Divirensols: Sesquiterpene Dimers from the Australian Termite Nest

A chemical investigation of the Australian termite nest-derived fungus Trichoderma virens CMB-TN16 yielded the known sesquiterpene gliocladic acid (1)...
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Article Cite This: J. Nat. Prod. 2019, 82, 87−95

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Divirensols: Sesquiterpene Dimers from the Australian Termite NestDerived Fungus Trichoderma virens CMB-TN16 Wei-Hua Jiao,†,‡ Pradeep Dewapriya,† Osama Mohamed,† Zeinab G. Khalil,† Angela A. Salim,† Hou-Wen Lin,‡ and Robert J. Capon*,†

J. Nat. Prod. 2019.82:87-95. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 01/25/19. For personal use only.

†

Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia ‡ Research Center for Marine Drugs, State Key Laboratory of Oncogenes and Related Genes, Department of Pharmacy, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, People’s Republic of China S Supporting Information *

ABSTRACT: A chemical investigation of the Australian termite nest-derived fungus Trichoderma virens CMB-TN16 yielded the known sesquiterpene gliocladic acid (1), together with two new acetylated analogues, 3-acetylgliocladic acid (2) and 14acetylgliocladic acid (3), and seven new dimeric congeners, divirensols A−G (4−10). All metabolites were identified by detailed spectroscopic analysis, supported by biosynthetic considerations, and were assessed for antibacterial and cytotoxic properties. The divirensols are examples of an exceptionally rare class of dimeric sesquiterpene, likely linked via a highly convergent biosynthetic pathway. HPLC-DAD-MS analysis of the crude fungal extract detected ions attributed to putative monomeric biosynthetic precursors.

F

the majority of which appeared to be new to science. What follows is an account of the production, isolation, and structure elucidation of 1−10, together with discussion on their biological properties and likely biosynthetic origins.

ungi are widely distributed in nature, and their chemistry has been studied extensively as a source of structurally diverse, bioactive secondary metabolites. To enhance our fungal biodiscovery program, we turned away from traditional soil-derived fungi, in favor of fungi isolated from, and closely associated with, other phyla. This strategy succeeded in revealing a diverse array of new chemistry, including polyketide viridicatumtoxins,1,2 chaunolidone A,3 chaunolidines A−C3 and chaunopyran A4 from mollusk-derived fungi; diketopiperazine talarazines5 and waspergillamide A6 from wasp-derived fungi; peptidic talarolide A7 and talaropeptides8 from tunicatederived fungi; hydrazine Schiff base prolinimines9 from fishderived fungi; and peptaibol trichodermamides10 from a termite nest-derived fungus. To further enhance this biodiscovery program, we implemented a miniaturized 24-well plate microbioreactor cultivation methodology, coupled to UHPLC-DAD and UHPLC-QTOF chemical profiling (known in-lab as the MATRIX). The MATRIX allows rapid and cost-effective cultivation of multiple fungal strains under ×11 media compositions, and ×3 culture conditions (solid agar and shaken and static broth), confirming and mapping the phenomena that different culture conditions stimulate the biosynthesis of different secondary metabolites. Particularly noteworthy applications of the MATRIX include the discovery of talarazines,5 waspergillamide A,6 talarolide A,7 talaropeptides,8 prolinimines,9 and trichodermamides.10 While the peptaibol trichodermamides were recovered from solid-phase PYG cultivation of an Australian termite nest-derived isolate of Trichoderma virens CMB-TN16,10 it was noted that rice media cultivations produced an extensive array of terpenes (1−10), © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Trichoderma virens CMB-TN16 was cultured on rice media for 4 weeks, after which the EtOAc extract was subjected to solvent partitioning and trituration, followed by reversed-phase fractionation, to yield the known fungal nor-sesquiterpene gliocladic acid (1), together with two new monoacetylated analogues, 3-acetylgliocladic acid (2) and 14-acetylgliocladic acid (3), and seven new dimeric congeners, divirensols A−G (4−10) (Figure 1). The structure of gliocladic acid (1) (relative configuration only) was first reported in 1982 as a metabolite produced by three Japanese fungi (Gliocladium virens SANK 12679, Chaetomium globosum SANK 13379, and Trichoderma viride SANK 13479).11 Although the original report failed to adequately document the NMR data or report an optical rotation, this oversight was in part remedied by a subsequent 2011 reisolation of (+)-1 from a Chinese termite nest-derived Xylaria sp.12 These latter authors assigned a 5R,10R absolute configuration to 1 based on CD comparisons to the cometabolite xylaric acid A, whose structure and absolute configuration they assigned by X-ray analysis. Our latest gliocladic acid installment, the reisolation of (+)-1 from an Australian termite nest-derived fungus, provides the first Received: September 2, 2018 Published: December 31, 2018 87

DOI: 10.1021/acs.jnatprod.8b00746 J. Nat. Prod. 2019, 82, 87−95

Journal of Natural Products

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Figure 1. Trichoderma virens CMB-TN16 metabolites 1−10.

