Peniisocoumarins AâJ: Isocoumarins from Penicillium commune QQF

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Cite This: J. Nat. Prod. 2018, 81, 1376−1383

Peniisocoumarins A−J: Isocoumarins from Penicillium commune QQF3, an Endophytic Fungus of the Mangrove Plant Kandelia candel Runlin Cai,† Yingnan Wu,† Senhua Chen,† Hui Cui,† Zhaoming Liu,† Chunyuan Li,‡ and Zhigang She*,†,§ †

School of Chemistry, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China College of Materials and Energy, South China Agricultural University, Guangzhou 510642, People’s Republic of China § South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou 510006, People’s Republic of China

J. Nat. Prod. 2018.81:1376-1383. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 06/22/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: Ten new isocoumarins, named peniisocoumarins A−J (1−9 and 11), along with three known analogues (10, 12, and 13) were obtained from the fermentation of an endophytic fungus, Penicillium commune QQF-3, which was isolated from a fresh fruit of the mangrove plant Kandelia candel. Their structures were elucidated through extensive spectroscopic analysis. The absolute configurations of 1−7 were determined by single-crystal X-ray diffraction and modified Mosher’s method, and those of 8, 9, and 11 were assigned on the basis of experimental and calculated electronic circular dichroism data. Compounds 1 and 2 were unusual dimeric isocoumarins with a symmetric four-membered core. These isolated compounds (1−13) were evaluated for their cytotoxicity and enzyme inhibitory activities against α-glucosidase and Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB). Among them, compounds 3, 7, 9, and 11 exhibited potent inhibitory effects against α-glucosidase with IC50 values ranging from 38.1 to 78.1 μM, and compound 7 was found to inhibit MptpB with an IC50 value of 20.7 μM.

T

fruit of the mangrove plant Kandelia candel. Bioassay screening results showed that the EtOAc extract of this fungal fermentation possessed inhibitory activity against α-glucosidase in vitro with 53% inhibition at a single dose (100 μg/mL). Subsequent chemical investigation led to the isolation of 10 new isocoumarins, named peniisocoumarins A−J (1−9 and 11), together with three known analogues (10, 12, and 13). In the bioactivity assays, the α-glucosidase inhibitory, cytotoxic, and MptpB inhibitory effects of 1−13 were assessed. Herein, details of the isolation, structure elucidation, and biological activity of these compounds are described.

ype 2 diabetes is a chronic metabolic disease characterized by relative insulin deficiency and has been considered as one of the main threats to human health in the 21st century.1 αGlucosidase plays an important role in the insulin signaling pathway, and its inhibitors such as acarbose, miglitol, and voglibose, all originating from natural products, are widely used to treat type 2 diabetes.2−4 In addition, tuberculosis (TB) is a major infectious disease caused by Mycobacterium tuberculosis (Mtb). As an important virulence factor, Mtb protein tyrosine phosphatase B (MptpB) is secreted by Mtb into the host cell and attenuates host immune defenses, and, thus, small-molecule inhibitors of MptpB are considered as potential anti-TB agents.5,6 Up to now, there is a lack of more potent, specific, and drug-like MptpB inhibitors for preclinical evaluation.6−8 Mangrove endophytic fungi, as plant mutualists, occurring at the tropical and subtropical intertidal estuarine zones, are widely recognized as a prolific source of structurally unique and biologically active natural products that could be used for the development of new medicinal agents.9−11 In our ongoing research on mangrove endophytic fungi from the South China Sea, we have found several new metabolites with enzyme inhibitory activities against α-glucosidase12−16 and Mptpb.17−19 More impressively, a series of bioactive isocoumarin derivatives with α-glucosidase inhibitory, antibacterial, and anti-inflammatory activities also have been isolated.15,20−22 Recently, a fungal strain, Penicillium commune QQF-3, was collected from a fresh © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

The EtOAc extract of cultures of Penicillium commune QQF-3 was fractionated and purified by various chromatographic methods, to afford 10 new (1−9 and 11) and three known isocoumarins (10, 12, and 13). The known compounds were identified as 3-[(R)-3,3-dichloro-2-hydroxypropyl]-8-hydroxy6-methoxy-1H-isochromen-1-one (10), 23 (+)-6-methylcitreoisocoumarin (12),24,25 and (+)-diaporthin (13),26,27 respectively. Received: December 4, 2017 Published: May 24, 2018 1376

