Arenicolins: C-Glycosylated Depsides from Penicillium arenicola

Abstract

During investigation of the secondary metabolism of four strains of Penicillium arenicola, two new depsides, arenicolins A (1) and B (2), were isolated and characterized. Their structures were established mainly by analysis of NMR and HRMS data and by comparison with known compounds. These depsides incorporate intriguing structural features, including dual alkyl side chains and a C-glycosyl unit, with 1 also containing an acylated 2-hydroxymethyl-4,5,6-trihydroxycyclohexenone moiety. Although the arenicolins were produced by all strains tested, arenicolin A (1) was obtained using only one of five medium compositions employed, while arenicolin B (2) was produced in all media tested. Neither compound showed antibacterial or antifungal activity, but 1 exhibited cytotoxicity toward mammalian cell lines, including colorectal carcinoma (HCT-116), neuroblastoma (IMR-32), and ductal carcinoma (BT-474), with IC₅₀ values of 7.3, 6.0, and 9.7 μM, respectively.

Graphical Abstract:

Penicillium arenicola (Eurotiales, Aspergillaceae) is a soil-dwelling fungus known from conifer forests in Canada and Ukraine.^1–3 P. arenicola has been long recognized as an atypical Penicillium species because of golden-brown pigments and septate, terverticillate conidiophores.^1,2 Phylogenetic analyses have now shown that P. arenicola is not a true Penicillium species and is phylogenetically close to Phialomyces macro-sporus.^2,3 However, at the time of writing, P. arenicola has yet to be formally reclassified as a species of Phialomyces; therefore, we are obliged to continue to use the name Penicillium arenicola.

A strain of P. arenicola (NRRL 8095) originally obtained from soil from British Columbia was described by investigators at the Swiss pharmaceutical company Sandoz as the producer of an echinocandin-type antifungal compound known as acrophiarin, or antibiotic S31794/F-1.^4,5 Reports of other secondary metabolites from P. arenicola include the γ-butyrolactone canadensolide, which was observed to inhibit germination of conidia of the plant pathogen Botryotinia fuckeliana,⁶ and mycophenolic acid from strain BICC 7673.⁷ During investigations on the production and biosynthesis of acrophiarin by different strains of P. arenicola (Table 1), LC-MS analysis of organic extracts indicated that these strains exhibited a rich secondary metabolism. Therefore, we further investigated some of the prominent metabolites. From extracts of P. arenicola strains grown on different media, we obtained two new depsides that we have named arenicolin A (1) and arenicolin B (2). Their structures show distinctive features, including a C-glycosyl unit, dual alkyl side-chains, and, in the case of 1, an acylated 2-hydroxymethyl-4,5,6-trihydroxy-cyclohexenone unit as a structural component. In this paper, we describe the isolation, structure elucidation, and biological evaluation of these two new highly modified depsides from P. arenicola.

Table 1.

Strains of Penicillium arenicola Examined in This Work and Their Geographic Origins

Strain number	habitat	geographic origin
NRRL 8095	soil	British Columbia, Canada
NRRL 3392a	soil, pine forest	near Kiev, Ukraine
NRRL 31507	mineral soil, under Pinus resinosa	Ontario, Canada
NRRL 31509	oil-soaked soil	Norman Wells, NW Territories, Canada

^aType strain.

RESULTS AND DISCUSSION

Initial LC-MS analysis of all the P. arenicola strains grown in medium I (see Experimental Section) showed the presence of two metabolites with similar UV data (λ_max at 218, 273, and 310 nm), but with different negative ion ESI mass spectra (m/z 803 and 647 for 1 and 2, respectively). The production of 1 was not observed when P. arenicola strains were tested in four other growth media, although compound 2 was detected in all four strains in all the media tested. Fractionation of the extracts by flash chromatography and further purification by semipreparative HPLC allowed isolation of 1 and 2.

