Prenylated Indole Diterpene Alkaloids from a Mine ... - ACS Publications

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

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Prenylated Indole Diterpene Alkaloids from a Mine-Soil-Derived Tolypocladium sp. Lu-Lin Xu,†,‡ Ping Hai,⊥ Shuai-Bing Zhang,† Jing-Fang Xiao,§ Yuan Gao,⊥ Bing-Ji Ma,∥ Hai-Yan Fu,*,† Ye-Miao Chen,*,§ and Xiao-Long Yang*,†,‡ †

School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan 430074, People’s Republic of China Chongqing Key Laboratory of Natural Product Synthesis and Drug Research, School of Pharmaceutical Sciences, Chongqing University, Chongqing 401331, People’s Republic of China § Institute of Pathology and Southwest Cancer Center, Southwest Hospital and Key Laboratory of Tumor Immunopathology of the Ministry of Education of China, Third Military Medical University, Chongqing 400038, People’s Republic of China ⊥ Department of Chemical Engineering, Yibin University, Yibin 644000, People’s Republic of China ∥ Department of Traditional Chinese Medicine, Henan Agricultural University, Wenhua Road 12, Zhengzhou 450002, People’s Republic of China

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S Supporting Information *

ABSTRACT: Ten new prenylated indole diterpene alkaloids, tolypocladin A−J (1−10), including four chlorinated metabolites, have been isolated from a culture of a mine-soil-derived fungus, Tolypocladium sp. XL115. The structures and absolute configurations of 1−10 were determined by spectroscopic analysis, ECD calculations, and comparison with known compounds. Compounds 1 and 8 displayed significant antimicrobial activities. In addition, compound 1 also showed weak cytotoxic activity against all tested human cancer cell lines and suppressed the growth and viability of the patient-derived HCC cells T1224.

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activity. Bioassay-guided fractionation of this extract led to the isolation of 10 new prenylated indole diterpene alkaloids, tolypocladin A−J (1−10). Herein, the isolation, structure elucidation, and biological activity of 1−10 are described.

ndole terpenoids, a structurally diverse class of natural products comprising the common cyclic diterpene backbone originating from geranylgeranyl diphosphate and an indole moiety originating from indole-3-glycerol phosphate, have played a vital role in the history of drug discovery, exemplified by reserpine for hypertension treatment.1−6 The indole diterpene alkaloids, including the paxilline-type and nonpaxilline-type, are one of the largest classes of indole terpenoids with diverse structures and biological activities.2 Over 100 indole diterpenoids have been discovered from fungi and have been shown to exhibit a range of pharmacological effects, including anti-H1N1, antibiotic, antifungal, antiinsectan, and cytotoxic activities, making these metabolites attractive for potential pharmaceutical applications.7−10 Recently indole diterpenoids have attracted attention as potential medicinal lead compounds, for example, the Maxi-K channel antagonists paspalinine and the BK channel antagonist paxilline.7,11−13 During our ongoing search for structurally unique biologically active compounds from fungi inhabiting unusual environments, an extract from an isolate of Tolypocladium sp. XL115 isolated from mine soil that was grown on a solid substrate exhibited significant cytotoxicity and antimicrobial © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Structure Elucidation. Compound 1 was isolated as a white, amorphous powder. Its molecular formula was determined as C32H43NO5 by HRESIMS, corresponding to 12 indices of hydrogen deficiency. The characteristic UV maximum peaks at λmax 230 and 285 nm, along with the typical IR absorptions at 3403, 1453, 1101, and 742 cm−1, revealed the presence of an indole chromophore.11−15 The 13C NMR and DEPT data of 1 (Table 1) revealed the presence of 32 carbons, which could be classified into six methyls, six methylenes, nine methines (including four sp2 carbons and four sp3 oxygenated carbons), and 11 nonprotonated carbons (including six sp2 carbons and three sp3 oxygenated carbons). The COSY correlations (Figure 1) between aromatic protons Received: July 18, 2018

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DOI: 10.1021/acs.jnatprod.8b00589 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

H-7/H-25, combined with the characteristic 13C NMR data [δC 77.8 (s, C-13)], are similar to those of known compounds drechmerins D and E and terpendoles A and C. The reported biosynthesis of analogues with a hydroxy group at C-13 further supports the relative configurations of H-7, H-9, OH-13, and Me-25 as α-oriented and H-10, H-11, H-16, and Me-26 as βoriented.2,14 The absolute configuration of 1 was determined as 3S,4R,7S,9S,10R,11R,12S,13S,16S by the calculated electronic circular dichroism (ECD) (Figure S91).16 Compound 2 was also obtained as a white, amorphous powder and shown to have the molecular formula C32H44ClNO6 from HRESIMS analysis, implying 11 degrees of unsaturation. Comprehensive analysis of its 13C NMR (Table 3), DEPT, and HSQC data displayed the presence of six methyls, six methylenes, eight olefinic or aromatic carbons (three of them protonated), six aliphatic methines (five of them attached to heteroatoms), and six aliphatic nonprotonated carbons (four of which are attached to

H-21, H-22, and H-23 indicated the presence of a 1,2,3trisubstituted aromatic ring. A detailed comparison of its NMR data with those of drechmerin F, a known indole diterpenoid alkaloid reported from Drechmeria sp.,15 revealed that the 3methyl-1-phenylbutane-2,3-diol at C-20 in drechmerin F was replaced by an isopentenyl group in 1. Evidence for this included the absence of signals of an sp3 oxygenated methine [δC 78.4 (d, C-31)] and an sp3 quaternary carbon [δC 79.0 (s, C-32)] seen in drechmerin F and the presence of signals of the C-31/C-32 trans-olefin [δC 123.7 (d, C-31), 131.8 (s, C-32)] in 1. This was further supported by the COSY correlation of H-30 with H-31, along with key HMBC correlations (Figure 1) of H-30 with C-19, C-21, and C-32, H-31 with C-20, H-33 with C-31 and C-32, and H-34 with C-31 and C-32 (Figure 1). The relative configuration of 1 was the same as those of drechmerin F deduced from the similar NMR data and proton coupling constants. The NOESY correlations (Figure 2) of H26/H-6b/H-11/H-16, H-10/H-11, H-7/H-6a/H-9, and H-5a/ B



