1.30, m 1.24, m 0.81, t (7.3) 1.44, m 1.40, m 1.502,f m 1.33, m 3.44, m

44

43, 44

62.6, CH2

CH3 CH3 CH3 CH2

1976

CH CH CH CH2

δ He 5.70 6.40 2.11 3.67 1.25 4.02 1.52 1.45 3.72 1.80 1.35 3.37 1.44 3.47 1.67 1.42 2.23 2.22 5.39 6.03 5.89 5.31 1.88 1.45 1.58 1.11 3.74 1.83 5.25 1.74 1.62 1.59 1.38 2.06 1.33 1.54 3.98 1.57 1.24 3.50 3.52 1.17 1.98 1.13 0.74 0.80 3.30 0.69 1.33 1.23 0.74 1.39 1.33 1.45 1.28 3.37



Article

Table 1. continued maclafungin (10) literature17

neomaclafungin A (1) experimental no.

δC,a multb

46

11.2, CH3

δH,c mult (J in Hz) 3.40, m 0.90, d (6.8)

COSY 30

HMBC 29, 30, 31

δCd

δ He

11.3, CH3

3.35 0.86

a

150 MHz. bAssignments by HSQC experiments. c600 MHz. d100 MHz. e400 MHz. fSignal partially obscured. Assignments of chemical shifts from the center of the HSQC cross-peak.

Figure 1. Key 2D-NMR correlations for neomaclafungins A (1) and D (4).

43-ethyl moieties agree with those of 4 and 6, respectively. All spectroscopic data were consistent with the proposed structure of 34,44-dimethylneomaclafungin E (neomaclafungin H) for 8. Neomaclafungin I (9), a white, amorphous powder, was similarly subjected to HRESITOFMS analysis, and its molecular formula was established as C46H80O10. A comparison of the 1H NMR data and MS data of 9 with those of 7 showed general similarities. An obvious difference found between 9 and 7 was the additional methyl group (δH 0.86, 3H, t, J = 7.3 Hz, δC 14.0 and Δ +14 amu, respectively) for 9. The combination of the correlations from both the COSY and HMBC spectra showed the connection of a C-24 n-pentyl moiety (H2-43−H348). The HMBC correlations from H3-48 to C-47 (δC 23.0)/C45 (δC 31.2), from H3-45 to C-44 (δC 29.2)/C-43 (δC 21.0), and from H2-43 to C-23 (δC 70.2)/C-24 (δC 39.6)/C-25 (δC 70.9) gave important information about the n-pentyl position in 9. Compound 9 was identified as 44-propylneomaclafungin E and named neomaclafungin I (Tables 2 and S1). The relative configuration of neomaclafungin A (1), the most abundant compound in the neomaclafungin family, was determined using NMR experiments, and the configurations of the remaining family members neomaclafungins B−I (2−9) were assumed to be the same as that of 1. The relative configurations of the tetrahydroxy chain (C-4 to C-13), two flexible side chains (C-31 to C-34 and C-20 to C-42), and three double bonds in 1 were determined using chemical shifts, coupling constants, and NOE interactions. The geometries of the three double bonds (C-16 to C-19 and C-2 to C-3) in the 26-membered ring were determined to be trans (E) on the basis of the characteristically large coupling constants (J > 14.0 Hz) observed in the 1H NMR spectrum. The relative configurations of three substructures (C-4 to C-13, C-31 to C-34, and C-20 to C42) were established using J-based configuration analysis,18 the method reported by Murata et al.19 3JH,H values were determined by 1D 1H NMR, 2D 1H−1H COSY, and HSQC experiments, and 3JC,H values by sensitivity- and gradientenhanced hetero (ω1) half-filtered TOCSY (HETLOC)20 experiments with a zero-quantum filter.21 For example, the relative configurations of C-4 to C-6 were determined through

to be C44H76O10 from a combination of HRESITOFMS and NMR data. The 1H and 13C NMR data (Tables 2 and S1) showed signals that closely resemble those of 5. The only difference was the presence of a propyl branch at C-24 in 6 instead of the ethyl branch in 5. The new methyl group was placed at C-44 by the H3-45 (δH 0.71, t, J = 7.6 Hz)/H2-44 (δH 1.22 and 1.14, m)/H2-43 (δH 1.42 and 1.25, m) spin system identified from the COSY spectrum and by the HMBC correlations of H3-45/C-44 (δC 22.5)/C-43 (δC 23.2). Consequently, the structure of 6 was identified as 44methylneomaclafungin E (neomaclafungin F). Neomaclafungin G (7) was obtained as a white, amorphous powder with a molecular ion peak at m/z 779.5675 [M + H]+ in the HRESITOFMS spectrum, which was consistent with the molecular formula C45H78O10 and implied seven degrees of unsaturation. The assignments of the 1H and 13C NMR spectroscopic data of 7 were assisted by a series of 2D NMR (COSY, HSQC, and HMBC) experiments. A comparison of the NMR data of 7 with those of 6 (Tables 2 and S1) showed that an additional methyl group was present at C-45 in 7 (nbutyl branch) compared to the n-propyl branch at C-24 in 6. Therefore, the structure of 7 was identified as 44-ethylneomaclafungin E (neomaclafungin G). Neomaclafungin H (8) was also isolated as a white, amorphous powder. A comparison of the NMR and MS data of 7 and 8 indicated that these molecules have the same molecular formula (C45H79O10) and substituent groups. The 1 H NMR spectrum of 8 lacked the methylene signals at H2-45 and the two methyl signals at H3-34 and H3-47 that were observed in 7. However, additional signals for geminal methylene protons at δH 1.53 and 1.47 (δC 31.1) and two methyl protons at δH 0.97 (δC 9.8) and 0.92 (δC 11.3) were observed. These spectroscopic characteristics and an initial inspection of both the 1H and 13C NMR spectra (Tables 2 and S1) indicated that 8 was a methylregioisomer of neomaclafungin G. HMBC correlations were observed between H-33/H2-34 and the methyl carbon at C-35 (δC 9.8) and between H2-43/H2-44 and the methyl carbon at C-46 (δC 11.3). These 1H and 13C NMR chemical shifts of the 33- and 1977



Article

Table 2. 13C NMR Data (δC) for Neomaclafungins A−I (1−9) in CDCl3

a

no.