Compound 1 was isolated as an off-white solid, with the molecular formula C₄₁H₅₆O₁₆ assigned on the basis of negative ion HRESIMS analysis. ¹H, ¹³C, and multiplicity-edited HSQC NMR data for 1 (Table 2) showed the presence of three carbonyls (one α,β-unsaturated ketone and two carboxyl carbons), 14 olefinic or aromatic carbons, eight oxymethines, two oxymethylenes, 12 other methylenes, and two methyl groups. ¹H and HSQC NMR data revealed the presence of two doublets corresponding to aromatic meta-coupled protons, an aromatic proton singlet, and an olefinic doublet of triplets (δ_H 7.07, J = 5.6, 1.7 Hz), correlating to four of the sp² carbons. These data, along with the remaining 10 (nonprotonated) aromatic/olefinic ¹³C NMR signals, were consistent with one 1,2,3,5-tetrasubstituted and one pentasubstituted aromatic ring, with both rings being meta-dioxygenated (based on ¹³C NMR shifts), as well as a trisubstituted double bond. These units left 10 exchangeable protons to be accounted for in the molecule. COSY and HMBC correlations (Figure 1) observed among the 12 nonoxygenated methylenes and the two methyl groups indicated the presence of two n-heptyl side-chains. HMBC correlations of the most deshielded methylene groups of each of these chains allowed the location of one such chain on each of the aromatic rings. Specifically, H₂-9 correlated with C-7 and H₂-8 correlated with C-2, C-6, and C-7, while H₂-9′ and H₂-8′ showed analogous correlations with C-2′, C-6′, and C-7′. The observation of strong HMBC correlations of H-6 to C-2, C-4, and C-8, from H-4′ to C-3′, C-5′, and C-6′, and from H-6′ to C-2′, C-4′, and C-8′, together with weak correlations from H-6 to C-1 and from H-4′ and H-6′ to C-1′, along with ¹³C NMR shift considerations, established the presence of two similarly decorated aromatic rings each bearing a 1-carboxy-2,4-dioxy-6-n-heptyl substitution pattern.

Table 2.

NMR Spectroscopic Data for 1 (¹H 500 MHz, ¹³C 125 MHz, CD₃OD)

position	δC, type	δH, mult. (J in Hz)	HMBC
1	170.8, C
2	105.8, C
3	164.3, C
4	110.9, C
5	163.0, C
6	112.5, CH	6.36, s	1w, 2, 4, 5, 8, 15w
7	148.1, C
8	37.8, CH2	a: 2.90, m; b: 2.87, m	2, 6, 7
9	33.1, CH2	1.64, m	7
10	33.0, CH2	1.35, m
11	32.9, CH2	1.25, m
12	30.8, CH2	1.29, m
13	23.7, CH2	1.27, m
14	14.5, CH3	0.88, t (6.9)
15	76.2, CH	4.93, d (10.0)	3, 4, 5, 16, 17, 19
16	73.2, CH	4.01, dd (8.9, 10.0)	4, 15, 17
17	80.0, CH	3.47, m
18	71.7, CH	3.47, m
19	82.6, CH	3.40, ddd (2.1, 5.2, 9.9)
20	62.7, CH2	a: 3.85, dd (2.1, 12.0); b: 3.75, dd (5.2, 12.0)	18, 19
1′	169.8, C
2′	115.3, C
3′	161.4, C
4′	109.0, CH	6.66, d (2.2)	1′, 2′, 3′, 5′, 6′
5′	154.6, C
6′	116.0, CH	6.62, d (2.2)	1′, 2′, 4′, 5′, 8′
7′	148.8, C
8′	36.1, CH2	a: 2.93, m; b: 2.86, m	2′, 6′, 7′
9′	33.5, CH2	1.64, m	7′, 8′
10′	32.3, CH2	1.29, m
11′	30.4, CH2	1.28, m
12′	23.8, CH2	1.33, m
13′	31.0, CH2	1.34, m
14′	14.5, CH3	0.85, t (7.0)
15′	194.0, C
16′	77.6, CH	5.94, d (10.7)	1′, 15′, 17′, 18′
17′	71.4, CH	4.13, dd (3.9, 10.7)	15′, 16′, 18′
18′	67.2, CH	4.56, ddd (0.9, 3.9, 5.6)	16′, 17′, 19′, 20′
19′	142.4, CH	7.07, ddd (1.7, 1.7, 5.6)	15′, 17′, 18′, 20 ′, 21′
20′	140.5, C
21′	59.4, CH2	a: 4.30, ddd (0.9, 1.7, 14); b: 4.26, dd (1.7, 14)	15′, 19′, 20′

Figure 1.

Key COSY (bold line, red) and selected HMBC (black arrows) correlations for 1. Analogous sets of correlations were observed for 2.

