Carboline alkaloids and isocoumarins from the wasp pathogenic fungus Ophiocordyceps sphecocephala BCC 2661

Phytochemistry Letters 27 (2018) 134–138

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Carboline alkaloids and isocoumarins from the wasp pathogenic fungus Ophiocordyceps sphecocephala BCC 2661

T

Jittra Kornsakulkarn, Siriporn Saepua, Sukitaya Veeranondha, Pranee Rachtawee, ⁎ Masahiko Isaka, Chawanee Thongpanchang National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand

A R T I C LE I N FO

A B S T R A C T

Keywords: Carboline alkaloids Insect-pathogenic fungi Ophiocordyceps sphecocephala Bioactive secondary metabolites

Ten β-carboline alkaloids, including five new and two new naturally occurring compounds, sphecolines A–G (1–7), together wit2h four known isocoumarins were isolated from a wasp-pathogenic fungus Ophiocordyceps sphecocephala BCC 2661. Structures of these compounds were determined by NMR spectroscopic and MS spectrometric analyses. Sphecolines A (1) and C (3) exhibited cytotoxic activity against human small-cell lung cancer NCI-H187 cell lines, with IC50 values of 79.9 and 75.1 μM, respectively.

1. Introduction β-Carboline alkaloids, commonly possess a tricyclic pyrido[3,4-b] indole ring structure, are a large group of indole alkaloids which have been found from various natural sources such as plants, animals tissues, marine creatures, and fungi (Hesse, 2002). Compounds in this class are known to have a wide variety of interesting biological properties, for examples, antimicrobial, antiviral, antitumor, antimalarial, and hallucinogenic activities (Cao et al., 2007; Patel et al., 2012). Insect pathogenic fungi on various substrates such as Homoptera, Coleoptera, Lepidoptera larvae, and Orthoptera have been reported to produce a diverse structure of bioactive substances, however, the study on wasp-pathogenic fungi is very rare (Isaka et al., 2005; Molnar et al., 2010). As part of our continuing research on the discovery of bioactive compounds from insect pathogenic fungi, we came across the crude extracts of a wasp-pathogen Ophiocordyceps sphecocephala, strain BCC 2661, showing prolific chemical profiles from HPLC and 1H NMR data. Therefore, the chemical constituents from the crude extracts were investigated. The study led to the isolation of five new β-carbolines, sphecolines A (1), B (2), and D–F (4–6), and two new naturally occurring β-carbolines, sphecolines C (3) and G (7) (Fig.1), together with three known carbolines, 1-acetyl-β-carboline (8) (Shin et al., 2010), 1acetyl-3-carbomethoxy-β-carboline (9) (Faini et al., 1978), and 1,3,4trioxo-1,2,3,4-tetrahydo-β-carboline (10) (Koike et al., 1990). Four known isocoumarins, cytogenin (11) (Kumagai et al., 1990), peyroisocoumarin D (12) (Zhao et al., 2016), diaportinol (13) (Ichihara et al., 1989; Larsen and Breinholt, 1999), and (+)-mucoricoumarin C (14) (Feng et al., 2014), were also isolated from the same extract.

