Reappraising the structures and distribution of metabolites from black aspergilli containing uncommon 2-benzyl-4H-pyran-4-one and 2-benzylpyridin-4(1H)-one systems

Abstract

To date, natural products containing 2-benzyl-4H-pyran-4-one and 2-benzylpyridin-4(1H)-one substructures have been encountered in relatively few fungi outside of the black aspergilli clade. While exploring the occurrence of these compounds among Aspergillus spp., it was determined that the structures of the unusual furopyrrols tensidols A and B (5 and 6) and JBIR-86 and JBIR-87 (9 and 10) were incorrect and should be reassigned as 2-benzyl-4H-pyran-4-ones (7, 8, 11e, and 12, respectively). The origin of the unique N-phenyl groups in the 2-benzylpyridin-4(1H)-ones nygerones A and B (1 and 2) was also examined and it was established that N-phenylamides added to the culture medium were suitable substrates for generating these metabolites; however, this phenomenon remained limited to a single fungus in our collection (Aspergillus niger ATCC 1015). A variety of 2-benzyl-4H-pyran-4-ones and 2-benzylpyridin-4(1H)-ones were detected among the black aspergilli, but only pestalamide B (13) was found in all eleven of the tested strains. These metabolites, as well as a group of synthetic analogues demonstrated weak antifungal activity against several Candida strains, Aspergillus flavus, and Aspergillus fumigatus.

Previously, we reported the discovery of the 2-benzylpyridin-4(1H)-one-containing metabolites nygerones A and B (1-2) from Aspergillus niger.^1-4 Subsequent ¹H NMR-guided efforts by our group to purify additional 2-benzylpyridin-4(1H)-one and related 2-benzyl-4H-pyran-4-one metabolites from A. niger yielded several compounds that we presumed were structurally similar to 1 and 2. However, during the course of dereplication studies, a variety of natural products possessing α-pyridone,⁵γ-pyrone,⁶ and furopyrrol⁷ skeletons were found in the literature and all of these compounds were reported to possess ¹H NMR chemical shifts that were virtually identical to the key diagnostic resonances in 1 and 2. Fortunately, the α-pyridone-containing variant had been previously examined by Ye and colleagues who successfully revised the proposed structure of aspernigrin A (3)⁵ as the more plausible 2-benzylpyridin-4(1H)-one shown for compound 4.⁸ Our analysis of the furopyrrol-containing molecules, which included tensidols A and B (5-6),⁷ led to the observation that these structures had arisen from a strikingly different interpretation of ¹H and ¹³C NMR data that were indistinguishable from the values reported for the 2-benzyl-4H-pyran-4-ones carbonarone A (7)⁶ and pestalamide A (8),⁹ respectively. In this report, we address the structure revision of the tensidols using a combination of NMR spectroscopy, ab initio carbon chemical shift calculations, and total synthesis. We have also analyzed the distribution of the 2-benzylpyridin-4(1H)-one and 2-benzyl-4H-pyran-4-one metabolites among black aspergilli, and explored the origins of the N-phenyl groups in 1 and 2, as well as investigated the biological activities of these compounds.

Results and Discussion

Structure Revision of the Furopyrrols

The tensidol family of metabolites, which includes 5-6, as well as JBIR-86 (9) and JBIR-87 (10),¹⁰ were reported from the fermentation broths of a marine isolate of A. niger and an unidentified Aspergillus sp., respectively. We were initially prompted to examine the spectroscopic data for 5 and 6 since the unusual furopyrrol skeleton in these structures was reportedly unprecedented.⁷ Focusing on 5, we noted several unusual chemical shift assignments that we could not reconcile. Namely, the ¹³C NMR data presented for C-2 (162.0), C-3 (178.0), C-3a (119.1), C-4 (164.3), C-5 (116.0), and C-6a (168.8) were inconsistent with our expectations for carbon resonances in isolated or fused furan and pyrrole systems (Supporting Information, Figure S1). At the same time, we purified a small molecule from A. niger whose MS and NMR data matched those reported for 5 (Supporting Information, Table S1); however, de novo structure characterization studies carried out by our group suggested that γ-pyrone 7 was a more reasonable alternative to consider. The molecular formula for 7 exhibited a low H:C ratio (0.85) which, along with the presence of ⁴J_H-C couplings in the HMBC data, might have played confounding roles in previous attempts to unambiguously assign the correct structure for this metabolite.^{11, 12} Further inspection of the literature demonstrated that nearly one year after the disclosure of 5 in 2006, Zhang and colleagues reported the alternative structure 7 from a marine isolate of Aspergillus carbonarius, although no mention of 5 was provided in their account.⁶ We propose that 2-benzyl-4H-pyran-4-one 7 represents the correct structure of this metabolite.

