γ-Butyrolactone, Cytochalasin, Cyclic Carbonate, Eutypinic Acid, and

Nov 6, 2014 - Purification of an extract from the broth of the soil fungus Aspergillus sp. PSU-RSPG185 resulted in the isolation of two new cyclic car...
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γ‑Butyrolactone, Cytochalasin, Cyclic Carbonate, Eutypinic Acid, and Phenalenone Derivatives from the Soil Fungus Aspergillus sp. PSURSPG185 Vatcharin Rukachaisirikul,*,† Nachamon Rungsaiwattana,† Saranyoo Klaiklay,‡ Souwalak Phongpaichit,§ Kawitsara Borwornwiriyapan,§ and Jariya Sakayaroj⊥ †

Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand ‡ Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani Campus, Surat Thani 84100, Thailand § Natural Products Research Center of Excellence and Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand ⊥ National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Klong Luang, Pathum Thani 12120, Thailand S Supporting Information *

ABSTRACT: Purification of an extract from the broth of the soil fungus Aspergillus sp. PSU-RSPG185 resulted in the isolation of two new cyclic carbonate derivatives, aspergillusols A (1) and B (2), and one new eutypinic acid derivative, aspergillusic acid (3), along with six known secondary metabolites. Compounds 1 and 2 contain an unusual cycliccarbonate functionality. In addition, the mycelial extract afforded two new phenalenones, aspergillussanones A (4) and B (5), one new cytochalasin, aspergilluchalasin (6), and one new γ-butyrolactone, aspergillulactone (7). Their structures were established by interpretation of spectroscopic evidence. Compound 4 exhibited weak activity toward KB and Vero cells with IC50 values of 48.4 and 34.2 μM, respectively.

■

S

oil fungi have gained prominence as a source of novel bioactive compounds.1−3 The fungi in the genus Aspergillus produced a wide range of biologically active metabolites, for example, the antibacterial butyrolactones,4 antifungal indole alkaloids,5 and antioxidant xanthones.6 Recently, we have reported the isolation of six new and three known indolediazepines from the mycelial extract of the soil fungus Aspergillus sp. PSU-RSPG185.7 In this paper, we describe the isolation and structure determination of secondary metabolites from the broth extract and further study of the mycelial extract of the fungus PSU-RSPG185, which displayed cytotoxic activity against human oral carcinoma (KB) cell lines. Two new cyclic carbonates, aspergillusols A (1) and B (2), and one new eutypinic acid derivative, aspergillusic acid (3), were obtained from the broth extract together with six known compounds, namely, (+)-asperpentyn (8),8,9 eutypinic acid (9),10 aspochalasin J (10),11 4-hydroxy-3-prenylbenzoic acid (11),10 2,2dimethyl-2H-1-chromene-6-carboxylic acid (12),12 and 2-(1′methylethenyl)benzofuran-5-carboxylic acid (13).10 Furthermore, two new phenalenones, aspergillussanones A (4) and B (5), one new cytochalasin, aspergilluchalasin (6), and one new γ-butyrolactone, aspergillulactone (7), were obtained from the mycelial extract. Their cytotoxic activity against KB and Vero (African green monkey kidney) cell lines was evaluated. © XXXX American Chemical Society and American Society of Pharmacognosy

RESULTS AND DISCUSSION

All compounds (1−13) (Figure 1) were purified using chromatographic techniques, and their structures were elucidated from analysis of their spectroscopic data. The relative configurations of compounds were assigned according to NOEDIFF results. The absolute configurations of the new compounds were proposed by a plausible biosynthetic pathway for compounds 1, 2, and 6 and assigned by circular dichroism and Snatzke’s method for compounds 4 and 5 and optical rotation for compound 7. The absolute configuration of the known compound 8 was determined by comparison of its optical rotation, [α]25D +25.4 (c 0.50, CHCl3), with that previously reported, [α]20D +24.2 (c 0.50, CHCl3).8 The literature search reveals that the stereochemistry of the cyclohexane and isoindole moieties in all isolated cytochalasins is the same.11 Therefore, the absolute configuration of compound 10 was assigned to be identical to that of aspochalasin J. Unfortunatedly, the optical rotation of aspochalasin J has never been reported. Received: April 12, 2014

A

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Figure 1. Compounds 1−13 isolated from the soil fungus Aspergillus sp. PSU-RSPG185.

Table 1. 1H and 13C NMR Spectroscopic Data for 1, 2, and 3 in CDCl3 1 position

δC, type

1 2 3 3-OH 4 4-OH 5 6 1′ 2′ 3′ 4′

116.5, C 143.2, CH 69.6, CH

5′ 6′ 7′ a

73.9, CH 79.1, 75.9, 85.6, 92.2, 127.2, 123.4,

CH CH C C C CH2

23.2, CH3 154.1, C

2 δH (J in Hz) 6.28, 4.19, 4.79, 3.58, 5.13, 4.79, 5.26,

dd, (2.0, 0.5) m brsa brt (8.0) brsa dd (8.3, 8.0) brd (8.3)

a: 5.36, quin (1.5) b: 5.34, quin (1.5) 1.90, t (1.5)

δC, type 130.2, C 124.5, CH 72.8, CH 77.3, CH 73.0, 69.0, 82.5, 96.7, 125.7, 124.3,

CH CH C C C CH2

3 δH (J in Hz)

δC, type

6.06, dd (3.9, 2.1) 5.15, ddd (7.8, 3.9, 2.1)

113.0, C 164.0, C 110.2, CH

6.94, d (8.5)

4.64, dd (8.7, 7.8)

132.1, CH

8.03, dd (8.5, 2.0)

3.76, t (8.7) 4.03, dt (8.7, 1.8)

