Penicilones AâD, Anti-MRSA Azaphilones from the ... - ACS Publications

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Penicilones A−D, Anti-MRSA Azaphilones from the Marine-Derived Fungus Penicillium janthinellum HK1‑6 Min Chen,*,† Nan-Xing Shen,† Zhi-Qi Chen,† Feng-Min Zhang,‡ and Yang Chen† †

Marine Science & Technology Institute, College of Environmental Science & Engineering, Yangzhou University, 196#, Huayang West Street, Yangzhou 225127, People’s Republic of China ‡ Testing Center, Yangzhou University, 48#, Wenhui East Street, Yangzhou 225009, People’s Republic of China S Supporting Information *

ABSTRACT: Four new azaphilones, penicilones A−D (1−4), were isolated from the mangrove rhizosphere soil-derived fungus Penicillium janthinellum HK1-6. Their planar structures and absolute configurations were determined by extensive analysis of NMR spectroscopic data, ECD spectra, the modified Mosher’s method, and chemical conversions. Interestingly, 1 and 2 had the opposite configuration at C-7 compared to the closely related chloro analogues 3 and 4. Ester hydrolysis of 2 and 4 afforded their parental azaphilones, named penicilones E (5) and F (6). Compounds 1−6 were evaluated for their antibacterial activities in vitro. Penicilones B−D (2−4) showed potent anti-MRSA (Staphylococcus aureus ATCC 43300, ATCC 33591) activities with MIC values ranging from 3.13 to 6.25 μg/mL.

E

also prepared. Herein, we report the isolation and structure characterization of 1−4 and the antibacterial activities of 1−6.

mergence of resistance to antibiotics in pathogenic bacteria has become a serious public health threat globally. Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of health-care-associated infections, resulting in extensive morbidity and mortality. Vancomycin is considered to be a last line of defense against serious MRSA infections; however, the emergence of less-susceptible strains and poor clinical outcomes are challenging its current role.1 Therefore, there is an urgent need to develop additional effective anti-MRSA agents. Marinederived fungi have been recognized as a growing source of new natural products and have already provided a number of promising leads against MRSA.2,3 The most prominent example of anti-MRSA compound is pestalone, isolated by Fenical and co-workers from coculturing a fungus of the genus Pestalotia and a unicellular marine bacterium (strain CNJ-328).4,5 In the course of our ongoing investigation on secondary metabolites from marine-derived fungi, we have isolated several new compounds with antibacterial activity.6−8 Recently, a marine fungal strain, Penicillium janthinellum HK1-6 isolated from the mangrove rhizosphere soil, attracted our attention because its EtOAc extract showed significant antibacterial activity against MRSA (Staphylococcus aureus ATCC 43300, ATCC 33591). Bioassay-guided separation of the fungal culture extract resulted in the isolation of four new azaphilones, penicilones A−D (1−4). Two hydrolytic derivatives from 2 and 4, named penicilones E and F (5, 6), were © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Penicilone A (1) was isolated as a yellow oil and has the molecular formula C29H36O7 (12 degrees of unsaturation) as Received: December 23, 2016 Published: March 1, 2017 1081

DOI: 10.1021/acs.jnatprod.6b01179 J. Nat. Prod. 2017, 80, 1081−1086

Journal of Natural Products

Article

Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data for 1 and 2 (CDCl3) 1 position

δC, type

1 3 4 4a 5 6 7 8 8a 9 10 11 12

153.9, CH 152.8, C 113.8, CH 141.7, C 107.5, CH 193.0, C 83.7, C 192.9, C 114.6, C 21.5, CH3 129.6, C 161.0, C 40.4, CH2

13 14

64.7, CH 45.5, CH2

15 16 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′

193.5, C 22.5, CH3 167.3, C 124.2, C 150.6, CH 33.0, CH 36.2, CH2 26.9, CH2 28.9, CH2 31.3, CH2 22.2, CH2 13.6, CH3 11.9, CH3 19.3, CH3

2 δH, mult. (J in Hz) 7.91, d (1.2) 6.23, s 5.59, d (1.2)