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Table 1. 1H NMR Data for Compounds 1−5 (δ in ppm, J in Hz) 1a

no. NH 5 6 7 9 10 11 14 15 16 17 20 21 22 23 25 26 28 29 30 31 33 34 35 36 38 39 32-MeO

2a

3b

7.76, 1.35, 1.80, 4.22, 3.36, 4.02, 3.63, 1.51, 1.62, 2.85, 2.58,

s m; 2.73, m m; 2.31, m t (8.8, 17.6) d (8.9) d (8.7) brs m; 1.62, m m; 1.95, m m t (11.7, 23.6); 2.85, m

7.86, 1.35, 1.77, 4.21, 3.36, 4.02, 3.63, 1.48, 1.63, 2.84, 2.60,

s m; 2.73, m m; 2.30, m t (8.8, 17.6) d (9.2) d (8.8) brs m; 1.63, m m; 1.95, m m t (12.0, 23.2); 2.84, m

1.65, 1.81, 4.18, 3.35, 3.93, 3.48, 1.47, 1.58, 2.86, 2.53,

m; 2.60, m m; 2.26, m t (9.0, 17.8) d (9.1) d (9.1) brs m; 1.65, m m; 1.93, m m t (11.2, 23.3); 2.81, m

6.87, 7.02, 7.16, 1.28, 1.15, 1.31, 1.27, 3.63, 5.42, 1.77, 1.77,

d (7.0) t (7.3, 8.0) d (8.1) s s s s m m s s

6.95, 7.06, 7.23, 1.27, 1.14, 1.30, 1.27, 2.91, 3.92, 1.73, 1.73,

d (7.2) t (7.2, 8.0) d (8.0) s s s s m; 3.40, m d (10.0) s s

6.85, 6.90, 7.14, 1.26, 1.10, 1.23, 1.22, 2.78, 3.80, 1.26, 1.24,

d (7.1) t (7.5, 7.8) d (7.9) s s s s m; 3.22, m d (8.0) s s

4a

5b

7.96, 1.35, 1.77, 4.20, 3.38, 4.00, 3.56, 1.36, 1.60, 2.71, 2.41, 7.40, 6.97,

s m; 2.71, m m; 2.29, m t (8.8, 17.6) d (9.2) d (8.8) brs m; 1.50, m m; 1.91, m m t (10.8, 22.4); 2.71, m d (8.0) d (8.0)

7.25, 1.26, 1.07, 1.29, 1.26, 2.71, 3.81, 1.68, 1.68,

s s s s s m; 3.19, d (13.2) dd (2.0, 10.4) s s

10.03, s 1.58, m; 2.56, 1.58, m; 2.19, 4.26, m 3.51, d (9.6) 3.97, d (9.2) 3.46, brs 1.10, m; 1.45, 1.45, m; 1.82, 2.61, m 2.52, m; 2.86, 6.85, 6.91, 7.15, 1.20, 0.91, 1.22, 1.29, 2.92, 3.88, 1.65, 1.63, 5.60, 5.20, 1.73, 1.73,

m m

m m m

d (7.2) t (7.2, 8.0) d (8.0) s s s s m; 3.43, m d (10.0) s s d (6.4) brs s s

3.29 (3H, s)

a

Measured in CDCl3 (400 MHz). bMeasured in CD3OD (400 MHz). cMeasured in DMSO-d6 (400 MHz).

Accordingly, the structure of compound 2, tolypocladin B, was assigned as shown. Compound 3 was obtained as a white, amorphous powder, and its molecular formula of C33H47NO7 determined by HRESIMS, indicating 11 degrees of unsaturation. Comparison of NMR data with those of 2 indicated that 3 had the same backbone. The chlorine atom at position C-32 in 2 was replaced by a methoxy group in 3, which was further confirmed by the HMBC correlation of the methoxy group with C-32, together with its molecular formula. The NOE correlative patterns (Figure 2) of 3 are similar to those of 2, which suggested the same relative orientations of H-7α, H-9α, H-10β, H-11β, OH-13α, H-16β, Me-25α, and Me-26β in 2 and 3. Similarly, both DP4+ probability and calculated ECD were used to determine the absolute configuration of 3 as 3S,4R,7S,9S,10R,11R,12S,13S,16S,31S (Table S6 and Figure S91). Compound 3 was named tolypocladin C. Compound 4 gave the same molecular formula C32H44ClNO6 as that of 2, by HRESIMS, suggesting 11 degrees of unsaturation. Comparison of the NMR data with those of 2 revealed that compound 4 differs only in the chemical shifts of the aromatic ring, implying that the substitution pattern for the benzene ring is different. The 1H NMR data (Table 1) exhibited three aromatic methines [δH 7.40 (1H, d, J = 8.0 Hz, H-20), 6.97 (1H, d, J = 8.0 Hz, H-21), 7.25 (1H, s, H-23)], suggesting the existence of a 1,2,4trisubstituted aromatic ring. The presence of a 3-chloro-3methyl-1-butan-2-ol moiety was supported by key HMBC correlations of H-30 with C-21 and C-23, H-31 with C-22, C33, and C-34, H-33 with C-31 and C-32, and H-34 with C-31