121.3, 135.8, 83.3, 95.4, 127.0, 122.6,

a: 5.36, quin (1.8) b: 5.34, quin (1.8) 1.88, t (1.2)

23.0, CH3 153.4, C

C CH C C C CH2

23.4, CH3 168.0, C 56.1, CH3

δH (J in Hz)

8.16, d (2.0)

a: 5.46, d (1.0) b: 5.34, quin (1.5) 2.03, t (1.5) 3.97, s

Interchangeable.

tively. The 1H NMR spectrum (Table 1) showed resonances for one olefinic proton of a trisubstituted alkene (δH 6.28, dd, J = 2.0 and 0.5 Hz, 1H), four oxymethine protons [δH 5.26 (brd, J = 9.0 Hz, 1H), 4.79 (dd, J = 9.0 and 8.0 Hz, 1H), 4.19 (m, 1H), and 3.58 (brt, J = 8.0 Hz, 1H)], and a 3-methyl-3-buten-1-

Aspergillusol A (1) was obtained as a colorless gum with the molecular formula C12H12O5 as determined from its HREIMS. The UV data displayed an absorption band at 244 nm, while the IR spectrum exhibited absorption bands at 3430 and 1776 cm−1 for hydroxy and carbonate carbonyl groups,13 respecB

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ynyl unit [δH 5.36 (quin, J = 1.5 Hz, 1H), 5.34 (quin, J = 1.5 Hz, 1H), and 1.90 (t, J = 1.5 Hz, 3H)]. The 13C NMR (Table 1) and DEPT spectra showed resonances for five quaternary (δC 154.1, 127.2, 116.5, 92.2, and 85.6, five methine (δC 143.2, 79.1, 75.9, 73.9, and 69.6), one methylene (δC 123.4) and one methyl (δC 23.2) carbons. The 3-methyl-3-buten-1-ynyl unit was established on the basis of the 1H−1H COSY correlations between Hab-4′ (δH 5.36 and 5.34) and H3-5′ (δH 1.90) and the HMBC correlations of both Hab-4′ and H3-5′ to C-2′ (δC 92.2) and C-3′ (δC 127.2) (Figure 2) as well as the chemical shifts of Figure 3. Possible biosynthetic pathway for compound 1.

identical to that of 1. Their UV, IR, and 1H and 13C NMR spectroscopic data were similar. The methine proton resonances at δH 5.15 (ddd, J = 7.8, 3.9, and 2.1 Hz, 1H) and 4.64 (dd, J = 8.7 and 7.8 Hz, 1H) (Table 1) were attributed to H-3 and H-4, respectively, on the basis of the 1H−1H COSY correlations from H-3 to H-2 (δH 6.06, dd, J = 3.9 and 2.1 Hz) and H-4. The HMBC correlation from H-4 to C-6′ (δC 153.4) together with the relatively deshielded chemical shifts of H-3 and H-4 established a carbonate moiety at C-3 (δC 72.8) and C4 (δC 77.3). In addition, the substituents at C-5 (δC 73.0) and C-6 (δC 69.0) were identified as hydroxy groups according to the chemical shifts. The methine protons H-4, H-5, and H-6 were located at pseudoaxial positions on the basis of the relatively large coupling constant of 8.7 Hz among these protons. Irradiation of H-4 affected the signal intensity of H-6, but not H-5, in the NOEDIFF experiment, supporting the above conclusion. The coupling constant of 7.8 Hz between H3 and H-4 established their cis relationship.13−15 The absolute configurations at C-3, C-5, and C-6 were proposed to be identical to those in 8, as 2 would be obtained from 8 by a similar biosynthetic pathway to 1. Consequently, 2 was identified as a new carbonate derivative of 8, and its absolute configuration was assigned as 3R, 4S, 5R, and 6S. Aspergillusic acid (3) was obtained as a colorless gum with the molecular formula C13H13O3 as determined from its HREIMS. The UV spectrum displayed absorption bands at λmax 239, 248, and 304 nm, while the IR absorption bands for hydroxy and carboxy groups were observed at 3285 and 1677 cm−1, respectively. The 1H NMR spectrum (Table 1) showed characteristic resonances for three aromatic protons of a 1,2,4trisubstituted benzene [δH 8.16 (d, J = 2.0 Hz), 8.03 (dd, J = 8.5 and 2.0 Hz), and 6.94 (d, J = 8.5 Hz), each 1H], a 3-methyl-3buten-1-ynyl unit [δH 5.46 (d, J = 1.0 Hz, 1H), 5.34 (quin, J = 1.5 Hz, 1H), and 2.03 (t, J = 1.5 Hz, 3H)], and a methoxy group at δH 3.97 (s, 3H). These spectroscopic data indicated that 3 differed from 9 by the presence of a methoxy group. The appearance of a methoxy carbon at δC 56.1 in the 13C NMR spectrum supported the 1H NMR data. As H3-7′ displayed a 3J HMBC correlation with C-2 (δC 164.0), the methoxy group was attached at this carbon. Therefore, 3 was determined as a new methyl ether derivative of the known natural product eutypinic acid.10 Aspergillussanone A (4) was obtained as a pale yellow gum with the molecular formula C31H40O8 as deduced from its HREIMS, revealing 12 degrees of unsaturation. The UV spectrum displayed absorption bands at 218, 273, 295, 360, and 384 nm. The IR spectrum exhibited an absorption band at 3450 cm−1 for a hydroxy group. The 1H NMR spectrum (Table 2) displayed resonances for one aromatic proton of a pentasub-

Figure 2. Selected HMBC correlations of compounds 1 and 4.

