Naphthacemycins, novel circumventors of β-lactam resistance in MRSA, produced by Streptomyces sp. KB-3346-5. II. Structure elucidation

Abstract

Seventeen new compounds, naphthacemycins A₁–A₁₁, B₁–B₄ and C₁–C₂, were isolated from a cultured broth of Streptomyces sp. KB-3346-5 during screening for circumventors of β-lactam resistance in methicillin-resistant Staphylococcus aureus. Their structures were elucidated by spectroscopic studies, including NMR and X-ray crystallographic analysis. The naphthacemycin A series has a new skeleton displaying a 7-phenylnaphthacene-5,6,11(12H)-trione. In contrast, the quinone moiety of the A series is changed to dehydroxyquinol in the B series and to a semiquinone-like structure in the C series.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of untreatable and potentially fatal hospital-associated infections. Community-acquired MRSA has also become a serious public health issue.¹ MRSA is resistant to β-lactam antibiotics and usually resistant to most other classes of antibiotics. There are a few antibiotics used for MRSA, for example, vancomycin, teicoplanin, arbekacin, linezolid, daptomycin and tigecycline, but microorganism strains resistant to these compounds are increasingly being reported. In the course of screening for new anti-MRSA compounds, we found cyslabdan, which enhances the activity of imipenem (a carbapenem type β-lactam antibiotic) against MRSA.^{2, 3} Further screening for microbial metabolites that circumvent the β-lactam resistance of MRSA led us to find the naphthacemycins A₁–A₁₁ (1–11), B₁–B₄ (12–15) and C₁–C₂ (16–17) (Scheme 1), reported as KB-3346-5 substances in the patent by our group,⁴ from a cultured broth of Streptomyces sp. KB-3346-5.⁵ Here we describe the structure elucidation of the naphthacemycins, based on NMR study and X-ray crystallographic analysis.

Results and discussion

Physicochemical properties

Naphthacemycins (1–17) were purified from a cultured broth of Streptomyces sp. KB-3346-5, isolated from a soil sample collected in Okinawa Prefecture, Japan.⁵ They are red powders. Compounds 2, 4-6, 8, 11 and 14–17 have one chlorine atom, while 7 and 10 have two chlorine atoms. The IR spectra showed they all have carbonyl (1600–1720 cm⁻¹) and hydroxyl (3350–3440 cm⁻¹) groups. UV spectra were observed at 274–280, 302–306 and 362–408 nm in the naphthacemycin A series (1–11), at 246–249, 287–288, 352–354 and 414–417 nm in the naphthacemycin B series (12–15), and 248 and 410 nm in the naphthacemycin C series (16–17). Naphthacemycins are soluble in chloroform, ethyl acetate and methanol and insoluble in n-hexane and H₂O.