1.60, s

2.82, dd (16.8, 4.2) 2.63, dd (16.8, 7.2) 4.38, m 2.79, dd (16.8, 4.2) 2.62, dd (16.8, 7.2) 2.09, s

6.73, dd (10.2, 1.8) 2.49, m 1.36, m 1.26, m 1.26, m 1.26, m 1.30, m 0.88, t (6.6) 1.86, d (1.2) 1.00, d (6.6)

determined by HRESIMS. The 1H NMR spectrum (Table 1) of 1 displayed the typical pattern of an azaphilone skeleton with three olefinic protons, attributed to H-1 (δH 7.91), H-4 (δH 6.23), and H-5 (δH 5.59), and a singlet methyl (H3-9, δH 1.60) connected to C-7. The 13C NMR data revealed that 1 contained three α,β-unsaturated keto groups (δC 193.5, 193.0, 192.9; corresponding with νmax 1703 cm−1 in the IR spectrum), one unsaturated ester carbonyl (δC 167.3), one oxygenated methine (δC 64.7), and one oxygenated nonprotonated carbon (δC 83.7). These spectroscopic features suggested that 1 is very similar to cohaerin B, an azaphilone isolated from stromata of the xylariaceous ascomycete Hypoxylon cohaerens.9 Interpretation of the 2D NMR spectrum indicated the subunit linked to the main azaphilone backbone at C-3 in 1 was 4-hydroxy-2-methyl-6oxocyclohex-1-enyl, i.e., a partial structure of cohaerin B. The significant differences between these two compounds were the units linked at C-7. The contiguous sequence of correlations from H-3′ to H-10′ in the COSY spectrum (Figure 1) confirmed the presence of an aliphatic subunit in 1. The HMBC correlations from H3-11′ to C-1′, C-2′, C-3′, and C-4′ and from H3-12′ to C-3′, C-4′, and C-5′ demonstrated the groups of CH3-11′ and CH3-12′ were anchored at C-2′ and C-4′, respectively. In the NOESY spectra, the correlations observed between H-4′ and H3-11′, and no cross-peaks observed between H-3′ and H3-11′, suggested the double bond between C-2′ and C-3′ had the E-configuration. Therefore, instead of the (E)-2methyloct-2-enoate at C-7 in cohaerin B, a lipid side chain of

δC, type 155.1, CH 156.2, C 114.3, CH 143.9, C 107.0, CH 194.3, C 84.1, C 193.6, C 115.2, C 22.1, CH3 118.5, C 138.8, C 122.2, CH 131.6, CH 113.9, CH 155.0, C 20.1, CH3 167.9, C 124.7, C 151.3, CH 33.5, CH 36.7, CH2 27.4, CH2 29.4, CH2 31.8, CH2 22.7, CH2 14.1, CH3 12.4, CH3 19.7, CH3

δH, mult. (J in Hz) 8.05, s 6.48, s 5.65, s

1.63, s

6.75, d (7.8) 7.15, t (7.8) 6.77, d (7.8)

2.27, s

6.74, d (10.8) 2.49, m 1.33, m 1.24, m 1.24, m 1.24, m 1.28, m 0.87, t (6.6) 1.85, s 0.98, d (6.6)

Figure 1. COSY and key HMBC correlations for 1.

(E)-2,4-dimethyldec-2-enoate was suggested to be connected to the azaphilone core at C-7 in 1. This lipid side chain is new to the azaphilone family. The absolute configuration of 1 was established by ECD spectroscopy as well as the modified Mosher’s method. The S-configuration was assigned to C-7 according to the ECD spectrum (Figure 2), showing positive Cotton effects at 356 and 229 nm and a negative Cotton effect at 270 nm that matched well with those of cohaerin B.9,10 In order to establish the absolute configuration of C-13, 1 was subjected to the modified Mosher’s method.11 When reacted with (R)- and (S)-MTPA chloride, 1 gave the corresponding (S)- and (R)-MTPA esters 1s and 1r, respectively. Because of the low stability of 1s and 1r, the reactions were performed in deuterated pyridine12,13 and 1H NMR measurements were carried out without purification. The observed chemical shift differences ΔδS−R (Figure 3) clearly defined the R-configuration at C-13. The absolute configuration of C-4′ was not determined. 1082



