Epigenetic modification in histone deacetylase deletion strain of Calcarisporium arbuscula leads to diverse diterpenoids

Abstract

Epigenetic modifications have been proved to be a powerful way to activate silent gene clusters and lead to diverse secondary metabolites in fungi. Previously, inactivation of a histone H3 deacetylase in Calcarisporium arbuscula had led to pleiotropic activation and overexpression of more than 75% of the biosynthetic genes and isolation of ten compounds. Further investigation of the crude extract of C. arbuscula ΔhdaA strain resulted in the isolation of twelve new diterpenoids including three cassanes (1−3), one cleistanthane (4), six pimaranes (5−10), and two isopimaranes (11 and 12) along with two know cleistanthane analogues. Their structures were elucidated by extensive NMR spectroscopic data analysis. Compounds 2 and 4 showed potent inhibitory effects on the expression of MMP1 and MMP2 (matrix metalloproteinases family) in human breast cancer (MCF-7) cells.

Graphical abstract

Epigenetic modifications have been proved to be a powerful technique to activate silent gene clusters and had led to diverse secondary metabolites in fungi. Investigation of the crude extract of Calcarisporium arbuscula ΔhdaA strain, in which a histone H3 deacetylase was deleted, resulted in the isolation of twelve new diterpenoids including three cassanes (1−3), one cleistanthane (4), six pimaranes (5−10), and two isopimaranes (11−12). Compounds 2 and 4 showed potent inhibitory effects on the expression of MMP1 and MMP2 (matrix metalloproteinases family) in human breast cancer cells.

1. Introduction

Calcarisporium arbuscula, an endophytic fungus lives in the flesh of healthy fruit-bodies of mushrooms¹, mainly produces the adenosine triphosphate synthase inhibitors aurovertins B and D, which structurally contain a 2,6-dioxabicyclo[3.2.1]-octane ring2, 3, 4. In our prior genome mining investigation⁵, genome sequencing of C. arbuscula revealed a large number of potential secondary metabolite gene clusters (68, of which 9 contain terpene synthases), which are significantly more than the two predominant metabolites aurovertins B and D, suggesting that most gene clusters are silent or lowly expressed in axenic cultures. Epigenetic modifications have been proved to be a powerful way to activate silent gene clusters in fungi6, 7, 8, 9, 10. Deletion of hdaA, which encodes a histone deacetylase (HDAC), has led to the globally pleiotropic activation and overexpression of more than 75% of the biosynthetic genes and the isolation of diverse ten compounds of which four possess new structures, including three tricyclic diterpenes and a novel meroterpenoid derived from the esterification between an anthraquinone and a cassane diterpene⁵. So far, diterpenoids have exhibited diverse structural features and a wide range of interesting biological activities11, 12, such as cytotoxic13, 14, antimicrobial15, 16, anti-inflammatory¹⁷, and antiviral18, 19, 20 properties, attracting more and more attention.

Careful analysis of the crude extract from the C. arbuscula ΔhdaA strain by UPLC—MS showed the presence of numerous minor ingredients that could not be identified due to sample limitations. To further explore the potential of the mutant strain ΔhdaA and ongoing search for diverse new bioactive natural products, we fermented in a larger scale using potato dextrose agar (PDA) culture. Subsequently, investigation of the PDA fermentation extract resulted in the isolation of twelve new diterpenoids including three cassanes (1--3), one cleistanthane (4), six pimaranes (5--10), and two isopimaranes (11 and 12), together with two known cleistanthanes analogues (13 and 14, Fig. 1). Herein, details of the isolation, structure characterization, and bioactivities of 1--14 are reported.

Figure 1.

Structures of compounds 1—14.

2. Results and discussion

Compound 1 was isolated as a colorless amorphous solid with [α]²⁰_D +30.0 (c 0.10, MeOH). The IR spectrum of 1 showed absorption bands for hydroxyl (3490 cm^—1), carbonyl (1724 cm^—1), and carbon-carbon double bonds moieties (1649 cm^—1). Its molecular formula was determined as C₂₀H₂₈O₅ by negative HR-ESI-MS at m/z 347.1864 [M–H]⁻ (Calcd. for C₂₀H₂₇O₅, 347.1864) combined with NMR spectroscopic data (Table 1, Table 2). The ¹H NMR spectrum of 1 showed signals attributable to a vinyl group [δ_H 6.25 (dd, J = 17.7 and 10.9 Hz, H-16), 5.09 (d, J = 17.7 Hz, H-17a), and 4.92 (d, J = 10.9 Hz, H-17b)], an olefinic proton [δ_H 5.66 (br s, H-12)], an oxygen-bearing methine [δ_H 4.34 (m, H-3)], a secondary methyl [δ_H 1.08 (d, J = 7.0 Hz, H-15)] and two tertiary methyl [δ_H 1.50 (s, H-18) and 0.89 (s, H-20)]. The ¹³C NMR and HSQC data revealed the presence of 20 carbons, including three methyls, four methylenes, four methines (including one O-methine), three sp³ quaternary carbons with one oxygenated, four olefinic carbons accounting for an olefin and a vinyl group, a carboxylic carbon (δ_C 175.1), and a ketone carbon (δ_C 211.7). These data accounted for all the NMR resonances of 1 and four of the seven unsaturation degrees, indicating 1 as a tricyclic compound.

Table 1.

¹³C NMR spectroscopic data (δ) for compounds 1−12^a (δ in ppm).

No.	1	2	3	4	5	6	7	8	9	10	11	12
1	28.2	29.21	71.1	28.6	25.9	25.6	31.1	30.8	31.6	28.4	31.3	30.0
2	24.3	26.3	26.45	26.0	27.7	27.8	20.1	20.1	27.7	19.7	27.7	20.0
3	79.4	73.2	26.43	72.9	73.7	73.8	32.8	32.9	71.3	33.5	71.4	32.9
4	59.3	49.1	48.6	51.0	51.1	51.2	49.7	49.8	48.7	50.4	49.0	49.9
5	82.2	78.9	77.7	78.0	80.5	80.1	77.1	76.9	46.5	81.2	46.7	76.9
6	211.7	69.8	27.8	69.2	26.4	26.4	26.3	26.3	21.3	30.0	21.7	26.8
7	42.0	32.7	24.2	35.0	26.8	26.7	27.2	27.1	31.4	27.8	32.0	26.7
8	39.1	29.17	33.5	35.4	128.8	128.1	129.0	128.2	129.8	137.5	128.7	128.0
9	35.8	35.8	29.3	45.8	138.8	138.5	139.2	139.2	140.5	78.5	140.3	137.9
10	45.2	41.6	42.8	41.1	44.6	44.6	44.4	44.6	39.4	43.4	39.2	44.4
11	26.9	24.9	24.4	26.6	22.1	22.2	22.1	22.1	21.7	27.6	22.0	22.6
12	128.8	129.3	128.7	35.7	30.1	31.6	30.2	31.5	32.3	32.9	27.6	28.4
13	142.6	141.5	141.1	151.0	40.6	41.1	40.6	41.1	40.7	40.0	40.4	40.4
14	33.2	31.6	31.6	53.6	77.2	88.5	77.2	88.6	77.9	132.8	76.0	76.3
15	14.8	15.0	14.6	139.8	145.8	146.4	145.8	146.3	147.7	146.6	147.9	147.5
16	139.6	139.0	138.7	116.7	112.3	112.3	112.3	112.4	111.8	113.7	111.8	112.0
17	110.5	110.1	109.6	106.2	23.0	21.8	22.9	21.9	19.6	29.7	21.1	21.5
18	19.4	20.6	24.0	19.9	20.4	20.5	24.5	24.5	24.6	24.2	24.6	24.6
19	175.1	178.4	179.0	177.5	178.7	178.9	181.0	181.1	181.3	180.2	181.4	181.2
20	15.5	15.1	14.5	15.0	22.9	22.9	22.6	22.6	17.9	19.2	17.9	22.5
OMe						62.0		62.1