Table 1. NMR Data (600 MHz, DMSO-d6) for 1−3 1 position

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 3-COCH3 3-COCH3 14-COCH3 14-COCH3

168.4, C 132.6, C 55.1, CH2 148.2, CH 38.1, CH 121.4, CH 139.8, C 25.3, CH2 21.2, CH2 44.8, CH 28.3, CH 16.8, CH3 20.9, CH3 64.6, CH2

a−c

2 δH (J in Hz)

4.18, 6.39, 3.19, 5.22,

δC, type

d (11.4); 4.13, d (11.4) d (10.5) br dd (10.5, 7.2) br s

1.99, m; 1.91, m 1.68, m; 1.23,a m 1.23,a m 1.59, m 0.77, d (7.2) 0.90, d (7.2) 3.78, s

167.4, C 127.5, C 57.8, CH2 152.0, CH 38.5, CH 120.1, CH 140.7, C 25.2, CH2 20.6, CH2 44.7, CH 28.4, CH 16.7, CH3 21.1, CH3 64.4, CH2 170.1, C 20.8, CH3

3 δH (J in Hz)

4.75, 6.63, 3.12, 5.16,

s d (10.7) dd (10.7, 8.5) br s

2.00,b m; 1.92, m 1.70, m; 1.23, m 1.28, m 1.57, m 0.76, d (6.6) 0.90, d (6.6) 3.78, s

δC, type 168.3, C 133.2, C 55.2, CH2 147.2, CH 38.2, CH 126.2, CH 134.3, C 25.4, CH2 20.67,d CH2 44.3, CH 28.1, CH 16.7, CH3 21.2, CH3 67.1, CH2

δH (J in Hz)

4.18, 6.38, 3.23, 5.35,

d (12.0); 4.13, d (12.0) d (10.4) br dd (10.4, 7.7) br s

2.01,c m; 1.97, m 1.71, m; 1.24, m 1.25, m 1.59, m 0.77, d (6.6) 0.90, d (6.6) 4.42, s

1.99,b s 170.2, C 20.65,d CH3

2.02,c s

d

Overlapping signals with the same superscript. Assignments with the same superscript may be interchanged.

(1). Comparison of the 1D NMR (DMSO-d6) data for 2 (Tables 1 and S2) with 1 indicated the principle difference as the appearance of an acetyl moiety (δH 1.99; δC 170.1, 20.8) and deshielding of H2-3 (ΔδH −0.6). A diagnostic HMBC correlation from H2-3 to COCH3 confirmed the structure as 3acetylgliocladic acid (2) (Figure 2). A comparable spectro-

detailed account of the 1D and 2D NMR (DMSO-d6) data for 1 (Tables 1 and S1) and associated new co-metabolites 2−10 (see below). HRESI(−)MS analysis of 2 revealed an [M − H]− ion attributed to a molecular formula of C16H24O5 (Δmmu −0.5), consistent with a monoacetylated analogue of gliocladic acid 88

DOI: 10.1021/acs.jnatprod.8b00746 J. Nat. Prod. 2019, 82, 87−95

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Figure 2. Selected 2D NMR (DMSO-d6) correlations and J values for 1−3.

Table 2.

a

13

C NMR Data (150 MHz, DMSO-d6) for 4−10 (δ, type)

pos.

4

5

6

7

8

9

10a

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

168.3, C 133.9, C 55.3, CH2 144.0, CH 40.1, CH 58.3, CH 71.2, C 34.6, CH2 20.2,b CH2 45.6, CH 27.8, CH 15.4, CH3 21.4, CH3 65.6, CH2 173.9, C 166.7, C 133.9, C 55.6, CH2 144.3, CH 33.6, CH 124.0, C 165.2, C 22.4, CH2 20.0,b CH2 44.7, CH 27.3, CH 17.7, CH3 21.4, CH3 71.5, CH2 173.0, C

166.8, C 129.4, C 60.9, CH2 145.2, CH 39.3, CH 51.8, CH 71.7, C 34.8, CH2 20.9, CH2 47.6, CH 27.0, CH 15.2, CH3 21.1, CH3 65.0, CH2 171.8, C 166.7, C 134.0, C 55.5, CH2 144.3, CH 33.6, CH 124.0, C 165.2, C 22.4, CH2 20.0, CH2 44.8, CH 27.3, CH 17.7, CH3 21.4, CH3 71.5, CH2 173.0, C

166.8, C 129.4, C 60.9, CH2 145.2, CH 39.5, CH 51.7, CH 71.8, C 34.8, CH2 21.0, CH2 47.6, CH 27.0, CH 15.2, CH3 21.3, CH3 65.0, CH2 171.8, C 166.7, C 133.4, C 55.4, CH2 144.1, CH 39.6, CH 52.0, CH 74.4, C 31.8, CH2 20.7, CH2 44.7, CH 28.0, CH 15.6, CH3 21.2, CH3 74.5, CH2 176.6, C

166.9, C 129.4, C 60.9, CH2 145.2, CH 39.5, CH 51.9, CH 71.8, C 34.8, CH2 20.9,c CH2 47.6, CH 27.0, CH 15.2, CH3 21.2, CH3 65.0, CH2 171.8, C 167.0, C 132.1, C 55.2, CH2 149.0, CH 38.1, CH 121.4, CH 144.0, C 25.2, CH2 21.1,c CH2 44.9, CH 28.5, CH 17.1, CH3 21.2, CH3 64.6, CH2

166.9, C 129.4, C 60.9, CH2 145.2, CH 39.5, CH 51.8, CH 71.7, C 34.8, CH2 20.9, CH2 47.6, CH 27.02, CH 15.2, CH3 21.3, CH3 64.7, CH2 171.9, C 164.6, C 129.1, C 65.4, CH2 141.9, CH 38.2, CH 71.8, CH 43.1, CH 23.4, CH2 23.3, CH2 44.7, CH 26.97, CH 15.9, CH3 21.3, CH3 62.5, CH2

