Immunomodulatory Polyketides from a Phoma-like Fungus Isolated

Article Cite This: J. Nat. Prod. 2017, 80, 2930-2940

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Immunomodulatory Polyketides from a Phoma-like Fungus Isolated from a Soft Coral Yi-Zhe Sun,† Tibor Kurtán,‡ Attila Mándi,‡ Hua Tang,† Yalan Chou,§ Keryea Soong,§ Li Su,† Peng Sun,† Chun-Lin Zhuang,† and Wen Zhang*,† †

Research Center for Marine Drugs and Pharmaceutical Analysis Center, School of Pharmacy, Second Military Medical University, 325 Guo-He Road, Shanghai 200433, People’s Republic of China ‡ Department of Organic Chemistry, University of Debrecen, POB 400, H-4002 Debrecen, Hungary § Department of Oceanography, National Sun Yat-sen University, 70 Lien-Hai Road, Kaohsiung 80424, Taiwan S Supporting Information *

ABSTRACT: Fourteen new polyketides with a trans-fused decalin ring system, libertalides A−N (3−16), together with two known analogues, aspermytin A and its acetate (1, 2), were isolated from the fermentation extract of a coral-derived Libertasomyces sp. fungus. Their relative configurations were elucidated on the basis of detailed spectroscopic analysis, and the absolute configurations were determined by TDDFT-ECD and optical rotation (OR) calculations. The OR of 1 and 2 were found to have opposite signs in CH3CN and CHCl3, which was in agreement with the OR calculations producing alternating signs for the optical rotation depending on the applied conditions. Because the signs of the OR for 1 and 2 showed high solvent dependence, they may not be used alone to correlate the absolute configurations. Compound 16 displayed structural novelty characterized by an α-enol ether bridge conjugated with an aldehyde group. In in vitro immunomodulatory screening, compounds 1, 4, and 10 significantly induced the proliferation of CD3+ T cells, while compounds 2, 7, 11, and 14 significantly increased the CD4+/CD8+ ratio at 3 μM. A preliminary structure−activity analysis revealed a crucial role of Δ7 and a terminal OH group in the regulation of CD3+ T cell proliferation. This is the first report of immunoregulatory activity for metabolites of this kind.

T

he fungal genus Libertasomyces was introduced as a Phoma-like genus collected from the twigs of Myoporum serratum on Robben Island off the west coast of Cape Town, South Africa.1,2 It belongs to the family Libertasomycetaceae, a new family to accommodate Libertasomyces and Neoplatysporoides.1,3 The genus currently contains only three species, namely, L. myopori,1 L. platani,2 and L. quercus.3 To date, there are no chemical reports on the fungi of this genus. As part of our continuing search for bioactive metabolites from marine invertebrates and associated fungi,4−7 a fungal strain (DYZ-027) was isolated from a specimen of the soft coral Sinularia sandensis collected from Dongsha Atoll in the South China Sea. Initial DNA identification of this strain suggested that it was a possible Phoma sp., as the ITS sequence had a 100% match with Phoma sp. MUT5178 (GenBank KM355985). As Phoma sp. associated with marine invertebrates are well known to produce a variety of bioactive metabolites,8−14 this strain was subjected to further study. Chemical investigation of this strain resulted in the isolation of 16 polyketides having a decalin skeleton. Their structures were elucidated on the basis of detailed spectroscopic analysis with the absolute configurations being determined by electronic circular dichroism (ECD) calculations. These compounds were evaluated for their tumor cell growth inhibitory and T cell © 2017 American Chemical Society and American Society of Pharmacognosy

immune regulatory activities. Interestingly, during the course of this study, the ITS sequence for another fungal strain, Libertasomyces platani, was deposited in GenBank, and it also had a 100% match with the sequence for our DYZ-027 strain, although with greater coverage than for the Phoma sp. MUT5178 strain. L. platani was isolated from a Platanus sp. tree in New Zealand.2 Because of the greater coverage with the ITS sequence of L. platani, and because the ITS sequence also had >90% identity with the ITS sequences for L. quercus and L. myopori, the DYZ-027 strain is tentatively identified as a Libertasomyces sp. Further taxonomic studies will be necessary to firmly establish a species assignment. Here, we report the isolation, structure elucidation, and bioactivity evaluation of these compounds.

■

RESULTS AND DISCUSSION

The Libertasomyces sp. was cultivated on biomalt agar medium at room temperature for 28 days and was then extracted with EtOAc. The EtOAc extract was subjected to repeated column Received: May 31, 2017 Published: October 19, 2017 2930

DOI: 10.1021/acs.jnatprod.7b00463 J. Nat. Prod. 2017, 80, 2930−2940

Journal of Natural Products

Article

Chart 1

oxygenated tertiary carbon was assigned at C-4 due to the change in the multiplicity of H3-11 from a doublet in 2 to a singlet in 3. The assignment was supported by HMBC correlations from H3-11 to C-3, C-4, and C-5. A β-orientation of 4-OH was deduced from NOE correlations of H3-11 with both H-3α and H-5α (Figure 2). The assigned β-orientation of 4-OH was supported by the remarkable pyridine-induced solvent shifts16,17 (Table S1) for H-2β (Δδ = 0.45) and H-6 (Δδ = 0.54). Compound 4 was isolated as colorless crystals. The HREIMS data gave its molecular formula as C14H22O3. NMR signals of 4 associated with the octahydronaphthalene unit were identical with those of 1, indicating the same relative configuration. The difference was observed for signals of the side chain. Signals for the terminal alcohol group disappeared and the keto group (δ 212.7) was replaced by a carboxyl group (δ 178.8) (Table 2). These observations indicated that 4 is the 14-carboxy analogue of 1. Compound 5, a colorless oil, had a molecular formula of C15H24O3 as determined by HREIMS. The compound showed identical NMR signals to those of 3 regarding the core structure of the octahydronaphthalene system. Signals for the terminal acetoxyethyl side chain in 3 were replaced by a methyl group in 5. The structure was supported by HMBC correlations from H3-15 to C-10 and C-14. The relative configurations of the core structure were preserved, as indicated by the NOE spectra (Figure 2). Compound 6 was obtained as a white powder and had a molecular formula of C16H24O3 based on the HRESIMS data, requiring five degrees of unsaturation. Three sp2 carbon signals were recognized for a double bond and an ester carbonyl group (Table 2), accounting for two degrees of unsaturation. The remaining degrees of unsaturation were attributed to the three rings of the molecule. Comparison of the NMR data of 6 with those of 3 and 5 revealed close similarity regarding signals of the octahydronaphthalene unit. The presence of the octahydronaphthalene unit was supported by the proton linkage from H-3 to H-5 and H-8 and long-range correlations from H3-11 to C-3, C-4, and C-5, from H3-12 to C-8, C-9, and C-10, and from H3-13 to C-1, C-9, and C-10 (Figure 3). The HMBC correlations from H2-14 to C-1, C-9, and C-10 and from H215 to C-10 and C-16 gave the connection from the carbonyl carbon to the octahydronaphthalene unit through the methylene bridge H2-14/H2-15 as established by a COSY experiment. The remaining degree of unsaturation in the molecule allowed the linkage from the ester carbon C-16 to the tertiary carbon C-9 by an oxygen bridge to form a δ-

