Asperaculanes A and B, Two Sesquiterpenoids from the Fungus Aspergillus aculeatus

Six sesquiterpenoids 1–6, including two new ones, an ent-daucane-type sesquiterpenoid, asperaculane A (1), and a nordaucane one, asperaculane B (2), and four known nordaucane derivatives, aculenes A–D 3–6, together with the known secalonic acid D (7), were isolated from a fermentation culture of the fungus Aspergillus aculeatus. Their structures and absolute configurations were established by analyses of their spectroscopic data, including 1D and 2D-NMR spectra, HR-ESIMS, electronic circular dichroism (ECD) data, and quantum chemical calculations. These metabolites were evaluated for in vitro cytotoxic activity against two cell lines, human cancer cell lines (HeLa) and one normal hamster cell line (CHO).

In order to identify new bioactive natural compounds from various Aspergillus fungi, we studied the chemical constituents of the cultures of A. aculeatus. Herein we report the isolation and structure elucidation of two new sesquiterpenoids, named asperaculanes A (1) and B (2).
Quantum chemical calculations of electronic circular dichroism (ECD) spectra have been proven to be reliable tools in determining the absolute configurations of organic molecules [19]. The CD spectrum of 1 showed two Cotton effects, including a negative first Cotton effect at 229 nm (-13.3) and the positive Cotton effect at 261 nm (∆ɛ = +1.56). To determine the absolute configuration of 1, a comparison was made between the experimental and calculated CD spectra by the time-dependent density functional theory (TDDFT) method. Conformational searches were performed by Conflex6.7 with the MMFF94S force field [20]. Conformers within 3 kcal/mol were saved and further optimized at B3LYP/6-311+G(d,p) level in Gaussian 09 software package [21]. The stable conformers with populations greater than 1% and without imaginary frequencies were submitted to ECD calculation by the TDDFT [CAM-B3LYP/TZVP] method associated with CPCM solvent model in methanol. As a result, the calculated CD spectrum for 1 agreed well with that experimental one for 1 (Figure 3), and the absolute configuration of 1 was assigned as (1R,5R,10S). Thus, the structure of 1 was elucidated as 4E(11)-(1R,5R,10S)-10,14-dihydroxydauca-4(11),7-dien-13-carboxylic acid.
Compounds 3-5, and 7 were characterized as aculenes A, B, C, and secalonic acid D, respectively, based on HR-ESIMS, NMR data, Marfey's method [22] and comparison of their spectroscopic data with those reported in the literature [15,23]. Aculene D (6) had the molecular formula C14H20O2, as deduced from HR-ESIMS data (m/z 243.1362 [M+Na] + ). The 1 H-and 13 C-NMR spectroscopic data of 6 ( Table 1) had the almost similar signals as 4, except for the absence of the signals of the proline moiety.
Its structure was confirmed by analysis of the COSY, HMQC, and HMBC experiments ( Figure 2). The NOESY cross-peaks between H-10α and Me-14α indicated that the OH group at C-10 was β-oriented. This compound had been detected previously in the same organism by LC-HRMS [24], but no NMR data were reported.
However, the absolute stereochemistries of 3-6 were unknown. To determine the absolute configuration of these compounds, we employed the experimental and calculated CD spectra. A comparison was made among the experimental and calculated CD spectra of (1R,10S,16S)-3 and (1S,10R,16S)-3. The calculated CD spectrum for 3 agreed well with its experimental one ( Figure 3). Thus, the absolute configuration of other chiral centers in 3 was assigned as 1R and 10S, and the structure of 3 was elucidated as (1R,10S,16S)-aculene A. Similarly, the absolute configuration of 5 was assigned as (1R,10S) from its well matched calculated and experimental CD spectra ( Figure 3). Thus, the structure of 5 was established as (1R,10S)-aculene C, while the absolute configuration of 6 was assigned as (1R,5R,10S) from its well matched calculated and experimental CD spectra ( Figure 3). Thus, the structure of 6 was established as (1R,5R,10S)-aculene D. Furthermore, theoretical CD spectra of 4 were calculated at CAM-B3LYP/SVP//B3LYP/6-31+G(d,p) level. The absolute configurations of chiral centers at C-1, C-5, C-10, and C-16 in 4 were established as 1R, 5R, 10S, and 16S from its well matched calculated and experimental CD spectra ( Figure 3). Thus, the structure of 4 was determined as (1R,5R,10S,16S)-aculene B.
The metabolites 1-6 were possibly biosynthesized from daucenes, [25] which could be sesquiterpenoid natural products of plants and fungi [24,25]. Compound 1 was proposed to be the precusor of the co-existing metabolites 2-6, which was formed via a series of decarboxylation, oxidations, and dehydrogenation of 1.