DOI: 10.1021/acs.jnatprod.7b01018 J. Nat. Prod. 2018, 81, 1376−1383

Journal of Natural Products

Article

methine unit at C-3. In addition, the methoxy protons 11-OMe (δH 3.76) revealed an HMBC correlation with ester carbonyl C11 (δC 171.6), allowing the establishment of a methoxycarbonyl group. The methoxycarbonyl group was then assigned at C-10 based on the HMBC correlation from H-10 to C-11. These data revealed half of the structure occupying eight of the 17 indices of hydrogen deficiency. The remaining index of hydrogen deficiency further confirmed that one additional four-membered ring system was determined to connect two identical structural moieties via C-9 (C-9′) and C-10 (C-10′). Considering no diagnostic NOE correlations among the densely substituted four-membered ring, the relative configuration of 1 could not be elucidated with confidence (Figure 1). Therefore, the structure of 1 was defined by X-ray diffraction analysis using Cu Kα radiation. The crystal of 1 had space group I2/a, representing a helical conformation with an intramolecular mirror symmetry (Figure 2). On the basis of these findings and the lack of optical activity, peniisocoumarin A (1) was determined as a mesomer, and its complete structure was established as shown. Peniisocoumarin B (2) was obtained initially as a white solid with the molecular formula of C28H24O12 based on HRESIMS. Similar to 1, the 13C NMR data of 2 revealed the presence of 14 carbons, including two methoxyls, two sp3 and three sp2 methines, and seven nonprotonated carbons (two carbonyls and five aromatic/olefinic carbons), which indicated a symmetric structure. Detailed comparison of their 1D and 2D NMR data suggested that peniisocoumarins A and B (1 and 2) shared the same planar structure. However, the NMR data for compound 2 differed from that of 1 with the chemical shifts of H-9 (δH 4.07 vs 4.11) and H-10 (δH 3.94 vs 4.00) in the 1H NMR spectrum and C-9 (δC 41.4 vs 42.1) and C-10 (δC 42.4 vs 41.4) in the 13C NMR spectrum (Table 1). These differences of their NMR data could be attributed to configurational variations and demonstrated that 1 and 2 were a pair of diastereoisomers. Similarly, an X-ray diffraction analysis (Cu Kα) was performed to confirm the structure of 2 (Figure 2). Therefore, peniisocoumarin B (2) was also determined as a mesomer, and its gross structure was assigned as shown. Analysis of the HRESIMS data for peniisocoumarin C (3) showed an ion peak at m/z 289.0716 [M − H]− consistent with a molecular formula of C15H14O6 (9 indices of hydrogen deficiency). The 1H NMR data (Table 2) revealed the presences of a hydroxy (δH 10.97, 8-OH), two aromatic protons (δH 6.49 and 6.34, H-7/5), an olefinic singlet (δH 6.29, H-4), a methoxy group (δH 3.87, 6-OMe), a methylene (δH 2.85 and 2.68, H-9), two methines (δH 3.78 and 3.59, H-11/

Peniisocoumarin A (1) had the molecular formula C28H24O12 as deduced from the HRESIMS spectrum, suggesting 17 indices of hydrogen deficiency. The 1H NMR spectroscopic data of 1 provided the resonances for a chelated phenolic hydroxy at δH 10.81, a set of meta-coupled aromatic protons at δH 6.43 (d, J = 2.2 Hz, 1H) and 6.32 (d, J = 2.2 Hz, 1H), an olefinic singlet proton of an α,β-unsaturated carbonyl group at δH 6.36 (s, 1H), two methoxy groups at δH 3.83 (s, 3H) and 3.76 (s, 3H), and two methine signals at δH 4.11 (d, J = 6.2 Hz, 1H) and 4.00 (d, J = 6.2 Hz, 1H). The combination of 13C NMR and HSQC spectra exhibited 14 carbon signals that could be assigned as 10 sp2-hybridized carbons including one ester carbonyl (δC 171.6) and one α,β-unsaturated lactone carbonyl (δC 165.4), two methoxy carbons (δC 55.9 and 52.7), and two aliphatic methine carbons (δC 42.1 and 41.4). On the basis of the above analysis, compound 1 could be proposed to be a completely symmetric framework and belongs to the isocoumarin class. The HMBC correlations from aromatic proton H-7 (δH 6.43) to C-5, C-6, C-8, and C-8a, from the methoxy protons 6-OMe (δH 3.83) to C-6, and from the chelated hydroxy group 8-OH (δH 10.81) to C-8 and C-8a showed an isocoumarin nucleus, structurally related to known compound (+)-6-methylcitreoisocoumarin (12).24,25 The 1H−1H COSY cross-peak of H-9/H-10 and HMBC correlations from the one aliphatic methine H-9 (δH 4.11) to C-3, and C-4 and from the other aliphatic methine H10 (δH 4.00) to C-3 confirmed the presence of a contiguous sp3

Figure 1. Key 2D NMR correlations for 1. 1377



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Figure 2. X-ray ORTEP drawings of 1 and 2.

10), and a methyl (δH 2.40, H-13), while the 13C NMR data (Table 3) exhibited eight aromatic/olefinic carbons and two carbonyl carbons for a lactone carbonyl (δC 165.9) and a ketone (δC 202.9). The 1D and 2D NMR data suggested a close structural relationship to (+)-6-methylcitreoisocoumarin (12).24,25 However, the side chain at C-3 was different from 12 due to the presence of an epoxy group attached at C-10/C-11, as supported by a decrease of 2 mass units, an increase of 1 index of hydrogen deficiency, and the 1H−1H COSY correlations from H-10 (δH 3.59, 1H) to H-9 (δH 2.85 and 2.68, 2H) and H-11 (δH 3.78, 1H) (Figure 3). Finally, the absolute configuration of peniisocoumarin C (3) was confirmed as 10S, 11S by X-ray single-crystal diffraction analysis (Figure 4). HRESIMS analysis of peniisocoumarin D (4) showed two ion peaks at m/z 285.0521 [M + H]+ (100%) and m/z 287.0493 [M + H]+ (33%) with the ratio 3:1, indicating one chlorine atom in the structure and corresponding to a molecular formula of C13H13ClO5. Apart from the similar NMR data of the isocoumarin skeleton in 4 and 3 (Tables 2 and 3), one methine group and two methylene groups were

Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data of 1 and 2 in CDCl3 1 position

δC, type

1/1′ 3/3′ 4/4′ 4a/4a′ 5/5′ 6/6′ 6/6′-OMe 7/7′ 8/8′ 8/8′-OH 8a/8a′ 9/9′ 10/10′ 11/11′ 11/11′-OMe