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 38 39 40 41 42 43 44 45 46 47 48

165.2 121.9 150.1 41.4 80.3 40.4 78.6 37.9 80.9 37.8 73.9 45.0 75.0 32.7 28.3 131.0 132.0 130.2 137.5 45.4 30.8 29.7 70.3 40.0 71.0 36.3 97.4 29.6 26.4 30.5 67.4 42.4 64.6 24.7

165.1 122.0 149.9 41.3 80.4 40.3a 78.6 37.9 80.9 38.0 73.2 45.2 75.1 32.5 28.4 132.1 131.5 130.5 136.9 44.9 30.8 28.7 70.6 39.7 71.0 36.5 97.4 29.7 26.5 30.6 67.3 42.4 64.5 24.8

165.2 122.0 150.1 41.4 80.3 40.5 78.7 37.9a 80.9 37.9a 73.9 45.1 75.1 32.8 28.3 131.0 132.0 130.2 137.6 45.5 30.8 29.7 70.3 40.0 71.0 36.4 97.4 29.8 26.5 30.7 67.4 40.1 69.6 31.1 9.7 17.7a 4.1 55.8 12.9 27.7 12.1 17.7a 32.9 62.6 11.3

165.1 122.0 148.2 41.3 80.4 40.3a 78.7 38.0a 81.0 38.0a 73.2 45.3 74.7 32.6 28.8 132.1 131.5 130.5 137.1 44.9 31.1 29.7 70.5 39.7 71.4 36.5 97.4 29.8 26.5 30.7 68.4 40.0 69.6 30.8 9.7 17.6a 4.3 55.8 12.6 27.1 12.2 17.6a 40.3a 70.0 11.3 22.4

164.9 121.8 150.1 41.4 80.4 40.6 78.4 38.3 80.8 38.4 73.7 45.3 76.3 33.5 28.0 129.7a 132.4 129.7a 138.3 46.4 31.8 31.7 70.8 43.5 70.5 36.3 97.2 29.8 26.5 30.6 67.4 42.7 64.5 24.8

164.9 122.0 149.9 41.5 80.4 40.6 78.4 38.2a 80.8 38.2a 73.8 45.1 76.4 33.5 28.0 129.7 132.4 129.8 138.2 46.1 31.2 31.3 70.3 39.6 70.8 36.4 97.2 29.8 26.5 30.6 67.4 42.6 64.5 24.7

165.0 121.9 149.8 41.4 80.4 40.7 78.2 38.4 80.8 38.2 73.8 45.1 76.3 33.4 28.1 129.6 132.6 129.8 138.4 45.7 30.9 31.1 70.1 39.8 70.9 36.4 97.2 29.8 26.5 30.6 67.4 42.5 64.5 24.7

165.0 121.8 150.0 41.4 80.5 40.7 78.3 38.5 80.8 38.3 73.8 45.1 76.5 33.5 28.2 129.6 132.7 129.8 138.6 45.8 31.1 31.2a 70.2 39.6 70.9 36.4 97.3 29.8 26.5 30.7 67.4 42.6 64.6 24.7

17.7 3.8 55.9 12.9 28.5 12.1 14.7 14.9

17.8 3.8 55.8 12.9 28.3 12.1 23.2 22.5 14.2 11.2

17.8 3.8 55.9 12.9 28.2 12.1 20.8 32.1 22.3 11.3 14.5

165.0 122.0 149.9 41.5 80.4 40.6 78.4 38.2a 80.8 38.2a 73.8 45.1 76.4 33.5 28.1 129.7 132.5 129.8 138.3 46.1 31.2 31.3 70.3 39.6 70.8 36.4 97.3 29.9 26.5 30.7 67.3 40.3 69.6 31.1 9.8 17.8 3.8 55.8 12.9 28.3 12.1 23.2 22.5 14.2 11.3

17.7a 4.1 55.8 12.9 27.4 12.1 17.7a 32.8 62.6 11.2

17.6 4.2 55.8 12.6 27.1 12.2 17.4 40.3a 68.3 11.2 22.4

11.2

17.7 3.8 55.9 12.9 28.2 12.1 21.0 29.2 31.2a 11.3 23.0 14.0

Assignments may be interchanged.

consider two major rotamers around the C-7 to C-8 carbon bond, with H-5/H2-8 exhibiting both anti and gauche orientations. The relative configurations of C-4 to C-13, C-34 to C-46, and C-20 to C-42 were elucidated using the same method (Figure 2). The proposed configuration of C-4/C-13, C-34/C-36, and C-20/C42 was in agreement with the NOE interactions observed among these protons (Figure S8). The relative configuration of the spiroketal moiety in 1 was also established by a NOESY experiment, in which the NOE correlation patterns were similar to other oligomycins.22 A growing body of evidence shows a common absolute

the assignment of the two methine protons of C-4 and C-5. The anti orientation of H-4/H-5 was suggested by the large 3J (H-4/H-5), small 3J (H-5/C-3), and small 3J (H-4/C-6) values. These interactions established the relative configurations of C-4 to C-6 as depicted in Figure 2. H-4 was found to be gauche to 5OH (δH 4.28, established by HMBC correlations) and anti to H-5 (Figures 2 and S8, Supporting Information). This observation is supported by the NOE correlation of H3-36/ H-5. The intermediate values for the homonuclear 3J (H-7/H8a, ca. 9 Hz) and 3J (H-7/H-8b, ca. 7 Hz) and heteronuclear coupling constants between H-8b/C-6 (ca. 2 Hz) led us to 1978



Article

Figure 2. Relative configuration determined for the C-4 to C-13, C-30 to C-34, and C-20 to C-42 segments: (A) Newman projection (H8a, H10a, H14a, H32a, and H41a, protons at low fields; H8b, H10b H14b, H32b, and H41b, protons at high fields; NA represents values that could not be measured due to overlapping signals). (B) Structural diagrams.

configurational assignment within the family of spiroketal macrolides.22−24 It is conceivable therefore that the configuration for 1 may be the same as that for oligomycin because 1 and oligomycin have similar stereocenters. In the oligomycin family, the configuration at C-23 has been assigned as S and that at C-20 as R.24 Neomaclafungins (1−9) and maclafungin (10) exhibit several distinct features that are absent in all other members of the oligomycin group. The specific features observed are the substitution patterns at C-7 and C-11 (hydroxy group instead of a ketone), C-8, C-10, and C-14 (nonsubstituted instead of a methyl group), C-9 (methyl ether instead of a hydroxy), C-12 (methyl instead of methyl and hydroxy groups), and C-24 (C2−C5 alkane or C3−C4 alcohol groups instead of a methyl group). Therefore, the maclafungins, which is the generic name for neomaclafungins (1−9) and maclafungin (10), are unique members in the oligomycin class. The neomaclafungins and oligomycin A (11) were evaluated for activity in a Trichophyton mentagrophytes (ATCC 9533) microbial assay at concentrations that ranged from 10 μg/mL to 10 ng/mL. Significant responses were noted for the neomaclafungins with minimal inhibitory concentration (MIC) values between 3 and 1 μg/mL (Table 3). In our research, the MIC values of compounds 1−9 are similar, and the inhibitory potency of compound 11 is the weakest. This distinction may be caused by the different structures, such as