168.2, C 134.3, C 55.5, CH2 142.7, CH 37.9, CH 48.9, CH 84.6, C 27.2, CH2 19.4, CH2 43.6, CH 27.7, CH 15.8, CH3 21.4, CH3 71.2, CH2 175.1, C 165.9, C 132.3, C 55.0, CH2 149.5, CH 38.2, CH 120.8, CH 140.2, C 25.2, CH2 20.9, CH2 44.6, CH 28.4, CH 16.9, CH3 21.2, CH3 64.5, CH2

169.8, C 132.8, C 57.0, CH2 145.2, CH 38.4, CH 49.3, CH 84.3, C 27.0, CH2 19.0, CH2 43.1, CH 28.0, CH 15.5, CH3 21.2, CH3 72.5, CH2 176.1, C 165.6, C 134.0, C 56.5, CH2 145.2, CH 34.4, CH 125.0, C 164.8, C 23.1, CH2 20.4, CH2 45.2, CH 28.0, CH 17.4, CH3 21.5, CH3 72.0, CH2 174.3, C

In CDCl3.

b,c

Assignments with the same superscript may be interchanged.

carboxylic acid, accounting for ×7 DBE and requiring that 4 be tricyclic. Diagnostic COSY and HMBC correlations permitted assembly of a complete planar structure for 4, dimeric to the co-metabolites 1−3, with an HMBC correlation from H2-14 to C-1′ establishing an ester linkage between the two monomeric subunits (Figure 3). Similarly, ROESY correlations between H5 and H-11, and between H-5′ and H-11′, required trans H-5/ H-10 and trans H-5′/H-10′ configurations in common with 1− 3. ROESY correlations between H2a-14 and both H-5 and H2b9, together with trans diaxial J5,10 10.6 Hz and J5,6 10.6 Hz, required a C-5 to C-10 chair configuration, with axially disposed H-5, H-6, H-10, and H2-14 (Figure 3). The absolute configuration of divirensol A (4) was assigned to be in common with 1−3, on biosynthetic grounds. HRESI(−)MS analysis of 5 revealed an [M − H]− ion attributed to a molecular formula of C30H40O10 (Δmmu −0.2), consistent with a dehydration analogue of 4. Comparison of

scopic analysis of the co-metabolite 3 (C16H24O5, Δmmu −0.9), including 1D NMR resonances for an acetyl moiety (Tables 1 and S3), deshielding of H2-14 in 3 relative to 1 (ΔδH −0.64), and a diagnostic 2D NMR correlation from H2-14 to COCH 3 (Figure 2), confirmed the structure as 14acetylgliocladic acid. ROESY correlations between H-5 and H-11 and comparable values for J5,10 established a common relative configuration across 1−3 (Figure 2), while biosynthetic considerations were used to assign absolute configurations to 2 and 3 in common with the co-metabolite 1. HRESI(−)MS analysis of 4 revealed an [M − H]− ion attributed to a molecular formula of C30H42O11 (Δmmu −0.9), requiring ×10 double-bond equivalents (DBE). Inspection of the 1D and 2D NMR (DMSO-d6) data for 4 (Tables 2, 3, and S4) revealed sp2 resonances and associated correlations consistent with an α,β-unsaturated lactone, an α,β-unsaturated ester, an α,β-unsaturated carboxylic acid, and an unconjugated 89

DOI: 10.1021/acs.jnatprod.8b00746 J. Nat. Prod. 2019, 82, 87−95

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Table 3. 1H NMR Data (600 MHz, DMSO-d6) for 4−6 [δ, mult (J in Hz)] pos.

4

5

4.11, d (11.4) 4.07, d (11.4) 6.28, d (10.6) 2.73, ddd (10.6, 10.6, 10.6) 2.40,a m 2.11, ddd (12.8, 3.0, 3.0) 1.31,b m

5.27, d (14.4) 4.83, d (14.4) 7.06, d (3.8) 2.59, ddd (12.4, 12.4, 4.2) 3.65, d (12.4) 2.15, ddd (12.8, 3.2, 3.2) 1.34, m

1.50,c m 1.09, m 1.30,b m 1.61, m 0.69, d (6.6) 0.84, d (6.6) 4.36, d (12.0) 4.24, d (12.0) 4.33, d (11.4) 4.30, d (11.4) 6.44, d (10.4) 3.49, br dd (10.4, 5.5)

1.59, m 1.16, m 1.51, m 1.95, m 0.83, d (6.6) 0.89, d (6.6) 4.97, d (12.2) 4.38, d (12.2) 4.35, d (12.0) 4.31, d (12.0) 6.44, d (10.4) 3.49, br dd (10.4, 6.5)

6′ 8′a

2.39,a m

2.41, m

8′b 9′a 9′b 10′ 11′ 12′ 13′ 14′a 14′b

2.39,a m 1.82, m 1.50,c m 1.46, m 1.64, m 0.83, d (6.6) 0.94, d (6.6) 4.82, m 4.82, m

2.37, 1.82, 1.48, 1.44, 1.64, 0.84, 0.95, 4.82, 4.82,

3a 3b 4 5 6 8a 8b 9a 9b 10 11 12 13 14a 14b 3′a 3′b 4′ 5′

a−e

m m m m m d (6.6) d (6.6) m m

11′ required trans H-5/H-10 and trans H-5′/H-10′ configurations, while correlations between H2b-14 and both of H2b-9 and H-5, together with J5,6 12.4 Hz and J5,10 12.4 Hz, required a C-5 to C-10 chair configuration with axially disposed H-5, H-6, H-10, and H2-14 (Figure 3). Biosynthetic considerations permitted assignment of the absolute configuration for divirensol B (5) as shown, in common with 1−4. HRESI(−)MS analysis of 6 revealed an [M − H]− ion attributed to a molecular formula of C30H42O11 (Δmmu −0.3), isomeric with 4 and a hydrated analogue of 5. Comparison of the 1D NMR (DMSO-d6) data for 6 (Tables 2, 3, and S6) with 5 supported the latter hypothesis, with principle differences

