Anti-inflammatory Mono- and Dimeric Sorbicillinoids from the Marine

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Anti-inflammatory Mono- and Dimeric Sorbicillinoids from the Marine-Derived Fungus Trichoderma reesei 4670 Panpan Zhang,† Yanlian Deng,§ Xiaojing Lin,⊥ Bin Chen,† Jing Li,† Hongju Liu,§ Senhua Chen,*,†,∥ and Lan Liu*,†,‡,∥ †

School of Marine Sciences, Sun Yat-Sen University, Guangzhou 510006, People’s Republic of China Key Laboratory of Functional Molecules from Oceanic Microorganisms, Department of Education of Guangdong Province, Sun Yat-Sen University, Guangzhou 510006, People’s Republic of China § School of Pharmacy, Guangdong Medical University, Dongguan 523808, People’s Republic of China ⊥ Research Center of Chinese Herbal Resource Science and Engineering, Guangzhou University of Chinese Medicine, Guangzhou 510006, People’s Republic of China ∥ Southern Laboratory of Ocean Science and Engineering, Zhuhai 519080, People’s Republic of China

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S Supporting Information *

ABSTRACT: Eight new dimeric sorbicillinoids (1−3, 5−9) and 12 new monomeric sorbicillinoids (10−20, 25), along with five known analogues (4 and 21−24), were isolated from the marine-derived fungus Trichoderma reesei 4670. Their structures were elucidated on the basis of extensive spectroscopic analyses (1D and 2D NMR, HR-ESIMS, and ECD) and X-ray crystallography. Compound 1, containing a pyrrolidin-2-one moiety, is reported for the first time in the sorbicillinoid family. Compounds 8 and 9 are the first examples of bisorbicillinoids possessing a benzofuro[2,3h]chromene scaffold from a natural source. Compounds 3− 11, 13−16, 18, 21, 22, 24, and 25 exhibited potent antiinflammatory activity by inhibiting the production of NO in RAW264.7 cells activated by lipopolysaccharide with IC50 values in the range from 0.94 to 38 μM. Structure−activity relationships of the sorbicillinoids were discussed.

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bioactivity, and structure−activity relationships of the isolated sorbicillinoid derivatives.

orbicillinoids are a family of polyketides with a cyclic hexaketide nucleus with a sorbyl side chain.1 Since the first sorbicillinoid was reported in 1948 by Cram et al.,2 more than 100 analogues have been discovered mainly in 10 genera of fungi: Acremonium, Aspergillus/Emericella, Clonostachys, Paecilomyces, Penicillium, Phaeoacremonium, Phialocephala, Scytalidium, Trichoderma, and Verticillium.3 According to the structural features, sorbicillinoids can be classified into monomers, bisorbicillinoids, trisorbicillinoids, and hybrid sorbicillinoids.1 The unique and diverse structural features of the sorbicillinoid family result in various pharmacological activities, including cytotoxic,4,5 antimicrobial,4 antiviral,6 antiinflammatory,7 and radical-scavenging activities.8 In our ongoing search for bioactive secondary metabolites from marine-derived fungi, the fungus Trichoderma reesei 4670 was found to be an abundant producer of diverse secondary metabolites. Chemical investigation of an extract of T. reesei 4670 from rice medium led to the discovery of 25 sorbicillinoids (1−25), including eight new dimeric sorbicillinoids (1−3, 5−9) and 12 new monomeric sorbicillinoids (10− 20, 25). Herein, we report the preparation, characterization, © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The EtOAc extract of T. reesei strain 4670 was subjected to repeated silica gel, Sephadex LH-20, and RP-C18 gel column chromatography, followed by semipreparative HPLC, to afford 20 new (1−3, 5−20, and 25) and five known sorbicillinoids (4 and 21−24) (Chart 1). Compound 1 was obtained as a colorless powder. The molecular formula of 1 was established as C19H23NO5 based on the HR-ESIMS data accounting for nine degrees of unsaturation. The IR spectrum of 1 indicated the presence of hydroxy (3362 cm−1) and carbonyl (1734 cm−1) groups. Its 1H NMR spectrum (Table 1) displayed signals corresponding to four olefinic protons (H-10 to H-13), three methines (H-4, H7, H-8), one methylene (H2-16), and four methyls (H3-14, H317, H3-18, H3-19). The 13C NMR and DEPT data (Table 1) of 1 showed the presence of 19 carbons. Six sp2-hybridized Received: December 7, 2018

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DOI: 10.1021/acs.jnatprod.8b01029 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

The relative configuration (1R*,4S*,5S*,7S*,8S*)-1 was determined by the NOESY correlations of H-4 with H-10 and H3-18 and of H-7 with H-8, H3-17, and H3-19 (Figure 2), as well as X-ray crystallographic analysis (Figure 3). The X-ray structure clearly showed that the configurations of the three double bonds in the typical sorbyl side chain are trans in accordance with the 1H NMR coupling constants (Table 1). The quality of the X-ray data was insufficient to determine the absolute configuration, so the absolute configuration of 1 was deduced by the comparison of experimental ECD and calculated ECD spectra. The theoretical ECD spectra were calculated by a quantum chemical method at the [RB3LYP/6311+G(2d,p)] level, and the predicted ECD curve of (1R,4S,5S,7S,8S)-1 was in good agreement with that of the experimental one (Figure S1). Therefore, the structure of 1 was established as (1R,4S,5S,7S,8S)-1 and named trichosorbicillin A. Compound 2 was obtained as a yellow amorphous powder. Its molecular formula was C28H34O10 according to the HRESIMS peak at m/z 529.2085 [M − H]−. The 1H and 13C NMR data of 2 showed 27 protons and 28 carbons, and the multiplicity of the carbon signals was classified into four

carbons (δC 170.9, 144.5, 141.1, 132.2, 119.0, 108.2) belonged to three double bonds, while three sp2 nonprotonated carbons (δC 210.1, 198.1, 177.8) were resonances for three carbonyl groups. The planar structure of 1 was identified by interpretation of the COSY and HMBC spectra (Figure 1). The key HMBC correlations from H-4 to olefinic carbon C-3 and keto carbon C-2, from H3-17 to C-1, C-2, and C-6, and from H3-18 to methine C-4, oxygenated carbon C-5, and C-6 indicated a substituted 1,5-dimethyl-5-hydroxycyclohexane-2,6-dione ring. The center bicycle[2.2.2]octane-2,6-dione ring was assigned by the COSY correlations of H-8 with H-4 and H-7, along with the HMBC correlation from H-17 to C-7. A pyrrolidin-2-one ring was fused to C-7 and C-8 of the center bridged bicyclic ring, which was established by the HMBC correlations from H7, H-8, and H2-16 to carbonyl C-15 and from N-methyl protons H3-19 to C-7 and C-15. A typical sorbyl side chain with three double bonds was linked to C-3 of the bicyclic ring, evidenced by a series of mutually coupled resonances from H10 to H3-14 in the COSY spectrum and from the HMBC correlations from H-10 to C-9 and H-4 to C-9. B