A similar set of circumstances surrounded compound 6, which was originally described as a methyl succinate derivative of 5.⁷ In this case, a 2008 report by Ding et al. unknowingly provided structure 8⁹ as a reasonable revision for compound 6. Our evaluation of both data sets confirmed that the ¹H and ¹³C NMR chemical shifts reported for the two compounds were identical and that 8 should be regarded as the correct structure of this metabolite.

In 2010, we encountered a new opportunity to further test our assertion that the furopyrrol metabolites should be revised as 2-benzyl-4H-pyran-4-ones. We noted that Takagi et al. had obtained what appeared to be two new furopyrrol-containing metabolites, JBIR-86 (9) and JBIR-87 (10);¹⁰ however, both compounds exhibited some problematic features. Among our initial concerns were the incongruous ¹³C NMR chemical shifts (Supporting Information, Figure S1) and presence of suspect ⁴J_H-C couplings within the furopyrrol ring system.¹⁰ Additionally, the spectrometric analysis of 9 and 10 was reported to have yielded HRESIMS data containing even-mass quasi-molecular ions at m/z 244.0986 [M + H]⁺ and 358.1286 [M + H]⁺, respectively. This would presumably necessitate the incorporation of an odd number of nitrogens in each metabolite. However, the published ¹³C NMR data for 9 clearly supported the presence of a γ-pyrone, thus eliminating candidate 11a from further consideration (Figure 1A). In addition, the observed chemical shifts for the methyl group in 9 (δ 52.8) suggested that it was attached to an oxygen atom, not the N-methyl amide shown for candidate 11b (Figure 1A). These restrictions left open the possibilities that the revised structure contained a primary ketimine (11c) or methyl imidate (11d) (Figure 1A), but both candidate compounds were also rejected based on biosynthetic considerations and evaluations of their respective predicted ¹³C NMR chemical shift data (Supporting Information, Figure S2).

Figure 1.

A) Candidate structures (11a-11e) evaluated as alternatives during the revision of 9. B) Mean average errors (MAE) were caluculated using Σ(δ_calc − δ_exp)/#. A smaller MAE value represents better overall agreement between the computational and published ¹³C-NMR data. C) ¹H→¹³C HMBC correlations observed for synthetic 11e.

With no plausible nitrogen-containing candidate structures left to consider for 9, we focused on formulating a reasonable alternative structure from the NMR data. This approach led us to propose 11e as a candidate structure (Figure 1A). We proceeded to test this hypothesis using gauge-including atomic orbital based ¹³C NMR chemical shift calculations on both 9 and 11e. Density functional theory calculations performed at the b3lyp/6-31+g(d,p) level yielded a poor mean absolute error value of 16.0 ppm for 9, but a reasonable value of 3.8 ppm for 11e (Figure 1B). Figure 2 illustrates that structure 9 exhibited several pronounced deviations in the calculated carbon NMR values for positions C-2, C-3, C-3a, C-4, C-5, and C-6a from the published data (Δ_δcalc-δexp −46.5, −20.0, −27.0, −41.1, −25.8, and −20.6 ppm, respectively). In contrast, 11e showed promise as a revised candidate structure with much less deviation in the predicted versus observed ¹³C NMR data for each of the corresponding carbon atoms (Δ_δcalc-δexp −5.8, −2.3, 6.6, −7.1, −2.7 and −1.7 ppm, respectively).

