Note pubs.acs.org/jnp
Macrolides from a Marine-Derived Fungus, Penicillium meleagrinum var. viridif lavum, Showing Synergistic Effects with Fluconazole against Azole-Resistant Candida albicans Miki Okabe,† Takashi Sugita,‡ Kaoru Kinoshita,† and Kiyotaka Koyama*,† †
Department of Pharmacognosy and Phytochemistry and ‡Department of Microbiology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose-shi, Tokyo 204-8588, Japan S Supporting Information *
ABSTRACT: Two new 13-membered macrolides (1, 7), along with known 13-membered macrolides PF1163A, B, D, H, and F (2−6), were isolated from a strain of a marine-derived fungus, Penicillium meleagrinum var. viridif lavum. The structures of 1 and 7 were elucidated from spectroscopic data (NMR, MS, IR). Compounds 1−7 showed synergistic effects with fluconazole against azole-resistant Candida albicans by a checkerboard assay.
measured the 1H NMR spectrum at −20 °C. As a result, the broadness of the signals was reduced. Sasaki et al. discussed the broad signals in the NMR spectrum of PF1136A and PF1163B. The N-methylamide can cause the existence of cis and trans conformers in solution, and the interconversion of the conformers may be restricted by the 13-membered rigid macrocyclic structure in PF1136A and -B. The broad signals in the NMR spectra of PF1136A and -B were therefore attributed to the restricted interconversion of conformers.9 However, Fodil Bouazza et al. reported that a conformational analysis of PF1163B revealed that amide bond isomerization was not adequate to explain the multiple signals that were observed in the NMR spectra.11 The broad signals and multiple signals observed in the 1H and 13C NMR spectra of 1−7 are then likely due to a complex mixture of conformers. Five known compounds, PF1163A (2), -B (3), -D (4), -H (5), and -F (6), were identified by comparisons to their published data.9,10,12 However, compounds 1 and 7 did not coincide with spectroscopic data for known compounds. The molecular formula for compound 1 was determined to be C30H47NO4 by HRFABMS, which indicated eight degrees of unsaturation. The 1H NMR signals were very broad at room temperature. However, at −20 °C signals derived from an aliphatic chain were observed in the shielded region. Additionally, an amide bond, an ester bond, a CH3 bound to a nitrogen atom, and a disubstituted benzene were deduced from 13C NMR and DEPT data. From this, we suspected that compound 1 has the same skeleton as PF1163A. In the 13C NMR analysis (Table 1), up to four signals were observed from one carbon atom (C-3′) due to conformational isomers. HRFABMS, 13C NMR, and DEPT results for compound 1 were compared with
O
pportunistic deep-seated fungal infections occur in immunocompromised hosts with serious underlying diseases, such as AIDS patients. Azole antifungal agents are widely used to treat patients with such systemic fungal infections. However, the increase in the number of azoleresistant fungal strains is problematic clinically; thus, it is necessary to develop novel antifungal agents. This can be challenging, however, as it is difficult to find a fungus-specific target because fungi and humans are both eukaryotes.1,2 In this study, we discovered new compounds