Characterization of Cytochalasins and Steroids From the Ascomycete Daldinia concentrica and Their Cytotoxicity

Abstract

One new cytochalasin daldinin (1), 2 known cytochalasins 2 and 3, along with 2 steroids 4 and 5, were characterized from the methanol extracts of the fruiting bodies of Daldinia concentrica. The structure of new compound 1 was elucidated using a combination of 1- and 2-dimensional nuclear magnetic resonance spectroscopic and high-resolution electrospray ionization mass spectrometric analyses. In addition, the isolated compounds were examined for their cytotoxicity against several tumor cell lines and the tested compounds demonstrated moderate-to-weak cytotoxicity.

The Xylariaceae is a large family and currently comprises more than 36 genera, of which Daldinia genus comprises about 20 species. ¹ Among these, the ascomycetes Daldinia (Xylariaceae) have been evidenced to be potential medicinal fungi sources, in which unique bioactive secondary metabolites were reported. ² In the recent studies on the chemical constituents of genus Daldinia various new compounds have been discovered, including cytochalasins, ^3-5 aromatic steroids, ⁶ terpenoids, ^7,8 polyketides, lactones, azaphilone derivatives, ⁹ benzophenones, ¹⁰ benzoquinones, ¹¹ binaphthyls. ¹⁰ These compounds exhibited significant cytotoxicity against various cancer cells, such as KB (human epidermal carcinoma), SK-LU-1 (human lung carcinoma), MCF7 (human breast carcinoma), and HepG2 (human hepatocellular carcinoma). ^12,13 In our continuing program aiming to explore bioactive substances from natural fungal materials in Vietnam, the fruiting bodies of Daldinia concentrica collected in Nghe An were extracted and isolated. Totally 5 compounds were characterized, including a new cytochalasin daldinin (1) (Figure 1). The structures of these isolated compounds were elucidated using a combination of UV, infrared, 1- and 2-dimensional nuclear magnetic resonance (NMR) techniques (¹H-, ¹³C-NMR, correlation spectroscopy [COSY], nuclear Overhauser effect spectroscopy [NOESY], heteronuclear single quantum coherence [HSQC], and heteronuclear multiple bond correlation [HMBC]) and MS analyses.

Figure 1

Significant COSY and HMBC correlations of 1.

Compounds 1-5 (Figure 2) were purified using the combination of conventional chromatographic techniques, and the structure of new compound 1 was elucidated as below. Compound 1 was isolated as optically active colorless needles, melting point (MP) 216°C to 217°C. The high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analytical data of 1 revealed the pseudomolecular formula as C₃₁H₄₁O₆NNa (m/z 546.2834 [M+Na]⁺). The ¹H-NMR spectrum of 1 displayed the characteristic resonances for amide NH (H-2 at δ_H 4.89), benzyl group (H-2′, -6′ at δ_H 7.19; H-3′, -5 at δ_H 7.28; H-4′ at δ_H 7.25), and a secondary alcoholic proton doublet (H-7 at δ_H 4.74). Moreover, the presence of 2 exomethylene olefinic protons (δ_H 5.12 and 5.45), 2 olefinic proton signals belonging to a macro ring system (δ_H 5.12 and 6.57), and 3 methyl groups (δ_H 0.89, 0.94, 1.51) were also supported by the ¹H-NMR spectral data (Table S1). Its ¹³C-NMR displayed the signals of 31 carbons, including 3 carbonyl carbons (C-21 at δ_C 213.2, C-1 at δ_C 175.2, OCO at δ_C 177.4), 6 aromatic carbons (δ_C 137.6, 130.1, 129.0, 127.1, 129.0, 130.1), 4 olefinic carbons (C-13 at δ_C 131.7, C-14 at δ_C 133.8, C-12 at δ_C 113.0, C-6 at δ_C 151.8), 3 oxygenated carbons (C-7 at δ_C 72.1, C-19 at δ_C 80.9, C-18 at δ_C 76.0), 2 quaternary carbons (C-9 at δ_C 64.8, C-18 at δ_C 76.0), 5 methine carbons (C-3 at δ_C 53.3, C-4 at δ_C 47.6, C-5 at δ_C 32.8, C-8 at δ_C 52.8, C-16 at δ_C 30.0), 4 methylene carbons (C-10 at δ_C 43.8, C-15 at δ_C 43.1, C-17 at δ_C 45.6, C-20 at δ_C 29.7), and 5 methyl carbons (C-22 at δ_C 25.1, C-23 at δ_C 24.3, C-11 at δ_C 13.1, CH₃COO at δ_C 24.0, and OCH₃ at δ_C 58.8), respectively. Comparison of the above spectral data with those of [11]-cytochalasa-6(12),13-diene-1,21-dione-7,18-dihydroxy-16,18-dimethyl-19-methoxy-10-phenyl-(7S*,13E,16S*,18S*,19R*), ³ the only difference observed was that one more acetoxy group in 1. The HMBC crosspeaks from H-10 to C-1′; from H-3′ to C-1′; from H-23 to C-18, C-19 and C-17; from OCH₃ to C-19; and from H-22 to C-15, C16 confirmed the basic skeleton and substitutions similar to those reported. The location of the acetoxy group was determined to be at C-18 rather than C-7 as compared to the chemical shifts of C-7 (δ_C 72.1) and C-18 (δ_C 76.0) with those of cytochalasin derivatives. ³ The successive 2-dimensional spectral experiments including COSY, NOESY, HSQC, and HMBC (Figure 1) accomplished the assignments of all the proton and carbon signals of 1, and therefore its chemical structure was established as [11]-cytochalasa-18-acetoxy-6(12),13-diene-1,21-dione-7,18-dihydroxy-16,18-dimethyl-9-methoxy-10-phenyl-(7S*,13E,16S*,18S*,19R*) (Figure 1) and named trivially as daldinin.