165.4, C 153.2, C 106.4, CH 138.3, C 102.3, CH 167.1, C 55.9, CH3 101.2, CH 163.7, C

2 δH (J in Hz)

6.36, s 6.32, d (2.2) 3.83, s 6.43, d (2.2)

δC, type 165.4, C 151.9, C 107.5, CH 138.7, C 102.3, CH 166.9, C 55.9, CH3 101.1, CH 163.7, C

10.81, s 99.9, C 42.1, CH 41.4, CH 171.6, C 52.7, CH3

4.11, d (6.2) 4.00, d (6.2) 3.76, s

δH (J in Hz)

6.46, s 6.26, d (2.2) 3.83, s 6.46, d (2.2) 10.94, s

100.1, C 41.4, CH 42.4, CH 169.9, C 52.4, CH3

4.07, d (10.0) 3.94, d (10.0) 3.73, s

Table 2. 1H NMR Data of 3−9 and 11 (500 MHz, δ in ppm) 3a

4a

5a

5b

10

6.29, s 6.34, d (2.2) 6.49, d (2.2) 2.85, dd (5.8, 15.3) 2.69, dd (6.5, 15.3) 3.59, m

6.33, s 6.34, d (1.7) 6.48, d (1.7) 2.79, dd (5.0, 14.7) 2.74, dd (7.8, 14.7) 4.32, m

6.32, s 6.36, d (2.2) 6.49, d (2.2) 3.06, dd (4.9, 14.9) 2.88, dd (8.9, 14.9) 4.43, m

11

3.78, d (4.6)

3.73, dd (3.8, 11.3) 3.61, dd (6.0, 11.3)

3.90, dd (4.2, 12.1) 3.82, dd (5.2, 12.1)

6.65, s 6.55, d (2.2) 6.63, d (2.2) 3.17, dd (3.8, 15.3) 2.78, dd (9.8, 15.3) 4.30, td (5.4, 9.6) 3.67, m

3.87, s

3.87, s

10.99, s 2.41, s

10.99, s

position 4 5 7 9

13 6-OCH3 5-OH 8-OH 10-OH 11-OH a

2.40, s 3.87, s 10.97, s

6c

7c

6.78, s

6.69, s

6.63, s 3.25, dd (4.3, 15.1) 2.87, dd (6.5, 15.1) 4.38, m 3.88, m

8c

5.34, t (5.9)

3.97, s 7.85, s 10.75, s 4.40, t (5.7)

11c

6.65, s

6.78, s

6.45, s 2.64, dd (5.1, 14.2) 2.60, dd (7.4, 14.2) 4.17, m

6.51, s 6.53, d (2.3) 6.46, d (2.3) 2.79, dd (3.8, 14.6) 2.56, dd (8.8, 14.6) 4.04, m

6.66, s 2.72, dd (3.7, 14.6) 2.41, dd (8.8, 14.6) 3.78, m

6.62, s 3.03, dd (3.4, 14.7) 3.82, dd (5.8, 14.7) 4.40, m

1.25, d (6.2)

3.58, m

3.40, m

6.21, d (3.4)

3.81, m

3.86, s

9b

3.29, m

3.90, s 11.15, s 4.13, d (3.8) 3.88, s

3.90, s 8.84, s 10.69, s 4.84, d (5.4) 4.69, t (5.5)

3.96, s 10.76, s 5.24, s

Recorded in CDCl3. bRecorded in DMSO-d6. cRecorded in acetone-d6. 1378



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Table 3. 13C NMR Data of 3−9 and 11 (125 MHz, δ in ppm)

a

position

3a

4a

5a

6c

7c

8c

9b

11c

1 3 4 4a 5 6 7 8 8a 9 10 11 12 13 6-OCH3

165.9, C 152.3, C 106.3, CH 138.7, C 102.2, CH 167.1, C 101.0, CH 163.9, C 100.1, C 31.8, CH2 55.5, CH 57.6, CH 202.9, C 29.1, CH3 55.9, CH3

166.1, C 153.3, C 106.8, CH 138.9, C 101.8, CH 167.1, C 100.9, CH 163.8, C 100.1, C 38.5, CH2 68.8, CH 49.5, CH2


167.0, C 152.8, C 101.8, CH 123.8, C 133.6, C 155.6, C 99.3, CH 157.7, C 98.8, C 39.2, CH2 60.7, CH 66.7, CH2




167.1, C 152.8, C 101.9, CH 124.0, C 133.5, C 155.5, C 99.1, CH 157.6, C 98.9, C 37.5, CH2 74.0, CH 77.3, CH

55.9, CH3

56.8, CH3

56.3, CH3

56.2, CH3

56.8, CH3

b

55.9, CH3 c

Recorded in CDCl3. Recorded in DMSO-d6. Recorded in acetone-d6.

chain and established the position of the hydroxy group (10OH) and the chlorine atom at C-10 and C-11, respectively. The linkage of the aliphatic side chain to C-3 was deduced by the HMBC correlations from H-9 to C-3 and C-4. Additionally, the absolute configuration of peniisocoumarin D (4) was determined by the modified Mosher’s method.28 The differences in 1H NMR chemical shifts between (S)- and (R)-MTPA esters (Δδ = δS − δR) (Figure 5) were calculated to assign the absolute configuration of C-10, which was found to be R.

Figure 3. 1H−1H COSY and key HMBC correlations for 3−9 and 11.

observed in the HSQC spectrum. The 1H−1H COSY crosspeaks from H-10 (δH 4.32, 1H) to H-9 (δH 2.79 and 2.74, 2H) and H-11 (δH 3.73 and 3.61, 2H) and from 10-OH (δH 2.41) to H-10 (Figure 3) confirmed the presence of an aliphatic side

Figure 5. Δδ (= δS − δR) values for (S)- and (R)-MTPA esters of 4.