^aNMR data (δ) were measured at 125 MHz in CD₃OD for 1, 5, 6, 8−12; at 150 MHz in CD₃OD for 7; and at 150 MHz in DMSO-d₆ for 2−4.

Table 2.

¹H NMR spectroscopic data (δ) for compounds 1−4^a (δ in ppm, J in Hz).

No.	1	2	3	4
1	1.94, overlap	1.70, td (13.8, 3.5)	3.57, d (3.0)	1.66, overlap
	1.59, m	1.14, d (12.5)		1.26, br d (12.8)
2	2.31, m	1.98, overlap	2.10, overlap	1.98, overlap
	2.03, overlap	1.56, d (11.6)	1.64, overlap	1.57, m
3	4.34, br d (5.5)	4.22, br s	1.72, td (13.7, 4.0)	5.21, br s
			1.50, overlap
6		4.04, br s	2.03, td (13.8, 2.4)	3.92, br s
			1.62, overlap
7	3.14, t (12.9)	2.11, m	1.87, overlap	1.62, overlap
	2.09, dd (13.0, 4.9)	1.35, overlap	1.24, br d (12.1)
8	1.94, overlap	1.77, m	1.52, overlap	1.41, m
9	2.71, m	2.09, m	2.56, m	1.72, overlap
11	2.22, m	2.01, overlap	2.12, overlap	1.70, overlap
	2.02, overlap	1.95, overlap	1.84, overlap	1.00, qd (13.0, 3.6)
12	5.66, br s	5.63, t (3.5)	5.63, t (3.8)	2.35, d (12.9)
				1.98, overlap
14	2.47, m	2.32, m	2.38, m	2.25, t (9.8)
15	1.08, d (7.0)	0.87, d (6.8)	0.92, d (6.9)	5.62, ddd (17.2, 9.8, 9.8)
16	6.25, dd (17.7, 10.9)	6.20, dd (17.6, 10.9)	6.20, dd (17.6, 10.9)	5.13, dd (10.2, 2.0)
				4.98, dd (17.2, 2.0)
17	5.09,d (17.7)	5.08,d (17.6)	5.07,d (17.6)	4.64, d (1.1)
	4.92, d (10.9)	4.90, d (10.9)	4.89, d (10.9)	4.48, br s
18	1.50, s	1.36, s	1.12, s	1.32, s
20	0.89, s	0.96, s	0.70, s	0.91, s
1-OH			5.50, d (4.0)
3-OH		6.23, br s		6.23, d (2.5)
5-OH		6.10, br s	5.15, s	6.20, br s
6-OH				7.21, br s
19-OH			12.11, s	14.32, br s

^aNMR data (δ) were measured at 500 MHz in CD₃OD for 1 and at 600 MHz in DMSO-d₆ for 2−4.

Analysis of the ¹H–¹H COSY NMR data (Fig. 2) of 1 identified the C-1–C-3 moiety, the C-16–C-17 vinyl group, and the C-7/8/9/11/12 unit with the C-14–C-15 fragment attached to C-8. In the HMBC spectrum (Fig. 2) of 1, correlations from the olefinic proton H-16 to C-12, C-13, and C-14 indicated that C-13 was connected to C-12, C-14, and C-16, respectively, establishing a cyclohexene ring with a vinyl and a methyl groups located at C-13 and C-14, respectively. HMBC correlations from the methyl H₃-20 to C-1, C-5, C-9, and C-10 led to the connections of C-1, C-5, C-9, and C-20 to C-10. The HMBC correlations of H₃-18/C-3, C-4, C-5, and C-19 and H₂-7/C-5 and C-6 located two hydroxy groups at C-3 and C-5, respectively, a carboxyl group at C-19, and a ketone group at C-6, establishing the tricyclic cassane type diterpene skeleton. Based on these data, the planar structure of 1 was established as shown in Fig. 1 which is closely resemble to sonomolide B¹⁵ and hawaiinolide F¹⁴.

Figure 2.

¹H—¹H COSY and key HMBC correlations of compounds 1, 4, 5 and 10.

The relative configuration of 1 was deduced by analysis of NOESY spectrum. NOESY correlations (Fig. 3) of H₃-20 with H-8 and H-2β (δ_H 2.03) and of H-3 with H-2β indicated that these protons were in the β-orientation, whereas those of H₃-15 with H-9 suggested the α-axial orientation of the methyl at C-14. NOESY cross peaks of OH-5 with H-9 and H₃-18, indicating the α-orientation for OH-5 and Me-18. Thus, the relative configuration of 1, which was very similar as those of hawaiinolides F and G¹⁴, was established.

Figure 3.

Key NOESY correlations of compounds 1, 4, 5 and 11.

The absolute configuration of 1 was determined by analysis of its CD spectrum. The negative Cotton effect observed at 320 nm in the CD spectrum (Fig. 4) is associated with the n→π* transition of the cyclohexanone chromophore13, 21, 22. On the basis of the octant rule for cyclohexanones, the 5 S absolute configuration was determined. Combining the established relative configuration, the absolute configuration of 1 was deduced to be 3 R, 4 S, 5 S, 8 S, 9 S, 10 R, 14 R. Therefore, 1 was determined as 3R, 5S-dihydroxy-6-oxo-14R-cassane-12, 16-dien-19-oic acid and named calcarisporic acid A.

Figure 4.

CD spectrum of 1 in MeOH.

Compound 2 possesses the molecular formula C₂₀H₃₀O₅ as determined by negative HR-ESI-MS at m/z 349.2030 [M–H]⁻ (Calcd. for C₂₀H₂₉O₅, 349.2020) and NMR spectroscopic data. Comparing the 1D NMR spectra (Table 1, Table 2) of 2 with those of 1, signals due to an oxygenated methine were observed at δ_H 4.04 (br s, H-6) and δ_C 69.8 in the NMR spectra of 2, with the disappearance of the ketone carbon at δ_C 211.7 (C-6) in 1. The HMBC correlations from the oxygenated methine proton at δ_H 4.04 (br s, H-6) to C-4, C-5, C-7, C-8, and C-10, indicated the location of the hydroxyl group at C-6. According to NOESY correlations, the relative configuration of 2 was same as that of 1, except for the presence of correlation of H-6 with H₃-18, which suggested the β-configuration of OH-6. Therefore, 2 was established as 3α, 5α, 6β-trihydroxy-15α-cassane-12, 16-dien-19-oic acid and named calcarisporic acid B.