heteroatoms). The data above accounted for all the NMR signals. Comparison of NMR data with those of the closely related compound drechmerin F revealed that compound 2 has the same indole diterpenoid skeleton,7 and its NMR data were almost identical to those of drechmerin F, differing only in the chemical shifts at C-32, C-33, and C-34. The chemical shift at C-32 (δC 73.9) was significantly upfield in 2 compared to drechmerin F [C-32 (δC 79.0)], and C-33 (δC 28.5) and C-34 (δC 28.5) were significantly downfield in 2 compared to drechmerin F [C-33 (δC 22.0) and C-34 (δC 20.6)]. Moreover, the proton chemical shifts at C-33 (δH 1.73) and C-34 (δH 1.73) are different from those of drechmerin F [H-33 (δH 1.26) and C-34 (δH 1.24)]. This information indicated that the hydroxy group at C-32 in drechmerin F was replaced by the chlorine atom in 2. The relative configurations of all chiral centers except for C-31 in 2 were determined to be the same as in drechmerin F by comparison of NMR data with those of drechmerin F, and this was further confirmed by the NOESY correlations (Figure 2) of H-5a/H-7/H-25, H-7/H-6a/H-9, H10/H-11, and H-26/H-6b/H-11/H-16. The use of the DP4+ probability was also required to determine the relative configuration of 2. Two stereoisomers, (3S,4R,7S,9S,10R,11R,12S,13S,16S,31S)-2 (2a) and (3S,4R,7S,9S,10R,11R,12S,13S,16S,31R)-2 (2b) existed on the basis of the relative configuration. As shown in Figure 2, the relative configuration was established as 3S*,4R*,7S*,9S*,10R*,11R*,12S*,13S*,16S*,31S* with confidence (Table S3).17−21 The calculated ECD revealed that the absolute configuration of 2 should be 3S,4R,7S,9S,10R,11R,12S,13S,16S,31S (Figure S91). C



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Figure 1. Key HMBC and 1H,1H COSY correlations of compounds 1−10.

Significant differences in the NMR data of 5 and terpendole L were found in the resonances assigned to both C-31 [(δC 79.8 (d) in 5; δC 124.0 (d) in terpendole L] and C-32 [(δC 74.5 (s) in 5; δC 130.6 (s) in terpendole L], suggesting that the substitution pattern for the isopentyl moiety at C-20 in 5 is different from that of terpendole L. Comparison of NMR data with those of 2, as well as analysis of the key HMBC correlations of H-30 with C-19 and C-21, H-31 with C-20, C33, and C-34, H-33 with C-31 and C-32, and H-34 with C-31 and C-32, revealed the presence of a 3-chloro-3-methyl-1butan-2-ol moiety connected to C-20 in the aromatic ring. The same relative configurations of all chiral centers except for C31 as in terpendole L could be deduced from the NOESY correlations (Figure 2) of H-5a with H-7/H-25, H-7 with H6a/H-9, H-10 with H-11/H-35, and H-26 with H-6b/H-11/H16, as well as by comparison of NMR data with those of terpendole L. Additionally, the relative configuration of 5 was elucidated by the DP4+ probability, and the absolute configuration was confirmed by the calculated ECD as 3S,4R,7S,9S,10R,11R,12S,13S,16S, 31S,35S (Table S12 and Figure S91). Compound 5 was named tolypocladin E.

and C-32, which was attached to C-22 in the aromatic ring. The NOESY spectrum showed correlations of H-5a with H-7/ H-25, H-7 with H-6a/H-9, H-10 with H-11, and H-26 with H6b/H-11/H-16, indicating the relative configurations of the chiral centers were the same as in 2 except for C-31. Correspondingly, the absolute configuration of 4 was determined to be 3S,4R,7S,9S,10R,11R,12S,13S,16S,31S by the calculated ECD based on the DP4+ probability (Table S9 and Figure S91). Compound 4 was named tolypocladin D. Compound 5 had a molecular formula of C37H50ClNO6 as deduced from HRESIMS, consistent with 13 hydrogen deficiencies. The 13C NMR data of 5 (Table 1) in conjunction with the DEPT-135 spectrum showed the presence of 37 carbon signals, which could be classified into 10 olefinic or aromatic carbons, eight methyls, six methylenes, seven aliphatic methines (six of which are attached to heteroatoms), and six aliphatic nonprotonated carbons (four of which are attached to heteroatoms). Direct comparison of its NMR data with those of the closely related compound terpendole L,22 a known indole diterpenoid reported from Albophoma yamanashiensis, suggested compound 5 to be a derivative of this compound. D



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Figure 2. Key NOESY correlations of compounds 1−10.

data revealed compound 6 has the same indole diterpenoid skeleton as 5, except that the chloride atom at C-32 in 5 is replaced by the hydroxy group in 6; this conclusion was supported by the upfield chemical shift values at C-33 [δH 1.28 (3H, s); δC 25.9 (q)] and C-34 [δH 1.25 (3H, s); δC 25.5 (q)] along with the difference of their molecular formula. The same relative configuration of 6 was determined as in 5 by analyses of the NOESY data, proton coupling constants, and by comparing its NMR data with those of 5. In addition, the absolute configuration of 6 was identified as

Compound 6, an amorphous powder, afforded a molecular formula of C37 H51 NO 7 based on HRESIMS analysis, corresponding to 13 degrees of hydrogen deficiency. Compared with the molecular formula of 5, compound 6 lacks a chloride atom and has an additional OH. The 1H and 13 C NMR and HSQC spectra revealed that 6 contained 10 olefinic or aromatic carbons, eight methyls, six methylenes, seven aliphatic methines (six of which are attached to heteroatoms), and six aliphatic nonprotonated carbons (four of which are attached to heteroatoms). Analysis of its NMR E



Article

Table 2. 1H NMR Data for Compounds 6−10 (δ in ppm, J in Hz) 6b

no. NH 5 6 7 9 10 11 14 15 16 17 20 21 22 23 25 26 28 29 30 31 33 34 35 36 38 39

10.01, s 1.63, m; 2.53, m 1.54, m; 2.21, m 4.29, t (9.0, 17.8) 3.52, d (9.4) 3.97, d (9.4) 3.48, brs 1.37, m; 1.63, m 1.54, m; 1.89, m 2.74, m 2.53, m; 2.85, m 6.84, 6.90, 7.15, 1.23, 1.04, 1.22, 1.28, 2.79, 3.71, 1.28, 1.25, 5.58, 5.19, 1.72, 1.72,

d, 7.1) t (7.4, 7.6) d (7.9) s s s s m; 3.23, d (13.6) d (9.8) s s d (6.2) d (5.2) s s

