PJS, a novel isocoumarin with hexahydropyrimidine ring from Bacillus subtilis PJS

Amicoumacin group of antibiotics, such as baciphelacin,¹ amicoumacins,² AI-77s,³ xenocoumacins,⁴ Y-05460M-A,⁵ PM-94128,⁶ Sg17-1-4,⁷ bacilosarcins A, B⁸ and lipoamicoumacins A–D,⁹ is a small family of isocoumarin, which possesses the common chromophore, 3, 4-dihydro-8-hydroxyisocoumarin. Most members of amicoumacin group of antibiotics are produced by the genus Bacillus and exhibit various important bioactivities. The chromophore shows specific UV absorbance at 247 and 314 nm in methanol. Thus, a project based on HPLC-diode array screening and HPLC-MS dereplication was carried out to find new amicoumacin analogues from the secondary metabolites of Bacillus spp. As a result, PJS (1), a new isocoumarin antibiotic was discovered from the fermentation broth of Bacillus subtilis PJS. In this study, we wish to report the fermentation, isolation, physico−chemical properties, structural elucidation and biological activities of (1) (Figure 1).

Figure 1

Structure of PJS (1).

The producing strain Bacillus subtilis PJS was isolated from the leaf of an unidentified plant collected at Luopu county, Hetian area, Xinjiang Province, People’s Republic of China. On the basis of analysis of 16S rRNA, it was identified as Bacillus subtilis subsp. inaquosorum. A stock culture of the strain Bacillus subtilis PJS was maintained on modified Gause's no. 1 agar slant consisting of soluble starch (Beijing Qi Te Xin Chemical Co. Ltd. China) 20.0 g, NaCl 50.0 g, K₂HPO₄ 0.5 g, KNO₃ 1.0 g, MgSO₄ 1.0 g, FeSO₄ 0.02 g, glucose 1.0 g, peptone 0.5 g, tryptone 0.3 g and agar 20.0 g in 1.0 l distilled water (pH 8.0) at 4 °C. The stock culture was inoculated into 250 ml Erlenmeyer flasks containing 50 ml of seed medium, which was the same modified Gause’s no.1 liquid medium as above, but no agar. The culture was incubated on a rotary shaker (180 r.p.m.) at 28 °C for 24 h. Fifty millilitres of the seed culture was transferred to a 5000 ml Erlenmeyer flask containing 1000 ml of the producing medium, which was the same as the seed medium. The fermentation was carried out at 28 °C for 48 h on a rotary shaker (180 r.p.m.).

Eighty liters of the fermentation broth was centrifuged at 4500 r.p.m. for 20 min, then, the supernatant was obtained and was extracted twice with 40 l of ethyl acetate each time. The organic layer was pooled and concentrated under reduced pressure at 37 °C to give yellow syrup (2.3 g). It was then separated by preparative thin layer chromatography on 10 × 10 cm plates (silica gel 60F₂₅₄, Merck KGaA, Darmstadt, Germany) using CHCl₃-MeOH, 65:35 (v/v) as the developing solvent. Under UV 365 nm, bands with light blue fluorescence at R_f =0.35 were scraped and then eluted with methanol to yield a semipurified sample (260 mg). After dissolved in 1 ml methanol, the sample was filtered through a 0.22 μm membrane and was further purified by HPLC on a shim-Pack PRC-ODS column (250 × 20 mm, Shimadzu Corp., Tokyo, Japan) with MeOH-H₂O, 55:45 (v/v) at 2 ml min⁻¹. Peak at R_t=26 min, showed UV absorbance at 247 and 314 nm detected by prominence diode array detector (SPD-M20A, Shimadzu), was collected and pooled to yield 10.4 mg of (1) as a white powder.