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compound bisvertinol (4).9 The main difference between them was that the two methyls of the two side chains in 4 were replaced by two hydroxymethylenes (δC 63.0 × 2, C-15, C-24; δH 4.19 × 2, H2-15, H2-24) in 2. COSY and HMBC experiments allowed the complete assignment for the planar structure of 2. The relative configuration of 2 was determined by a NOESY experiment. The correlations of H3-17/H-9a, H9a/H3-25, and H-20/H3-25 were observed in the NOESY spectrum (Figure 2), which indicated H-9a, H3-17, and CH325 were syn-oriented. According to the enhanced stability of a cis 5−6 ring junction over a trans 5−6 ring junction, the 4a-OH should be cis with H-9a, H-17, and CH3-25 as Andrade et al. reported.2 Also, the configuration of C-4 should be identical as shown in Chart 1 based on the biogenetic relationship to bisvertinol.2,9 The electronic circular dichroism (ECD) spectrum of 2 showed strong split Cotton effects at 345 (40.26) and 425 (−48.71) nm, which was similar to that of 4 (Figure S1). Hence, the absolute configuration of 2 was assigned as 4S, 4aR, 5aS, 9aR, and 9bR, and the trivial name was given as 15,24-dihydroxybisvertinol. Compound 3 was obtained as a yellow amorphous powder and was found to have a molecular formula of C28H34O9 based on HR-ESIMS. The NMR data of 3 were very similar to those of 2 except for a methyl at C-15 in 3 instead of the hydroxymethylene, which could be proved via a series of mutually coupled resonances of H2-15/H-14, H-14/H-13, H13/H-12, and H-12/H-11 in the COSY spectrum, along with the HMBC correlation from H-12 to C-10 (Figure 1). In light of quite similar NOESY correlations (Figure 2) and ECD

Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Spectroscopic Data for Compound 1 in CD3OD no.

δC, type

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

67.0, C 198.1, C 108.2, C 47.3, CH 75.2, C 210.1, C 69.8, CH 30.1, CH 170.9, C 119.0, CH 144.5, CH 132.2, CH 141.1, CH 18.9, CH3 177.8, C 35.2, CH2

17 18 19

11.7, CH3 24.2, CH3 32.1, CH3

δH, mult (J in Hz)

3.19, d (2.4)

3.88, d (8.8) 3.52, m 6.45, 7.37, 6.45, 6.26, 1.91,

m dd (14.8, 10.8) m m d (6.8)

2.70, 2.07, 1.44, 1.19, 2.83,

dd (17.6, 11.2) dd (17.6, 3.6) s s s

methyls, three methylenes, nine methines, and 12 nonprotonated carbons including two carbonyls according to the HSQC spectrum. Analysis of the NMR data of 2 (Table 2) revealed its structure possessed great similarity to the known

Figure 1. Key COSY and HMBC correlations of 1−3, 5−20, and 25. C



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Figure 2. Key NOESY correlations of 1−3, 5−9, and 12.

Figure 3. Single-crystal X-ray structures of 1, 10, 12, and 16. Thermal ellipsoids are drawn at the 50% probability level. Disordered positions of 1 are not included for clarity.

Compounds 6 and 7 were both obtained as yellow amorphous powders. The NMR spectroscopic data were very similar to those of 5, except for the presence of signals for a hydroxymethylene (δC 63.0, C-24; δH 4.21, d, J = 4.4, H-24) in 6 and two hydroxymethylenes (δC 63.5, C-15; δH 4.02, d, J = 5.2, H2-15; δC 63.0, C-24; δH 4.22, d, J = 4.4, H2-24) in 7. These proposed structures were verified by the coupling of H14/H2-15 and H-23/H2-24 in the COSY spectrum. At the same time, 6 and 7 shared the same absolute configurations of 4S, 4aR, 5aS, 9aR, and 9bR as those of compound 5 because of their similar NOESY correlations (Figure 2) and Cotton effects in the ECD spectra (Figure S1). Hence, the complete structures of 6 and 7 were identified and named trichobisvertinol B and trichobisvertinol C, respectively. Trichobisvertinol D and 12-epi-trichobisvertinol D (8 and 9) had the same molecular formula of C28H34O8 with 12 degrees of unsaturation and shared the same planar structure based on

spectra (Figure S1) of 3 and 4, the absolute configuration of 3 was established as 4S, 4aR, 5aS, 9aR, and 9bR. Thus, the new compound 3 was named 24-hydroxybisvertinol. Results from the HR-ESIMS analysis suggested that the molecular formula of compound 5 was C28H36O8. Comparison of the NMR data of 5 with those of the known compound 4 indicated that 5 possessed an identical bisvertinol skeleton to that of 4. However, the HMBC correlations of 5 were observed from H3-25 to C-1 (δC 182.0) and from H-11 to C-10 (δC 201.1), suggesting that a carbonyl is at C-10 of the side chain. The double bond (C-11 and C-12) in the side chain connected to C-2 in 4 was hydrogenated in 5, which was confirmed by the COSY correlation of H-11/H-12 and the HMBC correlation from H-11 to C-10. The absolute configuration of 5 was the same as for 4 based on comparing the NOESY (Figure 2) and ECD spectra of 5 and 4 (Figure S1). Therefore, 5 was identified as shown in Chart 1 and named trichobisvertinol A. D



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Table 2. 1H (400 MHz) and 13C NMR (100 MHz) Spectroscopic Data for Compounds 2, 3, and 5−7 in CD3OD 2 no.

δC, type

1 2 3

195.1, C 106.4, C 36.4, CH2

4 4a 5a 6 7 8 9 9a 9b 10 11

74.2, C 107.3, C 80.6, C 169.8, C 110.7, C 193.3, C 103.1, C 55.0, CH 60.5, C 178.7, C 123.6, CH

12 13

142.3, CH 143.1, CH

14 15 16 17 18 19 20 21

130.3, CH 63.0, CH2 22.7, CH3 25.8, CH3 7.1, CH3 167.8, C 123.7, CH 138.3, CH

22

140.0, CH

23 24 25

δH (J in Hz)

2.71, d (14.4), H-3a 2.43, d (14.4), H-3b

3.63, s

6.39, d (14.8) 7.26, m 6.24, dt (15.2, 4.8) 6.50, m 4.19, m 1.17, s 1.41, s 1.39, s 6.59, d (14.8) 7.19, m

3 δC, type 194.1, C 106.1, C 36.5, CH2

74.2, C 107.3, C 80.5, C 169.4, C 110.7, C 193.5, C 103.1, C 54.9, CH 60.4, C 179.8, C 132.2, CH

5

δH (J in Hz)

2.73, dd (14.4, 6.8), H-3a 2.46, dd (14.4, 6.0), H-3b

3.66, t (3.2)

6.44, m

143.5, CH 140.6, CH

7.24, m 6.20, m


6.31, 1.89, 1.21, 1.44, 1.42,

m d (6.4) s s s

6.62, d (14.8) 7.24, m

δC, type 182.0, C 105.6, C 37.0, CH2

74.1, C 107.0, C 80.3, C 170.3, C 109.6, C 193.3, C 102.6, C 54.3, CH 58.0, C 201.1, C 38.3, CH2

6

δH (J in Hz)

2.65, d (14.4), H-3a 2.36, d (14.4), H-3b

δC, type 182.0, C 105.6, C 37.0, CH2

7

δH (J in Hz)

2.67, d (14.4), H-3a 2.38, d (14.4), H-3b

δC, type 181.7, C 105.6, C 37.0, CH2

δH (J in Hz)