Figure 2.

Deviations in the calculated versus observed ¹³C NMR chemical shifts for 9 and 11e. Differences approaching zero demonstrate good agreement between calculated versus reported ¹³C NMR data, whereas large differences indicate poor agreement between calculated and reported ¹³C NMR experimental data.

We further tested the prospect of revising 9 to candidate structure 11e by engaging in its total synthesis. We prepared 11e using a modification of the procedure reported by McCombie et al. (Scheme 1).¹³ Upon purification of the product, HRESIMS yielded a quasi-molecular ion at m/z 245.0815 [M + H]⁺ (calcd for C₁₄H₁₃O₄, 245.0814). The ¹H and ¹³C NMR data of synthetic 11e was remarkably consistent with each of the chemical shift values reported for 9 (Table 1). Moreover, both the previously published and new ¹H→¹³C HMBC data that we examined showed that all but one of the observed heteronuclear couplings (H-5→C-3) were now accounted for as two-to-three bond correlations (Figure 1C). We are still unable to provide an explanation for the disagreement between the ESIMS data published by Takagi et al. and our own results. However, in light of the evidence, we are quite confident that 11e represents the correct structure for JBIR-86. Based on this finding, we also reevaluated compound 10. Upon inspection of its ¹H, ¹³C, and ¹H→¹³C HMBC NMR data (Supporting Information, Table S1 and Figure S3), reconsideration of its probable shared biosynthetic origins with 11e, and co-isolation of compound 8 from the same crude extract, we have concluded that 10 should be revised to the 2-benzyl-4H-pyran-4-one-containing structure 12.

Scheme 1.

Synthesis of 11e.

Table 1.

Comparisons of the observed ¹H and ¹³C NMR data for 11e versus the values reported for 9.

	Synthetic 11e		9a
Position	δ H	δ C	δ H	δ C
2	8.41, s	162.0	8.43, s	162.0
3a		119.3		119.1
4		175.0		175.0
5	6.19, s	116.9	6.21, s	117.3
6a		167.4	167.6
7	3.81, s	39.7	3.83, s	40.0
8		133.9		134.1
9,13	7.22, m	129.1	7.24, m	129.4
10,12	7.35, m	129.1	7.32, m	129.4
11	7.32, m	127.8	7.32, m	128.0
3		163.6		163.9
3-OCH3	3.85, s	52.7	3.87, s	52.8

^aData reproduced from Takagi, M.; Motohashi, K.; Hwang, J.-H.; Nagai, A.; Kazuo, S. J. Antibiotics. 2010, 63, 371-373.

Distribution of 2-Benzylpyridin-4(1H)-Ones and 2-Benzyl-4H-Pyran-4-Ones among Black Aspergilli

To date, the majority of secondary metabolites bearing 2-benzylpyridin-4(1H)-one and 2-benzyl-4H-pyran-4-one skeletons have been reported from black aspergilli.^{14, 15} Given our previous encounter with this family of metabolites from A. niger, as well as our desire to better understand its distribution among the members of this clade, we prepared a focused library of 11 Aspergillus strains for further investigation. The fungi were grown with and without epigenetic modifiers^2-4 and the extracts examined by LC-ESIMS. By probing extracted-ion chromatograms for each of the target compounds (1, 2, 4, 7, 8, and 13), we were able to determine which black aspergilli were producers of the 2-benzylpyridin-4(1H)-one and 2-benzyl-4H-pyran-4-one metabolites under our culture conditions (Supporting Information, Table S2). Interestingly, the 2-benzylpyridin-4(1H)-one-containing compound pestalamide B (13)⁹ was the only metabolite detected in all 11 of the black aspergilli strains (note – the ¹³C NMR data provided for 13 by Ding et al. have been revised¹⁶). In contrast, the 2-benzyl-4H-pyran-4-one-containing analog of 13, compound 8, was observed in a much more restricted group of five fungal strains and in some cases it was detected only under chemical epigenetic induction conditions. The unsubstituted amide analogs of 13 and 8, metabolites 4 and 7, respectively, were also found in relatively few fungi (two and four strains, respectively). At this time, the role of methyl succinate conjugation of these metabolites is unknown.