chromatography on silica gel, Sephadex LH-20, and RP-HPLC to yield 16 pure compounds. Compounds 1 and 2 were isolated as colorless crystals. Their molecular formulas were deduced from the HREIMS and 13C and DEPT NMR spectra. The planar structures and relative configurations of 1 and 2 were established by detailed NMR analysis and proven to be the same as those of aspermytin A and aspermytin A acetate,15 respectively. Aspermytin A is a metabolite obtained from an Aspergillus sp. isolated from Mytilus edulis, and aspermytin A acetate is the acetylation product of aspermytin A that was generated during the course of its structure elucidation.15 Compound 3 was obtained as colorless crystals. Its molecular formula was determined as C18H28O5 by HREIMS, requiring five degrees of unsaturation. The 1H and 13C NMR spectra of 3 showed significant similarities to those of 2 (Tables 1 and 2), revealing an octahydronaphthalene analogue as established by COSY and HMBC data (Figure 1). However, a methine group in 2 was replaced by an oxygenated tertiary carbon in 3. The Table 1. 1H NMR Data for Compounds 3−5 (in C5D5N, J in Hz) 3a

4b

5a

2α

2.18, ddd (13.4, 10.8, 2.8) 1.76, ov

2.16, ddd (13.3, 10.6, 3.1) 1.79, ov

2β

1.76, ov

3α

1.63, ov

3β 4 5α 5β 6

1.94, ov

2.13, ddd (11.9, 10.4, 2.8) 2.21, dddd (12.4, 3.5, 2.8, 2.8) 1.31, dddd (12.4, 12.4, 11.9, 2.7) 1.11, dddd (12.7, 12.7, 12.4, 3.5) 1.75, ov 1.44, m 0.66, ov 1.74, ov 1.89, dddd (12.1, 10.4, 4.7, 2.0) 5.46, dd (10.0, 2.0) 5.85, dd (10.0, 2.7) 0.89, d (6.5) 1.83, s 1.71, s

1

7 8 11 12 13 15 16 18 a

1.33, ov 2.01, ov 2.77, dddd (12.8, 10.8, 5.3, 2.0) 5.45, dd (10.0, 2.0) 5.77, dd (10.0, 2.7) 1.39, s 1.51, s 1.59, s a 3.69, dt (18.3, 6.4) b 2.97, dt (18.3, 6.4) 4.62, m 1.97, s

1.79, ov 1.62, ov 1.94, ov 1.34, ov 1.99, ov 2.77, dddd (15.5, 13.3, 5.3, 2.4) 5.46, dd (10.0, 2.4) 5.76, dd (10.0, 2.8) 1.39, s 1.52, s 1.59, s 2.49, s

400 MHz. b500 MHz. 2931



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Table 2. 13C NMR Data for Compounds 3−8 (in C5D5N) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 a

3a

4b

5a

6a

7b

8a

44.3, CH 24.2, CH2 40.7, CH2 69.3, C 46.9, CH2 34.6, CH 130.6, CH 136.0, CH 73.7, C 58.3, C 32.6, CH3 29.0, CH3 13.2, CH3 212.7, C 41.7, CH2 60.7, CH2 171.3, C 21.3, CH3

45.4, CH 29.0, CH2 36.4, CH2 34.0, CH 42.4, CH2 39.5, CH 130.1, CH 135.8, CH 74.3, C 54.5, C 23.2, CH3 29.7, CH3 13.9, CH3 178.8, C

44.4, CH 24.2, CH2 40.7, CH2 69.2, C 46.9, CH2 34.7, CH 130.6, CH 136.0, CH 73.5, C 58.3, C 31.3, CH3 29.1, CH3 13.7, CH3 213.3, C 32.6, CH3

46.8, CH 21.7, CH2 40.2, CH2 68.9, C 46.8, CH2 35.7, CH 132.6, CH 131.4, CH 85.1, C 38.2, C 32.5, CH3 26.4, CH3 15.1, CH3 27.2, CH2 28.2, CH2 172.3, C

42.7, CH 22.3, CH2 40.3, CH2 69.1, C 46.1, CH2 36.0, CH 132.0, CH 132.6, CH 85.4, C 43.2, C 32.6, CH3 28.4, CH3 15.9, CH3 68.6, CH 41.2, CH2 171.5, CH

41.2, CH 23.9, CH2 40.8, CH2 68.8, C 46.8, CH2 35.3, CH 134.8, CH 133.2, CH 78.0, C 43.8, C 32.7, CH3 19.5, CH3 16.6, CH3 74.4, CH 33.0, CH2 61.8, CH2

100 MHz b125 MHz.

Figure 1. Key COSY and HMBC correlations of compounds 3−5. Figure 3. Key COSY, HMBC, and NOE correlations of compound 6.

valerolactone ring. The significantly deshielded values of C-9 from δ 73.7 and 73.5 in 3 and 5 to δ 85.1 in 6 supported the established tricyclic structure. The relative configuration of 6 was determined by NOE correlations and pyridine-induced solvent shift experiments. The distinct NOE of H-6 with H-2β and H3-13 indicated the β-orientation of these protons. The NOE of H-1 and H3-11 with both H-3α and H-5α indicated the α-orientation of these protons (Figure 3). A β-orientation of 4OH was further supported by the remarkable deshielding of H2β (Δδ = 0.38) and H-6 (Δδ = 0.36) recorded in C5D5N relative to those in CDCl3 (Table S1). The α-orientation of H312 was deduced from its NOE with H-1, leading to the determination of the relative configuration of 6. Compound 7 was obtained as a white powder and had a molecular formula of C16H24O4 based on the HRESIMS data. The NMR data of 7 were almost identical to those of 6 with the exception of a methylene group in 6 being replaced by a secondary alcohol group in 7 (δH 4.26, ddd, J = 8.6, 4.7, 1.7; δC

68.6, CH). The hydroxy group was located at C-14 based on a COSY correlation of H-14 with H2-15 and HMBC correlations of H-14 with C-9, C-10, C-13, and C-16. The relative configuration in the decalin system of 7 was found to be the same as those in 6 as established by NOE correlations and pyridine-induced solvent shift experiments (Table S2). An αorientation of 14-OH was deduced from its NOE with H3-12 and further supported by an NOE of H-14 with H3-13. Compound 8 was obtained as a white powder. HREIMS gave the molecular formula of C16H26O3. The NMR signals of 8 were closely related to those of 7, except that the carbonyl group (δC 171.5) in 7 was replaced by an oxygenated methylene group (δH 3.94, ddd, J = 12.1, 5.4, 1.1; 3.80, ddd, J = 12.4, 12.1, 3.2; δC 68.6, CH2) in 8. The oxygenated methylene group was assigned at C-16 based on the proton sequence from H-14 to H2-16 and an HMBC correlation from

Figure 2. Key NOE correlations of compounds 3−5. 2932



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Table 3. 1H NMR Data for Compounds 6−8 (in C5D5N, J in Hz)

H2-16 to C-9. The relative configurations of 8 at C-1, C-4, C-6, C-10, and C-14 were the same as those of 7 as established by NOESY experiment (Figure 5). A β-orientation of H3-12 was

6a

Figure 4. Key COSY, HMBC, and NOE correlations of compound 8.