7c 10.49, s 1.67, m; 2.41, m 1.47, m; 2.14, m 4.31, t (9.2, 18.5) 3.41, d (9.6) 4.06, d (9.5) 3.53, brs 1.16, m; 1.55, m 1.47, m; 1.79, m 2.67, m 2.25, m; 2.55, m 6.78, d (8.0) 7.14, d (8.0) 7.13, 1.17, 1.04, 1.15, 1.24, 2.36, 3.29, 1.12, 1.09, 5.52, 5.11, 1.65, 1.67,

s s s s s m; 2.94, d (13.7) m s s d (6.4) d (5.5) s s

8b

9b

1.64, m; 1.99, m 2.55, m; 2.79, m 3.35, 3.37, 3.36, 1.60, 1.63, 2.85, 2.57,

d (6.8) d (6.8) brs m; 2.50, m m; 1.95, m m m; 2.80, m

1.65, 1.78, 4.31, 3.56, 3.94, 3.53, 1.40, 1.56, 2.76, 2.52,

6.70, 6.88, 7.12, 1.30, 1.40, 1.26, 1.26, 3.57, 5.36, 1.76, 1.74, 5.24, 5.06, 1.67, 1.70,

d (7.1) t (7.5, 7.7) d (8.0) s s s s m m s s d (6.4) d (6.1) s s

6.85, 6.91, 7.14, 1.23, 1.07, 1.24, 1.24, 2.76, 3.70, 1.28, 1.26, 4.71, 2.79, 1.29, 1.30,

m; 2.60, m m; 2.21, m t (9.2, 18.0) d (9.5) d (9.5) brs m; 1.61, m m; 1.91, m m m; 2.83, dd (12.5, 22.4) d (7.0) t (7.4, 7.8) d (7.8) s s s s m; 3.24, dd (2.0, 13.7) dd (2.2, 10.4) s s d (6.3) d (6.3) s s

10b 1.65, 1.81, 4.32, 3.57, 3.96, 3.59, 1.30, 1.57, 2.80, 2.34, 7.23, 6.88,

m; 2.61, m m; 2.23, m t (9.2, 18.0) d (9.5) d (9.5) brs m; 1.61, m m; 1.94, m m m; 2.63, m d (8.0) d (8.0)

7.21, 1.26, 1.12, 1.25, 1.24, 2.64, 3.71, 1.63, 1.61, 4.70, 2.78, 1.29, 1.30,

s s s s s m; 3.22, d (13.9) dd (1.5, 9.8) s s d (6.3) d (6.3) s s

a


aliphatic methines (four of which are attached to heteroatoms), five aliphatic nonprotonated carbons (three of which are attached to heteroatoms), and a ketocarbonyl carbon (δC 209.4). The above characteristic NMR signals suggested that the structure of 8 is similar to 5. The key differences between them were that the ether bond (C-7−O−C-9) in 5 was cleaved and subsequently an oxidation reaction occurred at the position C-7 to form a ketone carbonyl in 8, and the 3chloro-3-methyl-1-butan-2-ol moiety in 5 was replaced by the 3-methylbut-2-en-1-yl moiety in 8. This was further confirmed by the HMBC correlations (Figure 1) of H-5 with C-7 (δC 209.4), H-30 with C-19, C-21, and C-32, H-31 with C-20, and H-33 and H-34 with C-31 and C-32, together with the 1H,1H COSY correlations of H-5 with H-6 and of H-30 with H-31. The relative configuration of 8 was determined by comparison of its NMR with those of coisolated compounds, supported by the NOESY correlations (Figure 2) of H-5a with H-25, H-10 with H-11/H-9/35, and H-26 with H-6b/H-11/H-16. DP4+ probability validated the relative configuration, and the absolute configuration was identified as 3S,4R,9R,10R,11R,12S,13S,16S,35S (Table S21 and Figure S91). Compound 8 was named tolypocladin H. Compound 9 exhibited the molecular formula C37H51NO8 as determined by HRESIMS, containing 13 degrees of unsaturation. Its 1H and 13C NMR data (Table 2) resembled those of 6, except for the disappearance of the C-36/C-37 trans-olefin [δC 123.5 (d, C-36), 139.9 (s, C-37)] in 6 and the presence of one oxygenated methine [δC 64.4 (d, C-36)] and one oxygenated carbon [δC 59.0 (s, C-37)] in 9. The chemical shift values of two oxygenated carbons at C-36 and C-37, along with its molecular formula and degrees of unsaturation,

3S,4R,7S,9S,10R,11R,12S,13S,16S,31S,35S by the ECD calculations (Table S15 and Figure S91). Moreover, a dimolybdenum tetraacetate [Mo2(OAc)4]-induced electronic circular dichroism (IECD) experiment confirmed the absolute configuration of C-35 as S with a positive Cotton effect at 314 nm (Figure 3).23,24 Compound 6 was named tolypocladin F. Compound 7 was assigned the same molecular formula C37H51NO7 as 6 based on HRESIMS analysis, consistent with 13 degrees of unsaturation. The NMR spectroscopic data of 7 and 6 are very similar, apart from the chemical shifts of the aromatic ring, suggesting that the substitution pattern for the benzene ring in 7 is different. The 1H NMR data indicated the presence of a 1,2,4-trisubstituted aromatic ring [δH 6.78 (1H, d, J = 8.0 Hz, H-20), 7.14 (1H, d, J = 8.0 Hz, H-21), 7.13 (1H, s, H-23)]. The HMBC correlations from H-30 to C-21 and C23, from H-31 to C-22, C-33, and C-34, from H-33 to C-31 and C-32, and from H-34 to C-31 and C-32 displayed the presence of a 3-hydroxyl-3-methyl-1-butan-2-ol moiety, which was attached to C-22 in the aromatic ring. The same relative configuration of 7 as in 6 was confirmed by the NOESY data and proton coupling constants and by comparing NMR data. Finally, the absolute configuration of 7 was determined to be 3S,4R,7S,9S,10R,11R,12S,13S,16S,31S,35S by the ECD calculations (Figures 3 and S91). Compound 7 was named tolypocladin G. Compound 8, an amorphous powder, was assigned a molecular formula of C37H49NO6 (15 degrees of unsaturation) on the basis of HRESIMS analysis. As shown in Table 3, the 13 C NMR data exhibited 37 carbon signals, consisting of 12 olefinic or aromatic carbons, eight methyls, six methylenes, five F


Journal of Natural Products Table 3.

13

Article

C NMR Data for Compounds 1−10

no.