Table 3. Antifungal Activities of Neomaclafungins A−I (1− 9) MIC (μg/mL) Trichophyton mentagrophytes (ATCC 9533) neomaclafungin A (1) B (2) C (3) D (4) E (5) F (6) G (7) H (8) I (9) oligomycin A (11)

3 3 1 1 1 3 3 3 3 10

the absence of ketones in the 26-membered ring and the substituent at C-24.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a HORIBA SEPA-500 polarimeter with a 1 cm cell. UV spectra were measured with a JASCO V-560 UV/vis spectrophotometer. IR spectra were acquired on a JASCO FTIR VALOR-III spectrophotometer. The 1H, 13C, and 2D NMR spectroscopic data were obtained on a Varian NMR system 600NB 600 MHz NMR spectrometer. HRESITOFMS data were recorded on 1979



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H-10), 1.76 (1H, dd, J = 12.9, 4.8 Hz, H-26), 1.63 (1H, dd, J = 12.9, 12.2 Hz, H-26), 1.63 (1H, m, H-28), 1.61 (1H, m, H-32), 1.61 (1H, m, H-14), 1.58 (1H, m, H-8), 1.58 (1H, m, H-22), 1.58 (1H, m, H30), 1.52 (1H, m, H-8), 1.50 (1H, m, H-12), 1.49 (2H, m, H2-21), 1.44 (1H, m, H-14), 1.43 (1H, m, H-10), 1.43 (1H, m, H-28), 1.40 (1H, m, H-44), 1.39 (1H, d, J = 13.5 Hz, H- 29), 1.38 (1H, m, H-43), 1.37 (1H, m, H-41), 1.36 (1H, m, H-6), 1.34 (1H, m, H-43), 1.32 (1H, m, H-41), 1.31 (1H, m, H-44), 1.25 (1H, m, H-32), 1.24 (1H, m, H-22), 1.22 (3H, d, J = 6.3 Hz, H3-34), 1.15 (3H, d, J = 6.3 Hz, H336), 1.08 (3H, d, J = 6.3 Hz, H3-47), 0.90 (3H, d, J = 6.8 Hz, H3-46), 0.87 (3H, d, J = 6.9 Hz, H3-38), 0.81 (3H, t, J = 7.3 Hz, H3-42), 0.74 (3H, d, J = 6.8 Hz, H3-40); 13C NMR data, see Table 2; HRESITOFMS m/z 795.5624 [M + H]+ (calcd for C45H79O11, 795.5617). Neomaclafungin C (3): white, amorphous powder; [α]25D +11.9 (c 0.11, CHCl3); UV (MeOH) λmax (log ε) 224 (4.44), 231 (4.39), 240 (4.18); IR (KBr) 3421, 2962, 2926, 2876, 1717, 1700, 1647, 1458, 1385, 1281, 1131, 1082, 987 cm−1; 1H NMR (600 MHz, CDCl3) δ 6.59 (1H, d, J = 15.6, 10.4 Hz, H-3), 6.10 (1H, dd, J = 14.9, 10.5 Hz, H-17), 5.94 (1H, dd, J = 14.9, 10.5 Hz, H-18), 5.78 (1H, d, J = 15.6 Hz, H-2), 5.44 (1H, ddd, J = 14.9, 11.0, 3.9 Hz, H-16), 5.35 (1H, dd, J = 14.9, 9.7 Hz, H-19), 5.30 (1H, dt, J = 12.2, 4.8 Hz, H-25), 4.07 (1H, d, J = 9.7 Hz, H-7), 4.01 (1H, d, J = 10.4 Hz, H-31), 3.80 (1H, m, H23), 3.77 (1H, m, H-33), 3.77 (1H, t, J = 10.2 Hz, H-9), 3.65 (1H, d, J = 9.6 Hz, H-5), 3.45 (1H, m, H-45), 3.41 (1H, m, H-45), 3.54 (1H, dd, J = 11.2, 8.8 Hz, H-13), 3.43 (1H, dd, J = 11.6, 8.9 Hz, H-11), 3.37 (3H, s, OMe-39), 2.42 (1H, ddq, J = 10.4, 9.6, 6.3 Hz, H-4), 2.28 (2H, m, H2-15), 2.10 (1H, tt, J = 13.5, 4.1 Hz, H-29), 1.92 (1H, m, H-20), 1.88 (1H, m, H-24), 1.86 (1H, dd, J = 13.7, 11.6 Hz, H-10), 1.79 (1H, dd, J = 12.9, 4.8 Hz, H-26), 1.70 (1H, m, H-14), 1.67 (1H, dd, J = 12.9, 12.2 Hz, H-26), 1.64 (1H, m, H-32), 1.64 (1H, m, H-22), 1.64 (1H, m, H-28), 1.57 (1H, m, H-30), 1.56 (1H, m, H-8), 1.55 (1H, m, H-34), 1.51 (1H, m, H-44), 1.50 (1H, m, H-8), 1.50 (1H, m, H-12), 1.49 (1H, m, H-21), 1.48 (1H, m, H-34), 1.47 (1H, m, H-21), 1.46 (1H, m, H-14), 1.44 (1H, m, H-43), 1.43 (1H, m, H-28), 1.43 (1H, m, H-10), 1.41 (1H, m, H-43), 1.39 (1H, d, J = 13.5 Hz, H-29), 1.38 (1H, m, H-41), 1.33 (1H, m, H-6), 1.32 (1H, m, H-44), 1.31 (1H, m, H41), 1.25 (1H, m, H-32), 1.16 (3H, d, J = 6.3 Hz, H3-36), 1.16 (1H, m, H-22), 0.96 (3H, t, J = 7.2 Hz, H3-35), 0.91 (3H, d, J = 6.8 Hz, H3-46), 0.86 (3H, d, J = 6.9 Hz, H3-38), 0.82 (3H, t, J = 7.3 Hz, H3-42), 0.75 (3H, d, J = 6.8 Hz, H3-40); 13C NMR data, see Table 2; HRESITOFMS m/z 795.5626 [M + H]+ (calcd for C45H79O11, 795.5617). Neomaclafungin D (4): white, amorphous powder; [α]25D +30.9 (c 0.10, CHCl3); UV (MeOH) λmax (log ε) 224 (4.28), 231 (4.24), 240 (4.07); IR (KBr) 3412, 2962, 2926, 2856, 1717, 1700, 1653, 1457, 1385, 1281, 1131, 1084, 987 cm−1; 1H NMR (600 MHz, CDCl3) δ 6.60 (1H, d, J = 15.6, 10.4 Hz, H-3), 6.07 (1H, dd, J = 14.9, 10.5 Hz, H-17), 5.95 (1H, dd, J = 14.9, 10.5 Hz, H-18), 5.79 (1H, d, J = 15.6 Hz, H-2), 5.46 (1H, ddd, J = 14.9, 11.0, 3.9 Hz, H-16), 5.38 (1H, dd, J = 14.9, 9.7 Hz, H-19), 5.28 (1H, dt, J = 12.2, 4.8 Hz, H-25), 4.05 (1H, d, J = 9.7 Hz, H-7), 4.02 (1H, d, J = 10.4 Hz, H-31), 3.79 (1H, m, H23), 3.78 (1H, m, H-33), 3.78 (1H, t, J = 13.7 Hz, H-9), 3.65 (1H, m, H-45), 3.62 (1H, d, J = 9.6 Hz, H-5), 3.57 (1H, dd, J = 11.2, 8.8 Hz, H-13), 3.41 (1H, dd, J = 11.6, 8.9 Hz, H-11), 3.37 (3H, s, OMe-39), 2.43 (1H, ddq, J = 10.4, 9.6, 6.3 Hz, H-4), 2.30 (1H, m, H-15), 2.24 (1H, m, H-15), 2.12 (1H, tt, J = 13.5, 4.1 Hz, H-29), 1.99 (1H, m, H20), 1.94 (1H, m, H-24), 1.85 (1H, dd, J = 13.7, 11.6 Hz, H-10), 1.79 (1H, dd, J = 12.9, 4.8 Hz, H-26), 1.65 (1H, m, H-22), 1.65 (1H, m, H28), 1.64 (1H, m, H-32), 1.64 (1H, dd, J = 12.9, 12.2 Hz, H-26), 1.63 (1H, m, H-14), 1.59 (1H, m, H-30), 1.59 (1H, m, H-8), 1.53 (1H, m, H-8), 1.53 (1H, m, H-34), 1.52 (1H, m, H-12), 1.50 (H, m, H-21), 1.48 (1H, m, H-34), 1.48 (H, m, H-21), 1.47 (1H, m, H-14), 1.44 (1H, m, H-28), 1.44 (1H, m, H-10), 1.41 (1H, m, H-44), 1.40 (1H, d, J = 13.5 Hz, H-29), 1.37 (1H, m, H-43), 1.37 (1H, m, H-41), 1.36 (1H, m, H-6), 1.34 (1H, m, H-43), 1.33 (1H, m, H-41), 1.28 (1H, m, H-44), 1.24 (1H, m, H-32), 1.17 (1H, m, H-22), 1.17 (3H, d, J = 6.3 Hz, H3-36), 1.09 (3H, d, J = 6.3 Hz, H3-47), 0.97 (3H, t, J = 7.6 Hz, H3-35), 0.92 (3H, d, J = 6.8 Hz, H3-46), 0.89 (3H, d, J = 6.9 Hz, H338), 0.83 (3H, t, J = 7.6 Hz, H3-42), 0.76 (3H, d, J = 6.8 Hz, H3-40);