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Details of isolation, structure elucidation, and biological activities of these compounds are presented herein. 2. Results and discussion Sphecoline A (1) was obtained as a yellow solid and the molecular formula C13H12N2O2 was determined from the quasi-molecular ion peak at m/z 229.0961 [M+H]+ in the HRESIMS data. The absorptions at 423, 302, 294, 261, and 240 nm in UV spectrum suggested the βcarboline chromophore. The IR spectrum displayed the absorptions for amine/hydroxyl group at 3289 and 3182 cm−1. The 1H NMR spectrum showed the signals of five aromatic protons, one oxygenated methine proton, one methyl group, and three D2O exchangeable protons. The more down fielded chemical shifts at C-1 (δC 143.3), C-8a (δC 142.6), and C-9a (δC 134.3) indicated the presence of the amine group connected to these carbons. The COSY correlations between H-5 (δH 8.05) to H-8 (δH 7.52) together with the HMBC correlations from H-4 (δH 7.06) to C-3 (δC 156.4)/C-4a (δC 120.2)/C-4b (δC 127.0), H-5 (δH 8.05) to C-7 (δC 128.4)/C-8a (δC 142.6), H-6 (δH 7.08) to C-4b (δC 127.0), H-7 (δH 7.44) to C-5 (δC 121.8)/C-8a (δC 142.6), H-8 (δH 7.52) to C-6 (δC 118.1), and NeH (δH 10.66) to C-4a (δC 120.2)/C-4b (δC 127.0)/C-8a (δC 142.6)/C-9a (δC 134.3) (Fig. 2) revealed the presence of β-carboline unit. The attachment of the hydroxyethyl moiety at C-1 was established by the correlations between H-10 (δH 5.05), H-11 (δH 1.49), and 10eOH (δH 5.65) in COSY spectrum and the correlations from 10eOH (δH 5.65) to C-11 (δC 23.0) and H-11 (δH 1.49) to C-1 (δC 143.3)/C-10 (δC 68.2) in HMBC spectrum. The rest of the OH (δH 10.09) group situated at C-3 (δC 156.4) as deduced by its chemical shift. Comparison of

Corresponding author. E-mail address: [email protected] (C. Thongpanchang).

https://doi.org/10.1016/j.phytol.2018.07.020 Received 20 April 2018; Received in revised form 14 June 2018; Accepted 13 July 2018 1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

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Fig. 1. Chemical structures of compounds 1–14.

with known absolute configuration, dichotomines A–D (Sun et al., 2004; Zhang et al., 2012). The molecular formula of sphecoline C (3) was determined by HRESIMS, in combination with 13C NMR spectroscopy, as C15H14N2O3. Their spectroscopic data, including UV, IR, 1H, NMR, and 13C NMR, as well as optical rotation were identical to those of synthetic methyl(−)-1-(hydroxyethyl)pyrido[3,4-b]indole-3-carboxylate (Tagawa et al., 2013). The correlations from H-4 (δH 8.80) to C-4b (δC 122.6)/C-9a (δC 135.5)/C-12 (δC 167.2), H-5 (δH 8.36) to C-7 (δC 129.5)/C-8a (δC 142.3), H-6 (δH 7.34) to C-4b (δC 122.6)/C-8 (δC 113.5), H-7 (δH 7.60) to C-5 (δC 122.5)/C-8a (δC 142.3), H-8 (δH 7.79) to C-4b (δC 122.6)/C-6 (δC 121.3), H-11 (δH 1.64) to C-1 (δC 149.2)/C-10 (δC 71.2), and H-13 (δH 3.93) to C-12 (δC 167.2) in HMBC spectrum also supported the structural feature of sphecoline C (3) as shown in Fig. 1. Although compound 3 has previously been synthesized (Tagawa et al., 2013), it is now isolated and reported for the first time from natural source. The UV, IR, and 1H NMR spectra of sphecoline D (4) resembled those of the known co-metabolite 1-acetyl-β-carboline (8). Detailed analysis of 2D NMR spectroscopic data revealed the similar structure of these two compounds except for the presence of an additional methoxy group in sphecoline D (4). The molecular formula C14H12N2O2 as determined from HRESIMS also indicated a 30 amu higher than that of 8. The 1-acetyl-β-carboline moiety was confirmed by the cross peaks from H-5 (δH 8.23) to H-8 (δH 7.76) in COSY spectrum and the correlations from H-4 (δH 7.79) to C-3 (δC 157.3)/C-9a (δC 133.6)/C-4b (δC 121.5), H-5 (δH 8.23) to C-7 (δC 130.5)/C-8a (δC 144.8), and H-11 (δH 2.75) to C-1 (δC 132.1)/C-10 (δC 201.7) in HMBC spectrum. The HMBC correlation from the methoxy protons (δH 4.05) to C-3 (δC 157.3) indicated its position. Sphecoline D (4) was, therefore, identified as 3-methoxy derivative of 1-acetyl-β-carboline. Sphecolines E (5), F (6), and G (7) were also identified as β-carboline derivatives since they were found to exhibit the same characteristic UV absorption of β-carboline. The 1H and 13C NMR spectroscopic data of sphecolines E (5), F (6), and G (7) were similar to those of sphecoline D (4), suggesting the related structural feature of these compounds. The 1-acetyl-β-carboline core structure of these compounds were indicated on the basis of COSY and HMBC correlations as previously described for sphecoline D (4). The difference between these compounds were the attachment of the hydroxy or methoxy group at C-3 and/or C-6, which were established on the basis of their chemical shifts and the correlations from these protons to their corresponding carbons in HMBC

