New Polyketides and New Benzoic Acid Derivatives from the Marine Sponge-Associated Fungus Neosartorya quadricincta KUFA 0081

Two new pentaketides, including a new benzofuran-1-one derivative (1) and a new isochromen-1-one (5), and seven new benzoic acid derivatives, including two new benzopyran derivatives (2a, b), a new benzoxepine derivative (3), two new chromen-4-one derivatives (4b, 7) and two new benzofuran derivatives (6a, b), were isolated, together with the previously reported 2,3-dihydro-6-hydroxy-2,2-dimethyl-4H-1-benzopyran-4-one (4a), from the culture of the marine sponge-associated fungus Neosartorya quadricincta KUFA 0081. The structures of the new compounds were established based on 1D and 2D NMR spectral analysis, and in the case of compounds 1, 2a, 4b, 5, 6a and 7, the absolute configurations of their stereogenic carbons were determined by an X-ray crystallographic analysis. None of the isolated compounds were active in the tests for antibacterial activity against Gram-positive and Gram-negative bacteria, as well as multidrug-resistant isolates from the environment (MIC > 256 μg/mL), antifungal activity against yeast (Candida albicans ATTC 10231), filamentous fungus (Aspergillus fumigatus ATTC 46645) and dermatophyte (Trichophyton rubrum FF5) (MIC > 512 µg/mL) and in vitro growth inhibitory activity against the MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma) cell lines (GI50 > 150 µM) by the protein binding dye SRB method.


Introduction
Aspergillus section Fumigati and its teleomorph Neosartorya include many important species because they can be pathogenic or allergenic to man, as well as causing food spoilage and producing mycotoxins. Certain species are also found to produce interesting bioactive secondary metabolites mycotoxins. Certain species are also found to produce interesting bioactive secondary metabolites that can be considered to have potential for drug development [1]. For this reason, we have investigated the bioactive secondary metabolites produced from the cultures of four Neosartorya species collected from soil in Thailand, i.e., Neosartorya glabra KUFC 6311 [2], N. pseudofischeri KUFC 6422 [3], N. siamensis KUFC 6349 [4] and N. fischeri KUFC 6344 [5], as well as six marine-derived species of Neosartorya, including N. paulistensis KUFC 7898 [6], N. laciniosa KUFC 7896 [5], N. spinosa KUFC 8104, N. tsunodae KUFC 9213 [5], N. siamensis KUFA 0017 and N. takakii KUFC 7898 [7], as well as one marine-derived Aspergillus species (Aspergillus similanensis KUFA 0013) [8,9]. Recently, we have also reported the antifungal activity of the crude extract of N. quadricincta KUFA 0064, isolated from an agricultural soil in Southern Thailand, against plant pathogenic fungi, which are causative agents of diseases of economically-important plants of Thailand [10]. So far, the only report on secondary metabolites of N. quadricincta was by Ozoe et al., who described the isolation of dihydroisocoumarin derivative, PF1223, from the culture of N. quadricincta strain PF1223 (unidentified source). This compound was shown to inhibit the [ 3 H] EBOB binding by 65% [11]. Thus, in our ongoing search for bioactive secondary metabolites from marine-derived fungi from Thai waters, we have investigated the culture of N. quadricincta KUFA 0081, isolated from the marine sponge Clathria reinwardti, which was collected from the Coral reef at Samae San Island in the Gulf of Thailand. The ethyl acetate extract of the culture of this fungus yielded, besides the previously described 2,3-dihydro-6-hydroxy-2,2-dimethyl-4H-1-benzopyran-4-one (4a) [12], two new polyketide derivatives (1, 5) and seven new benzoic acid derivatives (2a, 2b, 3, 4b, 6a, 6b and 7) (Figure 1). All of the isolated compounds were tested for their antibacterial activity against Gram-positive and Gramnegative bacteria, as well as multidrug-resistant isolates from the environment and for their antifungal activity against yeast (Candida albicans ATCC 10231), filamentous fungus (Aspergillus fumigatus ATCC 46645) and dermatophyte (Trichophyton rubrum FF5). Additionally, these compounds were also evaluated for their in vitro growth inhibitory activity against the MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma) cell lines by the protein binding dye SRB method.