Structure elucidation of the naphthacemycin A series

The molecular formula of naphthacemycin A₉ (9) was established as C₃₀H₂₆O₈ by HR-FAB-MS. ¹H and ¹³C NMR spectra and HSQC analysis revealed the presence of 30 carbons, including six methyl, six sp² methine, one sp³ quaternary and 17 sp² quaternary carbons (Table 1). The long-range couplings of HMBC correlation revealed fragments I and II (Figure 1) as follows; the cross peaks from H-1 (δ_H 6.55) to C-2 (δ_C 163.9), C-3 (δ_C 101.6), C-4a (δ_C 110.0) and C-12a (δ_C 154.7), from H-3 (δ_H 6.25) to C-1 (δ_C 105.5), C-2, C-4 (δ_C 165.0) and C-4a, and from 4-OH (δ_H 12.86) to C-3, C-4 and C-4a indicated a 2,3,5-trisubstituted phenol (ring A). The cross peaks from H₃-13 (δ_H 1.77) and H₃-14 (δ_H 1.83) to C-11a (δ_C 155.4), C-12 (δ_C 39.2) and C-12a indicated a dimethyl moiety being connected to C-12a via C-12. This was confirmed by the coupling between H-1 and C-12, and thus the fragment I was established. An oxygen atom is suggested to be attached to C-2 by its chemical shift. The cross peaks from H-8 (δ_H 6.99) to C-6a (δ_C 125.8), C-7 (δ_C 140.1), C-9 (δ_C 162.6) and C-10 (δ_C 109.5), from H-10 (δ_H 7.52) to C-6a, C-8 (δ_C 124.6), C-9, C-10a (δ_C 135.8) and C-11 (δ_C 185.7), and from 9-OCH₃ (δ_H 3.94) to C-9 indicated a 3,4,5-trisubstituted anisole (ring D). The cross peaks from H-17 (δ_H 6.39) to C-15 (δ_C 120.9), C-16 (δ_C 156.7), C-18 (δ_C 159.9) and C-19 (δ_C 106.9), from H-19 (δ_H 6.44) to C-15, C-17 (δ_C 96.4) and C-18, from H₃-21 (δ_H 2.06) to C-15, C-19 and C-20 (δ_C 137.0), from 16-OCH₃ (δ_H 3.65) to C-16, and from 18-OCH₃ (δ_H 3.80) to C-18 indicated a 2-monosubstituted 3,5-dimethoxytoluene (ring E). The coupling between H-8 and C-15 suggested a connection of rings D and E at C-7 and C-15, respectively, and thus the fragment II was established. Among the remaining three carbons, C-5 (δ_C 185.0) had couplings with H-1 and H-3 and C-6 (δ_C 182.6) had a coupling with H-10. They are considered to be ⁴J_CH W-couplings, and C-5 and C-6 are suggested to bond to C-4a and C-6a, respectively (Figure 2). The chemical shifts of C-6 (δ_C 182.6) and C-11 (δ_C 185.7) indicated that they form a quinone. If the remaining C-5a (δ_C 135.5) bonds to C-5 and C-11a to form ring B, and C-5a–C-6 and C-11–C-11a bondings form ring C, the naphthacenequinone structure is constructed. C-2 is suggested to be hydroxylated by the molecular formula. The carbon connections unrevealed by HMBC were clarified by INADEQUATE analysis (Table 1). Thus the structure of 9 was elucidated as shown in Scheme 1. It has a 7-phenylnaphthacene-5,6,11(12H)-trione skeleton. Most structurally related compounds, tetarimycin A⁶ and fasamycins⁷, were recently reported by Brady and co-workers as antibacterial agents. The structure was confirmed by X-ray crystallographic analysis as shown in Figure 3. The naphthacenequinone forms a planar skeleton.

Table 1 NMR spectroscopic data for 8, 9, 12 and 17^a

Figure 1

Partial structures of 9.

Figure 2

Structure elucidation of 9.

Figure 3

ORTEP (Oak Ridge thermal ellipsoid plot) plot of the X-ray crystallographic structure of 9.

The molecular formula of naphthacemycin A₈ (8) was established as C₃₀H₂₅ClO₈ by HR-FAB-MS, and this suggested that 8 was chlorinated analog of 9. ¹H and ¹³C NMR spectra of 8 were similar to those of 9, except for ring D (Table 1). The HMBC correlations from H-8 (δ_H 6.93) to C-6a (δ_C 127.1), C-7 (δ_C 137.4), C-9 (δ_C 158.4) and C-10 (δ_C 120.4) and from 9-OCH₃ (δ_H 3.95) to C-9 were observed, and C-10 was a quaternary carbon (Figure 4). Thus, the structure of 8 was elucidated as 10-chloro-9, which was confirmed by X-ray crystallographic analysis (Figure 5). In contrast to 9, the naphthacenequinone of 8 bent about 40° at two quinone carbonyl atoms of ring C.

Figure 4

Structure elucidation of 8 and 12 by HMBC.

Figure 5

ORTEP plot of the X-ray crystallographic structure of 8.