Fig. 2. Selected COSY and HMBC correlations of compound 1.

the optical rotation observed for sphecoline A (1) ([α]24 D −31.3, c 0.5 in MeOH) to those of similar 1-(1-hydroxyethyl)-β-carboline analogues such as dichotomine A (Sun et al., 2004; Zhang et al., 2012) ([α]26 D −22.3, c 0.5 in MeOH) suggested that the absolute configuration at C10 of sphecoline A (1) should be the same as those analogues, which was assigned as S. Sphecoline B (2) with the molecular formula C15H14N2O4 from HRESIMS, in combination with 13C NMR spectroscopy, displayed characteristic UV absorption of β-carboline chromophore at 360, 315, 279, and 240 nm. The similarity of the 1H NMR spectra of sphecolines B (2) and A (1), except for the presence of an additional methoxy signal at δH 3.92 and the missing of one aromatic proton in sphecoline B (2), suggested that these two compounds shared the same core structure. The presence of a carbonyl signal at δC 167.3 in the 13C NMR spectrum, which attributed in HMBC spectrum to H-4 (δH 8.68) and methoxy protons, established the position of the carbomethoxy group at C-3 of the carboline unit. The assignment of the hydroxyl group at C-6 (δC 153.0) was deduced on the basis of its chemical shift. The 10S configuration was indicated by comparing the optical rotation of sphecoline B (2) ([α]24 D −34.5, c 0.5 in MeOH) to those of other similar analogues 135

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3.3. Fermentation, extraction, and isolation

spectrum. Accordingly, sphecolines E (5), F (6), and G (7) were identified as 1-acetyl-3,6-dihydroxy-β-carboline, 1-acetyl-3-hydroxy-6methoxy-β-carboline, and 1-acetyl-3-hydroxy-β-carboline, respectively. Sphecoline G (7) has been obtained as a biosynthetic product from the biosynthetic gene cluster of marine carboline alkaloid (Chen et al., 2013), however, it has not previously been isolated from the nature. The seven known compounds were identified as 1-acetyl-β-carboline (8) (Shin et al., 2010), 1-acetyl-3-carbomethoxy-β-carboline (9) (Faini et al., 1978), 1,3,4-trioxo-1,2,3,4-tetrahydo-β-carboline (10) (Koike et al., 1990), cytogenin (11) (Kumagai et al., 1990), peyroisocoumarin D (12) (Zhao et al., 2016), diaportinol (13) (Ichihara et al., 1989; Larsen and Breinholt, 1999), and (+)-mucoricoumarin C (14) (Feng et al., 2014), respectively, by analysis of their spectroscopic data including NMR and MS, together with the specific rotations which were identical to those reported in the literature data. All isolated β-carbolines were subjected to assays for antimycobacterial (Mycobacterium tuberculosis H37Ra) and antibacterial (Bacillus cereus and Enterococcus faecium) activities and cytotoxic activity against cancer cell-lines (KB, MCF-7, and NCI-H187) and noncancerous Vero cells. Only sphecolines A (1) and G (3) exhibited cytotoxic activity against human small-cell lung cancer, NCI-H187 cell lines, with IC50 values of 79.9 and 75.1 μM, respectively, while the others were inactive in our biological assays at the maximum tested concentrations (50 μg/ml). It was reported that 1-acetyl-β-carboline (8) exhibited antibacterial activity against various strains with MICs range from 304.4 to 1217.7 μM and also it showed synergistic activity with ampicillin against MRSA strains (Shin et al., 2010). 1-Acetyl-3-carbomethoxy-β-carboline (9) was reported to display cytotoxic activity against the HCT116 cell lines with IC50 36.0 μM (Tian et al., 2012). In conclusion, ten β-carboline alkaloids (1–10) and four isocoumarins (11–14) were isolated from a wasp-pathogenic fungus Ophiocordyceps sphecocephala BCC 2661. Among all isolated β-carbolines, only sphecolines A (1) and C (3) showed cytotoxic activity against human small-cell lung cancer, NCI-H187 cell lines (IC50 79.9 and 75.1 μM, respectively).