Since Compound 2b could not be obtained as a suitable crystal for X-ray analysis, the absolute configuration of its C-3 could not be determined with certainty. However, as compound 2b is the acetate derivative of compound 2a, it was speculated that the stereochemistry of its C-2 should be the same as that of C-2 of compound 2a, i.e., 2S. In order to confirm this hypothesis, the NOESY experiments were carried out. The NOESY spectrum of compound 2b showed a weak correlation of H-8 to H3-9 and not to H2-10 ( Figure 6b, Supplementary Information, Figure S18), similar to what has been observed for compound 2a. Acid hydrolysis of 2b gave the product whose structure was confirmed as 2a by 1 H and 13 C NMR data, as well as the optical rotation. Therefore, the absolute configuration of C-2 of 2b is assigned as 2S. Compound 2b is also a new compound, thus we named it quadricinctapyran B. carboxyl carbonyl (δ C 166.7), three quaternary sp 2 (δ C 161. 2, 125.3, 125.0), five methine sp 2 (δ C 139. 5, 134.6, 129.8, 123.5, 119.8), one oxy-quaternary sp 3 (δ C 70.7), one oxymethylene sp 3 (δ C 77.0) and one methyl (δ C 26.1) groups. The 1 H NMR spectrum (Table 3, Supplementary Information, Figure S19) revealed the existence of three aromatic protons of the 1,2,4-trisubstituted benzene ring, similar to that of compound 2a, at δ H 7.05, d (J = 8.4 Hz), 7.74, dd (J = 8.4, 2.1 Hz), 7.89, d (J = 2.1 Hz), two olefinic protons of the cis-double bond at δ H 5.95, dd (J = 12.0, 1.2 Hz) and 6.31, d (J = 12.0 Hz), two oxymethylene protons at δ H 3.84, d (J = 11.1 Hz) and 4.02, dd (J = 11.1, 1.6 Hz), and a methyl singlet at δ H 1.26. That the carboxylic acid functionality was on C-7, and the substituent with a cis-double bond was on C-5a was substantiated by the HMBC correlations of H-6 (7.89, d, J = 2.1 Hz) to C-10 (δ C 166.7), C-9a (δ C 161.2), C-8 (δ C 129.8) and C-5 (δ C 123.5), of H-5 (δ H 6.31, d, J = 12.0 Hz) to C-6 (δ C 134.5) and C-9a and of H-4 (δ H 5.95, dd, d, J = 12.0, 1.2 Hz) to C-5a (δ C 125.0) ( Table 3 and Figure 7a, Supplementary Information, Figure S23). However, contrary to compound 2a, compound 3 showed the HMBC correlations of H 2 -2 (δ H 3.84, d, J = 11.1 Hz and 4.02, dd, J = 11.1, 1.6 Hz) to not only C-4 (δ C 139.5), but also to C-9a (Table 3 and Figure 7a). Consequently, the benzoic acid moiety was fused with the 2,3,6,7-tetrahydro-oxepine ring through C-5a and C-9a. That the methyl group and the hydroxyl group were on C-3 of the oxepin ring was confirmed by the HMBC correlations of the methyl singlet at δ H 1.26 (H-11) to C-3 (δ C 70.7), C-2 (δ C 77.0) and C-4, as well as of H 2 -2 to C-11 (δ C 26.1) and C-3 (Table 3 and Figure 7). This was also corroborated by the NOESY correlations of H 3 -11 to OH-3 (δ H 3.39, br), H 2 -2 and H-4 (Table 3 and Figure 7b, Supplementary Information, Figure S24). Therefore, Compound 3 was identified as 3-hydroxy-3-methyl-2,3-dihydro-1-benzoxepine-7-carboxylic acid. with the 2,3,6,7-tetrahydro-oxepine ring through C-5a and C-9a. That the methyl group and the hydroxyl group were on C-3 of the oxepin ring was confirmed by the HMBC correlations of the methyl singlet at δH 1.26 (H-11) to C-3 (δC 70.7), C-2 (δC 77.0) and C-4, as well as of H2-2 to C-11 (δC 26.1) and C-3 (Table 3 and Figure 7). This was also corroborated by the NOESY correlations of H3-11 to OH-3 (δH 3.39, br), H2-2 and H-4 (Table 3 and Figure 7b, Supplementary Information, Figure  S24). Therefore, Compound 3 was identified as 3-hydroxy-3-methyl-2,3-dihydro-1-benzoxepine-7-carboxylic acid.  