The molecular formula of compound 3 was determine as C₂₀H₃₀O₄ on the basis of negative HR-ESI-MS at m/z 333.2083 [M–H]⁻ (Calcd. for C₂₀H₂₉O₄, 333.2071). The ¹H NMR and ¹³C NMR spectroscopic data (Table 1, Table 2) of 3 were similar to those of 2. The major difference was that signals for a methylene at δ_C 27.8 (C-6), δ_H 2.03 (td, J = 13.8, 2.4 Hz, H-6a), and δ_H 1.62 (m, H-6b) in 3 replaced resonances for an oxygenated methine at δ_C 69.8 (C-6) and δ_H 4.04 (br s, H-6) in 2, which were verified by HMBC correlations from H₂-6 to C-5, C-7, C-8, and C-10. HMBC correlations from H-1 (δ_H 3.57) to C-3, C-5, C-10, and C-20, suggested that C-1 was substituted by a hydroxy group. In addition, the relative configuration of 3 was deduced from NOESY correlations and by comparison to that of 2. NOESY correlations of H₃-20 with H-1 and H-8 indicated the β-configuration of H-1, whereas those of OH-5 with H₃-18, OH-1, and H-9, and of H₃-15 with H-9 revealed that they were in the α-orientation. Thus, 3 was elucidated as 1α, 5α-dihydroxy-15α-cassane-12, 16-dien-19-oic acid and designated as calcarisporic acid C.

Compound 4, colorless needles with [α]²⁰_D +10.2 (c 0.17, MeOH), was determined to have the molecular formula of C₂₀H₃₀O₅ by negative HR-ESI-MS at m/z 349.2031 [M–H]⁻ (Calcd. for C₂₀H₂₉O₅, 349.2020). The ¹H NMR and ¹³C NMR spectroscopic data (Table 1, Table 2) showed the presence of two tertiary methyl, five methylenes, five methines (including two O-methines), three sp³ quaternary carbons with one oxygenated, four olefinic carbons accounting for an exocyclic olefin [δ_H 4.64 (d, J = 1.1 Hz, H-17a), and 4.48 (br s, H-17b); δ_C 106.2 (C-17) and 151.0 (C-13)] and a vinyl group [δ_H 5.62 (ddd, J = 17.2, 9.8 and 9.8 Hz, H-15), 5.13 (dd, J = 10.2 and 2.0 Hz, H-16a), and 4.98 (dd, J = 17.2 and 2.0 Hz, H-16b); δ_C 139.8 (C-15) and 116.7 (C-16)], and one carboxylic carbon [δ_C 177.5 (C-19)]. Comparison of the NMR spectroscopic data of 4 with those of 2 suggested that 4 possessed the same A and B ring units as those in 2. Analysis of the ¹H—¹H COSY NMR data (Fig. 2) of 4 revealed the C-6/7/8/9/11/12 unit with the C-14–C-16 fragment attached to C-8. HMBC correlations (Fig. 2) of H₂-17/C-12, C-13, and C-14, and H-15/C-13, C-14, and C-8 confirmed that the olefinic methylene and vinyl group connected to C-13 and C-14, respectively, establishing a cyclohexane of C ring moiety with an exocyclic olefin at C-13/C-17 and a vinyl group attached to C-13 fused to the B ring unit at C-8/C9. Thus, the planar structure of 4 was established (Fig. 1), a tricyclic cleistanthane diterpene, which closely resembled that of zythiostromic acid A isolated from Zythiostroma sp. with [α]²⁰_D −31.7 (c 0.35, MeOH)²³. This suggested that the two compounds are diastereomers. Comparison of the chemical shifts and coupling constants in the ¹H NMR spectra of 4 and zythiostromic acid A indicated difference only in the signals attribute to H-14 and H-15. This suggested that 4 was the C-14 epimer of zythiostromic acid A, which were confirmed by NOESY correlations (Fig. 3) of H-14 with H-9, and of H-8 with H-15 and H₃-20, while other NOESY correlations of 4 were the same as those of zythiostromic acid A. Therefore, 4 was established as 3α, 5α, 6β-trihydroxy-15β-cleistanthane-13(17), 15-dien-19-oic acid and named calcarisporic acid D.

Compound 5 was obtained as colorless needles. Its molecular formula was C₂₀H₃₀O₅, as determined by negative HR-ESI-MS at m/z 349.2036 [M–H]⁻ (Calcd. for C₂₀H₂₉O₅, 349.2020). Analysis of its ¹H and ¹³C NMR data (Table 1, Table 3) revealed the presence of three tertiary methyls, six methylenes, two oxygenated methines, four sp³ quaternary carbons with one oxygenated, four olefinic carbons accounting for a tetra-substituted olefin and a vinyl group, and a carboxylic carbon (δ_C 178.7). Comparison of the NMR spectroscopic data of 5 with those of 2 suggested that 5 possessed the same A ring moiety as 2. Interpretation of the ¹H—¹H COSY NMR data (Fig. 2) of 5 identified the C-6–C-7 moiety, the C-11–C-12 unit, and the C-15–C-16 vinyl group. HMBC correlations (Fig. 2) from H-6 to C-4, C-5, and C-10 suggested that the C-6–C-7 moiety was attached to C-5. HMBC correlations from H-1, H₃-20, and H-7 to C-9, and from H-6 and H-7 to C-8 suggested that the tetra-substituted olefin C-8 and C-9 were connected to C-7 and C-9, respectively, establishing a cyclohexene of B ring moiety fused to the A ring unit at C-5/C10. HMBC correlations from H-14 to C-7, C-8, and C-9, and from H-11 to C-8, C-9, and C-10 indicated that the oxygenated C-14 and C-11 were attached to C-8 and C-9, respectively. HMBC cross peaks from H₃-17 to C-12, C-13, C-14, and C-15 led to the connections of C-12, C-14, C-15, and C-17 to C-13, establishing the C-8/C-9 fused cyclohexene of C ring unit with a methyl and a vinyl group both attached to C-13. Thus, the planar structure of 5 was established as shown in Fig. 1, a tricyclic pimarane diterpene. The relative configuration of 5 was confirmed by NOESY spectrum. NOESY correlations (Fig. 3) of H-11β (δ_H 1.96) with H₃-20 and H-15, and of H-2β (δ_H 2.31) with H-3 and H₃-20 suggested that these protons were in the β-configuration, whereas those of H-14 with H₃-17 and H-12α (δ_H 1.44) indicated the α-axial configuration of H-14. NOESY correlation of Me-18 with H-3 and H-6α (δ_H 1.92) and the absence of NOESY correlation between Me-18 and the axial Me-20 revealed that the carboxylic group at C-4 was in the β-axial orientation. Thus, 5 was elucidated as 3α, 5α, 14β-trihydroxy-pimarane-8(9), 15-dien-19-oic acid and designated as calcarisporic acid E.