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7S-configuration is suggested for 3.16 Unfortunately, an attempt to prepare the (S)- and (R)-MTPA esters of 3 for determining the absolute configuration of C-13 in the cyclohexenone ring by the modified Mosher’s method was not successful. Instead, the reaction yielded compound 4, with a 3-methylphenol connected to C-3 of the azaphilone core. Penicilone D (4) was also obtained as a bright yellow oil with the molecular formula C29H33ClO6, which indicated the formal loss of H2O in comparison to 3 and a chlorine atom in place of a proton in comparison to 2. Its 1H and 13C spectra (Table 2) closely resembled those of 2. The obvious difference in the 1H NMR spectrum compared to 2 was the missing H-5 signal, suggesting that a chlorine atom was anchored at C-5 in 4. Similar to that of 3, the ECD spectrum of 4 displayed a negative Cotton effect at 366 nm and positive Cotton effects around 316, 277, and 231 nm (Figure 2), indicating a 7S configuration for 4. It is interesting that penicilones A and B (1, 2) and the chlorine-substituted penicilones C and D (3, 4), co-isolated from the same fungal cultures, had opposite configurations at C-7. To further confirm the absolute configurations of C-7 in 1−4, ester hydrolyses of 2 and 4 were performed.17 Consequently, two azaphilone parent structures, named penicilones E (5) and F (6) with only one stereogenic carbon at C-7, were obtained, respectively. It is noteworthy that 5 and 6 showed opposite signs of specific rotations (5 [α]D +134; 6 [α]D −180), suggesting the opposite configurations at C-7 for 5 and 6.10,18 In the ECD spectra (Figure 4), 5 and 6 displayed opposite ECD curves, confirming the opposite configurations at C-7. Thus, combined with the specific rotations and ECD data of 1−6, compounds 1 and 2 were unambiguously determined to have the opposite C-7 configurations compared to 3 and 4. Compound 3 was found to be unstable under the Mosher’s ester formation conditions, converting to 4, preventing formation of the Mosher’s esters from 3. Therefore, it might be possible that 4 is an artifact formed from 3 during the process of extraction and separation. However, the EtOAc extract freshly prepared from the fungus P. janthinellum HK1-6 was examined by HPLC (Supporting Information, Figure S39), revealing all of the isolated azaphilones in the HPLC profile. As a result, penicilone D (4) is more likely to be produced by the fungus rather than by the separation process. The azaphilones, a large group of fungal pigments, are structurally variable fungal polyketide metabolites.19,20 Azaphilones have been reported with a wide range of biological activities, such as antimicrobial,21,22 cytotoxic,21,22 anti-inflammatory,23,24 and inhibition against several enzyme activities.25,26 In the present study, the antibacterial activities of 1−6 were evaluated with Gram-positive S. aureus (ATCC 43300), S. aureus (ATCC 33591), S. aureus (ATCC 29213), S. aureus (ATCC 25923), Enterococcus faecalis (ATCC 51299), E. faecium (ATCC 35667), and Gram-negative Escherichia coli (ATCC 25922). The results (Table 3) indicated that 2−4 showed significant antibacterial activities against the tested Gram-positive bacteria including both antibiotic-resistant and -susceptible strains with MIC values ranging from 3.13 to 12.5 μg/mL, while none of the tested compounds exhibited activity against E. coli. It is worth noting that 2−4 displayed potent antibacterial activities against two strains of methicillin-resistant S. aureus, ATCC 43300 and ATCC 33591, with MIC values ranging from 3.13 to 6.25 μg/mL (positive control vancomycin, MIC 0.39− 0.78 μg/mL). Compounds 2−4 also showed significant antibacterial activities against two strains of susceptible S. aureus. Additionally, the antibacterial activities of 2 and 3 exhibited no

Figure 2. ECD spectra of compounds 1−4.

Figure 3. Δδ (=δS − δR) values for (S)- and (R)-MTPA esters of 1.

