New Andrastin-Type Meroterpenoids from the Marine-Derived Fungus Penicillium sp.

Three new andrastin-type meroterpenoids penimeroterpenoids A–C (1–3) together with two known analogs (4 and 5) were isolated from the cultures of the marine-derived Penicillium species (sp.). The structures of the new compounds were elucidated on the basis of 1- and 2-dimensional (1D/2D) Nuclear Magnetic Resonance (NMR) spectroscopic and mass spectrometric analysis. The absolute configurations of 1–3 were determined by comparison of experimental and calculated electronic circular dichroism (ECD) spectra. Compound 1 showed moderate cytotoxicity against A549, HCT116, and SW480 cell lines.

Marine-derived fungi, living under extreme environmental conditions such as high salinity, intensely high pressure, absence of sunlight, and deficiency of nutrients, are considered to be a new reservoir of structurally diverse and biologically active metabolites for drug discovery [20,21]. In our ongoing search for new bioactive secondary metabolites from marine-derived fungi, the fungus Penicillium sp. (A18), isolated from a deep-water sediment sample that was collected at a depth of 5115 m in the East Pacific, was selelected for chemical investigations. As a result, three new andrastin-type meroterpenoids, which have been named penimeroterpenoids A-C (1-3), together with two known compounds, andrastone E (4) [22] and citreohybridonol (5) [11] (Figure 1), were isolated and identified from the culture extract of the fungus. Their structures were established by a detailed interpretation of 1D/2D NMR spectroscopic and mass spectrometric data, and the absolute configurations of 1-3 were determined by electronic circular dichroism (ECD) calculations. All of these compounds were evaluated for cytotoxicity against a panel of six human tumor

Results
Penimeroterpenoid A (1) was obtained as a colorless oil. Its molecular formula C 28 H 38 O 8 was established by high-resolution electrospray ionisation mass spectrometry (HRESIMS) analysis (m/z 525.2454 [M + Na] + ), indicating ten degrees of unsaturation. The infrared (IR) spectrum showed the presence of hydroxy (3445 cm −1 ) and carbonyl (1752 cm −1 ) groups. Analysis of its NMR data (Table 1) revealed the presence of eight methyl groups, four methylenes, three methines (including one oxymethine), six sp 3 quarternary carbons (one oxygenated), one trisubstituted olefin unit, two carboxylic carbons (δ C 167.3, 170.7), one aldehyde group (δ C 204.5; δ H 10.1), and two ketone carbons (δ C 210.6, 206.8, respectively). These data accounted for all 1 H and 13 C NMR resonances except for one exchangeable proton, suggesting that 1 was a tetracyclic compound. Analysis of the 1 H-1 H correlation spectroscopy (COSY) NMR data ( Figure 2) led to the identification of three isolated spin-systems of C-1-C-2-C-3, C-5-C-6-C-7, and C-9-C-11. Heteronuclear multiple bond correlations (HMBC) from H 2 -1 to C-10, H-3 to C-5, H-5 to C-1, C-4, C-10, C-24, and C-25, and from the geminal methyl groups H 3 -24 and H 3 -25 to C-3, C-4, and C-5 completed the cyclohexane ring (ring A). HMBC cross-peaks from H-3 and H 3 -23 to the carboxylic carbon C-22 (δ C 170.7) established the location of the acetyl group at C-3. Other correlations from H-5 to the aldehyde carbon C-21, and from the aldehyde proton H-21 to C-1 and C-10 indicated that C-21 was attached to C-10. While the HMBC cross-peaks from H 2 -1 and H-5 to C-9, H 2 -7 to C-8 and C-26, H-9 to C-8, C-10, C-21, and C-26, and from H 3 -26 to C-7, C-8, C-9, and C-14 permitted the completion of another cyclohexane unit (ring B) with the methyl carbon C-26 and the sp 3 quarternary carbon C-14 (δ C 70.6) attached at C-8. HMBC correlations from H-9 to C-12, H-11 to C-8, C-10, C-13, C-20, H 3 -19 to C-12, C-13, C-14, and C-17, and from H 3 -20 to C-11, C-12, and C-13, as well as from H 3 -18 to C-16 and two ketone carbons (C-15 and C-17: δ C 210.6 and 206.8, respectively) permitted the completion of the tetrahydro-1H-indene-1,3(2H)-dione moiety (rings C and D), fused with the cyclohexane ring B at C-8 and C-9. In addition, HMBC correlations from H 3 -28 to the carboxylic carbon C-27 (δ C 167.3) located the methoxy group at C-27. The exchangeable proton was located at C-16 by default, an identification supported by the chemical shift value for C-16 (δ C 72.1). Thus, the planar structure of 1 was established as shown ( Figure 1), and has the same planar structure as compound 4. value for C-16 (δC 72.1). Thus, the planar structure of 1 was established as shown ( Figure  1), and has the same planar structure as compound 4.  The relative configuration of 1 was assessed by analysis of the nuclear overhauser effect spectroscopy (NOESY) correlations ( Figure 3). NOESY correlations of H-5 with H-7a, H-9, and H3-25 indicated that these protons are all on the same side of the ring system. While NOESY correlations of H-3 with H3-24, H3-26 with H-7b and H-21, and of H3-28 with H3-19 and H3-18 placed these protons on the opposite side of the tetracyclic system. Futhermore, the NOESY correlation between H-9 and H3-18 in 1 disappeared compared to 4, and the carborn signal for C-16 and C-18 was shifted upfield by 3.8 and 5.8 ppm, suggesting an inversion of the C-16 stereocenter. These observations led to the assignment of the relative configuration of 1, indicating that 1 was the C-16 epimer of compound 4.  Figure S22). The overall calculated ECD spectra of 1a and 1b were then generated by Gaussian broadening (Figure 4). The experimental ECD spectrum of 1 was nearly identical to the calculated ECD spectrum for 1a, clearly indicating the 3S,5R,8S,9R,10S,13R,14R,16R absolute configuration for 1.
Two known compounds 4 and 5 were identified as andrastone E (4) [22] and citreohybridonol (5) [11], respectively, by comparing their spectroscopic data with those reported previously in the literature. Compounds