Figure 2

Structure of compounds 1-5.

In addition, 4 known compounds were identified as [11]-cytochalasa-6(12),13-diene-1,21-dione-7,18,19-trihydroxy-16,18- dimethyl-10-phenyl-(7S*,13E,16S*,18S*,19R*) (2), ³ [11]-cytochalasa-6(12),13-diene-1,21-dione-7,18-dihydroxy-16,18- dimethyl-10-phenyl-(7S*,13E,16S*,18R*) (3), ³ ergosterol (4), ¹⁴ and ergosterol peroxide (5) ¹⁵ (Figure 2), respectively, based on the comparison of their spectroscopic and spectrometric profiles (NMR, UV, IR, and MS) with the reported values in the cited literature.

These isolates were screened for in vitro cytotoxicity against SK-LU-1 (human lung carcinoma), HepG2 (human hepatocellular carcinoma), Hep3B (human hepatocellular carcinoma), SW480 (human colon adenocarcinoma), and MCF-7 (human breast carcinoma) tumor cell lines as described previously, ¹⁶ and the data are given in Table 1. Compound 1 exhibited moderate cytotoxicity against 5 cell lines with IC₅₀ values of 11.4 ± 0.5, 13.5 ± 1.3, 13.3 ± 1.4, 13.1 ± 0.9, and 13.5 ± 1.2 µM, respectively, compared with the positive control ellipticine (IC₅₀ values ranged between 0.4 ± 0.1 and 0.5 ± 0.1 µM). Compounds 2 and 3 showed weak cytotoxicity against all the tested tumor cell lines with the IC₅₀ values ranging between 23.0 ± 1.1 and 58.2 ± 2.3 µM. In contrast, compounds 4 and 5 showed only weak cytotoxicity against HepG2 and Hep3B cells with IC₅₀ values ranging between 21.5 ± 5.1 and 46.9 ± 3.7 µM, and they did not show any significant effect at the highest tested concentrations toward SK-LU-1 and SW480 cell lines.

Table 1

Cytotoxicity of Isolated Compounds 1-5.