1

1.33, ov

2.14, ov

2α 2β

2.08, ov 1.93, ov

3α

1.42, ov 1.76, dddd (13.0, 13.0, 12.6, 3.8) 1.42, ov

3β 5α 5β 6

1.95, 1.22, 1.90, 2.74,

7 8 11 12 13

15α

5.41, dd (10.0, 2.1) 5.69, dd (10.0, 3.0) 1.40, s 1.45, s 0.99, s α 1.65, ddd (15.3, 12.4, 8.4) β 1.54, ddd (12.4, 7.5, 6.3) 2.79, ov

15β

2.77, ov

14

Figure 5. Key COSY and HMBC correlations of compounds 9 and 10.

7b

m dd (12.8, 12.7) dd (12.7, 2.9) ov

1.55, ddd (12.7, 12.4, 4.0) 1.99, ov 1.36, ov 1.98, ov 2.85, dddd (15.0, 12.3, 4.9, 2.5) 5.46, dd (10.1, 2.5) 5.77, dd (10.1, 3.0) 1.40, s 1.97, s 1.02, s

a

5.61, 5.55, 1.39, 1.33, 1.26,

dd (10.0, 1.6) dd (10.0, 2.4) s s s

4.36, dd (12.3, 3.9)

3.53, dd (19.0, 8.6)

2.33, dddd (12.7, 12.4, 12.3, 5.4) 1.81, dddd (12.7, 3.9, 3.2, 1.1) 3.94, ddd (12.1, 5.4, 1.1) 3.80, ddd (12.4, 12.1, 3.2)

3.13, dd (19.0, 1.7)

16β 14OH

2.46, ddd (12.3,10.3, 3.1) 2.83, ov 2.19, dddd (13.4, 13.2, 12.3, 3.6) 1.55, ddd (13.4, 13.3, 4.3) 2.02, ov 1.37, ov 1.98, ov 2.83, ov

4.26, ddd (8.6, 4.7, 1.7)

16α

deduced from its NOE with H-14 and H3-13, indicating a cisfused junction between the pyran and decalin units (Figure 4). This assignment was supported by the observation of the γgauche shift effect18−21 for Me-12, shielded from δC 28.4 in 7 to δC 19.5 in 8 (Table 2). Compound 9, a white solid, had a molecular formula of C16H24O4 as determined by HRESIMS. The compound showed similar NMR signals to those of 6 except for the replacement of the Δ7 double bond in 6 by an epoxide moiety in 9 (Tables 2−4, Figure 5). Both protons of the epoxide were β-oriented due to their NOE with H3-13. The relative configuration at other stereogenic centers remained intact based on NOE data (Figure 6). Compound 10 was obtained as colorless crystals and had a molecular formula of C16H24O5 based on the HREIMS data. Compound 10 differed from 9 in replacement of H-6 with a hydroxy group. This substitution resulted in deshielding of H2β from δ 1.45 in 9 to δ 1.88 in 10, suggesting a β-orientation of 6-OH (Table 4). The assignment was supported by the observation of a gauche shift effect for C-2, shielded from δ 20.5 in 9 to δ 16.3 in 10 (Table 4), meaning a trans-fused decalin ring system. The relative configurations at the other stereocenters remained intact based on NOE analysis (Figure 6). Compound 11 was isolated as a colorless oil and had a molecular formula of C16H27ClO4 as established by HRESIMS. An isotopic ratio of 3:1 observed in the sodium adduct ion peak at m/z 341/343 ([M + Na]+) indicated the presence of a chlorine atom in the molecule. The NMR data of 11 resembled those of 1 (Experimental, Tables 5 and 6). However, the Δ7 double bond in 1 was substituted by a hydroxy group and a chlorine atom in 11. The proton sequence from H-6 to H-8 and HMBC correlations from H3-12 to C-8, C-9, and C-10 supported the above conclusion (Figure 7). The observed NOEs between H-6 and H-7 and between H-8 and H3-12

8b

6.93, d (4.7)

400 MHz. b500 MHz.

indicated a β-orientation for H-6 and an α-orientation for H-8, respectively. The relative configurations for the remainder of 11 were the same as that of 1 based on the NOE data (Figure 8). Compound 12 was obtained as a colorless oil. Its molecular formula was determined as C18H29ClO5 by the HRESIMS and displayed a similar chlorine isotopic ion peak to that in 11. The 1 H and 13C NMR data of 12 were almost identical to those of 11 except for the presence of an additional acetyl group (δH 1.99, s; δC 171.2 C, 21.3 CH3) (Tables 5 and 6). The location of the acetyl group at C-16 was deduced from the HMBC correlation of H2-16 with the acetylcarbonyl carbon (Figure 7). This assignment was in agreement with the deshielded signals for H2-16 in 12 (δ 4.65, 4.58, each 1H) with respect to those in 11 (δ 4.31, 2H). Compound 12 was thus identified as the 16acetate of 11. Compound 13, obtained as colorless crystals, had the molecular formula of C15H25ClO3 established by HRESIMS. Its MS spectrum also demonstrated the presence of chlorine. The compound displayed identical NMR signals to those of 11 and 12 regarding the decalin ring system. A difference was recognized in the side chain. Signals for the ethyl alcohol group in 11 were replaced by a methyl group in 13 (Tables 5 and 6). HMBC correlations from H3-15 to C-10 and C-14 supported the above conclusion (Figure 7). The relative configuration of 13 was proven the same as those 11 and 12 based on NOE data (Figure 8). Compound 14 was obtained as colorless crystals. The HREIMS spectrum gave the same molecular formula as for 5. The 1H and 13C NMR data of 14 also showed similarities to 2933



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Table 4. 1H and 13C NMR Data for Compounds 9 and 10 (in CDCl3) 9 δ Ca 1

39.3, CH

2α 2β 3α

20.5, CH2

3β 4 5α 5β 6 7 8 9 10 11 12 13 14α

39.0, CH2

33.0, 57.2, 57.8, 81.7, 37.8, 31.8, 20.5, 15.0, 25.5,

CH CH CH C C CH3 CH3 CH3 CH2

14β 15α

27.3, CH2

15β 16 a

10

δHb (J in Hz) 0.86, ddd (11.5, 11.5, 2.7) 1.37, ov 1.45, ov 1.38, ov

δCa

1.43, 1.81, 2.20, 3.15, 3.20,

ov dd (13.5, 2.9) m dd (3.3, 2.9) d (3.3)

1.28, s 1.44, s 0.91, s 1.63, ddd (12.8, 10.5, 6.5) 1.55, ddd (12.8, 9.9, 2.6) 2.64, ddd (19.7, 10.5, 2.6) 2.72, ddd (19.7, 9.9, 6.5)

172.0, C

δHb (J in Hz)

42.3, CH

1.04, ov

16.3, CH2

1.39, m 1.88, ov 1.48, ddd (13.8, 13.6, 4.9) 1.85, ov

40.1, CH2

1.73, m 69.8, C 41.7, CH2

Table 5. 1H NMR Data for Compounds 11−13 (400 MHz, in C5D5N, J in Hz)