Figure 4. X-ray ORTEP drawings of 3 and 5−7. 1379



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Peniisocoumarin E (5) was identified as an isomer of 4 with the same molecular formula based on HRESIMS. Comparison of the 1D and 2D NMR data from 4 and 5 revealed evidence for the same isocoumarin subunit, but differences in the signals associated with the side chain at C-3. In contrast to the data for 4, the 1H−1H COSY cross-peaks (in DMSO-d6) from 11-OH (δH 5.34, t, 5.9 Hz) to H-11 (δH 3.66, 2H) and from H-10 (δH 4.30, 1H) to H-9 (δH 3.17 and 2.78, 2H) and H-11 (Figure 3) strongly suggested the presence of a terminal hydroxy group (11-OH) in C-11 and the location of the chlorine atom to C10. An X-ray single-crystal diffraction analysis was performed to verify the results, from which the structure and 10Rconfiguration of peniisocoumarin E (5) were defined (Figure 4). The molecular formula of peniisocoumarin F (6) was calculated as C13H13ClO6 from HRESIMS and found to differ from 5 by 16 mass units. The 1H NMR spectrum of 6 was different from 5 by a hydroxy group at δH 7.85, an aromatic proton at δH 6.63, an olefinic proton at δH 6.78, and the absence of a signal of an aromatic proton (H-5). The linkage of the hydroxy group (δH 7.85, 5-OH) to C-5 in the isocoumarin subunit was further established by the HMBC correlations from the hydroxy group 5-OH to C-5 (δC 133.6), C-6 (δC 155.6), and C-4a (δC 123.8) and from the aromatic proton H-7 (δH 6.63) to C-5 and C-8a (δC 98.8) (Figure 3). Similar to 5, the structure and 10R-configuration of peniisocoumarin F (6) were also determined by X-ray single-crystal diffraction analysis (Figure 4). Peniisocoumarin G (7) was isolated as an amorphous power with a molecular formula of C12H12O6 derived from HRESIMS. The 1H and 13C NMR and HSQC spectra of compound 7 closely resembled those of the known compound orthosporin27,29 with an isocoumarin core structure, but compound 7 lacked an aromatic proton signal at C-5. The aromatic proton (H-5) in orthosporin was replaced by a hydroxy group (5-OH) in 7, which was supported by an increase of 16 mass units in the HRESIMS analysis and HMBC correlations from the other aromatic proton H-7 (δH 6.45) to C-5 (δC 132.1), C-6 (δC 153.9), and C-8a (δC 98.6) (Figure 3). Since peniisocoumarin G (7) developed suitable crystals from methanol, the absolute configuration of C-10 was designated as S by X-ray diffraction analysis (Figure 4). Peniisocoumarin H (8) was obtained as pale yellow solid with a molecular formula of C13H14O6 based on HRESIMS. Analysis of the NMR data led to the identification of 8 as diaportinol, a previously reported and structurally characterized compound with a 10S-configuration isolated from Penicillium nalgiovense.24 However, comparisons of their optical rotations and CD spectra showed the opposite results and indicated that there is a pair of enantiomers. The absolute configuration of C10 in peniisocoumarin H (8) was further confirmed as R by comparing the experimental and theoretical electronic circular dichroism (ECD) spectra (Figure 6). The NMR data of peniisocoumarins I (9) and J (11) were found to have a similar structure relationship to 8 (Tables 2 and 3). The structure of 9 was determined to be a 5-hydroxylated analogue of 8, based on the close similarity of their NMR data with the exception of a hydroxy group (δH 8.84, 5-OH) in 9 replacing the aromatic proton (H-5) of 8. In addition, compound 11 was assigned a molecular formula of C13H12Cl2O6 from the HRESIMS. Further comparison of the 1 H and 13C NMR data of 11 with those of 9 demonstrated the presence of a terminal methine group (1H, δH 6.21, H-11) for

Figure 6. Experimental ECD spectra of 8, 9, and 11 in MeOH and the calculated ECD spectra of 8 at the B3LYP/6-311+G(d,p) level.

11, instead of a methylene group (2H, δH 3.40 and 3.29, H-11) in 9. The COSY and HMBC data were identical to this deduction and established the positions of two additional chlorine atoms at C-11 (Figure 3). Accordingly, the experimental ECD spectra of compounds 9 and 11 were consistent with the experimental and calculated ECD spectra of 8 in MeOH (Figure 6). Therefore, their absolute configurations of the C-10 chiral center were also determined as R. The isolated compounds 1−13 were tested for their inhibitory activities against α-glucosidase in vitro, and acarbose was selected as a positive control (IC50 = 478.4 μM). Compounds showing inhibitory effects against α-glucosidase with values greater than 45% at 200 μM were further evaluated against calculated IC50 values. Compounds 3, 7, 9, and 11 were more potent than acarbose, with IC50 values ranging from 38.1 to 78.1 μM, while compounds 5, 6, and 10 exhibited moderate activity, with IC50 values between 102.4 and 158.4 μM (Table 4). These results suggested that the epoxy group attached at C10/C-11 was crucial for the α-glucosidase inhibitory effects of peniisocoumarins, as exemplified by compound 3 showing 95% inhibition of α-glucosidase at 200 μM, whereas compound 12 exerted 38% inhibition at the same dose. In addition, compounds 6, 7, 9, and 11 (with a hydroxy substitution at Table 4. Inhibitory Effects of Compounds 1−13 against αGlucosidase

a

1380

compd

% inhibition (200 μM)