As compound 3 could not be obtained as a suitable crystal for X-ray analysis, an effort to tentatively determine the relative configuration of the stereogenic carbon (C-3) by molecular mechanics conformation analysis and the NOESY experiments was carried out. Stochastic conformational search on the computational models of the structure of 3 with C-3 in R configuration, followed by energy minimization, converged to two half-chair conformations for the As compound 3 could not be obtained as a suitable crystal for X-ray analysis, an effort to tentatively determine the relative configuration of the stereogenic carbon (C-3) by molecular mechanics conformation analysis and the NOESY experiments was carried out. Stochastic conformational search on the computational models of the structure of 3 with C-3 in R configuration, followed by energy minimization, converged to two half-chair conformations for the seven-membered ring C1 and C2, as depicted in Figure 8, regardless of the modelling level of the theory used (MP2/6-311G, PM3, MMFF and MM2). All methods, except for PM3, also agree that conformation C2, with the methyl group in the equatorial position, is more stable by ca. 2 kcal/mol. However, this difference can be attributed to a weak intramolecular hydrogen bond in conformation C2, between HO-3 and O-1, which is not possible in conformation C1. The semi-empirical PM3 method gives less weight to non-ideal intramolecular hydrogen bonds, as compared to the other methods, and assigns virtually the same energy to both conformations of 3, while still orienting HO-3 towards the seven-membered ring. Since DMSO solvent molecules compete for HO-3 hydrogen bonding, it is more likely that the intramolecular bond is not an important feature of ring conformation C2 and that, in reality, both conformations have approximately the same energy. bonding, it is more likely that the intramolecular bond is not an important feature of ring conformation C2 and that, in reality, both conformations have approximately the same energy. Both model conformations of 3 predict hydrogen-hydrogen distances that are similar to within 0.2 Å , with the exception of some distances to the methyl group (H3-11). The most notable are to the diastereotopic hydrogens (H2-2), partially presented in Figure 8. While both conformations show almost the same distance between H-2a and H-11, a difference is predicted between H-2b and H-11 if the conformation C1 predominates, which should be apparent in the build-up rate of NOESY cross-peaks for small mixing times. Alternatively, the predominance of the conformation C2 would be indicated by two equal strength cross-peaks for H-2a and H-2b in cross-relaxation with H-11. It is observed that the H-2b (δH 3.84, d, J = 11.1 Hz)/H-11 NOESY cross-peak is weak while the H-2a (δH 4.02, dd, J = 11.1, 1.6 Hz)/H-11 is medium, suggesting that the conformation C1 predominates. NOE effective distances, reff, are calculated by [13] : The average effective positions of the three methyl H-11i protons are relative to H-2a or H-2b. The predicted ratio reff (H-2a/H-11)/reff (H-2b/H-11) is 1.40 (1.36, if r −3 averages are used instead of r −6 ). Assuming that the cross-relaxation rate is similar in both cases, the NOE intensities should also have a similar ratio [14]. Since the observed intensities ratio is actually closer to two, the evidence points Both model conformations of 3 predict hydrogen-hydrogen distances that are similar to within 0.2 Å, with the exception of some distances to the methyl group (H 3 -11). The most notable are to the diastereotopic hydrogens (H 2 -2), partially presented in Figure 8. While both conformations show almost the same distance between H-2a and H-11, a difference is predicted between H-2b and H-11 if the conformation C1 predominates, which should be apparent in the build-up rate of NOESY cross-peaks for small mixing times. Alternatively, the predominance of the conformation C2 would be indicated by two equal strength cross-peaks for H-2a and H-2b in cross-relaxation with H-11. It is observed that the H-2b (δ H 3.84, d, J = 11.1 Hz)/H-11 NOESY cross-peak is weak while the H-2a (δ H 