Table 3.

¹H NMR spectroscopic data (δ) for compounds 5−8^a (δ in ppm, J in Hz).

No.	5	6	7	8
1	1.97, overlap	1.96, overlap	1.78, dd (12.9, 4.1)	1.77, overlap
	1.41, br d (12.5)	1.38, overlap	1.42, overlap	1.41, overlap
2	2.31, m	2.30, m	1.97, overlap	1.96, overlap
	1.73, dd (14.4, 3.0)	1.71, m	1.46, overlap	1.45, overlap
3	4.10, br s	4.07, br s	1.83, d (12.7)	1.83, d (12.8)
			1.74, overlap	1.72, overlap
6	2.48, m	2.47, m	2.49, m	2.50, m
	1.92, overlap	1.94, overlap	1.93, overlap	1.94, overlap
7	2.55, m	2.51, m	2.57, m	2.51, m
	1.93, overlap	1.93, overlap	1.95, overlap	1.96, overlap
11	2.02, overlap	1.99, overlap	1.94, overlap	1.92, overlap
	1.96, overlap	1.94, overlap
12	1.67, m	1.59, m	1.66, m	1.58, m
	1.44, overlap	1.47, overlap	1.43, overlap	1.46, overlap
14	3.47, br s	3.24, br s	3.48, br s	3.24, br s
15	5.76, dd (17.7, 11.0)	5.80, dd (17.7, 11.0)	5.77, dd (17.7, 11.0)	5.80, dd (17.7, 11.0)
16	4.96, br d (17.7)	4.99, dd (17.7, 1.4)	4.96, dd (17.7, 1.4)	4.99, dd (17.7, 1.0)
	4.93, br d (11.0)	4.95, dd (11.0, 1.4)	4.94, dd (10.9, 1.4)	4.95, dd (11.0, 1.0)
17	1.01, s	1.05, s	1.01, s	1.05, s
18	1.46, s	1.45, s	1.23, s	1.24, s
20	1.01, s	1.01, s	1.00, s	1.00, s
OMe		3.51, s		3.52, s

^aNMR data (δ) were measured at 500 MHz in CD₃OD for 5, 6, and 8, and at 600 MHz in CD₃OD for 7.

The molecular formula of compound 6 was established as C₂₁H₃₂O₅ by negative HR-ESI-MS at m/z 363.2182 [M–H]⁻ (Calcd. for C₂₁H₃₁O₅, 363.2177). The ¹H and ¹³C NMR spectra (Table 1, Table 3) of 6 showed close resemblance to those of 5, except for the presence of an additional methoxy group [δ_H 3.51 (3 H, s), δ_C 62.0] relative to 5. It was observed that the resonance for C-14 was downfield-shifted from δ_C 77.2 in 5 to δ_C 88.5 in 6, and the protons of methoxy group showed HMBC correlation with C-14, which suggested that 6 was the 14-methoxy derivative of 5. The relative configuration of 6 was the same as that of 5 according to NOESY correlations. Therefore, 6 was elucidated as 3α,5α-dihydroxy-14β-methoxy-pimarane-8(9), 15-dien-19-oic acid and designated as calcarisporic acid F.

Compound 7 was assigned the molecular formula C₂₀H₃₀O₄ by negative HR-ESI-MS at m/z 333.2077 [M–H]⁻ (Calcd. for C₂₀H₂₉O₄, 333.2071), one less oxygen than that of 5. Comparison of the NMR data of 7 with those of 5 indicated that an O-methine [δ_H 4.10, δ_C 73.7] at C-3 in 5 was replayed by a methylene [δ_H 1.83 and 1.74, δ_C 32.8] in 7. This suggested that 7 was the 3-dehydroxy analog of 5, which was confirmed by HMBC correlations from H₂-3 to C-1, C-2, C-4, and C-5. NOESY correlations of H-11β (δ_H 1.94) with H₃-20 and H-15, of H-14 with H₃-17 and H-12α (δ_H 1.43), and of H₃-18 with H-3α (δ_H 1.83), H-3β (δ_H 1.74), and H-6α (δ_H 1.93) indicated that the relative configuration of 7 was similar as that of 5. Thus, 7 was established as 5α,14β-dihydroxy-pimarane-8(9), 15-dien-19-oic acid and named calcarisporic acid G.

Compound 8 was determined to have the molecular formula of C₂₁H₃₂O₄ by negative HR-ESI-MS at m/z 347.2239 [M–H]⁻ (Calcd. for C₂₁H₃₁O₄, 347.2228). The NMR data (Table 1, Table 3) of 8 were closely similar to those of 7. The major differences were that an additional methoxy group was presented in 8, and the resonance for C-14 was downfield-shifted by 11.4 ppm in comparison with 7. This indicated that 8 was the 14-methoxy derivative of 7, which was verified by HMBC correlations from H-14 to the additional methoxy group (δ_C 62.1), C-8, C-9, C-13, and C-15. The relative configuration of 8 was identical with that of 7 according to NOESY spectrum. Consequently, 8 was elucidated as 5α-hydroxy-14β-methoxy-pimarane-8(9), 15-dien-19-oic acid and designated as calcarisporic acid H.

The molecular formula of compound 9 was deduced to be C₂₀H₃₀O₄ by negative HR-ESI-MS at m/z 333.2077 [M–H]⁻ (Calcd. for C₂₀H₂₉O₄, 333.2071). The ¹H and ¹³C NMR spectra (Table 1, Table 4) of 9 showed close resemblance to those of 5, with the exception of the signals assigned to C-5, where the oxygenated quaternary carbon (δ_C 80.5) in 5 was replayed by a methine [δ_H 1.79 (d, J = 12.2 Hz), and δ_C 46.5] in 9. This suggested that 9 was the 5-dehydroxy analog of 5, which was confirmed by HMBC correlations from H-5 (δ_H 1.79) to C-1, C-4, C-6, C-10, C-18, C-19, and C-20. NOESY correlations of H₃-18 with H-5 (δ_H 1.79) and H-3, of H-11β (δ_H 1.92) with H₃-20 and H-15, and of H-14 with H₃-17 and H-12α (δ_H 1.43) indicated that the relative configuration of 9 was in accordance with that of 5. Therefore, 9 was elucidated as 3α,14β-dihydroxy-pimarane-8(9), 15-dien-19-oic acid and designated as calcarisporic acid I.

Table 4.

¹H NMR spectroscopic data (δ) for compounds 9−12^a (δ in ppm, J in Hz).