Cytotoxicity Assay
The isolated compounds 1-6 were all tested for their in vitro cytotoxic activities against one human cancer cell lines HeLa and one normal hamster cell line (CHO) using the SRB colorimetric assay [26]. None of the tested compounds showed any cytotoxicity below IC50 values of 50 μM.

General Procedures
Optical rotations were measured on an Autopol III automatic polarimeter. 1 H-and 13 C-NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer or a Bruker Avance III 500 spectrometer. Chemical shifts are reported in ppm relative to DMSO-d6 ( 1 H, δ 2.50; 13 C, δ 40.08) or MeOH-d4 ( 1 H, δ 3.30; 13 C, δ 49.1). 1 H-NMR data is reported as: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant, and integration. Infrared (IR) spectra were run on a Bruker Tensor spectrophotometer. High resolution electrospray ionization mass (HR ESIMS) was obtained on an Agilent 6210 time of flight LC-MS. Reactions were monitored by analytical thin-layer chromatography on EMD silica gel-60F254 plates. Flash chromatography was performed on EMD silica gel 60, 70-230 mesh. All reagents were used without further purification unless otherwise noted.

Fungal Material and Fermentation
The fungal strain Aspergillus aculeatus ATCC16872 v1.1 was provided by the U.S. Department of Energy Joint Genome Institute (DOE JGI). The fungus was cultivated at 30 °C on 40 solid YES plates at 2.25 × 10 6 spores per 15-cm plates (~0.1 mL of medium per plate). After 5 days, agar was cut into small pieces and put in two 2-liter flasks.

Extraction, Isolation and Purification
The solid agar was chopped into pieces and saturated twice in around 750 mL of 1:1 CH2Cl2/MeOH for 24 h. After filtration, the combined extract was evaporated in vacuo to obtain a residue, which was suspended in water (400-500 mL) and then partitioned with EtOAc three times. The combined EtOAc layer was evaporated in vacuo to yield a crude extract. The crude extract was applied to a silica gel column (Merck, 230 to 400 mesh, ASTM, 20 × 80 mm) and eluted with 400 mL CH2Cl2/MeOH mixtures of increasing polarity (fraction A, 1:0; fraction B, 19:1; fraction C, 9:1; fraction D, 7:3). The LC-MS profiles of these fractions suggests that fraction C (947.7 mg) had most of the compouds.
Fraction C was further purified by reverse phase HPLC [Phenomenex Luna 5 μm C18 (2), 250 × 10 mm] with a flow rate of 5.0 mL/min and measured by a UV detector at 254 nm. The gradient system was MeCN (solvent B) in 5% MeCN/H2O (solvent A) both containing 0.05% TFA. The HPLC gradient condition is 0% to 40% B from 0 to 20 min, 40% to 100% B from 20 to 40 min, maintained at 100% B from 40 to 42 min, 100% to 0% B from 42 to 44 min.

Absolute Configuration Determination
The stereochemistry of the proline residue was determined by the advanced Marfey's method (see the Supporting Information) [22].

Computational Section
A preliminary conformational search was performed on the basis of a molecular mechanical method by CONFLEX6.7 with the MMFF94S force field [20]. Stable Conformers (within 3 kcal/mol) of compound 3 were saved and further optimized at B3LYP/6-31+G(d,p) level in Gaussian 09 software package [21]. Other compounds (1, 4, 5 and 6) conformers were further optimized at B3LYP/6-31+G(d,p) level. Frequency was calculated at the same level of theory. The stable conformers with populations greater than 1% and without imaginary frequencies were submitted to ECD calculation by the TDDFT (CAM-B3LYP) method associated with CPCM solvent model in methanol. Compound 3 was calculated at SVP level, and the compounds 1, 4, 5 and 6 were calculated at TZVP level. The excitation energies (E), oscillator strength (f), rotatory strength in velocity form (Rvel), and rotatory strength in length form (Rlen) of the lowest 30 excited states were calculated. ECD spectra of different conformers were simulated using SpecDis [27] with a half-bandwidth of 0.3 eV. The final ECD spectra were generated according to the Boltzmann-calculated distribution of each conformer.

Cytotoxicity Assay
In vitro cytotoxicity of these isolated compounds was assessed by the sulforhodamine B (SRB) assay as described previously by our group [26].

Conclusions
In conclusion, six sesquiterpenoids, including a new ent-daucane sesquiterpenoid, asperaculane A (1) and five nordaucanes, a new asperaculane B (2) and four aculenes A-D (compounds 3-6), have been identified from the fungus A. aculeatus. The absolute configurations of 1-6 were determined for the first time by ECD. The present study revealed the asperaculane or aculene family is the antipode of most daucanes isolated from plant species. Asperaculane A (1) is the first example of an ent-daucane-type sesquiterpene found in natural products.