C-1′ (δC 85.6) and C-2′. The ene-yne unit was attached at C-1 (δC 116.5) from the 3J HMBC correlation between H-6 (δH 5.26) and C-1′. The NMR data of 1 were similar to those of 8. However, 1 had an additional resonance for a carbonate carbonyl carbon at δC 154.1,13 which was only observed in the HMBC spectrum. The methine proton resonances at δH 5.26 and 4.19 were attributed to H-6 and H-3, respectively, on the basis of the HMBC correlations from H-6 to C-1, C-2 (δC 143.2), and C-1′ and those from H-3 to C-1 and C-2. The remaining methine protons resonating at δH 3.58 and 4.79 were assigned as H-4 and H-5 on the basis of the 1H−1H COSY correlations from H-4 to H-3 and H-5 and those from H-5 to H-4 and H-6. The HMBC correlation from H-5 to C-6′ (δC 154.1) together with the relatively deshielded chemical shifts of H-5 and H-6 established a carbonate moiety at C-5 (δC 79.1) and C-6 (δC 75.9). The substituents at C-3 (δC 69.6) and C-4 (δC 73.9) were identified as hydroxy groups according to the chemical shifts. The hydroxy proton at δH 5.13 belonged to the 4-OH due to its 1H−1H COSY cross-peak with H-4. Since H-4 was coupled with H-3 and H-5 with a relatively large coupling constant of 8.0 Hz, they were located at pseudoaxial positions. This assignment was confirmed by signal enhancement of H-5, but not H-4, upon irradiation of H-3 in the NOEDIFF experiment. The cis relationship between H-5 and H-6 was established according to signal enhancement of H-5 after irradiation of H-6. This conclusion was confirmed by the coupling constant of 8.3 Hz between H-5 and H-6, which was similar to that observed in phomoxin,13 theobroxide derivatives,14 and phomoxins B and C.15 Therefore, 1 contained a rare carbonate functionality and was a carbonate derivative of 8. The absolute configurations at C-3, C-4, and C-6 were proposed to be identical to those in the co-metabolite 8. The biosynthetic pathway of 1 could involve ring opening of the epoxy moiety in 8 by a carbonate anion, derived from the carbonyl carbon of an acetate,14,16 at C-5 via trans-diaxial ring opening16 and subsequent intramolecular esterification of the resulting monocarbonate intermediate with 6-OH (Figure 3). Alternatively, the 6-OH in 1 could react with carbon dioxide followed by intramolecular epoxide ring opening to provide 1. Consequently, the absolute configuration of 1 was proposed as 3R, 4S, 5R, and 6S. Aspergillusol B (2) was obtained as a colorless gum with the molecular formula C12H12O5 as deduced from its HREIMS, C

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Table 2. 1H and 13C NMR Spectroscopic Data for 4 in CDCl3 and 5 in Acetone-d6 4 position 1 2 3 4 5 6 7 7-OH 8 9 10 11 12 13 13-OH 14 15 16 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′

δC, type 135.9, 107.8, 201.8, 88.5, 199.8, 104.7, 166.3,

C C C C C C C

106.7, 161.5, 112.2, 149.5, 118.9, 164.8,

C C C C CH C

26.4, 7.2, 55.0, 41.6, 114.5, 141.3, 39.4, 26.2, 124.8, 134.6, 36.5,

CH3 CH3 CH3 CH2 CH C CH2 CH2 CH C CH2

5 δH (J in Hz)