No.	9	10	11	12
1	1.56, overlap	1.61, dd (13.6, 3.7)	1.54, overlap	1.76, overlap
	1.52, overlap	1.31, br d (13.6)		1.45, overlap
2	2.17, m	1.99, dd (13.5, 3.8)	2.19, m	1.98, overlap
	1.63, m	1.48, overlap	1.63, dd (14.4, 3.0)	1.45, overlap
3	4.00, t (2.50)	1.88, d (14.0)	4.01, br s	1.85, d (13.2)
		1.52, overlap		1.69, m
5	1.79, d (12.2)		1.79, dd (9.3, 3.9)
6	1.90, overlap	2.54, td (13.9, 6.0)	2.02, overlap	2.45, overlap
	1.84, td (12.1, 5.3)	1.92, dd (14.7, 5.2)	1.90, overlap	1.95, overlap
7	2.34, m	2.32, tdd (13.5, 5.8, 2.2)	2.47, br d (13.5)	2.43, overlap
	1.95, dd (18.0, 4.5)	2.22, dd (15.5, 4.9)	1.86, overlap	2.08, overlap
11	2.07, m	1.71, m	2.03, overlap	2.02, overlap
	1.92, overlap	1.50, overlap	1.91, overlap	1.93, overlap
12	1.57, overlap	1.59, overlap	1.73, m	1.74, m
	1.43, m	1.49, overlap	1.36, br d (12.3)	1.38, dd (12.9, 5.0)
14	3.63, br s	5.37, br s	3.22, br s	3.30, br s
15	5.86, dd (17.7, 10.9)	5.70, dd (17.4, 10.5)	6.00, dd (18.0, 10.5)	6.01, dd (17.4, 10.4)
16	4.98, dd (17.7, 1.1)	4.96, dd (10.5, 1.7)	5.00, dd (18.0, 1.0)	5.01, dd (17.4, 1.5)
	4.94, dd (10.9, 1.1)	4.91, dd (17.4, 1.7)	4.99, dd (10.5, 1.0)	5.00, dd (10.4, 1.5)
17	0.98, s	1.03, s	0.89, s	0.92, s
18	1.27, s	1.28, s	1.28, s	1.23, s
20	0.92, s	0.95, s	0.96, s	1.06, s

^aNMR data (δ) were measured at 500 MHz in CD₃OD for 9−12.

Compound 10 was assigned the molecular formula C₂₀H₃₀O₄ by negative HR-ESI-MS at m/z 333.2069 [M–H]⁻ (Calcd. for C₂₀H₂₉O₄, 333.2071). The ¹H and ¹³C NMR data (Table 1, Table 4) showed the presence of three tertiary methyls, seven methylenes, five sp³ quaternary carbons with two oxygenated, four olefinic carbons accounting for a trisubstituted olefin and a vinyl group, and a carboxylic carbon (δ_C 180.2). Comparison of the NMR spectroscopic data of 10 with those of 7 suggested that 10 possessed the same A ring unit as 7. Analysis of the ¹H—¹H COSY spectrum (Fig. 2) of 10 revealed the C-6–C-7 moiety, the C-11–C-12 unit, and the C-16–C-17 vinyl group. HMBC correlations (Fig. 2) from H₃-20 to C-1, C-5, C-10, and an oxygenated quaternary carbon at C-9 (δ_C 78.5) indicated that C-9 was connected to C-10. HMBC cross peaks from H₂-6 to C-4, C-5, and C-10, and from H₂-7 to C-8 (δ_C 137.5), C-14 (δ_C 132.8), and C-9 suggested that C-6 was attached to C-5, and the olefinic C-8 was connected to both C-7 and C-9, establishing a cyclohexane of B ring moiety with an exocyclic olefin at C-8/C-14 fused to the A ring unit at C-5/C10. Further HMBC correlations from H₂-11 to C-8, C-9, and C-10 indicated that C-11 was connected to C-9. HMBC correlations from H₃-17 to C-12, C-13, C-14, and C-15 (δ_C 146.6) led to the connections of C-12, C-14, C-15, and C-17 to C-13, establishing the C-8/C-9 fused cyclohexene of C ring unit with a methyl and a vinyl group both attached to C-13. The relative configuration of 10 was similar as that of 7 according to NOESY correlations of H-11β (δ_H 1.71) with H₃-20 and H-15 and of H₃-18 with H-3α (δ_H 1.88), H-3β (δ_H 1.52), and H-6α (δ_H 1.92). Thus, 10 was established as 5α, 9α-dihydroxy-pimarane-8(14), 15-dien-19-oic acid and named calcarisporic acid J.

Compound 11 possessed the same molecular formula as 9, C₂₀H₃₀O₄, deduced from negative HR-ESI-MS at m/z 333.2077 [M–H]⁻ (Calcd. for C₂₀H₂₉O₄, 333.2071). The NMR spectroscopic data (Table 1, Table 4) of 11 closely resembled those of 9 except for minor differences in signals due to C-12, C-14, and C-17, suggesting that 11 was the C-13 and C-14 diastereomer of 9. This speculation was supported by NOESY experiment. NOESY cross peaks (Fig. 3) of H-11β (δ_H 2.03) with H₃-20 and H₃-17 implied that the methyl at C-13 had a β-configuration. Correlations of H-14 (δ_H 3.22) with H₃-17 indicated that H-14 was in the β-equatorial configuration. Therefore, 11 was established as 3α,14α-dihydroxy-isopimarane-8(9), 15-dien-19-oic acid and designated as calcarisporic acid K.

Compound 12 possessed the same molecular formula of C₂₀H₃₀O₄ as 7, which were determined by negative HR-ESI-MS at m/z 333.2084 [M–H]⁻ (Calcd. for C₂₀H₂₉O₄, 333.2071). A detailed comparison of the NMR spectroscopic data (Table 1, Table 4) of 12 with those of 7 revealed that they possessed the identical planar structure, while weak differences were attributed to C-12, C-14, C-15, and C-17, suggesting that the two compounds were diastereomers. This was further verified by NOESY correlations of H-11β (δ_H 2.02) with H₃-20 and H₃-17, and of H-14 (δ_H 3.30) with H₃-17. Thus, 12 was elucidated as 5α,14α-dihydroxy-isopimarane-8(9), 15-dien-19-oic acid and designated as calcarisporic acid L.

The known compounds were identified as hawaiinolide G¹⁴ (13) and 14-epi-zythiostromic acid B¹⁶ (14), respectively, by comparing their NMR spectroscopic data with those reported in literatures.

It was worth mentioning that the pimarane and isopimarane type diterpenes (5−12) were isolated from the genus Calcarisporium for the first time. This discovery further supported the proposed pathway for the biosynthesis of these diterpenes (Scheme 1), where they could be synthesized from the same diterpene biosynthetic gene cluster, and the cassane and cleistanthane type diterpenoids both derived from the (iso)pimaranes, in which migration of either the C-13 methyl or vinyl group to C-14 led to the cassane or cleistanthane type diterpenoids5, 24, 25. Subsequently, three types of diterpenoids skeleton were heavily oxidized to form the characteristic diterpenoids above, encouraging us to search more diverse structures in this mutant strain.

Scheme 1.

Proposed biosynthetic pathways for compounds 1−14.

Cytotoxic activities of compounds 1—14 were evaluated against two human cancer cell lines (MCF-7 and HepG-2) in the MTT assay, with taxol as a positive control (IC₅₀ 5.0±0.6, 3.5±0.4 nmol/L, respectively). None of them exhibited significant activities in the concentration range of 10^—5–10^—7 mol/L.

