Antiviral Alkaloids Produced by the Mangrove ... - ACS Publications

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Antiviral Alkaloids Produced by the Mangrove-Derived Fungus Cladosporium sp. PJX-41 Jixing Peng,† Tao Lin,‡ Wei Wang,† Zhihong Xin,*,‡ Tianjiao Zhu,† Qianqun Gu,† and Dehai Li*,† †

Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ Key Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China S Supporting Information *

ABSTRACT: Six new indole alkaloids including five new glyantrypine derivatives (1, 2a, 2b, 3, 4) and a new pyrazinoquinazoline derivative (5), together with eight known alkaloids (6−13), were isolated from the culture of the mangrove-derived fungus Cladosporium sp. PJX-41. Their structures were elucidated primarily by spectroscopic and physical data. The absolute configurations of compounds 1−9 were established on the basis of CD, NOESY data, and singlecrystal X-ray diffraction analysis. Compounds 2b, 5, 7−9, and 11 exhibited significant activities against influenza virus A (H1N1), with IC50 values of 82−89 μM.

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he investigation of structurally novel and biologically active compounds from marine-derived microorganisms has become an important research area in drug discovery.1 Our group has recently examined marine-derived fungi for the presence of novel bioactive natural products.2 In particular, with the pandemic of influenza, we have initiated a program of screening for inhibitors against influenza A virus (H1N1), and consequently we have discovered a series of new antiviral natural products including six isoindolone derivatives,3 one pyronepolyene C-glucoside (iso-D8646-2-6),4 and one butenolide, isoaspulvinone E.5 In the context of our ongoing search for anti-influenza compounds, an active mangrove-derived fungal strain, identified as a Cladosporium sp., was selected for further research. A chemical investigation of the EtOAc extracts of both the fermentation broth and mycelia of the fungus, which resulted in the isolation of six new indole alkaloids including 3hydroxyglyantrypine (1), oxoglyantrypine (2a, 2b), cladoquinazoline (3), epi-cladoquinazoline (4), and norquinadoline A (5), together with eight known quinazoline-containing indole alkaloids (6−13), is described here. The structures and absolute configurations of all new compounds were determined. Furthermore, we also report the absolute configurations of the known quinadoline A (6), deoxynortryptoquivaline (7), and deoxytryptoquivaline (8), as well as provide the first X-ray diffraction analysis of tryptoquivaline (9). All compounds were evaluated for their antiviral activities against influenza virus A (H1N1), and compounds 2b, 5, 7−9, and 11 exhibited significant activities with IC50 values of 85, 82, 87, 85, 89, and 82 μM, respectively. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 12, 2013

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dx.doi.org/10.1021/np400200k | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H and 13C NMR Data for Compounds 1 and 2 1a position

δC

δH (J in Hz)

1 2-NH 3 3-OH 4 6 7 8 9 10 11 12 14 15

168.9, C

16 17 18-NH 19 20 21 22 23 24

109.8, C 124.5, CH

76.0, CH 150.9, C 147.5, C 127.7, CH 135.3, CH 127.9, CH 126.9, CH 120.8, C 160.3, C 56.8, CH 30.6, CH2

136.7, 111.9, 121.4, 118.8, 118.9, 128.0,

2b

C CH CH CH CH C

δC

δH (J in Hz)

168.8, C 7.62, d (4.4) 5.58, t (4.9) 9.26, d (4.9)

7.72, 7.87, 7.56, 8.13,

156.6, C

brd (8.3) td (8.3, 1.1) td (8.3, 1.1) dd (8.3, 1.1)

5.30, m 3.65, dd (14.3, 9.9) 3.41, dd (14.3, 5.5)

105.9, C 124.9, CH

7.12, d (2.2) 10.82, brs 7.33, 7.05, 6.95, 7.79,

139.9, C 146.0, C 128.6, CH 135.2, CH 129.1, CH 126.4, CH 121.2, C 159.4, C 57.2, CH 27.9, CH2

d (8.2) td (8.2, 1.1) td (8.2, 1.1) brd (8.2)

135.9, 111.5, 121.2, 118.7, 117.4, 127.1,

C CH CH CH CH C

7.78, 7.95, 7.74, 8.33,

brd (8.3) td (8.3, 1.1) td (8.3, 1.1) dd (8.3,1.1)

5.53, m 3.55, dd (14.8, 5.5) 3.48, dd (14.9, 2.8) 6.68, d (2.8) 10.96, brs 7.26, 6.97, 6.71, 7.05,

brd (8.2) td (8.2, 1.1) td (8.2, 1.1) brd (8.2)

a

1

Spectra were recorded at 600 MHz for 1H NMR and at 150 MHz for 13C NMR using DMSO-d6 as solvent. bSpectra were recorded at 600 MHz for H NMR and at 100 MHz for 13C NMR using DMSO-d6 as solvent.