Ophiocordyceps sphecocephala BCC 2661 was developed on potato dextrose agar at 25 °C for 18 days, and the agar was cut into pieces (1 × 1 cm) and inoculated into 4 × 250 ml Erlenmeyer flasks containing 25 ml of yeast extract broth (YES: yeast extract 20.0 g/l, sucrose 150 g/l). After incubation at 25 °C for 10 days on a rotary shaker (200 rpm), each primary culture was transferred into 1 l Erlenmeyer flask containing 250 ml of the same medium and incubated under the same conditions for 10 days. Each 25 ml portion of the secondary culture was transferred into 40 × 1 l Erlenmeyer flasks containing 250 ml of YES medium and was statically incubated at 25 °C for 30 days. After filtration of the culture, the culture broth was extracted with EtOAc (3 × 10 l) and evaporated to dryness, leaving an orange gum (4.26 g). Trituration of the crude extract in MeOH followed by filtration gave orange solid (470.3 mg), in which compounds 7 (tR 9.8 min, 188.0 mg) and 9 (tR 18.3 min, 60.0 mg) were obtained after further purification by preparative HPLC using reverse phase column (SunFire C18 OBD, 10 μm, 19☓250 mm, 30 °C, step gradient elution with 30 − 55% MeCN/H2O, flow rate 15 mL/min, 30 min). The filtrate (3.78 g) was fractionated using Sephadex LH 20 (7.0 ☓ 35 cm), eluted with 100% MeOH, to provide five fractions (A1–A5). Fraction A2 was subjected to preparative HPLC (step gradient elution with 15 − 60% MeCN/H2O, flow rate 15 mL/min, 35 min) to give six subfractions (A21–A2-6). Compound 1 (49.7 mg) was obtained after further purification of subfractions A2-1 and A2-2. Trituration of subfractions A2-3 with MeOH followed by filtration yielded 1,3,4-trioxo-1,2,3,4-tetrahydeo-βcarboline as a yellow solid (1.1 mg). After purification by preparative thin layer chromatography, using 100% EtOAc as an eluent, peyroisocoumarin D (Rf 0.56, 1.8 mg) and diaportinol (Rf 0.38, 6.1 mg) were obtained from subfraction A2-4 while compound 3 (3.5 mg) and mucoricoumarin C (4.0 mg) were provided from subfraction A2-6. Fraction A3 was subjected to preparative HPLC (step gradient elution with 15 − 50% MeCN/H2O, flow rate 15 mL/min, 50 min) to furnish compounds 1 (tR 15.3 min, 4.6 mg), 2 (tR 18.4 min, 3.9 mg), 3 (tR 33.8 min, 2.7 mg), 9 (tR 47.3 min, 2.3 mg), and mucoricoumarin C (tR 32.7 min, 8.0 mg). Purification of fraction A4 by preparative HPLC (step gradient elution with 10 − 80% MeCN/H2O, flow rate 15 mL/min, 50 min) afforded compounds 2 (tR 13.8 min, 18.0 mg), 7 (tR 20.1 min, 7.8 mg), 8 (tR 29.6 min, 0.8 mg), 9 (tR 31.6 min, 4.4 mg), and cytogenin (tR 20.1 min, 3.7 mg). Fraction A5 was purified by preparative HPLC (step gradient elution with 20 − 60% MeCN/H2O, flow rate 12 mL/min, 40 min) to provide compounds 5 (tR 11.9 min, 1.8 mg) and 6 (tR 21.5 min, 2.4 mg). The cells were macerated in MeOH (1.0 l) for 3 days, and then in CH2Cl2 (1.0 l) for 3 days. The MeOH and CH2Cl2 extracts were combined and evaporated under reduced pressure. Water (200 ml) was added and the mixture was extracted with hexanes (3 × 200 ml), followed by EtOAc (3 × 200 ml). Trituration of the EtOAc extract (an orange solid, 2.22 g) in MeOH followed by filtration provided a yellow solid (380.6 mg). The concentrated filtrate (1.84 g) was fractionated using Sephadex LH 20 (7.0 ☓ 35 cm), eluted with 100% MeOH, to give four fractions (B1–B4). Compound 3 (tR 28.8 min, 9.4 mg) was obtained from fractions B1 and B2 after purification by preparative HPLC (step gradient elution with 15 − 60% MeCN/H2O flow rate 15 mL/min, 50 min). Trituration of fraction B3 in MeOH followed by filtration gave compound 9 as an orange solid (93.0 mg). Purification of the filtrate by preparative HPLC (step gradient elution with 25 − 70% MeCN/H2O, flow rate 15 mL/min, 35 min) afforded compounds 7 (tR 11.3 min, 5.8 mg), 8 (tR 18.7 min, 1.3 mg), 9 (tR 19.9 min, 6.1 mg), and 4 (tR 29.6 min, 0.6 mg). Fraction B4 was further purified by preparative HPLC (step gradient elution with 25 − 70% MeCN/H2O, flow rate 12 mL/min, 35 min) to furnish compounds 7 (tR 8.9 min, 78.4 mg), 9 (tR 13.1 min, 5.7 mg), and 4 (tR 20.9 min, 1.7 mg). Compounds 3 (5.8 mg), 4 (2.5 mg), 7 (10.9 mg), and 9 (100.3 mg) were obtained from the hexanes extraction of the mycelium (4.45 g) after consecutive