δC, type 130.0, 110.6, 199.2, 58.7, 199.5, 98.4, 162.9,

C C C C C C C

130.0, 172.1, 113.6, 146.1, 112.1, 167.6,

C C C C CH C

23.5, 59.5, 21.0, 38.7, 119.7, 137.0, 39.7, 26.2, 123.9, 134.9, 36.6,

CH3 CH3 CH3 CH2 CH C CH2 CH2 CH C CH2

14.34, s

9′

29.4, CH2

10′

78.0, CH

11′ 12′ 13′ 14′ 15′

73.3, 26.4, 23.2, 15.6, 15.9,

C CH3 CH3 CH3 CH3

HMBC correlations from 7-OH to C-5, C-6 (δC 104.7), C-7, and C-8 (δC 106.7) and the correlations from 13-OH to C-2 (δC 107.8), C-3, C-12 (δC 118.9), and C-13 (Figure 2). The aromatic proton resonance at δH 6.78 was identified with H-12 due to its HMQC correlation to C-12. The substituent at C-11 (δC 149.5) was assigned as a methyl group according to the HMBC correlations from the resonance of H3-14 (δH 2.86) to those of C-10 (δC 112.2), C-11, and C-12. The HMBC correlations from H3-15 (δH 2.25) to C-7, C-8, C-9 (δC 161.8), and C-10 (δC 112.2) established the attachment of a methyl group at C-8. The chemical shift of C-9 indicated that a hydroxy group was attached to this carbon. These data together with the remaining eight degrees of unsaturation, apart from four degrees of unsaturation from two carbonyl groups and two trisubstituted double bonds, established a naphthalene skeleton (seven degrees of unsaturation) with ketone carbonyl groups at C-3 and C-5. A (6E,10E)-2,6,10-trimethyldodeca-6,10-diene2,3-diol unit was established according to the HMBC correlations demonstrated in Figure 2 as well as the 1H−1H COSY correlations from H2-1′ (δH 2.76) to H-2′ (δH 4.88), from H2-5′ (δH 1.62) to H2-4′ (δH 1.71) and H-6′ (δH 4.94), and from Hab-9′ (δH 1.57 and 1.32) to Hab-8′ (δH 2.10 and 2.02) and H-10′ (δH 3.36). The substituents at C-10′ (δC 78.0) and C-11′ (δC 73.3) were hydroxy groups according to their chemical shifts. Signal enhancement of H2-1′ upon irradiation of H3-15′ revealed that the double bond at C-2′ and C-3′ had an E configuration. In addition, the configuration of the other trisubstituted double bond at C-6′ and C-7′ was assigned as E on the basis of signal enhancement of H2-5′ after irradiation of H3-14′. The HMBC correlations from H2-1′ to C-3, C-4 (δC 88.5), and C-5 and that from H3-16 (δH 3.34) to C-4 established a phenalene-1,3-dione with a methoxy group and the dodecadienediol unit attached at C-4. Consequently, 4 was a new phenalene-1,3-dione derivative. The negative Cotton effect at 271 nm (Δε −0.6) in the CD spectrum of 4, which was similar to that of sculezonones A and B,17 established the S configuration at C-4. Moreover, the absolute configuration of C-10′ in the 1,2-diol moiety was assigned using Snatzke’s method.18 The negative Cotton effect observed at 316 nm (Δε −3.0) allowed assignment of the R configuration at C-10′. Aspergillussanone B (5) was obtained as a pale yellow gum and possessed the same molecular formula as 4 from HRESIMS. The UV and IR data were almost identical to those of 4. The 1H and 13C NMR (Table 2), HMBC, and 1 H−1H COSY correlations of 5 were also similar to those of 4. These data indicated that 5 had all substituents identical to those of 4. However, 5 had a methyl group, instead of the methoxy group, at C-4 (δC 58.7) on the basis of the HMBC correlations from H3-16 (δH 1.34) to C-3 (δC 199.2), C-4, C-5 (δC 199.5), and C-1′ (δC 38.7). The more shielded chemical shift of C-4 supported the above conclusion. In addition, the substituent at C-8 was a methoxy group due to the HMBC correlations from 7-OH (δH 15.49) to C-6 (δC 98.4), C-7 (δC 162.9), and C-8 (δC 130.0) and that from H3-15 (δH 3.85) to C-8. In addition, the absolute configurations at C-4 and C-10′ of 5 were assigned to be identical to those of 4 on the basis of the negative Cotton effects at 265 (Δε −11.7) and 314 nm (Δε −3.5) of 5 and the Mo24+ complex of 5, respectively. Consequently, 5 differed from 4 in the substituents at C-4 and C-8. Aspergilluchalasin (6) was obtained as a colorless gum with the molecular formula C24H37NO5 as deduced from its HREIMS. The IR spectrum exhibited absorption bands at

6.78, s 13.31, s 2.86, s 2.25, s 3.34, s 2.76, d (7.8) 4.88, t (7.8) 1.71, t (6.9) 1.62, m 4.94, brt (7.8) a: 2.10, m b: 2.02, m a: 1.57, m b: 1.32, m 3.36, dd (10.5, 1.5) 1.21, 1.18, 1.47, 1.37,

s s s s

δH (J in Hz)

15.49, s

29.8, CH2 77.6, CH 72.2, 25.3, 23.9, 15.1, 15.4,

C CH3 CH3 CH3 CH3

6.24, s

2.61, 3.85, 1.34, 2.57, 4.94,

s s s d (7.8) t (7.8)

1.79, m 1.78, m 4.97, m a: 2.09, m b: 1.86, m a: 1.59, m b: 1.29, m 3.25, dd (10.5, 1.8) 1.13, 1.12, 1.48, 1.52,

s s s s

stituted benzene (δH 6.78, s, 1H), two chelated hydroxy protons (δH 14.34 and 13.31, each, s, 1H), two sets of nonequivalent methylene protons [δH 2.10 (m, 1H)/2.02 (m, 1H) and 1.57 (m, 1H)/1.32 (m, 1H)], three sets of equivalent methylene protons [δH 2.76 (d, J = 7.8 Hz, 2H), 1.71 (t, J = 6.9 Hz, 2H), and 1.62 (m, 2H)], two olefinic protons of two trisubstituted alkenes [δH 4.94 (brt, J = 7.8 Hz, 1H) and 4.88 (t, J = 7.8 Hz, 1H)], one methoxy group (δH 3.34, s, 3H), one methine proton (δH 3.36, dd, J = 10.5 and 1.5 Hz, 1H), and six methyl groups (δH 2.86, 2.25, 1.47, 1.37, 1.18, and 1.21, each, s, 3H). The 13C NMR spectrum (Table 2) showed resonances for two ketone carbonyls (δC 201.8 and 199.8) and 13 quaternary (δC 166.3, 164.8, 161.5, 149.5, 141.3, 135.9, 134.6, 112.2, 107.8, 106.7, 104.7, 88.5, and 73.3), four methine (δC 124.8, 118.9, 114.5, and 78.0), five methylene (δC 41.6, 39.4, 36.5, 29.4, and 26.2), and six methyl (δC 55.0, 26.4, 23.2, 15.9, 15.6, and 7.2) carbons. Two chelated hydroxy protons with resonances at δH 14.34 (7-OH) and 13.31 (13-OH) were sequentially placed at C-7 (δC 166.31) and C-13 (δC 164.77), respectively, which were at peri-positions to the carbonyl groups resonating at δC 199.8 (C-5) and 201.8 (C-3), respectively, on the basis of the D

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3348, 1702, and 1686 cm−1 for hydroxy, ketone carbonyl, and amide carbonyl groups, respectively. The 1H and 13C NMR spectra (Table 3) consisted of signals for a pentahydro-3Table 3. 1H and 13C NMR and HMBC Data for 6 in CDCl3 position