Compound (1) was soluble in dimethyl sulfoxide (DMSO), MeOH, CHCl₃ and pyridine. Its m.p. was 79–80 °C. MW of (1) was found to be 435 by high-resolution ESI-MS, which showed [M+H]⁺ at m/z 436.2089 (calcd 436.2079). The molecular formula was then established as C₂₁H₂₉O₇N₃, which has nine degrees of unsaturation. ¹³C-NMR and DEPT spectra of (1) indicated 21 carbon signals could be attributed to three carbonyl carbons, six aromatic carbons and 12 aliphatic carbons including six carbons bonded to nitrogen or oxygen. The UV absorption of (1) at λ_max^MeOH nm (ɛ): 202(29 930), 246(6491) and 314(3491) was almost identical with that of isocoumarin compounds, such as Sg17-1-4, PM-94128, Y-05460M-A, bacilosarcins and AI-77-B. The IR spectrum (film) of (1) exhibited absorption bands at 3343, 2956, 1668, 1620, 1539, 1231, 807, 697 cm⁻¹, which indicated the presence of a benzoic acid moiety with a phenolic hydroxyl group and an amide group. All data above revealed (1) had a chromophore similar to the 3, 4-dihydro-8-hydroxyisocoumarin skeleton in its structure. NMR data of (1) in DMSO-d₆ (I) and CD₃OD (II) were listed in Table 1. By careful analysis of ¹H and ¹³C-NMR, ¹H–¹H COSY, DEPT, ¹³C–¹H COSY, HMBC spectra in DMSO-d₆ and in CD₃OD together with NOESY and D₂O exchange experiment in DMSO-d₆, chemical structure of (1) was elucidated. It consisted of structural fragment L and R, as shown in Figure 2.

Table 1 NMR data of 1 in DMSO-d₆ (I) and CD₃OD (II)

Figure 2

Summary of ¹H–¹H COSY and HMBC experiments of (1) in DMSO-d₆ (I) and CD₃OD (II).

Identification of structural fragment L was started from H-5, H-6 and H-7. The three aromatic protons were readily observed and displayed the coupling patterns for 1, 2, 3-trisubstituted benzenoid ring in ¹H-NMR. Their corresponding carbons were assigned by ¹³C–¹H COSY. Other three aromatic carbons were observed and assigned by tracing cross peaks from H-5 and H-7 to C-8a, from H-6 to C-4a and C-8 in HMBC. Chemical shift of C-8 at 161.5 suggested a hydroxyl group should attach to C-8, which was confirmed by NOESY correlation from 8-OH proton at δ 7.24 to H-7 at δ 6.82 in DMSO-d₆. Identification of isopentyl group in structural fragment L was started from two methyl protons, H-1′ at δ 0.84 and H-2′ at δ 0.89 in ¹H-NMR in DMSO-d₆, which showed HMBC correlation to one another, meanwhile, to C-4′ at δ 39.1. H-1′, H-2′ and H-4′ showed cross peaks with a methine proton H-3′ at δ 1.61 in ¹H–¹H COSY. These observations suggested 3′-CH linked with 1′-CH₃, 2′-CH₃ and 4′-CH₂. A cross peak observed between H-4′ and H-5′, a methine proton at δ 4.20 in ¹H–¹H COSY confirmed the presence of the isopentyl group. H-5′ had a cross peak with H-3, a methine proton at δ 4.66 in ¹H–¹H COSY in DMSO-d₆, in turn, H-3 showed COSY correlation with H-4. H-4 showed HMBC correlation with C-8a in DMSO-d₆, meanwhile, H-4 also showed HMBC correlation with C-5 in CD₃OD. Thus, connectivity between C-4 and C-4a was established. Chemical shift of H-3 and its corresponding carbon, C-3 at δ 81.0 in low field indicated C-3 should attach to the oxygen of lactone ring to form 3, 4-dihydro-8-hydroxyisocoumarin skeleton. Furthermore, a cross peak observed between H-5′ and 6′-XH proton at δ 7.95 in ¹H–¹H COSY in DMSO-d₆ revealed 6′-XH bound with C-5′ at δ 48.1. Chemical shift of H-5′and C-5′ in downfield, as well as a cross peak observed between 6′-XH proton and 7′-C=O at δ 169.7 in HMBC in DMSO-d₆ revealed XH was NH, which bound to 7′-C=O to form an amide group. All data above revealed (1) had methylbutylamino-3, 4-dihydro-8-hydroxyisocoumarin, which was the common structural moiety of amicoumacin group of antibiotics. It was further confirmed by comparison of ¹H and ¹³C-NMR data of (1) with that of known amicoumacin group of compounds such as PM-94128, Y-05460M-A, xenocoumacins and AI-77-B.