2.68, d (14.4), H-3a 2.40, d (14.4), H-3b

2.44, dd (15.2, 8.0) 28.6, CH2 2.22, m 130.9, CH 5.46, m

74.1, C 107.1, C 80.3, C 167.4, C 109.8, C 193.6, C 103.1, C 54.3, CH 58.1, C 201.2, C 38.3, CH2

2.46, m

74.1, C 107.0, C 80.3, C 170.1, C 109.7, C 193.5, C 103.1, C 54.3, CH 58.0, C 201.1, C 37.9, CH2

28.6, CH2 130.9, CH

2.24, m 5.50, m

28.1, CH2 131.3, CH

2.31, m 5.70, m



5.50, 1.65, 1.21, 1.46, 1.45,


5.70, 4.02, 1.22, 1.46, 1.47,

3.65, s

5.46, 1.63, 1.19, 1.43, 1.45,

m d (4.8) s s s

140.0, CH

6.20, m

129.6, CH

6.15, dt (15.2, 4.8) 6.50, m

6.47, d (14.8) 7.16, dd (14.8, 10.8) 137.0, CH 6.09, m

130.3, CH

6.53, m

132.5, CH 6.32, m

139.9, CH

63.0, CH2 20.1, CH3

4.19, m 1.25, s

63.0, CH2 20.0, CH3

4.22, d (4.0) 1.28, s

18.7, CH3 19.3, CH3

63.0, CH2 19.2, CH3

1.86, d (6.0) 1.28, s

130.4, CH

3.67, s

m d (4.8) s s s

6.61, d (14.8) 7.22, dd (14.8, 10.8) 6.53, m 6.17, dt (15.2, 5.2) 4.21, d (4.4) 1.30, s

130.3, CH 139.9, CH 63.0, CH2 19.2, CH3

3.69, s

2.52, m

m d (5.2) s s s

6.62, d (14.8) 7.24, dd(14.8, 10.8) 6.54, m 6.18, dt(15.2, 5.2) 4.22, d (4.4) 1.30, s

at C-12 were 12R* for 8 and 12S* for 9, respectively. The absolute configurations of 8 and 9 were established by comparing the experimental ECD data with the calculated ones. The ECD spectra of four configurations, (4S, 4aR, 5aS, 9aR, 9bR, 12R), (4S, 4aR, 5aS, 9aR, 9bR, 12S), (4R, 4aS, 5aR, 9aS, 9bS, 12S), and (4R, 4aS, 5aR, 9aS, 9bS, 12R), were calculated. The theoretical ECD curves of (4S, 4aR, 5aS, 9aR, 9bR, 12R) and (4S, 4aR, 5aS, 9aR, 9bR, 12S) agreed well with the experimental curves of 8 and 9. Although the quite similar experimental ECD spectra of 8 and 9 suggest that the C-12 configuration has a minimal effect on the ECD spectrum, the absolute configurations of (4S,4aR,5aS,9aR,9bR,12R)-8 and (4S,4aR,5aS,9aR,9bR,12S)-9 could be confidently determined through a combination of NOESY and ECD experiments. Compound 10 was isolated as a white powder. The molecular formula of 10 was C14H16O4 according to the deprotonated molecule ion peak at m/z 247.0977 [M − H]− in the HR-ESIMS spectrum. Its 1H NMR and 13C NMR data displayed signals corresponding to two methyls, two methylenes, four methines, and six nonprotonated carbons including one carbonyl, indicating the presence of a chromanone skeleton, which was similar to the known compound (2R)-2,3-dihydro-7-hydroxy-6,8-dimethyl-2-[(E)propenyl] chromen-4-one.10 The main difference between

the HR-ESIMS and NMR data (Table 3). The different 20 specific rotations ([α ]20 D −150 (c 0.33, MeOH) of 8, [α ]D −130 (c 0.15, MeOH) of 9) suggested that 9 was a stereoisomer of 8. Their 1H and 13C NMR spectra had similar features compared to those of 5, which suggested that 8 and 9 possessed a similar bisvertinol core with 11 degrees of unsaturation. In consideration of the remaining degree of unsaturation, compounds 8 and 9 should have an additional ring. Characteristic chemical shifts of an oxymethine at C-12 (δC 79.2 in 8; δC 80.6 in 9) and the HMBC correlations from H-11 to C-1, C-2, and C-10 (Figure 1) indicated an ether linkage between C-1 and C-12, forming a 2,3-dihydro-4H-pyran-4-one ring. The relative configurations, (4S*,4aR*,5aS*,9aR*,9bR*)-8 and (4S*,4aR*,5aS*,9aR*,9bR*)-9, were determined by the NOESY correlations of H-9a/H3-17 and H-9a/H3-25, as well as the enhanced stability of a cis 5−6 ring junction and biosynthetic considerations.2,9 Their 13C NMR data were almost identical, except for the chemical shift of C-11 and C12, whose chemical shifts were δC 41.1 (C-11) and δC 79.2 (C12) in 8, but δC 42.3 (C-11) and δC 80.6 (C-12) in 9. It is suggested that the epimers (8 and 9) have a different configuration at C-12. The NOESY correlations of H-12/H318 in 8 and H-12/H3-25 in 9 confirmed that the configurations E



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Table 3. 1H (400 MHz) and 13C (100 MHz) NMR Spectroscopic Data for Compounds 8 and 9 in CD3OD 8 no.

δC, type

1 2 3

172.1, C 110.1, C 33.1, CH3

4 4a 5a 6 7 8 9 9a 9b 10 11

74.4, C 107.5, C 80.3, C 172.1, C 108.6, C 193.7, C 102.8, C 53.9, CH 57.7, C 193.7, C 41.1, CH2

12 13

79.2, CH 128.6, CH

14 15 16 17 18 19 20 21 22 23 24 25

131.7, CH 18.0, CH3 22.2, CH3 26.6, CH3 7.8, CH3 168.0, C 122.0, CH 139.1, CH 132.5, CH 137.2, CH 18.7, CH3 19.7, CH3

9

δH (J in Hz)

2.54, d (15.2), H-3a 2.26, d (15.2), H-3b

3.67, s

2.44, dd (16.8, 13.2) 2.32, dd (16.8, 4.0) 4.26, m 5.47, ddd (15.6, 6.10, 1.6) 5.79, m 1.72, d (6.4) 1.10, s 1.45, s 1.61, s 6.45, 7.14, 6.32, 6.07, 1.85, 1.31,