1a

2a

3b

4a

5b

6b

7c

8b

9b

10b

2 3 4 5 6 7 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 32-MeO

151.1, C 50.3, C 42.3, C 27.3, CH2 27.7, CH2 71.4, CH 76.7, CH 67.3, CH 64.5, CH 70.0, C 77.8, C 30.3, CH2 20.6, CH2 50.2, CH 29.0, CH2 117.1 C 124.5, C 133.0, C 119.0, CH 120.9, CH 109.3, CH 139.6, C 16.0, CH3 18.8, CH3 73.5, C 28.2, CH3 23.9, CH3 32.0, CH2 123.7, CH 131.8, C 25.8, CH3 18.0, CH3

151.8, C 50.3, C 42.2, C 27.3, CH2 27.7, CH2 71.3, CH 76.5, CH 67.3, CH 64.5, CH 70.0, C 77.7, C 30.2, CH2 20.5, CH2 50.1, CH 29.3, CH2 116.5, C 125.0, C 129.1, C 120.8, CH 120.9, CH 110.2, CH 139.8, C 16.0, CH3 18.8, CH3 73.4, C 28.1, CH3 23.8, CH3 35.8, CH2 78.6, CH 73.9, C 28.5, CH3 28.5, CH3

153.3, C 51.6, C 43.6, C 27.2, CH2 29.3, CH2 72.8, CH 77.6, CH 68.6, CH 64.9, CH 70.5, C 78.8, C 30.6, CH2 21.8, CH2 51.5, CH 30.5, CH2 116.6, C 126.3, C 131.5, C 120.8, CH 120.9, CH 110.7, CH 141.8, C 16.4, CH3 18.9, CH3 73.6, C 27.4, CH3 25.0, CH3 36.0, CH2 78.2, CH 78.5, C 21.9, CH3 20.6, CH3

151.8, C 50.6, C 42.2, C 27.1, CH2 27.7, CH2 71.3, CH 76.3, CH 67.4, CH 64.4, CH 70.0, C 77.6, C 30.1, CH2 20.5, CH2 50.0, CH 27.2, CH2 117.3, C 123.8, C 118.6, CH 120.8, CH 130.6, C 112.3, CH 140.0, C 16.0, CH3 18.7, CH3 73.4, C 28.0, CH3 23.8, CH3 38.5, CH2 80.2, CH 74.3, C 28.7, CH3 28.2, CH3

153.4, C 51.6, C 43.6, C 27.2, CH2 29.6, CH2 73.0, CH 72.5, CH 72.5, CH 61.1, CH 68.7, C 78.6, C 30.4, CH2 21.7, CH2 51.2, CH 30.6, CH2 116.5, C 126.4, C 130.8, C 120.8, CH 121.0, CH 110.8, CH 141.8, C 16.5, CH3 19.0, CH3 76.0, C 28.7, CH3 17.1, CH3 36.7, CH2 79.8, CH 74.5, C 30.3, CH3 28.5, CH3 94.1, CH 123.5, CH 139.9, C 18.8, CH3 25.7, CH3


151.9, C 50.2, C 42.2, C 25.6, CH2 28.5, CH2 70.7, CH 71.1, CH 70.2, CH 59.0, CH 67.1, C 76.5, C 28.6, CH2 20.5, CH2 49.5, CH 26.7, CH2 114.6, C 122.5, C 120.2, CH 116.9, CH 132.3, C 112.2, CH 140.2, C 16.1, CH3 18.0, CH3 74.1, C 28.3, CH3 16.7, CH3 37.8, CH2 79.8, CH 71.8, C 27.0, CH3 24.4, CH3 92.0, CH 122.6, CH 137.5, C 18.4, CH3 25.1, CH3

152.8, C 52.1, C 45.1, C 29.9, CH2 39.4, CH2 209.4, C 76.2, CH 74.6, CH 64.7, CH 70.5, C 80.1, C 30.2, CH2 21.6, CH2 51.2, CH 30.0, CH2 116.6, C 125.6, C 133.5, C 119.2, CH 121.1, CH 110.6, CH 141.8, C 16.6, CH3 19.4, CH3 77.6, C 28.5, CH3 17.1, CH3 33.2, CH2 123.6, CH 132.0, C 25.9, CH3 18.1, CH3 93.0, CH 123.2, CH 140.1, C 18.7, CH3 25.6, CH3


153.4, C 52.0, C 43.7, C 27.3, CH2 29.7, CH2 73.1, CH 72.6, CH 72.5, CH 61.1, CH 68.8, C 78.7, C 30.5, CH2 21.8, CH2 51.4, CH 28.1, CH2 116.9, C 124.8, C 118.3, CH 121.2, CH 132.3, C 113.4, CH 142.1, C 16.4, CH3 19.0, CH3 76.3, C 28.3, CH3 16.9, CH3 39.8, CH2 81.8, CH 74.4, C 29.7, CH3 28.1, CH3 96.6, CH 64.3, CH 59.0, C 19.3, CH3 24.6, CH3

49.6 CH3

a


Figure 3. Mo2(OAc)4-induced ECD spectra of compounds 6, 7, and 9 in DMSO solution.

suggested the presence of a 36,37-epoxide, which was confirmed by the 1H,1H COSY correlation between H-35 and H-36 and key HMBC correlations from H-36 to C-38 and C-39, from H-35 to C-37, from H-38 to C-36 and C-37, and from H-39 to C-36 and C-37. The NOESY data, proton coupling constants, and comparison of its NMR with those of

6 and the related known compound terpendole A reported from Albophoma yamanashiensis7 revealed that the relative configurations of a 36,37-epoxide are the same as that found in terpendole A. The remaining chiral carbon centers are the same as those found in 6. In order to further confirm the relative configuration, four stereoisomers, (3S,4R,7S,9S,10R,G