In structural fragment L of (1), there were seven degrees of unsaturation. Thus, two degrees of unsaturation were left in structural fragment R. Elucidation of structural fragment R was started from H-8′, a methine proton, which was readily observed as a doublet signal at δ 3.69 in ¹H-NMR in DMSO-d₆. By tracing the cross peaks from H-8′ to H-9′, from H-9′ to H-10′, from H-10′ to H-11′ in ¹H–¹H COSY, structural moiety as -CH(8′)-CH(9′)-CH(10′)-CH₂(11′)- was identified, and was further confirmed by cross peaks in HMBC between H-8′ and C-10′, between H-11′ and C-9′ in DMSO-d₆, in addition, between H-10′ and C-8′ in CD₃OD. A broad single signal in downfield at δ 10.8 in ¹H-NMR and a quaternary carbon signal in downfield at δ 173.1 in ¹³C-NMR in DMSO-d₆ suggested the presence of 12′-COOH. A cross peak observed between H-11′ and C-12′ in HMBC suggested 12′-COOH was attached to C-11′.

On the basis of chemical shift of H-9′ at 3.17, C-9′ at 68.1 and H-10′ at 2.78, C-10′ at 57.3 in DMSO-d₆, it was speculated that a hydroxyl group attached to C-9′ and a NH attached to C-10′. It was confirmed by D₂O exchange experiment. When drops of D₂O added into DMSO-d₆, a proton at δ 5.15 (d, J=5.0), showed COSY correlation with H-9′ and HMBC correlation with C-8′ in DMSO-d₆, was absent. Thus, the proton was attributed to 9′-OH. Meanwhile, another proton at δ 2.85 (dt, J=12.5, 12.5, 5.0), showed COSY correlation to H-14′ and HMBC correlation to C-9′ in DMSO-d₆, was also absent. Thus, the proton was attributed to 15′-NH. The absence of 15′-NH proton in D₂O exchange experiment led the multiplicity of H-10′ at δ 2.78(m), H-14′ at δ 3.99 (dd, J=10.5, 12.5) and δ 4.41 (dd, J=10.5, 3.5) simplified as the corresponding data in ¹H-NMR in CD₃OD. The phenomena suggested that 15′-NH not only attached to C-14′, but also attached to C-10′. By the calculation of element composition and degrees of unsaturation of (1), it was speculated that a hexahydropyrimidine ring must exist in structural fragment R and was composed of 8′-CH, 9′-CH, 10′-CH, 15′-NH, 14′-CH₂, and left 13′-NH at δ 6.82 overlapped by the aromatic proton of H-7. Cross peaks between H-14′ and C-8′, between H-14′and C-10′ in HMBC spectrum in CD₃OD further supported the speculation. Thus, the structural fragment R was elucidated.

HMBC correlations from H-9′ to C-7′ in CD₃OD revealed the linkage between the structural fragment L and structural fragment R was through C-8′ to C-7′ and completed the elucidation of the planar chemical structure of (1), which was further confirmed by downfield shift of 13′-NH proton, which could form H-bonding with 7′-C=O.

By analysis of ¹H–¹H coupling constants and cross peaks observation in NOESY, the relative configuration of H-3, H-8′, H-9′, H-10′ and H-15′ were elucidated. In the ¹H-NMR in CD₃OD, large diaxial coupling constants between H-3 and H-4, between H-8′ and H-9′, between H-9′ and H-10′ were observed as 12.5, 9.5 and 9.5 Hz, separatively, which indicated all protons including H-3, H-8′, H-9′, H-10′ had axial orientation. In the ¹H-NMR in DMSO-d₆, two large diaxial coupling constants between H-10′ and H-15′, between H-14′ and H-15′ were observed as 12.5 and 12.5 Hz, respectively. It indicated H-15′ also had axial orientation. As for H-13′overlapped by H-7, careful analysis of its vicinal protons in DMSO-d₆ in ¹H-NMR showed H-13′ had no contribution to multiplicity and coupling constants of H-8′ and H-14′. In addition, no cross peak can be observed between H-13′ and H-8′ or H-14′ in NOESY in DMSO-d₆. Therefore, H-13′ either had an equatorial orientation or couldn’t be defined, due to rapid interchange in DMSO-d₆.

PJS was active against Gram-positive and less active against Gram-negative bacteria and fungi. MICs were determined by broth micro-dilution method according to National Committee for Clinical Laboratory Standards guidelines.^{10, 11} The MIC values of PJS were listed in Table 2. The cytotoxicities in vitro against murine leukaemia P388 cells, human lung adenocarcinoma epithelial cell lines A549, human liver hepatocellular carcinoma cell lines HepG2, human pancreatic adenocarcinoma cell lines SW1990, human cervical cancer HeLa cells were measured by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay.¹² PJS exhibited activities with IC₅₀ values of 103.6, 70.7, 39.5, 34.2, and 4.0 μM, separatively.

Table 2 Antimicrobial activity of PJS