73.4, C 45.8, CH2 71.5, 59.2, 58.5, 81.7, 37.8, 32.1, 20.8, 17.0, 26.0,

C CH CH C C CH3 CH3 CH3 CH2

1 2α 2β 3α 3β 4 5α 5β 6

1.60, ov 1.91, ov 3.18, d (3.5) 3.27, d (3.4)

1.28, 1.34, 1.04, 1.56,

7 8 11 12 13 15a 15b 16a 16b 18

s s s ov

1.56, ov 27.5, CH2

2.63, ddd (19.7, 9.8, 2.8) 2.74, ddd (19.7, 9.9, 6.3)

11

12

2.56, ddd (11.4, 11.3, 3.2) 1.76, ov

2,51, ddd (11.6, 11.5, 3.2) 1.70, ov

13

1.08, dddd (13.0, 11.8, 11.4, 2.9) 0.97, dddd (12.9, 11.8, 10.9, 2.9) 1.57, ov 1.33, m 1.46, ov 1.54, ov 2.38, dddd (15.2, 11.3, 4.1, 3.4) 4.55, dd (2.8, 2.4) 4.23, d (2.8) 0.86, d (6.4) 1.69, s 1.74, s 3.64, dt (17.7, 6.2) 3.11, dt (17.7, 6.2) 4.31, ov 4.31, ov

1.08, dddd (12.1, 11.5, 11.4, 2.8) 0.97, dddd (12.9, 11.4, 10.9, 2.8) 1.59, ov 1.33, m 1.47, ov 1.54, ov 2.37, dddd (15.0, 11.6, 4.0, 3.5) 4.54, ov 4.22, d (2.6) 0.86, d (6.4) 1.64, s 1.68, s 3.67, dt (18.3, 6.0) 2.99, dt (18.3, 6.0) 4.65, dt (11.0, 6.0) 4.58, ov 1.99, s

2.51, ov 1.73, dddd (13.3, 4.4, 3.8, 1.3) 1.07, dddd (13.3, 12.9, 12.2, 2.3) 0.94, ov 1.57, ov 1.31, m 1.47, ov 1.52, ov 2.37, dddd, (14.8, 11.4, 4.0, 3.1) 4.54, dd, (3.1, 2.7) 4.22, d (2.7) 0.86, d (6.4) 1.62, s 1.65, s 2.47, s

Compound 16 was obtained as a colorless oil. HRESIMS established the molecular formula C16H24O4, requiring five degrees of unsaturation. IR absorptions at 2854 and 1732 cm−1 indicated the presence of a CHO group in the molecule. This was in agreement with the presence of typical signals for CHO groups (δH 9.87, d, J = 8.3; δC 189.6 CHO) in the NMR spectra. Two additional sp2 carbon signals were recognized for a trisubstituted double bond (Table 6), which accounted for two degrees of unsaturation. The remaining degrees of unsaturation were attributed to three rings in the molecule. The planar structure of 16 was elucidated on the basis of detailed analysis of NMR spectra. Analysis of the COSY spectra led to establishment of the proton sequence from both H2-3 and H3-11 to H-7. HMBC correlations from H3-11 to C-3, C-4, and C-5 led to connection of CH2-3 and CH-4 to form a sixmembered ring as shown in Figure 13. HMBC correlations from H-7 to C-8 and C-9 led to the extension of the sixmembered ring fragment to the oxygenated tertiary carbon C-9. HMBC correlations from H3-13 to C-1, C-9, and C-10 resulted in the linkage of C-1 and C-9 by the quaternary C-10 to form the decalin unit. HMBC correlations from H-8, H3-13, and the olefinic proton H-15 to C-14 led to the formation of the third ring in the structure (Figure 11). An NOE of H-6 with the axial proton H-4 indicated the βorientation of these protons. NOE correlations of H-1 with H3α, H-5α, H-7, and H-15 indicated the α-orientation of these protons. The α-bridge for the ether ring was thus assigned. The Z configuration of Δ14 was deduced from the NOE correlation between H-1 and H-15 (Figure 11). The remarkable pyridineinduced solvent shift (Table S3) for H-6 (Δδ = 0.92) indicated a boat conformer of ring A and a β-orientation of 9-OH. The Me-12 was consequently assigned as α-orientated. The substructure of the enol ether bridge conjugated with an aldehyde group was reported in coicenal A, obtained from the plant pathogenic fungus Bipolaris coicis. The similar carbon shift

172.0, C

600 MHz, J in Hz. b100 MHz.

Figure 6. Key NOE correlations of compounds 9 and 10.

those of 5. The disubstituted double bond and one of the oxygenated tertiary carbons in 5 (Tables 1 and 2) were replaced by a trisubstituted double bond and a secondary oxygenated carbon in 14 (Tables 6 and 7). The assignment of the trisubstituted double bond at Δ8 and the secondary oxygenated carbon at C-7 was indicated by the proton connection from H-8 to H2-3 and H2-5. The conclusion was supported by HMBC correlations of H3-12 with C-8, C-9, and C-10 and of H-8 with C-10 (Figure 9). The α-orientation of H7 was indicated by its NOE with H-1 (Figure 10), leading to the determination of the relative configuration of 14. Compound 15 was obtained as a colorless oil. The HREIMS data gave the molecular formula as C16H26O4 , which corresponded to an HCl unit less with respect to that of 11. A loss of HCl from 11 was in agreement with the presence of a trisubstituted double bond (Tables 6 and 7). This double bond was readily assigned as Δ6 on the basis of the COSY correlation from the olefinic proton H-7 to H-8. HMBC correlations from H-7 to C-1, C-5, and C-9 (Figure 9) further confirmed the assignment. The relative configuration of 15 was determined as shown based on NOE data (Figure 10). 2934



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Table 6. 13C NMR Data for Compounds 11−16 (100 MHz) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 a

11a

12a

13a

14a

15b

16b

36.9, CH 29.2, CH2 35.5, CH2 33.2, CH 40.4, CH2 35.4, CH 67.6, CH 81.3, CH 72.7, C 59.3, C 23.2, CH3 27.9, CH3 14.5, CH3 213.7, C 45.3, CH2 58.4, CH2

36.8, CH 29.1, CH2 35.4, CH2 33.7, CH 40.3, CH2 35.3, CH 67.5, CH 81.2, CH 72.5, C 59.4, C 23.2, CH3 27.7, CH3 14.3, CH3 211.6, C 40.8, CH2 60.7, CH2 171.2, C 21.3, CH3

36.8, CH 29.1, CH2 35.5, CH2 33.2, CH 40.4, CH2 35.4, CH 67.6, CH 81.2, CH 72.3, C 59.4, C 23.2, CH3 27.9, CH3 14.7, CH3 212.2, C 30.4, CH3

41.9, CH 23.0, CH2 38.9, CH2 67.9, C 44.8, CH2 37.4, CH 73.3, CH 131.5, CH 136.6, C 58,0, C 32.5, CH3 19.8, CH3 16.1, CH3 211.7, C 25.6, CH3

41.4, CH 28.5, CH2 34.4, CH2 33.4, CH 43.7, CH2 142.7, C 118.0, CH 73.8, CH 73.2, C 57.1, C 22.5, CH3 23.1, CH3 15.1, CH3 215.7, C 43.0, CH2 59.0, CH2