Penicilone B (2) was also obtained as a yellow oil with the molecular formula C29H34O6 from HRESIMS data, which indicated the formal loss of H2O in comparison to 1. Careful comparison of the 1H and 13C NMR spectra of 2 (Table 1) with those of 1 showed a close structural relationship. The obvious differences were the signals for the substituent connected to C-3 of the azaphilone core. An aromatic system was indicated in 2 by signals in the 1H NMR spectrum at δH 7.15 (t, J = 7.8 Hz), 6.77 (d, J = 7.8 Hz), and 6.75 (d, J = 7.8 Hz) and in the 13C NMR spectrum at δC 155.0 (C-15), 138.8 (C-11), 131.6 (C-13), 122.2 (C-12), 118.5 (C-10), and 113.9 (C-14), coincidental disappearance of aliphatic signals of the moiety. Furthermore, the HMBC correlations from CH3-16 to C-10, C-11, C-12, and C-15 showed this methyl group was located at C-11 of the aromatic system. Thus, the side group at C-3 in 2 was assigned as 3-methylphenol, identical to the corresponding moiety of cohaerin A.9 The absolute configuration of 7S was deduced by comparison of the Cotton effects in the ECD spectrum of 2 with that of cohaerin A,9,10 which showed positive (358 nm) and negative (274 nm, 237 nm) Cotton effects (Figure 2). Penicilone C (3), with the molecular formula C29H35ClO7 as determined by HRESIMS, was isolated as a bright yellow oil. Its positive ESIMS spectrum exhibited protonated molecule peaks at m/z 531 and 533 [M + H]+ with a ratio of 3:1, indicating the presence of one chlorine atom. The 1H NMR spectrum (Table 2) was very similar to that of 1, with the notable difference being the absence of the H-5 signal in 3, which suggested that in 3 a chlorine atom replaced the hydrogen at C-5 in 1. Compared to those of 1, significant changes in the chemical shifts of C-5, C-6, C-4a, and C-4 in the 13C NMR spectrum as well as H-4 in the 1H NMR spectrum of 3 are attributed to the chlorine atom at C-5 in 3. The ECD spectrum of 3 showed a negative Cotton effect at 365 nm and positive maxima at 312 and 231 nm (Figure 2). These Cotton effects are opposite those in the ECD spectrum of 1. According to earlier studies the chlorine atom at C-5 has no significant effect on the signs of the ECD curves.14,15 Therefore, a 1083



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Table 2. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data for 3 and 4 (CDCl3) 3 position

δC, type

1 3 4 4a 5 6 7 8 8a 9 10 11 12

153.0, CH 154.5, C 110.9, CH 137.3, C 111.2, C 186.3, C 84.0, C 191.5, C 114.3, C 21.5, CH3 129.7, C 161.2, C 40.5, CH2

13 14

64.8, CH 45.6, CH2

15 16 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′


4 δH, mult. (J in Hz)

δC, type

7.96, brd 6.69, s

1.64, s

2.86, dd (16.8, 4.2) 2.67, dd (16.8, 8.4) 4.41, m 2.83, dd (16.2, 4.2) 2.65, dd (16.2, 7.8) 2.13, s

6.73, dd (10.2, 1.2) 2.50, m 1.36, m 1.26, m 1.26, m 1.26, m 1.30, m 0.89, t (6.6) 1.87, d (1.2) 1.01, d (6.6)

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δH, mult. (J in Hz)

154.0, CH 157.4, C 111.3, CH 138.8, C 111.1, C 187.1, C 84.5, C 192.2, C 114.9, C 22.2, CH3 118.7, C 138.9, C 122.6, CH

8.07, s

131.8, CH 113.8, CH

7.22, t (7.8) 6.76, d (7.8)


6.96, s

1.67, s

6.84, d (7.8)

2.32, s

6.74, dd (10.2, 1.2) 2.49, m 1.34, m 1.24, m 1.24, m 1.24, m 1.28, m 0.88, t (6.6) 1.87, d (1.2) 1.00, d (6.6)