We were struck by the limited distribution of compound 1, which was detected only in A. niger ATCC 1015 treated with suberoylanilide hydroxamic acid (SAHA) (Supporting Information, Table S2). The origin of the N-phenyl group in 1 is an intriguing feature of this metabolite and its biosynthetic source was not readily apparent to us. Our original investigation of 1,¹ including follow-up feeding studies involving the incorporation of aniline in the fungal growth medium did not support the idea that the phenyl ring of SAHA was the N-phenyl group precursor. To further verify the role SAHA plays in the production of nygerones, a subsequent test employing p-fluoro SAHA (Supporting Information, Scheme S1A) was used as the eliciting agent. The addition of p-fluoro SAHA resulted in the production of p-fluoro nygerone B (14) from A. niger ATCC 1015 (Supporting Information, Table S1). This led us to use p-fluoro aniline for further feeding studies (Supporting Information, Scheme S2), but we did not observe any production of 14 when the substrate was administered alone or with SAHA present (Supporting Information, Table S1B and S1C). These data indicated that aniline was not a suitable substrate for generating the N-phenyl group in 1. We next considered the possibility that an acetanilide precursor might be utilized in establishing the N-phenyl group in compound 1. Accordingly, we prepared p-fluoro acetanilide (Supporting Information, Scheme S1D and S1E) from p-fluoro aniline and acetyl chloride and added it alone or with SAHA to the growth medium. Under both conditions, 14 was obtained, which allowed us to purify the compound and confirm its structure using ESIMS, ¹H, ¹³C, and ¹⁹F 1D and 2D NMR (Supporting Information, Table S1). These data indicate that acetanilides and SAHA are suitable precursor substrates for generating the N-phenyl group of the nygerones.

Evaluation of Antimicrobial Activity

In light of the sparse biological activity data reported for this family of metabolites, we prepared a group of several naturally occurring and synthetic γ-pyrone. and γ-pyridone-containing compounds for investigation (1, 8, 11e, and 14). We also took this opportunity to make 4-oxo-6-phenyl-4H-pyran-3-carboxylic acid (15) and associated ester derivatives (16, 17) using the synthetic approach presented in Scheme 1 (Supporting Information, Scheme S2 and Table S1). None of the compounds exhibited any inhibitory effects against bacteria (Acinetobacter baumannii ATCC 19606, A. baumannii ATCC BAA-1605, Burkholderia cepacia ATCC 25608, Enterobacter cloacae ATCC 13047, Escherichia coli ATCC 10798, E. coli ATCC 11775, Pseudomonas aeruginosa ATCC 10145, Klebsiella pneumonia ATCC 33495, K. pneumonia ATCC 51503, Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 25923, and S. aureus ATCC 700787) at a concentration of 100 μg/mL.

Previously, Fukuda et al.⁷ and Takagi et al.¹⁰ had reported that tensidols (7, 8, 11e, and 12) were capable of potentiating the activity of a sub-inhibitory concentration of miconazole (0.06 μM) against Candida albicans in a disk diffusion assay. Our attempts to confirm this activity in a broth microdilution assay against C. albicans ATCC 38245 failed to detect any imidazole potentiation. However, we did observe that compounds 11e, 16, and 17 provided complete growth inhibition against five Candida spp. (C. albicans, ATCC 38245, Candida glabrata NRRL Y-65, Candida krusei ATCC 27803, Candida parapsilosis ATCC 12969, and Candida tropicalis ATCC 12968) and two non-black aspergilli (Aspergillus fumigatus NRRL A1100 and Aspergillus flavus NRRL A1120) at a concentration of 100 μg/mL. At 10 μg/mL, 11e was only capable of inhibiting A. flavus growth, whereas 16 and 17 exhibited broader ranges of activities against C. krusei, A. flavus, and A. fumigatus at this reduced concentration.