δC, type

1 2-NH 3 4

173.6, C

5 6 7 8 9 10

35.1, 139.9, 127.1, 36.4, 64.1, 47.4,

CH C CH CH C CH2

11 12 13 14 15

13.6, 20.2, 38.9, 82.7, 42.6,

CH3 CH3 CH C CH2

16

35.2, CH2

17

18 19 20 21 22 23 24 25

51.8, CH 51.8, CH

24.7, CH2

66.7, 42.6, 84.2, 210.5, 25.3, 23.8, 21.2, 23.3,

CH CH CH C CH CH3 CH3 CH3

δH (J in Hz)

HMBC

6.22, brs 3.10, dt (10.5, 3.3) 2.66, t (4.5) 2.37, m

C-3, C-4, C-9 C-1, C-4, C-5, C-9, C-22 C-1, C-3, C-5, C-6, C-8, C-9, C-10, C-11, C-21 C-3, C-4, C-6, C-9

5.43, s 2.42, m

C-12 C-1, C-13, C-19, C-21

Figure 4. Selected 1H−1H COSY (−) and NOEDIFF (↔) data of 6.

a: 1.72, m b: 1.31, ddd (13.5, 10.2, 3.3) 1.16, d (7.2) 1.78, d (1.3) 2.99, m

C-3, C-4, C-22, C-23, C-24 C-3, C-4, C-22, C-23, C-24

a: 2.96, m b: 2.55, m a: 1.91, td (13.2, 4.5) b: 1.44, dd (13.2, 5.1) a: 2.04, ddd (13.2, 3.6, 1.5) b: 1.72, m 3.57, brs 2.56, m 3.72, s

C-13, C-14, C-16, C-17, C-25 C-13, C-14, C-16, C-17, C-25 C-14, C-15, C-17, C-18

1.57, 0.95, 0.92, 1.23,

C-3, C-10, C-23, C-24 C-10, C-22, C-24 C-10, C-22, C-23 C-13, C-14, C-15

between C-8 (δC 36.4) and C-13 was established on the basis of the HMBC correlations from H-13 to C-7 (δC 127.1) and C-8 to form a fused tetracyclic ring. The relative configuration of 6 was established by the following NOEDIFF data (Figure 4). Irradiation of H-4 affected the signal intensity of H-5 (δH 2.37) and H-8, suggesting their cis relationship. In addition, irradiation of H-3 (δH 3.10) enhanced the signal intensity of H3-11 (δH 1.16), indicating their cis relationship and trans orientation to H-4, H-5, and H-8. Upon irradiation of H-13, the signal intensities of H-8, H-18, and H-20 were enhanced, indicating the cis relationship of H-8, H-13, H-18, and H-20 and their trans orientation to H-19 and H3-25. In addition, the assigned location of H3-25 was further confirmed by its signal enhancement upon irradiation of H-7. From these results, the relative configuration of the isoindolone unit and C-18 in 6 was identical to that of 10. In general, the biosynthesis of the cytochalasans involves the formation of a polyketide chain and the attachment of an amino acid such as leucine for aspochalasins.19 The absolute configuration at C-3 in 10, derived from the α-carbon of leucine, was assigned as S. As both 6 and 10 would be constructed from leucine, C-3 in 6 would also have the S configuration. Consequently, the remaining absolute configurations were proposed to be 4R, 5S, 8R, 9S, 13S, 14S, 18S, 19S, and 20S. Therefore, 6 was a new tetracyclic cytochalasin derivative. Aspergillulactone (7) was obtained as a colorless gum with the molecular formula C29H29O7 as determined from its HREIMS, which requires 15 degrees of unsaturation. The UV spectrum displayed absorption bands at 224, 269, 277, and 306 nm. The IR spectrum exhibited absorption bands at 3369 and 1736 cm−1 for hydroxy and γ-lactone carbonyl groups, respectively. The 1H NMR spectrum (Table 4) displayed resonances for four aromatic protons of a 1,4-disubstituted benzene (δH 7.53 and 6.85, each d, J = 9.0 Hz, 2H), two metacoupled aromatic protons (δH 6.30 and 6.28, each d, J = 2.0 Hz, 2H), two cis-olefinic protons (δH 6.05 and 5.45, each d, J = 10.0 Hz, 1H), one set of nonequivalent methylene protons (δH 3.44 and 3.37, each d, J = 14.5 Hz, 1H), one methoxy group (δH 3.71, s, 3H), two methyl groups (δH 1.28, s, 6H), and one prenyl group [δH 4.97 (brt, J = 7.5 Hz, 1H), 3.00 (d, J = 7.5 Hz, 2H), 1.60 (s, 3H), and 1.55 (s, 3H)]. The 13C NMR (Table 4) and HMBC spectra indicated resonances for one conjugated γlactone (δC 169.8), one ester carbonyl (δC 169.7), 11 quaternary (δC 156.3, 149.6, 137.0, 132.0, 128.5, 127.2, 124.1, 122.5, 120.6, 85.9, and 76.0), nine methine [δC 131.2, 130.4, 129.6 (×2), 126.1, 122.6, 122.5, and 115.9 (×2)], two methylene (δC 38.7 and 27.9), and five methyl (δC 53.5, 27.8