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Shanghai Shenguang wzz-2s automatic polarimeter. UV spectra were obtained on a Beckman DU 640 spectrophotometer. ECD spectra were recorded on a JASCO J-810 circular dichroism spectrometer. IR spectra were recorded on a Cary 610/670 spectrometer using KBr pellets. NMR spectra were acquired using an AVANCE 600 NMR spectrometer (600 MHz for 1H and 150 MHz for 13C) or a Bruker AV-400 NMR spectrometer (400 MHz for 1H and 100 MHz for 13C), using tetramethylsilane as an internal standard. HRESIMS spectra were obtained from a maXis spectrometer or a Micromass Q-TOF spectrometer. Semipreparative HPLC was performed on a Hitachi system using a semipreparative C18 (Kromasil, 5 μm, 10 × 250 mm) column coupled with a 2400 UV detector. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200−300 mesh), Sephadex LH-20 (Amersham Biosciences), and octadecylsilyl silica gel (Unicorn; 45−60 μm) were used for column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin-layer chromatography (TLC). Fungal Material. The fungus Penicillium janthinellum HK1-6 was isolated from the mangrove rhizosphere soil, which was collected from Dongzhaigang mangrove natural reserve in Hainan Island in September 2015. The strain was deposited at the Marine Science & Technology Institute, College of Environmental Science & Engineering, Yangzhou University, Yangzhou, PR China. The fungus was identified according to its morphological traits and a molecular protocol by amplification and sequencing of the DNA of the ITS region of the rRNA gene as described previously.27 The fungus was identified as a P. janthinellum, whose 563 base pair ITS sequence had 99% sequence identity to that of

Figure 4. ECD spectra of compounds 5 and 6.

differences between vancomycin-resistant E. faecalis and susceptible E. faecium, while 4 showed stronger activity against vancomycin-resistant E. faecalis than susceptible E. faecium. These findings suggest that 2−4 might possess the capability to effectively circumvent antibiotic cross-resistance. Comparing the antibacterial activities of 2 and 5 (or 4 and 6), the 7-O-(E)-2,4dimethyldec-2-enoyl group plays a critical role in the antibacterial activities. 1084



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Table 3. Antibacterial Activities of 1−6a MIC (μg/mL)

a

pathogenic bacteria

antibiotic resistant (−)/susceptible (+)

1

2

3

4

5

6

Ob

Vc

S. aureus ATCC 43300 S. aureus ATCC 33591 S. aureus ATCC 25923 S. aureus ATCC 29213 E. faecalis ATCC 51299 E. faecium ATCC 35667 E. coli ATCC 25922

methicillin (−) methicillin (−) methicillin (+) methicillin (+) vancomycin (−) vancomycin (+)

>50 >50 >50 >50 >50 >50 >50

3.13 3.13 3.13 3.13 3.13 3.13 >50

6.25 6.25 12.5 6.25 12.5 12.5 >50

6.25 6.25 12.5 3.13 6.25 12.5 >50

>50 >50 >50 >50 >50 >50 >50

>50 50 25 >50 50 50 >50

>100 >100 0.78 3.13 50 >100 >100

0.39 0.78 1.56 0.39 6.25 0.39 >100

Data are expressed in MIC values. bO = oxacillin sodium. cV = vancomycin·HCl.