a Waters LCT-Premier XE mass spectrometer. Preparative HPLC separations were performed using a JAI LC-9104 system with a UV variable-wavelength detector set at 224 nm. A JAIGEL-ODS-AP-30, 15 μm, 120 Å (30 mm × 250 mm) column was used. NMR Method for Measuring 3JC,H. Long-range C−H coupling constants 3JC,H were measured by using the HETLOC (ω1-hetero halffiltered TOCSY)20 experiment with a zero-quantum filter21 for artifact suppression: The DIPSI2 spin-lock period was set for 80 ms. The HETLOC spectra were acquired with 616 scans per increment for a 4K (F2) × 2K (F1) data matrix for the spectral width of 5434 Hz for both dimensions on a 600 MHz spectrometer at 20 °C. Bacterial Isolation and Identification. Strain NPS702 was obtained from a marine sediment sample collected at a depth of ca. 20 m from Usa Bay, Kochi Prefecture, Japan. The sediment was first heatshocked at 60 °C for 6 min and then directly plated onto medium C1 (2 g of colloidal chitin (Sigma), 150 mg of KH2PO4, 210 mg of K2HPO4, 250 mg of MgSO4·7H2O, 18 g of Daigo’s artificial seawater powder, 18 g of agar, 1 L of distilled water, pH 7.2). The strain was identified as an Actinoalloteichus sp. based on 16S rRNA gene sequence analysis (DDBJ accession number AB643798) and is most similar (99.2%) to the marine sponge-derived species Actinoalloteichus hymeniacidonis strain HPA177 (accession number DQ144222). Fermentation and Extraction. Actinoalloteichus strain NPS702 was cultured in 500 mL baffled Erlenmeyer flasks that contained 100 mL of the production-modified KG medium with 0.8% glucose, 0.8% maltose monohydrate, 0.8% soluble starch, 1.5% soytone, 0.2% yeast extract, and 1.8% Daigo’s artificial seawater powder. The flasks were shaken at 220 rpm and 28.5 °C for 8 days. At the end of the fermentation period, the whole culture broth was extracted with an equal volume of EtOAc, after which the EtOAc was removed under vacuum to give 3.2 g of solid material from a 16.2 L culture. Isolation of Neomaclafungins A−I (1−9). The EtOAc extract (3.2 g) was fractionated by silica gel open column chromatography by eluting with a step gradient from CHCl3 to MeOH to give three fractions. Fraction two (eluted with CHCl3−MeOH, 9:1), which contained the macrolides, was subjected to reversed-phase (ODS) open column chromatography with a step gradient from 70% MeOH in H2O to 10% MeOH in 2-propanol to yield four fractions. Fraction 2 (eluted with MeOH−H2O, 9:1, the first and second column volumes) contained the macrolides with six hydroxy groups and was subjected to HPLC (JAIGEL-ODS-AP-30, 30 mm × 250 mm, 15 μm) and eluted with 82.5% aqueous MeOH at a 15.0 mL/min flow rate. Neomaclafungins A (1, 22.8 mg), B (2, 1.7 mg), C (3, 12.7 mg), and D (4, 2.3 mg) eluted at 37.4, 42.7, 47.1, and 53.3 min, respectively. Fraction 3 (eluted with MeOH−H2O, 9:1, the third and fourth column volumes) contained the macrolides with five hydroxy groups and was subjected to the same HPLC separation procedure as fraction 2. Neomaclafungins E (5, 6.7 mg), F (6, 11.9 mg), G (7, 8.2 mg), H (8, 4.1 mg), and I (9, 2.2 mg) eluted at 59.1, 67.0, 77.7, 86.4, and 96.3 min, respectively. Neomaclafungin A (1): white, amorphous powder; [α]25D +16.5 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 224 (4.40), 231 (4.33), 240 (4.13); IR (KBr) 3395, 2927, 2858, 1717, 1653, 1459, 1385, 1281, 1131, 1082, 988 cm−1; NMR data, see Table 1; HRESITOFMS m/z 781.5470 [M + H]+ (calcd for C44H77O11, 781.5460). Neomaclafungin B (2): white, amorphous powder; [α]25D +21 (c 0.06, CHCl3); UV (MeOH) λmax (log ε) 223 (4.31), 231 (4.28), 240 (4.05); IR (KBr) 3421, 2961, 2926, 2856, 1717, 1699, 1671, 1457, 1385, 1282, 1131, 988 cm−1; 1H NMR (600 MHz, CDCl3) δ 6.58 (1H, d, J = 15.6, 10.4 Hz, H-3), 6.05 (1H, dd, J = 14.9, 10.5 Hz, H-17), 5.93 (1H, dd, J = 14.9, 10.5 Hz, H-18), 5.78 (1H, d, J = 15.6 Hz, H-2), 5.44 (1H, ddd, J = 14.9, 11.0, 3.9 Hz, H-16), 5.39 (1H, dd, J = 14.9, 9.7 Hz, H-19), 5.29 (1H, dt, J = 12.2, 4.8 Hz, H-25), 4.04 (1H, d, J = 9.7 Hz, H-7), 4.03 (1H, m, H-33), 4.00 (1H, d, J = 10.4 Hz, H-31), 3.78 (1H, dd, J = 6.2, 4.2 Hz, H-23), 3.76 (1H, t, J = 10.1 Hz, H-9), 3.63 (1H, m, H-45), 3.61 (1H, d, J = 9.6 Hz, H-5), 3.56 (1H, dd, J = 11.2, 8.8 Hz, H-13), 3.40 (1H, dd, J = 11.6, 8.9 Hz, H-11), 3.36 (3H, s, OMe-39), 2.42 (1H, ddq, J = 10.4, 9.6, 6.3 Hz, H-4), 2.30 (1H, m, H15), 2.23 (1H, m, H-15), 2.11 (1H, tt, J = 13.5, 4.1 Hz, H-29), 1.97 (1H, m, H-20), 1.92 (1H, m, H-24), 1.84 (1H, dd, J = 13.7, 11.6 Hz, 1980