4.02, dd, J = 11.1, 1.6 Hz)/H-11 is medium, suggesting that the conformation C1 predominates. NOE effective distances, r eff , are calculated by [13] : The average effective positions of the three methyl H-11 i protons are relative to H-2a or H-2b. The predicted ratio r eff (H-2a/H-11)/r eff (H-2b/H-11) is 1.40 (1.36, if r´3 averages are used instead of r´6). Assuming that the cross-relaxation rate is similar in both cases, the NOE intensities should also have a similar ratio [14]. Since the observed intensities ratio is actually closer to two, the evidence points towards the predominance of the conformation C1. Since, as stated previously, both conformations have similar conformational energy, the higher stability of 3R-C1 of 3 (or of its stereoisomer 3S-C2) must originate from the ready interaction of the equatorial HO-3 with the hydrogen-bonding solvent and also from a higher entropic rotational freedom of the hydroxyl and methyl groups. The NOESY spectrum also revealed the correlations of both H 2 -2 to H-9. However, only one of the H 2 -2, i.e., the doublet at δ H 3.84 (J = 11.1 Hz), showed a weak cross-peak to H-4, while the double doublet at δ H 4.02 (J = 11.1, 1.6 Hz) did not give any cross-peak to H-4. This observation led to the conclusion that the plucked oxepin ring should adopt the conformation in which H-2 at δ H 3.84 is near H-4, i.e., in the α (axial), while H-2 at δ H 4.02 is in β (equatorial) positions, which is in agreement with our conformational analysis. A literature search revealed that compound 3 is also a new compound, so we named it quadricinctoxepine.
Mar. Drugs 2016, 14,x FOR PEER REVIEW 19 of 29 the methyl group on C-1 in compound 6a was replaced by the hydroxymethyl group in compound 6b, it is legitimate to postulate that compounds 6b is derived from compound 6a. Consequently, the absolute configuration of C-2 of compound 6b should be the same as that of compound 6a, i.e., 2R. However, it is not yet possible to determine the absolute configuration of C-1′. Compound 6b is also a new compound, and we have named it quadricinctafuran B. Compound 7 was also isolated as white crystals (mp. 196-197 °C), and its molecular formula C14H12O5 was established on the basis of the (+)-HRESIMS m/z 237.0792 [M + H] + (calculated 237.0763), indicating seven degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3404 cm −1 ), conjugated ketone carbonyl (1708 cm −1 ), conjugated carboxyl carbonyl (1670 cm −1 ) and aromatic (1558, 1540 cm −1 ) groups. The 13 C NMR, DEPT and HSQC spectra (Table 7, Supplementary  Information, Figures S48 and S50)  Compound 7 was also isolated as white crystals (mp. 196-197˝C), and its molecular formula C 14 H 12 O 5 was established on the basis of the (+)-HRESIMS m/z 237.0792 [M + H] + (calculated 237.0763), indicating seven degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3404 cm´1), conjugated ketone carbonyl (1708 cm´1), conjugated carboxyl carbonyl (1670 cm´1) and aromatic (1558, 1540 cm´1) groups. The 13 C NMR, DEPT and HSQC spectra (Table 7, Supplementary  Information, Figures S48 and S50) exhibited the signals of one conjugated ketone carbonyl (δ C 191.1), one conjugated carboxyl carbonyl (δ C 166.4), one oxy-quaternary sp 2 (δ C 162.9), two quaternary sp 2 (δ C 123.0, 119.6), three methine sp 2 (δ C 136.4, 127.5, 118.5), one oxy-quaternary sp 3 (δ C 83.0), one oxymethylene sp 2 (δ C 66.8), one methylene sp 2 (δ C 43.4) and one methyl (δ C 21.2) carbons. The 1 H NMR spectra (Table 7, Supplementary Information, Figure S47) exhibited, besides the signals of three aromatic protons of the 1,2,4-trisubstituted benzene ring at δ H 8.26,d (J = 2.3 Hz),8.04,dd (J = 8.7,2.3 Hz),7.07,d (J = 8.7 Hz), a methyl singlet at δ H 1.30, two pairs of geminally-coupled methylene protons at δ H 2.74,d (J = 16.7 Hz)/3.00,d (J = 16.7 Hz) and δ H 3.49,dd (J = 11.6,4.5 Hz)/3.59,dd (J = 11.6,4.2 Hz); the latter showed COSY correlations with the broad triplet of the hydroxyl proton at δ H 5.26 (Table 7, Figure 16a, Supplementary Information, Figure S49). As the HMBC spectrum exhibited correlations of the aromatic proton signal at δ H 8.26,d (J = 2.3 Hz, to the oxy-quaternary sp 2 carbon at δ C 162.9 (C-8a), as well as to the conjugated ketone carbonyl carbon at δ C 191.1 (C-4) and the methine sp 2 at δ C 136.4 (C-7) (Table 7, Figure 16a, Supplementary Information, Figure S51), the ketone moiety was placed on C-4a. Additionally, the HMBC spectrum also showed correlations of H-8 (δ H 7.07, d, J = 8.7 Hz) to C-8a and the quaternary sp 2 carbons at δ C 123.0 and 119.6; they were therefore assigned to C-4a and C-6, respectively (Table 7, Figure 16a). The presence of the 1-hydroxy-2-methyl-2-oxypropyl moiety was supported by the HMBC correlations of the methyl singlet at δ H 1.30 (CH 3 -9) to the oxy-quaternary sp 3 carbon at δ C 83.0 (C-2), the oxymethylene sp 3 carbon at δ C 66.8 (C-10) and the methylene sp 3 carbon at δ C 43.4 (C-3), as well as of the H [2][3]d,J = 16.7 Hz/3.00,d,J = 16.7 Hz) to C-2, C-9 (δ C 21.2) and C-10 (Table 7, Figure 16a). Since H 2 -3 also gave a HMBC cross-peak to C-4, the 1-hydroxy-2-methyl-2-oxypropyl moiety was linked to C-4. Due to the fact that these two moieties accounted for only C 11 H 12 O 4 , it was concluded that the carboxyl group (δ C 166.4, δ H 12.69 br) was on C-6. Therefore, compound 7 was identified as 2-(hydroxymethyl)-2-methyl-4-oxo-3,4-dihydro-2H-chromene-6-carboxylic acid. As compound 7 was obtained as a suitable crystal for an X-ray diffraction, its X-ray analysis was carried out. The ORTEP view of compound 7, shown in Figure 17, revealed the absolute configuration for C-2 as 2S. A literature search revealed that compound 7 is also a new compound, so we named it quadricinctone D.  8,CH2 3.49,dd (11.6,4.5) OH- 10 3.59, dd (11.6, 4.2) 11 166.4, CO -OH- 10 -5.26, brt (5.4) -OH-11 -12.69, br -  Compounds 1 and 5 can be hypothesized as originating from the pentaketide intermediate (I). Methylation (by SAM) gives II, which undergoes cyclization and enolization to give the intermediate III. Methylation of the phenolic hydroxyl groups and the α-carbon of the carbonyl ketone leads to the intermediate IV. Enolization, followed by a lactonization, originates V, which undergoes hydration to give VI, and oxidation of one of the methyl groups gives rise to compound 5. Alternatively, oxidation of the α-carbon of the side chain of IV leads to the intermediate VII, which, after lactonization and reduction of the ketone carbonyl, gives rise to compound 1 ( Figure 18). In order to establish the conformation of the 2,3-dihydro-4H-pyran-4-one, analysis of the NOESY correlations was carried out. The NOESY spectrum (Table 7, Figure 16b, Supplementary Information, Figure S52) exhibited not only a strong correlation of H-7 to H-8, but also week correlations of H 3 -9 to H-5 and H-8. Therefore, CH 3 -9 is in the α-axial position. Additionally, since H 3 -9 also exhibited a strong cross-peak with the signal of the methylene proton at δ H 2.74, d (J = 16.7 Hz), and a weak cross-peak with the proton signal at δ H 3.00, d (J = 16.7 Hz), the former was assigned to H-3α and the latter to H-3β. It is interesting to observe that the structure of compound 7 is analogous to the structure of compound 3 in that it also exhibits the same methyl-methylene bridge structural feature (at C-9 and C-3), with the same relative intensity of NOESY cross-peaks between the protons of the two groups and with similar conformational energies for the two half-chair conformations of the non-aromatic ring. Therefore, the conclusions drawn for compound 3 are also valid for compound 7, whose stereochemistry is unequivocally defined by X-ray analysis.