47.8, CH 30.4, CH2 36.5, CH2 33.1, CH 40.9, CH2 45.8, CH 74.3, CH 86.9, CH 79.9, C 49.1, C 22.3, CH3 25.2, CH3 11.0, CH3 185.8, C 98.9, CH 189.6, CHO

In C5D5N. bIn CDCl3.

the 16-OH group. The ECD spectra computed at various levels for all sets of conformers gave moderate to good mirror-image agreement with the experimental spectrum of 1, allowing d e t e r m i n a t i o n o f t h e a b s o l u t e c o n fig ur a t io n a s (1S,4S,6R,9R,10R) (Figure 12A). OR calculations performed for 1 at the same levels as the ECD calculations for all sets of conformers produced alternating signs for the optical rotation depending on the applied conditions (including theoretical levels), because the low-energy individual conformers showed diverse OR values and opposite signs (Tables S4−S6). OR calculations performed for the CAM-B3LYP/TZVP PCM/MeCN conformers matched well with the experimental OR value, but with opposite sign, in accordance with the ECD results (e.g., the Boltzmann-averaged BH&HLYP/TZVP PCM/CH3CN OR value is +5.9 vs the −5.2 experimental value; see Table S6). The optical rotations of 1 and 2 were then measured in CHCl3 for 1 and 2 ([α]25 D +6.5 and +2.8, respectively), which were in agreement with the literature value.22,23 Because the conformational ensemble of 1 seems to be very sensitive to the applied conditions (e.g., solvent of OR measurement), it is not recommended to determine its absolute configuration on the basis of simple comparison of specific rotation values. The absolute configuration of 2 was assigned as (1S,4S,6R,9R,10R) on the basis of its similar ECD spectrum to that of 1 and based on biosynthetic considerations. The relative and absolute configurations of the core structure were preserved for 5, as indicated by the NOE and ECD spectra. The TDDFT-ECD computational protocol was also

Figure 7. Key COSY and HMBC correlations of compounds 11−13.

values for C-14, C-15, and C-16 in coicenal A (δ 186.8, 101.6, 190.6) and in 16 (δ 185.8, 98.9, 189.6) further confirmed the established structure.22 Compound 1 displayed a negative specific rotation ([α]25 D = −5.2) in CH3CN, opposite the reported small positive values of 8 23 (+)-aspermytin A ([α]25 D = +1.2, +7.6 ) in CHCl3, which suggested either an enantiomeric relationship or solvent dependence of the specific rotation of 1.24,25 To elucidate the absolute configuration of 1, the solution TDDFT-ECD26,27 and OR 4,28 calculation methods were performed on the (1R,4R,6S,9S,10S) enantiomer. The initial Merck Molecular Force Field (MMFF) conformers were reoptimized with three different DFT methods affording 6 to 10 low-energy conformers over 1% Boltzmann population (Figure S174). The rotation along the σ bond between C-10 and C-14 was identified as a crucial factor in determining the ECD curves, as it changes the relative orientation of the carbonyl group. Meanwhile, OR values are also influenced by the orientation of

Figure 8. Key NOE correlations of compounds 11−13. 2935



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Table 7. 1H NMR Data for Compounds 14−16 (500 MHz, J in Hz) 14a

15b

1 2α 2β

1.91, ov 1.23, ov 2.12, ov

3α

1.37, ov

3β

1,86, ov

2.75, dd (10.8, 3.9) 1.48, ov 1.20, dddd (12.4, 12.0, 12.0, 3.1) 1.07, dddd (14.0, 13.6, 13.6, 2.7) 1.73, ov

4 5α

1.28, ov

1.20, m 1.70, ov

5β 6 7 8 9 10 11 12 13 14 15

1.43, ov 1.90, ov 1.24, dddd (12.6, 12.5, 12.5, 3.1) 0.95, dddd (13.2, 12.6, 12.6, 3.7) 1.81, dddd (13.2, 3.3, 3.3, 3.1) 1.45, ov 0.78, ddd (12.2, 11.8, 11.5) 2.09, ddd (12.2, 4.4, 3.7) 2.18, ddd (12.5, 11.5, 10.1, 3.7) 3.55, dd (10.1, 1.0)

2.90, ddd (13.4, 3.6, 3.5) 2.38, ddd (12.0, 11.6, 10.3, 3.5) 4.21, ddd (10.3, 3.6, 1.7) 5.97, d (1.7)

2.30, ddd (14.4, 3.2, 2.8)

1.42, s 1.57, s 1.27, s

0.99, d (6.4) 1.01, s 1.32, s

0.9, d (6.6) 1.34, s 1.06, s

2.20, s

a 3.15, dt (17.8, 5.3) b 2.64, dt (17.8, 5.3) 3.88, m 2.01, s

4.98, d (8.3)

16 8OH a

16b

5.49, ddd (5.0, 2.8, 2.2) 3.72, d (2.2)

Figure 11. Key COSY, HMBC, and NOE correlations of compound 16.

though occurrence of both positive and negative OR values for the low-energy conformers indicated the possibility of sign inversion for the different conformers. Similarly to 1, the best combination for OR calculation was the BH&HLYP/TZVP PCM/CH3CN level applied for the CAM-B3LYP/TZVP PCM/CH3CN conformers (i.e., −20.9 vs the +25.8 experimental value, Table S9). Conformational analysis (Figure S176) and ECD calculations of (1R,4S,6S,9S,10S)-6 gave moderate to good mirror-image spectra of the experimental ECD curve, allowing elucidation of the absolute configuration as (1S,4R,6R,9R,10R) (Figure 12C). ECD spectra computed at various levels for all sets of conformers of (1R,4S,6R,7R,8R,9R,10S)-9 gave good overall mirror-image agreement with the experimental spectrum, allowing elucidation of the absolute configuration of 9 as (1S,4R,6S,7S,8S,9S,10R) (Figure 12D). ECD spectra computed for all sets of conformers of (1R,4S,6R,7S,10S)-14 gave moderate to good mirror-image agreement with the experimental spectrum, allowing elucidation of the absolute configuration as (1S,4R,6S,7R,10R) (Figure 12E). As 14 had the largest absolute [α]25 D value (+80.7), OR calculations were performed to check the applicability of this approach. OR calculations performed at various levels for the same sets of conformers resulted in large negative average values, and all low-energy conformers gave a negative sign, allowing application of OR calculations and verifying the ECD results (Tables S10−S12). Similarly to 1 and 5, the best mirrorimage agreement was achieved with the BH&HLYP functional, but this time for the gas phase (−78.9, Table S10) and for the B97D PCM (−83.8, Table S11) conformers. In contrast to 1, compound 14 presumably does not change sign of specific rotation in different solvents, because the ketone carbonyl moiety has the same orientation in all of the low-energy conformers. The absolute configuration of the other derivatives could be determined by comparing the experimental ECD spectra with those of the above related derivatives, which afforded the absolute configurations depicted in accordance with a common biosynthetic origin. Due to the low sample amount, a highquality ECD spectrum could not be recorded for 16, and thus the absolute configuration was assigned on the basis of a shared biosynthetic origin. The isolated compounds were evaluated in vitro for their tumor cell inhibitory activity and immune regulation activity. These compounds were not active at 30 μM toward A549, HCT-116, MCF-7, and DAOY tumor cells in in vitro tests. The in vitro immunomodulatory activity of these compounds was examined on concanavalin A (Con A)-induced splenocyte activation using flow cytometric analysis. An apoptosis analysis of these compounds to splenocytes was first conducted, and the compounds were found to have low cytotoxicty to the tested

4.08, d (1.0)

9.87, d (8.3)

In C5D5N. bin CDCl3.