Strain and Fermentation
The strain Penicillium sp. was isolated from a deep-water sediment sample that was collected at a depth of 5115 m in the East Pacific (145 • 2 W, 07 • 37 N). The isolate was identified as Penicillium sp. by sequencing the internal transcribed spacer (ITS) region of the rDNA (GenBank Accession No. MW767028). Penicillium sp. was grown on potato dextrose agar (PDA) at 27 • C for five days, and then several pieces of agar plugs (about 0.5 × 0.5 × 0.5 cm 3 ) were added into 250 mL Erlenmeyer flasks containing 50 mL of media (glucose 4 g/L; malt extract 10 g/L and yeast extract 4 g/L) at 27 • C with shaking (170 rpm) for five days to produce the seed culture. Finally, Erlenmeyer flasks (500 mL) containing Mar. Drugs 2021, 19, 189 7 of 9 80 g of rice and 120 mL of distilled H 2 O and 4.0 mL seed culture incubated at 25 • C for 30 days.

Bioassays for Cytotoxic Activity
Cytotoxic assay was performed as previously described [23].

ECD Calculation
Conformational analyses for compounds 1-3 were performed using Maestro 10.2 in the OPLS3 molecular mechanics force-field within an energy window of 5.0 or 3.0 kcal/mol. The conformers were then further optimized with the software package Gaussian 09 at the B3LYP/6-311G(2d,p), B3LYP/6-31G(d), and B3LYP/6-311G(d,p) level for compounds 1-3, respectively [24], and the harmonic vibrational frequencies were also calculated to confirm their stability. The time-dependent density functional theory (TD-DFT) methods at the CAM-B3LYP/6-31G(2d,p), B3LYP/6-31G(d), and B3LYP/6-311G(d,p) were applied to calculate the 60 lowest electronic transitions which obtained conformers in vacuum, respectively. The Gaussian function was applied to simulate the ECD spectrum of the conformers. The calculated ECD spectra were obtained according to the Boltzmann weighting of each conformer's ECD spectrum in MeOH solution.

Conclusions
In conclusion, three new andrastin-type meroterpenoids penimeroterpenoids A-C (1-3) together with two known compounds (4 and 5) were isolated from the fermentation broth of the marine-derived fungi Penicillium sp. The structures of the new compounds were elucidated by mass spectrometry (MS), NMR, and ECD spectroscopic data. Compound 1 showed moderate cytotoxicity against A549, HCT116, and SW480 cell lines. Our findings Mar. Drugs 2021, 19, 189 8 of 9 also suggest that the fungal genus Penicillium is a rich source of bioactive secondary metabolites, and thus worthy of in-depth investigations.