with six degrees of unsaturation from the HR-ESIMS data. The 1 H NMR spectrum showed resonances for two aromatic AB spin system protons [δH 6.36 (d, J = 8.8 Hz, H-5); 7.60 (d, J = 8.8 Hz, H-6)], three oxygenated methines [δH 4.37 (m, H-9); 3.70 (m, H-12); 3.70 (m, H-13)], three methylenes [δH 3.18 (dd, J = 15.2, 7.2 Hz, H-8) and 3.06 (dd, J = 15.2, 5.6 Hz, H8); 2.07 (m, H-10) and 1.66 (m, H-10); 1.89 (dd, J = 14.4, 6.8 Hz, H2-11)], and two methyls [δH 2.01 (s, 3-CH3); 1.09 (s, H3-14)]. Except for six signals (δC 164.5, 164.5, 131.0, 113.8, 112.3, 108.3) attributed to one aromatic ring, the other carbons were identified as one keto carbonyl (δC 204.3), three oxygenated methines (δC 85.1, 77.8, and 69.9), three methylenes, and two methyls. Analysis of the COSY spectrum suggested the presence of one isolated proton spin system: CH2(8)−CH(9)−CH2(10)−CH2(11)−CH(12)−CH(13)− CH3(14) (Figure 1). The HMBC correlations from H-6 to C-1 and C-2, from H-5 to C-4, and from 3-CH3 to C-2, C-3, and C-4 indicated the presence of a 2,4-dihydroxy-3-methylphenyl ring. The HMBC correlations from H-6 and H-8 to the keto carbon C-7 suggested a combination of the phenyl ring and the C-8−C-14 subunit through C-7. In consideration of the remaining unsaturation and the chemical shift values of two oxymethines at C-9 (δC 77.8) and C-12 (δC 85.1), an ether linkage connected C-9 and C-12, forming a substituted tetrahydrofuran ring. Finally, the absolute configuration of 12 was assigned as (9S, 12R, 13S) by a single-crystal X-ray diffraction experiment using the anomalous scattering of Cu Kα irradiation with a Flack parameter = 0.01 (9). 12-Hydroxysorbicillin (13) gave a molecular formula of C14H16O4 as determined from HR-ESIMS analysis. The 1H and 13C NMR spectroscopic data closely resembled those of the known compound sorbicillin,2 except for the absence of the methyl C-12 and the presence of a hydroxymethylene (δH 4.23, 2H; δC 62.9) in 13. The chemical shift of the hydroxymethylene (δC 62.9) and the COSY cross-peaks between H212 and H-11 were observed, which indicated that 13 is a 12hydroxy derivative of sorbicillin. According to HR-ESIMS analysis, 8,9-dihydro-12-hydroxysorbicillin (14) was found to have a molecular formula of C14H18O4, which was two hydrogens more than that of 13. The 1D NMR data of 14 were closely comparable to those of 13, except that the C-8 (δC 125.2)/C-9 (δC 144.4) double bond in 13 was hydrogenated, resulting in two methylenes (δC 38.2, C-8; 28.4, C-9) in 14. The gross structure of 14 was further evidenced by analysis of the 2D NMR spectra (Figure 1). The molecular formula of trichosorbicillin E (15) was established as C13H16O4 by HR-ESIMS, lacking a methyl substituent in comparison with 8,9-dihydro-12-hydroxysorbicillin (14). The NMR spectra of 15 were closely related to those of 14 except for the presence of one additional aromatic proton (δH 6.23, s) and the disappearance of an aromatic methyl group. The deduction was further confirmed by the HMBC correlations from the aromatic proton H-3 to C-2 and C-4. Thus, compound 15 was elucidated as (E)-1-(2,4dihydroxy-5-methylphenyl)-6-hydroxyhex-4-en-1-one. The structure of trichosorbicillin F (16) was directly identified by a single-crystal X-ray diffraction experiment using Cu Kα radiation (Figure 3), in good agreement with its HR-ESIMS and NMR spectra. Compound 16 was the only monomeric sorbicillinoid with a trihydroxy aromatic ring isolated from T. reesei strain 4670.

d (14.8) dd (14.8, 10.8) m m d (6.7) s

δC, type 171.6, C 110.2, C 34.2, CH2 74.1, C 107.7, C 80.2, C 171.6, C 108.7, C 194.3, C 102.7, C 53.9, CH 57.8, C 194.3, C 42.3, CH2 80.6, CH 129.2, CH 130.7, CH 17.9, CH3 22.6, CH3 26.6, CH3 7.8, CH3 168.2, C 121.9, CH 139.3, CH 132.5, CH 137.3, CH 18.7, CH3 19.0, CH3

δH (J in Hz)

2.49, d (16.0), H-3a 2.31, d (16.0), H-3b

3.70, s

2.24, d (16.8, 3.6) 2.14, d (16.8, 14.8) 4.58, m 5.47, ddd (15.6, 6.4, 2.0) 5.79, m 1.75, d (6.4) 1.19, s 1.47, s 1.60, s 6.47, d (14.8) 7.18, dd (14.8, 10.8) 6.34, m 6.10, m 1.88, d (6.8) 1.31, s

the two compounds was that the methyl of the side chain in the known compound was replaced by a hydroxymethylene group in 10. The chemical shift of the methylene (δC 62.7, C11; δH 4.11, H2-11) and the COSY correlation of H2-11 and H10 further determined the structure of 10. A single-crystal Xray diffraction experiment using Cu Kα radiation confirmed the relative configuration, but was not able to assign the absolute configuration (Figure 3). The absolute configuration was established as S by comparing the experimental ECD spectrum with the calculated one (Figure S1). Thus, compound 10 was assigned as (S,E)-7-hydroxy-2-(3-hydroxyprop-1-en-1-yl)-6,8-dimethylchroman-4-one, and the trivial name was given as trichosorbicillin B. The molecular formula of compound 11 was determined to be C14H18O4 from HR-ESIMS and NMR data. The 1H and 13C NMR spectra of 11 were similar to those of trichosorbicillin B (10), except that the NMR resonances of the olefinic carbons (C-9 and C-10) were replaced by two sp3-hybridized methylene (δC 32.6, δH 1.89; δC 29.4, δH 1.76) signals. Analysis of the 2D NMR spectra (Figure 1) further indicated that 11 is a dihydro analogue of 10. Compounds 10 and 11 showed quite similar ECD spectra (Figure S3), which indicates they share the same 2S absolute configuration. Hence, compound 11 was named trichosorbicillin C. Trichosorbicillin D (12) was obtained as a white powder and was determined to have a molecular formula of C15H20O5 F



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should derive from sorbicillinol like other sorbicillinoids. Thus, we hypothesized that 1 could arise from a net [4+2] cycloaddition or double Michael reaction between sorbicillinol (ii) and 1-methyl-1,3-dihydro-2H-pyrrol-2-one (iii) (Scheme S1). In this work, the cytotoxic activities of all of the isolated compounds 1−25 were evaluated. Compounds 1−25 displayed no cytotoxicity against three human cancer cell lines, MCF-7 (breast cancer), HeLa (cervical cancer), and HepG2 (liver cancer), with paclitaxel as the positive control (Table S4). In addition, all compounds (1−25) were evaluated for the inhibition of nitric oxide (NO) production in RAW264.7 cells activated by lipopolysaccharide. Compounds 3−5, 10, 13−16, and 24 showed strong anti-inflammatory activity with IC50 < 10 μM, compared to the positive control indomethacin, whose IC50 was 41 μM. Compounds 6−9, 11, 18, 21, 22, and 25 exhibited considerable anti-inflammatory activity (IC50 values of 12−30 μM). However, compounds 1, 2, 12, 17−19, and 22 were not active (IC50 > 50 μM). In order to investigate whether the anti-inflammatory effects of the active compounds were impacted by their cytotoxicity, antiproliferative effects were also evaluated by an MTT assay, and no compound showed significant cytotoxic activity against the RAW264.7 cells (IC50 > 50 μM). A preliminary structure−activity relationship analysis indicated that the anti-inflammatory activities of the sorbicillinoids mainly depended on the structural types and the functional groups of the sorbyl side chain. For monomeric sorbicillinoids, double bonds of the sorbyl side chain played an important role in their anti-inflammatory action, as compounds 10, 13−16, 18, 21, 22, 24, and 25 with at least one double bond in the side chain were much more active than compounds 11, 12, 17, 19, and 20 without a double bond in the side chain. Compounds 15 and 21 exhibited stronger antiinflammatory effects than those of 19 and 23, indicating that the terminal carboxylic acid group of the sorbyl side chain is a disadvantage for the anti-inflammatory activity. The keto carbonyl group at C-7 made no difference to the antiinflammatory activity, because compound 21, with a keto carbonyl group at C-7, showed the same level of activity as compound 25, containing a methylene group at C-7. For the dimeric sorbicillinoids, the sorbyl side chain possessing a lipophilic terminus made a more positive contribution to the anti-inflammatory activity than those with a hydrophilic group. For example, when the lipophilic methyl group replaced the hydrophilic hydroxymethylene group in the sorbyl side chain, the activity increased to some extent (2 vs 4; 7 vs 5).