Figure 9. Key COSY and HMBC correlations of compounds 14 and 15.

Figure 10. Key NOE correlations of compounds 14 and 15.

performed on (1R,4S,6S,9S,10S)-5 (Figure S175). ECD spectra computed for all sets of conformers gave moderate to good mirror-image agreement with the experimental spectrum (Figure 12B), allowing unambiguous elucidation of the absolute configuration as (1S,4R,6R,9R,10R). In contrast to 1, all combinations of OR calculations resulted in negative Boltzmann-averaged OR values for 5 (Tables S7−S9), even 2936



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Figure 12. Experimental ECD spectra of (A) 1, (B) 5, (C) 6, (D) 9, and (E) 14 in CH3CN (black curves) compared with the Boltzmann-weighted ECD spectra of (A) (1R,4R,6S,9S,10S)-1 (purple curve), (B) (1R,4S,6S,9S,10S)-5 (purple curve), (C) (1R,4S,6S,9S,10S)-6, (blue curve), (D) (1R,4S,6R,7R,8R,9R,10S)-9 (red curve), and (E) (1R,4S,6R,7S,10S)-14 (olive curve) computed for the B97D/TZVP PCM/CH3CN (A−C, E) and CAM-B3LYP/TZVP PCM/CH3CN (D) conformers. Bars represent the rotational strength values of the lowest-energy conformers, respectively.

Figure 13. Effects of compounds 1−16 on the proliferation of T cells (*p < 0.05, **p < 0.01).

cells at the concentration of 3 μM (Figure S179). The percentage of CD3+ cells and CD4+/CD8+ ratio were then analyzed by flow cytometry under this condition (compounds at 3 μM), using Con A as a positive control (5 μg/mL). We found that compounds 1, 4, and 10 significantly induced CD3+ T cell proliferation (Figure 13A). Their effects would increase when incubating together with ConA (Figure S182). Compounds 2, 7, 11, and 14 significantly increased the

CD4+/CD8+ ratio (Figure 13B), compared to the Con A control group (Figure 13). In recent years, polyketides structurally related to 1−16 have been reported from various fungi: Phomopsis sp.,29,30 Penicillium sp.,31 Craterellus odoratus,32 and Aspergillus sp.33,34 More recently, six analogues were reported from the mangrove endophytic fungus Penicillium aurantiogriseum 328#, showing a weak inhibitory activity against human aldose reductase.35 This 2937



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is the first report of immunoregulatory activity for metabolites in this structure class. For compounds 1 and 4, having a C-10 side chain, the co-occurring Δ7 and terminal OH group likely play a crucial role in the regulation of CD3+ T cell proliferation. This activity disappeared when the Δ7 was absent (1 vs 11 and 15) or the terminal hydroxy group at the side chain was acetylated (1 vs 2), etherified (1 vs 8), esterified (1 vs 6 and 7), or substituted by a methyl group (4 vs 5). The length of the side chain and the 4-OH seemed to have a minor contribution to the activity considering the activities of 1 and 4, 2 and 3, and 4 and 5, respectively. When the terminal hydroxy group (16OH) was esterified to a six-membered lactone, Δ7 was no longer crucial to the activity (6 and 7), whereas the 6-OH was recognized to be important (9 vs 10). The above-mentioned crucial factors for the CD3+ T cell proliferation did not significantly affect the ratio of CD4+/CD8+. This interesting discovery may promote an ongoing investigation on these metabolites and their immune system regulation activities.

■

H2O, 68:32, 2 mL/min) afforded 1 (44.3 mg, tR = 32 min). Fr.14 was separated by CC on Sephadex LH-20 (CH2Cl2/MeOH, 2:1) to give five subfractions (Fr.14.1−14.5). Fr.14.3 was purified by HPLC (MeOH/H2O, 49:51, 2 mL/min) to afford 6 (9.2 mg, tR = 58 min). Fr.14.4 was separated by CC on silica gel (petroleum ether/2propanol, 20:1) to yield nine additional subfractions (Fr.14.4.1− 14.4.9). Fr.14.4.3 was purified by HPLC (MeOH/H2O, 58:42, 2 mL/ min) to afford 12 (11.2 mg, tR = 30 min) and 4 (8.3 mg, tR = 45 min), and Fr.14.4.6 was purified by HPLC (MeOH/H2O, 47:53, 2 mL/min) to afford 10 (0.8 mg, tR = 51 min) and 5 (4.9 mg, tR = 25 min), respectively. Fr.15 was separated by CC on Sephadex LH-20 (CH2Cl2/MeOH, 2:1) to give eight subfractions (Fr.15.1−15.8). Fr. 15.4 was further purified by CC on silica gel (petroleum ether/ acetone, 5:1) and HPLC (MeOH/H2O, 55:45, 2 mL/min) to afford 9 (14.0 mg, tR = 47 min) and 3 (10.2 mg, tR = 66 min). Fr.15.5 was separated by HPLC (MeOH/H2O, 53:47, 2 mL/min) to afford 14 (2.6 mg, tR = 55 min), 8 (1.6 mg, tR = 42 min), and 16 (0.8 mg, tR = 25 min). Fr.15.6 was purified by CC on RP-18 silica gel (MeOH/H2O, 55:45) and HPLC (MeOH/H2O, 45:55, 2 mL/min) to give 7 (4.0 mg, tR = 33 min), 15 (2.9 mg, tR = 50 min), and 11 (12.6 mg, tR = 65 min). Aspermytin A (1): colorless crystals (MeOH); mp 148−149 °C; Rf = 0.50 (PE/Me2CHOH, 10:1); [α]25 D −5 (c 0.0045, CH3CN), +6.5 (c 0.0090, CHCl3); ECD (CH3CN, c 0.0043 M) λmax (Δε) 294.5 (−1.07), 211.5 (+1.60), 196 (−3.37), 192 (−1.99), 190 (−1.45) nm; HREIMS m/z 266.1870 [M]+ (calcd for C16H26O3, 266.1876). Aspermytin A acetate (2): colorless crystals (MeOH); mp 131− 132 °C; Rf = 0.50 (PE/Me2CO, 3:1); [α]25 D −10 (c 0.0039, CH3CN), +2.8 (c 0.0078, CHCl3); ECD (CH3CN, c 0.0033 M) λmax (Δε) 294.5 (−0.90), 211 (+1.33), 196 (−2.36), 190 (−1.11) nm; HREIMS m/z 308.1982 [M]+ (calcd for C18H28O4, 308.1982). Libertalide A (3): colorless crystals (MeOH); mp 174−175 °C; Rf = 0.60 (CH2Cl2/Me2CO 4:1); [α]25 D +8 (c 0.0037, CH3CN); ECD (CH3CN, c 0.0037 M) λmax (Δε) 294.5 (−0.38), 211 (+0.65), 196.5 (−1.13), 190 (−0.15) nm; IR (film) νmax 3360, 2922, 1732, 1372, 1248, 1091, 1040, 804,730 cm−1; 1H and 13C NMR data, Tables 1 and 2; HREIMS m/z 324.1925 [M]+ (calcd for C18H28O5, 324.1931). Libertalide B (4): colorless crystals (MeOH); mp 163−164 °C; Rf = 0.50 (PE/Me2CO, 10:1); [α]25 D +14 (c 0.0050, CH3CN); ECD (CH3CN, c 0.0050 M) λmax (Δε) 221 (+0.24), 191.5 (−4.41), 190 (−4.22) nm; IR (film) νmax 3460, 2920, 1699, 1454, 1377, 1238, 1121, 735 cm−1; 1H and 13C NMR data, Tables 1 and 2; HREIMS m/z 238.1563 [M]+ (calcd for C14H22O3, 238.1563). Libertalide C (5): colorless oil; Rf = 0.50 (CH2Cl2/MeOH, 10:1); [α]25 D +26 (c 0.0048, CH3CN); ECD (CH3CN, c 0.0048 M) λmax (Δε) 294.5 (−2.85), 212.5 (+0.53), 197.5 (−1.70), 190 (−0.15) nm; IR (film) νmax 3406, 2962, 2925, 1688, 1260, 1096, 802, 735 cm−1; 1H and 13 C NMR data, Tables 1 and 2; HREIMS m/z 252.1703 [M]+ (calcd for C15H24O3, 252.1720). Libertalide D (6): white powder; Rf = 0.50 (PE/Me2CHOH, 8:1); [α]25 D +21 (c 0.0045, CH3CN); ECD (CH3CN, c 0.0045 M) λmax (Δε) 190 (−3.50) nm; IR (film) νmax 3452, 2926, 1722, 1271, 1133, 1092, 1056, 968 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 287.1622 [M + Na]+ (calcd for C16H24O3Na, 287.1623). Libertalide E (7): white powder, Rf = 0.50 (CH2Cl2/Me2CO, 5:1); [α]25 D +28 (c 0.0043, CH3CN); ECD (CH3CN, c 0.0043 M) λmax (Δε) 231.5 (+0.14), 198 (−1.37), 190 (−2.79) nm; IR (film) νmax 3429, 2924, 1708, 1291, 1135, 1087, 1043, 975 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 303.1564 [M + Na]+ (calcd for C16H24O4Na, 303.1572). Libertalide F (8): white powder, Rf = 0.60 (CH2Cl2/Me2CO, 2:1); [α]25 D −51 (c 0.0045, CH3CN); ECD (CH3CN, c 0.0045 M) λmax (Δε) 190 (−2.87) nm; IR (film) νmax 3424, 2925, 2860, 1451, 1372, 1133, 1084, 886, 851, 616 cm−1; 1H and 13C NMR data, Tables 2 and 3; HREIMS m/z 266.1879 [M]+ (calcd for C16H26O3, 266.1876). Libertalide G (9): white solid; mp 166−167 °C; Rf = 0.60 (CH2Cl2/ Me2CO, 4:1); [α]25 D +15 (c 0.0043, CH3CN); ECD (CH3CN, c 0.0043 M) λmax (Δε) 216.5 (+0.60), 202 (+0.39), 190 (+1.28) nm; IR (film) νmax 3449, 2923, 1711, 1263, 1143, 1091, 1058, 972 cm−1; 1H and 13C NMR data, Table 4; HRESIMS m/z 303.1566 [M + Na]+ (calcd for C16H24O4Na, 303.1572).

