Eremophilane Sesquiterpenes and Polyketones Produced by an

Aug 4, 2015 - A cultured endophytic fungus, Guignardia mangiferae, isolated from the toxic plant Gelsemium elegans yielded five new sesquiterpenes (1â...
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Eremophilane Sesquiterpenes and Polyketones Produced by an Endophytic Guignardia Fungus from the Toxic Plant Gelsemium elegans Yunbao Liu, Yong Li, Jing Qu, Shuanggang Ma, Caixia Zang, Yutian Zhang, and Shishan Yu* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: A cultured endophytic fungus, Guignardia mangiferae, isolated from the toxic plant Gelsemium elegans yielded five new sesquiterpenes (1−5), two new polyketones (6 and 7), and two known terpene polyketones (8 and 9). Their structures were elucidated using spectroscopic methods. On the basis of circular dichroism, the absolute configurations of the new compounds were determined. Compounds 1, 3, 4, and 9 inhibited lipopolysaccharide-induced NO production in BV2 cells with IC50 values of 15.2, 6.4, 4.2, and 4.5 μM, respectively (positive control curcumin, IC50 = 3.9 μM).

M

Bel-7402, BGC-823, A549, and A2780 cell lines. Herein, we report the structural elucidation and bioactivity of 1−9.

icroglia are a type of glial cell that are the resident macrophages of the central nervous system (CNS). Activation of microglia plays a critical role in the neural inflammatory process by releasing a variety of inflammatory mediators including nitric oxide (NO).1,2 Thus, secondary metabolites that inhibit NO production may have antineuroinflammatory effects. Gelsemium elegans Benth., historically used in traditional Chinese medicine as an analgesic and antispasmodic and as a remedy for certain kinds of skin ulcers,3 can be lethal to humans and livestock due to its toxic alkaloid content.4 We have previously discovered some bisindole alkaloids with potential antineuroinflammatory activity by suppressing lipopolysaccharide (LPS)-induced NO production in BV2 microglial cells from G. elegans.2 We have continued this research program on the discovery of bioactive secondary metabolites with NO production inhibition from endophytic fungi associated with the toxic plant G. elegans. Guignardia mangiferae is an endophytic fungus isolated from the leaves of G. elegans. A series of bioactive secondary metabolites including the cytotoxic tricycloalternarenes,5,6 the cytotoxic vermistatin derivatives,7 and the phytotoxic dioxolanone-type secondary metabolites8 have been reported from Guignardia fungi. In our study, five new eremophilane sesquiterpenes named guignarderemophilanes A−E (1−5), two new benzyl derivatives named guignardene A (6) and guignarlactone A (7), and two known metabolites (8 and 9) were isolated from G. mangiferae. The compounds obtained were assayed for their inhibitory activity on NO production. Compounds 1, 3, 4, and 9 inhibited LPS-induced NO production in BV2 cells with IC50 values of 15.2, 6.4, 4.2, and 4.5 μM, respectively (positive control curcumin, IC50 = 3.9 μM). None of the compounds were active against the HCT-8, HCT-116, © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The fungus G. mangiferae was cultured in potato dextrose agar (PDA) medium (100 L). The filtrate of the culture was loaded onto a macroporous resin column and eluted with water and 95% alcohol, respectively. The alcohol elution, after concentration, was fractionated by chromatography, and compounds were purified by preparative HPLC, to afford compounds 1−9. Compound 1 was obtained as a colorless oil. Its molecular formula, C14H18O5, was determined by HR-ESIMS. The IR Received: November 12, 2014

A

DOI: 10.1021/np5009027 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR and 13C NMR Data of Compounds 1−5 1a

2a

3b

4b

5b

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

37.9, CH2

2.84, t (12.0)

76.3, CH

4.22, d (3.0)

70.1, CH

4.47, dd (10.2, 1.8)

70.1, CH

4.43, dd (10.2, 1.8)

128.6, CH

2

72.3, CH

71.6, CH

3.39, dd (10.2, 3.0) 3.85, t (3.0)

77.6, CH

3.39, dd (10.2, 3.6) 3.85, t (3.0)