that showed synergistic effects with fluconazole (an antifungal agent) against azole-resistant Candida albicans, from the extract of a marinederived fungus. Marine-derived fungi have great potential as a source of drugs because they produce a variety of natural products.3 We previously reported several novel compounds from marine-derived fungi including phomactins,4−6 didimellamides,7 and myrocin D.8 In this paper, we report two new 13membered macrolides (1, 7) and five known 13-membered macrolides (2−6),9,10 which showed antifungal activity against azole-resistant C. albicans in combination with fluconazole. Penicillium meleagrinum var. viridif lavum was grown on a marine wheat medium, then extracted with CHCl3. The CHCl3 extract showed synergistic effects with fluconazole against azole-resistant C. albicans. The CHCl3 extract was subjected to silica gel column chromatography (CC), Sephadex LH-20 CC, and HPLC. Two new and five known compounds were obtained. Compounds 1−7 were obtained as colorless, amorphous solids. The chemical shift patterns for 1−7 in the 1H and 13C NMR spectra were very similar to those for PF1163A isolated from a Penicillium sp.,9,10 suggesting that compounds 1−7 may be analogues of PF1163A having a 13-membered macrolide. It was reported that the signals were very broad in the 1H and 13C NMR spectra of PF1163A and PF1163B.9 Consequently, we © XXXX American Chemical Society and American Society of Pharmacognosy
Received: January 8, 2016
A
DOI: 10.1021/acs.jnatprod.6b00019 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Table 1. NMR Spectroscopic Data (δ in ppm) for Compounds 1 and 7 (1H NMR: 500 MHz, 13C NMR: 125 MHz) melearoride A (1)a position 1 2 3 4
5 6 7 8
9
10 11 12
13 14 15 1′ 2′
3′
4′ 5′ 9′ 6′ 8′ 7′ 10′
11′ 12′ 13′
δC, type 173.3, 173.8, 32.9, 34.1, 23.9, 24.0, 33.7, 33.7, 34.1, 26.5,
C C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2
28.2, CH 29.4, CH 25.3, CH 28.3, CH2 28.7, CH2 29.0, CH2 29.7, CH 74.5, CH 75.2, CH 75.8, CH2 23.4, CH2 23.7, CH2 24.7, CH2 25.0, CH2 31.6, CH2 31.7, CH2 31.8, CH2 22.5, CH2 22.9, CH3 14.0, CH3 20.0 CH3 20.5, CH3 170.2, C 171.1, C 55.4, CH 61.6, CH 67.3, CH 33.0, CH2 33.5, CH2 CH2 35.0, CH2 35.1, C 128.1, C 128.4, C 129.5, CH 130.0, CH 114.5, CH 114.9, CH 157.5, C 157.9, C 64.6, CH2 64.7, CH2 119.5, CH 119.7, CH 138.0, C 138.3, C 25.8,c CH3
melearoride B (7)a
δH (J in Hz)b
δC, type 173.7, C 173.8, C 34.0, CH2
2.44, t (13.1) 2.18, m 1.33, m 1.33, m 2.69, m 2.69, m 2.19, m, 2.69, m 1.87−1.94, m 1.37−1.53, m 1.19−1.40, m 1.06, m 1.37−1.53, m 1.37−1.53, m 1.37−1.53, m 1.37−1.53, m 4.85, m 4.85, m 4.85, m 1.53, m 1.27, m 1.24, m 1.27, m 1.27, m 1.19, m 1.19, m 1.19−1.40, m 1.37, m 0.88 (t, 6.9) 0.84 (d, 6.9) 0.84 (d, 6.9)
5.81, 4.58, 3.52, 2.83, 3.20, 3.43, 3.01, 3.01,
7.09, 7.18, 6.83, 6.85,
t (8.3) dd (10.3, dd (11.2, dd (14.3, dd (14.3, dd (14.5, m m
d d d d
1.42−1.51, m 1.53−1.69, m 2.73, m
25.5, CH2 27.0, CH2 27.9, CH
1.84−1.93, m 1.84−1.93, m 1.39−1.47, m
34.1, CH2 30.3, CH2
1.14−1.26, m 1.53−1.69, m
72.3, 73.0, 73.1, 42.5, 43.8, 66.9,
5.02, m 5.02, m 5.02, m 1.53−1.69, m 1.53−1.69, m 3.00, m, 3.21−3.29, m
4.45, d (7.2) 4.78, d (7.2) 5.51, t (7.2) 5.50, t (7.2)
CH CH CH CH2 CH2 CH
39.1, CH2