6 5.29, 4.84, 7.07, 2.59,

d (14.4) d (14.4) d (3.6) m

3.67, d (12.5) 2.20, ddd (12.9, 3.3, 3.3) 1.35, ddd (12.9, 12.9, 3.3) 1.62,d m 1.18e, m 1.52, m 1.97, m 0.85, d (6.6) 0.90, d (6.6) 5.03, d (12.0) 4.35, d (12.0) 4.10, d (12.0) 3.96, d (12.0) 6.56, d (10.6) 2.62, m

attributed to H2O addition to Δ6′,7′ in 5, to deliver a 7′-OH (δC 74.4) and an sp3 C-6′ methine (δC 52.0; δH 2.03) in 6. Remaining 1D and 2D NMR data for 6 compared favorably with 4 and 5, with diagnostic ROESY correlations between H5 and H-11 and between H-5′ and H-11′, requiring trans H-5/ H-10 and trans H-5′/H-10′ configurations (Figure 3). Similarly, ROESY correlations between H2b-14 and both of H2b-9 and H-5 and between H2a-14′ and both H2b-9′ and H-5′, together with J5,6 12.5 Hz and J5′,6′ 11.0 Hz, required C-5 to C10, and C-5′ to C-10′, chair configurations, with axially disposed H-5/H-5′, H-6/H-6′, H-10/H-10′, and H2-14/H214′ (Figure 3). The absolute configuration of divirensol C (6) was assigned to be the same as 1−5, as shown, on biosynthetic grounds. HRESI(+)MS analysis of 7 revealed an [M + Na]+ adduct ion attributed to a molecular formula of C29H42O9 (Δmmu −0.2), requiring ×9 DBE. The 1D and 2D NMR (DMSO-d6) data for 7 (Tables 2, 4, and S7) revealed two distinct structure fragments, C-1 to C-15, in common with divirensol C (6), and C-1′ to C-14′ in common with gliocladic acid (1), accounting for all DBE. An HMBC correlation from H2-14 to C-1′ established an ester linkage between these two fragments, while diagnostic ROESY correlations between H-5 and H-11, and between H-5′ and H-11′, indicated trans H-5/H-10 and trans H-5′/H-10′ configurations (Figure 4). Similarly, ROESY correlations between H2b-14 and both H2b-9 and H-5, and between H-6 and H-10, together with trans diaxial J5,6 12.6 Hz and J5′,6′ 10.8 Hz, indicated a C-5 to C-10 chair configuration, with axially disposed H-5, H-6, H-10, and H2-14 (Figure 4). Biosynthetic considerations permitted assignment of the partial absolute configuration to divirensol D (7) as shown, in common with 1−6. HRESI(−)MS analysis of 8 revealed an [M − H]− ion attributed to a molecular formula of C29H42O9 (Δmmu −0.6), isomeric with 7. The 1D and 2D NMR (DMSO-d6) data for 8

2.03, d (11.0) 1.89, ddd (13.4, 2.8, 2.8) 1.56, m 1.61,d m 1.19,e m 1.23, m 1.64, m 0.71, d (6.6) 0.88, d (6.6) 4.43, d (9.6) 3.94, d (9.6)

Overlapping signals with the same superscript.

the 1D NMR (DMSO-d6) data for 5 (Tables 2, 3, and S5) with 4 indicated the principle difference as deshielding of resonances for H2-3/C-3 in 5 (δH 5.27 and 4.83; δC 60.9) compared to 4 (δH 4.11 and 4.07; δC 55.3). This, together with an HMBC correlation from H2-3 to C-15, and other 2D NMR correlations (Figure 3), was consistent with a 3-OH to 6CO2H lactonization of 4, to yield 5. Diagnostic ROESY correlations between H-5 and H-11 and between H-5′ and H-

Figure 3. Selected 2D NMR (DMSO-d6) correlations and J values for divirensols A−C (4−6). 90

DOI: 10.1021/acs.jnatprod.8b00746 J. Nat. Prod. 2019, 82, 87−95

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Table 4. 1H NMR Data (600 MHz, DMSO-d6) for 7−10 [δ, mult (J in Hz)] pos.

7

8

3a 3b 4 5

5.27, d (14.4) 4.83, d (14.4) 7.07, br s 2.59, dd (12.6, 11.5)

5.27, d (14.1) 4.84, d (14.1) 7.06, d (3.0) 2.58, ddd (12.7, 12.7, 3.0)

6 8a

3.65, d (12.6) 2.17, ddd (12.6, 3.0, 3.0) 1.34, m 1.58,a m 1.16, m 1.52, m 1.97, m 0.83, d (6.6) 0.88, d (6.6) 4.95, d (12.0) 4.38, d (12.0) 4.24, d (11.6) 4.19, d (11.6) 6.52, d (10.8) 3.22, dd (10.8, 7.7) 5.24, br s

8b 9a 9b 10 11 12 13 14a 14b 3′a 3′b 4′ 5′ 6′ 7′ 8′a 8′b 9′a 9′b 10′ 11′ 12′ 13′ 14′a