202.2, C

75.1, CH

3.65, dt (7.8, 3.0) 1.95, overlap

77.4, CH

3

2.58, dd (12.0, 4.2) 3.74, d (12.0) 5.24, brs

1.50, m

44.7, CH 43.0, C 43.6, CH2

no. 1

34.6, CH2

75.6, CH

75.6, CH

42.7, CH2

1.55, m 4 5 6

41.8, CH 43.9, C 124.0, CH

1.72, m 6.23, s

41.5, CH 38.9, C 43.8, CH2

1.50, m 1.95, overlap

45.8, CH 40.8, C 44.3, CH2

1.86, m 7

147.4, C

8 9

182.0, C 124.2, CH

10 11 12

168.6, C

51.7, CH

6.26, s

166.7, C 145.1, C 113.7, CH2

13 14 15 3-CH3CO 3-CH3CO

199.0, C 128.4, CH

20.4, CH3 21.9, CH3 12.6, CH3 21.0, CH3

1.34, s 1.14, d (7.1) 2.14, s

18.1, CH3 15.1, CH3

3.27, dd (15.0, 4.8) 5.78, s

4.86, dd (3.0, 1.2) 4.78, dd (3.0, 0.8) 1.68, dd (1.2, 0.8) 1.33, s 0.97, d (6.0)

51.3, CH 201.8, C 122.0, CH

172.6, C 144.9, C 114.7, CH2

1.99, dd (12.0, 4.8) 1.88, dd (12.0, 9.0) 3.23, dd (15.0, 4.8)

1.56, m 2.97, d (13.8) 2.11, d (13.8)

145.6, C

6.19, d (1.8)

194.2, C 124.2, CH

4.93, t (1.8)

171.2, C 128.6, C 22.8, CH3

41.4, CH 40.3, C 42.8, CH2

δH (J in Hz) 5.78, s

2.57, dd (12.4, 4.8) 2.12, dd (12.4, 3.6) 1.96, m 2.60, dd (13.8, 12.4) 2.51, m

50.0, CH

2.42, m

6.19, s

72.1, CH 43.7, CH2

3.56 1.86, dd (15.0, 13.8) 1.31, m

2.07, s

171.8, C 147.3, C 111.5, CH2

4.81, brs

4.88, s 4.62, s

20.2, CH3

1.69, s

22.4, CH3

1.88, s

23.0, CH3

1.73, s

19.7, CH3 11.8, CH3

1.41, s 1.13, d (6.6)

20.1, CH3 12.1, CH3

1.18, s 1.16, d (6.0)

20.0, CH3 17.4, CH3

1.17, s 0.97, d (7.2)

170.9, C

a

Data were recorded at 600 MHz for proton and at 150 MHz for carbon in acetone-d6. bData were recorded at 600 MHz for proton and at 150 MHz for carbon in methanaol-d4.

spectrum showed absorption bands for hydroxy (3402 cm−1), enone carbonyl (1676 cm−1), and ester carbonyl groups (1731 cm−1). The 1H NMR spectrum (Table 1) indicated the presence of three methyl signals at δH 1.14 (d, J = 7.1 Hz), 1.34 (s), and 2.14 (s) and two olefinic signals at δH 6.23 (s) and 6.26 (s). The 13C NMR spectrum (Table 1) displayed 14 signals including two carbonyls (δC 182.0, 170.9), four olefinic carbons (δC 124.0, 124.2, 147.4, 168.6), three methyls (δC 21.9, 12.6, 21.0), one methylene (δC 37.9), three methines (two of which were oxygenated: δC 72.3, 75.1, 41.8), and one quaternary carbon (δC 43.9). Two double bonds and two carbonyl groups accounted for four of six degrees of unsaturation, indicating the presence of two rings in the structure. The two ring systems were then established based on 1H−1H COSY, HSQC, and HMBC correlations (Figure 1). Inspection of the 1H−1H COSY and HSQC data led to the assignment of a C(1)H2−C(2)H−C(3)H−C(4)H− C(15)H3 unit. The assigned spin system, together with HMBC correlations from H2-1 to C-3/C-5, from H-2 to C-4/C-10, from H-3 to C-1/C-5/C-15, from H-4 to C-2/C-10, and from H-15 to C-3/C-5, permitted the complete elucidation of ring A. HMBC correlations from H-6 to C-8/C-10/C-4/C-14 and from H-9 to C-1/C-5/C-7/C-8 allowed the elucidation of ring B. The 1 H NMR signal for H-3 (δH 5.24) was downfield, indicating that