The matrix metalloproteinases (MMPs) family can degrade various protein components in the extracellular matrix and plays a key role in tumor invasion and metastasis26, 27. Therefore, we tested the inhibitory effects of the isolated compounds on the expression of MMP1 and MMP2 in human breast cancer (MCF-7) cells by Western blot assay. Due to the sample limitation, compounds 2, 4, 6, and 7 were evaluated for this inhibitory activity firstly. Experimental results (Fig. 5) indicated that the four tested compounds had no significant inhibitory effects on the viability of MCF-7 cells, but the expression level of MMP1 and MMP2 could be affected obviously. Western blot showed that both 2 and 4 could reduce MMP2 expression significantly at 200 μmol/L for 24 h with significant difference from that in DMSO control group. Meanwhile, 4 could also reduce MMP1 expression significantly. This indicated that the activities of 2 and 4 may be associated with expression inhibition of MMP proteinases family, and although 2 and 4 exhibited no cytotoxicities against tested tumor cell lines, they could function in suppressing tumor invasion and metastasis and possesses potential antitumor value. Comparing the structure of 4 with that of 2, the major difference was that a vinyl group at C-14 in 4 and a methyl group at C-14 in 2 were observed. Both 4 and 2 possessed an unsaturated unit connected to C-13. These suggested that the vinyl group attached to C-14 contributed as a functional group and the substituted group at C-14 may play an important role in the inhibitory effect on the expression level of MMP1 and MMP2.

Figure 5.

Effects of compounds 2, 4, 6, and 7 on the expressions of MMP1 and MMP2 in MCF-7 cells. (A) Cells were treated with different test compounds at 200 μmol/L or DMSO as control for 24 h. The expressions of MMP1 and MMP2 were determined by Western blot. Representative immunoblots are shown and GAPDH is the loading control. (B) Relative expression level of MMP1 is calibrated by GAPDH. (C) Relative expression level of MMP2 is calibrated by GAPDH. Each value represents the mean±SD of three independent experiments. *P<0.05, **P<0.01 versus DMSO control.

In addition, compounds 1—14 were assessed for their anti-inflammatory inhibitory activities against the lipopolysaccharide (LPS)-induced NO production in murine microglial BV2 cells as well as their antioxidant activities against rat liver microsomal lipid peroxidation induced by Fe²⁺-cystine in vitro. All compounds were inactive for anti-inflammatory and antioxidant activities at the concentrations of 10^—5 and 10^—4 mol/L, respectively.

3. Conclusions

Twelve new diterpenoids, calcarisporic acids A--L, including three cassanes (1−3), one cleistanthane (4), six pimaranes (5--10), and two isopimaranes (11 and 12) along with two know cleistanthanes analogues (13 and 14), were isolated from the mutant strain ΔhdaA of Calcarisporium arbuscula, in which an encoding histone deacetylase gene (and hdaA) was deleted. Compounds 5--12 are the first examples of pimarane and isopimarane type diterpenes isolated from the genus Calcarisporium. In addition, the inhibitory effects of four compounds (2, 4, 6 and 7) on the expression of MMP1 and MMP2 in MCF-7 cells were evaluated. Compounds 2 and 4 significantly inhibited the expression level of MMP1 and MMP2, and are promising as a potential anti-metastatic agent for the treatment of human breast cancer. Our results further present the potential of genome mining-guided discovery of bioactive natural products in fungi.

4. Experimental

4.1. General experimental procedures

Optical rotations were measured on an Autopol IV automatic polarimeter (Rudolph Research Co.). UV spectra were measured on a JASCO V650 spectrophotometer. CD spectra were measured on a JASCO J-815 spectrometer. IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer using a FT-IR microscope transmission method. NMR spectra were recorded at 600 or 500 MHz for ¹H NMR and 150 or 125 MHz for ¹³C NMR, respectively, on a Bruker AVIIIHD 600 (Bruker Corp., Karlsruhe, Germany) or a VNS-600 or an Inova 500 instrument (Varian Associates Inc., Palo Alto, CA, USA) in CD₃OD or DMSO-d₆ with solvent peaks used as references at 25 °C. ESI-MS and analytical HPLC were performed on a Waters ACQUITY H-Class UPLC—MS with QDA mass detector (ACQUITY UPLC® BEH, 1.7 μm, 50 mm × 2.1 mm, C18 column) using positive and negative mode electrospray ionization. HR-ESI-MS data were taken on an Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer (Agilent Technologies, Ltd., Santa Clara, CA, USA). Semi-preparative HPLC was performed on a LabAlliance Series III pump equipped with a LabAlliance model 201 UV detector, using YMC-Pack ODS-A column (250 mm × 10 mm, 5μm). Column chromatography (CC) was carried out on silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), or reversed phase C18 silica gel (50 µm, YMC, Kyoto, Japan). TLC was carried out on glass precoated silica gel GF₂₅₄ plates (Qingdao Marine Chemical Co., Ltd., China). Spots were visualized under UV light or by spraying with 10% H₂SO₄ in 95% EtOH followed by heating.

4.2. Fungal material

Fungus Calcarisporium arbuscula NRRL 3705 (ATCC^® 46034^TM) is the wild type strain. The ΔhdaA mutants were constructed by replacement of hdaA by hph (Hygromycin resistance gene)⁵. The mutant strains were grown on 20 L of solid PDA media (BD) divided into 200 large 150 × 15 mm² Petri dishes at room temperature for 40 days.

4.3. Extraction and isolation

The fermented material was extracted repeatedly with EtOAc (3 × 5.0 L), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (14.2 g), which was partitioned between MeOH and hexane. The MeOH fraction (13.5 g) was evaporated to dryness under vacuum, and separated by reversed phase C18 silica gel vacuum liquid chromatography with a gradient of MeOH (20%–100%) in H₂O to give eleven fractions (F1−F11).

Fractions F3−F5 were combined (4.1 g) and subjected to reversed phase C18 silica gel vacuum liquid chromatography with a gradient of MeOH (5–80%) in H₂O to afford twenty fractions (F3-1−F3-20). Fraction F3-15 (307 mg) was separated by reverse-phase HPLC (YMC-ODS-A, 5 µm, 250 mm × 20 mm), eluted by 42% MeCN−H₂O (v/v, 0.02% formic acid) at a flow rate of 5.0 mL/min detected at 210 nm, to give six fractions (F3-15-1−F3-15-6). Fraction F3-15-2 (30.2 mg) was further purified by reverse-phase HPLC, eluted by 72% MeOH−H₂O (v/v, 0.01% trifluoroacetic acid) at a flow rate of 2.0 mL/min detected at 210 nm, to yield 5 (16.1 mg, t_R = 22 min). Fraction F3-15-3 (18.2 mg) was further purified by reverse-phase HPLC, eluted by 75% MeOH−H₂O (v/v, 0.01% trifluoroacetic acid) at a flow rate of 2.0 mL/min detected at 210 nm, to yield 11 (1.9 mg, t_R = 14 min) and 9 (8.9 mg, t_R = 19 min). Fraction F3-18 (472 mg) was subjected to CC over Sephadex LH-20 eluted by MeOH to afford nine fractions (F3-18-1−F3-18-9). Fraction F3-18-5 (90 mg) was purified by reverse-phase HPLC, eluted by 63% MeCN−H₂O (v/v, 0.02% formic acid) at a flow rate of 2.0 mL/min detected at 210 nm, to yield 7 (21.8 mg, t_R = 17 min), 6 (12.6 mg, t_R = 19 min), 8 (8.3 mg, t_R = 34 min), and 13 (2.3 mg, t_R = 42 min). Fraction F3-18-7 (65 mg) was purified by reverse-phase HPLC, eluted by 57% MeCN−H₂O (v/v, 0.02% formic acid) at a flow rate of 2.0 mL/min detected at 210 nm, to yield 1 (1.5 mg, t_R = 36 min).