The structures of the other naphthacemycin A series were elucidated by comparison with NMR data of 8 or 9 or other A series compounds (Supplementary Tables S1 and S2) and analyses of HMBC correlations (Supplementary Figure S1).

Structure elucidation of the naphthacemycin B series

The molecular formula of naphthacemycin B₁ (12) was established as C₂₇H₂₂O₇ by HR-FAB-MS, which indicated 12 was 28 mass units (CO) less than 1. ¹H and ¹³C NMR spectra and HSQC analysis revealed the presence of 27 carbons, including three methyl, seven sp² methine, one sp³ quaternary and sixteen sp² quaternary carbons (Table 1). The structure was elucidated by long-range couplings of HMBC correlation (Figure 4). The cross peaks from H-1 (δ_H 6.65) to C-2 (δ_C 166.7), C-3 (δ_C 102.1) and C-4a (δ_C 108.6), from H-3 (δ_H 6.20) to C-1 (δ_C 107.1), C-2, C-4 (δ_C 166.6) and C-4a, and from 4-OH (δ_H 12.87) to C-3, C-4 and C-4a indicated a 2,3,5-trisubstituted phenol (ring A). The cross peaks from H₃-13 (δ_H 1.91) and H₃-14 (δ_H 1.70) to C-11a (δ_C 146.5), C-12 (δ_C 39.7) and C-12a (δ_C 155.5) indicated a dimethyl moiety connected to C-12a via C-12. This was confirmed by the coupling between H-1 and C-12. The cross peaks from H-8 (δ_H 6.72) to C-6a (δ_C 118.2), C-9 (δ_C 166.9) and C-10 (δ_C 110.1) and from H-10 (δ_H 7.05) to C-6a, C-8 (δ_C 122.5), C-9, C-10a (δ_C 142.9) and C-11 (δ_C 116.1) indicated a 3,4,5-trisubstituted phenol (ring D). The cross peaks from H-11 (δ_H 7.36) to C-5a (δ_C 107.6), C-6a and C-12 and from 6-OH (δ_H 14.53) to C-5a, C-6 (δ_C 160.1) and C-6a indicated a 2,3,5,6-tetrasubstituted phenol (ring C), which condensed with ring D and connected to ring A via C-12. Comparing NMR data and structures of 12 with 1, it is appropriate to form 4,4-dimethylcyclohexadienone (ring B) by C-4a, 5, 5a, 11a, 12 and 12a, though C-5 had no coupling with neighboring protons. The resemblance of the NMR data also suggested that a hydroxyl residue is attached to C-2 of 12 as in 1. The cross peaks from H-17 (δ_H 6.23) to C-15 (δ_C 124.5), C-16 (δ_C 155.4), C-18 (δ_C 157.5) and C-19 (δ_C 108.8), from H-19 (δ_H 6.26) to C-15, C-17 (δ_C 100.8) and C-18, and from H₃-21 (δ_H 1.88) to C-15, C-19 and C-20 (δ_C 138.3) indicated 2-monosubstituted 3,5-dihydroxytoluene (ring E). The coupling between H-8 and C-15 indicated that ring E connects to ring D at C-7. Thus, the structure of 12 was elucidated as 6,11-didehydro-O¹⁶-demethyl-6,11-dideoxo-11-hydroxy-1.