1.99, m 1.91, m 1.71, m 1.26,b m 1.26,b m 1.59,a m 0.78, d (6.6) 0.89, d (6.6) 3.79, m

14′b

3.79, m

a−e

9

4.09, d (11.9) 3.98, d (11.9) 6.53, d (10.3) 2.86, ddd (10.3, 9.6, 9.6) 3.64, d (12.7) 2.75, d (9.6) 2.10, ddd (12.8, 2.39, ddd 2.3, 2.3) (14.0, 5.0, 5.0) 1.33, m 1.89, m 1.60, m 1.62,d m 1.18, m 1.29, m 1.51,c m 1.38, m 1.95, m 1.63,d m 0.84, d (6.6) 0.72, d (6.8) 0.88, d (6.6) 0.90, d (6.1) 4.99, d (11.9) 4.65, d (10.8) 4.33, d (11.9) 4.62, d (10.8) 4.39, d (16.2) 4.16, d (11.7) 4.26, d (16.2) 4.13, d (11.7) 7.18, d (5.0) 6.43, d (10.5) 1.99, m 3.20, dd (10.5, 7.7) 3.60, s 5.20, br s 1.54,c m 1.58, m 1.98, m 1.03, m 1.90, m 1.52,c m 1.68, m 1.12, m 1.23, m 1.48, m 1.28, m 1.85, m 1.55, m 0.85, d (6.6) 0.76, d (6.6) 0.88, d (6.6) 0.89, d (6.6) 3.48, dd (10.2, 3.79, d (15.1) 7.8) 3.27, dd (10.2, 3.75, d (15.1) 6.6)

between H-6 and H-10, confirmed a common C-1 to C-15 structure fragment (inclusive of configurations about chiral centers). The principle differences in the NMR data for the C1′ to C-15′ structure fragment in 8 compared to 7 were the

10f 4.32, d (11.8) 4.29, d (11.8) 6.81, d (9.7) 2.74, ddd (9.7, 9.7, 9.0)

absence of resonances for Δ6′,7′ and the appearance of resonances for a C-6′/H-6′ oxymethine (δC 71.8; δH 3.60) and a C-7′/H-7 alkylmethine (δC 43.1; δH 1.54), with ROESY correlations between H-5′ and H-6′ necessitating a cis relationship (Figure 4). These observations, together with HMBC correlations from both H2-3′ and H2-14′ to C-6′, were consistent with a β-facial intramolecular electrophilic addition of the 3′-OH to C-6′ in 7, to yield a didehydropyran heterocycle in 8. A ROESY correlation between H2-14 and H2a-9 suggests that the didehydropyran formation proceeds via a syn-addition to Δ6′,7′, in which C-14′ is driven to adopt an axial, α-orientation in 8. An HMBC correlation from H2-14 to C-1′ established an ester linkage between the C-1 to C-15 and C-1′ to C-15′ structure fragments. Biosynthetic considerations permitted assignment of the absolute configuration to divirensol E (8) as shown. HRESI(+)MS analysis of 9 revealed an [M + Na]+ adduct ion attributed to a molecular formula of C29H42O9 (Δmmu +0.4), isomeric with divirensol D (7). Indeed, comparison of the 1D and 2D NMR (DMSO-d6) data for 9 (Tables 2, 4, and S9) with 7 indicated a common C-1′ to C-15′ structure fragment, inclusive of relative configurations, the latter evidenced by a ROESY correlation between H-5′ and H-11′. By contrast, comparison of the NMR data for the C-1 to C-15 structure fragment in 9 with 7 revealed replacement of the seven-membered lactone (3-O to 6-CO) moiety in 7 with an alternate five-membered (14-O to 6-CO) moiety in 9 (Figure 5). This alternate lactonization was validated by diagnostic changes in the 1H NMR chemical shifts for H2-3 (9 δH 3.98, 4.09; 7 δH 4.83, 5.27) and H2-14 (9 δH 4.62, 4.65; 7 δH 4.38, 4.95) and the appearance in 9 of an HMBC correlation from H2-14 to C-15 (Figure 5). A ROESY correlation between H-5 and H-11 indicated a trans H-5/H-10 configuration, while correlations between H2a-14 and both H2b-9 and H-5, together with trans diaxial J5,6 9.6 Hz, indicated a C-5 to C-10 chair configuration, with axially disposed H-5, H-6, H-10, and H2-14 (Figure 5). By a process of elimination, the two structure fragments were deemed to be connected via a 7-OH to 2′CO2H ester linkage, while biosynthetic considerations permitted assignment of the absolute configuration to divirensol F (9) as shown, in common with 1−8. HRESI(−)MS analysis of 10 revealed an adduct ion [M − H]− attributed to a molecular formula of C30H40O10 (Δmmu

2.76, d (9.0) 2.28, m 2.15, m 1.72, m 1.25, m 1.54, m 1.67, m 0.68, d (6.6) 0.92, d (6.6) 4.78, d (12.6) 4.49, d (12.6) 4.46, d (12.1) 4.40, d (12.1) 6.40, d (10.9) 3.52, dd (10.9, 5.6)

2.40, m 2.40, m 1.93, m 1.51,e m 1.50,e m 1.62, m 0.85, d (6.6) 0.98, d (6.6) 4.80, d (12.6) 4.72, d (12.6)

Overlapping signals with the same superscript. fIn CDCl3.