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RESULTS AND DISCUSSION Compound 1 was obtained as a yellow powder. Its molecular formula of C20H16N4O3 was established on the basis of the HRESIMS ion at m/z 361.1300 [M + H]+, with 16 amu more than that of glyantrypine (13), previously isolated from Aspergillus clavatus.6 Comparison of the 1H and 13C NMR data (Table 1) with those of 13 showed the presence of the same pyrazinoquinazoline-containing indole skeleton, except for one of the C-3 methylene hydrogens in 13 being replaced by a hydroxy group in 1. The alcohol functionality at C-3 was confirmed by COSY correlations between H-3 and NH-2, as well as the key HMBC correlations from H-3 (δH 5.58) to C-1 (δC 168.9) and C-4 (δC 150.9) and from OH-3 (δH 9.26) to C3 (δC 76.0) and C-4 (δC 150.9) (Figure 1). The relative spatial relationship between C-15 and OH-3 of 1 was deduced as (3S*, 14R*) from the NOESY correlation between H-3 and H-15 (δH 3.65) (Figure 1). Compound 1 exhibited quite similar CD Cotton effects (Figure 2) to glyantrypine (13) at 234.9 nm (Δε +7.43) and 211 nm (Δε

Figure 2. Experimental ECD spectra of compounds 1, 2a, 2b, and 13.

−12.48), implying an identical R-configuration at C-14. The absolute configuration of compound 1, named 3-hydroxyglyantrypine, was thus determined as 3S, 14R. Oxoglyantrypine (2), isolated as a yellow powder, was assigned the molecular formula C20H14N4O3 by the HRESIMS ion at m/z 357.0996 [M − H]−. The IR spectrum showed absorption bands for amine (3403 cm−1) and amide (1716 cm−1) groups. A direct comparison of the 1H and 13C NMR data (Table 1) with those of 1 revealed that they share the same skeleton with the sole difference being the presence of a carbonyl carbon (C-3, δC 156.6) in 2 instead of the oxymethine carbon (C-3, δC 76.0) in 1. The structure of 2 was further confirmed by the COSY and HMBC correlations (Figure 1) and by comparing the 1D NMR data with those of brevianamide N7, containing the same pyrazinoquinazoline moiety (C-1 to C-14). This oxidized derivative of glyantrypine was named oxoglyantrypine.

Figure 1. Key COSY, HMBC, and NOESY correlations of 1 and 2. B



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Table 2. 1H (400 MHz) and 13C (100 MHz) NMR Data for Compounds 3 and 4 in DMSO-d6 3 position 1 2-NH 3 4 6 7 8 9 10 11 12 14 15 16 17 18-NH 19 20 21 22 23 24 25 26 27

δC

4 δH (J in Hz)

166.6, C 61.2, 150.7, 146.6, 126.8, 134.6, 126.7, 126.2, 119.7, 160.0, 51.8, 38.3, 73.5, 177.6,

CH C C CH CH CH CH C C CH CH2 C C

143.1, 109.6, 129.2, 120.9, 124.2, 130.4, 35.2, 19.1, 19.9,

C CH CH CH CH C CH CH3 CH3

δC

δH (J in Hz)

168.4, C 8.88, s 3.99, d (8.3)

7.63, 7.81, 7.54, 8.12,

d (8.1) td (8.1, 1.2) t (8.1) d (8.1)

4.97, dd (8.7, 5.9) 2.47, m

57.3, 150.7, 146.4, 126.8, 134.4, 126.4, 126.3, 119.3, 159.6, 51.6, 35.6, 73.5, 177.5,

CH C C CH CH CH CH C C CH CH2 C C

142.6, 109.5, 129.0, 120.8, 123.8, 129.7, 27.9, 15.2, 18.5,

C CH CH CH CH C CH CH3 CH3

10.16, brs 6.81, 7.17, 6.93, 7.49,

8.39, s 4.77, d (1.6)

7.56, 7.76, 7.43, 7.78,

d (8.1) td (8.1, 1.2) t (8.1) d (8.1)

5.17, dd (8.1, 5.9) 2.53, m

10.20, brs

d (7.4) t (7.4) t (7.4) d (7.4)

2.28, m 1.07, d (6.5) 1.09, d (5.0)

6.63, 6.79, 6.51, 7.22,

d (7.4) t (7.4) t (7.4) d (7.4)

2.96, m 0.86, d (6.8) 1.18, d (7.3)

indicates that compounds 3 and 4 have the same planar structure.