C-3 was acetylated, which was confirmed by HMBC correlations from H-3 to the carbonyl carbon at δC 170.9. Thus, compound 1 was established as a nor-eremophilane sesquiterpene. The relative configuration of 1 was determined by analysis of NOE correlations and the splitting patterns of related protons (Figure 2). The NOE correlation between H-2 and H-4 revealed that H-2 and H-4 were both in axial orientations in ring A. In the 1 H NMR, H-3 (δH 5.24, brs) was a broad singlet adjacent to the axial H-2 and H-4, indicating an equatorial orientation. The NOE correlation between the equatorial Me-15 and H-6 revealed that C5−6 was equatorial and that Me-14 was axial. Thus, we assigned the relative configuration of 1 as shown in Figure 2. Compound 1 possessed a chiral cyclohexadienone chromophore (C5−10). As a result, the absolute configuration at C-5 could be determined from the Cotton effect (CE) of the cyclohexadienone unit. The electronic circular dichroism (ECD) spectrum of 1 showed a positive CE at 350 nm and a negative one at 299 nm. This is consistent with that of the model compound cyclohexadienone.9,10 Thus, the absolute configuration at C-5 was determined to be S. On the basis of the relative configuration described above, the absolute configurations at C-2, -3, and -4 were determined to be S, R, and R, respectively. Therefore, the structure of 1 was characterized and named guignarderemophilane A. B

DOI: 10.1021/np5009027 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. 1H−1H COSY and key HMBC correlations of compounds 1−7.

Figure 2. Key NOE correlations of compounds 1−7.

Me-14 and the C7−11 bond were both in the axial orientation on ring B. The established relative configuration is shown in Figure 2. The CD spectrum of 2 showed the characteristic Cotton effect of the α,β-unsaturated ketone (C8−10 unit); the configuration at C-7 was determined to be R based on a negative CE at 328 nm (the n → π* transition of the α,β-unsaturated ketone).11 On the basis of this relative configuration, the absolute configuration at C-1, -2, -4, and -5 was assigned as S, R, S, and R, respectively. Thus, the structure of compound 2 was established and named guignarderemophilane B. Compound 3 was assigned the molecular formula C15H22O4 based on HR-ESIMS. The 1H NMR data (Table 1) displayed three methyl signals at δH 1.69 (3H, s, Me-13), 1.41 (3H, s, Me-14), and 1.13 (d, J = 6.6 Hz, Me-15) and three olefinic signals at δH 6.19 (1H, d, J = 1.2 Hz, H-9), 4.93 (1H, t, J = 1.2 Hz, H-12a), and 4.81(1H, brs, H-12b). The 13C NMR data (Table 1) of 3 contained 15 signals including three methyls, one sp3 methylene, five sp3 methines (three oxygenated), one sp3 quaternary carbon, one α,β-unsaturated ketone (δC 122.0, 172.6, and 201.8), and two terminal olefinic carbons. Comparison of NMR and HR-ESIMS data with those of compound 2 revealed that the structures were similar except for an additional hydroxy group in 3 and the absence of a methylene in 3. Therefore, the structure was proposed for 3, which was unambiguously confirmed by comprehensive analysis of its 1D and 2D NMR data. Based on the 1H−1H COSY and HSQC data, two spin systems [C(1)H−C(2)H−C(3)H−C(4)H−C(15)H3 and C(6)H2−C(7)H] and one terminal double bond [C(11)− C(12)H2] were established as shown in Figure 1. HMBC correlations (Figure 1) from H-1 to C-3/C-5/C-9, from H-2 to

The molecular formula of 2 was assigned as C15H22O3 based on HR-ESIMS. The 1H NMR data (Table 1) exhibited the resonances of three olefinic protons at δH 5.78 (s), 4.86 (dd, J = 3.0 and 1.2 Hz), and 4.78 (dd, J = 3.0 and 1.2 Hz) and three methyl signals at δH 1.68, 1.33, and 0.97. The 13C NMR data (Table 1) showed three methyls (δC 15.1, 18.1, and 20.4), two sp3 methylenes (δC 34.6 and 43.8), four sp3 methines (including two oxygenated methines) at δC 76.3 and 71.6, one sp3 quaternary carbon (δC 38.9), four olefinic carbons (δC 128.4, 166.7, 145.1, and 113.7), and an α,β-unsaturated ketone at δC 199.0. Of 15 total carbons, one carbonyl and four olefinic carbons accounted for three degrees of unsaturation, suggesting that 2 was a sesquiterpene possessing a dicyclic ring system. The connectivity of 2 was deduced by comprehensive analysis of its 1D and 2D NMR spectra. On the basis of the 1H−1H COSY and HSQC spectra, the structural units C(1)H−C(2)H− C(3)H2−C(4)H−C(15)H3, C(6)H2−C(7)H, and the terminal double bond C(11)−C(12)H2 were established as shown in Figure 1. HMBC correlations (Figure 1) from H-1 to C-3/C-5/ C-10, from H-2 to C-10, from H-3 to C-5, and from H-15 to C-3/ C-5, together with the spin system described above, established the structure of ring A. On the basis of HMBC correlations from H-9 to C-7/C-5, from H-7 to C-5, from H-14 to C-6, and from H-6 to C-4/C10, the structure of ring B was also established. HMBC correlations from H-7 to C-12/C-11/C-13 and from H-13 to C-12/C-7 indicated a C7−11 bond. An NOE correlation between H-2 and H-4 in ring A indicated that both were axial. The small coupling constant between H-1 (d, J = 3.0 Hz) and H-2 (axial) revealed that H-1 was equatorial. The NOE correlation between Me-14 and Me-13 suggested that C