P. janthinellum HN07 (JQ796872.1). The sequence data have been submitted to GenBank with accession number KY412802. Fermentation, Extraction, and Isolation. The fungus P. janthinellum HK1-6 was cultivated in potato glucose liquid medium (20 g of glucose and 30 g of artificial sea salt (China National Salt Industry Corporation) in 1 L of potato infusion; 1 L Erlenmeyer flasks each containing 400 mL of culture broth) at 25 °C without shaking for 4 weeks. The culture (10 L) was filtered to separate the broth from the mycelia. Then the broth was extracted three times with an equal volume of EtOAc. The organic extracts were concentrated under vacuum to afford an EtOAc extract (13.0 g), which was separated by silica gel CC using a step gradient elution with EtOAc−petroleum ether (0−100%) and then with MeOH−CHCl3 (0−100%) to provide 10 fractions (Fr.1−Fr.10). Fr.4 was subjected to Sephadex LH-20 CC eluting with a mixture of CHCl3−MeOH (v/v, 1:1) and then was further purified by an ODS column eluting with 85% MeOH−H2O to give 4 (10.9 mg). Fr.5 was isolated on silica gel CC by using repeated gradient elution of EtOAc−petroleum ether and further purified by HPLC (80% MeOH− H2O) to give 2 (28.2 mg). Fr.8 was subjected to Sephadex LH-20 CC eluting with a mixture of CHCl3−MeOH (v/v, 1:1) and then was subjected to an ODS column eluting with 75% MeOH−H2O to provide Fr.8-1 and Fr.8-2, which were further purified by HPLC to afford 1 (23.7 mg) and 3 (8.8 mg), respectively. Penicilone A (1): yellow oil; [α]15D +160 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 224 (5.1), 332 (3.0) nm; ECD (0.08 mM, MeOH) λmax (Δε) 356 (7.3), 270 (−3.3), 229 (7.5) nm; IR (KBr) νmax 3415, 2925, 2854, 1703, 1635, 1453, 1377, 1321, 1099, 873 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), Table 1; HRESIMS m/z 497.2536 (calcd for C29H37O7, 497.2534), 519.2353 (calcd for C29H36O7Na, 519.2353). Penicilone B (2): yellow oil; [α]15D +140 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 221 (5.2), 336 (3.8) nm; ECD (0.09 mM, MeOH) λmax (Δε) 358 (9.4), 274 (−4.8), 237 (−0.8) nm; IR (KBr) νmax 3265, 2924, 2853, 1703, 1614, 1462, 1366, 1321, 1289, 1232, 1124, 1086, 872 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), Table 1; HRESIMS m/z 479.2437 (calcd for C29H35O6, 479.2428). Penicilone C (3): bright yellow oil; [α]15D −70 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 222 (4.9), 340 (2.8) nm; ECD (0.05 mM, MeOH) λmax (Δε) 365 (−7.2), 312 (2.1), 231 (9.7) nm; IR (KBr) νmax 3293, 2925, 2854, 1703, 1640, 1531, 1378, 1244, 1129, 1069, 891 cm−1; 1 H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), Table 2; HRESIMS m/z 531.2152 (calcd for C29H3635ClO7, 531.2144), 533.2155 (calcd for C29H3637ClO7, 533.2115), 553.1973 (calcd for C29H3535ClO7Na, 553.1964), 555.1966 (calcd for C29H3537ClO7Na, 555.1934). Penicilone D (4): bright yellow oil; [α]15D −170 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 220 (4.8), 347 (3.3) nm; ECD (0.08 mM, MeOH) λmax (Δε) 366 (−9.1), 316 (4.1), 277 (4.5), 231 (11.1) nm; IR (KBr) νmax 3408, 2925, 2853, 1705, 1627, 1527, 1465, 1326, 1132, 1096, 997 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), Table 2; HRESIMS m/z 513.2057 (calcd for C29H3435ClO6, 513.2038), 515.2039 (calcd for C29H3437ClO6, 515.2009). Ester Hydrolysis of 2 and 4. To a solution of 2 (2.2 mg) in MeOH (2.0 mL) was added NaOH (320 mg), and the reaction mixture was stirred at room temperature (rt) overnight, neutralized with 3 M HCl to