Because 2 displayed a baseline ECD curve, it was assumed to be a racemate, which was confirmed by the baseline separation of the enantiomers on HPLC using a chiral-phase column (Figure S58, Supporting Information). The pair of enantiomers was isolated, and their absolute configurations were assigned on the basis of their ECD spectra (Figure 2). The ECD spectrum of the faster eluting peak (2a) was similar to that of glyantrypine (13), which allowed the determination of the absolute configuration of 2a as 14R. Literature precedent exists for the base-induced epimerization of similar compounds at C14;8 therefore compounds 2a and 2b were each treated with 0.4% KOH in MeOH at 28 °C for 24 h, with both affording a mixture of 2a and 2b.8 Additionally, both 2a and 2b were dissolved individually in DMSO at 4 °C for three months, with both affording a mixture of 2a and 2b (Figure S59, Supporting Information). Thus the enantiomer (2b) of 2a, which contains 6 L-tryptophan, was proposed as an unnatural substance. Cladoquinazoline (3) and epi-cladoquinazoline (4) have the same molecular formula, C23H22N4O4, established on the basis of the HRESIMS ions detected at m/z 419.1704 [M + H]+ and 419.1707 [M + H]+, respectively. Analysis of their 1D NMR data (Table 2) suggested the presence of 22 proton and 23 carbon resonances including three amide carbonyls, eight aromatic sp2 methines, three sp3 methines, one sp3 methylene, five sp2 quaternary carbons, one sp3 oxygenated quaternary carbon, and two methyls. These data revealed that the structures of 3 and 4 closely resembled that of previously reported fiscalin B.9 The main difference was in the indole moiety and was determined by the HMBC correlations from H15 to C-16, C-17, and C-24 and from NH-18 to C-16, C-19, and C-24 (for compound 4, the NH-18 showed HMBC correlations only to C-16) (Figure 3). This information coupled with the requirements of the molecular formula

Figure 3. Key COSY, HMBC, and NOESY correlations of 3 and 4.

The relative configuration of 3 was determined as 3R*, 14R*, 16R* from the X-ray diffraction analysis (Figure 4), and the absolute configuration was further established to be 3R, 14R, 16R based on the Cotton effects at 260.9 nm (Δε +2.57), 237.4 nm (Δε −9.97), and 211.8 nm (Δε +25.30) (Figure 5), which were similar to those of fumiquinazoline L.10 The 1H and 13C NMR spectra of 3 and 4 were very similar but not identical. The 1H NMR spectrum of 3 (Table 2) has a resonance H-3 at δH 3.99 appearing as a doublet (J = 8.3), while the signal of H-3 in 4 appeared as a doublet at a higher frequency with a lower coupling constant (δH 4.77, J = 1.6). Conversely, the resonances of C-3, C-15, and C-25 in 4 appeared at lower frequencies (δC 57.3, 35.6, and 27.9, respectively) than those in 3 (δC 61.2, 38.3, and 35.2, respectively) (Table 2). These differences are very similar to those observed in fiscalin C and epi-fiscalin C,11 implying that compounds 3 and 4 are diastereomers at C-3. The relative configurations at C-3 and C-14 of 4 was deduced as (3S*, C



Article

Figure 4. X-ray ORTEP diagram of compound 3. Figure 6. X-ray ORTEP diagram of compound 6.

Compounds 7, 8, and 9 were assigned as deoxynortryptoquivaline (7),13 deoxytryptoquivaline (8),13 and tryptoquivaline (9)14 based on comparison of spectroscopic data with those reported in the literature. These three alkaloids were isolated as tremorgenic mycotoxins from A. clavatus as early as 1975, although their absolute configurations remained doubtful until 1979, when Springer et al. established the correct absolute configuration of tryptoquivaline (9) by a crystal X-ray diffraction experiment performed on its derivative nortryptoquivaline.15 Later, Nakagawa et al. reported the first total synthesis of tryptoquivaline in 1984.16 We have been able to isolate crystallographic quality crystals of 9, and therefore we carried out an X-ray diffraction study of tryptoquivaline (Figure S61, Supporting Information), allowing unambiguous confirmation of its absolute configuration directly. Because the CD spectra of quinazoline-containing indole alkaloids are frequently correlated with their absolute configurations,17,18 CD experiments were performed on compounds 7, 8, and 9. The results (Figure S57, Supporting Information) showed the ECD curves of 7 and 8 closely resembled that of 9, which assigned the absolute configurations of 7 and 8 as 2R, 3S, 12R and 27S, respectively. Furthermore, the absolute configuration of C-15 in 7 was determined as 15S by a NOESY correlation (Table S1, Supporting Information) between H-2 and H-34. Fungal quinazolinone metabolites such as glyantrypine, as well as the fiscalins, fumiquinazolines, and tryptoquialanines, have been reported to be derived from anthranilic acid, tryptophan, and an additional incorporated amino acid.12,19 We therefore propose that the glyantrypine derivatives (1 and 2) are biosynthesized from anthranilic acid, tryptophan, and glycine, while in the biosynthesis of the cladoquinazolines (3 and 4), valine is incorporated instead of glycine for generation of the pyrazinoquinazoline intermediate. In terms of the quinadoline A analogue (5), the S-configuration at C-22 may be consistent with L-alanine incorporation into the imidazoindolone moiety.20 Compounds 10−13 were identified as CS-C (10),18 quinadoline B (11),12 prelapatin B (12),21 and glyantrypine (13),6 respectively, by comparison of their spectroscopic and physical data (1H and 13C NMR, MS, and [α]D) with those reported in the literature. Among them, compound 12 has been