Compounds 1 and 5 can be hypothesized as originating from the pentaketide intermediate (I). Methylation (by SAM) gives II, which undergoes cyclization and enolization to give the intermediate III. Methylation of the phenolic hydroxyl groups and the α-carbon of the carbonyl ketone leads to the intermediate IV. Enolization, followed by a lactonization, originates V, which undergoes hydration to give VI, and oxidation of one of the methyl groups gives rise to compound 5. Alternatively, oxidation of the α-carbon of the side chain of IV leads to the intermediate VII, which, after lactonization and reduction of the ketone carbonyl, gives rise to compound 1 (Figure 18 Biosynthetically, compounds 2a, 2b, 4a, 4b, 6a, 6b and 7 are of mixed origin, i.e., shikimic acid and mevalonic acid pathways, similar to that proposed for fomannoxin [19], as depicted in Figure 19. Elimination of pyruvate from chorismate (IX) by chorismate pyruvate lyase leads to the formation of p-hydroxybenzoic acid (X), which after prenylation by DMAPP (XI), originates the intermediate XII.
Epoxidation and cyclization of XII, via Route a, leads to a formation of the furan ring in compound 6a and after oxidation of one of the methyl groups leads to compound 6b. On the other hand, cyclization via Route b leads to the formation of the pyran ring in XIV. Dehydration and oxidation of one of the methyl groups originates compound 2a, which after acetylation of the primary alcohol function of the side chain will originate compound 2b. Alternatively, the intermediate XIV can also undergo dehydration, reduction and oxidation to give the ketone function in compound 7. Oxidative decarboxylation of compound 7 leads to the formation of compound 4a, which after sulfinylation of the benzene ring originates compound 4b. However, it is possible that the introduction of the methyl sulfoxide group to the aromatic ring could happen before cyclization. Biosynthetically, compounds 2a, 2b, 4a, 4b, 6a, 6b and 7 are of mixed origin, i.e., shikimic acid and mevalonic acid pathways, similar to that proposed for fomannoxin [19], as depicted in Figure 19. Elimination of pyruvate from chorismate (IX) by chorismate pyruvate lyase leads to the formation of p-hydroxybenzoic acid (X), which after prenylation by DMAPP (XI), originates the intermediate XII.
Epoxidation and cyclization of XII, via Route a, leads to a formation of the furan ring in compound 6a and after oxidation of one of the methyl groups leads to compound 6b. On the other hand, cyclization via Route b leads to the formation of the pyran ring in XIV. Dehydration and oxidation of one of the methyl groups originates compound 2a, which after acetylation of the primary alcohol function of the side chain will originate compound 2b. Alternatively, the intermediate XIV can also undergo dehydration, reduction and oxidation to give the ketone function in compound 7. Oxidative decarboxylation of compound 7 leads to the formation of compound 4a, which after sulfinylation of the benzene ring originates compound 4b. However, it is possible that the introduction of the methyl sulfoxide group to the aromatic ring could happen before cyclization. Compound 3 is also derived from a prenylation of p-hydroxybenzoic acid (X); however, it can occur with IPP (XVI) instead of DMAPP (XI). Epoxidation of the double bond of the side chain of XVII, followed by cyclization of XVIII leads to the formation of an oxepin ring in XIX. Oxidation and dehydration of the oxepin ring will lead to the formation of compound 3, as depicted in Figure 20. Compound 3 is also derived from a prenylation of p-hydroxybenzoic acid (X); however, it can occur with IPP (XVI) instead of DMAPP (XI). Epoxidation of the double bond of the side chain of XVII, followed by cyclization of XVIII leads to the formation of an oxepin ring in XIX. Oxidation and dehydration of the oxepin ring will lead to the formation of compound 3, as depicted in Figure 20.