pH 6.0, and then concentrated in vacuo. The residue was diluted with EtOAc, and the organic layer was washed with water and brine and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by HPLC (50% MeOH−H2O) to afford 5 as a yellow oil (1.2 mg, 87.7%). To a solution of 4 (1.5 mg) in MeOH (1.0 mL) was added NaOH (160 mg), and the reaction mixture was stirred at rt overnight, neutralized with 3 M HCl to pH 6.0, and then concentrated in vacuo. The residue was diluted with EtOAc, and the organic layer was washed with water and brine and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by HPLC (60% MeOH−H2O) to afford 6 as a bright yellow oil (0.8 mg, 82.2%). Penicilone E (5): yellow oil; [α]15D 134 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 218 (1.8), 256 (0.8), 334 (1.0) nm; ECD (0.84 mM, MeOH) λmax (Δε) 368 (6.8), 280 (−6.8) nm; 1H NMR (400 MHz, CDCl3) δH 8.00 (1H, s, H-1), 7.21 (1H, t, J = 8.0 Hz, H-13), 6.83 (1H, d, J = 8.0 Hz, H-12), 6.79 (1H, d, J = 8.0 Hz, H-14), 6.43 (1H, s, H-4), 5.57 (1H, s, H-5), 2.28 (3H, s, H-16), 1.59 (3H, s, H-9); 13C NMR (CDCl3, 100 MHz) δC 196.3 (C-6), 195.4 (C-8), 155.8 (C-3), 154.2 (C-1), 153.7 (C-15), 144.0 (C-4a), 138.9 (C-11), 131.8 (C-13), 122.8 (C-12), 118.5 (C-10), 115.6 (C-8a), 113.9 (C-4), 113.7 (C-14), 105.9 (C-5), 83.6 (C-7), 28.6 (C-9), 20.0 (C-16); HRESIMS m/z 299.0914 (calcd for C17H15O5, 299.0914). Penicilone F (6): bright yellow oil; [α]15D −180 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (1.8), 252 (0.8), 341 (1.1) nm; ECD (0.75 mM, MeOH) λmax (Δε) 370 (−8.2), 299 (5.6) nm; 1H NMR (400 MHz, CDCl3) δH 8.03 (1H, s, H-1), 7.25 (1H, t, J = 8.0 Hz, H-13), 6.88 (1H, d, J = 8.0 Hz, H-12), 6.76 (1H, d, J = 8.0 Hz, H-14), 6.89 (1H, s, H4), 2.31 (3H, s, H-16), 1.63 (3H, s, H-9); 13C NMR (CDCl3, 100 MHz) δC 157.3 (C-3), 155.4 (C-1), 153.9 (C-15), 139.1 (C-4a), 139.1 (C-11), 132.0 (C-13), 123.1 (C-12), 117.9 (C-10), 114.7 (C-8a), 113.7 (C-14), 112.1 (C-4), 110.8 (C-5), 84.2 (C-7), 28.6 (C-9), 20.0 (C-16) (the signals for C-6 and C-8 were not detected due to the limited sample size); HRESIMS m/z 333.0520 (calcd for C17H1435ClO5, 333.0524), 335.0490 (calcd for C17H1437ClO5, 335.0495). Preparation of the (S)- and (R)-MTPA Esters of 1 by the Modified Mosher’s Method. For (S)-MTPA ester (1s), (R)-MPTA chloride (20 mg) was added to a solution of 1 (1.3 mg) and pyridine-d5 (500 μL). After stirring at rt for 1.5 h, the mixture was analyzed by 1H NMR spectroscopy without purification. (S)-MTPA ester (1s): 1H NMR (pyridine-d5, 600 MHz) δH 3.097 (1H, brd, J = 16.8 Hz, H-12a), 3.001 (1H, dd, J = 16.8, 5.4 Hz, H-12b), 3.001 (1H, dd, J = 19.2, 5.4 Hz, H-14a), 2.778 (1H, dd, J = 19.2, 4.2 Hz, H-14b), 2.157 (3H, s, H-16). The (R)-MTPA ester (1r) was prepared in the same manner by the addition of (S)-MPTA chloride. (R)-MTPA ester (1r): 1H NMR (pyridine-d5, 600 MHz) δH 3.069 (1H, brd, J = 16.8 Hz, H-12a), 3.069 (1H, brd, J = 19.2 Hz, H-14a), 2.944 (1H, dd, J = 16.8, 7.2 Hz, H-12b), 2.866 (1H, dd, J = 19.2, 4.2 Hz, H-14b), 2.156 (3H, s, H-16). Antibacterial Assays. Seven bacterial strains, including Grampositive Staphylococcus aureus (ATCC 43300), S. aureus (ATCC 33591), S. aureus (ATCC 29213), S. aureus (ATCC 25923), Enterococcus faecalis (ATCC 51299), E. faecium (ATCC 35667), and Gram-negative Escherichia coli (ATCC 25922), were used for the antibacterial assay. The specific antibacterial assay was carried out as described previously.28 1085