1 2 3 4 5 6 7 8 9 10 11 12 13 acarboseb

18 23 95 41 58 66 91 43 83 69 91 38 33 19

IC50 (μM)a

38.1 ± 1.0 158.4 ± 1.1 110.3 ± 1.0 40.5 ± 1.0 78.1 ± 1.0 102.4 ± 1.2 45.1 ± 1.0

478.4 ± 1.0

IC50: 50% inhibitory concentration. bPositive control. DOI: 10.1021/acs.jnatprod.7b01018 J. Nat. Prod. 2018, 81, 1376−1383


Article

eluting with CH2Cl2−CH3OH (1:1, v/v) and purified by a semipreparative reversed-phase (RP) HPLC column (80% CH3OH−H2O) to afford compounds 1 (3.9 mg, tR 27.5 min) and 2 (3.4 mg, tR 25.6 min). Fraction F3 was separated into two subfractions (F3a and F3b) by CC on silica gel eluting with a step gradient of PE− EtOAc (80:20 to 70:30, v/v). F3a was subjected to an RP-HPLC column (70% CH3OH−H2O) to obtain 3 (5.1 mg, tR 23.8 min) and 5 (6.8 mg, tR 27.6 min). F3b was submitted to silica gel CC (CH2Cl2− CH3OH, 100:2, v/v) to yield 10 (10.5 mg) and 13 (4.8 mg). Fraction F4 was subjected to CC on silica gel to obtain two subfractions (F4a and F4b) eluting with gradient CH2Cl2 and CH3OH (100:2 to 100:3, v/v). F4b was purified by RP-HPLC column (70% CH3OH−H2O) to afford 4 (5.6 mg, tR 26.8 min), 6 (6.7 mg, tR 21.2 min), and 12 (5.8 mg, tR 24.5 min). Compounds 8 (3.8 mg) and 11 (4.1 mg) were obtained from F4b using silica gel CC eluting with CH2Cl2 and CH3OH (100:3, v/v) and further purified by Sephadex LH-20 eluting with CH2Cl2−CH3OH (1:1, v/v). Fraction F6 was chromatographed on an RP-HPLC column (65% CH3OH−H2O) to obtain 7 (6.9 mg, tR 20.3 min) and 9 (3.5 mg, tR 24.1 min). Peniisocoumarin A (1): colorless crystals; mp 142−143 °C; [α]25 D 0 (c 0.15 MeOH); UV (MeOH) λmax (log ε) 240 (4.82), 327 (3.72) nm; IR (KBr) νmax 3376, 2985, 2748, 1639, 1621, 1497, 1347, 1167, 1037, 790 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 551.1195 [M − H]− (calcd for C28H23O12: 551.1195). Peniisocoumarin B (2): colorless crystals; mp 148−149 °C; [α]25 D 0 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 240 (4.73), 327 (3.65) nm; IR (KBr) νmax 3373, 2987, 2746, 1640, 1621, 1495, 1345, 1169, 1031, 798 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 553.1335 [M + H]+ (calcd for C28H25O12: 553.1341). Peniisocoumarin C (3): white needles; mp 128−129 °C; [α]25 D +26.9 (c 0.15 MeOH); UV (MeOH) λmax (log ε) 240 (4.82), 327 (3.71) nm; ECD (MeOH) λmax (Δε) 240 (+6.65), 280 (−2.26) nm; IR (KBr) νmax 3299, 2948, 2854, 1690, 1633, 1463, 1439, 1171, 1077, 861 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 289.0716 [M − H]− (calcd for C15H13O6: 289.0718). Peniisocoumarin D (4): light brown oil; [α]25 D +46.8 (c 0.15 MeOH); UV (MeOH) λmax (log ε) 240 (4.83), 327 (3.74) nm; ECD (MeOH) λmax (Δε) 2.07 (+1.49), 221 (−0.88), 240 (+16.2), 283 (+1.84), 322 (+0.79) nm; IR (KBr) νmax 3477, 2904, 2853, 1684, 1638, 1505, 1384, 1202, 1165, 689 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 285.0521 [M + H]+ (100%) and m/z 287.0493 [M + H]+ (33%) (calcd for C13H14ClO5: 285.0524). Peniisocoumarin E (5): light brown crystals; mp 107−108 °C; [α]25 D +69.2 (c 0.15 MeOH); UV (MeOH) λmax (log ε) 240 (4.84), 327 (3.72) nm; ECD (MeOH) λmax (Δε) 225 (−2.35), 240 (+12.8), 285 (+4.06), 327 (+2.06) nm; IR (KBr) νmax 3433, 2919, 2850, 1691, 1641, 1505, 1382, 1105, 1068, 739 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 285.0524 [M + H]+ (100%) and m/z 287.0497 [M + H]+ (33%) (calcd for C13H14ClO5: 285.0524). Peniisocoumarin F (6): light brown crystals; mp 154−155 °C; [α]25 D +44.3 (c 0.15 MeOH); UV (MeOH) λmax (log ε) 240 (4.38), 349 (3.63) nm; ECD (MeOH) λmax (Δε) 214 (−1.43), 241 (+3.51), 272 (+1.72), 330 (−0.46) nm; IR (KBr) νmax 3299, 2948, 2854, 1680, 1633, 1500, 1439, 1238, 1171, 861 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 299.0327 [M − H]− (100%) and m/z 301.0297 [M − H]− (33%) (calcd for C13H12ClO6: 299.0328). Peniisocoumarin G (7): light brown crystals; mp 206−207 °C; [α]25 D +26.3 (c 0.15 MeOH); UV (MeOH) λmax (log ε) 240 (4.45), 349 (3.69) nm; ECD (MeOH) λmax (Δε) 240 (+1.61), 284 (+0.74) nm; IR (KBr) νmax 3429, 2988, 2248, 1693, 1621, 1497, 1380, 1167, 1037, 759 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 251.0563 [M − H]− (calcd for C12H11O6: 251.0556). Peniisocoumarin H (8): pale yellow solid; [α]25 D −18.6 (c 0.15 MeOH); UV (MeOH) λmax (log ε) 240 (4.75), 327 (3.67) nm; ECD (MeOH) λmax (Δε) 240 (+9.07), 285 (−1.30), 332 (−1.09) nm; IR (KBr) νmax 3288, 2933, 2853, 1684, 1625, 1570, 1407, 1200, 1070, 848 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 265.0715 [M − H]− (calcd for C13H13O6: 265.0718). Peniisocoumarin I (9): pale yellow solid; [α]25 D −30.2 (c 0.15 MeOH); UV (MeOH) λmax (log ε) 240 (4.78), 327 (3.67) nm; ECD

