Verrulactones D and E with unprecedented skeletons, new inhibitors of Staphylococcus aureus enoyl-ACP reductase, from Penicillium verruculosum F375

Enoyl-ACP reductase catalyzes the final and rate-limiting step in the bacterial fatty acid synthesis (FAS), and a target for the development of new anti-bacterial agents,¹ as well as having been identified as the target of triclosan, the broad-spectrum biocide in a wide range of consumer goods, and isoniazid, which is used in the treatment of tuberculosis for 50 years.² Among four isoforms, FabI, FabK, FabL and FabV, in bacterial enoyl-ACP reductase, FabI is highly conserved among important pathogenic bacteria including methicillin-resistant Staphylococcus aureus.³ So far, several inhibitors of FabI have been reported,^{4, 5, 6} but inhibitors with new chemical skeletons are needed. In the course of screening for FabI inhibitors, we previously discovered verrulactones A (3), B (4) and C (5), together with the known compound, altenuisol (6), from the fermentation broth of Penicillium verruculosum F375 (Figure 1).^{7, 8} Compounds 3 and 4 were dimeric compounds of the alternariol class and compound 5 was a dispiro compound. Further investigation of minor fractions from this fermentation led to the discovery of two unique compounds with a high quaternary carbon content named verrulactones D (1) and E (2). In this paper, we present the isolation, structure determination and FabI-inhibitory and anti-bacterial activities of 1 and 2.

Figure 1

Chemical structures of verrulactones A (3), B (4), C (5), D (1), E (2) and altenuisol (6).

Fermentation was carried out in 1-L Erlenmeyer flasks containing glucose 2%, polypeptone 0.5%, yeast extract 0.2%, KH₂PO₄ 0.1% and MgSO₄·7H₂O 0.05% (adjusted to pH 5.7 before sterilization). A piece of agar from the mature plate culture of the producing strain was inoculated into a 500 ml Erlenmeyer flask containing 80 ml of sterile seed liquid medium with the above composition and cultured on a rotary shaker (150 r.p.m.) at 28 °C for 3 days. For the production of verrulactones, 5 ml of the seed culture was transferred into 1-L Erlenmeyer flasks containing 100 ml of the above medium, and cultivated for 14 days using the same conditions. The culture broth (12 l) was added to the same volume of acetone and the cell debris removed by filtration. After the filtrate was evaporated to remove acetone, the resultant water phase was partitioned with EtOAc three times and the EtOAc layer was concentrated in vacuo. The resultant residue (2.8 g) was subjected to Sephadex LH-2 column (24 × 900 mm²; GE Healthcare, Uppsala, Sweden) followed by elution with MeOH to give two active fractions, Fr. I and II, followed by 3-, 4- and 5-containing fractions. The Fr. I was applied to the second Sephadex LH-20 eluted with MeOH. The resultant active fraction was purified by TLC on RP-18 F₂₅₄ plates (Merck No. 1.15389.0001; Merck, Darmstadt, Germany) developed with CH₃CN-H₂O (55:45) containing 0.1% trifluoroacetic acid. The active band was further purified by a second RP-18 TLC step developed with MeOH-H₂O (70:30) containing 0.1% trifluoroacetic acid to afford 2 (3.7 mg) with an R_f value of 0.58. The Fr. II was also applied to the second Sephadex LH-20 eluted with MeOH. The resultant active fraction was purified by RP-18 TLC with CH₂CN-H₂O (55:45) containing 0.1% trifluoroacetic acid. The active band was finally purified by the second RP-18 TLC with MeOH-H₂O (80:20) containing 0.1% trifluoroacetic acid to give 1 (2.0 mg) with an R_f value of 0.2. NMR spectra were recorded with Bruker Biospin Avance 500 and 700 spectrometers (Korea Basic Science Institute, Daejeon, Korea). HRESI-MS data was recorded on a SYNAPT G2 mass spectrometer (Waters Corporation, Manchester, UK).

Verrulactone D ( 1): Yellow powder; mp 203 °C: λ_max (nm) (log ɛ) in MeOH: 208 (4.68), 257 (4.37), 299 (4.02), 362 (3.47); IR (KBr): 3393, 2954, 1651, 1462, 1021 cm⁻¹; HR-ESI-MS: m/z 573.1390 (M+H)⁺, C₃₁H₂₅O₁₁ requires 573.1397.

Verrulactone E ( 2): Yellow powder; mp 197 °C: [α]_D=+21.7° (c 0.35, MeOH); λ_max (nm) (log ɛ) in MeOH: 210 (4.79), 256 (4.53), 299 (4.04), 336 (3.88); IR (KBr): 3300, 2922, 1675, 1463, 1049 cm⁻¹; HR-ESI-MS: m/z 561.1042 (M+H)⁺, C₂₉H₂₁O₁₂ requires 561.1033.