DOI: 10.1021/np5009027 J. Nat. Prod. XXXX, XXX, XXX−XXX

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including three methyls, three sp3 methylenes, three sp3 methines (one oxygenated), one sp3 quaternary carbon, one α,βunsaturated ketone (δC 128.6, 171.8, 202.2), and one terminal olefinic carbon (δC 111.5). On the basis of the 1H−1H COSY and HSQC spectra, two spin systems [C(3)H2−C(4)H−C(15)H3 and C(6)H2−C(7)H− C(8)H−C(9)H2] were established, as shown in Figure 1. Two double bonds and one carbonyl group accounted for three degrees of unsaturation, indicating the presence of two rings in 5. We elucidated the structure of ring A from the spin system C(3)H2−C(4)H−C(15)H3, the α,β-unsaturated ketone, and finally from HMBC correlations from H-1 to C-10/C-3, from H-3 to C-2, from H-4 to C-2/C-10, from H-15 to C-3/C5, and from H-14 to C-4/C-5/C-10. The structure of ring B was determined based on HMBC correlations from H-6 to C-10/C-5 and from H-9 to C-7/C-5/C-10, along with the spin system C(6)H2− C(7)H−C(8)H−C(9)H2 described above. On the basis of HMBC correlations from H-13 to C-11/C-7/C-12 and from H-12 to C-13/C-7, the connection position of the C12−C11− C13 unit with ring B was determined to be via the C7−C11 bond. Thus, the structure of compound 5 was established to be as shown. The NOE correlations H-1/H-8, H-4/Me-13, and Me-15/H-7 gave a relative configuration of 5 as shown in Figure 2. The absolute configuration at C-8 was determined as R from the CD spectrum of the in situ Rh complex of 5.12 Accordingly, the absolute configuration at C-4, -5, and -7 was assigned to be S, R, and S, respectively. Thus, compound 5 was characterized and named guignarderemophilane E. Compound 6, C17H21NO2, showed IR absorptions consistent with hydroxy (3358 cm−1) and ketone groups (1676 cm−1). Its 1H NMR data (Table 2) displayed three methyls [two singlets (δH 2.10 and 2.20) and one triplet (δH 1.11)], a terminal