Figure 5. Experimental ECD spectra of compounds 3 and 4.

14R*) by the NOESY correlation between H-3 and H-15 (Figure 3). Compound 4 showed the same signs for the corresponding ECD transitions as those of 3, which established the absolute configuration of compound 4 as 3S, 14R, and 16R. It should be noted that when 3 and 4 were each treated with 0.4% KOH in MeOH, both underwent epimerization to afford a mixture of 3 and 4 (Figure S60, Supporting Information). Norquinadoline A (5) was obtained as a white, amorphous solid, and its molecular formula was determined to be C26H25N5O4 on the basis of HRESIMS, exhibiting 14 amu less than that of compound 6. The IR and UV spectra of 5 were very similar to those of 6. Quinadoline A (6) has been reported previously from the culture broth of Aspergillus sp. FKI-1746.12 However, the absolute configuration of quinadoline A was not established in that study. To determine the absolute configuration of 6, crystallographic quality crystals were successfully made and analyzed by X-ray diffraction with Cu Kα irradiation. The ORTEP diagram of compound 6 (Figure 6) confirmed the absolute configuration of C-14, C-19, and C20 as 14R, 19S, and 20R. Comparison of the 1D and 2D NMR data for 5 and 6 (Table 3 and Figure 7) revealed that one of the methyls (CH3-32) on the imidazolone ring of 6 was replaced by a hydrogen in 5. The relative configuration of (19S*, 20R*, 22S*) was deduced by the NOESY correlations between H-31 (δH 1.29) and H-20 (δH 5.31) and between H-20 (δH 5.31) and H-15 (δH 2.34) as well as between NH-21 (δH 3.36) and OH19 (δH 5.48) (Figure 7). The absolute configuration of 5 was further determined as (14R, 19S, 20R, 22S) by comparing the ECD spectrum of 5 with that of compound 6 (Figure 8). D



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Table 3. 1H and 13C NMR Data for Compounds 5 and 6 5a position

6b

δC

1 2-NH 3 4 6 7 8 9 10 11 12 14 15

166.9, C 121.4, 146.9, 146.9, 127.3, 134.7, 127.1, 126.4, 119.6, 159.8, 52.1, 37.8,

C C C CH CH CH CH C C CH CH2

16 17 18 19 19-OH 20 21-NH 22 23 25 26 27 28 29 30 31 32

131.4, 21.2, 21.5, 75.2,

C CH3 CH3 C

δH (J in Hz)

δC 166.9, C

10.15, s

81.4, CH 60.2, 174.8, 137.9, 115.0, 129.6, 124.7, 124.6, 137.9, 18.2,

CH C C CH CH CH CH C CH3

δH (J in Hz)

7.68, 7.85, 7.56, 8.17,

10.09, s

d (8.1) td (8.1, 1.1) t (8.1) dd (8.1, 1.1)

5.51, dd (8.4, 5.5) 2.34, dd (14.9, 8.2) 2.52, dd (14.9, 5.4) 1.98, s 2.32, s 5.48, 5.31, 3.36, 3.80,

s d (7.7) s m

7.35, 7.34, 7.11, 7.33,

d (7.1) t (7.1) td (7.1, 2.0) d (7.1)

121.5, 146.8, 146.9, 127.1, 134.6, 126.9, 126.2, 119.7, 159.9, 52.4, 38.0,

C C C CH CH CH CH C C CH CH2

130.8, 21.2, 21.6, 74.5,

C CH3 CH3 C

78.7, CH

1.29, d (7.1)