Compounds 1-7 were evaluated for their antibacterial activity against Gram-positive and Gram-negative bacteria, as well as multidrug-resistant isolates from the environment, according to the previously described protocol [6], as well as for their antifungal activity against yeast (Candida albicans ATCC 10231), filamentous fungus (Aspergillus fumigatus ATCC 46645) and dermatophyte (Trichophyton rubrum FF5) in the antifungal assay [20]. The results showed that none of the tested compounds exhibited significant antibacterial activity (MIC > 256 µg/mL) or antifungal activity (MIC > 512 µg/mL). These compounds were also evaluated for their in vitro growth inhibitory activity against the MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma) cell lines by the protein binding dye SRB method [21], and they did not show any activity in this assay (GI 50 > 150 mM). Compounds 1-7 were evaluated for their antibacterial activity against Gram-positive and Gramnegative bacteria, as well as multidrug-resistant isolates from the environment, according to the previously described protocol [6], as well as for their antifungal activity against yeast (Candida albicans ATCC 10231), filamentous fungus (Aspergillus fumigatus ATCC 46645) and dermatophyte (Trichophyton rubrum FF5) in the antifungal assay [20]. The results showed that none of the tested compounds exhibited significant antibacterial activity (MIC > 256 μg/mL) or antifungal activity (MIC > 512 μg/mL). These compounds were also evaluated for their in vitro growth inhibitory activity against the MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma) cell lines by the protein binding dye SRB method [21], and they did not show any activity in this assay (GI50 > 150 mM).

General Procedure
Melting points were determined on a Bock monoscope and are uncorrected. Optical rotations were measured on an ADP410 Polarimeter (Bellingham + Stanley Ltd., Tunbridge Wells, Kent, U.K.). Infrared spectra were recorded in a KBr microplate in a FTIR spectrometer Nicolet iS10 from Thermo Scientific (Waltham, MA, USA) with the Smart OMNI-Transmission accessory (Software 188 OMNIC 8.3). UV spectra were taken in CHCl3 and were recorded on a Varian CARY 100 spectrophotometer. 1 H and 13 C NMR spectra were recorded at ambient temperature on a Bruker AMC instrument (Bruker Biosciences Corporation, Billerica, MA, USA) operating at 300. 13 and 75.4 MHz, respectively. High resolution mass spectra were measured with a Waters Xevo QToF mass spectrometer (Waters Corporations, Milford, MA, USA) coupled to a Waters Acquity UPLC system. A Merck (Darmstadt, Germany) silica gel GF254 was used for preparative TLC, and a Merck Si gel 60 (0.2-0.5 mm) was used for column chromatography.

General Procedure
Melting points were determined on a Bock monoscope and are uncorrected. Optical rotations were measured on an ADP410 Polarimeter (Bellingham + Stanley Ltd., Tunbridge Wells, Kent, U.K.). Infrared spectra were recorded in a KBr microplate in a FTIR spectrometer Nicolet iS10 from Thermo Scientific (Waltham, MA, USA) with the Smart OMNI-Transmission accessory (Software 188 OMNIC 8.3). UV spectra were taken in CHCl 3 and were recorded on a Varian CARY 100 spectrophotometer. 1 H and 13 C NMR spectra were recorded at ambient temperature on a Bruker AMC instrument (Bruker Biosciences Corporation, Billerica, MA, USA) operating at 300.13 and 75.4 MHz, respectively. High resolution mass spectra were measured with a Waters Xevo QToF mass spectrometer (Waters Corporations, Milford, MA, USA) coupled to a Waters Acquity UPLC system. A Merck (Darmstadt, Germany) silica gel GF 254 was used for preparative TLC, and a Merck Si gel 60 (0.2-0.5 mm) was used for column chromatography.

Extraction and Isolation
The strain KUFA 0081 was isolated from the marine sponge Clathria reinwardti, which was collected, by scuba diving at a depth of 15-20 m, from the coral reef at Samae San Island (12˝34 1 36.64" N 100˝56 1 59.69" E) in the Gulf of Thailand, Chonburi Province, in July 2013. The sponge was washed with 0.06% sodium hypochlorite solution for 1 min, followed by sterilized seawater 3 times and then dried on sterile filter paper, cut into small pieces (5ˆ5 mm) and placed on a malt extract agar (MEA) medium containing 70% seawater and 300 mg/L of streptomycin sulphate, then incubated at 28˝C for 7 days, after which the hyphal tips were transferred onto a slant MEA and maintained as pure culture for further identification. The fungus was identified as Neosartorya quadricincta (E. Yuill)