(Tables 2, 4, and S8) revealed a C-1 to C-15 structure fragment in common with divirensols C and D (6 and 7) and a C-1′ to C-14′ structure fragment isomeric with that in 7. Excellent concordance in the NMR data for the C-1 to C-15 structure fragment in 8 with 7, together with ROESY correlations between H2b-14 and both H2a-9 and H-5 and

Figure 4. Selected 2D NMR (DMSO-d6) correlations and J values for divirensols D and E (7 and 8). 91

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Figure 5. Selected 2D NMR correlations and J values for divirensol F (9) in DMSO-d6 and diverensol G (10) in CDCl3.

−0.6), isomeric with divirensol B (5). Comparison of the 1D and 2D NMR (CDCl3) data for 10 (Tables 2, 4, and S10) with 9 indicated a common C-1 to C-15 structure fragment, inclusive of relative configurations for all chiral centers, as evidenced by ROESY correlations between H2a-14 and H2a-9 and between H2b-14 and H-5 and a trans diaxial J5,6 9.0 Hz (Figure 5). Likewise, comparison of the NMR data for 10 with 5 indicated a common C-1′ to C-15′ structure fragment, inclusive of relative configurations, as evidenced by a ROESY correlation between H-5′ and H-11′ (Figure 5). Biosynthetic considerations permitted assignment of the absolute configuration to divirensol G (10) as shown, in common with 1−9. The divirensols A−G (4−10) are new examples of a rare class of dimeric sesquiterpene fungal secondary metabolites. To the best of our knowledge, members of this class are limited to a single known example, trichoderonic acid B (11), the planar structure of which was reported in 2010 from a Japanese strain of Trichoderma virens.13 Of note, these same authors described (and patented) 11 as a selective inhibitor of a family of X DNA polymerases (IC50 30.8 μM), capable of inhibiting the growth of human cervix (HeLa, LD50 33.0 μM) and breast (MCF-7, LD50 31.6 μM) carcinoma cells. In our hands, 1−10 did not inhibit the growth of human colorectal (SW620) or lung (NCIH460) carcinoma cells, at concentrations up to 30 μM. In further testing, we determined that 1−10 did not inhibit the growth of Gram-positive Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633 or Gramnegative Escherichia coli ATCC 25922 at concentrations up to 30 μM. The ecological role and possibly pharmacological potential of the divirensols 4−10 remain unknown.

putative monomers A−G exist in the CMB-TN16 crude extract, we acquired UHPLC-DAD-ESI(−)MS data with selected single-ion extraction (SIE) (Figure 7). This analysis successfully detected a single species with an m/z 279 [M − H]−, which we tentatively attributed to A, reasoning that in CMB-TN16 the isomeric subfragment D was likely produced late in the biosynthesis. Our analysis also detected three species with an m/z 297 [M − H]− tentatively attributed to B, C, and E, and a single species with an m/z 315 [M − H]− attributed to F. Finally, we detected a single species with an m/ z 253 [M − H]−, which we assigned to G, based on HPLC comparison to an authentic sample (i.e., 1). Supportive of these conclusions, several of these putative monomers have been previously reported as fungal metabolites. For example, A was first reported in 198014 as heptelidic acid from the same three fungi that were the original source of gliocladic acid (1), which is itself G; B was described in 2016 from Aspergillus oryzae;15 C and D were described in 2011 (along with A and G) from a Xylaria sp.;12 while F and H have yet to be described as natural products. Even though it was not isolated from the CMB-TN16 cultivation extract, the biosynthetic relationship linking 1−10, as shown in Scheme 1, positions heptelidic acid (A) as a pivotal precursor.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation values ([α]D) were measured with a JASCO P-1010 polarimeter in a 100 × 2 mm cell at room temperature. NMR spectra were recorded in DMSOd6 or CDCl3 with referencing to residual 1H (δH 2.50 and 7.24, respectively) or 13C (δC 39.51 and 77.23, respectively) NMR resonances on a Bruker Avance 600 MHz spectrometer equipped with either a 5 mm PASEL 1H/D-13C Z-Gradient probe or a 5 mm CPTCI 1H/19F−13C/15N/DZ-Gradient cryoprobe, controlled by TopSpin 2.1 software. HRESIMS data were obtained on a UHPLC-QTOF instrument comprising an Agilent 1290 Infinity II UHPLC equipped with a Zorbax C8 column (2.1 mm × 50 mm, 1.8 μm particles), with a linear gradient of 90% H2O/MeCN to 100% MeCN with isocratic 0.5% formic acid at 0.45 mL/min over 6.0 min. HPLC-DAD-ESIMS data were acquired on an Agilent 1100 series separation module equipped with an Agilent 1100 series HPLC/MSD mass detector and diode array multiple wavelength detector using a Zorbax SB-C8 column (4.9 mm x 150 mm, 5 μm particles) with a linear gradient of 90% H2O/ MeCN to 100% MeCN inclusive of an isocratic 0.05% HCO2H at 1 mL/min over 15 min. Semipreparative and preparative HPLCs were performed using Agilent 1100 series HPLC instruments with corresponding detectors, fraction collectors, and software inclusively. All solvents used for HPLC separation and purification were chromatographic grade. Collection and Isolation of Trichoderma virens CMB-TN16. The fungus CMB-TN16 was isolated from samples of termite nest, collected in 2016 from an urban location in Pullenvale, Queensland. The sample was dissected under aseptic conditions and placed on

Figure 6. Known Trichoderma virens metabolite trichoderonic acid B (11).