m d (6.6) d (6.6) s

C-4, C-5, C-6 C-5, C-6, C-7 C-7, C-8, C-15, C-19, C-25

C-14, C-15, C-17, C-18 C-15, C-16, C-18, C-19 C-15, C-16, C-13, C-13,

C-16, C-18, C-19 C-20 C-17, C-18, C-20, C-21 C-18, C-19, C-21

isobutyl-4,5-dimethylisoindol-1-one unit, which were similar to those of 10.11 The 1H NMR spectrum also displayed resonances for three sets of nonequivalent methylene protons [δH 2.96 (m, 1H)/2.55 (m, 1H), 2.04 (ddd, J = 13.2, 3.6, and 1.5 Hz, 1H)/1.72 (m, 1H), and 1.91 (td, J = 13.2 and 4.5 Hz, 1H)/1.44 (dd, J = 13.2 and 5.1 Hz, 1H)], four methine protons [δH 3.72 (s, 1H), 3.57 (brs, 1H), 2.99 (m, 1H), and 2.56 (m, 1H)], and one methyl group (δH 1.23, s, 3H). A 2,3disubstituted 1-methylcycloheptane-1,4-diol was constructed on the basis of the following correlations in the 1H−1H COSY spectrum: Hab-16 (δH 1.91 and 1.44) to Hab-15 (δH 2.96 and 2.55) and Hab-17 (δH 2.04 and 1.72); H-18 (δH 3.57) to Hab-17 and H-19 (δH 2.56); and H-19 to H-13 (δH 2.99) (Figure 4) and the HMBC correlations from H3-25 (δH 1.23) to C-13 (δC 38.9), C-14 (δC 82.7), and C-15 (δC 42.6) (Table 3) as well as the chemical shifts of C-14 and C-18 (δC 66.7). In addition, H19 gave a 1H−1H COSY cross-peak with H-20 (δH 3.72) and a HMBC correlation with the ketone carbonyl carbon at δC 201.5 (C-21). These results together with the chemical shift of C-20 (δC 84.2) thus connected the hydroxymethine carbon of a −CH(OH)CO− unit with C-19. The 3J HMBC correlations from both H-4 (δH 2.66) and H-8 (δH 2.42) of the isoindolone unit with C-21 connected C-9 with C-21. The connection E

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Table 4. 1H and 13C NMR and HMBC Data for 7 in CDCl3 position

δC, type

1 2-OH 2 3 4 5

169.8, C 137.0, 127.2, 85.9, 38.7,

C C C CH2

6 7 1′ 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 11″ 12″ 13″ 14″ 15″, 16″

169.7, 53.5, 122.5, 129.6, 115.9, 156.3, 124.1, 126.1, 120.6, 149.6, 128.5, 131.2, 27.9, 122.5, 132.0, 25.7, 17.7, 122.6, 130.4, 76.0, 27.8,

C CH3 C CH CH C C CH C C C CH CH2 CH C CH3 CH3 CH CH C CH3

δH (J in Hz)

C-1, C-2, C-3

C-3, C-4, C-6, C-1′, C-1″, C-2″, C-6″ C-3, C-4, C-6, C-1′, C-1″, C-2″, C-6″

3.71, s

C-6

7.53, d (9.0) 6.85, d (9.0)

C-3, C-1′, C-4′ C-1′, C-4′

6.28, d (2.0)

C-5, C-4″, C-6″, C-12″

6.30, d (2.0) 3.00, d (7.5) 4.97, brt (7.5)

C-5, C-2″, C-4″, C-7″ C-4″, C-5″, C-6″, C-8″, C-9″ C-7″, C-10″, C-11″

1.60, 1.55, 6.05, 5.45,

C-8″, C-8″, C-2″, C-3″,

s s d (10.0) d (10.0)

1.28, s

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 polarimeter. The ultraviolet (UV) absorption spectra were measured in MeOH on a PerkinElmer Lambda 45 spectrophotometer. The infrared (IR) spectra were recorded neat using a PerkinElmer 783 FTS165 FT-IR spectrometer. 1 H and 13C NMR spectra were recorded on a 300 or 500 MHz Bruker FTNMR Ultra Shield spectrometer. Chemical shifts are expressed in δ (ppm) and are referenced to tetramethylsilane. Mass data were obtained on a MAT 95 XL mass spectrometer (ThermoFinnigan) except for those of compounds 3, 5, and 7, which were recorded on a Bruker MicrOTOF mass spectrometer. Thin-layer chromatography (TLC) and preparative TLC were performed on silica gel 60 GF254 (Merck). Column chromatography was carried out on Sephadex LH20 with MeOH, on silica gel (Merck) type 60 (230−400 mesh ASTM) or type 100 (70−230 mesh ASTM), or on reversed-phase C18 silica gel. Petroleum ether has a bp of 40−60 °C. Fungal Materials. The soil fungus PSU-RSPG185 was isolated from soil samples collected from Surat Thani Province, Thailand. This fungus was deposited as PSU-RSPG185 (BCC56854) at Biotec Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. PSU-RSPG185 was identified based on morphology and the internal transcribed spacer (ITS1-5.8S-ITS2) rDNA analysis using universal fungal primers.22 Colonies on potato dextrose agar (PDA) at 25 °C grew slowly, granular to rather floccose. Colony color is yellow with a white colonial edge. Conidiophores are upright, simple, terminating in a globose conidial head bearing phialides, which are the characteristics of the genus Aspergillus.23 From the maximum parsimonious tree, PSURSPG185 (GenBank accession no. KC478518) formed a sister group to unknown species of Aspergillus (Aspergillus sp. HQ288052, Aspergillus sp. DQ092528, Aspergillus sp. HQ832962, and Aspergillus sp. EF669595) and had no closely related species with moderate bootstrap support (70%). Therefore, it can be named as an Aspergillus sp. Extraction and Isolation. The extracts obtained from the culture broth and mycelia of the fungus PSU-RSPG185 were prepared using the same procedure as described for Fusarium spp. PSU-F14 and PSUF135. The broth extract (1.6 g) was subjected to column chromatography (CC) over Sephadex LH-20 using MeOH as a mobile phase to afford four fractions (A−D). Fraction B (516.3 mg) was dissolved in CHCl3 to give a soluble part (fraction B2). Fraction B2 (440.0 mg) was fractionated by CC over silica gel using a gradient of acetone−CH2Cl2 (1:19 to 1:0) as a mobile phase to provide four fractions (B2A−B2H). Fraction B2C (12.7 mg) was purified by preparative TLC using EtOAc−petroleum ether (1:1) (2 runs) as a mobile phase to afford 2 (2.3 mg). Fraction B2D (17.5 mg) was subjected to CC over silica gel using a gradient of MeOH−CH2Cl2 (1:49 to 1:1) to give four fractions. The second fraction (4.0 mg) was further purified by preparative TLC using EtOAc−petroleum ether (3:7) as a mobile phase to afford 8 (1.3 mg) and 10 (1.1 mg). Compound 1 (1.2 mg) was obtained from the third fraction after purification by preparative TLC using acetone−CH2Cl2 (1:19) (9 runs). Fraction B2E (104.6 mg) was fractionated by CC over Sephadex LH-20 using MeOH−CH2Cl2 (1:1) to afford three fractions. The second fraction (71.1 mg) was purified by CC over Sephadex LH20 using a mixture of MeOH−CH2Cl2−EtOH in a ratio of 10:10:1 followed by preparative TLC using EtOAc−CH2Cl2 (1:9) (9 runs) to give 11 (1.1 mg). Fraction B2F (133.6 mg) was subjected to CC over reversed-phase C18 silica gel using a gradient of MeOH−H2O (1:1 to 1:0) as a mobile phase followed by preparative TLC using acetone− petroleum ether (3:17) (3 runs) to provide 3 (1.1 mg) and 12 (1.3 mg). Fraction B2G (133.4 mg) was purified using the same procedure as fraction B2F to afford six fractions. The third fraction (27.9 mg) was purified using the same procedure as fraction B2E to afford 9 (10.6 mg) and 13 (1.2 mg). The mycelial extract (3.5 g) was separated by CC over Sephadex LH-20 using MeOH to afford five fractions (CA− CE). Fraction CB (791.0 mg) was subjected to CC over Sephadex LH20 using MeOH−CH2Cl2 (1:1) to give three fractions. The second