EXPERIMENTAL SECTION

General Experimental Procedures

All solvents were of ACS grade or better. NMR data were obtained on Varian 300, 400, and 500 spectrometers and processed using VNMRj software. LC-ESIMS data were collected using a Thermo-Finnigan Surveyor LC system and a Finnigan LCQ Deca mass analyzer. HRESIMS data were obtained by electrospray ionization employing an Agilent 6538 UHD Accurate-Mass Quadrupole TOF mass analyzer. Initial separations were performed by normal phase flash chromatography using a Biotage Isolera system. Further isolation and purification was performed using preparative and semi-preparative Shimadzu HPLC systems with Phenomenex C₁₈ Gemini columns.

Density Functional Theory Calculations (DFT)

Full geometry optimization and carbon chemical shift calculations for the proposed 11e and JBIR-86 (9) were performed on eight nodes of the Pentium-4 Xeon54 Quad Core Linux Cluster (running Red Hat Linux) using Gaussian03. Geometry optimization with frequency check and GIAO methods for determining carbon chemical shifts were performed using the basis set b3lyp/6-31+g(d,p). DFT calculations of tetramethylsilane (TMS) were also performed to determine the carbon chemical shift to be used as a reference. Carbon chemical shifts were referenced by subtracting the calculated δ_C of each carbon from the calculated δ_C of TMS. Analysis of results was performed by comparing the calculated chemical shifts with the published chemical shifts of JBIR-86.

Synthesis of 3-carbomethoxy-6-benzyl-4-pyrone (11e)

The general scheme for syntheszing 11e followed the methodology developed by McCombie et al.¹³ The enaminone, methyl 2-[(dimethylamino) methylene]-3-oxobutanonte, was synthesized as described with the application of methyl acetoacetate as a starting material instead of ethyl acetoacetate. Methyl acetoacetate (5.4 mL, 50 mmol) was added to the DMF-Me₂SO₂ adduct in CH₂Cl₂ (75 mL) at 0 °C and triethylamine (10 mL) was added slowly over 3 min. The mixture was stirred for 2 h at room temperature, washed with aqueous tartaric acid (10% w/v, 100 mL) and H₂O (100 mL × 2), and dried over anhydrous MgSO₄. The sample was gravity filtered (Whatman #1 filter paper) and the solvent from the filtrate was evaporated under vacuum. The crude material was subjected to silica gel flash chromatography using a CHCl₃-EtOAc gradient (100:0 to 0:100). The solvent was evaporated from the enaminone under vacuum and further purified by flash chromatography using a hexane/EtOAc gradient (90:10 to 0:100). The solvent was dried to give the white solid product (Z)-methyl 2-((dimethylamino)methylene)-3-oxobutanoate.

To construct the pyrone ring, a solution of hexamethldisilazine (1 mL) in THF (6 mL) was stirred at −78 °C and n-BuLi in hexane (3 mL of a 1.6 M solution) was added slowly over 2 min. After the mixture cooled, a solution of the enaminone ((Z)-methyl 2-((dimethylamino)methylene)-3-oxobutanoate) (2 mmol) and phenyl acetyl chloride (2.4 mmol) in THF (5 mL) was added over 1 min. The dry ice/acetone bath was removed and the solution was allowed to warm for 5 min. Diethyl ether (20 mL) was added to the reaction, followed by 3 N HCl (8 mL). The solution was stirred rapidly overnight. The following day, the organic phase was washed with saturated sodium bicarbonate (100 mL) and H₂O (100 mL × 2) then dried over anhydrous MgSO₄. The sample was gravity filtered (Whatman #1 filter paper) and the filtrate dried under vacuum. The residue was subjected to silica gel flash chromatography using a CH₂Cl₂/EtOAc gradient (100:0 to 0:100). Final purification was performed by preparative HPLC using a MeOH/H₂O gradient (50:50 to 100:0). The resulting product appeared as a red-brown amorphous solid (16 mg, 6%); ¹H (CD₃OD, 400 MHz) δ_H 8.45 (1H, s, H-2), 7.32 (3H, m, H-10, H-11, H-12), 7.24 (2H, m, H-9, H-13), 6.22 (1H, s, H-5), 3.88 (3H, s, 14-OCH₃), 3.84 (2H, s, H-7); ¹³C (CD₃OD, 100 MHz) δ_C 175.1 (C-4), 167.7 (C-6), 163.9 (C-14), 161.8 (C-2), 134.0 (C-8), 129.4 (C-9, C-13), 129.3 (C-10, C-12), 128.0 (C-11), 119.7 (C-3), 117.2 (C-5), 52.8 (14-OCH₃), 39.72 (C-7); HRESIMS m/z 245.0815 [M + H]⁺ (calcd for C₁₄H₁₃O₄, 245.0814).