A COSY-correlated contiguous spin-system of five oxy-methines and one oxymethylene (H-15 to H₂-20) was suggestive of a hexose. However, the relatively shielded chemical shifts of H-15 (δ_H 4.93) and C-15 (δ_C 76.2) revealed the absence of an anomeric carbon, indicative of a C-glycosyl sugar unit. An HMBC correlation from H-15 to C-19 confirmed assignment of a pyranose-type six-membered ring. The coupling between H-15 and H-16 (J = 10.0 Hz) revealed a trans-diaxial relationship. Although the signals for H-17 and H-18 overlapped, trans-diaxial J-values were observed in the other methine signals that enabled establishment of trans-diaxial relationships throughout the ring, enabling assignment of a β-substituted C-glucopyranosyl unit. A NOESY correlation between H-15 and H-19 supported this assignment (Figure S7). Carbon-15 of this C-glucopyranosyl unit was linked to C-4 of the pentasubstituted aromatic ring on the basis of HMBC correlations of H-15 to aromatic ring carbons C-3, C-4, and C-5 and of H-16 to C-4. H-6 also showed a weak correlation with C-15.

COSY and HMBC correlations allowed the straightforward assembly of the remaining subunit of the molecule as a 2-hydroxymethyl-4,5,6-trihydroxycyclohexenone unit. Coupling values for H-17′ with H-16′ (J = 10.7 Hz) and H-18′ (J = 3.9 Hz) and comparison with model compounds containing this unit indicated trans and cis relative orientations for the H16′–H17′ and H17′–H18′ relationships, respectively.^8–13 An HMBC correlation between H-16′ and carboxyl carbon C-1′ indicated that C-1′ was esterified to the C-16′ oxygen atom of this unit. Notably, H-16′ was the only oxymethine in the molecule with a significant downfield shift increment suggestive of acylation (δ_H 5.94).

At this point, completion of the structure required only connection between the two substituted aromatic units. There were no explicit NMR correlations between the two rings, but structural and NMR comparison to depsides described in the literature strongly supported the connection of both rings through an ester linkage joining the C-5′ oxygen and carboxyl carbon C-1.^14–16 Connection of the two aromatic rings via an ester rather than an ether linkage was supported by major ions in the original LC-ESIMS data at m/z 397 (in the positive ion spectrum) and m/z 413 (in the negative ion spectrum), corresponding to the C-1–C-20 acylium ion and the C-1–C-20 unit plus the ester oxygen atom, respectively. This conclusion was later supported by negative ion HRESIMS fragmentation data collected using purified compounds 1 and 2. The UV spectrum also matched well with similarly substituted depside structures.¹⁷ These data led to assignment of the structure of 1 as shown. The name arenicolin A is proposed for compound 1.

Compound 2 was obtained as a white solid with the molecular formula C₃₄H₄₈O₁₂, again assigned on the basis of negative ion HRESIMS analysis. As was the case for 1, an intense pseudomolecular species was observed only in the negative ion spectrum. 1H NMR analysis of 2 in CD₃OD (Table 3) showed a pattern very similar to that of 1, but lacking the signals for the cyclohexanone moiety, suggesting that 2 is a truncated analogue of 1 containing a free C-1′ carboxylic acid group in place of the acylated cyclohexenone unit. This conclusion was consistent with the molecular formula of 2, together with the appearance of a fragment ion at observed m/z 647.3076 corresponding to the formula of 2 in the negative ion HRESIMS data for 1.

Table 3.

NMR Spectroscopic Data for 2 (¹H 500 MHz, ¹³C 125 MHz, CD₃OD)

position	δC, type	δH, mult. (J in Hz)	HMBC
1	171.3, C
2	105.8, C
3	164.5, C
4	110.8, C
5	162.9, C
6	112.5, CH	6.35, s	1w, 2, 4, 5, 8, 15w
7	149.0, C
8	37,9 CH2	2.89, m	2, 6, 7, 9, 10
9	33.6, CH2	1.66, m	8, 10
10	31.0, CH2	1.38, m
11	30.5, CH2	1.33, m
12	33.2, CH2	1.29, m
13	23.8, CH2	1.30, m
14	14.4, CH3	0.88, m	12, 13
15	76.2, CH	4.92, d (9.5)	4, 5, 16, 17, 19
16	73.2, CH	4.03, br t (9.5)	4, 15, 17
17	80.2, CH	3.46, mc	18
18	71.7, CH	3.47, mc	17, 19
19	82.8, CH	3.40, m
20	62.7, CH2	a: 3.84, dd (2.0, 11.8); b: 3.75, dd (5.0, 11.8)	18, 19
1′	–a
2′	115.2, Cb
3′	162.2, Cb
4′	107.8, CHb	6.58 br sb,d	5′, 6′
5′	152.8, Cb
6′	114.0, CHb	6.50 br sb,d	4′, 5′
7′	149.5, Cb
8′	36.5, CH2b	3.06, m	2′, 6′, 7′, 10′
9′	33.2, CH2	1.64, m	8′, 10′
10′	30.9, CH2	1.36, m
11′	30.4, CH2	1.31, m
12′	33.1, CH2	1.24, m
13′	23.8, CH2	1.25, m
14′	14.5, CH3	0.84, m	12, 13′

^aNot observed.