64.6, 175.3, 137.9, 114.5, 129.7, 124.7, 124.5, 137.5, 24.6, 24.1,

C C C CH CH CH CH C CH3 CH3

7.67, 7.85, 7.54, 8.17,

d (8.1) td (8.1, 1.6) t (8.1) dd (8.1, 1.6)

5.46, dd (8.3, 4.4) 2.44, dd (14.9, 8.4) 2.60, dd (14.6, 4.5) 1.95, s 2.31, s 5.59, s 5.09, d (8.0)

7.34, 7.35, 7.11, 7.39,

d (7.1) t (7.1) td (7.1, 2.3) d (7.1)

1.21, s 1.15, s

a

1

Spectra were recorded at 600 MHz for 1H NMR and at 100 MHz for 13C NMR using DMSO-d6 as solvent. bSpectra were recorded at 400 MHz for H NMR and at 100 MHz for 13C NMR using DMSO-d6 as solvent.

Figure 7. Key COSY, HMBC, and NOESY correlations of 5. Figure 8. Experimental ECD spectra of compounds 5 and 6.

reported as an intermediate in the chemical synthesis of (−)-lapatin B,21 with this study being the first report of its isolation from a natural source. The antiviral activities of compounds 1−13 against influenza A virus (H1N1) were evaluated by the CPE inhibition assay.4,22 Compounds 2b, 5, 7−9, and 11 exhibited notable anti-H1N1 activities (ribavirin as positive control, IC50 87 μM) with IC50 values of 85, 82, 87, 85, 89, and 82 μM, respectively, while the other compounds 1, 2a, 3, 4, 6, 10, and 13 were weakly active (IC50 values between 100 and 150 μM), while compound 12 showed no activity (IC50 > 200 μM) (Table S2, Supporting Information).

In summary, 14 indole alkaloids, including six new ones, were isolated from the mangrove-derived fungus Cladosporium sp. PJX-41. All of the chemical structures, including absolute configurations, were established. We also showed that these compounds had differing antiviral activities against influenza virus A (H1N1). This class of alkaloids, which has unique structural features including prenylated indole derivatives, quinazoline-containing indole derivatives, and pyrazinoquinazoline-containing indole derivatives, is frequently isolated from Aspergillus species. Conversely, few derivatives are known from Neosartorya or Penicillium species, let alone Cladosporium E



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species.11,23 This study has revealed new carbon skeleton leads for investigating antiviral mechanisms and for developing antiviral agents against influenza virus A (H1N1).

