A New Cyclic Hexapeptide and a New Isocoumarin Derivative from the Marine Sponge-Associated Fungus Aspergillus similanensis KUFA 0013

A new isocoumarin derivative, similanpyrone C (1), a new cyclohexapeptide, similanamide (2), and a new pyripyropene derivative, named pyripyropene T (3) were isolated from the ethyl acetate extract of the culture of the marine sponge-associated fungus Aspergillus similanensis KUFA 0013. The structures of the compounds were established based on 1D and 2D NMR spectral analysis, and in the case of compound 2 the stereochemistry of its amino acid constituents was determined by chiral HPLC analysis of the hydrolysate by co-injection with the d and l amino acids standards. Compounds 2 and 3 were evaluated for their in vitro growth inhibitory activity against MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A373 (melanoma) cell lines, as well as antibacterial activity against reference strains and the environmental multidrug-resistant isolates (MRS and VRE). Only compound 2 exhibited weak activity against the three cancer cell lines, and neither of them showed antibacterial activity.

In our ongoing search for new natural products with antibacterial and anticancer activities produced by the marine-derived fungi of the genera Neosartorya and Aspergillus, we have recently reported the isolation of new isocoumarins similanpyrones A and B, a new chevalone (chevalone E), and a new natural product pyripyrone S; besides the previously reported chevalone B and C, a meroterpenoid S14-95, and pyripyropene E, from the crude ethyl acetate extract of the undescribed marine sponge-associated fungus Aspergillus similanensis KUFA 0013 [29]. Reexamination of the fractions remaining from the previous study of this fungus led to the isolation of a new 8-hydroxy-3-methylisocoumarin derivative, which we have named similanpyrone C (1), a new cyclic hexapeptide, similanamide (2), and a new pyripyropene analog, pyripyropene T (3) (Figure 1). Hydrolysis of compound 2, followed by HPLC analysis of its hydrolysate using a chiral column, led to the elucidation of its amino acid constituents. Compounds 2 and 3 were evaluated for their antibacterial activity as well their cytotoxicity against three human cancer cell lines.

Results and Discussion
Compound 1 was isolated as pale yellow viscous mass, and its molecular formula C19H20O6 was established on the basis of the (+)-HRESIMS m/z 345.1342 [M + H] + , indicating ten degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3443 cm −1 ), ketone carbonyl (1730 cm −1 ), conjugated lactone carbonyl (1683 cm −1 ), olefin (1647 cm −1 ) and aromatic (1625, 1572 cm −1 ) groups. The 13 C NMR (Supplementary Information, Figure S3), DEPTs and HSQC spectra (Table 1, Supplementary Information, Figure S4) Figure S1) revealed, besides a singlet of the hydrogen bonded hydroxyl proton at δH 11.35, one doublet at δH 6.13 (J = 0.9 Hz) and one singlet at δH 6.26, two multiplets at δH 4.15 and δH 1.98, two double double doublets at δH 2.48 (J = 14.7, 2.9, 1.9 Hz) and δH 2.28 (J = 14.7, 11.3, 0.7 Hz), two double doublets at δH 2.81 (J = 16.8, 5.6 Hz) and δH 2.55 (14.0, 1.9), two broad doublets at δH 2.81 (J = 14.0 Hz) and δH 2.57 (J = 16.8 Hz), one methyl singlet at δH 2.24, and two methyl doublets at δH 1.23 (J = 6.2 Hz) and δH 1.21 (J = 6.2 Hz).   