Fraction F6 (1.6 g) was subjected to CC over Sephadex LH-20 eluted by MeOH to afford seven fractions (F6-1−F6-7). Fraction F6-6 (1.1 g) was recrystallized with MeOH to give a white solid, which was further purified by reverse-phase HPLC, eluted by 50% MeCN−H₂O (v/v, 0.01% trifluoroacetic acid) at a flow rate of 2.0 mL/min detected at 210 nm, to give 4 (49.8 mg, t_R = 34 min). Fraction F6-7 (88 mg) was purified by reverse-phase HPLC, eluted by 50% MeCN−H₂O (v/v, 0.01% trifluoroacetic acid) at a flow rate of 2.0 mL/min detected at 210 nm, to give 2 (13.7 mg, t_R = 32 min) and subfraction F6-73 (13.4 mg), which was further purified by reverse-phase HPLC, eluted by 80% MeOH−H₂O (v/v, 0.01% trifluoroacetic acid) at a flow rate of 2.0 mL/min detected at 210 nm, to give 3 (3.8 mg, t_R = 29 min).

Fraction F8 (1.1 g) was further purified by reverse-phase HPLC (YMC-ODS-A, 5 µm, 250 × 20 mm), eluted by 47% MeCN−H₂O (v/v, 0.02% formic acid) at a flow rate of 5.0 mL/min detected at 210 nm, to give six fractions (F8-1−F8-6). Fraction F8-4 (7.0 mg) was purified by reverse-phase HPLC, eluted by 47% MeCN−H₂O (v/v, 0.02% formic acid) at a flow rate of 2.0 mL/min detected at 210 nm, to give 10 (3.6 mg, t_R = 53 min). Fraction F8-5 (270 mg) was further purified by Sephadex LH-20 eluted by MeOH to give 14 (8.0 mg). Fraction F8-6 (89 mg) was further purified by reverse-phase HPLC, eluted by 50% MeCN−H₂O (v/v, 0.02% formic acid) at a flow rate of 2.0 mL/min detected at 210 nm, to give 12 (5.2 mg, t_R = 24 min).

4.3.1. Calcarisporic acid A (1)

Colorless amorphous solid; [α]²⁰_D +30.0 (c 0.10, MeOH); UV (MeOH) λ_max (logε) 229 (4.68) nm; CD (c 1.4 × 10^—3 mol/L, MeOH) λ_max (Δε) 240 (−0.29), 288 (+0.13), 320 (−0.12) nm; IR ν_max 3490, 3086, 2930, 1809, 1724, 1649, 1603, 1457, 1391, 1223, 1099, 994, 897, 852, 760 cm^—1; ¹H NMR (CD₃OD, 500 MHz) data see Table 2, ¹³C NMR (CD₃OD, 125 MHz) data see Table 1; ESI-MS m/z 347 [M–H]⁻; HR-ESI-MS: m/z 347.1864 [M–H]⁻ (Calcd. for C₂₀H₂₇O₅, 347.1864).

4.3.2. Calcarisporic acid B (2)

Colorless granules (MeOH); [α]²⁰_D +51.7 (c 0.19, MeOH); UV (MeOH) λ_max (logε) 231 (4.96) nm; IR ν_max 3307, 3198, 2938, 2612, 1694, 1652, 1602, 1516, 1468, 1436, 1276, 1081, 1004, 955, 893, 769, 732, 592 cm^—1; ¹H NMR (DMSO-d₆, 600 MHz) data see Table 2, ¹³C NMR (DMSO-d₆, 150 MHz) data see Table 1; ESI-MS m/z 349 [M–H]⁻; HR-ESI-MS: m/z 349.2030 [M–H]⁻ (Calcd. for C₂₀H₂₉O₅, 349.2020).

4.3.3. Calcarisporic acid C (3)

Colorless amorphous solid; [α]²⁰_D +83.3 (c 0.19, MeOH); UV (MeOH) λ_max (logε) 231 (4.81) nm; IR ν_max 3309, 3183, 2974, 2930, 1688, 1652, 1606, 1462, 1271, 1117, 962, 949, 929, 893, 798, 711, 647, 535 cm^—1; ¹H NMR (DMSO-d₆, 600 MHz) data see Table 2, ¹³C NMR (DMSO-d₆, 150 MHz) data see Table 1; ESI-MS m/z 333 [M–H]⁻; HR-ESI-MS: m/z 333.2083 [M–H]⁻ (Calcd. for C₂₀H₂₉O₄, 333.2071).

4.3.4. Calcarisporic acid D (4)

Colorless needles (MeOH); [α]²⁰_D +10.2 (c 0.17, MeOH); UV (MeOH) λ_max (logε) 204 (4.50) nm; IR ν_max 3456, 3367, 3073, 2965, 2320, 1694, 1645, 1509, 1465, 1421, 1297, 1166, 1003, 965, 918, 771, 725, 608, 568 cm^—1; ¹H NMR (DMSO-d₆, 600 MHz) data see Table 2, ¹³C NMR (DMSO-d₆, 150 MHz) data see Table 1; ESI-MS m/z 349 [M–H]⁻; HR-ESI-MS: m/z 349.2031 [M–H]⁻ (Calcd. for C₂₀H₂₉O₅, 349.2020).

4.3.5. Calcarisporic acid E (5)

Colorless amorphous solid; [α]²⁰_D −12.0 (c 0.20, MeOH); UV (MeOH) λ_max (logε) 206 (4.37) nm; IR ν_max 3339, 3080, 2935, 1701, 1638, 1460, 1380, 1267, 1009, 948, 912, 855, 706, 672 cm^—1; ¹H NMR (CD₃OD, 500 MHz) data see Table 3, ¹³C NMR (CD₃OD, 150 MHz) data see Table 1; ESI-MS m/z 349 [M–H]⁻; HR-ESI-MS: m/z 349.2036 [M–H]⁻ (Calcd. for C₂₀H₂₉O₅, 349.2020).