Article

Scheme 1. Proposed Formation of 1−10 Derived from Terpendole I and Terpendole C

this is the first report of the occurrence of indole diterpenoids in the genus Tolypocladium. In particular, the chlorinated compounds 2, 4, 5, and 10 are the first representatives of indole diterpenes in fungi. The structures of compounds 1−10 are similar to two related known compounds, terpendole I and terpendole C, which have been reported to be biosynthesized from the key intermediate paspaline.6,22 Based on previous findings, the formation of 1−10 from terpendole I and terpendole C is proposed as shown in Scheme 1. Antifungal Activity. All isolated compounds were tested for their activities against the agricultural pathogenic fungi A. f ragariae, C. cassiicola, A. alternata, B. cinereal Pers. C. personata, V. dahliae Kleb, S. sclerotiorum, R. solani, F. oxysporum Schlecht, and A. solani. Sorauer. The results (Table S33) indicated that compounds 1−10 showed no inhibitory activity against three pathogenic fungi (F. oxysporum Schlecht, A. solani.Sorauer, and R. solani), but were selectively active against other fungal strains. Significantly, compound 1 showed obvious inhibitory activities against seven pathogenic fungi (A. f ragariae, C. cassiicola, A. alternata, B. cinereal Pers. C. personata, V. dahliae Kleb, and S. sclerotiorum) with MIC values of 6.25−25 μg/mL (ketoconazole: 0.78−1.56 μg/mL), and all isolated compounds except for 8 were active against A. f ragariae with MIC values of 6.25−50 μg/mL (ketoconazole: 0.78 μg/mL). Antibacterial Assay. Compounds 1−10 were also evaluated for their antibacterial activity toward Gram-positive (B. cereus, methicillin-resistant S. aureus, M. lysodeikticus, B. paratyphosum, and B. subtilis) and Gram-negative (E. aerogenes, S. typhi, and P. vulgaris) human pathogenic bacterial strains. The results (Table S34) indicated that compound 8 was active against all tested bacteria with MIC values of 12.5−25 μg/mL (ciprofloxacin: 0.78−1.56 μg/mL), and compound 1 was

11R,12S,13S,16S,31S,35S,36S)-9 (9a), (3S,4R,7S,9S,10R,11R,12S, 13S,1 6S, 31S, 35S, 36R )-9 (9b), (3S, 4R ,7S,9 S,10R,11R,12S,13S,16R,31S,35S,36R)-9 (9c), and (3S,4R,7S,9S,10R,11R,12S,13S,16R,31S,35S,36S)-9 (9d), were analyzed with DP4+ probability (Table S26), which suggested that the relative configuration of 9 should be 3S*,4R*,7S*,9S*,10R*,11R*,12S*,13S*,16S*,31S*,35S*,36S*. Both IECD and calculated ECD suggested that the absolute configuration of 9 was 3S,4R,7S,9S,10R,11R,12S,13S,16S,31S,35S, 36S (Figures 3 and S91). Compound 9 was named tolypocladin I. Compound 10 was assigned a molecular formula of C37H50ClNO7 as deduced from HRESIMS analysis, indicating 13 degrees of unsaturation. Comparison of NMR data with those of 9 revealed that the skeleton of 10 was the same, except that the substitution pattern for the benzene ring in 10 is different. Further analysis of the 1H NMR data revealed the presence of a 1,2,4-trisubstituted aromatic ring [δH 7.23 (1H, d, J = 8.0 Hz, H-20), 6.88 (1H, d, J = 8.0 Hz, H-21), 7.21 (1H, s, H-23)]. The HMBC correlations of H-30 with C-21 and C23, H-31 with C-22, C-33, and C-34, H-33 with C-31 and C32, and H-34 with C-31 and C-32, combined with the 1H,1H− COSY correlation of H-30/H-31, suggest the presence of a 3chloro-3-methyl-1-butan-2-ol moiety attached to C-22 in the aromatic ring. The relative configuration of 10 was determined to be the same as in 9 by a NOESY experiment and proton coupling constants and by comparing its NMR with those of 9. The absolute configuration of 10 was confirmed as 3S,4R,7S,9S,10R,11R,12S,13S,16S,31R,35S,36S by the ECD calculations (Table S31 and Figure S91). Compound 10 was named tolypocladin J. Structurally, compounds 1−10 belonged to prenylated indole diterpene diterpenoids. To the best of our knowledge, H