C-10, from H-3 to C-5, and from H-15 to C-3/C-5, together with the spin system described above, allowed the complete elucidation of ring A. As with compound 2, HMBC correlations in 3 from H-9 to C-7/C-5, from H-7 to C-5, from H-14 to C-6, and from H-6 to C-4/C10 allowed the elucidation of ring B. HMBC correlations from H-13/H-12 to C-7, from H-6 to C-11, and from H-13 to C-12 established the connectivity of the C7−11 bond. HMBC correlations from H-3 to C-1/C-5 and the chemical shift of C-3 (δC 75.6) confirmed that the extra hydroxy group was substituted at C-3, as proposed. The H-1 and Me-14 protons showed strong NOE correlations with each other, indicating that H-1 and Me-14 were in axial orientations on the same face of ring A. The large coupling constant between the axial H-1 and H-2 (J = 10.2 Hz) demonstrated that H-2 was also axial, with the orientation opposite of H-1. The NOE correlation between H-2 and H-4 confirmed that both protons were axial and on the opposite side from H-1/Me-14. The splitting pattern of H-3 (t, J = 3.0 Hz) indicated that it was equatorial. On the basis of the n → π* transition of the α,β-unsaturated ketone (negative Cotton effect at 293 nm), the absolute configuration at C-7 was determined to be S.11 On the basis of the relative configuration, the absolute configurations at C-1, -2, -3, -4, and -5 were all assigned as R. Thus, the structure of compound 3 was established and named guignarderemophilane C. Compound 4 was assigned the same molecular formula (C15H22O4) as 3 based on HR-ESIMS. The NMR data for these compounds were similar, suggesting that the structure of 4 possesses the same skeleton as 3. Careful comparison of the NMR data of 4 and 3 revealed that the terminal double bond was not present in 4. Instead, two additional quaternary carbons and one additional methyl carbon were evident in the 13C NMR spectrum. This was assigned as a C7−C11−C12−C13 unit based on HMBC correlations from Me-12/Me-13 to C-7 and from H-6 to C-11. The proposed structure of 4 was confirmed by 2D NMR. The same spin system as in 3, C(1)H−C(2)H−C(3)H− C(4)H−C(15)H3, was also inferred from the 1H−1H COSY and HSQC spectra (Figure 1). HMBC correlations (Figure 1) from H-1 to C-3/C-5/C-10, from H-3 to C-5/C-1, and from H-15 to C-3/C-5, together with the spin system described above, permitted the elucidation of the structure of ring A. As described for compound 3, the HMBC correlations in 4 from H-9 to C-7/C-5, from H-7 to C-5, from H-14 to C-6, and from H-6 to C-4/C10 allowed the elucidation of the structure of ring B. Thus, compound 4 was established to be as shown. The relative configuration of the three hydroxy groups at C1−3 and of two methyl groups (Me-14 and -15) in 4 was confirmed to be identical to those in 3 based on splitting patterns and NOE correlations as described (Figure 2). The absolute configuration of 4 could not be directly assigned from the Cotton effect of the α,β-unsaturated ketone as for 3 because C-7 in 4 was olefinic. Therefore, the absolute configurations of 4 as C-1 to -5 were tentatively assigned as R (as in 3) based on their assumed common biogenesis and similar NMR resonances at C1−5. Thus, the structure of compound 4 was established and named guignarderemophilane D. The molecular formula of 5 was determined to be C15H22O2 from HR-ESIMS analysis. The IR spectrum (KBr) showed absorption bands for hydroxy (3422 cm−1) and ketone groups (1658 cm−1). The 1H NMR data (Table 1) displayed three methyls [two singlets (δH 1.73, 1.17) and one doublet (δH 0.97, J = 7.2 Hz)], along with three olefinic signals at δH 5.78, 4.62, and 4.57. The 13C NMR data (Table 1) of 5 contained 15 signals

Table 2. 1H NMR (600 MHz) and 13C NMR (150 MHz) Data of Compounds 6 and 7 in Acetone-d6 6 no.

D

δC, type

1 2 3

130.1, C 136.0, C 137.3, C

4

130.1, CH

5 6

7 δH (J in Hz)

no.

δC, type

1 2 3

168.7, C 32.2, CH2

7.05, d (7.2)

4

22.5, CH2

125.9, CH 127.9, CH

6.93, t (7.2) 6.86, d (7.2)

5 5a

28.3, CH 72.8, CH

7 8

127.1, CH 137.6, C

6.62, s

7a 7

157.1, C 107.7, CH

9 10

145.0, C 198.5, C

8 9

137.6, CH 111.4, CH

11 12

26.6, CH3 130.3, CH2

10 10a

161.5, C 117.6, C

13 14 15 16 17

137.5, C 162.3, CH 37.2, CH3 13.2, CH3 14.3, CH3

11 11a 5-Me

183.9, C 66.1, CH 17.4, CH3

2.10, s 6.24, brs 5.86, brs 8.32, s 3.65, q (7.2) 1.11, t (7.2) 2.20, s

δH (J in Hz)

2.61, d (13.8, 7.8) 2.60, d (13.8, 9.6) 2.16, m 2.23, m 2.42, m 3.83, dd (3.8, 2.8) 6.94, dd (8.4, 0.6) 7.59, t (8.4) 6.62, dd (8.4, 0.6)

4.83, d (3.0) 1.17, d (7.0)

DOI: 10.1021/np5009027 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Article

between H-5a and H-11a (JH‑5a,H‑11a = 3.8 Hz) and NOE correlations (H-5a/H-5 and H-a/H-11a) allowed the establishment of the relative configuration of 7, as shown in Figure 2. On the basis of the octant rule for α,β-unsaturated ketone groups (a negative Cotton effect at 319.5 nm), the absolute configuration at C-11a was determined to be S.11 On the basis of the relative configuration described above, the absolute configurations at C-5 and C-5a were both assigned as S. Thus, the structure of compound 7 was established and named guignarlactone A. In addition to the seven new compounds (1−7) described above, two known compounds, guignardone C (8)13 and 11-(5′epoxy-4′-hydroxy-3′-hydroxymethylcyclo-2′-hexenone)-Δ8(12)drimene (9),14 were identified by comparison with published data. All the compounds were assayed for their anti-inflammatory and cytotoxic activities. Compounds 1, 3, 4, and 9 exhibited antiinflammatory activities indirectly by suppressing LPS-induced NO production in BV2 cells with IC50 values of 15.2, 6.4, 4.2, and 4.5 μM, respectively. Meanwhile, curcumin (positive control) gave 50% inhibition at 3.9 μM. However, all test compounds were inactive against the HCT-8, HCT-116, Bel-7402, BGC-823, A549, and A2780 cell lines.