Figure S5) of H2-9 to C-10 and C-11 (δC 102.9), of H2-12 (δH 2.81, brd, J = 14.0 Hz and 2.55, dd, J = 14.0, 1.9 Hz) to C-10, 11, 13 (δC 205.3), and of H-14 to C-13 and 15, respectively ( Figure 2). That the 1,7-dioxaspiro [5.5]undec-8-en-4-one ring system was fused with the 3-methyl-1H-isochromen-1-one nucleus, through C-8 and C-9 of the methyldihydropyran ring of the former and C-6 and C-7 of the latter, was supported by the HMBC correlations of H2-9 to C-6, 7 (Table 1, Supplementary Information, Figure S5). Literature search revealed that 1 is a new compound, and in compliance with our previous work, we have therefore named it similanpyrone C. Since 1 was isolated as viscous mass, it was not possible to obtain suitable crystals for the X-ray analysis. Consequently, the absolute configuration of C-10, C-11 and C-15 is still undetermined. In an attempt to determine the stereochemistry of C-10 and C-15, we have sorted out the coupling constants of both H2-9 and H2-14. The fact that H2-9 appeared as a double doublet at δH 2.81 with a germinal coupling of 16.8 Hz, and a small coupling at δH 5.6 Hz, typical of axial-equatorial coupling, and a broad doublet at δH 2.57 with a germinal coupling constant of 16.8 Hz, we concluded that CH3-10 was in the β-axial position of the half-chair conformation of the tetrahydropyran ring. This was corroborated by the correlations of H-10, which was in α equatorial position, to both H-9β (δH 2.57, brd, J = 16.8 Hz) and H-9α (δH 2.81, dd, J = 16.8, 5.6 Hz) in the NOESY spectrum (Table 1, Supplementary Information, Figure S6). On the contrary, one of H-14 appeared as a double double doublet at δH 2.28, with a germinal coupling of 14.7 Hz and a diaxial coupling of 11.3 Hz, as well as a small coupling (long range) of 0.7 Hz, while another appeared also as a double double doublet at δH 2.48, with a germinal coupling of 14.7 Hz and an axial-equatorial coupling of 2.9 Hz, as well as a small coupling (long range) of 1.9 Hz. These patterns of couplings revealed that CH3-15 was in α equatorial position of the chair conformation of the tetrahydro-4H-pyran-4-one ring. This analysis was corroborated by the NOESY spectrum (Table 1, Supplementary Information, Figure S6), which exhibited a correlation only between H-15 and H-14β (δH 2.48, ddd, J = 14.7, 2.9, 1.9 Hz) and not between H-15 and H-14α (δH 2.28, ddd, J = 14.7, 11.3, 0.7 Hz). This assignment was also supported by the higher chemical shift value of H-14β than that of H-14α since the former is under the anisotropic deshielding of the C-13 carbonyl group. Based on the same reasoning, we assigned the broad doublet at δH 2.81 (J = 14.0 Hz) as H-12β, and the double doublet at δH 2.55 (J = 14.0, 1.9 Hz) as H-12α. Consequently, the relative configuration of C-10 and C-15 was tentatively assigned as 10S* and 15R*. The relative configuration of C-11 was tentatively assigned as 11S* based on the fact that the NOESY spectrum (Table 1, Supplementary Information, Figure S6) did not exhibit any correlation between H-10 and H-15. According to the molecular model, when the configuration of C-11 is S*, the substituents on C-11 are arranged in a way that H-10 and H-15 are pointing toward the opposite directions. On the contrary, the R* configuration of C-11 would have H-10 and H-15 close enough to give a strong NOESY correlation.
Similanpyrone C (1) can be assumed to be derived from the acetate-malonate pathway (Scheme 1). Cyclization and enolization of the pentaketide (I) leads to the formation of 6,8-dihydroxy-3-methylisocoumarin (II), which, after Claisen condensation with the tetraketide (III), gives rise to IV. Enolization of the side chain, together with a formation of the hemiketal by the phenolic hydroxyl group on C-6 of the isocoumarin nucleus and the ketone carbonyl of the side chain, leads to the formation of a hemiketal V. Formation of the ketal and methylation by SAM in the side chain finally gives rise to similanpyrone C (1).