Trichosorbicillin G (17) was isolated as a white powder. The molecular formula C13H18O4 was assigned by HR-ESIMS, containing two more hydrogen atoms than 15. The 1H and 13C NMR were nearly identical to those of 15, except that the signals of two methylenes (δH 1.42, m; δC 26.7; δH 1.56, m; δC 33.5) in 17 replaced those of a double bond in 15, suggesting that 17 was a hydrogenated derivative of 15. Therefore, compound 17 was elucidated as 1-(2,4-dihydroxy-5-methylphenyl)-6-hydroxyhexan-1-one. Isotrichosorbicillin E (18) was isolated as a yellow powder. Its molecular formula C13H16O4 was the same as that of 15, determined by observing a deprotonated molecule at m/z 235.0977 [M − H]− in the HR-ESIMS spectrum, indicating 18 was the isomer of 15. A detailed analysis of the NMR data revealed that the location of the hydroxy group in the sorbyl side chain was different, being at C-9 in 18 rather than C-12. The absolute configuration of 18 was established by comparison of the experimental and theoretical ECD spectra (Figure S1). The calculated ECD spectrum of (S)-18 was in agreement with the experimental one. Consequently, compound 18 was identified as (S,E)-1-(2,4-dihydroxy-5-methylphenyl)-3-hydroxyhex-4-en-1-one. Trichosorbicillin H (19) was obtained as a white powder. Comparing the spectroscopic data of 19 and 17 showed that they share a similar sorbicillin skeleton, except 19 has a modified sorbyl side chain. Comprehensive analysis of 2D NMR data (Figure 1) indicated that the modified sorbyl side chain of 19 was a propionic acid side chain instead of a pentanol side chain in 17. Hence, 19 was elucidated as 4-(2,4dihydroxy-5-methylphenyl)-4-oxobutanoic acid. Comparison of the spectroscopic data of 19 and 20 suggested that they have the same sorbicillin skeleton with a propionic acid side chain, except that an additional methyl (δH 2.03, s; δC 8.0) is substituted at C-3 of the phenyl ring in 20, which was evidenced by the HMBC correlations from 3-CH3 to C-2, C-3, and C-4. Thus, 20 was characterized as shown in Chart 1 and named trichosorbicillin H. Trichosorbicillin I (25) had a molecular formula of C13H17O2 as deduced by HR-ESIMS analysis. The 1H and 13 C NMR spectroscopic data closely resembled those of the known compound sohirnone A (21), except for the absence of keto carbonyl C-7 and the presence of an additional methylene (δH 2.48, t, J = 8.0 Hz; δC 28.6) in 25. A series of COSY correlations from H2-7 to H3-12 indicated the presence of an aliphatic side chain connected to C-1, which was established by the HMBC correlation from H-6 to C-7. Therefore, compound 25 was determined as (E)-4-(hex-4-en-1-yl)-6-methylbenzene1,3-diol. Additionally, the known compounds were identified as bisvertinol (4),9 sohirnone A (21),11 2′,3′-dihydrosorbicillin (22),11 (E)-6-(2,4-dihydroxyl-5-methylphenyl)-6-oxo2-hexenoic acid (23), 12 and (2E,4E)-1-(2,6-dihydroxy-3,5dimethylphenyl)hexa-2,4-dien-1-one (24)13 by comparing their spectroscopic data with published literature values. The sorbicillinoid family normally includes monomeric sorbicillinoids, dimeric sorbicillinods, bisvertinols, trichodimerols, and bridged bicyclic bisorbicillinoids.1 However, only a few studies have reported the bridged bisorbicillinoids incorporating nitrogen like 1.14 Additionally, the pair of epimers 8 and 9 represent a new bisorbicillinoid core (Chart 1). Although a similar structure has been synthesized by the Gulder group,15 they are the first examples as natural products. Among all of the compounds, compound 1 was an isolated congener, but it

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EXPERIMENTAL SECTION

General Experimental Procedures. The melting points were determined on an SGW X-4 micro melting point instrument (Shanghai Jingke Scientific Instrument Co., Ltd.). Optical rotations were measured on an MCP 200 (Anton Paar) polarimeter. UV spectra were recorded on a Blue Star A spectrophotometer. For experimental ECD data analysis, Chirascan and Chirascan-Plus circular dichroism spectrometers (Applied Photophysics Ltd.) were used. A Fourier transformation infrared spectrometer coupled with an EQUINOX 55 infrared microscope (Bruker) was used to record the IR spectra. NMR spectra were obtained with a Bruker Avance 400 MHz spectrometer with tetramethylsilane as the internal standard. HR-ESIMS data were determined using an LTQ-Orbitrap LC-MS spectrometer (Thermo Corporation). ESIMS was carried out on an ACQUITY QDA (Waters Corporation). Column chromatography (CC) was performed using silica gel (200−300 mesh, Qingdao G