double bond (δH 6.24 and 5.86), two olefinic singlets at δH 6.62 and 8.32, and three aromatic protons (δH 7.05, 6.93, and 6.86). The 13C NMR data (Table 2) of 6 contained 17 signals: three methyls, one sp3 methylene, six olefinic carbons (one terminal olefinic carbon at δC 130.3), one ketone (δC 198.5), and six aromatic carbons. The chemical shifts and splitting patterns of the aromatic protons at δH 7.05 (d, J = 7.2 Hz), 6.93 (t, J = 7.2 Hz), and 6.86 (d, J = 7.2 Hz) indicated the presence of one ortho-trisubstituted aromatic ring, which was unambiguously confirmed by 1H−1H COSY, HSQC, and HMBC (Figure 1). HMBC correlations from Me-16 to C-13, from H-14 to C-15, from H-15 to C-14/C-8, from Me-11 to C-9, from H-12 to C-8/ C-10, and from H-7 to C-9/C-8 allowed the assignment of the C7−16 fragment. HMBC correlations from H-7 to C-2/C-6 indicated that this fragment linked with the aromatic ring through the C1−C7 bond. An HMBC correlation from Me-17 to C-3 revealed the position of the methyl groups at C-3. HMBC correlations from Me-17 to C-3/C-2/C-4 revealed that Me-17 was located at C-3. Two exchangeable protons (NH2) showed HMBC correlations with C-2, indicating that C-2 is substituted by NH2. The chemical shifts of H-14 (δH 8.32) and C-14 (δC 163.2) suggested that C-14 was oxygenated (an enol). Thus, the structure of 7 was established as shown in Figure 1. The NOE correlations H-14/H-7, H-12a/H-11, and H-12b/H-15 led to the assignment of the configurations 7(Z) and 13(E), as shown in Figure 2. Thus, the structure of compound 6 was established and named guignardene A. Compound 7 was isolated as a white powder. Its molecular formula, C14H14O5, was deduced from HR-ESIMS, suggesting the presence of eight degrees of unsaturation. The IR spectrum showed a broad absorption at 3376 cm−1, suggesting the presence of hydroxy group(s) and an absorption at 1677 cm−1, consistent with the presence of a carbonyl group. The presence of one ortho-trisubstituted aromatic ring (ring A) was recognized from the splitting patterns of three aromatic protons at δH 6.94 (1H, dd, J = 8.4, 0.6 Hz), 7.59 (1H, t, J = 8.4 Hz), and 6.62 (1H, dd, J = 8.4, 0.6 Hz). A resonance of one methyl group (δH 1.17, d, J = 7.0 Hz) was also observed in the 1H NMR spectrum of 7 (Table 2). The 13C NMR data of 7 (Table 2) showed resonances for one methyl, two sp3 methylenes, three sp3 methines, six aromatic carbons, and two carbonyl carbons. The 1H−1H COSY spectrum of 7, aided by HSQC experiments, revealed two spin systems (bolded in Figure 1): units A [C(7)H−C(8)H−C(9)H] and B [C(3)H2−C(4)H2− C(5)H(Me)−C(5a)H−C(11a)H]. The presence of an orthotrisubstituted aromatic ring (A) was confirmed by the assigned unit A and HMBC correlations from H-7 to C-9/C-10a, from H-8 to C-10, and from H-9 to C-10a. HMBC correlations from H-5a to C-6a, from H-11a to C-10a, and from H-5a to the ketone group at C-11 (δC 183.9), along with the C5a−C11a fragment in unit B and the chemical shifts of C-5a (δH 72.8) and C-6a (157.1), allowed for the elucidation of ring B. The presence of a seven-membered lactone (ring C) was inferred from HMBC correlations from H-11a to C-2 and from H-4 to C-2, along with the chemical shifts of the lactone group (δC 168.7) and a methine carbon at δC 66.1 (C-11a). Together with unit B described above, these data indicate that C-11a must be attached to the lactone, forming ring C. Thus, all NMR data for 7 were unambiguously assigned in accordance with the structure of 7 (Table 2). NOE experiments and the coupling constants of related protons in 7 permitted the assignment of the relative configurations of H-5, -5a, and -11a. The small coupling constant