Compound 2 was isolated as pale yellow viscous mass, and its molecular formula C34H52N6O6 was established on the basis of the (+)-HRESIMS m/z 641.4053 [M + H] + , indicating twelve degrees of unsaturation. The IR spectrum showed absorption bands for amine (3335 cm −1 ), carbonyl (1682, 1644 cm −1 ) and aromatic (1594, 1519 cm −1 ). The 13 C NMR (Supplementary Information, Figure S8), DEPTs and HSQC spectra (  Figure S7 Figure S11). The anthranilic acid residue was linked to the valine residue, through the amino group of the former and the carboxyl group of the latter, since the HMBC spectrum ( Supplementary Information, Figure S11) showed correlations of the NH signal at δH 9.41, brs to the carbonyl carbon at δC 170.7 (C-8), of the methine proton at δH 4.32 dd, J = 7.4, 3.3 Hz (H-9) to the methine carbon at δC 29.9 (C-10), the methyl carbon at δC 16.2 (C-12) and C-8, and of the NH signal at δH7.43, d (J = 7.5 Hz) to C-9 (δC 59.3) and C-10 (Table 2, Figure 3). The presence of the leucine residue was supported by the coupling system from CH-14 (δH 4.57, m; δC 50.9) through CH3-17 (δH 0.97, d, J = 6.5 Hz; δC 23.3) and CH3-18 (δH 0.88, d, J = 6.4 Hz; δC 21.1), and of NH at δH 8.02 d (J = 7.9 Hz) to H-14, as observed in the COSY spectrum (Table 2, Supplementary Information, Figure S9), as well as by the HMBC correlations of the NH signal at δH 8.02 d (J = 7.9 Hz) to C-14 (Table 2, Figure 3). That the valine residue was linked to the leucine residue was supported by the HMBC cross peak between the NH signal of the former (δH7.43, d, J = 7.5 Hz) to the signal of the carbonyl carbon (δC174.2, C-13) of the latter. In turn, the leucine residue was linked to the alanine residue, as evidenced by the HMBC cross peaks of the NH signal of the former to the carbonyl carbon signal (δC174.  (Table 2, Supplementary Information, Figure S9). Since both H-30 and CH3-28 gave HMBC cross peaks to C-29 (δC 168.9), the pipecolic acid residue was linked to the N-methyl leucine residue through the carboxyl group of the former and a nitrogen atom of the latter. Since 2 presents twelve degrees of unsaturation, the nitrogen atom of the piperidine ring of the pipecolic acid residue was linked to the carbonyl of the anthranilic acid residue. The proposed structure was supported by the NOESY correlations which showed cross peaks of NH at δH 9.41, brs to H-9, CH3-12, of NH at δH 7.43 (d, J = 7.5 Hz) to H-9, CH3-11 (δH 1.06, d, J = 6.9 Hz), CH3-12, H-14, of NH at δH 8.02 (d, J = 7.9 Hz) to H-14, H-15 (δH 2.02, m; 1.77, m), CH3-17, of NH at δH 7.64 (d, J = 7.9 Hz) to CH3-21, H-23, CH3-28, of H-3 to H-34β (δH 4.14, dd, J = 14.4, 2.4 Hz), and of H-30 to CH3-28 (Table 2, Supplementary Information, Figure S12). Combining this information, it was possible to conclude that 2 was cyclo (anthranilic acid-Val-Leu-Ala-N-methyl-Leu-pipecolic acid).    The 1 H and 13 C NMR data of compound 2 resembled those of PF1171C, a cyclic hexapeptide isolated from extracts of the unidentified ascomycete OK-128 fermented with okara by Kai et al. [30], and later by a total synthesis by Masuda et al. [31]; however, its value of optical rotation was quite different from that of PF1171C [30,31]. Moreover, PF1171C was reported as white solid (mp 138-140 °C) [31], while compound 2 is pale yellow viscous mass. This observation suggested that compound 2 and PF1171C could be diastereomers.

Scheme 1. Proposed biogenesis of similanpyrone C (1).
The stereochemistry of the amino acid residues of compound 2 was determined by chiral HPLC analysis of its acidic hydrolysate, using appropriate D and L amino acids standards, according to the previously described method [32]. The enantioseparations of the standard amino acids were successfully performed with the Chirobiotic T column under reversed-phase elution conditions [33]. Actually, the teicoplanin selector of this column has several characteristic features that make it suitable for amino acid analysis [33][34][35]. The elution order of the enantiomers of all the standard amino acids was confirmed by injecting the solutions of the racemic or enantiomeric mixtures and then each enantiomer separately at a flow rate of 1 mL/min ( Supplementary Information, Figure S18). As predicted, the D enantiomer was always more strongly retained than the corresponding L enantiomer on the Chirobiotic T column [33]. Based on mix HPLC analyses of the acidic hydrolysate with standard amino acids (co-injection) (Supplementary Information, Figure S19 and Table S1), compound 2 was elucidated unambiguously as cyclo (anthranilic acid-L-Val-D-Leu-L-Ala-N-methyl-L-Leu-D-pipecolic acid). Although the amino acid sequence of compound 2 is the same as that of PF1171C, the stereochemistry of its amino acid constituents is different from that of the amino acids constituent of PF1171C. While PF1171C contains D-Ala, L-Leu, D-Val, and L-pipecolic acid, compound 2 contains L-Ala, D-Leu, L-Val and D-pipecolic acid. Thus compound 2 is a new compound, and we have named it similanamide.