HMBC

a: 3.44, d (14.5) b: 3.37, d (14.5)

Article

C-9″, C-11″ C-9″, C-10″ C-3″, C-4″, C-14″ C-14″, C-15″, C-16″

C-13″, C-14″

(×2), 25.7, and 17.7) carbons. A detailed comparison of the NMR data of 7 with those of butyrolactone 120 and aspernolide E21 indicated the presence of the aromatic butenolide moiety in 7. The significant difference observed in the 1H NMR spectrum was the absence of the resonance for H-5″ (δH 6.52, d, J = 8.0 Hz) found in the spectrum of aspernolide E due to the C-5″ prenyl substituent. The prenyl group was established on the basis of the 1H−1H COSY correlations between the resonances for H2-7″ (δH 3.00) and H-8″ (δH 4.97) and the HMBC correlations of the resonances of H3-10″ (δH 1.60) and H3-11″ (δH 1.55) with both C-8″ (δC 122.5) and C-9″ (δC 132.0) (Table 4). As H2-7″ of the prenyl substituent displayed HMBC correlations with C-4″ (δC 149.6), C-5″ (δC 128.5), and C-6″ (δC 131.2), this group was attached at C-5″ of the benzopyran unit. The absolute configuration at C-4 of the γ-butyrolactone unit was assigned as R, as 7 had a positive optical rotation, the same sign as that of the related natural γ-butyrolactones.20,21 Consequently, 7 was the 5″-prenyl derivative of aspernolide E. The broth extract exhibited cytotoxicity toward KB cell lines with an IC50 value of 33.1 μg/mL, while the ethyl acetate extract from the mycelia had an IC50 value of 18.6 μg/mL. Compounds 4−6 and 9, which were obtained in sufficient amounts, were tested for cytotoxic activity against the same KB and Vero cell lines. Only compound 4 exhibited weak activity toward KB and Vero cells with IC50 values of 48.4 and 34.2 μM, respectively. The remaining compounds were inactive against the tested cell lines. In addition, none of them showed antimalarial (against Plasmodium falciparum, K1 strain) or antimycobacterial (against Mycobacterium tuberculosis) activities. F

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(GFP)-based fluorescent detection.26 Isoniazid, the standard drug, displayed a MIC value of 0.094 μg/mL. Antimalarial Assay. The activity was evaluated against the parasite P. falciparum (K1, multi-drug-resistant strain), using the microculture radioisotope technique based on the method described.27 Dihydroartemisine, the standard drug, displayed an IC50 value of 2.23 nM.