3. Experimental 3.1. General procedures Melting points were measured using a Metler MP90 melting point apparatus and are uncorrected. Optical rotation measurements were conducted by using a JASCO P-1030 digital polarimeter. UV and FT-IR spectra were recorded on an Analytic Jena Spekol 1200 spectrophotometer and a Bruker Alpha spectrometer. NMR spectra were recorded on Bruker Advance III 400 and Bruker Advance 500 spectrometers. ESITOF MS data were obtained on a Bruker micrOTOF mass spectrometer. Column chromatography was performed on silica gel 60 (70–230 Mesh ASTM, Merck). HPLC were performed using DionexUltimate 3000 series equipped with a binary pump, an autosampler, and a diode array detector. 3.2. Fungal material The fungus used in this study was isolated from a wasp (Hymenoptera) associated with the leaf litter, collected at Nam Nao National Park, Phetchabun Province, Thailand. The living culture was deposited in the BIOTEC Culture Collection (BCC) as BCC 2661 on June 19, 2000. This fungus was identified as Ophiocordyceps sphecocephala of the Class Sordariomycetes, order Hypocreales, family Ophicordycipitaceae, on the basis of morphology, by Dr. Nigel HywelJones, BIOTEC, which was confirmed by the molecular identification using the rDNA internal transcribed spacer region (ITS: GenBank accession number MH161351), analyzed by Dr. Nattawut Boonyuen, BIOTEC. 136

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Table 1 NMR spectroscopic data for compounds 1–3. Position

Table 2 NMR spectroscopic data for compounds 4–7.

1 (in DMSO-d6)a

2 (in acetone-d6)b

3 (in acetone-d6)b

δC

δH mult(J in Hz)

δC

δH mult(J in Hz)

δC

149.2 130.3 117.2 137.7 122.6 122.5 121.3 129.5

– – 8.80 s – – 8.36 d (7.9) 7.34 t (7.6) 7.60 t (7.6)

113.5 142.3 135.5 71.2

7.79 d (8.3) – – 5.36 brq (6.0)

23.7 167.2 52.2 – – – –

1.64 d (6.0) – 3.93 s 10.97 brs – – 5.16 brs

1 3 4 4a 4b 5 6 7

143.3 156.4 96.7 120.2 127.0 121.8 118.1 128.4

– – 7.06 s – – 8.05 d (7.8) 7.08 t (6.8) 7.44 t (7.4)