Article

Trichosorbicillin A (1): white powder; mp 150−151 °C; [α ]20 D +11.5 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 359 (4.09) nm; ECD (c 0.33 mg/mL, MeOH), λmax (Δε) 311 (−43.14); 349 (42.85) nm; IR (neat) νmax 3362, 2914, 1734, 1664, 1603, 1528, 1371, 1312, 1155, 1107, 1050, 1000 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table 1; HR-ESIMS m/z 344.1506 [M − H]− (calcd for C19H22NO5, 344.1504). 15,24-Dihydroxybisvertinol (2): yellow, amorphous powder; [α ]20 D −310 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 227 (4.11), 270 (4.24), 298 (4.20), 391 (4.31) nm; ECD (c 0.33 mg/mL, MeOH), λmax (Δε) 225 (−15.53), 274 (13.62), 345 (40.26), 425 (−48.71) nm; IR (neat) νmax 3439, 2926, 1626, 1444, 1084, 881, 575, 478 cm−1; 1 H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table 2; HR-ESIMS m/z 529.2085 [M − H]− (calcd for C28H33O10, 529.2068). 24-Hydroxybisvertinol (3): yellow, amorphous powder; [α ]20 D −380 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 273 (4.17); 360 (4.05) nm; ECD (c 0.13 mg/mL, MeOH), λmax (Δε) 216 (−13.94), 259 (−20.63), 314 (32.83), 406 (−33.32) nm; IR (neat) νmax 3741, 3383, 3261, 2927, 1701, 1616, 1549, 1348, 1014 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table 2; HRESIMS m/z 513.2132 [M − H]− (calcd for C28H33O9, 513.2130). Bisvertinol (4): yellow, amorphous powder; [α ]20 D −1354 (c 0.10, MeOH) (ref 2: [α]D −1274 (c 0.99, MeOH)); UV (MeOH), λmax (log ε) 274 (4.20), 381 (4.21) nm; ECD (c 0.33 mg/mL, MeOH), λmax (Δε) 224 (−13.51), 273 (10.50), 314 (23.90), 354 (35.39), 408 (−51.00) nm; IR (neat) νmax 3389, 2983, 2940, 2361, 2326, 1612, 1558, 1452, 1377, 1344, 1064, 1020, 669, 597 cm−1; HR-ESIMS m/z 497.2184 [M − H]− (calcd for C28H33O8, 497.2181). Trichobisvertinol A (5): yellow, amorphous powder; [α ]20 D −450 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 230 (3.79), 276 (3.75), 302 (3.71), 384 (3.79) nm; ECD (c 0.20 mg/mL, MeOH), λmax (Δε) 225 (−13.50), 273 (10.51), 313 (23.72), 352 (35.45), 410 (−50.92) nm; IR (neat) νmax 3375, 3205, 2922, 1630, 1558, 1431, 1350, 1209, 1095, 1011 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data,Table 2; HR-ESIMS m/z 499.2342 [M − H]− (calcd for C28H35O8, 499.2337). Trichobisvertinol B (6): yellow, amorphous powder; [α ]20 D −470 (c 0.40, MeOH); UV (MeOH) λmax (log ε) 229 (4.00), 271 (4.16), 308 (4.02), 382 (4.20) nm; ECD (c 0.33 mg/mL, MeOH), λmax (Δε) 223 (−24.34), 274 (31.42), 305 (43.19), 389 (−31.73) nm; IR (neat) νmax 3359, 2926, 1616, 1564, 1346, 1213, 1082, 1022 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table 2; HR-ESIMS m/z 515.2284 [M − H]− (calcd for C28H35O9, 515.2287). Trichobisvertinol C (7): yellow, amorphous powder; [α ]20 D −380 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 227 (3.95), 269 (4.16), 300 (4.02), 379 (4.20) nm; ECD (c 0.33 mg/mL, MeOH), λmax (Δε) 225 (−21.73), 275 (29.66), 305 (39.00), 384 (−30.69) nm; IR (neat) νmax 3430, 2922, 1695, 1622, 1456, 1365, 1070, 884, 5701, 484 cm−1; 1 H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data,Table 2; HR-ESIMS m/z 533.2380 [M + H]+ (calcd for C28H37O10, 533.2381). Trichobisvertinol D (8): yellow, amorphous powder; [α ]20 D −150 (c 0.33, MeOH); UV (MeOH) λmax (log ε) 272 (4.06), 376 (3.95) nm; ECD (c 0.20 mg/mL, MeOH), λmax (Δε) 223 (−11.62), 282 (11.14), 318 (7.35), 386 (−13.21) nm; IR (neat) νmax 3406, 2929, 1724, 1599, 1450, 1406, 1379, 1346 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table 3; HR-ESIMS m/z 497.2182 [M − H]− (calcd for C28H33O8, 497.2181). 12-epi-Trichobisvertinol D (9): yellow, amorphous powder; [α ]20 D −130 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 273 (4.00), 371 (3.79) nm; ECD (c 0.20 mg/mL, MeOH), λmax (Δε) 215 (−9.78), 277 (11.71), 324 (3.58), 379 (−8.49) nm; IR (neat) νmax 3406, 2929, 1724, 1599, 1450, 1406, 1379, 1346 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table 3; HRESIMS m/z 497.2185 [M − H]− (calcd for C28H33O8, 497.2181).

Marine Chemical Factory). HPLC was carried out on an Essentia LC16 (Shimadzu). Fungal Material. Strain Trichoderma reesei 4670 was isolated from a sponge collected in Shantou, Guangdong Province, China. It was identified as Trichoderma reesei on the basis of ITS sequence, and the sequence has been deposited in GenBank under accession number MH542677. The fungal strain has been preserved at the School of Marine Sciences, Sun Yat-Sen University. Extraction and Isolation. The fungus T. reesei 4670 was cultivated on rice medium in 1 L Erlenmeyer flasks containing 60 mL of rice and 2.4 g of artificial sea salt (Guangdong Province Salt Industry Group Company Limited) dissolved in 80 mL of H2O. A total of 113 flasks were incubated at room temperature for 30 days. The fermented material was extracted exhaustively with MeOH three times to obtain a crude extract. The extract was suspended in MeOH/ H2O (8:2) and extracted three times with n-hexane and EtOAc successively. The EtOAc extract (105 g) was subjected to CC on silica gel (200−300 mesh) and was eluted with PE (petroleum ether)/ EtOAc of increasing polarity (from 8:2 to 0:1) to afford six fractions (A−F). Fr.A was fractionated on a Sephadex LH-20 column with MeOH to afford 10 fractions (Fr.A.1 to Fr.A.10). Fr.A.5 was further subjected to Sephadex LH-20 chromatography with MeOH and then purified by RP-HPLC (35% CH3CN/H2O, flow rate 1 mL/min, Ultimate AmySR column 10 × 250 mm, 5 μm) to give 18 (3.0 mg). Fr.A.6 was purified by RP-HPLC with MeOH/H2O (75:35) to afford 21 (1.5 mg), 22 (1.7 mg), and 25 (4.4 mg). Fr.B was fractionated on a Sephadex LH-20 column with MeOH to afford 21 fractions (Fr.B.1 to Fr.B.21). Fr.B.2 was purified on a Sephadex LH-20 column to give 13 (50 mg). Fr.B.3 was applied to a RP-C18 column with MeOH/H2O (from 70:30 to 100:0) to afford 14 fractions (Fr.B.3.1 to Fr.B.3.14). Fr.B.3.9 was subjected to a silica gel column eluting with CH2Cl2/ MeOH (98:2) to give 4 (300 mg). Fr.B.3.13 was purified by RPHPLC with MeOH/H2O (85:15) to afford 5 (30 mg). Fr.B.6 was applied to a Sephadex LH-20 column with MeOH and then purified by RP-HPLC with MeOH/H2O (60:40) to give 17 (21 mg). Fr.B.7 was subjected to a Sephadex LH-20 column to give 15 (47 mg). Fr.B.8 was purified by RP-HPLC with MeOH/H2O (55:45) to afford 23 (12 mg). Fr.B.9 was purified by RP-HPLC with MeOH/H2O (60:40) to afford 14 (5 mg), 19 (3 mg), and 20 (3 mg). Fr.B.13 was successively subjected to Sephadex LH-20 and RP-C18 chromatography, then finally purified by RP-HPLC with MeOH/H2O (60:40) to afford 12 (4 mg). Fr.B.16 was purified by RP-HPLC with MeOH/ H2O (60:40) to afford 10 (6 mg). Fr.B.19 was applied to a silica gel column eluting with CH2Cl2/MeOH (98:2) and then purified using normal-phase HPLC with n-hexane/2-propanol (96:4) to give 8 (20 mg, tR = 46 min) and 9 (4 mg, tR = 48 min). Fr.C was further fractionated on a silica gel column with PE/EtOAc (from 7:3 to 2:8) to give four fractions (Fr.C.1 to Fr.C.4). Fr.C.1 was applied to an RPC18 column and then purified by normal-phase HPLC with n-hexane/ 2-propanol (90:10) to give 11 (4 mg). Fr.C.2 was successively subjected to a Sephadex LH-20 column and RP-C18 column with MeOH/H2O (80:20) to give 16 (20 mg). Fr.C.3 was successively applied to an RP-C18 column and a silica gel column eluting with CH2Cl2/MeOH (95:5) and finally purified by normal-phase HPLC with n-hexane/2-propanol (78:22) to give 6 (17 mg). Fr.D was further fractionated on an RP-C18 column with MeOH/H2O (from 60:40 to 100:0) to give five fractions (Fr.D.1 to Fr.D.5). Fr.D.2 was applied to an RP-C18 column with MeOH/H2O (65:35) and then purified by a silica gel column with CH2Cl2/MeOH (96:4) to give 1 (18 mg). Fr.D.3 was subjected to a Sephadex LH-20 column and further purified by normal-phase HPLC with n-hexane/2-propanol (85:15) to give 3 (10 mg). Compound 24 (5 mg) was isolated from Fr.D.4 by normal-phase HPLC with n-hexane/2-propanol (95:5). Fr.E was subjected to a Sephadex LH-20 column to give four fractions (Fr.E.1 to Fr.E.4). Fr.E.2 was purified on an RP-C18 column with MeOH/H2O (60:40) and by RP-HPLC with MeOH/H2O (67:33) to give 7 (25 mg). Compound 2 (95 mg) was purified from Fr.E.3 using Sephadex LH-20 with MeOH. H