The sesquiterpenes 1−11 are likely biosynthetically derived from a core “cadinene scaffold”, which undergoes oxidation to form the epoxy-lactone-acid A (Scheme 1). Subsequent hydrolysis of A, supported by relactonization and dehydration, can deliver the monomeric units B−F, whereas F can decarboxylate to G, which can undergo an intramolecular addition to the ether H. To determine which (if any) of the 92

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Scheme 1. Plausible Biosynthetic Relationship among 1−11

phase was concentrated in vacuo to yield a crude extract (2.32 g), which was sequentially partitioned between H2O (100 mL) and equal volumes of n-hexane and CH2Cl2. The dried CH2Cl2-soluble fraction (0.93 g) was subjected to reversed-phase SPE chromatography (GracePure C18-Max with a 10% stepwise gradient elution from 10% MeOH/H2O to 100% MeOH), and the peaks of interest (HPLCDAD-MS) concentrated in two fractions, F2 (0.23 g) and F3 (0.45 g). F2 was further fractionated by preparative HPLC (Phenomenex C18 column, 250 × 21.2 mm, 10 μm, 20 mL/min with a linear gradient of 30% to 80% MeCN/H2O over 20 min, with a constant 0.01% TFA/ MeCN modifier) to yield subfractions F2A and F2B. The subfractions were subjected to reversed-phase HPLC (YMC C18 RS column, 250 × 10 mm, 5 μm, 3.0 mL/min,). F2A (15.5 mg) was eluted with a linear gradient of 35%−40% MeCN/H2O, with a constant 0.01% TFA/MeCN modifier over 30 min, to afford 3 (tR = 14.5 min, 1.4 mg). F2B (45.6 mg) was eluted with a linear gradient of 42%−58% MeCN/H2O, with a constant 0.01% TFA/MeCN modifier over 35 min, to yield 1 (tR = 6.5 min, 2.8 mg), 4 (tR = 11.2 min, 7.2 mg), 2 (tR = 11.5 min, 2.4 mg), 5 (tR = 17.8 min, 3.1 mg), and 10 (tR = 18.9 min, 2.6 mg). F3 was also further fractionated by preparative HPLC (Phenomenex C18 column, 250 × 21.2 mm, 10 μm, 20 mL/min linear with a linear gradient of 40−80% MeCN/H2O, with a constant 0.01% TFA/MeCN modifier over 30 min) to yield subfractions F3A−F3F. These fractions were further fractionated by semipreparative HPLC (YMC C18 RS column, 250 × 10 mm, 5 μm, 3.0 mL/min). F3A (25 mg) (isocratic 35% MeCN/H2O) yielded 6 (tR = 22.1 min, 2.5 mg); F3B (29.2 mg) (isocratic 45% MeCN/H2O) yielded 7 (tR = 15.6 min, 3.5 mg) and 9 (tR = 17.7 min, 8.4 mg); and F3D (16.2 mg) (isocratic 45% MeCN/H2O) yielded 8 (tR = 34.1 min, 2.0 mg). Gliocladic acid (1): colorless gum; [α]25D +139.5 (c 0.17, MeOH); UV−vis (DAD from MeCN/H2O with 0.01% TFA/MeCN) λmax 221

Figure 7. UHPLC-ESI(−)MS analysis of the crude extract of CMBTN16, with single-ion extraction (SIE) detecting putative monomeric precursors where (i) G m/z 253; (ii) F m/z 315; (iii) B, C, and E m/z 297; and (iv) A m/z 279. PYG agar plates (comprising 2% glucose, 1% peptone, 0.5% yeast extract, 0.02% chloramphenicol, and 1.5% agar in distilled water). The plates were wrapped in Parafilm and incubated at 26.5 °C for 3−4 weeks. A pure culture of fungus CMB-TN16 was obtained by singlecolony serial transfer on agar plates and cryopreserved at −80 °C in 15% aqueous glycerol. Taxonomic analysis identified CMB-TN16 as a T. virens.10 Cultivation and Isolation of Trichoderma virens CMB-TN16. The fungus of T. virens was cultivated in rice medium (70 g of rice, 0.3 g of peptone, 0.3 g of yeast extract, 0.1 g of monosodium glutamate, prepared in 100 mL of distilled water) at room temperature for 30 days. The resulting cultivation was repeatedly extracted with EtOAc until the organic phase was almost colorless. The combined organic 93