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO P-2000 automatic digital polarimeter. CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer. NMR spectra were recorded on INOVA SX-600 spectrometers. HR-ESIMS data were recorded on an Agilent Technologies 6250 Accurate-Mass Q-TOF LC/MS spectrometer. Preparative HPLC was performed on a Shimadzu LC-6AD instrument with an SPD-10A detector, using a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Sephadex LH-20 (Amersham Pharmacia Biotech AB, Sweden), ODS (45−70 μm, Merck), macroporous adsorptive resins (XAD-D101, Tianjin Nankai Chemical Inc. China), and silica gel (200−300 mesh, Qingdao Marine Chemical Inc. China) were used for column chromatography (CC). TLC was conducted on glass precoated with silica gel GF254 (Qingdao Marine Chemical Inc. China). Plant Material. Fresh leaves of G. elegans were collected in Guangxi Province, China, in May 2013. The plant was identified by Prof. Song-Ji Wei, Guangxi College of Chinese Traditional Medicine. A voucher specimen (specimen No. S2505) was deposited in the herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, China. Fungal Material. The fungus stain was separated from the fresh leaves of G. elegans. The isolate was assigned the accession number 2013Y-3 in Professor J. Dai’s culture collection in the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. The fungus Guignardia mangiferae 2013-Y-3 was identified based on colony morphology and the 18S rDNA-ITS sequence analysis. From the analysis of the ITS1-5.8S-ITS2 region of 18S rDNA (GenBank accession no. KP903462), this strain was found to be a very close relative to strains of the Guignardia genus and most similar to G. mangiferae CGMCC3.14345 (100% identity, based on 18S rDNA-ITS sequence). Thus, the fungus was identified as G. mangiferae. Extraction and Isolation. Plugs of agar supporting mycelial growth were cut and transferred aseptically to a 1000 mL Erlenmeyer flask containing 400 mL of PDA medium. After incubation for 3 days at 28 °C on a rotary shaker at 110 rpm, 20 mL of culture liquid was transferred as seed culture into each 5000 mL flask containing culture liquid (2000 mL). The culture was then incubated for 21 days at 28 °C. The filtrate of the culture (100 L) was loaded onto a macroporous resin column. The resin was washed with water, then eluted with 95% alcohol. The alcohol fraction was concentrated to obtain 8.5 g of a crude material. Of this, 8 g was applied to a silica gel column, eluted with CHCl3−MeOH (20:1−1:1), E

DOI: 10.1021/np5009027 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

give final concentrations of 10 μM. The cell growth was evaluated by an MTT assay procedure after 72 h. To each well was added 100 μL of MTT, and the plate incubated for 4 h at 37 °C. To dissolve the formazan crystals, 200 μL of DMSO was added, and the absorbances at 570 nm were measured. Camptothecin was used as a positive control.2