149.1 136.6 117.3 129.8 123.3 106.8 153.0 119.7

8 8a 9a 10

112.0 142.6 134.3 68.2

114.1 136.8 135.9 70.9

11 12 13 NH 3-OH 6-OH 10-OH

23.0 – – – – – –

7.52 d (8.1) – – 5.05 dq (3.8, 6.4) 1.49 d (6.4) – – 10.66 s 10.09 brs – 5.65 d (3.8)

– – 8.68 – – 7.71 – 7.19 8.8) 7.62 – – 5.34

23.6 167.3 52.1 – – – –

1.61 d (6.6) – 3.92 s 10.76 brs – 9.26 sc 5.83 d (4.1)c

a b c

s

d (2.3) dd (2.3, d (8.8)

q (6.6)

4 (in acetone -d6)a

5 (in DMSOd6)b

6 (in DMSOd6)b

7 (in DMSO -d6)b

δC

δH mult(J in Hz)

δC

δH mult(J in Hz)

δC

δH mult(J in Hz)

δC

δH mult(J in Hz)

1 3 4 4a 4b 5

132.1 157.3 106.8 137.4 121.5 123.0

131.5 154.4 105.0 135.6 120.1 106.2

– – 7.67 s – – 7.76 d (2.4) –

130.8 155.1 105.1 135.8 119.6 122.1

120.9

– – 7.57 s – – 7.46 d (2.4) –

131.1 154.5 105.1 135.7 119.9 104.2

6 7

130.5

– – 7.79 s – – 8.23 d (7.9) 7.26 t (7.5) 7.58 t (7.3)

– – 7.68 s – – 8.19 d (7.8) 7.18 t (7.5) 7.52 t (7.3)

8

113.4

8a 9a 10 11 NH

144.8 133.6 201.7 25.7 –

3-OH/OCH3 6-OH/OCH3

Position

δH mult(J in Hz)

500 MHz for 1H and 125 MHz for 13C. 400 MHz for 1H and 100 MHz for 13C. appear in DMSO d6.

purification by Sephadex LH 20, eluted with 100% MeOH, and then preparative HPLC (step gradient elution with 20 − 60% MeCN/H2O, flow rate 15 mL/min, 50 min).

a b

150.9 118.9

137.3 130.9 200.3 25.7 –

54.1

7.76 d (8.2) – – – 2.75 s 10.88 brs 4.05 s

113.2

–

–

–

–

400 MHz for 1H and 100 MHz for 500 MHz for 1H and 125 MHz for

7.03 dd (2.4, 9.0) 7.48 d (9.0) – – – 2.69 s 11.12 s 9.21 brs 10.32 brs 13

153.3 118.7

113.4 138.1 131.3 200.3 25.7 – – 55.6

7.16 dd (2.4, 8.8) 7.57 d (8.8) – – – 2.70 s 11.26 s 10.36 brs 3.83 s

119.4 129.2

112.7 143.4 131.2 200.6 25.9 – – –

7.67 d (9.5) – – – 2.71 s 11.44 s 10.44 brs –

C. C.

13

1376, 1352, 1207, 1193, 1152, 1123, 1089, 1051, 1026 cm−1; 1H NMR and 13C NMR data, see Table 2; HRMS (ESITOF) m/z241.0615 [M−H]− (calcd. for C13H9N2O3, 241.0619).

3.3.1. Sphecoline A (1) Yellow solid; m.p. 146.0–148.0 °C; [α]24 D −31.3 (c 0.51, MeOH); UV (MeOH) λmax (log ε) 240 (4.22), 261 (4.13), 294 (3.82), 302 (3.74), 423 (3.70) nm; IR (ATR) νmax 3289, 3182, 1653, 1612, 1542, 1495, 1435, 1320, 1222, 1127, 1067 cm−1; 1H NMR and 13C NMR data, see Table 1; HRMS (ESITOF) m/z 229.0961 [M+H]+ (calcd. for C13H13 N2O2, 229.0972).