C-5) displayed a higher inhibitory effect than that of compounds 5, 8, 10, and 13, respectively. Compounds 1−13 were also tested for their inhibitory activity of MptpB and cytotoxicity using A549 (lung cancer), HepG2 (liver cancer), HeLa (cervical cancer), MCF-7 (breast cancer), and HEK293T (embryonic kidney) human cell lines. Compound 7 showed inhibitory activity of MptpB with an IC50 value of 20.7 μM compared to the positive control (oleanolic acid, IC50 = 22.1 μM), while the other compounds revealed weak or no inhibitory effects at a concentration of 50 μM. In the cytotoxicity assay, none of the compounds displayed cytotoxicities against five tested human cell lines at 100 μM. In conclusion, 10 new isocoumarin derivatives, peniisocoumarins A−J, with structural diversity were isolated from the mangrove endophytic fungus P. commune QQF-3. The absolute configurations of all new compounds were unambiguously established by a combination of X-ray diffraction analysis, modified Mosher’s method, and TDDFT-ECD calculations. Structurally, compounds 1 and 2 are a pair of unusual dimeric isocoumarin-type diastereoisomers containing a four-membered core at C-9/9′ and C-10/10′. Compounds 5 and 6 possess a chlorine atom group at C-10 in the side chain, which are described for the first time from isocoumarin derivatives. This work provides additional evidence to support mangrove endophytic fungi as a sustainable source of chemical diversity.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Fisher-Johns hot-stage apparatus and were uncorrected. Optical rotations were determined using an Anton Paar (MCP 300) polarimeter at 25 °C, and ECD spectra were recorded using a Chirascan CD spectrometer. UV data were recorded on a PERSEE TU-1900 spectrophotometer in CH3OH solution. IR spectra were recorded on a Nicolet Nexus 670 spectrophotometer in KBr discs. All NMR experiments were measured with Bruker Avance 500 spectrometers (500 and 125 MHz), and the residual solvent peaks of acetone-d6 (δC 29.8 and 206.3/δH 2.05), CDCl3 (δC 77.1/δH 7.26), or DMSO-d6 (δC 39.52/δH 2.50) were used as references. ESIMS and HRESIMS data were measured on a Thermo LCQ-DECA mass spectrometer and a Thermo Fisher LTQ Orbitrap Elite high-resolution mass spectrometer, respectively. Column chromatography (CC) was carried out on silica gel (200−300 mesh, Qingdao Marine Chemical Factory) and Sephadex LH-20 (Amersham Pharmacia). Semipreparative HPLC was performed on a Hitachi (Primaide) HPLC system using an Ultimate XB-C18 column (10 × 250 mm, 10 μm). X-ray crystallographic analysis was performed on an Agilent Gemini Ultra diffractometer (Cu Kα radiation). Fungal Strain and Identification. The fungus Penicillium commune QQF-3 was isolated from a fresh fruit of the mangrove plant Kandelia candel, which were collected in August 2015 from Zhuhai Mangrove Nature Reserve in Guangdong Province, China. The fungal strain was identified according to sequencing of the internal transcribed spacer (ITS), and a BLAST search result showed it was most similar (99%) to the sequence of Penicillium commune (compared to KJ194124.1). The sequence data have been deposited at GenBank with accession no. MG193677. The fungus specimen was kept in our laboratory at −20 °C, and its working stocks were prepared on potato dextrose agar slants stored at 4 °C. Fermentation, Extraction, and Isolation. The fungus was cultured on autoclaved rice medium in 30 Erlenmeyer flasks (1 L), each containing 50 g of rice and 50 mL of 0.3% saline water. After incubation for 28 days at 25 °C, the mycelia and solid rice medium were dried and soaked in EtOAc three times to afford an EtOAc extract (18 g). The EtOAc extract was subjected to silica gel (200−300 mesh) column chromatography using a petroleum ether (PE, 60−90 °C) and EtOAc gradient system (from 1:0 to 0:1) to give seven fractions (F1 to F7). Fraction F2 was applied to Sephadex LH-20 1381



