The ether-soluble fraction of the crude extract acquired for this study was successively subjected to column chromatography to yield benzocamphorin F (1). The structure of compound 1 was elucidated by the methods of UV, IR, HR-ESI/MS, ESI-MS/MS and NMR.

Benzocamphorin F (1) was isolated as colorless powder and showed a [M + Na]⁺ ion peak at m/z 255.0997 in its HRESIMS, corresponding to the molecular formula C₁₄H₁₆O₃Na. The UV spectrum of 1 displayed absorption maxima at 246, 258, 282 and 317 nm, and the IR spectrum exhibited strong absorption peaks for carbon-carbon triple bond (2183 cm⁻¹), and carbon-carbon double bond (1605 cm⁻¹), respectively. The ¹H NMR (CDCl₃) spectra of 1 showed signals assignable to a set of single aromatic protons at δ 6.91 (1H, s, H-3), 6.48 (1H, s, H-6), terminal methylene protons at δ 5.38 (1H, s, H-4′) and δ 5.26 (1H, s, H-4′), three methoxy singlets at δ 3.90 (3H, H-5), 3.88 (3H, H-1) and 3.84 (3H, H-2), and a methyl singlet at δ 2.02 (3H, s, H-3′), respectively. The ¹³C NMR and DEPT spectra combined with heteronuclear multiple-quantum correlation (HMQC) experiment indicated 14 signals including an olefinic carbon resonances at δ 121.2, three methoxy groups at δ 56.0, 56.4 and 56.9, two aromatic methines at δ 97.4 and 115.9, a methyl group at δ 23.6, seven quaternary carbons at δ 155.3, 150.3, 142.9, 127.1, 103.4, 93.5 and 84.7. The heteronuclear multiple-bond correlations (HMBC) (Figure 2) from OCH₃-7(δ 3.88) to C-1(δ 155.3), from OCH₃-8(δ 3.84) to C-2 (δ 142.9), from H-3(δ 6.91) to C-1(δ 155.3)/C-2(δ 142.9)/C-4(δ 103.4)/C-5(δ 150.3), from OCH₃-9(δ 3.90) to C-5 (δ 150.3), from H-6 (δ 6.48) to C-1 (δ 155.3)/C-2 (δ 142.9)/C-4 (δ 103.4)/C-5 (δ 150.3), from CH₃-5′ (δ 2.00) to C-2′ (δ 93.5)/C-3′ (δ 127.0)/C-4′ (δ 121.2), from H-4′ (δ 5.38, 5.26) to C-2′ (δ 93.5)/C-3′ (δ 127.0)/CH₃-3′ (δ 23.6) constructed the substituted pattern of this enynyl-benzenoid. On the basis of these spectral data (Table 1), the chemical structure of 1 was identified as shown in Figure 1. It is the first report of this compound from the natural sources and it was given the trivial name, benzocamphorin F, proposed following a previous convention [9].

In the previous literature, Wu et al. reported a total synthesis of antrocamphin A with six steps and an overall yield of 3.7% [10]. However, the low yield and high cost of the reagents for this method reduce the application efficiency. Herein we wish to explore a more efficient and economic method to prepare the analogs possessing the same skeleton as that of antrocamphin A. The retro-synthetic analysis of benzocamphorin F (1) was displayed in Figure 3 and thus we initiated the preparation of 1 from 1,2,4-trimethoxybenzene (3). The N-Bromosuccinimide (NBS)-assisted bromination of 3 resulted in the 1-bromo-2,4,5-trimethoxybenzene 2 as shown in Figure 4 [11]. Compound 2 was coupled with 2-methyl-3-butyn-2-ol by Sonogashira reaction [12], which successively led to compound 4. Finally the dehydration of compound 4 in toluene by methanesulfonyl chloride to produce benzocamphorin F (1) in good yield (92%) in Figure 4 [13]. The chemical structure of synthetic compound 1 was elucidated by 1D and 2D-NMR and mass spectrometry and compared with those of the purified natural compound. The present synthetic protocol of benzocamphorin F (1) provides a more efficient synthetic pathway with a satisfactory overall yield (three steps, 68.9%). In addition, the reagents used in this synthesis were of comparatively low cost, thus it would be more suitable for the large-scale production of benzocamphorin F and the analogs with similar skeleton.