1.14−1.26, m
19.1, CH2
1.30, m
CH3 CH3 CH3 C
0.80, t (6.5), 0.92, t (7.0) 0.85, d (6.9) 0.89, d (6.9)
55.9, CH 61.7, CH
5.86, t (8.2) 4.62, dd (9.6, 4.8)
32.6, CH2 33.2, CH2
3.44, m 3.59, dd (10.9, 4.1)
35.3, CH2 128.8, C 130.0, 130.5, 114.6, 115.1, 157.2, 157.7, 69.2,
(8.6) (8.6) (8.6) (8.6)
2.18−2.27, m, 2.44, m
23.1, CH2 23.7, CH2 28.2, CH2
14.2, 20.0, 20.5, 171.9,
5.5) 4.1) 7.7) 8.9) 11.5)
δH (J in Hz)b
CH2
2.81, dd (13.8, 7.1), 3.00, m
CH2 CH CH CH CH C C CH2
69.3, CH2 75.7, CH
7.15, 7.22, 6.83, 6.86,
d d d d
(8.6) (8.6) (8.6) (8.6)
3.98, 4.09, 4.03, 3.81,
dd (9.4, 2.6) dd (9.4 2.6) m, 4.12, dd (9.4, 2.6) t (9.4)
71.6, C 25.1,c CH3
1.81, s
B
1.34, s, 1.35, s
DOI: 10.1021/acs.jnatprod.6b00019 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Table 1. continued melearoride A (1)a position 14′ N-CH3
a
δC, type 18.2, 29.3, 30.7, 40.3,
c
CH3 CH3 CH3 CH2
melearoride B (7)a
δH (J in Hz)b 1.74, 3.03, 2.95, 2.71,
δC, type
s s s s
26.7, 29.2, 29.3, 30.9,
c
CH3 CH3 CH3 CH3
δH (J in Hz)b 1.28, 2.69, 3.02, 2.98,
s, 1.30, s s s s
In CDCl3 bAt −20 °C cMay be interchanged.
suggested binding of the aliphatic side chain (C-10−14) to the macrolide at C-9. From the above, the structure of 1 has no hydroxy group at C-11 and has a prenyl group at the 7′-O position compared with compound 2. Hence, the planar structure of 1 was established, and the compound was named melearoride A. The molecular formula for compound 7 was determined to be C30H49NO7 by HRFABMS, which indicated seven degrees of unsaturation.. From 1H and 13C NMR and DEPT we suspected that compound 7 had the same skeleton as PF1163A as well as 1. In the 13C NMR analysis (Table 1), up to three signals were observed from one carbon atom (C-9, 3′) due to conformational isomers. HSQC-TOCSY suggested the presence of a hydroxy group at C-11 (δC 66.9: CH) as in 2 (Figure 1). The COSY correlation from H-10′ to H-11′, the NOESY correlation from H-8′ to H-10′, and the HMBC correlations from H-13′ to C-11′, C-12′, and C-14′ and from H-14′ to C11′, C-12′, and C-13′ suggested the presence of a 2,3dihydroxy-2-methylbutyl moiety attached to the oxygen at C-7′ (Figure 1). Therefore, the structure of 7 was determined to have a hydroxy group at C-11 and a 2,3-dihydroxy-2methylbutyl moiety at the 7′-O position. Hence, the planar structure of 7 was determined, and the compound was named melearoride B. The relative configurations for the macrolide ring portions of compounds 1 and 7 were determined to be the same as those of 3 and 2, respectively, because the macrolide ring NMR shifts of both 1H and 13C of compounds 1 and 3 and compounds 7 and 2 were similar. Additionally, because the absolute configurations of 2 and 3 have been confirmed by synthesis11,12 and 2 and 3 were co-isolated from the fungus with compounds 1 and 7, the relative and absolute configurations at C-6, -9, and -2′ of compound 1 and at C-6, -9, -11, and -2′ of compound 7 have been suggested to be the same as those of compounds 3 and 2, respectively. However, the relative configuration at C-11′ of compound 7 could not be elucidated. Compounds 1−7 were evaluated for antifungal activities against azole-resistant C. albicans J2-36. Compounds 2, 3, 5, and 6 inhibited the growth of