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13

C NMR data, see Table 2; HRESITOFMS m/z 809.5770 [M + H]+ (calcd for C46H81O11, 809.5773). Neomaclafungin E (5): white, amorphous powder; [α]25D +11.3 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 224 (4.40), 231 (4.37), 240 (4.13); IR (KBr) 3422, 2961, 2935, 2879, 1717, 1700, 1654, 1457, 1386, 1282, 1131, 1087, 988 cm−1; 1H NMR (600 MHz, CDCl3) δ 6.57 (1H, d, J = 15.6, 10.3 Hz, H-3), 6.15 (1H, dd, J = 14.9, 10.5 Hz, H-17), 5.94 (1H, dd, J = 14.9, 10.5 Hz, H-18), 5.77 (1H, d, J = 15.6 Hz, H-2), 5.46 (1H, ddd, J = 14.9, 11.0, 3.4 Hz, H-16), 5.30 (1H, dd, J = 14.9, 9.8 Hz, H-19), 5.26 (1H, dt, J = 12.3, 4.6 Hz, H-25), 4.10 (1H, d, J = 9.1 Hz, H-7), 4.04 (1H, m, H-33), 3.99 (1H, d, J = 10.6 Hz, H31), 3.80 (1H, t, J = 10.2 Hz, H-9), 3.79 (1H, d, J = 10.2 Hz, H-23), 3.64 (1H, d, J = 9.6 Hz, H-5), 3.50 (1H, dd, J = 11.2, 8.8 Hz, H-13), 3.38 (3H, s, OMe-39), 3.36 (1H, dd, J = 11.6, 8.9 Hz, H-11), 2.39 (1H, ddq, J = 10.3, 9.6, 6.4 Hz, H-4), 2.34 (1H, br d, J = 14.4 Hz, H15), 2.22 (1H, m, H-15), 2.09 (1H, tt, J = 13.5, 4.1 Hz, H-29), 1.82 (1H, m, H-14), 1.82 (1H, m, H-20), 1.82 (1H, dd, J = 13.7, 11.6 Hz, H-10), 1.74 (1H, dd, J = 13.1, 4.6 Hz, H-26), 1.71 (1H, m, H-24), 1.62 (1H, m, H-28), 1.62 (1H, m, H-22), 1.62 (1H, dd, J = 13.1, 12.3 Hz, H-26), 1.60 (1H, m, H-32), 1.57 (1H, m, H-30), 1.52 (1H, m, H-21), 1.51 (1H, m, H-8), 1.48 (1H, m, H-21), 1.47 (1H, m, H-8), 1.45 (1H, m, H-12), 1.44 (1H, m, H-14), 1.43 (1H, m, H-28), 1.41 (1H, m, H10), 1.40 (1H, m, H-43), 1.39 (1H, m, H-41), 1.38 (1H, d, J = 13.5 Hz, H-29), 1.33 (1H, m, H-43), 1.31 (1H, q, J = 6.8 Hz, H-6), 1.28 (1H, m, H-41), 1.26 (1H, m, H-32), 1.23 (3H, d, J = 6.3 Hz, H3-34), 1.16 (3H, d, J = 6.5 Hz, H3-36), 1.02 (1H, dd, J = 13.9, 11.4 Hz, H22), 0.91 (3H, d, J = 7.0 Hz, H3-46), 0.85 (3H, d, J = 6.8 Hz, H3-38), 0.83 (3H, t, J = 7.3 Hz, H3-42), 0.77 (3H, t, J = 7.6 Hz, H3-44), 0.73 (3H, d, J = 6.8 Hz, H3-40); 13C NMR data, see Table 2; HRESITOFMS m/z 751.5388 [M + H]+ (calcd for C43H75O10, 751.5355). Neomaclafungin F (6): white, amorphous powder; [α]25D +18.3 (c 0.11, CHCl3); UV (MeOH) λmax (log ε) 224 (4.48), 231 (4.45), 240 (4.21); IR (KBr) 3439, 2960, 2931, 2878, 1717, 1700, 1653, 1457, 1386, 1282, 1131, 1088, 989 cm−1; 1H NMR (600 MHz, CDCl3) δ 6.56 (1H, d, J = 15.6, 10.4 Hz, H-3), 6.15 (1H, dd, J = 14.9, 10.5 Hz, H-17), 5.93 (1H, dd, J = 14.9, 10.5 Hz, H-18), 5.77 (1H, d, J = 15.6 Hz, H-2), 5.45 (1H, ddd, J = 14.9, 11.0, 3.4 Hz, H-16), 5.31 (1H, dd, J = 14.9, 9.8 Hz, H-19), 5.25 (1H, dt, J = 12.2, 4.6 Hz, H-25), 4.10 (1H, t, J = 5.0 Hz, H-7), 4.03 (1H, m, H-33), 3.99 (1H, d, J = 10.4 Hz, H31), 3.80 (1H, t, J = 10.2 Hz, H-9), 3.79 (1H, d, J = 9.8 Hz, H-23), 3.64 (1H, d, J = 9.7 Hz, H-5), 3.50 (1H, dd, J = 11.2, 8.8 Hz, H-13), 3.37 (3H, s, OMe-39), 3.35 (1H, dd, J = 11.6, 8.9 Hz, H-11), 2.40 (1H, ddq, J = 10.4, 9.7, 6.4 Hz, H-4), 2.34 (1H, br d, J = 13.4 Hz, H15), 2.21 (1H, m, H-15), 2.10 (1H, tt, J = 13.5, 4.1 Hz, H-29), 1.86 (1H, m, H-24), 1.84 (1H, m, H-20), 1.82 (1H, dd, J = 13.8, 11.7 Hz, H-10), 1.81 (1H, m, H-14), 1.75 (1H, dd, J = 12.9, 4.6 Hz, H-26), 1.62 (1H, m, H-28), 1.62 (1H, m, H-22), 1.61 (1H, dd, J = 12.9, 12.0 Hz, H-26), 1.60 (1H, m, H-32), 1.57 (1H, m, H-30), 1.55 (1H, m, H-8), 1.51 (1H, m, H-21), 1.49 (1H, m, H-8), 1.47 (1H, m, H-21), 1.46 (1H, m, H-12), 1.44 (1H, m, H-14), 1.43 (1H, m, H-28), 1.42 (1H, m, H-43), 1.41 (1H, m, H-10), 1.38 (1H, m, H-41), 1.38 (1H, d, J = 13.5 Hz, H-29), 1.31 (1H, q, J = 7.2 Hz, H-6), 1.29 (1H, m, H-41), 1.26 (1H, m, H-32), 1.25 (1H, m, H-43), 1.23 (3H, d, J = 6.2 Hz, H3-34), 1.22 (1H, m, H-44), 1.14 (1H, m, H-44), 1.16 (3H, d, J = 6.3 Hz, H336), 1.04 (1H, dd, J = 14.9, 11.4 Hz, H-22), 0.91 (3H, d, J = 6.8 Hz, H3-46), 0.85 (3H, d, J = 7.2 Hz, H3-38), 0.83 (3H, t, J = 7.3 Hz, H342), 0.73 (3H, d, J = 6.7 Hz, H3-40), 0.71 (3H, t, J = 7.6 Hz, H3-45), 13 C NMR data, see Table 2; HRESITOFMS m/z 765.5542 [M + H]+ (calcd for C44H77O10, 765.5511). Neomaclafungin G (7): white, amorphous powder; [α]25D +24.4 (c 0.10, CHCl3); UV (MeOH) λmax (log ε) 224 (4.46), 231 (4.41), 240 (4.16); IR (KBr) 3422, 2960, 2936, 2874, 1717, 1699, 1653, 1457, 1386, 1282, 1131, 1082, 988 cm−1; 1H NMR (600 MHz, CDCl3) δ 6.57 (1H, d, J = 15.5, 10.3 Hz, H-3), 6.15 (1H, dd, J = 14.9, 10.5 Hz, H-17), 5.93 (1H, dd, J = 14.9, 10.5 Hz, H-18), 5.77 (1H, d, J = 15.5 Hz, H-2), 5.44 (1H, ddd, J = 14.9, 11.0, 3.4 Hz, H-16), 5.32 (1H, dd, J = 14.9, 9.6 Hz, H-19), 5.24 (1H, dt, J = 12.2, 4.5 Hz, H-25), 4.12 (1H, t, J = 5.0 Hz, H-7), 4.04 (1H, m, H-33), 3.98 (1H, d, J = 10.4 Hz, H31), 3.81 (1H, m, H-9), 3.79 (1H, d, J = 9.8 Hz, H-23), 3.64 (1H, d, J