LC-ESIMS analysis of extracts from black aspergilli grown under chemical epigenetic induction conditions

Freeze-dried spores from 11 strains of black aspergilli (Aspergillus aculeatus NRRL 5094, Aspergillus basiliensis NRRL 35542, A. carbonarius NRRL 346, A. carbonarius NRRL 369, Aspergillus ellipticus NRRL 5120, Aspergillus ibericus NRRL 35644, Aspergillus japonicus NRRL 2053, A. niger NRRL 326, A. niger NRRL 1015, Aspergillus parasiticus NRRL 465, and Aspergillus tubingensis NRRL 4875) were obtained from the ARS mycology collection. All strains were cryopreserved in 15% (vol/vol) glycerol solution at −80 °C. Cryopreserved samples were removed and plated on potato-dextrose agar and grown for 14 d at 25 °C. Hyphae from each strain were suspended in sterile H₂O and used to inoculate three 1 L Erlenmeyer flasks containing 250 mL of sterile potato-dextrose broth. Each culture flask was treated with either 50 μM suberoylanilide hydroxamic acid (dissolved in 50:50 DMSO/H₂O), 50 μM 5-azacytidine (dissolved in 50:50 DMSO/H₂O), or bolus of 50:50 DMSO/H₂O. Cultures were grown for 21 d at room temperature on a rotary shaker at 150 rpm. After 21 d, 2% (wt/vol) Diaion HP-20 resin was added to the flasks, which were further shaken for 4 h, followed by filtration of the cell mass and resin. The cell mass and resin from each flask was extracted in 250 mL aliquots of MeOH over a 48 h period (2×). The MeOH extracts were combined and solvent removed by rotary evaporation.

Extracts were prepared for LC-ESIMS analysis by suspending samples in a minimal amount of MeOH and adsorbing the solution onto C₁₈. The dried C₁₈ stationary phases loaded with samples were then placed in SPE cartridges and rinsed with 5 mL of deionized H₂O followed by 5 mL of MeOH (each collected separately on a vacuum manifold). The MeOH fractions were evaporated in pre-weighed vials. The vials which contained the dried material from the MeOH fractions were weighed and the dried material was resuspended in a 90:10 MeOH/H₂O solution at a concentration of 20 mg/mL. A portion of each sample was then transferred to a 2 mL Eppendorf tube and centrifuged at 14,000 rpm for 5 min. The supernatants were placed in vials for LC-ESIMS analysis using a MeOH/H₂O gradient (25:75 to 100:0). The mass detector was set to scan a range from m/z 120-1500 in positive mode.