Article

solution. Each flask was inoculated with 5.0 mL of the seed culture and then incubated at room temperature for 60 days. Extraction and Isolation. The fermented material was harvested after 60 days and then extracted three times with CH2Cl2−MeOH (1:1, 10 L/each time) at room temperature. The combined solvent extract was evaporated to obtain a dark brown residue, which was subsequently suspended in H2O and then extracted three times with EtOAc to afford 230 g of crude extract. The crude extract was fractionated by silica gel column chromatography (CC) eluting with a CH2Cl2−MeOH gradient system to afford five fractions (A1−A5). Fraction A3 (8.0 g) was further fractionated by CC eluting with petroleum ether−EtOAc (15:1−1:1) to yield five fractions (B1−B5). Fraction B2 (1.0 g) was further separated by silica gel CC with petroleum ether−EtOAc (8:1), followed by semipreparative HPLC using a C18 column (5 μm, 10 × 250 mm, MeOH−H2O, 2 mL/min) to yield 1 (6 mg, tR = 26.9 min, 87% MeOH in H2O), 3 (7 mg, tR = 20.7 min, 78% MeOH in H2O), 7 (17 mg, tR = 31.3 min, 83% MeOH in H2O), and 9 (9 mg, tR = 15.4 min, 85% MeOH in H2O). Compounds 2 (18 mg, tR = 16.8 min, 87% MeOH in H2O), 5 (17 mg, tR = 36.8 min, 87% MeOH in H2O), and 6 (6 mg, tR = 19.8 min, 87% MeOH in H2O) were isolated from fraction B3 (1.7 g) by Sephadex LH-20 (CH2Cl2−MeOH, 1:1), silica gel (petroleum ether−EtOAc, 10:1−1:1), and semipreparative HPLC (5 μm, 10 × 250 mm, MeOH−H2O, 2 mL/min). Fraction B4 (0.8 g) was subjected to chromatography over silica gel (petroleum ether−acetone, 3:1) and then by semipreparative HPLC using a C18 column (5 μm, 10 × 250 mm, MeOH−H2O, 2 mL/min) to obtain 4 (10 mg, tR = 17.8 min, 87% MeOH in H2O), 8 (4 mg, tR = 17.8 min, 81% MeOH in H2O), and 10 (5 mg, tR = 19.0 min, 83% MeOH in H2O). Tolypocladin A (1): white, amorphous powder; [α]24 D −51.5 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 230 (3.42), 285 (3.91) nm; IR (KBr) νmax 3403 (OH), 2933, 1453, 1376, 1101, 819, 788, 742 cm−1; 1 H and 13C NMR data, see Tables 1 and 3; positive HRESIMS ion at m/z 522.32084 [M + H]+ (calcd for C32H44NO5, 522.32140). Tolypocladin B (2): white, amorphous powder; [α]26 D −45.0 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 205 (3.98), 230 (4.14), 290 (3.54) nm; IR (KBr) νmax 3419 (OH), 2936, 1455, 1375, 1098, 821, 786, 746 cm−1; 1H and 13C NMR data, see Tables 1 and 3; negative HRESIMS ion at m/z 572.28056 [M − H]− (calcd for C32H43ClNO6, 572.27844). Tolypocladin C (3): white, amorphous powder; [α]31 D −22.8 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 210 (4.44), 230 (4.52), 284 (3.94) nm; IR (KBr) νmax 3412 (OH), 2936, 1457, 1374, 1094, 929, 739 cm−1; 1H and 13C NMR data, see Tables 1 and 3; negative HRESIMS ion at m/z 568.32958 [M − H]− (calcd for C33H46NO7, 568.32798). Tolypocladin D (4): white, amorphous powder; [α]27 D −25.8 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 205 (4.31), 235 (4.64), 280 (3.96) nm; IR (KBr) νmax 3386 (OH), 2931, 1458, 1374, 1107, 829, 800, 737 cm−1; 1H and 13C NMR data, see Tables 1 and 3; positive HRESIMS ion at m/z 574.29181 [M + H]+ (calcd for C32H45ClNO6, 574.29299). Tolypocladin E (5): white, amorphous powder; [α]24 D −27.0 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 205 (4.23), 230 (4.33), 285 (3.76) nm; IR (KBr) νmax 3379 (OH), 2931, 1455, 1375, 1117, 978, 825, 742 cm−1; 1H and 13C NMR data, see Tables 1 and 3; negative HRESIMS ion at m/z 638.32582 [M − H]− (calcd for C37H49ClNO6, 638.32539). Tolypocladin F (6): white, amorphous powder; [α]25 D −64.0 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 205 (4.51), 230 (4.69), 285 (4.11) nm; IR (KBr) νmax 3360 (OH), 2934, 1456, 1379, 1050, 926, 819, 747 cm−1; 1H and 13C NMR data, see Tables 2 and 3; negative HRESIMS ion at m/z 620.36185 [M − H]− (calcd for C37H50NO7, 620.35928). Tolypocladin G (7): white, amorphous powder; [α]26 D −37.0 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 205 (3.90), 230 (4.11), 280 (3.43) nm; IR (KBr) νmax 3373 (OH), 2929, 1457, 1378, 1098, 1047, 925, 818, 736 cm−1; 1H and 13C NMR data, see Tables 2 and 3; positive HRESIMS ion at m/z 644.35390 [M + Na]+ (calcd for C37H51NNaO7, 644.35577).

active against B. cereus and methicillin-resistant S. aureus with MIC values of 25 and 12.5 μg/mL, respectively (ciprofloxacin: 0.78 and 1.56 μg/mL, respectively). Moreover, compound 2 exhibited weak activity against methicillin-resistant S. aureus with an MIC value of 50 μg/mL. In Vitro Cytotoxic Evaluation. Compounds 1−10 were also evaluated for their cytotoxicity against the A549, Huh7, LN229, MGC and MHCC97H, LOVO, and MDA231 cell lines (Table S35). Compound 1 displayed weak cytotoxicity against all tested cancer cell lines with IC50 values from 16.32 to 37.80 μM (positive control camptothecin: 0.32−31.8 nM) and suppressed the growth and viability of the HCC cells T1224 in the patient-derived organoids (PDOs) model (Figure S92).

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MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were acquired using a Rudolph Autopol III automatic polarimeter (NJ, USA). UV spectra were obtained on an Agilent spectrophotometer (Agilent Cary60). CD spectra were measured with a Bio-Logic electronic circular dichroism (MOS-450) spectrometer. IR spectra were performed on a Bruker spectrophotometer (TENSOR-27). HRESIMS spectra were obtained on a Bruker FTMS instrument (Bruker, Karlsruhe, Germany). NMR measurements were acquired using an Agilent DD2 (400 MHz) instrument. Column chromatography was carried out on Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) and silica gel (Qingdao Marine Chemical Inc., Qingdao, China). Semipreparative HPLC purifications were performed using an Lab Alliance instrument (Systems Inc., State College, PA, USA) equipped with a Series III pump (flow rate: 2 mL/min) and a UV detector (mode 201) using a Prevail C18 column (250 mm × 10 mm, 5 μm, GRACE Corporate, Columbia, MD, USA). Fungal Material. The strain was isolated from the mine soil collected from Lianyuan, Hunan Province, P. R. China, in May 2015. The isolate was identified to be Tolypocladium sp. (GenBank Accession No. MH260264) by sequence analysis of the ITS region of 18S rDNA. The accession number was assigned as XL115, and a voucher specimen is preserved at the School of Pharmaceutical Sciences, Chongqing University (Huxi Campus). All strains used for antimicrobial activity assays in this study were gifts from Dr. Fei Cao (Pharmaceutical Sciences, Hebei University). Cells and Culture. Huh7 (human hepatocellular carcinoma), MGC803 (gastric cancer), and PBMC (normal peripheral blood mononuclear cell) cell lines were purchased from Shanghai Cell Research Institute of the Chinese Academy of Sciences (Shanghai, China), while LN229 (human GBM), MDA231 (breast cancer), HCT116 (colon cancer), and A549 (lung adenocarcinoma epithelial) cell lines from the American Type Culture Collection (ATCC). All cells were cultivated at 37 °C with 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. Patient-Derived Organoid Culture. The patient-derived HCC cells T122425 (50 000 cells) were mixed with 120 μL of Matrigel (cat. 356230, Corning, NY, USA) and seeded in a 48-well plate to incubate at 37 °C for 20 min. Then, the cells was incubated with 500 μL of medium/well (DMEM/F12 from Thermo Fisher Scientific, supplemented with 1× B27 from Thermo Fisher Scientific, 0.01% BSA from Roche, 2 mM L-glutamine from Thermo Fisher Scientific, 100 units/ mL penicillin/streptomycin from Thermo Fisher Scientific, 10 ng/mL FGF basic from PeproTech, 50 ng/mL EGF from PeproTech, and 50 ng/mL HGF from PeproTech) for 6−8 days to form the PDOs,26 and the medium was changed every 2 days. Preparative-Scale Culture. The strain Tolypocladium sp. XL115 was cultivated on potato dextrose agar (PDA) at 28 °C for 1 week. Then, small pieces were transferred into 10 Erlenmeyer flasks (250 mL) containing 100 mL of PDB medium and incubated at 28 °C and 180 rpm on a rotary shaker for 1 week to afford the seed culture. The scale-up fermentation culture was performed in 200 Erlenmeyer flasks (500 mL), containing 60 g of rice and 100 mL of 2.0% dextrose I