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respectively. Fraction 4.2.4 was purified by Sephadex LH-20, eluting with MeOH to provide two fractions, fractions 4.2.4.1 and 4.2.4.2. These two fractions were further purified by semipreparative HPLC (50:50 MeOH−H2O, 4 mL/min) to give compound 3 (4.5 mg, tR 11.5 min) and compound 4 (5.6 mg, tR 13.8 min). Fraction 3 was purified by Sephadex LH-20, eluting with MeOH, to provide two fractions, fractions 3.1 and 3.2. Fraction 3.1 was rechromatographed on a silica gel column, eluting with petroleum ether−acetone (2:1), and then on a semipreparative HPLC column (65:35 MeOH−H2O, 4 mL/min) to afford compound 8 (27.0 mg, tR 8.5 min) and compound 7 (8.7 mg, tR 13.7 min). Fraction 3.2 was chromatographed on a silica gel column using a step gradient elution of petroleum ether−acetone to provide two subfractions, fractions 3.2.1 and 3.2.2. Fraction 3.2.1 was purified by Sephadex LH-20, eluting with MeOH, and then by semipreparative HPLC (70:30 MeOH−H2O, 4 mL/min) to afford compound 10 (30.0 mg, tR 11.2 min) and compound 9 (13.3 mg, tR 13.7 min). Fraction 3.2.2 was purified by Sephadex LH-20, eluting with MeOH, and then by semipreparative HPLC (65:35 MeOH−H2O, 4 mL/min) to afford compound 12 (3 mg, tR 13.5 min). 3-Hydroxyglyantrypine (1): yellow powder; [α]25D +160 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 216 (4.61), 257 (4.26) nm; CD (1.54 × 10−3 M, CHCl3) λmax (Δε) 309.8 (+2.04), 255.8 sh (+0.57), 234.9 (+7.43), 211 (−12.48) nm; IR (KBr) νmax 3203, 2359, 1649, 1608, 1471 cm−1; 1H and 13C NMR see Table 1; HRESIMS m/z 361.1300 [M + H]+ (calcd for C20H17N4O3, 361.1301). Oxoglyantrypine (2): yellow powder; UV (MeOH) λmax (log ε) 215 (4.60), 265 (4.27), 291 (3.57) nm; IR (KBr) νmax 3403, 2359, 1716, 1468, 1325, 1166 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 357.0996 [M − H]− (calcd for C20H13N4O3, 357.0982). (14R)-2 (2a): [α]25D +230 (c 0.1, CHCl3); retention time (tR) 18.1 min (Chiralpak IB, hexane−2-propanol, 60:40); CD (1.12 × 10−3 M, CHCl3) λmax (Δε) 336.6 (+5.67), 260.3 sh (−4.91), 221.6 (+6.24), 198.4 (−7.98) nm. (14S)-2 (2b): [α]25D −230 (c 0.1, CHCl3); retention time (tR) 24.2 min (Chiralpak IB, hexane−2-propanol 60:40); CD (1.67 × 10−3 M, CHCl3) λmax (Δε) 335.6 (−4.25), 259.3 sh (+3.98), 217.2 (−2.96), 197.3 (+0.86) nm. Cladoquinazoline (3): colorless crystals (MeOH−H2O); mp >300 °C; [α]25D −28 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 213 (4.64), 249 (4.18), 288 (3.99) nm; CD (0.84 × 10−3 M, CHCl3) λmax (Δε) 260.9 (+2.57), 237.4 sh (−9.97), 211.8 (+25.30), 196.5 (−2.56) nm; IR (KBr) νmax 3244, 2964, 1719, 1603, 1472, 760 cm−1; 1H and 13 C NMR data see Table 2; HRESIMS m/z 419.1704 [M + H]+ (calcd for C23H23O4N4, 419.1714). epi-Cladoquinazoline (4): white solid; [α]25D −71 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 214 (4.64), 247 (4.18), 289 (3.99) nm; CD (0.84 × 10−3 M, CHCl3) λmax (Δε) 263.3 (−1.56), 233.7 sh (−17.92), 209.8 (+26.29), 192.8 (−11.41) nm; IR (KBr) νmax 3361, 2921, 1669 cm−1; 1H and 13C NMR data see Table 2; HRESIMS m/z 419.1707 [M + H]+ (calcd for C23H23N4O4, 419.1714). Norquinadoline A (5): white solid; [α]25D −2.7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 223 (4.41), 349 (4.01) nm; CD (1.06 × 10−3 M, CHCl3) λmax (Δε) 364 (+0.02), 335 (−2.32), 295 (−0.45), 270 (−1.34), 202 (+0.09) nm; IR (KBr) νmax 3307, 3065, 1600, 1585, 1482, 1389, 760 cm−1; 1H and 13C NMR see Table 3; HRESIMS m/z 494.1790 [M + Na]+ (calcd for C26H25N5O4Na, 494.1804). Quinadoline A (6): colorless crystals (MeOH−H2O); mp 205 °C; [α]25D −32 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 225 (4.41), 350 (4.01) nm; CD (1.03 × 10−3 M, CHCl3) λmax (Δε) 364 (−0.008), 337 (−2.43), 296 (−0.69), 270 (−1.25), 203 (+0.21) nm; 1H and 13C NMR see Table 3; ESIMS m/z 486.5 [M + H]+. Deoxynortryptoquivaline (7): white solid; [α]25D +60 (c 0.1, CHCl3) [lit. [α]25D +69.5 (c 0.82, CHCl3)];13 UV (MeOH) λmax (log ε) 229 (4.64), 235 (4.60), 253 (4.19), 265 (4.07), 277 (4.03), 305 (4.60), 314 (3.53) nm; CD (1.36 × 10−3 M, CHCl3) λmax (Δε) 312.8 (0.64), 272 (−3.44), 253.4 (−1.74), 238.4 (−3.91), 222.4 (12.23), 203.3 (−0.83) nm; IR (KBr) νmax 3365, 2973, 2933, 2885, 1795, 1723, 1677, 1610, 1485 cm−1; 1H and 13C NMR, Table S1; ESIMS m/z 517.2 [M + H]+.