Compound	SK-LU-1	HepG2	Hep3B	SW480	MCF7
	IC50 (μM) a	IC50 (μM) a	IC50 (μM) a	IC50 (μM) a	IC50 (μM) a
1	11.4 ± 0.5	13.5 ± 1.3	13.3 ± 1.4	13.1 ± 0.9	13.5 ± 1.2
2	23.4 ± 2.1	32.8 ± 1.4	41.5 ± 2.3	23.0 ± 1.1	31.7 ± 3.6
3	40.2 ± 2.6	55.7 ± 4.0	53.1 ± 5.1	58.2 ± 2.3	35.2 ± 1.5
4	>50	21.5 ± 5.1	21.7 ± 2.8	>100	43.6 ± 5.1
5	>50	46.9 ± 3.7	35.2 ± 3.4	>100	>100
Ellipticine b	0.4 ± 0.1	0.4 ± 0.1	0.4 ± 0.1	0.5 ± 0.1	0.4 ± 0.1

^aConcentration necessary for 50% inhibition (IC₅₀). Results are presented as means±SD (n = 3).

^bEllipticine was used as a positive control.

Experimental

General

Melting points were determined using Yanagimoto MP-S3 apparatus without corrections. Optical rotations were measured using a Jasco DIP-370 polarimeter. The UV spectra were obtained on a Agilent UV-Vis spectrophotometer, and IR spectra were recorded on a Bruker Optics Vertex 80 spectrophotometer. ¹H- and ¹³C-NMR, COSY, NOESY, HMQC, and HMBC spectra were obtained on the Bruker AV-III 700 NMR and 500 NMR spectrometers, with tetramethylsilane as the internal standard, and chemical shifts were reported in δ values (ppm). The HR-ESI mass spectra were determined using an Agilent 1200 LC-MSD Trap spectrometer. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, E. Merck). The preparative and semipreparative highperformance liquid chromatography (HPLC) was conducted on the Agilent 218 purification system using a Shim-pack XR-ODS II column (2.0 × 100 mm, inner diameter 2.2 µm, Shimadzu Co., Kyoto, Japan). Thin layer chromatography (TLC) was conducted on precoated Kieselgel 60 F 254 plates (Merck) and the compounds were visualized by spraying with 10% (v/v) H₂SO₄ followed by heating at 110°C for 10 minutes.

Fungus Material

The fruiting bodies of D. concentrica (Bolton Fr. Ces. & De Not) were collected at the Pumat National Park of Nghe An Province, Vietnam, in August 2017 and identified by Prof Dr Ngo Anh, Department of Biology, Hue University. A voucher specimen (Vinh-TSWu 20170815) was deposited at the School of Chemistry, Biology and Environment, Vinh University, Vinh City, Vietnam.

Extraction and Isolation

The dried fruiting bodies of D. concentrica (2.0 kg) was extracted with 70% methanol at ambient temperature, and concentrated under reduced pressure to give a deep brown syrup (185 g). The crude extract was suspended in water and partitioned with ethyl acetate and butanol to afford ethyl acetate (95 g), butanol (47 g) and water-soluble (43 g) fractions, respectively. The ethyl acetate soluble extracts were applied to silica gel CC with a mixture of chloroform and methanol step gradient system (100:0, 40:1: 30:1; 20:1; 10:1: 4:1; 2:1) to afford minor fractions. These fractions were monitored by TLC to combine into 7 major fractions (Frs. DC1-DC7). Fraction DC1 (1.2 g) was separated by preparative HPLC (CH₃CN/H₂O, 60:40 to 90:10, 30 minutes, 18 mL/min), further purified by semipreparative HPLC (CH₃CN/H₂O, 60:40 to 90:10, 25 minutes, 7 mL/min) to afford compound 1 (23 mg). Fraction DC3 (2.6 g) was subjected to the silica gel CC (300 g, 60 × 3 cm) eluting with a hexane and ethyl acetate solvent mixture (15:1) to yield compound 4 (138 mg) and 5 (24 mg). Fraction DC4 (2.5 g) was subjected to the preparative (Prep) TLC (typical plate dimensions: 20 cm × 20 cm, 2.5 mm SiO₂ thickness) eluting with a mixture of chloroform and methanol (9:1) to produce compounds 2 (15 mg) and 3 (34 mg).

Daldinin (1)

Colorless needles (CHCl₃).

MP: 216°C to 217°C.

${\left[\alpha \right]}_{\mathrm{D}}^{25}$ : + 47 (c 0.05, MeOH).