azole-resistant C. albicans at concentrations of 1, 2, 16, and 8 μg/mL, respectively, while compounds 1, 4, and 7 did not exhibit any antifungal activity at a concentration of 32 μg/mL (Table 2). Compounds 2 and 3 were reported to have antifungal activity against C. albicans TIMM1768,13 whereas the prenyl derivative and dihydroxy derivative (compounds 1 and 7) had decreased antifungal activity. From these results, the hydroxyethyl group is important for the antifungal activity of 2 and 3. We investigated synergistic effects with fluconazole of compounds 1−7 against azole-resistant C. albicans by a checkerboard assay. As a result, all compounds, including compounds 1, 4, and 7, which did not show any antifungal activity, showed a synergistic effect with fluconazole against azole-resistant C. albicans J2-36. It is necessary to perform a
those for compound 2, and the following differences were observed: Two carbons attached to oxygenated C-11 (δC 66.7, 66.8: CH) and C-11′ (δC 61.4: CH2) and a hydroxy group of 2 had disappeared in 1, whereas two CH3 (C-13′: δC 25.8 and C14′: δC 18.2), one olefinic bond (C-11′: δC 119.5, 119.7; C12′: δC 138.0, 138.3), and one CH2 (C-11: δC 24.7, 25.0) were present in 1. The presence of an olefinic bond is also supported from an increase in the degree of unsaturation. The COSY correlation from H-10′ to H-11′ and HMBC correlations from H-13′ to C-11′, C-12′, and C-14′ and from H-14′ to C-11′, C12′, and C-13′ suggested the presence of a prenyl group (Figure 1). The HSQC-TOCSY correlations (Figure 1)
Figure 1. Key correlations for 1 and 7: COSY, HSQC-TOCSY (bold) and HMBC, 1D selective TOCSY (arrows), NOESY (dashed arrows) in 7. C
DOI: 10.1021/acs.jnatprod.6b00019 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Table 2. Synergistic Effects with Fluconazole of Compounds 1−7 against Azole-Resistant C. albicans (J2-36)a fluconazole (μg/mL)
compounds (μg/mL) compound
MICbalone
MICccombnation
FICAd
MlCealone
MICfcombnation
FICBg
FICIh
1 2 3 4 5 6 7
>32 1 2 >32 16 8 >32
1 0.0078 0.0016 0.5 0.125 0.031 0.5
0.031 0.0078 0.000 78 0.016 0.0078 0.0039 0.016
>64 >64 >64 >64 >64 >64 >64
1 0.5 1 1 1 0.5 0.5
0.016 0.0078 0.016 0.016 0.016 0.0078 0.0078
0.05 0.02 0.02 0.03 0.02 0.01 0.02
Clinical isolates, incubated for 48 h at 37 °C. bMIC of compounds 1−7 for growth was determined as the lowest antifungal concentration that caused ≥90% reduction compared with control (no drug). cMIC of compounds 1−7 in combination with fluconazole. dFractional inhibitory concentration (FIC)A value is calculated as MIC of compounds 1−7 in combination/MIC of compounds 1−7 alone. eMICs of fluconazole for growth were determined as the lowest antifungal concentrations that caused ≥90% reduction compared with control (no drug). fMIC of fluconazole in combination with compounds 1−7. gFICB value is calculated as the MIC of fluconazole in combination/MIC of fluconazole alone. hFractional inhibitory concentration index (FICI) values are calculated as follows: (MIC of compounds 1−7 in combination/MIC of compounds 1−7 alone) + (MIC of fluconazole in combination/MIC of fluconazole alone). The interpretation of the FICI is determined as follows: ≤0.5, synergistic effect; >0.5 but 0.5 but