3.3.6. Sphecoline F (6) Orange solid; UV (MeOH) λmax (log ε) 225 (3.88), 277 (3.76), 308 (3.84), 423 (3.58) nm; IR (ATR) νmax 3366, 1621, 1596, 1556, 1492, 1478, 1449, 1310, 1285, 1208, 1161, 1054, 1033 cm−1; 1H NMR and 13 C NMR data, see Table 2; HRMS (ESITOF) m/z 279.0736 [M + Na]+ (calcd. for C14H12N2O3Na, 279.0740).

3.3.2. Sphecoline B (2) Light brown solid; m.p. 148.5–150.0 °C; [α]24 D −34.5 (c 0.51, MeOH); UV (MeOH) λmax (log ε) 240 (4.31), 279 (4.31), 315 (3.91), 360 (3.77) nm; IR (ATR) νmax 3269, 1705, 1581, 1503, 1468, 1437, 1337, 1286, 1242, 1056, 1033, 1018 cm−1; 1H NMR and 13C NMR data, see Table 1; HRMS (ESITOF) m/z 287.1000 [M+H]+ (calcd. for C15H15N2O4, 287.1026).

3.3.7. Sphecoline G (7) Orange solid; m.p. 174.0–177.0 °C; UV (MeOH) λmax (log ε) 229 (3.94), 271 (3.94), 297 (4.00), 411 (3.81) nm; IR (ATR) νmax 3358, 1630, 1593, 1577, 1492, 1468, 1454, 1315, 1216, 1186, 1146, 1054, 1033 cm−1; 1H NMR and 13C NMR data, see Table 2; HRMS (ESITOF) m/z 249.0639 [M + Na]+ (calcd. for C13H10N2O2Na, 249.0634).

3.3.3. Sphecoline C (3) Light brown solid; [α]24 D −31.7 (c 0.48, MeOH); UV (MeOH) λmax (log ε) 238 (4.31), 267 (4.37), 303 (3.95), 346 (3.70) nm; IR (ATR) νmax 3339, 1709, 1626, 1570, 1500, 1434, 1351, 1253, 1242, 1111, 1065 cm−1; 1H NMR and 13C NMR data, see Table 1; HRMS (ESITOF) m/z 271.1079 [M+H]+ (calcd. for C15H15N2O3, 271.1077).

3.4. Biological assays Antibacterial activity against B. cereus and E. faecium, and cytotoxic activities against KB cells (oral human epidermoid carcinoma, ATCC CCL-17), MCF-7 cells (human breast cancer, ATCC HTC-22), and NCIH187 cells (human small-cell lung cancer, ATCC CRL-5804) were evaluated by using the resazurin microplate assay (O’Brien et al., 2000). The IC50 values of standard compound, doxorubicin, against KB, MCF-7, and NCI-H187 cells were 1.25, 14.62, and 0.13 μM, respectively. The standard compound for antibacterial against B. cereus and E. faecium, rifampicin, exhibited MIC values of 0.15 and 1.89 μM, respectively. Growth inhibitory activity against M. tuberculosis H37Ra and cytotoxicity to Vero cells (African green monkey kidney fibroblasts, ATCC CCL81) was performed by using the green fluorescent protein (GFP) based method (Changsen et al., 2003; Hunt et al., 1999). Isoniazid and

3.3.4. Sphecoline D (4) Yellow solid; UV (MeOH) λmax (log ε) 223 (3.83), 272 (3.82), 297 (3.84), 409 (3.66) nm; IR (ATR) νmax 3313, 1651, 1626, 1605, 1581, 1493, 1467, 1454, 1376, 1206, 1193, 1167, 1147, 1032 cm−1; 1H NMR and 13C NMR data, see Table 2; HRMS (ESITOF) m/z 263.0798 [M + Na]+ (calcd. for C14H12N2O2Na, 263.0791). 3.3.5. Sphecoline E (5) Red solid; UV (MeOH) λmax (log ε) 221 (3.73), 278 (3.55), 309 (3.61), 427 (3.36) nm; IR (ATR) νmax 3365, 1631, 1592, 1547, 1468, 137

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