The molecular formula of 1 was determined to be C₃₁H₂₄O₁₁ on the basis of high-resolution ESI-MS ((M+H)⁺, 573.1390 m/z (−0.7 mmu error)) in combination with ¹H and ¹³C NMR spectral data. The IR absorption at 1651 cm⁻¹ suggested that at least one carbonyl moiety was present. The ¹H and ¹³C NMR data (Table 1) of 1 with COSY and HMQC spectra exhibited resonances for three singlet aromatic methines (δ_H 7.02, s, δ_C 121.6; δ_H 7.45, s, δ_C 118.5; and δ_H 7.83, s, δ_C 125.8), two sets of meta-coupled aromatic methines (δ_H 6.23, d, J=2.1, δ_C 111.6; δ_H 6.51, d, J=2.1, δ_C 101.2 and δ_H 6.26, d, J=2.1, δ_C 111.6; δ_H 6.54, d, J=2.1, δ_C 101.5), two methoxyls, two aromatic methyls, seventeen sp² quaternary carbons and three carbonyls.

Table 1 ¹H and ¹³C NMR data^a of verrulactone D (1)

The presence of an 1, 2, 4, 5-tetrasubstituted benzene (ring A) in 1 was determined by the HMBC correlations (Figure 2a) of a singlet aromatic proton at δ 7.83 (H-9) with two sp² quaternary carbons at δ 146.1 (C-7) and 155.8 (C-5a); of another singlet aromatic proton at δ 7.45 (H-6) with three sp² quaternary carbons at δ 141.5 (C-8), 121.5 (C-9a) and C-5a; and of one methyl protons at δ 2.25 (H₃-12) with C-6, C-7 and C-8. In addition, the HMBC correlation of H-6 with C-12 together with the NOE between H₃-12 and H-6, and the HMBC correlation of H-9 with a carbonyl carbon at δ_C 181.2 (C-10) indicated the attachment of the methyl (C-12) and the carbonyl (C-10) at C-7 and C-9a, respectively. The presence of an 1, 2, 3, 4, 5-pentasubstituted benzene (ring C) was determined by the HMBC correlations of another singlet aromatic proton at δ 7.02 (H-3) with three sp² quaternary carbons at δ 128.9 (C-1), δ 144.6 (C-4a) and δ 147.4 (C-4), and of the other methyl protons at δ 2.50 (H₃-11) with three sp² quaternary carbons at δ 121.3 (C-10a), δ 140.7 (C-2), C-1 and C-4a. The chemical shifts of C-5a at δ_C 155.8 and also C-4a at δ 144.6 also indicated the presence of xanthone system. Relatively high-field shifted chemical shift of C-4a was because of the presence of a hydroxyl group at C-4; that is, ortho position C-4a.

Figure 2

HMBC and NOE correlations of 1 (a) and 2 (b).

The presence of two 1, 2, 3, 5-tetrasubstituted benzenes (rings D and E) in 1 was also determined by the HMBC correlations. The ring D was determined by the HMBC correlations (Figure 2a) from one of the meta-coupled aromatic protons at δ 6.26 (H-2') to two sp² quaternary carbons at δ 106.9 (C-6') and 165.4 (C-3') and an aromatic methine at δ 101.5 (C-4'); from the other of the meta-coupled aromatic protons at δ 6.54 (H-4') to two sp² quaternary carbons at δ 166.6 (C-5') and C-6', and an aromatic methine at δ 111.6 (C-2'). The HMBC correlation of a methoxyl at δ 3.86 (3'-OMe) with C-3' together with the NOEs between 3'-OMe/H-2' and 3'-OMe/H-4' indicated the attachment of the methoxyl (3'-OMe) at C-3'. The ring E was also determined by the HMBC correlations in the same way as the ring D. The connectivity of ring D with ring A was established by the HMBC and NOEs. The HMBC correlations from H-2' to C-8 indicated the linkages of C-1' with C-8. Additionally, the NOEs between H-2'/H-9 and H-2'/H₃-12 suggested the free rotation of ring D. The connectivity of ring E with ring C was also established in the same way. These spectral data with the molecular formula indicated the attachments of the carboxylic acid groups of δ 174.0 and δ 174.1 at either C-6' or C-6", and the assignments of the remaining low-field shifted sp² quaternary carbons of δ 147.2 and 145.9 at either C-1' or C-1". Thus, the structure of 1 was established as shown in Figure 1.

The molecular formula of 2 was determined to be C₂₉H₂₀O₁₂ on the basis of high-resolution ESI-MS ((M+H)⁺, 561.1042 m/z (+0.9 mmu error)) in combination with ¹H and ¹³C NMR data. The IR absorption at 1675 cm⁻¹ suggested the presence of carbonyl groups. The ¹H and ¹³C NMR spectra (Table 2) of 2 with COSY and HMQC indicated the presence of two meta-coupled aromatic methines (δ_H 6.55, d, J=1.9; δ_C 102.5 and δ_H 6.96, d, J=1.9; δ_C 103.8), two isolated aromatic methines (δ_H 6.48, s; δ_C 97.8 and δ_H 6.82, s; δ_C 101.1), an isolated olefinic methine (δ_H 6.96, s; δ_C 124.0), an isolated methine (δ_H 3.99, s; δ_C 46.2), two methoxyls (δ_H 3.71, s; δ_C 56.3 and δ_H 3.86, s; δ_C 56.1), an isolated methyl (δ_H 1.58, s; δ_C 28.7), five exchangeable protons, fifteen sp² quaternary carbons, two oxygenated sp³ quaternary carbons (δ_C 72.3 and δ_C 80.6), two ester carbonyls (δ_C 166.2 and δ_C 164.5) and a ketone carbonyl (δ_C 199.2).