Synthesis of p-fluoro SAHA

Suberic acid monomethyl ester (2.01 g, 10.7 mmol), p-fluroraniline (1.42 g, 12.8 mmol), and hydroxybenzotriazole (1.73 g, 12.8 mmol) were dissolved in 25 mL of DMF. To this solution, diisopropylcarbodiimide (1.62 g, 12.8 mmol) was added and the reaction mixture was stirred at room temperature for 3 h. The reaction was quenched by pouring the mixture over 450 mL of ice stirred in H₂O. After the ice had melted, the resulting precipitate was filtered using vacuum assisted filtration, followed by purification via flash silica chromatography using a hexane/EtOAC gradient. The procedure provided the suber-p-fluoroanilic acid methyl ester in 82% yield (2.47 g). Fresh methanolic hydroxyl amine was prepared by dissolving hydroxylamine hydrochloride (9.43 g, 135.6 mmol) in MeOH (25 mL) followed by the addition of KOH (7.60 g, 135.6 mmol). The mixture was allowed to stir under an atmosphere of nitrogen in an ice bath to aid the precipitation of the resulting salt, which was subsequently removed by filtration. The filtrate was transferred to a round bottom flask and a solution of suber-p-fluoroanilic acid methyl ester (2.07 g, 7.36 mmol) in 10 mL of MeOH was added. This reaction mixture was stirred under nitrogen at 50 °C for 12 h. The reaction was quenched via dilution in MeOH with ice H₂O (250 mL). The resulting aqueous solution was neutralized with acetic acid resulting in the precipitation of p-fluoro SAHA. The white precipitate was filtered and evaporated under vacuum to provide an overall product yield of 61%. Analysis of the product by ¹H, ¹³C, ¹⁹F NMR revealed that the p-fluoro SAHA was obtained in high chemical purity without need for further purification. ¹H ((CD₃)₂SO, 500 MHz) δ_H 10.35 (1H, br s, N-H-12), 9.91 (1H, br s, N-H-2), 7.60 (2H, m, H13, H-17), 7.10 (2H, m, H14, H-16), 2.27 (1H, t, J = 7.5 Hz, H-9), 1.93 (2H, t, J = 7.5 Hz, H-4), 1.55 (2H, m, H-8), 1.48 (2H, m, H-5), 1.26 (4H, m, H-6 and 7); ¹³C ((CD₃)₂SO, 125 MHz): δ 171.7 (C-10), 169.8 (C-3), 159.6 (C-15), 136.3 (C-12), 121.1 (C-13, C-17), 115.7 (C-14, C-16), 36.7 (C-9), 32.8 (C-4), 28.9 (C-6, C-7), 25.5 (C-5, C-8); ¹⁹F ((CD₃)₂SO, 282 MHz) δ_C −119.96 (F-15); HRESIMS m/z 305.1274 [M + Na]⁺ (calcd for C₁₄H₁₉FNaO₃, 305.1277).

Isolation and purification of p-fluoro nygerone B (14)

A cryopreserved sample of A. niger ATCC 1015 was plated on potato-dextrose agar and grown for 14 d at 25 °C. Hyphae were suspended in sterile H₂O and used to inoculate a set of 20 Erlenmeyer flasks (1 L) each containing 250 mL of sterile potato-dextrose broth. Each flask was treated with 50 μM p-fluoro suberoylanilide hydroxamic acid (p-F-SAHA) (dissolved in 50:50 DMSO/H₂O). Controls were created by inoculating four 1 L Erlenmeyer flasks containing 250 mL of sterile potato-dextrose broth and 0.5% (vol/vol) of 50:50 DMSO/H₂O. Cultures were grown on a rotary shaker (150 rpm) for 14 d at room temperature and the combined culture broth were partitioned (3×) against an equivalent volume of CH₂Cl₂. The organic phase was collected and evaporated under vacuum. Treated and control extracts were analyzed by LC-ESIMS as described above. Further fractionation was performed on the p-F-SAHA culture extract by preparative HPLC using a MeOH/H₂O gradient (35:65 to 100:0) with final purification being performed by semi-preparative HPLC to yield 2 mg of light-yellow amorphous solid; ¹H (CDCl₃, 400 MHz) δ_H 9.89 (1H, br s, N-H_b-15), 8.49 (1H, s, H-2), 7.23 (1H, m, H-11), 7.21 (2H, m, H-10, H-12), 7.10 (2H, m, H-3′, H-5′), 7.05 (2H, m, H-2′, H-6′), 6.83 (2H, m, H-9, H-13), 6.61 (1H, s, H-5), 6.06 (1H, br s, N-H_a-15), 3.71 (2H, s, H-7); ¹³C (CDCl₃, 100 MHz) δ_C 177.7 (C-4), 166.0 (C-14), 164.1 (C-4′), 162.0 (C-1′), 151.3 (C-6), 146.9 (C-2), 134.8 (C-8), 128.9 (C-2′, C-6′, C-10, C-12), 128.5, C-9, C-13), 127.7 (C-11), 121.9 (C-5), 117.7 (C-3), 116.9 (C-3′, C-5′), 39.74 (C-7); ¹⁹F (CDCl₃, 376 MHz) δ −112.36 (F-11); HRESIMS m/z 323.1192 [M + H]⁺ (calcd for C₁₉H₁₆FN₂O₂, 323.1196).