= 9.7 Hz, H-5), 3.53 (1H, dd, J = 11.2, 8.8 Hz, H-13), 3.44 (1H, dd, J = 10.4, 9.5 Hz, H-11), 3.38 (3H, s, OMe-39), 2.40 (1H, ddq, J = 10.4, 9.7, 6.3 Hz, H-4), 2.35 (1H, br d, J = 14.5 Hz, H-15), 2.22 (1H, m, H15), 2.10 (1H, tt, J = 13.5, 4.1 Hz, H-29), 1.89 (1H, m, H-24), 1.86 (1H, m, H-20), 1.84 (1H, dd, J = 13.8, 11.6 Hz, H-10), 1.81 (1H, m, H-14), 1.76 (1H, dd, J = 12.8, 4.6 Hz, H-26), 1.63 (1H, m, H-22), 1.63 (1H, m, H-28), 1.61 (1H, dd, J = 12.8, 12.0 Hz, H-26), 1.60 (1H, m, H-32), 1.57 (1H, m, H-30), 1.53 (1H, m, H-8), 1.51 (1H, m, H-8), 1.50 (1H, m, H-21), 1.48 (1H, m, H-12), 1.46 (1H, m, H-21), 1.45 (1H, m, H-14), 1.44 (1H, m, H-28), 1.43 (1H, m, H-43), 1.43 (1H, m, H-10), 1.39 (1H, m, H-41), 1.38 (1H, d, J = 13.5 Hz, H-29), 1.32 (1H, q, J = 7.2 Hz, H-6), 1.31 (1H, m, H-41), 1.26 (1H, m, H-32), 1.24 (1H, m, H-43), 1.23 (3H, d, J = 6.2 Hz, H3-34), 1.17 (1H, m, H-45), 1.17 (1H, m, H-44), 1.16 (3H, d, J = 6.3 Hz, H3-36), 1.10 (1H, m, H44), 1.09 (1H, dd, J = 14.9, 11.4 Hz, H-22), 1.02 (1H, dd, J = 14.5, 7.6 Hz, H-45), 0.91 (3H, d, J = 6.8 Hz, H3-46), 0.85 (3H, d, J = 7.2 Hz, H3-38), 0.82 (3H, t, J = 7.3 Hz, H3-42), 0.82 (3H, t, J = 7.6 Hz, H347), 0.74 (3H, d, J = 6.8 Hz, H3-40); 13C NMR data, see Table 2; HRESITOFMS m/z 779.5675 [M + H]+ (calcd for C45H79O10, 779.5668). Neomaclafungin H (8): white, amorphous powder; [α]25D +29.0 (c 0.10, CHCl3); UV (MeOH) λmax (log ε) 224 (4.45), 231 (4.39), 240 (4.13); IR (KBr) 3421, 2959, 2930, 2874, 1717, 1699, 1653, 1457, 1386, 1282, 1131, 1109, 988 cm−1; 1H NMR (600 MHz, CDCl3) δ 6.56 (1H, d, J = 15.6, 10.4 Hz, H-3), 6.15 (1H, dd, J = 14.9, 10.5 Hz, H-17), 5.94 (1H, dd, J = 14.9, 10.5 Hz, H-18), 5.77 (1H, d, J = 15.6 Hz, H-2), 5.45 (1H, ddd, J = 14.9, 10.8, 3.4 Hz, H-16), 5.31 (1H, dd, J = 14.9, 9.7 Hz, H-19), 5.24 (1H, dt, J = 12.2, 4.7 Hz, H-25), 4.11 (1H, t, J = 5.2 Hz, H-7), 4.00 (1H, d, J = 10.4 Hz, H-31), 3.81 (1H, m, H9), 3.78 (1H, d, J = 9.8 Hz, H-23), 3.77 (1H, m, H-33), 3.65 (1H, d, J = 9.8 Hz, H-5), 3.50 (1H, dd, J = 11.2, 8.7 Hz, H-13), 3.38 (1H, dd, J = 11.6, 8.9 Hz, H-11), 3.38 (3H, s, OMe-39), 2.40 (1H, ddq, J = 10.4, 9.8, 6.3 Hz, H-4), 2.35 (1H, br d, J = 14.5 Hz, H-15), 2.22 (1H, m, H15), 2.11 (1H, tt, J = 13.5, 4.1 Hz, H-29), 1.88 (1H, m, H-24), 1.85 (1H, m, H-20), 1.83 (1H, dd, J = 13.8, 11.6 Hz, H-10), 1.82 (1H, m, H-14), 1.76 (1H, dd, J = 12.6, 4.7 Hz, H-26), 1.63 (1H, m, H-28), 1.62 (1H, m, H-22), 1.62 (1H, m, H-32), 1.61 (1H, t, J = 12.6 Hz, H-26), 1.58 (1H, m, H-30), 1.54 (1H, m, H-8), 1.53 (1H, m, H-8), 1.53 (1H, m, H-34), 1.51 (1H, m, H-21), 1.48 (1H, m, H-21), 1.47 (1H, m, H12), 1.47 (1H, m, H-34), 1.45 (1H, m, H-14), 1.44 (1H, m, H-28), 1.43 (1H, m, H-43), 1.41 (1H, m, H-10), 1.39 (1H, m, H-41), 1.39 (1H, d, J = 13.5 Hz, H-29), 1.32 (1H, q, J = 7.2 Hz, H-6), 1.29 (1H, m, H-41), 1.25 (1H, m, H-43), 1.24 (1H, m, H-32), 1.23 (1H, m, H-44), 1.16 (3H, d, J = 6.3 Hz, H3-36), 1.15 (1H, m, H-44), 1.05 (1H, dd, J = 14.9, 11.4 Hz, H-22), 0.97 (3H, d, J = 7.4 Hz, H3-35), 0.92 (3H, d, J = 6.8 Hz, H3-46), 0.85 (3H, d, J = 7.2 Hz, H3-38), 0.83 (3H, t, J = 7.3 Hz, H3-42), 0.73 (3H, d, J = 6.8 Hz, H3-40), 0.72 (3H, t, J = 7.3 Hz, H3-45); 13C NMR data, see Table 2; HRESITOFMS m/z 779.5699 [M + H]+ (calcd for C45H79O10, 779.5668). Neomaclafungin I (9): white, amorphous powder; [α]25D +17 (c 0.09, CHCl3); UV (MeOH) λmax (log ε) 224 (4.39), 231 (4.35), 240 (4.11); IR (KBr) 3422, 2959, 2930, 2873, 2857, 1717, 1700, 1653, 1457, 1386, 1280, 1131, 988 cm−1; 1H NMR (600 MHz, CDCl3) δ 6.57 (1H, d, J = 15.6, 10.3 Hz, H-3), 6.16 (1H, dd, J = 14.9, 10.6 Hz, H-17), 5.92 (1H, dd, J = 14.9, 10.6 Hz, H-18), 5.77 (1H, d, J = 15.6 Hz, H-2), 5.44 (1H, ddd, J = 14.9, 10.8, 3.5 Hz, H-16), 5.32 (1H, dd, J = 14.9, 9.7 Hz, H-19), 5.24 (1H, dt, J = 12.2, 4.7 Hz, H-25), 4.12 (1H, t, J = 5.1 Hz, H-7), 4.04 (1H, m, H-33), 3.98 (1H, d, J = 10.4 Hz, H31), 3.81 (1H, m, H-9), 3.79 (1H, d, J = 9.7 Hz, H-23), 3.64 (1H, d, J = 9.5 Hz, H-5), 3.53 (1H, dd, J = 11.2, 8.7 Hz, H-13), 3.43 (1H, dd, J = 11.4, 8.9 Hz, H-11), 3.38 (3H, s, OMe-39), 2.40 (1H, ddq, J = 10.3, 9.5, 6.3 Hz, H-4), 2.35 (1H, br d, J = 14.8 Hz, H-15), 2.22 (1H, m, H15), 2.10 (1H, tt, J = 13.5, 4.1 Hz, H-29), 1.91 (1H, m, H-24), 1.87 (1H, m, H-20), 1.85 (1H, dd, J = 13.8, 11.6 Hz, H-10), 1.83 (1H, m, H-14), 1.76 (1H, dd, J = 12.6, 4.7 Hz, H-26), 1.64 (1H, m, H-22), 1.63 (1H, m, H-28), 1.63 (1H, dd, J = 12.8, 12.0 Hz, H-26), 1.62 (1H, m, H-32), 1.58 (1H, m, H-30), 1.55 (1H, m, H-8), 1.53 (1H, m, H-8), 1.52 (1H, m, H-45), 1.50 (1H, m, H-21), 1.50 (1H, m, H-45), 1.48 (1H, m, H-21), 1.48 (1H, m, H-12), 1.47 (1H, m, H-14), 1.45 (1H, m, H-28), 1.43 (1H, m, H-43), 1.43 (1H, m, H-10), 1.39 (1H, m, H-41), 1981