Table 2 ¹H and ¹³C NMR data^a of verrulactone E (2)

The presence of 1-methoxy-3-hydroxy-4-carbonyl-tetrasubstituted benzene moiety (partial structure I) in 2 was determined by the HMBC spectrum (Figure 2b) of one meta-coupled aromatic proton at δ 6.55 (H-8) with three sp² quaternary carbons at δ 100.0 (C-6a), δ 162.8 (C-7) and δ 166.0 (C-9), an aromatic methine at δ 103.8 (C-10) and a carbonyl carbon at δ 166.2 (C-6); of the other meta-coupled aromatic proton at δ 6.96 (H-10) with three sp² quaternary carbons at δ 136.9 (C-10a), C-6a and C-9, and an aromatic methine at δ 102.5 (C-8); of one methoxyl protons at δ 3.86 (9-OMe) with C-9; and of the hydroxyl proton at δ 10.76 (7-OH) with C-6a, C-7 and C-8. The position of the methoxyl group was supported by the NOEs between 9-OMe/H-8 and 9-OMe/H-10. The presence of an altenuisol moiety (partial structure II) was also determined by the HMBC correlations (Figure 2b) of a singlet aromatic proton at δ 6.48 (H-3′) with four sp² quaternary carbons at δ 97.2 (C-4a′), δ 111.7 (C-1′), δ 162.3 (C-2′) and 162.9 (C-4′), and a carbonyl carbon at δ 164.5 (C-5'); of the other singlet aromatic proton at δ 6.82 (H-7') with four sp² quaternary carbons at δ 108.1 (C-10a′), δ 141.7 (C-9'), δ 142.9 (C-6a'), and δ 149.1 (C-8'); of the other methoxyl protons (2'-OMe) at δ 3.71 with C-2'; and of the hydroxyl proton at δ 11.08 (4′-OH) with C-3′, C-4′ and C-4a′. The last partial structure III was determined by the HMBC correlations (Figure 2b) of the olefinic proton at δ 6.96 (H-1) with an sp² quaternary carbon at δ 146.8 (C-10b) and two sp³ quaternary carbons at δ 72.3 (C-3) and δ 80.6 (C-4a); of the isolated methyl protons at δ 1.58 (H₃-11) with C-4a, C-10b and the methine carbon at δ 46.2 (C-4); of the isolated methine proton at δ 3.99 (H-4) with C-3, C-4a, C-10b and the ketone carbonyl carbon at δ 199.2 (C-2); and of the hydroxyl proton at 5.68 (3-OH) with C-4 and C-1′. The connectivity of the partial structures II and III was established by the long-range couplings from H-4 to C-1', C-9', C-10' and C-10a', and from 3-OH to C-1′. Taken together with the molecular formula of 2, the HMBC correlations from both H-1 and H-10 to both C-10a and C-10b indicated the connectivity of the partial structures I and III through the linkage of C-10a with C-10b and the ester bond of C-4a and C-6a. The NOEs between H₃-11 and H-4 in CD₃OD, and between H-4 and 3-OH in DMSO-d₆ were observed. These data indicated that H₃-11, H-4 and 3-OH are syn configuration. Thus, the structure of 2 was elucidated as shown in Figure 1.

The inhibitory activity of 1 and 2 against Staphylococcus aureus FabI and FabG, and anti-bacterial activity was evaluated according to our previously reported method.^{9, 10, 11} Compounds 1 and 2 inhibited S. aureus FabI in a dose-dependent manner with IC₅₀s (half-maximal inhibitory concentrations) of 6.8 and 24.1 μm, respectively (Table 3). Also, 1 exhibited anti-bacterial activity with minimal inhibitory concentrations (MICs) of 64 μg ml⁻¹ against S. aureus RN4220, including methicilline-resistant S. aureus CCARM 3167 and quinolone-resistant S. aureus (QRSA) CCARM 3505, whereas 2 showed the weaker anti-bacterial activity with with MICs of 128 μg ml⁻¹. FabI inhibitory activity and anti-bacterial activity of 1 and 2 were weaker compared with those of 3 and 4, but similar with those of 5. The MIC of triclosan as a positive control was 0.01 μg ml⁻¹. Compounds 1 and 2 did not inhibit S. aureus FabG, another reductase of bacterial FAS, even at 300 μm, suggesting their selective inhibition for FabI similar to 3–6.

Table 3 Inhibitory effects of compounds 1–6 on FabI and cell viability of S. aureus