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Tolypocladin H (8): white, amorphous powder; [α]30 D +51.0 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 205 (4.49), 230 (4.50), 285 (3.97) nm; IR (KBr) νmax 3402 (OH), 2978, 1716 (CO), 1451, 1380, 1162, 1077, 975, 746, 706 cm−1; 1H and 13C NMR data, see Tables 2 and 3; positive HRESIMS ion at m/z 626.34544 [M + Na]+ (calcd for C37H49NNaO6, 626.34521). Tolypocladin I (9): white, amorphous powder; [α]27 D −23.0 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 205 (4.10), 230 (4.34), 285 (3.76) nm; IR (KBr) νmax 3415 (OH), 2929, 1456, 1377, 1116, 1051, 924, 803, 739 cm−1; 1H and 13C NMR data, see Tables 2 and 3; negative HRESIMS ion at m/z 636.35625 [M − H]− (calcd for C37H50NO8, 636.35419). Tolypocladin J (10): white, amorphous powder; [α]28 D −26.0 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 205 (3.25), 235 (4.56), 280 (3.87) nm; IR (KBr) νmax 3387 (OH), 2931, 1457, 1376, 1117, 803, 736 cm−1; 1H and 13C NMR data, see Tables 2 and 3; negative HRESIMS ion at m/z 654.32065 [M − H]− (calcd for C37H49ClNO7, 654.32030). Antifungal Asssay. Compound 1−10 were evaluated for their antifungal activity against 10 agricultural pathogenic fungi, including Alteranira f ragariae, Corynespora cassiicola, Alternaria alternata, Botrytis cinereal Pers., Cercospora personata, Verticillium dahliae Kleb, Sclerotinia sclerotiorum, Fusarium oxysporum Schlecht, Alternaria solani Sorauer, and Rhizoctonia solani, by using the microbroth dilution method.27−29 Briefly, the concentration of fungal cultures was prepared to be 104 mycelia fragments/mL, and the stock solutions of compound 1−10 and positive control (ketoconazole) were made to achieve the final concentration of 1 mg/mL in DMSO. Then, 180 μL of fungal cultures was added into each 96-well plate in triplicate, and 20 μL stock solutions of samples were added into the first line well and mixed well to achieve the final concentration of 100 μg/mL. After serially diluting the mixture, the plates were incubated at 28 °C for 3−7 days to determine the minimum inhibitory concentration (MIC). Antibacterial Assay. Compounds 1−10 were also evaluated for their antibacterial activity against Bacillus cereus, methicillin-resistant Staphylococcus aureu, Micrococcus lysodeikticus, Bacterium paratyphosum, Bacillus subtilis, Enterobacter aerogenes, Salmonella typhi, and Proteusbacillm vulgaris, by using the modified broth dilution test.30 Briefly, the suspension solutions of tested bacteria were prepared to achieve the concentration of 106 CFU/mL, and the concentrations of compound 1−10 and positive control (ciprofloxacin) were made to be 1 mg/mL in DMSO. The detailed procedures were similar to those mentioned above in the antifungal activity assay. Cell Viability Assays. The cytotoxic activities of compounds 1− 10 were also evaluated. The cancer cells were seeded in 96-well flatbottomed plates (the cells of A549, Huh7, LN229, and MGC were seeded as 5000 cells/well, while HCT116 and MDA231 were seeded as 10 000 cells/well) and subsequently incubated at 37 °C in a 5% CO2 cell culture incubator for 16 h. Then, cells were treated with different concentrations of tested samples in the growth medium for 48 h. Finally, the cell viability was assessed by the Cell Counting Kit-8 (Beyotime, China) according to the manufacturer’s instruction, and the cell growth inhibitory activity was determined through the absorbance values from treated cells as percentage of control cells. The half-maximal inhibitory concentration (IC50) was further determined by generating a sigmoidal curve. Camptothecin was used as the positive control. For proliferation assays, tumor cells were transferred into 96-well plates (5000 cells per well) and incubated for 1 to 5 days. At the indicated intervals, 10 μL of Cell Counting Kit-8 (Beyotime, China) was added into each well and incubated at 37 °C for 1 h. The absorbance at 450 nm was measured using the Thermo Multiskan spectrum reader (Thermo Scientific).

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00589.

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Spectroscopic data, biological activity results, and calculated data for compounds 1−10 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*(H.-Y. Fu) E-mail: [email protected]. *(Y.-M. Chen) E-mail: [email protected]. *(X.-L. Yang) E-mail: [email protected]. ORCID

Hai-Yan Fu: 0000-0003-3853-0464 Ye-Miao Chen: 0000-0001-5397-1407 Xiao-Long Yang: 0000-0002-0932-6889 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 81872755, 21776321, 21576297, 81500479, U1604169, and 21702181), the Start-up Fund for the “Hundred Young-Talent Scheme” Professorship provided by Chongqing University in China (No. 0247001104410), and the Southwest Hospital Grants (Nos. SWH2016JCYB-44 and SWH2016ZDCX3009).

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