4.3.6. Calcarisporic acid F (6)

Colorless amorphous solid; [α]²⁰_D −21.0 (c 0.13, MeOH); UV (MeOH) λ_max (logε) 204 (4.58) nm; IR ν_max 3495, 3274, 3079, 2934, 1701, 1635, 1461, 1383, 1238, 1089, 1005, 939, 907, 864, 690, 665 cm^—1; ¹H NMR (CD₃OD, 500 MHz) data see Table 3, ¹³C NMR (CD₃OD, 125 MHz) data see Table 1; ESI-MS m/z 363 [M–H]⁻; HR-ESI-MS: m/z 363.2182 [M–H]⁻ (Calcd. for C₂₁H₃₁O₅, 363.2177).

4.3.7. Calcarisporic acid G (7)

Colorless needles (MeOH); [α]²⁰_D +16.0 (c 0.20, MeOH); UV (MeOH) λ_max (logε) 204 (4.47) nm; IR ν_max 3544, 3353, 2934, 1686, 1621, 1467, 1433, 1377, 1280, 1255, 1065, 941, 914, 800, 675 cm^—1; ¹H NMR (CD₃OD, 600 MHz) data see Table 3, ¹³C NMR (CD₃OD, 150 MHz) data see Table 1; ESI-MS m/z 333 [M–H]⁻; HR-ESI-MS: m/z 333.2077 [M–H]⁻ (Calcd. for C₂₀H₂₉O₄, 333.2071).

4.3.8. Calcarisporic acid H (8)

Colorless amorphous solid; [α]²⁰_D +12.0 (c 0.15, MeOH); UV (MeOH) λ_max (logε) 205 (4.51) nm; IR ν_max 3570, 3147, 2937, 1724, 1638, 1606, 1458, 1383, 1274, 1200, 1073, 973, 937, 854, 796, 675 cm^—1; ¹H NMR (CD₃OD, 500 MHz) data see Table 3, ¹³C NMR (CD₃OD, 125 MHz) data see Table 1; ESI-MS m/z 347 [M–H]⁻; HR-ESI-MS: m/z 347.2239 [M–H]⁻ (Calcd. for C₂₁H₃₁O₄, 347.2228).

4.3.9. Calcarisporic acid I (9)

Colorless amorphous solid; [α]²⁰_D +56.9 (c 0.19, MeOH); UV (MeOH) λ_max (logε) 206 (4.50) nm; IR ν_max 3427, 3082, 2933, 1694, 1456, 1379, 1250, 1197, 1046, 966, 915, 858, 698 cm^—1; ¹H NMR (CD₃OD, 500 MHz) data see Table 4, ¹³C NMR (CD₃OD, 125 MHz) data see Table 1; ESI-MS m/z 333 [M–H]⁻; HR-ESI-MS: m/z 333.2077 [M–H]⁻ (Calcd. for C₂₀H₂₉O₄, 333.2071).

4.3.10. Calcarisporic acid J (10)

Colorless amorphous solid; [α]²⁰_D +103.9 (c 0.22, MeOH); UV (MeOH) λ_max (logε) 205 (4.41) nm; IR ν_max 3382, 2950, 2875, 1700, 1637, 1553, 1453, 1377, 1254, 1143, 1090, 999, 917, 882, 802, 656 cm^—1; ¹H NMR (CD₃OD, 500 MHz) data see Table 4, ¹³C NMR (CD₃OD, 125 MHz) data see Table 1; ESI-MS m/z 333 [M–H]⁻; HR-ESI-MS: m/z 333.2069 [M–H]⁻ (Calcd. for C₂₀H₂₉O₄, 333.2071).

4.3.11. Calcarisporic acid K (11)

Colorless amorphous solid; [α]²⁰_D +85.9 (c 0.16, MeOH); UV (MeOH) λ_max (logε) 204 (4.52) nm; IR ν_max 3430, 3082, 2928, 2871, 1693, 1455, 1413, 1378, 1357, 1199, 1146, 1005, 980, 913, 802, 698, 621 cm^—1; ¹H NMR (CD₃OD, 500 MHz) data see Table 4, ¹³C NMR (CD₃OD, 125 MHz) data see Table 1; ESI-MS m/z 333 [M–H]⁻; HR-ESI-MS: m/z 333.2077 [M–H]⁻ (Calcd. for C₂₀H₂₉O₄, 333.2071).

4.3.12. Calcarisporic acid L (12)

Colorless amorphous solid; [α]²⁰_D +126.9 (c 0.12, MeOH); UV (MeOH) λ_max (logε) 204 (4.59) nm; IR ν_max 3498, 2930, 2865, 1707, 1639, 1458, 1391, 1308, 1278, 1002, 981, 917, 798, 685 cm^—1; ¹H NMR (CD₃OD, 500 MHz) data see Table 4, ¹³C NMR (CD₃OD, 125 MHz) data see Table 1; ESI-MS m/z 333 [M–H]⁻; HR-ESI-MS: m/z 333.2084 [M–H]⁻ (Calcd. for C₂₀H₂₉O₄, 333.2071).

4.4. Cytotoxicity assay

Compounds 1–14 were tested for cytotoxic activities against MCF-7 (human breast cancer) and HepG-2 (human liver cancer) cell lines by the MTT method, as described in the literature²⁸.

4.5. Inhibitory effects on the expression of MMP1 and MMP2 in MCF-7 cells

Compounds 2, 4, 6, and 7 were tested for the inhibitory effects on the expression of MMP1 and MMP2 in human breast carcinoma cell line MCF-7 by Western blot. MCF-7 cells was purchased from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science, and was cultured in DMEM medium (Gibco, Invitrogen Corporation) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL benzyl penicillin G, and 100 mg/L streptomycin under a humidified atmosphere of 5% CO₂ at 37 °C for 24 h. MCF-7 cells were treated with different test compounds at 200 μmol/L or DMSO as control for 24 h. Then, cells were collected and lysed. The lysates were clarified by 12,000 × g centrifugation at 4 °C for 30 min. Total proteins were quantified with a microplate spectrophotometer using the bicinchoninic acid assay (BCA assay). Then equal amount of proteins were separated by SDS-polyacrylamide gel (SDS-PAGE) and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The blots were incubated with primary antibodies overnight at 4 °C, followed by second antibodies at the room temperature for 0.5 h. Immunoreactive protein bands were detected by the ECL detection system (Amersham Bioscience, Piscataway, NJ, USA).

4.6. Anti-inflammatory activity assay

Compounds 1–14 were assessed for their anti-inflammatory inhibitory activities against the lipopolysaccharide (LPS) induced NO production in murine microglial BV2 cell line using the Griess method, as described in the literature²⁹. Curcumin was used as the positive control (inhibitory rate 66.0±2.5% at 10^—5 mol/L).

4.7. Antioxidant assay

Compounds 1–14 were assessed for their antioxidant activities against rat liver microsomal lipid peroxidation induced by Fe²⁺--cystine in vitro, as described in the literature³⁰. Curcumin was used as the positive control (inhibitory rate 95.0±3.5% at 10^—4 mol/L).

Appendix A. Supplementary material

Supplementary material

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