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H.; Kim, B. Y.; Ueki, M.; Takahashi, S.; Osada, H.; Jang, J. H.; Ahn, J. S. Phytochemistry 2016, 122, 154−164. (16) Due to priority rules for assigning absolute configurations, (7S)-1 and (7S)-3 have opposite configurations at C-7 even though they both have the 7S designation. (17) Nalli, Y.; Mirza, D. N.; Wani, Z. A.; Wadhwa, B.; Mallik, F. A.; Raina, C.; chaubey, A.; Riyaz-Ul-Hassan, S.; Ali, A. RSC Adv. 2015, 5, 95307−95312. (18) Chen, F. C.; Manchand, P. S.; Whalley, W. B. J. Chem. Soc. C 1971, 3577−3579. (19) Gao, J. M.; Yang, S. X.; Qin, J. C. Chem. Rev. 2013, 113, 4755− 4811. (20) Osmanova, N.; Schultze, W.; Ayoub, N. Phytochem. Rev. 2010, 9, 315−342. (21) Gao, S. S.; Li, X. M.; Zhang, Y.; Li, C. S.; Cui, C. M.; Wang, B. G. J. Nat. Prod. 2011, 74, 256−261. (22) Wang, W. X.; Kusari, S.; Laatsch, H.; Golz, C.; Kusari, P.; Strohmann, C.; Kayser, O.; Spiteller, M. J. Nat. Prod. 2016, 79, 704−710. (23) Akihisa, T.; Tokuda, H.; Yasukawa, K.; Ukiya, M.; Kiyota, A.; Sakamoto, N.; Suzuki, T.; Tanabe, N.; Nishino, H. J. Agric. Food Chem. 2005, 53, 562−565. (24) Hsu, L. C.; Liang, Y. H.; Hsu, Y. W.; Kuo, Y. H.; Pan, T. M. J. Agric. Food Chem. 2013, 61, 2796−2802. (25) Huang, H.; Feng, X.; Xiao, Z.; Liu, L.; Li, H.; Ma, L.; Lu, Y.; Ju, J.; She, Z.; Lin, Y. J. Nat. Prod. 2011, 74, 997−1002. (26) Stierle, A. A.; Stierle, D. B.; Girtsman, T.; Mou, T. C.; Antczak, C.; Djaballah, H. J. Nat. Prod. 2015, 78, 2917−2923. (27) Zheng, C. J.; Shao, C. L.; Guo, Z. Y.; Chen, J. F.; Deng, D. S.; Yang, K. L.; Chen, Y. Y.; Fu, X. M.; She, Z. G.; Lin, Y.-C.; Wang, C.-Y. J. Nat. Prod. 2012, 75, 189−197. (28) Pierce, C. G.; Uppuluri, P.; Tristan, A. R.; Wormley, F. L.; Mowat, E.; Ramage, G.; Lopez-Ribot, J. L. Nat. Protoc. 2008, 3, 1494−1500.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01179. 1 H NMR, 13C NMR, HSQC, COSY, HMBC, NOESY, and HRESIMS spectra of 1−4, 1H NMR, 13C NMR, and HRESIMS spectra of 5 and 6, and 1H NMR spectra of 1s and 1r (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-514-89795882. E-mail: [email protected], [email protected]. ORCID

Min Chen: 0000-0001-6702-3993 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Prof. C.-Y. Wang, School of Medicine and Pharmacy, Ocean University of China, for testing of MS data; Dr. R.-Z. Liu, College of Chemistry and Chemical Engineering, Yangzhou University, for testing of NMR and optical rotation data; and Dr. C.-J. Zheng, College of Chemistry and Chemical Engineering, Hainan Normal University, for collection of the mangrove rhizosphere soil sample. The Testing Center of Yangzhou University is acknowledged for the testing assistance. This work was supported by the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 16KJB350005) and the Financial Support from Yangzhou University.

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REFERENCES

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