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Beckman DU 640 spectrophotometer. CD spectra were measured on a JASCO J-715 (JASCO) or Chirascan CD (Applied Photophysics) spectropolarimeter. IR spectra were recorded on a Nicolet Nexus 470 spectrophotometer in KBr discs. NMR spectra were recorded on JEOL JNMECP 600 and Bruker-400 spectrometers using TMS as an internal standard, with chemical shifts recorded as δ values. ESIMS spectra were measured on a Micromass Q-TOF Ultima Global GAA076 LC mass spectrometer. HRESIMS spectra were measured on a Micromass EI-4000 (Autospec-Ultima-TOF). X-ray crystal data were measured on a Bruker APEX DUO diffractometer (Cu Kα radiation). Semipreparative HPLC was performed using an ODS column (YMC-Pack ODS-A, 5 μm, 10 × 250 mm, 4 mL/min). Racemic mixtures were resolved on a Chiralpak IB column (5 μm, 4.6 × 250 mm, hexane−2-propanol eluent, 1 mL/min). TLC and column chromatography were performed on plates precoated with silica gel GF254 (10−40 μm) or over silica gel (200−300 mesh, Qingdao Marine Chemical Factory). Size exclusion chromatography was performed using Sephadex LH-20 (GE Healthcare). Fungal Material. The fungal strain Cladosporium sp. PJX-41 was isolated from soil around a mangrove collected in Guangzhou, China, and was identified by ITS sequence. The ITS1-5.8S-ITS2 rDNA sequence of the fungus PJX-41 has been submitted to GenBank with the accession number KC589122. A voucher specimen is deposited in our laboratory at −20 °C. The working strain was prepared on potato dextrose agar slants and stored at 4 °C. Fermentation and Extraction. The fungus PJX-41 was cultured under static conditions at 28 °C in 1 L Erlenmeyer flasks containing 300 mL of liquid culture medium, composed of maltose (20.0 g/L), mannitol (20.0 g/L), glucose (10.0 g/L), monosodium glutamate (10.0 g/L), MgSO4·7H2O (0.3 g/L), KH2PO4 (0.5 g/L), yeast extract (3.0 g/L), corn steep liquor (1.0 g/L), and seawater (Huiquan Bay, Yellow Sea), then adjusting the pH to 7.0. After 30 days of cultivation, 30 L of whole broth was filtered through cheesecloth to separate the supernatant from the mycelia. The former was extracted three times with EtOAc, while the latter was extracted three times with acetone and concentrated under reduced pressure to afford an aqueous solution, which was extracted three times with EtOAc. Both EtOAc solutions were combined and concentrated under reduced pressure to give the organic extract (10.5 g). Purification. The organic extract was subjected to vacuum liquid chromatography over a silica gel column using a gradient elution with petroleum ether−CHCl3−MeOH to give four fractions (fractions 1− 4). Fraction 4 was separated by Sephadex LH-20 eluting with CHCl3− MeOH (1:1) to provide four subfractions (fractions 4.1−4.4). Fraction 4.3 was rechromatographed on a silica gel column with petroleum ether−acetone (5:1) and by semipreparative HPLC (50:50 MeOH− H2O, 4 mL/min) to afford compound 13 (5.2 mg, tR 14.5 min) and compound 2 (5.5 mg, tR 15.7 min). Compound 2 was a racemic mixture; therefore 2.0 mg was resolved into 2a (0.6 mg) and 2b (0.4 mg) by HPLC on a chiral phase. Fraction 4.4 was rechromatographed on a silica gel column with petroleum ether−acetone (3:1) and then on a semipreparative HPLC column (55:45 MeOH−H2O, 4 mL/min) to afford compound 1 (9.5 mg, tR 17.5 min). Fraction 4.2 was separated by Sephadex LH-20 eluting with CHCl3−MeOH (1:1) to provide four subfractions (fractions 4.2.1-4.2.4). Fraction 4.2.2 was further separated by semipreparative HPLC (55:45 MeOH−H2O, 4 mL/min) to give compound 11 (48.5 mg, tR 10.1 min). Fraction 4.2.3 was chromatographed on a silica gel column using a step gradient elution of petroleum ether−acetone to provide two fractions, fractions 4.2.3.1 and 4.2.3.2. These two fractions were further purified by semipreparative HPLC (60:40 MeOH−H2O, 4 mL/min) to give compounds 6 (16.1 mg, tR 10.5 min) and 5 (10.3 mg, tR 13.8 min), F