Article

1.39 (1H, d, J = 13.5 Hz, H-29), 1.33 (1H, q, J = 7.2 Hz, H-6), 1.31 (1H, m, H-41), 1.29 (1H, m, H-47), 1.26 (1H, m, H-32), 1.26 (1H, m, H-43), 1.26 (1H, m, H-44), 1.23 (3H, d, J = 6.2 Hz, H3-34), 1.22 (1H, m, H-47), 1.16 (1H, m, H-44), 1.16 (3H, d, J = 6.3 Hz, H3-36), 1.10 (1H, m, H-22), 0.91 (3H, d, J = 6.8 Hz, H3-46), 0.86 (3H, t, J = 7.3 Hz, H3-48), 0.85 (3H, d, J = 7.2 Hz, H3-38), 0.83 (3H, t, J = 7.3 Hz, H3-42), 0.74 (3H, d, J = 6.8 Hz, H3-40); 13C NMR data, see Table 2; HRESITOFMS m/z 793.5830 [M + H]+ (calcd for C46H81O10, 793.5824).

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Okino, T.; Nogle, L. M.; Marquez, B. L.; Wiliamson, R. T.; Sitachitta, N.; Berman, F. W.; Murray, T. F.; McGough, K.; Jacobs, R.; Colsen, K.; Asano, T.; Yokokawa, F.; Shioiri, T.; Gerwick, W. J. Am. Chem. Soc. 2000, 122, 12041−12042. (c) Kawahara, T.; Izumikawa, M.; Takagi, M.; Shin-ya, K. Org. Lett. 2012, 14, 4434−4437. (19) (a) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana, K. J. Org. Chem. 1999, 64, 866−876. (b) Matsumori, N.; Nonomura, T.; Sasaki, M.; Murata, M.; Tachibana, K.; Satake, M.; Yasumoto, T. Tetrahedron Lett. 1996, 37, 1269−1272. (20) Uhrin, D.; Batta, G.; Hruby, V. J.; Barlow, P. N.; Koever, K. E. J. Magn. Reson. 1998, 130, 155−161. (21) Thrippleton, M. J.; Keeler, J. Angew. Chem., Int. Ed. 2003, 42, 3938−3941. (22) (a) Gareth, A.; Morris, A. G.; Richards, S. M. Magn. Reson. Chem. 1985, 23, 676−683. (b) Nakata, M.; Ishiyama, T.; Hirose, Y.; Maruoka, H.; Tatsuta1, K. Tetrahedron Lett. 1993, 34, 8439−8442. (c) Wagenaar, M. M.; Williamson, R. T.; Ho, D. M.; Carter, G. T. J. Nat. Prod. 2007, 70, 367−371. (23) (a) Kirst, H. A.; Larsen, S. H.; Paschal, J. W.; Occolowitz, J. L.; Creemer, L. C.; Steiner, J. L.; Lobkovsky, E.; Clardy, J. J. Antibiot. 1995, 48, 990−996. (b) Kirst, H. A.; Mynderse, J. S.; Martin, J. W.; Baker, P. J.; Paschal, J. W.; Rios Steiner, J. L.; Lobkovsky, E.; Clardy, J. J. Antibiot. 1996, 49, 162−167. (c) Hayashi, K.; Ogino, K.; Oono, Y.; Uchimiya, H.; Nozaki, H. J. Antibiot. 2001, 54, 573−581. (24) (a) Evans, D. A.; Rieger, L. D.; Jones, K. T.; Kaldor, W. S. J. Org. Chem. 1990, 55, 6260−6268. (b) Evans, D. A.; Ng, P. H.; Rieger, L. D. J. Am. Chem. Soc. 1993, 115, 11446−11459. (c) Palmer, A. R.; Potter, S. B. J. Chem. Crystallogr. 2008, 38, 243−253.

ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR, COSY, HSQC, and HMBC spectra of 1−9 and HETLOC and NOESY spectrum of 1 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-42-638-0549. Fax: +81-42-638-0685. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. M. Kuramoto (Ehime University) for previewing the manuscript. We wish to thank Dr. T. Mukai and Dr. M. Aoki for support with sample collection and culture. We greatly appreciate Dr. J. Kurita (Agilent Technologies Japan) for technical support with the HETLOC experiment.

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REFERENCES

(1) Fenical, W. Chem. Rev. 1993, 93, 1673−1684. (2) Bugni, T. S.; Ireland, C. M. Nat. Prod. Rep. 2004, 21, 143−163. (3) Bhakuni, D. S.; Rawat, D. S. Bioactive Marine Natural Products; Springer: New York, 2005; pp 13−17. (4) Fenical, W.; Jensen, P. R. Nat. Chem. Biol. 2006, 2, 666−673. (5) Cragg, G. M.; Grothaus, P. G.; Newman, D. J. Chem. Rev. 2009, 109, 3012−3043. (6) Bhatnagar, I.; Kim, S. K. Mar. Drugs 2010, 8, 2673−2701. (7) Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep, M. R. Nat. Prod. Rep. 2011, 28, 196−268. (8) Kieser, T.; Bibb, M. J.; Buttner, M. J.; Chater, K. F.; Hopwood, D. A. Practical Streptomyces Genetics, 2nd ed.; John Innes Foundation: England, 2000; pp 354−356. (9) Yamazaki, M.; Yamashita, T.; Harada, T.; Nishikiori, T.; Saito, S.; Shimada, N.; Fujii, A. J. Antibiot. 1992, 45, 171−179. (10) Masamune, S.; Sehgal, J. M.; van Tamelen, E. E.; Strong, F. M.; Peterson, W. H. J. Am. Chem. Soc. 1958, 80, 6092−6095. (11) Enomoto, Y.; Shiomi, K.; Matsumoto, A.; Takahashi, Y.; Iwai, Y.; Harder, A.; Kölbl, H.; Woodruff, H. B.; Ohmura, S. J. Antibiot. 2001, 54, 308−313. (12) Laatsch, H.; Kellner, M.; Wolf, G.; Lee, Y.; Hansske, F.; Konetschny-Rapp, S.; Pessara, U.; Scheuer, W.; Stockinger, H. J. Antibiot. 1993, 46, 1334−1341. (13) Kim, H. S.; Han, S. B.; Kim, H. M.; Kim, Y. H.; Lee, J. J. J. Antibiot. 1996, 49, 1275−1277. (14) Lardy, H. A.; Johnson, D.; McMurray, W. C. Arch. Biochem. Biophys. 1958, 78, 587−597. (15) Lardy, H. A.; Witoncky, P.; Johnson, D. Biochemistry 1965, 4, 552−554. (16) Tzagoloff, A.; Meagher, P. J. Biol. Chem. 1972, 274, 594−603. (17) Mukhopadhyay, T.; Nadkarni, S. R.; Patel, M. V.; Bhat, R. G.; Desikan, K. R.; Ganguli, B. N.; Rupp, R. H.; Fehlhaber, H.-W.; Kogler, H. Tetrahedron 1998, 54, 13621−13628. (18) (a) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Corbett, T. H. J. Am. Chem. Soc. 2001, 123, 5418−5423. (b) Wu, M.; 1982


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