fraction (564.4 mg) was purified by CC over silica gel using a gradient of MeOH−CH2Cl2 (1:99 to 1:0) to afford 4 (20.4 mg) and 6 (3.0 mg). Fraction CC (1717.9 mg) was purified using the same procedure as fraction CB to afford five fractions. The fourth fraction (1016.6 mg) was purified using CC over silica gel using a gradient of MeOH− CH2Cl2 (1:99 to 1:0) to provide 10 fractions. The fifth fraction (113.0 mg) was subjected by CC over silica gel using a gradient of MeOH− CH2Cl2 (1:49 to 1:0) to afford five fractions. The first fraction (7.0 mg) was further purified by preparative TLC using acetone−CH2Cl2 (1:19) (5 runs) as a mobile phase to give 7 (1.2 mg). Fraction CD (615.7 mg) was purified using the same procedure as fraction B2F to provide three fractions. The second fraction was purified by CC over reversed-phase C18 silica gel using a gradient of MeOH−CH2Cl2 followed by CC over Sephadex LH-20 using MeOH−CH2Cl2 (1:1) to give 5 (153.8 mg). Aspergillusol A (1): colorless gum; [α]25D −4.6 (c 0.49, MeOH); UV (MeOH) λmax (log ε) 206 (3.52), 244 (3.91) nm; IR (neat) νmax 3430, 1776 cm−1; 1H and 13C NMR see Table 1; HREIMS [M]+ m/z 236.0679 (calcd C12H12O5, 236.0679). Aspergillusol B (2): colorless gum; UV (MeOH) λmax (log ε) 206 (3.52), 244 (3.91) nm; IR (neat) νmax 3430, 1776 cm−1; 1H and 13C NMR see Table 1; HREIMS [M]+ m/z 236.0678 (calcd C12H12O5, 236.0679). Aspergillusic acid (3): colorless gum; UV (MeOH) λmax (log ε) 239 (3.23), 248 (3.21), 304 (2.64) nm; IR (neat) νmax 3285, 1677 cm−1; 1H and 13C NMR see Table 1; HRESIMS m/z [M − H]+ 215.0714 (calcd C13H13O3, 215.0708). Aspergillusanone A (4): pale yellow gum; [α]25D −6.5 (c 1.00, MeOH); UV (MeOH) λmax (log ε) 218 (3.30), 273 (2.93), 295 (2.45), 360 (2.88), 384 (2.61) nm; CD (c 8.87 × 10−5 mol/L, EtOH) Δε −0.6 (271 nm), +1.2 (280 nm), −1.4 (313 nm), +5.4 (334 nm), −3.3 (382 nm); IR (neat) νmax 3450, 1700, 1688 cm−1; 1H and 13C NMR see Table 2; HREIMS [M]+ m/z 540.2720 (calcd C31H40O8, 540.2718). Aspergillusanone B (5): pale yellow gum; [α]25D +20.4 (c 1.00, MeOH); λmax (log ε) 217 (3.28), 271 (2.78), 293 (2.54), 360 (2.98), 384 (2.56) nm; CD (c 8.87 × 10−5 mol/L, EtOH) Δε −11.7 (265 nm), +1.4 (284 nm), +1.4 (313 nm), −8.8 (330 nm), +5.0 (396 nm); IR (neat) νmax 3447, 1710, 1688 cm−1; 1H and 13C NMR see Table 2; HRESIMS m/z 563.2621 (calcd C31H40O8Na, 563.2621). Aspergilluchalasin (6): colorless gum; [α]25D +63.3 (c 1.00, MeOH); UV (MeOH) λmax (log ε) 205 (4.70), 256 (1.81) nm; IR (neat) νmax 3348, 1702, 1686 cm−1; 1H and 13C NMR see Table 3; HREIMS [M]+ m/z 401.2567 (calcd C24H37NO5, 401.2561). Aspergillulactone (7): colorless gum; [α]25D −12.8 (c 1.00, MeOH); UV (MeOH) λmax (log ε) 224 (3.23), 269 (2.68), 277 (2.73), 306 (2.92) nm; IR (neat) νmax 3369, 1736 cm−1; 1H and 13C NMR see Table 4; HRESIMS m/z [M − H]− 489.1919 (calcd C29H29O7, 489.1913). (+)-Asperpentyn (8): [α]25D +25.4 (c 0.50, CHCl3); [lit. [α]20D +24.2 (c 0.50, CHCl3).8 Determination of the Absolute Configuration of C-10′ in 4 and 5. According to the published procedure,18 a mixture of 4 (1.5 mg) or 5 (1.5 mg) with Mo2(OAc)4 (1.2 mg) in AR grade DMSO was subjected to CD measurement. The mixture was kept for 30 min to form a stable chiral metal complex, after which the CD spectrum was recorded. The observed sign of the diagnostic band at around 310 nm in the induced CD spectrum was correlated to the absolute configuration of C-10′ in 4 and 5. Compound 4: CD (c 1.70 × 10−3 mol/L, DMSO) Δε −3.0 (316 nm). Compound 5: CD (c 1.70 × 10−3 mol/L, DMSO) Δε −3.5 (314 nm). Cytotoxicity Assay. Cytotoxic activity against Vero cells was assessed employing the colorimetric method,24 while that against KB cells was conducted using the method described by O’Brien and coworkers.25 The standard compound for Vero and KB cell lines was ellipticine, exhibiting IC50 values of 4.5 and 4.1 μM against Vero and KB cell lines, respectively. Antimycobacterial Assay. Antimycobacterial activity was assessed against M. tuberculosis H37Ra using green fluorescent protein

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra for aspergillusols A (1) and B (2), aspergillusic acid (3), aspergillussanones A (4) and B (5), aspergilluchalasin (6), and aspergillulactone (7) are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(V. Rukachaisirikul) Tel: +66-74-288-435. Fax: +66-74-558841. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS V.R. thanks the Thailand Research Fund (TRF) for the TRF Senior Research Scholar (Grant No. RTA5480002). N.R. thanks the Science, Mathematics and Technology Excellence Development Project, Teacher Professional Development Project (TPDP), for a scholarship. The Center of Excellence for Innovation in Chemistry (PERCH−CIC), the Office of Higher Education Commission, and Prince of Songkla University are acknowledged for partial support. We thank Dr. P. Pittayakhajonwut for HRESIMS data. Finally, the National Center for Genetic Engineering and Biotechnology (BIOTEC) is acknowledged for antimycobacterial, antimalarial, anticancer, and cytotoxic assays.

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REFERENCES

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