Article

(MeOH) λmax (Δε) 2.08 (−1.64), 240 (+11.2), 285 (−1.97), 332 (−1.51) nm; IR (KBr) νmax 3299, 2977, 2854, 1680, 1633, 1439, 1362, 1238, 1032, 741 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 281.0663 [M − H]− (calcd for C13H13O7: 281.0661). Peniisocoumarin J (11): pale yellow solid; [α]25 D −17.1 (c 0.15 MeOH); UV (MeOH) λmax (log ε) 240 (4.67), 345 (3.68) nm; ECD (MeOH) λmax (Δε) 217 (−5.18), 240 (+8.95), 281 (−3.80) nm; IR (KBr) νmax 3085, 2919, 2848, 1691, 1625, 1566, 1382, 1197, 1150, 691 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 332.9938 [M − H]− (calcd for C13H11Cl2O6: 333.9938). X-ray Crystal Structure Analysis of Compounds 1−3 and 5− 7. Crystals of 1−3 and 5−7 were obtained from MeOH−H2O (10:1, v/v) and MeOH, respectively. Crystal X-ray diffraction data were collected on an Agilent Gemini Ultra diffractometer with Cu Kα radiation (λ = 1.541 78 Å). The structure was solved by direct methods (SHELXS-97) and refined using full-matrix least-squares difference Fourier techniques. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were placed in the ideal geometrical positions and refined isotropically with a riding model. Crystallographic data of 1−3 and 5−7 have been deposited with the Cambridge Crystallographic Data Centre. Crystal data of 1: C28H24O12, Mr = 552.13, monoclinic, a = 27.9933(4) Å, b = 9.57268(8) Å, c = 22.8556(4) Å, α = γ = 90°, β = 118.274°, V = 5393.89(16) Å3, space group I2/a, Z = 150, Dc = 1.340 g/cm3, μ = 1.097 mm−1, and F(000) = 2250.0. Crystal dimensions: 0.35 × 0.21 × 0.15 mm3. Independent reflections: 5408 (Rint = 0.0203). The final R1 values were 0.0384, wR2 = 0.1061 (I > 2σ(I)). The goodness of fit on F2 was 1.067. CCDC number: 1580261. Crystal data of 2: C28H24O12, Mr = 552.13, triclinic, a = 8.189 40(10) Å, b = 10.3069(2) Å, c = 15.1113(2) Å, α = 82.6270(10)°, β = 87.4920(10)°, γ = 76.4570(10)°, V = 1229.66(3) Å3, space group P1̅, Z = 2, Dc = 1.492 g/cm3, μ = 1.005 mm−1, and F(000) = 576.0. Crystal dimensions: 0.21 × 0.19 × 0.18 mm3. Independent reflections: 4881 (Rint = 0.0252). The final R1 values were 0.0359, wR2 = 0.1008 (I > 2σ(I)). The goodness of fit on F2 was 1.030. CCDC number: 1580268. Crystal data of 3: C15H14O6, Mr = 290.08, orthorhombic, a = 4.55420(10) Å, b = 11.4674(3) Å, c = 25.3578(7) Å, α = β = γ = 90°, V = 1324.31(6) Å3, space group P212121, Z = 4, Dc = 1.456 g/cm3, μ = 0.961 mm−1, and F(000) = 608.0. Crystal dimensions: 0.60 × 0.10 × 0.05 mm3. Independent reflections: 2603 (Rint = 0.0252). The final R1 values were 0.0319, wR2 = 0.0818 (I > 2σ(I)). The goodness of fit on F2 was 1.032. Flack parameter value was 0.01(13). CCDC number: 1580269. Crystal data of 5: C13H13ClO5, Mr = 284.05, monoclinic, a = 22.0660(6) Å, b = 9.1694(3) Å, c = 6.5996(2) Å, α = γ = 90°, β = 107.302(2)°, V = 1274.89(7) Å3, space group C2, Z = 4, Dc = 1.483 g/ cm3, μ = 2.804 mm−1, and F(000) = 592.0. Crystal dimensions: 0.021 × 0.019 × 0.018 mm3. Independent reflections: 2499 (Rint = 0.0229). The final R1 values were 0.0336, wR2 = 0.0926 (I > 2σ(I)). The goodness of fit on F2 was 1.044. Flack parameter value was −0.02(2). CCDC number: 1580270. Crystal data of 6: C13H13ClO6, Mr = 300.04, triclinic, a = 5.0238(4) Å, b = 5.5679(4) Å, c = 12.8812(14) Å, α = 99.398(7)°, β = 94.679(8)°, γ = 105.645(7)°, V = 339.31(5) Å3, space group P1, Z = 1, Dc = 1.560 g/cm3, μ = 2.814 mm−1, and F(000) = 166.0. Crystal dimensions: 0.30 × 0.15 × 0.10 mm3. Independent reflections: 2489 (Rint = 0.0895). The final R1 values were 0.0779, wR2 = 0.2170 (I > 2σ(I)). The goodness of fit on F2 was 1.108. Flack parameter value was 0.06(2). CCDC number: 1580271. Crystal data of 7: C12H12O6, Mr = 252.06, orthorhombic, a = 14.7901(4) Å, b = 19.0871(5) Å, c = 8.0499(2) Å, α = β = γ = 90°, V = 2272.49(10) Å3, space group P212121, Z = 67, Dc = 1.474 g/cm3, μ = 1.025 mm−1, and F(000) = 1056.0. Crystal dimensions: 0.021 × 0.019 × 0.018 mm3. Independent reflections: 4468 (Rint = 0.0207). The final R1 values were 0.0348, wR2 = 0.0982 (I > 2σ(I)). The goodness of fit on F2 was 1.080. Flack parameter value was 0.13(12). CCDC number: 1580272. Preparation of MTPA Esters of 4 by the Modified Mosher’s Method. Compound 4 (2.1 mg) was reacted with (S)-α-methoxy-α-