Structure elucidation of the naphthacemycin C series

The molecular formula of naphthacemycin C₂ (17) was established as C₃₃H₃₁ClO₉ by HR-FAB-MS, which indicated 17 was 58 mass units (C₃H₆O) more than 11. ¹H and ¹³C NMR spectra and HSQC analysis revealed the presence of 33 carbons, including seven methyl, one sp³ methylene, five sp² methine, two sp³ quaternary and eighteen sp² quaternary carbons (Table 1). The structure was elucidated by long-range couplings of HMBC correlation (Figure 6). The cross peaks from H-1 (δ_H 6.46) to C-2 (δ_C 162.6), C-3 (δ_C 102.1) and C-4a (δ_C 108.6), from H-3 (δ_H 6.26) to C-1 (δ_C 105.4), C-2, C-4 (δ_C 165.4) and C-4a, and from 4-OH (δ_H 12.52) to C-3, C-4 and C-4a indicated a 2,3,5-trisubstituted phenol (ring A). The cross peaks from H₃-13 (δ_H 1.59) and H₃-14 (δ_H 1.01) to C-11a (δ_C 53.2), C-12 (δ_C 43.3) and C-12a (δ_C 152.9) indicated a dimethyl moiety was connected to C-12a via C-12. This was confirmed by the coupling between H-1 and C-12. The cross peaks from H-8 (δ_H 6.98) to C-6a (δ_C123.8), C-9 (δ_C 161.9) and C-10 (δ_C 110.1), H-10 (δ_H 7.73) to C-6a, C-8 (δ_C 124.6), C-9, C-10a (δ_C 137.3) and C-11 (δ_C 197.8), and from 9-OCH₃ (δ_H 3.94) to C-9 indicated a 3,4,5-trisubstituted anisole (ring D). The cross peaks from 6-OH (δ_H 14.74) to C-5a (δ_C 107.6), C-6 (δ_C 168.8) and C-6a and from H₂-22 (δ_H 2.89, 3.52) to C-5a, C-11 and C-11a indicated a 2,3,5,6-tetrasubstituted 4-hydroxy-2,4-cyclohexadienone (ring C), condensed with ring D and connected to ring A via C-12. The cross peaks from H₃-24 (δ_H 1.89) to C-22 (δ_C 52.4) and C-23 (δ_C 205.4) and from H₂-22 to C-23 indicated that a 2-oxopropyl residue was attached to C-11a. Comparing NMR data and structures of 17 with 11, it is appropriate to form 4,4-dimethyl-2-cyclohexenone (ring B) by C-4a, 5, 5a, 11a, 12 and 12a, though C-5 had no coupling with neighboring protons. The chemical shifts of ring E of 17 is quite similar to 11, which suggests ring E of 17 is 2-monosubstituted 4-chloro-3,5-dimethoxytoluene. This was confirmed by the cross peaks from H-19 (δ_H 6.61) to C-15 (δ_C 130.2), C-17 (δ_C 113.5) and C-18 (δ_C 154.7), from H₃-21 (δ_H 1.96) to C-15, C-19 (δ_C 108.6) and C-20 (δ_C 134.9), from 16-OCH₃ (δ_H 3.63) to C-16 (δ_C 153.1), and from 18-OCH₃ (δ_H 3.94) to C-18. The coupling between H-8 and C-15 indicated that ring E connects to ring D at C-7. Thus, C-11a of 11 was substituted with a 2-oxopropyl group and the C-6 ketone was reduced in 17.

Figure 6

Structure elucidation of 16 and 17 by HMBC.

The molecular formula of naphthacemycin C₁ (16) was established as C₃₂H₂₉ClO₉ by HR-FAB-MS, which indicated 16 was 14 mass units (CH₂) less than 17. ¹H and ¹³C NMR spectra of 16 were quite similar to those of 17, except for rings D and E (Supplementary Table S3). The H-10 signal of 17 disappeared in 16 and the cross peaks of HMBC from H-8 (δ_H 7.01) to C-6a (δ_C 126.7), C-9 (δ_C 158.6) and C-10 (δ_C 120.8) and from 9-OCH₃ (δ_H 4.03) to C-9 were observed, which indicated that a chlorine was attached to the C-10 of ring D (Figure 6). The 18-OCH₃ signal of 17 was also absent in 16 and an H-17 (δ_H 6.37) signal appeared in 17. The cross peaks from H-17 (δ_H 6.37) to C-15 (δ_C 123.7), C-16 (δ_C 158.1), C-18 (δ_C 158.4) and C-19 (δ_C 109.4), from H-19 (δ_H 6.39) to C-15, C-17 (δ_C 97.4) and C-18, from H₃-21 (δ_H 2.01) to C-15, C-19 and C-20 (δ_C 137.0), and from 16-OCH₃ (δ_H 3.64) to C-16 indicated a ring E structure of 2-monosubstituted 3,5-dimethoxytoluene. Thus 16 was 10-chloro-17-dechloro-O¹⁸-demethyl-17. We have not elucidated the configurations of 16 and 17 yet.