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nm; 1H and 13C NMR (DMSO-d6) data, see Tables 1 and S1; HRESIMS m/z 253.1441 [M − H]− (calcd for C14H21O4, 253.1445). 3-Acetyl gliocladic acid (2): colorless gum; [α]25D +161.4 (c 0.16, MeOH); UV−vis (DAD from MeCN/H2O with 0.01% TFA/MeCN) λmax 223 nm; 1H and 13C NMR (DMSO-d6) data, see Tables 1 and S2; HRESIMS m/z 295.1556 [M − H]− (calcd for C16H23O5, 295.1551). 14-Acetyl gliocladic acid (3): colorless gum; [α]25D +148.5 (c 0.24, MeOH); UV−vis (DAD from MeCN/H2O with 0.01% TFA/MeCN) λmax 225 nm; 1H and 13C NMR (DMSO-d6) data, see Tables 1 and S3; HRESIMS m/z 295.1560 [M − H]− (calcd for C16H23O5, 295.1551). Divirensol A (4): colorless gum; [α]25D +53.4 (c 0.07, MeOH); UV−vis (DAD from MeCN/H2O with 0.01% TFA/MeCN) λmax 221 nm; 1H and 13C NMR (DMSO-d6) data, see Tables 2, 3, and S4; HRESIMS m/z 577.2663 [M − H]− (calcd for C30H41O11, 577.2654). Divirensol B (5): colorless gum; [α]25D +78.7 (c 0.19, MeOH); UV−vis (DAD from MeCN/H2O with 0.01% TFA/MeCN) λmax 222 nm; 1H and 13C NMR (DMSO-d6) data, see Tables 2, 3, and S5; HRESIMS m/z 559.2551 [M − H]− (calcd for C30H39O10, 559.2549). Divirensol C (6): colorless gum; [α]25D +15.8 (c 0.09, MeOH); UV−vis (DAD from MeCN/H2O with 0.01% TFA/MeCN) λmax 224 nm; 1H and 13C NMR (DMSO-d6) data, see Tables 2, 3, and S6; HRESIMS m/z 577.2657 [M − H]− (calcd for C30H41O11, 577.2654). Divirensol D (7): colorless gum; [α]25D +74.3 (c 0.07, MeOH); UV−vis (DAD from MeCN/H2O with 0.01% TFA/MeCN) λmax 223 nm; 1H and 13C NMR (DMSO-d6) data, see Tables 2, 4, and S7; HRESIMS m/z 557.2723 [M + Na]+ (calcd for C29H42NaO9, 557.2721). Divirensol E (8): colorless gum; [α]25D +23.6 (c 0.13, MeOH); UV−vis (DAD from MeCN/H2O with 0.01% TFA/MeCN) λmax 223 nm; 1H and 13C NMR (DMSO-d6) data, see Tables 2, 4, and S8; HRESIMS m/z 533.2762 [M − H]− (calcd for C29H41O9, 533.2756). Divirensol F (9): colorless gum; [α]25D +88.6 (c 0.07, MeOH); UV−vis (DAD from MeCN/H2O with 0.01% TFA/MeCN) λmax 221 nm; 1H and 13C NMR (DMSO-d6) data, see Tables 2, 4, and S9; HRESIMS m/z 557.2717 [M + Na]+ (calcd for C29H42NaO9, 557.2721). Divirensol G (10): colorless gum; [α]25D +89.1 (c 0.15, MeOH); UV−vis (DAD from MeCN/H2O with 0.01% TFA/MeCN) λmax 226 nm; 1H and 13C NMR (CDCl3) data, see Tables 2, 4, and S10; HRESIMS m/z 559.2543 [M − H]− (calcd for C30H39O10, 559.2549). Cytotoxicity Assay. Adherent human colorectal (SW620) and lung (NCIH-460) carcinoma cells were cultured in RPMI medium 1640. All cells were cultured as adherent monolayers in flasks supplemented with 10% fetal bovine serum, L-glutamine (2 mM), penicillin (100 unit/mL), and streptomycin (100 μg/mL), in a humidified 37 °C incubator supplied with 5% CO2. Briefly, cells were harvested with trypsin and dispensed into 96-well microtiter assay plates at 3000 cells/well, after which they were incubated for 18 h at 37 °C with 5% CO2 (to allow cells to attach as adherent monolayers). The compounds to be tested were dissolved in DMSO and diluted with PBS to give a concentration of 600 μM in 20% DMSO. An aliquot (10 μL) was applied to cells to give a final concentration of 30 μM in 1% DMSO. After 48 h of incubation at 37 °C with 5% CO2 an aliquot (20 μL) of MTT in PBS (5 mg/mL) was added to each well (final concentration 0.5 mg/mL), and microtiter plates were incubated for a further 4 h at 37 °C with 5% CO2. After a final incubation, the medium was aspirated and precipitated formazan crystals were dissolved in DMSO (100 μL/well). The absorbance of each well was measured at 580 nm with a PowerWave XS microplate reader from Bio-Tek Instruments Inc. (Vinooski, VT, USA). IC50 values were calculated using Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA), as the concentration of analyte required for 50% inhibition of cancer cell growth (compared to negative controls). Negative controls comprised 1% aqueous DMSO, while positive controls used doxorubicin (30 μM) as the test sample. All experiments were performed in duplicate. Antibacterial Assays. The bacteria to be tested was streaked onto a tryptic soy agar plate and incubated at 37 °C for 24 h. One colony

was then transferred to fresh tryptic soy broth (15 mL), and the cell density adjusted to 104−105 CFU/mL. The compounds to be tested were dissolved in DMSO and diluted with H2O to give a 600 μM stock solution in 20% DMSO. An aliquot (10 μL) from the stock solution was transferred to a 96-well microtiter plate, and freshly prepared microbial broth (190 μL) was added to each well to give a final concentration of 30 μM in 1% DMSO. The plates were incubated at 37 °C for 24 h, and the optical density of each well was measured spectrophotometrically at 600 nm using a POLARstar Omega plate (BMG LABTECH, Offenburg, Germany). Each test compound was screened against the Gram-negative bacteria Escherichia coli ATCC 25922 and the Gram-positive bacteria Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633. Rifampicin was used as a positive control (30 μM 1% DMSO). The IC50 value was calculated as the concentration of the compound or antibiotic required for 50% inhibition of the bacterial cells using Prism 7.0 from GraphPad Software Inc. (La Jolla, CA, USA).

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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.8b00746. Additional information (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Hou-Wen Lin: 0000-0002-7097-0876 Robert J. Capon: 0000-0002-8341-7754 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 41576130, U1605221, 41476121, and 31600014) and the Fund of the Science and Technology Commission of Shanghai Municipality (No. 14431901300), as well as The University of Queensland and the Institute for Molecular Bioscience.

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REFERENCES

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