Article

400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table S3; HRESIMS m/z 237.0770 [M − H]− (calcd for C12H13O5, 237.0769). Trichosorbicillin I (25): yellow powder; UV (MeOH) λmax (log ε) 208 (3.95), 285 (3.32) nm; IR (neat) νmax 3392, 2943, 2837, 2515, 1724, 1456, 1246, 1026, 669 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table S3; HR-ESIMS m/z 205.1235 [M − H]− (calcd for C13H17O2, 205.1234). X-ray Crystallographic Analysis of Compounds 1, 10, 12, and 16. Colorless crystals of compound 1 were obtained from a solvent mixture of MeOH/EtOAc. Colorless crystals of compounds 10, 12, and 16 were obtained from a mixed solvent of MeOH/CHCl3. Crystal data were acquired using the hemisphere technique on a Rigaku Oxford Diffraction diffractometer with graphite monochromated Cu Kα radiation, λ = 1.541 78 Å. Structure analysis and refinement: The structure was solved by direct methods using SHELXS-97; refinement was done by full-matrix least-squares on F2 using the SHELXL-97 program suite on Olex2 Launcher.16,17 All nonhydrogen positions were refined with anisotropic displacement parameters. In the crystal structure of compound 1, the alkyl chain in one of the fragments in the symmetric unit is disordered over two positions, the atoms of which were split into two parts with equal occupancy rate in order to obtain good refined parameters. Crystallographic Data of 1. C19H19O5, M = 341.35, monoclinic, C2, a = 31.2722(16) Å, b = 9.0328(3) Å, c = 13.4285(5) Å, α = 90°, β = 101.879(3)°, γ = 90°, V = 3712.0(3) Å3, T = 150.0(10) K, Z = 8, Dcalcd = 1.222 g/m3, crystal size 0.3 × 0.2 × 0.04 mm3, F(000) = 1440.0, absorption coefficient 0.736 mm−1, reflections collected 11 079, independent reflections 7092 [Rint = 0.0370, Rsigma = 0.0456], final R indices [I > 2σ(I)] R1 = 0.0768, wR2 = 0.2200, R indices (all data) R1 = 0.0934, wR2 = 0.2463; Flack parameter 0.32(14); Hooft parameter 0.35(13) (CCDC 1843253). Crystallographic Data of 10. C14H16O4, M = 248.27, monoclinic, P21, a = 4.9332(2) Å, b = 16.4077(5) Å, c = 7.9261(2) Å, α = 90°, β = 103.439(3)°, γ = 90°, V = 623.99(4) Å3, T = 293(2) K, Z = 2, Dcalcd = 1.321 g/m3, crystal size 0.350 × 0.210 × 0.005 mm3, F(000) = 264.0, absorption coefficient 0.797 mm−1, reflections collected 3902, independent reflections 2383 [Rint = 0.0494, Rsigma = 0.1354], final R indices [I > 2σ(I)] R1 = 0.0768, wR2 = 0.2200, R indices (all data) R1 = 0.0502, wR2 = 0.1364; Flack parameter 0.33(10); Hooft parameter 0.33(10) (CCDC 1862677). Crystallographic Data of 12. C15H20O5, M = 280.31, orthorhombic, P212121, a = 4.84130(10) Å, b = 12.6161(2) Å, c = 22.6509(3) Å, α = β = γ = 90°, V = 1383.48(4) Å3, T = 293(2) K, Z = 4, Dcalcd = 1.346 g/m3, crystal size 0.520 × 0.130 × 0.050 mm3, F(000) = 600.0, absorption coefficient 0.833 mm−1, reflections collected 4757, independent reflections 2692 [Rint = 0.0220, Rsigma = 0.0286], final R indices [I > 2σ(I)] R1 = 0.0334, wR2 = 0. 0910, R indices (all data) R1 = 0.0344, wR2 = 0.0923; Flack parameter 0.01(9); Hoof parameter 0.01(9) (CCDC 1840342). Crystallographic Data of 16. C13H16O5, M = 252.26, monoclinic, P21/c, a = 4.7038(2) Å, b = 26.6414(8) Å, c = 9.5760(3) Å, α = 90°, β = 94.010(3)°, γ = 90°, V = 1197.09(7) Å3, T = 293(2) K, Z = 4, Dcalcd = 1.400 g/m3, crystal size 0.40 × 0.12 × 0.05 mm3, F(000) = 536, absorption coefficient 0.902 mm−1, reflections collected 7560, independent reflections 2389 [Rint = 0.0332, Rsigma = 0.0260], final R indices [I > 2σ(I)] R1 = 0.0537, wR2 = 0. 1468, R indices (all data) R1 = 0.0606, wR2 = 0.1514 (CCDC 1877279). Cytotoxicity Assay. Three human cancer cell lines, HeLa, MCF7, and HepG2, were provided by the cell bank of the Chinese Academy of Sciences. The cytotoxic activities of the tested compounds were assayed by the MTT method using 96-well plates (Nest) according to a previous report.18 Anti-inflammatory Assay. RAW264.7 cells were purchased from the cell bank of the Chinese Academy of Sciences. All the tested compounds were prepared as stock solutions in DMSO. RAW264.7 cells were seeded in 96-well plates at a density of 5 × 104 cells per well and incubated for 24 h. Then the cells were treated with lipopolysaccharide (LPS) (1 μg/mL) and various concentrations of the compounds for 24 h. After that, 50 μL of the cell culture supernatant solution was removed to new 96-well plates. Sub-