Circumvention of β-lactam resistance in MRSA

The circumvention of β-lactam resistance in MRSA was measured by enhancement of imipenem activity on MRSA using the paper disk method. Naphthacemycins alone showed no antibacterial activity against a clinically isolated MRSA strain K24 at 0.01–1 μg per disk. When the agar plates contained 10 μg ml⁻¹ of imipenem, which also did not affect the growth of MRSA, 0.01–1 μg per disks of naphthacemycins showed inhibition zones (Table 2). Compounds 3 and 5–10 inhibited the growth of MRSA at 0.01 μg per disk in the presence of imipenem. Among them, 6 and 10 showed the largest inhibition zones (11 mm) at 0.01 μg per disk. The detailed activity of naphthacemycins against MRSA will be reported in an accompanying paper.⁵

Table 2 Enhancement of imipenem anti-MRSA activity by naphthacemycins

Conclusion

In conclusion, 17 new compounds designated naphthacemycins were isolated from the culture broth of Streptomyces sp. KB-3346-5. They are circumventors of β-lactam resistance and enhanced imipenem activity against β-lactam resistant MRSA. Naphthacemycins are 1-phenylnaphthacene antibiotics produced by Streptomyces sp. Many naphthacene type compounds have been isolated from actinomycetes, but most of them have partially unsaturated rings, such as tetracyclines and anthracyclines. Some highly unsaturated naphthacene compounds have been reported to be produced by actinomycetes, such as tetracenomycin D, galtamycin and tetracenoquinocin.^{8, 9, 10} A biosynthetic intermediate of tetracycline, pretetramid and an aglycone of anthracycline, η-pyrromycinone, are classified in the latter.^{11, 12} Naphthacemycins A and B series also belong to the latter group, but they are the first compounds having a 1-phenylnaphthacene skeleton isolated from a natural origin.

Methods

General experimental procedure

¹H NMR (300 MHz) and ¹³C NMR (75 MHz) spectra were recorded on a Varian XL-300 spectrometer. Chemical shifts are shown in δ values (p.p.m.) relative to the solvents (acetone-d₆ at 2.05 p.p.m. for ¹H NMR and at 29.8 p.p.m. for ¹³C NMR; CDCl₃ at 7.26 p.p.m. for ¹H NMR and at 77.0 p.p.m. for ¹³C NMR; DMSO-d₆ at 2.50 p.p.m. for ¹H NMR and at 39.5 p.p.m. for ¹³C NMR). INADEQUATE experiment of 9 (170 mg) was carried out for 100 h by 125 MHz NMR using methanol-d₄ as a solvent. Mass spectrometry was conducted on a JEOL JMS-AX505 HA spectrometer. The UV and IR spectra were measured with a Hitachi U-2810 spectrophotometer and a Horiba FT-710 Fourier transform infrared spectrometer, respectively. Optical rotations were recorded on a JASCO model DIP-1000 polarimeter.

Assay of antibacterial activity

Measurement of inhibition zone of naphthacemycins, with or without imipenem, to evaluate circumvention activity of β-lactam resistance were carried out by paper disk method, as reported previously.³

Data availability

The X-ray crystallographic data have been deposited at the Cambridge Crystallographic Data Centre (CCDC) under deposition numbers CCDC 1536220 (8) and CCDC 1536223 (9).

Structures of naphthacemycins A₁–A₁₁ (1–11), B₁–B₄ (12–15) and C₁–C₂ (16–17).