The quantity of purified benzocamphorin F (1) from natural sources was too small to be examined for bioactivity. Thus only the inhibitory effect of synthetic benzocamphorin F (1) on LPS-induced NO production in murine microglial cells (BV2) was investigated. Nitrite accumulated in the culture medium was estimated by the Griess reaction as an index for NO release from the cells [12]. When BV2 were treated with different concentrations of 1 together with LPS (0.5 μg/mL) for 24 h, a significant concentration-dependent inhibition of nitrite production was detected. The IC₅₀ value for inhibition of nitrite production of 1 was 8.6 ± 2.7 μM. It was more potent than L-NAME (IC₅₀: 12.0 ± 0.6 μM), a non-specific NOS inhibitor. To further understand whether 1 also exerted anti-oxidative properties, its effect on NOX activity was examined. Our data suggest that compound 1 (IC₅₀: 74.4 ± 9.5 μM) was an ordinary inhibitor of NOX, as compared to the specific NOX inhibitor, DPI (IC₅₀: 0.96 ± 0.06 μM) (Table 2), indicating that NOX might not be the direct target. The free radical-scavenging capacity of compound 1 was also examined in a cell-free DPPH solution and it did not show considerable free radical-scavenging activity. Trolox, a vitamin E analogue included as a positive control, displayed a stronger free radical-scavenging effect than the examined compound (Table 2).

Melting points were determined using the Yanagimoto MP-S3 micro melting point apparatus without correction. Optical rotations were measured using a Jasco DIP-370 digital polarimeter. UV spectra were obtained on a Hitachi UV-3210 spectrophotometer, and IR spectra were recorded on a Shimadzu FT-IR DR-8011 spectrophotometer. ¹H NMR (400 MHz) and ¹³C NMR (75 MHz) spectra were recorded on Bruker AMX-400 spectrometers using CDCl₃ as the solvents. Chemical shifts are shown in δ values (ppm) with tetramethylsilane as an internal standard. ESI and HR-ESI mass spectra were measured on a Bruker APEX II mass spectrometer. Microwave irradiation was carried out using the CEM LabMate microwave apparatus. Reversed-phase column chromatography was accomplished with Diaion HP-20 and Sephadex LH-20 columns. Silica gel column chromatography was carried out using Kieselgel 60 (70–230 and 230–400 mesh, Merck). Thin-layer chromatography (TLC) was executed on pre-coated Kieselgel 60 F₂₅₄ plates (Merck), with compounds visualized by UV light or spraying with 10% (v/v) H₂SO₄ followed by charring at 110 °C for 10 min.

The fruiting body of T. camphoratus (1.0 kg) was extracted with Et₂O (4 × 10 L) for three days. The Et₂O extract was concentrated to afford a brown syrup (360 g) and then partitioned between H₂O and Et₂O. The ether layer was chromatographed on silica gel and eluted with MeOH in chloroform (0–100% of MeOH, gradient) to obtain eight fractions, (Fr. A–H) monitored by TLC. Fr. C was chromatographed on a column of silica gel, eluted successively with a gradient of petroleum n-hexane–EtOAc (3:1 to 1:2) as eluent to yield 1 (18.0 mg). Fr. H was chromatographed on a column of silica gel, eluted successively with a gradient of petroleum i-Pr₂O–EtOAc (15:1) as eluent to yield 2 (24.0 mg).

A murine microglial cell line (BV2) was cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, USA) supplemented with 5% fetal bovine serum (Hyclone, Logan, UT, USA). The production of NO was determined by measuring the accumulation of nitrite in the culture medium 24 h after stimulation with LPS (0.5 μg/mL) by the Griess reagent as in our previous report [14].

NADPH oxidase activity was measured as described previously [14]. Test compounds were added to the wells of a bioluminescence plate and incubated with 50 μg of cell homogenate for 20 min at 37 °C in the dark. O₂⁻ production was stimulated with 200 μM NADPH, and the chemiluminescence was monitored for 30 min, after which the AUC (area under the curve) was calculated to represent reactive oxygen species production (NADPH activity).

A DPPH radical scavenging capacity assay was performed as in our previous report [12]. The compound was diluted with MeOH into a range of concentrations (0.1–50 μM). DPPH (Sigma-Aldrich, USA) solution (200 μL, final concentration: 200 μM in MeOH) was added to 10 μL of each diluted sample in a 96-well microplate, and the resulting solution was allowed to react for 30 min in the dark at ambient temperature. The absorbance caused by the DPPH radical at 517 nm was determined by a microplate-spectrophotometer. The radical scavenging capacity is expressed as delta OD₅₁₇ (ΔOD₅₁₇), and values are the means of three replicates. An antioxidant, Trolox (OXIS, USA), was included as a reference.