to afford four fractions (A, 3.6 g; B, 1.2 g; C, 0.9 g; D, 0.8 g). Fraction A (3.0 g) was chromatographed over an ODS column, eluted with a gradient of increasing methanol in water (5−100%), to afford four subfractions (A1, 0.8 g; A2, 0.9 g; A3, 0.4 g; A4, 0.5 g). Fraction A3 was purified by RP-HPLC using 30% acetonitrile in water to afford 1 (4.3 mg), 2 (1.7 mg), 6 (1 mg), and 8 (10.4 mg). Fraction A2 was purified by RP-HPLC [solvent system: CH3CN−H2O (4:1)] to afford compounds 5 (2.1 mg) and 7 (3 mg). Fraction A1 was purified by RPHPLC using 30% acetonitrile in water to yield 9 (13 mg). Fraction B was chromatographed over an ODS column, eluting with a gradient of increasing methanol in water (5−100%), to afford four subfractions (B1, 0.5 g; B2, 0.2 g; B3, 0.3 g; B4, 0.1 g). From fraction B1, compounds 3 (2.3 mg) and 4 (5.4 mg) were isolated via RP-HPLC using 35% acetonitrile in water. Guignarderemophilane A (1): colorless oil; [α]20D +34 (c 0.01, MeOH); UV (MeOH) λmax 235, 272 nm; CD (MeOH) λmax (Δε) 211 (52), 252 (−12), 299 (−8), 350 (−1) nm; IR νmax 3402, 2918, 1731, 1676, 1639, 1245 cm−1; HRESIMS m/z 267.1223 [M + H]+ (C14H19O5, calcd [M + H]+ 267.1227), 289.1048 [M + Na]+ (C14H18NaO5, calcd [M + Na]+ 289.1046); 1H and 13C NMR (Table 1). Guignarderemophilane B (2): colorless gum; [α]20D +65 (c 0.03, MeOH); UV (MeOH) λmax 214, 232 nm; CD (MeOH) λmax (Δε) 218 (20), 255 (−2), 328 (−1.5) nm; IR νmax 3394, 1677, 1207, 1142 cm−1; HRESIMS m/z 251.1639 [M + H]+ (C15H23O3, calcd [M + H]+ 251.1642), 273.1463 [M + Na]+ (C15H22NaO3, calcd [M + Na]+ 273.1461); 1H and 13C NMR (Table 1). Guignarderemophilane C (3): white powder; [α]20D −175 (c 0.01, MeOH); UV (MeOH) λmax 245 nm; CD (MeOH) λmax (Δε) 243 (35), 293 (−8) nm; IR νmax 3498, 2918, 1651 cm−1; HRESIMS m/z 267.1590 [M + H]+ (C15H23O4, calcd [M + H]+ 267.1591), 289.1409 [M + Na]+ (C15H22NaO4, calcd [M + Na]+ 289.1410); 1H and 13C NMR (Table 1). Guignarderemophilane D (4): white powder; [α]20D −121 (c 0.12, MeOH); UV (MeOH) λmax 248, 285 nm; CD (MeOH) λmax (Δε) 247 (130), 286 (−50) nm; IR νmax 3392, 1679, 1441, 1206, 1141 cm−1; HRESIMS m/z 267.1589 [M + H]+ (C15H23O4, calcd [M + H]+ 267.1591), 289.1404 [M + Na]+ (C15H22NaO4, calcd [M + Na]+ 289.1410); 1H and 13C NMR (Table 1). Guignarderemophilane E (5): colorless oil; [α]20D +102 (c 0.03, MeOH); UV (MeOH) λmax 240 nm; CD (MeOH) λmax (Δε) 231 (−32), 313 (5) nm; IR νmax 3422, 2920, 1717, 1658, 1609 cm−1; HRESIMS m/z 235.1690 [M + H]+ (C15H23O2, calcd [M + H]+ 235.1693), 257.1512 [M + Na]+ (C15H22NaO2, calcd [M + Na]+ 257.1512); 1H and 13C NMR (Table 1). Guignardene A (6): yellow powder; UV (MeOH) λmax 230, 252, 325 nm; IR νmax 3358, 2929, 1676, 1451, 1204 cm−1; HRESIMS m/z 272.1640 [M + H]+ (C17H22NO2, calcd [M + H]+ 272.1645), 294.1466 [M + Na]+ (C17H21NNaO2, calcd [M + Na]+ 294.1465); 1H and 13C NMR (Table 2). Guignarlactone A (7): white powder; [α]20D +19 (c 0.01, MeOH); UV (MeOH) λmax 230 nm; CD (MeOH) λmax (Δε) 210 (+11), 319.5 (−1) nm; IR νmax 3376, 2917, 1731, 1677, 1660, 1471 cm−1; HRESIMS m/z 263.0915 [M + H]+ (C14H15O5, calcd [M + H]+ 263.0914), 285.0736 [M + Na]+ (C14H14NaO5, calcd [M + Na]+ 285.0733); 1H and 13 C NMR (Table 2). Anti-inflammatory Activity. The BV2 macrophage cell line was obtained from the Cell Culture Center at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. LPS (from Escherichia coli 055:B5) was obtained from Sigma-Aldrich. After preincubation for 24 h in a 96-well plate, the cells were treated with test compounds, followed by stimulation with LPS for 24 h. Production of NO was determined by measuring the concentration of nitrite in the supernatant. Absorbance was measured at 550 nm. Curcumin was used as a positive control.15,16 Cytotoxicity Assay. The cytotoxicity of the compounds against the human cancer cell lines (HCT-8, HCT-116, Bel-7402, BGC-823, A549, and A2780) was measured using the MTT assay. The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and cultured in 96-well plates. After incubation of the culture medium at 37 °C for 24 h, a DMSO solution of the samples was added to

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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/np5009027. UV, IR, MS, 1D and 2D NMR, and CD spectra for 1−8 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-10-63165324. Fax: +86-10-63017757. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (No. 21132009) and the National Science and Technology Project of China (No. 2012ZX09301002002). The authors are grateful to the Department of Instrumental Analysis in our institute for measurement of UV, IR, NMR, and MS spectra.

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REFERENCES

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DOI: 10.1021/np5009027 J. Nat. Prod. XXXX, XXX, XXX−XXX