Trichosorbicillin B (10): white powder; mp 178−180 °C; [α ]20 D +8.4 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 215 (3.29), 235 (3.01), 285 (3.05), 324 (2.63) nm; ECD (c 0.33 mg/mL, MeOH), λmax (Δε) 213 (9.16), 303 (−5.18), 337 (4.68) nm; IR (neat) νmax 3329, 2922, 1655, 1599, 1458, 1347, 1296, 1182, 1086, 974 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table S1; HR-ESIMS m/z 247.0977 [M − H]− (calcd for C14H15O4, 247.0976). Trichosorbicillin C (11): white powder; [α ]20 D +2.4 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 217 (4.11), 232 (3.83), 283 (3.88), 322 (3.50) nm; ECD (c 0.33 mg/mL, MeOH), λmax (Δε) 211 (8.46), 303 (−3.63), 336 (4.28) nm; IR (neat) νmax 3298, 2924, 1659, 1605, 1462, 1362, 1306, 1184, 1093, 889, 810 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table S1; HRESIMS m/z 251.1277 [M + H]+ (calcd for C14H19O4, 251.1278). Trichosorbicillin D (12): white powder; mp 149−150 °C; [α ]20 D +8.8(c 0.07, MeOH); UV (MeOH) λmax (log ε) 215 (4.05), 286 (3.91) nm; ECD (c 0.33 mg/mL, MeOH), λmax (Δε) 220 (1.54), 274 (−0.79), 305 (1.38) nm; IR (neat) νmax 3118, 2931, 1618, 1503, 1431, 1331, 1093, 798 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table S2; HR-ESIMS m/z 279.1236 [M − H]− (calcd for C15H19O5, 279.1238). 12-Hydroxysorbicillin (13): yellow powder; UV (MeOH) λmax (log ε) 206 (4.22), 321 (4.07) nm; IR (neat) νmax 3375, 2921, 2857, 1706, 1618, 1561, 1483, 1420, 1368, 1286, 1221, 1145, 1074, 992, 860, 765 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table S2; HR-ESIMS m/z 247.0977 [M − H]− (calcd for C14H15O4, 247.0976). 8,9-Dihydro-12-hydroxysorbicillin (14): white powder; UV (MeOH) λmax (log ε) 215 (4.26), 284 (4.05), 330 (3.71) nm; IR (neat) νmax 3421, 3153, 2947, 1593, 1506, 1352, 1265, 1149, 1028, 962, 845, 762 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table S2; HR-ESIMS m/z 249.1136 [M − H]− (calcd for C14H17O4, 249.1132). Trichosorbicillin E (15): white powder; UV (MeOH) λmax (log ε) 213 (3.14), 234 (2.86), 280 (3.00), 328 (2.76) cm; IR (neat) νmax 3400, 2951, 2837, 1649, 1452, 1417, 1028, 665 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table S2; HR-ESIMS m/z 235.0977 [M − H]− (calcd for C13H15O4, 235.0976). Trichosorbicillin F (16): yellow powder; mp 154−156 °C; UV (MeOH) λmax (log ε) nm 216 (4.51) 238 (4.26), 287 (4.38), 353 (4.11) nm; IR (neat) νmax 3737, 3489, 3296, 2926, 1572, 1514, 1414, 1333, 1240, 1182, 1024, 750 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table S2; HR-ESIMS m/z 251.0932 [M − H]− (calcd for C13H15O5, 251.0925). Trichosorbicillin G (17): white powder; UV (MeOH) λmax (log ε) 213 (3.93), 235 (3.68), 279 (3.80), 328 (3.57) nm; IR (neat) νmax 3412, 3157, 2939, 1624, 1512, 1390, 1259, 1147, 1032, 968, 845, 756 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table S3; HR-ESIMS m/z 237.1134 [M − H]− (calcd for C13H17O4, 237.1132). Isotrichosorbicillin E (18): yellow powder; [α ]20 D +8.1 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 213 (3.87), 225 (3.51), 282 (3.70), 327 (3.48) nm; ECD (c 0.33 mg/mL, MeOH), λmax (Δε) 214 (3.95) nm; IR (neat) νmax 3356, 3201, 2922, 1616, 1367, 1255, 1140, 976, 841 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table S3; HR-ESIMS m/z 235.0977 [M − H]− (calcd for C13H15O4, 235.0976). Trichosorbicillin H (19): white powder; UV (MeOH) λmax (log ε) 213 (3.63), 234 (3.40), 278 (3.50), 324 (3.25) nm; IR (neat) νmax 3367, 2926, 1713, 1633, 1508, 1419, 1373, 1267, 1147 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, Table S3; HR-ESIMS m/z 223.0613 [M − H]− (calcd for C11H11O5, 223.0612). 3-Methyltrichosorbicillin H (20): white powder; UV (MeOH) λmax (log ε) 216 (4.11), 283 (3.96), 329 (3.59) nm; IR (neat) νmax 3354, 3180, 2914, 2843, 1624, 1367, 1213, 1147 cm−1; 1H NMR (CD3OD, I



Article

Table 4. Inhibition of NO Production in LPS-Induced RAW264.7 Cells and Cytotoxicity against RAW264.7 Cells of Compounds 1−25a compound

inhibition of NO production, IC50 (μM)

cytotoxicity against RAW264.7

compound

inhibition of NO production, IC50 (μM)

cytotoxicity against RAW264.7

1 2 3 4 5 6 7 8 9 10 11 12 13

>50 >50 6.1 9.9 5.9 22 24 22 32 8.5 38 >50 6.8

>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50

14 15 16 17 18 19 20 21 22 23 24 25 indomethacin

2.9 0.94 6.1 >50 12 >50 >50 14 13 >50 3.3 13 41

>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50

a

The IC50 values were calculated from the normalized response-variable slope curves (GraphPad Prism). Result are presented as mean (n = 3).

sequently, 50 μL of the nitric oxide detection reagents I and II were successively added to each well. Then the absorbance was measured at 540 nm with a Multiskan GO (Thermo Scientific). Calculation of ECD Spectra. The theoretical calculations for the ECD spectra of compounds 1, 8−10, and 18 were performed using Gaussian 09.19 Conformational analysis was initially carried out based on the Molecular Mechanics method by Spartan’14. The conformers were then optimized at the B3LYP/6-31G(d,p) level. Roomtemperature equilibrium populations were calculated according to the Boltzmann distribution law. The theoretical calculations of the ECD spectra were performed using time-dependent density functional theory (TDDFT) at the RB3lYP/6-311+G(2d,p) level in MeOH with the polarizable continuum model (PCM) model and in the gas phase, respectively. For comparisons of the calculated curves and experimental ECD spectra, the programs SpecDis 3.0 (University of Würzburg) and Origin Pro 8.5 (Origin Lab, Ltd.) were used.

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41606167, 41706148, 41806155, and 51709287), the National Science and Technology Major Project of the Ministry of Science and Technology of China (2018ZX09735010), the Natural Science Foundation of Guangdong Province, China (2018A030310304), Special Fund for Economic Development of Guangdong Province (Uses for Marine Economic Development) (GDME-2018C004), and the Guangzhou Science and Technology Project (Grant No. 201804010476).

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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.8b01029. 1 H and 13C NMR data of 10−20 and 25; 1H, 13C, and 2D NMR spectra of 1−20 and 25; ECD spectra of 1−12 and 18; normal-phase HPLC chromatogram of compounds 8 and 9; cytotoxicity data of compounds 1−25; biosynthesis hypothesis of compound 1 (PDF) X-ray crystallographic data (ZIP)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*(S. Chen) E-mail: [email protected]. *(L. Liu) E-mail: [email protected]. Tel: +86-2084725459. ORCID

Panpan Zhang: 0000-0003-0081-7320 Senhua Chen: 0000-0002-5498-0206 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research program is financially supported by the National Natural Science Foundation of China (Grant Nos. 21272286, J



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(18) Zhang, P. P.; Jia, C. X.; Lang, J. J.; Li, J.; Luo, G. Y.; Chen, S. H.; Yan, S. J.; Liu, L. Mar. Drugs 2018, 16, 173−182. (19) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresmaz, J. B.; Fox, D. J. Gaussian 09; Gaussian, Inc.: Wallingford, CT, 2016.

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Anti-inflammatory Mono- and Dimeric Sorbicillinoids from the Marine

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