Article

Deoxytryptoquivaline (8): [α]25D +50 (c 0.1, CHCl3) [lit. [α]25D +56.8 (c 0.78, CHCl3)];13 CD (0.94 × 10−3 M, CHCl3) λmax (Δε) 307.2 (+1.53), 269.9 sh (−3.54), 218.4 (+19.85), 202 (−9.07) nm. Tryptoquivaline (9): colorless crystals (MeOH−H2O); mp 215 °C; [α]25D +120 (c 0.1, CHCl3) [lit. [α]25D +130 (c 0.22, CHCl3)];13 CD (0.92 × 10−3 M, CHCl3) λmax (Δε) 308.3 (+1.42), 254.8 (−6.20), 219.4 (+21.84), 203 (−11.35) nm. CS-C (10): [α]25D +130 (c 0.05, CHCl3) [lit. [α]30D +138 (c 0.020, CHCl3)].18 Prelapatin B (12): white powder; [α]25D +170 (c 0.1, EtOAc); UV (MeOH) λmax (log ε) 209 (4.64), 217 (4.73), 268 (4.13), 285 (4.19) nm; IR (KBr) νmax 3148, 3035, 2830, 1694, 1623, 1422, 1336, 1169, 778, 671 cm−1; ESIMS m/z 343 [M + H]+. Glyantrypine (13): [α]25D −520 (c 0.1, CHCl3) [lit. [α]30D −535 (c 0.028, CHCl3)];6 CD (2.03 × 10−3 M, CHCl3) λmax (Δε) 290 (+5.41), 264.6 sh (+3.09), 231 (+24.96), 208.8 (−7.73) nm. X-ray Crystallographic Analysis of Compounds 3, 6, and 9. Single-crystal X-ray diffraction data were collected on an Agilent Gemini Ultra diffractometer with Cu Kα radiation (λ = 1.54184 Å). The structure was solved by direct methods (SHELXS-97) and refined using full-matrix least-squares difference Fourier techniques. Carbon, oxygen, and nitrogen atoms were refined anisotropically. Hydrogen atoms were either refined freely with isotropic displacement parameters or positioned with an idealized geometry and refined riding on their parent C atoms. Crystals suitable for X-ray diffraction were obtained by slow evaporation of a solution in MeOH−H2O. Crystallographic data (excluding structure factors) for 3, 6, and 9 have been deposited with the Cambridge Crystallographic Data Centre: CCDC reference numbers 922839 (for compound 3), 922840 (6), and 922841 (9). These data can be obtained, free of charge, from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac. uk/data_request/cif. Crystal data for 3: monoclinic, C23H22N4O4, space group P21 with a = 10.8171(4) Å, b = 6.2921(3) Å, c = 15.0930(5) Å, α = 90°, β = 96°, γ = 90°, V = 1021.46(7) Å3, Z = 2, T = 291(2) K, Dc = 1.360 mg/ m3, μ = 0.781 mm−1, and F(000) = 440. Crystal size: 0.32 × 0.2 × 0.2 mm3. Independent reflections: 3227 with Rint = 0.0228. The final agreement factors are R1 = 0.0476 and wR2 = 0.1282 [I > 2σ(I)]. Flack parameter = −0.1(3). Crystal data for 6: orthorhombic, C27H27N5O4.CH3OH, space group P212121 with a = 12.4363(2) Å, b = 13.6529(2) Å, c = 15.6264(2) Å, α = β = γ = 90°, V = 2653.23(7) Å3, Z = 4, T = 291(2) K, Dc = 1.296 mg/m3, μ = 0.742 mm−1, and F(000) = 1096.0. Crystal size: 0.38 × 0.36 × 0.32 mm3. Independent reflections: 4898 with Rint = 0.0215. The final agreement factors are R1 = 0.0411 and wR2 = 0.1203 [I > 2σ(I)]. Flack parameter = 0.1(2). Crystal data for 9: orthorhombic, C29H30N4O7, space group P212121 with a = 9.58100(10) Å, b = 10.93600(10) Å, c = 26.3804(2) Å, α = β = γ = 90°, V = 2764.08(4) Å3, Z = 4, T = 290(2), Dc = 1.313 mg/m3, μ = 0.788 mm−1, and F(000) = 1152. Crystal size: 0.38 × 0.35 × 0.3 mm3. Independent reflections: 5369 with Rint = 0.0259. The final agreement factors are R1 = 0.0281 and wR2 = 0.0754 [I > 2σ(I)]. Flack parameter = 0.01(12).

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Fundation of China (No. 41176120), the National High Technology Research and Development Program of China (No. 2013AA092901), the Program for New Century Excellent Talents in University (No. NCET-12-0499), the Public Projects of State Oceanic Administration (No. 2010418022-3), the Promotive Research Fund for Excellent Young and Middle-aged Scientists of Shandong Province (No. BS2010HZ027), and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0944). We also thank Dr. R. A. Keyzers (Victoria University of Wellington, New Zealand) for helping with the manuscript preparation.

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra for compounds 1−7, CD spectra for compounds 7−9, HPLC chromatogram of 2 on a chiral phase, HPLC analysis of 2a, 2b, 3, and 4 in base solvent, X-ray ORTEP diagram of compound 9, 1D NMR and 2D NMR data of 7, as well as antiviral activities data of compounds 1−13 against H1N1. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail: [email protected] (D. Li); [email protected] (Z. Xin). G



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