It is interesting to note that Kai et al. [30] has firstly assigned the stereochemistry of the amino acid constituents of PF1171C using the reversed phase HPLC analysis of the Marfey derivatives of the amino acids. Since the retention times for the Marfey derivatives of D-Ala (19.4 min) and L-Ala (20.0 min) were too close for resolution, they had wrongly assigned D-Ala for L-Ala. However, in our chiral HPLC analysis using the Chirobiotic T column under reversed-phase elution conditions, not only the retention time of L-Ala (7.16 min) was very different from that of D-Ala (9.36 min), but also the retention times of the D/L pair of other amino acid constituents ( Supplementary  Information, Table S1).
So far, only few cyclohexapeptides have been reported from marine-derived microorganisms. Wu et al. [36] have isolated two cyclohexapeptides, nocardiamides A and B, from the culture broth of the marine-derived actinomycete Nocardiopsis sp. CNX037, isolated from sediment. Cai et al. [37] have isolated two anti-Mycobacterium tuberculosis cyclohexapeptides from a Streptomyces hygroscopicus Strain, while Song et al. [38] reported isolation of three new cyclopeptides, desotamides B-D, from the deep South China Sea-derived Streptomyces scopuliridis SCSIO ZJ46. To the best of our knowledge, compound 2 is the first cyclopeptide containing D-pipecolic acid residue ever isolated from marine fungi.
Compound 3 was isolated as pale yellow viscous mass, and its molecular formula C29H33NO8 was established on the basis of (+)-HRESIMS m/z 524.2287 [M + H] + , indicating fourteen degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3418 cm −1 ), ester carbonyl (1732 cm −1 ), conjugated ester carbonyl (1667cm −1 ), olefin (1643 cm −1 ), aromatic (1557, 1507 cm −1 ). The 13 C NMR ( Supplementary Information, Figure S15), DEPTs and HSQC spectra (Table 3, Supplementary Information, Figure S16 carbons. The general feature of 13 C and 1 H spectra of compound 3 closely resembled those of pyripyropene S, previously isolated from the same fungus [29]. Analysis of the 1 H ( Supplementary Information, Figure S13), 13 C, HSQC and HMBC spectra (Table 3, Supplementary Information, Figure S17) revealed the presence of the ring system comprising of the decahydronaphthalene fused, on C-5 and C-6, with the 2H,5H-pyrano [4,3-b]pyran-5-one, which connected to the pyridine ring through C-6′ of the former and C-3″ of the latter, similar to pyripyropene S [29]. However, there were only two acetoxyl groups (δC 170.1, CO; δC 20.5, CH3, δH 2.00, s and δC 169.8, CO; δC 20.8, CH3; δH 2.00, s) in compound 3. That the acetoxyl groups were on C-1 and C-11 was supported by the fact that the chemical shift values of H-1 (δH 4.64, t, J = 8.5 Hz) and H-11 (δH 3.75, s) were very similar to those of pyripyropene S, whereas the chemical shift of H-7 (δH 3.85, dd, J = 10.6, 4.2 Hz) was nearly 1.4 ppm less than that of pyripyropene S. On the other hand, the 13 C chemical shift values of C-6 (δH 85.9) and C-8 (δC 27.4) of compound 3 were 2.00 and 3.00 ppm, respectively, higher than those of the corresponding carbons in pyripyropene S, while the 13 C chemical shift value of C-7 (δC 75.5) of compound 3 was 2.00 ppm lower than that of C-7 of pyripyropene S. Since H-7 appeared as a double doublet with coupling constants of 10.6 and 4.2 Hz, the position of the hydroxyl group on C-7 was β. Thus, compound 3 is 7-deacetylpyripyropene S. In order to prove the stereochemistry of compound 3, the NOESY experiment was carried out. As the NOESY spectrum (Table 3) clearly exhibited correlations of CH3-15 to CH3-12, but not to H-1 and H-9; of CH3-12 to CH3-14 and CH3-15, and of CH3-14 to CH3-12, but not to H-7 (Table 3, Supplementary Information, Figure S18), the stereochemistry of compound 3 is the same as that of pyripyropene S [29], i.e., 1S*, 4R*, 6S*, 7S*, 9R*, 10R*. Since it is a new compound we