3-carboethoxy-6-phenyl-4-pyrone (16)

Compound 16 was prepared and purified based on a previously reported method.¹³ The resulting product was a red crystalline solid; ¹H-NMR was in agreement with the literature;¹³ HRESIMS m/z 245.0816 [M + H]⁺ (calcd for C₁₄H₁₃O₄, 245.0814).

3-carbomethoxy-6-phenyl-4-pyrone (17)

Compound 17 was prepared and purified based on a previously reported method,¹³ with the exception that methyl acetoacetate was utilized instead of ethyl acetoacetate. The resulting product was a red crystalline solid; ¹H (CD₃OD, 500 MHz) δ_H 8.82 (1H, s, H-2), 7.89 (2H, m, H-8, H-12), 7.55 (2H, m H-10), 7.53 (2H, m, H-9, H-11), 6.94 (1H, s, H-5), 3.86 (3H, s, H-14); ¹³C (CD₃OD, 125 MHz) δ_C 175.6 (C-4), 166.0 (C-2), 164.1 (C-6), 163.0 (C-13), 130.8 (C-9, C-10, C-11), 129.6 (C-7), 126.2 (C-8, C-12), 118.3 (C-3), 112.4 (C-5), 52.3 (C-14); HRESIMS m/z 231.0659 [M + H]⁺ (calcd C₁₃H₁₁O₄, 231.0657).

3-carboxylic-6-phenyl-4-pyrone (15)

A 10 mg sample of 17 was reacted with 1 N NaOH in distilled THF for 3 h at room temperature then adjusted to pH 3 with 1 N HCl. The resulting solution was partitioned against EtOAc and the solvent removed under vacuum. Purification was performed by preparative HPLC yielding 6.2 mg of a yellow amorphous solid. ¹H ((CD₃)₂CO, 500 MHz) δ_H 9.88 (1H, s, H-2), 8.04 (2H, m, H-8, H-12), 7.66 (1H, m, H-10), 7.60 (2H, m, H-9, H-11), 6.95 (1H, s, H-5); ¹³C ((CD₃)₂CO, 125 MHz) δ_C 193.2 (C-2), 178.5 (C-4), 167.1 (C-6), 161.7 (C-13), 132.8 (C-10), 130.4 (C-7), 129.2, (C-9, C-11), 126.7 (C-8, C-12), 100.6 (C-3), 97.6 (C-5); HRESIMS m/z 215.0358 [M + H]⁻ (calcd for C₁₂H₇O₄, 215.0350).

Pestalamide A (8)

¹H- and ¹³C-NMR were in agreement with published values;⁹ HRESIMS m/z [M + Na]⁺ 366.0956 (calcd for C₁₈H₁₇NO₆Na, 366.0954).

Biological Assays

Twelve bacteria and seven fungi were used in the antimicrobial assays. Stock cultures of strains were delivered into presterilized 96-well plates, and compounds prepared in DMSO were added to each well of the 96-well plate (final DMSO concentration of 1%). Plates were shaken orbitally for 5 s and an initial OD₆₀₀ value recorded. Bacteria and fungi in 96-well plates were incubated for 24 h and 48 h, respectively, in a humidified incubator at 37 °C. Plates were removed, orbitally shaken, and a final OD₆₀₀ value recorded. The final OD₆₀₀ reading was subtracted from the initial OD₆₀₀ reading to obtain the change in OD₆₀₀ (antimicrobial activity was determined by the change in optical density). Chloramphenicol and ketoconazole were used as antibacterial and antifungal positive controls, respectively.