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in MeOH with an Autopol IV polarimeter at the sodium D line (590 nm). UV absorption spectra were recorded with a Hitachi U-3010 spectrophotometer. ECD spectra were recorded with a Jasco715 spectropolarimeter. Infrared spectra were recorded in thin polymer films on a Nexus 470 FT-IR spectrophotometer (Nicolet, USA). NMR spectra were recorded at 300 K on Bruker DRX 600, DRX 500, and Varian Inova-400 spectrometers. Chemical shifts were referenced to the residual CDCl3 signal (δH 7.26; δC 77.0), CD3OD signal (δH 3.31; δC 49.0), and C5D5N signal (δH 8.74, 7.58, 7.22; δC 150.3, 135.9, 123.9) as internal standards for 1H and 13C NMR. The 1 H and 13C NMR assignments were supported by COSY, HMQC, HMBC, and NOESY experiments. The mass spectra and highresolution mass spectra were acquired on a Waters Q-TOF Micro mass spectrometer and Thermo Scientific DFS Magnetic Sector GCHRMS. Semipreparative HPLC was performed on an Agilent 1100/ 1200 system equipped with a refractive index detector using a YMC Pack ODS-A column (5 μm, 250 × 10 mm). Commercial silica gel (Yantai, 200−300 and 400−500 mesh) was used for column chromatography (CC). Precoated silica gel plates (Yantai, HSGF254) were used for analytical thin-layer chromatography (TLC). Spots were detected on TLC under UV or by heating after spraying with anisaldehyde sulfuric acid reagent. Fungal Material. The fungus was isolated from the internal tissues of the soft coral Sinularia sandensis collected by Dr. Yalan Chou from the Dongsha Atoll in the South China Sea at a depth of 15 m, in September 2015, and was identified as Libertasomyces sp. (GenBank accession number MF975701) by sequence analysis of the ITS region of the rDNA. A voucher strain of this fungus (internal strain no. DYZ027) was deposited at the Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University. Culture, Extraction, and Isolation. The fungus Libertasomyces sp. was cultivated on 12 L of 5% w/v biomalt (Villa Natura, Germany) solid agar medium (20 g/L biomalt, 15 g/L agar, 800 mL/L ASW) at room temperature for 28 days.6,7 The culture medium was extracted with EtOAc (5 × 3 L) to afford a residue (7.8 g) after removal of the solvent under reduced pressure. The extract was subjected to CC on silica gel eluting with a gradient of EtOAc in petroleum ether (EtOAc/ PE, 1:80 to 1:1) to give 15 fractions (Fr.1−15). Fr.12 was purified by CC on Sephadex LH-20 (CH2Cl2/MeOH, 2:1) to give six subfractions (Fr.12.1−12.6). Fr.12.4 was further separated by CC on silica gel (petroleum ether/acetone, 10:1) followed by HPLC (MeOH/H2O, 65:35, 2 mL/min) to afford 2 (43.8 mg, tR = 40 min), while Fr.12.6 was purified by HPLC (MeOH/H2O, 60:40, 2 mL/min) to afford 13 (3.1 mg, tR = 54 min). Fr.13 was first subjected to CC on Sephadex LH-20 (CH2Cl2/MeOH, 2:1). Subsequent CC on silica gel (petroleum ether/2-propanol, 30:1) followed by HPLC (MeOH/ 2938