IR (KBr) ν_max: 3386, 2950, 2847, 1723, 1664, 1450, 1037 cm⁻¹.

UV (MeOH) λ _max (log ε): 206 nm (2.90).

¹H NMR (700 MHz, Pyridine-d ₅): 7.28 (m, H-3′, 5′), 7.25 (m, H-4′), 7.19 (m, H-2′, 6′), 6.57 (m, H-13), 5.45 (m, H-12a), 5.12 (m, H-12b), 5.12 (m, H-14), 4.89 (d, 19.6, H-2), 4.75 (d, 9.8, H-7), 3.75 (s, OCH₃), 3.66 (d, 5.6, H-19), 3.63 (m, H-3), 3.21 (t, 9.8, H-8), 3.15 (t, 6.3, H-4), 3.11 (d, 7.0, H-5), 2.92 (m, H-17a), 2.69 (d, 6.3, H-10), 2.26 (s, COCH₃), 1.93 (m, H-15a), 1.67 (m, H-15b), 1.51 (s, H-23), 1.41 (m, H-17b), 1.20 (m, H-20), 1.17 (m, H-16), 0.94 (d, 7.0, H-22), 0.89 (d, 7.0, H-11).

¹³C NMR (175 MHz, Pyridine-d ₅): 213.2 (C-21), 177.4 (OCO), 175.5 (C-1), 151.8 (C-6), 137.6 (C-1′), 133.8 (C-14), 131.7 (C-13), 130.1 (C-2′, 6′), 129.0 (C-3′, 5′), 127.1 (C-4′), 113.0 (C-12), 80.9 (C-19), 76.0 (C-18), 72.1 (C-7), 64.8 (C-9), 58.8 (OCH₃), 53.3 (C-3), 52.8 (C-8), 47.6 (C-4), 45.6 (C-17), 43.8 (C-10), 43.1 (C-15), 32.8 (C-5), 30.0 (C-16), 29.7 (C-20), 25.1 (C-22), 24.3 (C-23), 24.0 (COCH₃), 13.1 (C-11).

HRESIMS m/z 546.2834 [M+Na]⁺ (cal. for C₃₁H₄₁O₆NNa, m/z 546.2832).

Cell Lines and Culture

MCF-7 (human breast carcinoma), SW480 (human colon adenocarcinoma), SK-LU-1 (human lung carcinoma), HepG2 (human hepatocellular carcinoma), and Hep3B (human hepatocellular carcinoma) cells were obtained from the American Type Culture Collection. The cells were maintained in RPMI or IMDM (GibcoBRL, NY, USA) with 10% fetal bovine serum (FBS) supplemented with 2% penicillin and 100 µg/mL of ellipticine at 37°C in a humidified atmosphere containing 5% CO₂.

Cytotoxic Activity

The cytotoxicity against 5 human tumor cell lines (SK-LU-1, HepG2, Hep3B, SW480, MCF7) were maintained in RPMI 1640 or IMDM that included l-glutamine (GIBCO) with 10% FBS (GIBCO) and 2% penicillin-streptomycin (GIBCO). Cells were cultured at 37°C in a 5% CO₂ incubator. Cytotoxic activity was measured using a modified MTT assay. ¹⁶ Viable cells were seeded in the growth medium into 96-well microtiter plates (1 × 10⁴ cells/well) and were incubated at 37°C in a 5% CO₂ incubator. A test sample was dissolved in dimethyl sulfoxide (DMSO) and was adjusted to final sample concentrations ranging from 5 to 100 µg/mL by diluting with the growth medium. Each sample was prepared in triplicate. The final DMSO concentration was adjusted to <0.1%. After standing for 24 hours, the test sample was added to each well. The same volume of medium with 0.1% DMSO was added to the control wells. Forty-eight hours after the addition of the test sample, MTT reagent was added to each well (final concentration: 5 µg/mL). After 4 hours, the plate was centrifuged for 5 minutes at 1500 rpm, the medium was removed, and the resulting formazan crystals were dissolved in DMSO. The optical density was measured at 570 nm using a Titertek microplate reader (Multiskan MCC/340, Flow). The cytotoxicity was expressed as IC₅₀ values (50% inhibitory concentration).