(trifluoromethyl)phenylacetyl chloride ((S)-MTPA-Cl, 20 μL) in anhydrous pyridine (400 μL) for 8 h at room temperature. Then the solvent from the reaction mixture was removed under reduced pressure to furnish a residue, which was purified by preparative TLC (with CH2Cl2 eluant) to give (R)-MTPA ester (4a, 1.8 mg). Through the same procedure, (S)-MTPA ester (4b, 1.7 mg) was obtained from 4 (2.2 mg) using (R)-MTPA-Cl. (R)-MTPA ester for 4a: 1H NMR (CDCl3, 500 MHz) δH 10.96 (1H, s, 8-OH), 6.50 (1H, d, J = 2.2 Hz, H-7), 6.17 (1H, d, J = 2.2 Hz, H-5), 6.01 (1H, s, H-4), 5.62 (1H, m, H-10), 3.95 (1H, dd, J = 12.3, 3.9 Hz, H-11a), 3.74 (1H, dd, J = 12.3, 4.7 Hz, H-11b), 2.88 (2H, dd, J = 6.6, 8.8 Hz, H-9); ESIMS m/z 499.0 [M − H]−. (S)-MTPA ester for 4b: 1H NMR (CDCl3, 500 MHz) δH 10.98 (1H, s, 8-OH), 6.51 (1H, d, J = 2.2 Hz, H-7), 6.28 (1H, d, J = 2.2 Hz, H-5), 6.23 (1H, s, H-4), 5.60 (1H, m, H-10), 3.84 (1H, dd, J = 10.1, 4.5 Hz, H-11a), 3.72 (1H, dd, J = 10.1, 4.3 Hz, H-11b), 2.98 (2H, dd, J = 3.4, 6.5 Hz, H-9); ESIMS m/z 499.1 [M − H]−. Biological Assays. α-Glucosidase inhibitory activity was assayed in the 96-well plates using 0.01 M KH2PO4−K2HPO4 (pH 7.0) buffer solution. Enzyme solutions were prepared to give 2.0 units/mL in buffers. The assay was conducted in a 200 μL reaction system containing 168 μL of buffers, 10 μL of diluted enzyme solution, and 2 μL of DMSO or sample (dissolved in DMSO). After 20 min of incubation at 37 °C, 20 μL of substrate (p-nitrophenyl glycoside, 3 mg/mL) was added to start the enzymatic reaction, which was measured by a BIO-RAD (iMark) microplate reader at 410 nm in 37 °C immediately. Inhibitory activity was calculated according to the equation η (%) = [(B − S)/B] × 100% (B stands for the assay medium with DMSO; S stands for the assay medium with inhibitor). αGlucosidase from Saccharomyces cerevisiae was purchased from SigmaAldrich Co. (CAS number: 9001-42-7, E.C 3.2.1.20). All assays were performed in three replicates, and acarbose was used as the positive control. The phosphatase activity test of MptpB was performed in triplicates in 96-well microplates in reaction buffer (50 mM Tris/100 mM NaCl, pH 7.0) using p-nitrophenyl phosphate as a substrate, and oleanolic acid was used as the positive control. The detailed methodology for overexpression, purification, and inhibition assays of MptpB has already been described in a previous report.19 Cytotoxicity assays were evaluated by an MTT method using A549, HepG2, HeLa, MCF-7, and HEK293T human cell lines. The detailed methodology for biological testing has already been described in a previous report.30

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b01018. 1D and 2D NMR spectra, HRESIMS spectra for new compounds, experimental ECD spectra for compounds 3−9 and 11, ECD calculation details (Methods and Results) for compound 8 (PDF) Crystallographic data for compound 1 (CIF) Crystallographic data for compound 2 (CIF) Crystallographic data for compound 3 (CIF) Crystallographic data for compound 5 (CIF) Crystallographic data for compound 6 (CIF) Crystallographic data for compound 7 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86 020 84113356. E-mail: [email protected]. cn (Z. She). ORCID

Runlin Cai: 0000-0001-7388-8096 1382



Article

(26) Ichihara, A.; Hashimoto, M.; Hirai, T.; Takeda, I.; Sasamura, Y.; Sakamura, S.; Sato, R.; Tajimi, A. Chem. Lett. 1989, 18, 1495−1498. (27) Harris, J. P.; Mantle, P. G. Phytochemistry 2001, 57, 165−169. (28) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092−4096. (29) Hallock, Y. F.; Clardy, J.; Kenfield, D. S.; Strobel, G. Phytochemistry 1988, 27, 3123−3125. (30) Chen, S.; Chen, D.; Cai, R.; Cui, H.; Long, Y.; Lu, Y.; Li, C.; She, Z. J. Nat. Prod. 2016, 79, 2397−2402.

Senhua Chen: 0000-0002-5498-0206 Hui Cui: 0000-0003-4281-016X Chunyuan Li: 0000-0001-8817-8994 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (21472251, 41276146, and 41404134), the Key Project of Natural Science Foundation of Guangdong Province (2016A040403091), the Fundamental Research Funds for the Central Universities (141gjc16), the Special Promotion Program for Guangdong Provincial Ocean and Fishery Technology (2017), and System Resource of Tianhe-2 of National Supercomputer Center in Guangzhou for generous support.

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REFERENCES

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Peniisocoumarins AâJ: Isocoumarins from Penicillium commune QQF

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