Article

Libertalide H (10): colorless crystals (MeOH); mp 144−145 °C; Rf = 0.40 (CH2Cl2/MeOH, 10:1); [α]25 D +43 (c 0.0027, CH3CN); ECD (CH3CN, c 0.0041 M) λmax (Δε) 215 (+0.59), 202 (+0.33), 190 (+1.05) nm; IR (film) νmax 3353, 2923, 1704, 1459, 1272, 1057, 969, 868 cm−1; 1H and 13C NMR data, Table 4; HREIMS m/z 296.1623 [M]+ (calcd for C16H24O5, 296.1618). Libertalide I (11): colorless oil; Rf = 0.60 (CH2Cl2/MeOH, 10:1); [α]25 D −27 (c 0.0038, CH3CN); 199 nm; ECD (CH3CN, c 0.0043 M) λmax (Δε) 294 (−0.94), 194.5 (+1.88), 190 (+1.56) nm; IR (film) νmax 3380, 2925, 1693, 1454, 1386, 1032, 733 cm−1; 1H and 13C NMR data, Tables 5 and 6; HRESIMS m/z 341.1521 [M + Na]+ (calcd for C16H2735ClO4Na, 341.1517). Libertalide J (12): colorless oil; Rf = 0.60 (PE/Me2CHOH, 10:1); [α]25 D −10 (c 0.0033, CH3CN); ECD (CH3CN, c 0.0033 M) λmax (Δε) 294.5 (−1.00), 194 (+2.05), 190 (+1.83) nm; IR (film) νmax 3453, 2925, 1740, 1458, 1385, 1243, 1035, 804 cm−1; 1H and 13C NMR data, Tables 5 and 6; HRESIMS m/z 383.1584 [M + Na]+ (calcd for C18H2935ClO5Na, 383.1601). Libertalide K (13): colorless crystals (MeOH); mp 136−137 °C; Rf = 0.60 (CH2Cl2/Me2CO, 3:1); [α]25 D −30 (c 0.0042, CH3CN); ECD (CH3CN, c 0.0041 M) λmax (Δε) 294.5 (−0.50), 194.5 (+1.26), 190 (+1.01) nm; IR (film) νmax 3451, 2925, 1688, 1455, 1385, 1355, 1115, 1029, 736, 699 cm−1; 1H and 13C NMR data, Tables 5 and 6; HRESIMS m/z 311.1387 [M + Na]+ (calcd for C15H2535ClO3Na, 311.1390). Libertalide L (14): colorless crystals (MeOH); mp 170−171 °C; Rf = 0.50 (CH2Cl2/Me2CO, 3:1); [α]25 D +81 (c 0.0048, CH3CN); ECD (CH3CN, c 0.0048 M) λmax (Δε) 293 (+1.41), 201 (+2.26), 190 (+0.37) nm; IR (film) νmax 3329, 2924, 1703, 1450, 1375, 1259, 1107, 999, 909, 810 cm−1; 1H and 13C NMR data, Tables 6 and 7; HREIMS m/z 252.1696 [M]+ (calcd for C15H24O3, 252.1720). Libertalide M (15): colorless oil; Rf = 0.40 (CH2Cl2/Me2CO, 5:1); [α]25 D −31 (c 0.0042, CH3CN); ECD (CH3CN, c 0.0043 M) λmax (Δε) 294 (−2.23), 211 (+0.37), 197.5 (+0.08), 190 (+0.89) nm; IR (film) νmax 3405, 2925, 1693, 1454, 1381, 1034 cm−1; 1H and 13C NMR data, Tables 6 and 7; HREIMS m/z 282.1832 [M]+ (calcd for C16H26O4, 282.1826). Libertalide N (16): colorless oil, Rf = 0.50 (CH2Cl2/Me2CO, 2:1); [α]25 D +12 (c 0.0029, CH3CN); UV (CH3CN) λmax (log ε) 264 (2.85) nm; IR (film) νmax 3374, 2924, 2854, 1732, 1641, 1459, 1396, 1311, 1041, 1001 cm−1; 1H and 13C NMR data, Tables 6 and 7; HRESIMS m/z 303.1576 [M + Na]+ (calcd for C16H24O4Na, 303.1572). Cytotoxicity Assay. The cytotoxicities of the isolated compounds were evaluated against A549 human lung carcinoma cells, HCT-116 human colon carcinoma cells, MCF-7 human breast carcinoma cells, K562 human erythroleukemia cells, and DAOY medulloblastoma cells. A549, HCT116, and MCF-7 cell lines were grown in DMEM medium supplemented with 10% FBS (fetal bovine serum), K562 was cultured in RPMI 1640 supplemented with 10% FBS, and DAOY cells were cultivated in MEM. All cell lines were incubated at 37 °C with 5% CO2 in air atmosphere and treated with the isolated compounds (30 μM). Adriamycin was used as a positive control. Relative cell viability was assayed using a CellTiter-Blue Cell viability assay kit at 530/590 nm after 48 h.36 Immune Regulation Activity Assay. Splenocytes (2 × 106 cell/ mL) from C57BL/6 mice were incubated with compounds for 24 h, using Con A (5 μg/mL) as a positive control.37,38 The cells were collected and washed with PBS, and then samples were immediately detected by a FACScan flow cytometer (BD) for apoptosis. To investigate the effects of compounds on the proliferation of T cell subtypes, Con A-stimulated splenocytes were incubated with compounds (3 μM) for 24 h. The cells were collected and stained with PE-CD3, FITC-CD4, and Percp/cy5.5-CD8, respectively. The percentage of CD3+T, CD4+T, and CD8+T cells was analyzed by flow cytometry.39 The values are presented as means ± SD (n = 3). *p < 0.05 and **p < 0.01 versus Con A control treatment. Computational Section. Mixed torsional/low-frequency mode conformational searches were carried out by means of the Macromodel 10.8.011 software using the MMFF with an implicit solvent model for CHCl3.40 Geometry reoptimizations were carried out at the

B3LYP/6-31G(d) level in vacuo and at the B97D/TZVP7,41 and the CAMB3LYP/TZVP42 levels with the PCM solvent model for MeCN. TDDFT ECD calculations and OR calculations were run with various functionals (B3LYP, BH&HLYP, CAM-B3LYP, PBE0) and the TZVP basis set as implemented in the Gaussian 09 package with the same or no solvent model as in the preceding DFT optimization step.43 ECD spectra were generated as sums of Gaussians with 3000 and 3900 cm−1 widths at half-height (corresponding to ca. 13 and 17 at 210 nm), using dipole-velocity-computed rotational strength values.44 Boltzmann distributions were estimated from the ZPVE-corrected B3LYP energies in the gas-phase calculations and from the uncorrected B97D/TZVP and CAM-B3LYP/TZVP energies in the PCM ones. The MOLEKEL software package was used for visualization of the results.45

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00463. Supplementary tables and figures of the MS and NMR spectra for 3−16; OR values of the low-energy conformers for 1, 5, and 14 of the computed structures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86 21 81871257. E-mail: [email protected]. ORCID

Attila Mándi: 0000-0002-7867-7084 Chun-Lin Zhuang: 0000-0002-0569-5708 Wen Zhang: 0000-0002-5747-4413 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research work was financially supported by NSFC (U1405227, 41576157, 81573342, 81502978), the Program of Shanghai Subject Chief Scientist (15XD1504600), the key project of STCSM (14431902900, 14401902600), and the Shanghai “ChenGuang” project. T.K. and A.M. thank the National Research, Development and Innovation Office (NKFI K120181 and PD121020) for financial support and the Governmental Information-Technology Development Agency (KIFÜ ) for CPU time.

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REFERENCES

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Immunomodulatory Polyketides from a Phoma-like Fungus Isolated

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