have named it pyripyropene T. Since compound 1 was isolated in a very small amount, only compounds 2 and 3 were evaluated for their cytotoxicity and antibacterial activity. Compounds 2 exhibited weak in vitro growth inhibitory activity, by Sulforhodamine B (SRB) assay [39], against the MCF-7 (breast adenocarcinoma, GI50 = 125 ± 0), NCI-H460 (non-small cell lung cancer, GI50 = 117.50 ± 3.55) and A373 (melanoma, GI50 = 115 ± 7.07) cell lines. Compounds 2 and 3 were also tested for their antibacterial activity against four reference strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa), as well as the environmental multidrug-resistant isolates, according to the previously described method [40], and neither of them showed activity (MIC values higher than 256 µg/mL). The stereochemistry of the amino acids was determined by analysis of the acidic hydrolysate from compound 2. Compound 2 (5.0 mg) was dissolved in 6 N HCl (5 mL) and heated at 110 °C, in a furnace, for 24 h in a sealed glass tube. After cooling to room temperature, the solution was dried under N2 for 24 h, reconstituted in methanol for HPLC-MS (200 µL), filtered through a 4 mm PTFE Syringe Filter F2504-4 of 0.2 µm pore size (Thermo Scientific, Mumbai, India), and then analyzed by HPLC equipped with a chiral column.

Chiral HPLC Analysis
The HPLC system consisted of Shimadzu LC-20AD pump, equipped with a Shimadzu DGV-20A5 degasser, a Rheodyne 7725i injector fitted with a 20 µL loop, and a SPD-M20A DAD detector (Kyoto, Japan). Data acquisition was performed using Shimadzu LCMS Lab Solutions software, version 3.50 SP2. The chiral column used in this study was Chirobiotic T (15 cm × 4.6 mm I.D., particle size 5 µm) manufactured by ASTEC (Whippany, NJ, USA). The mobile phase composition was MeOH:H2O:CH3CO2H (70:30:0.02, v/v/v), all were LC-MS grade solvents obtained from Sigma-Aldrich Co (St. Louis, MO, USA). The flow rate was 0.5 mL/min and the UV detection wavelength was 210 nm. Analyses were performed at room temperature in an isocratic mode.
All standards of racemic amino acids and pure amino acid enantiomers were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). The elution order of the enantiomers of all the standards amino acids was confirmed by injecting the solutions of the racemic or enantiomeric mixtures, and then each enantiomer separately (or only L-amino acid in the case of N-methyl leucine) at a flow rate of 1 mL/min or 0.5 mL/min. Working solutions of single enantiomeric amino acids were prepared by dissolution in MeOH at the concentration of 1 mg/mL (10 µL sample injection), while the enantiomeric mixtures were prepared by mixing equal aliquots of each enantiomer (20 µL sample injection). Mix HPLC analyses of the acidic hydrolysate with standard amino acids (co-injection) confirmed the stereochemistry of the amino acids of compound 2.

Conclusions
Following our first report of the isolation of new isocoumarin derivatives and merotepenoids from the ethyl acetate crude extract of the culture of the undescribed marine sponge-associated fungus Aspergillus similanensis KUFA 0013, we have reexamined its remaining column fractions and have isolated a new isocoumarin derivative containing an unusual 1,7-dioxaspiro-undecenone moiety, together with a new cyclohexapeptide and a new pyripyropene analog. Although several cyclopeptides have been reported from many fungi of the genus Aspergillus, this is the first report of isolation of cyclohexapeptide from the marine-derived fungus. The fact that these new cyclohexapeptide and pyripyropene analog did not exhibit relevant antibacterial and the in vitro growth inhibitory activities on human cancer cell lines does not mean that they are void of other interesting biological activities. In order to prove this hypothesis, it is necessary to explore their potential in a broader biological or pharmacological assay system.