^bThese signals varied in appearance and/or precise shifts depending on concentration and pH.

^cThese signals resolved better in CD₃OD/Na₂CO₃, both appearing as triplets with J = 9.5 Hz.

^dThese signals sharpened in CD₃OD/Na₂CO₃, both appearing as doublets with J = 2.3 Hz.

Interestingly, however, there was one other difference, as the ¹H NMR signals associated with the tetrasubstituted aromatic ring (i.e., those for H-4′ and H-6′) were more complicated than expected for such a simple system. Specifically, three sets of signals of differing intensity appeared that collectively integrated for the requisite two hydrogens, including two pairs of meta-coupled doublets and two additional broad singlets. This feature hindered observation of a complete set of signals or correlations for the corresponding aromatic ring carbons in ¹³C and 2D NMR spectra, although all three sets showed correlations to very similar carbon shift positions. This behavior was enigmatic, as no such phenomenon was observed for 1, nor was it reported for precedents containing similar units in the literature. Compounds containing similarly substituted aromatic C-glycosides are known to display signal doubling due to rotational isomerism about the glycoside bond in some instances,¹⁸ but the observed issue in this case was remote from the region where such an effect would be most prominent. Analysis in acetone-d₆ did not resolve the issue. Only two sets of signals were observed in the region of interest (in approximately a 2:1 ratio), but in this case, several other signals also showed doubling, which would be more consistent with a mixture of diastereomeric rotamers. Analysis in DMSO-d₆ proved to be of limited value. While it appeared that only one set of lines was observed, the lines were broad, and elevated temperature failed to achieve any significant difference up to the point of partial decomposition. Given the structural difference between 1 and 2, it was postulated that the presence of the free acid group near the affected signals might be associated with the observed NMR behavior in CD₃OD. With this in mind, ¹H NMR data were collected using a saturated solution of Na₂CO₃ in CD₃OD, leading to complete disappearance of the broad, extra set of signals for the tetrasubstituted aromatic ring and replacement with a pair of sharp, meta-coupled, one-proton doublets, as was originally expected. These data also displayed slightly better resolution of signals for the C-linked β-glucopyranose, enabling clear observation of trans-diaxial coupling between all of the adjacent ring methine hydrogens (J = ca. 9.5 Hz in each case) and confirming the assigned relative configuration for this unit to match that of 1. Notably, some very minor companion signals or shoulders on some of the peaks were observed under these conditions, which supported the possibility that a minor rotamer was present. As noted above, some aromatic C-glycosides are known to display signal doubling due to rotational isomerism when ortho-substituents are present on the ring to which the glycoside is attached.¹⁸ Such an effect is not always observed, even in seemingly similar examples. In most of these cases, one form dominates, sometimes in a greater than 10:1 ratio, as would be the case here, and the ratio (or the appearance of the effect at all) is solvent-dependent. All of these features are consistent with what was observed for 2, with the free acid group being an additional complicating feature. Given all of these results, the structure of compound 2 is proposed as shown, and we have named it arenicolin B.

The relative configurations of the two stereogenic moieties in 1 could not be definitively correlated because of their distance from one another, but absolute configurations are proposed for them individually based on biogenetic precedents. The absolute configuration of the 2-hydroxymethyl-4,5,6-trihydroxycyclohexenone substructure is proposed as (16′S, 17′R, 18′R) based on its likely origin from the pathway leading to the known fungal metabolite epoxydon, as was judged to be the case for the structurally analogous fungal metabolites epoxydon 6-methylsalicylate ester,⁸ epoxydine B,⁹ epoxydon 3-chlorosalicylate ester,¹⁰ and 6S-acetoxy-4R,5R-dihydroxy-2-hydroxymethylcyclohex-2-en-1-one.¹¹ The latter compound and epoxydine B differ from 1 by having much simpler acyl groups linked at the analogous position of the substituted cyclohexenone unit. Efforts to obtain a degradation product of 1 corresponding to the cyclohexenone moiety by acid or base hydrolysis were unsuccessful. Verification of the absolute configuration of the C-glucopyranosyl unit found in both 1 and 2 was not attainable by hydrolytic methods, but as is the presumption with most molecules containing such a unit,¹⁹ it is proposed to be derived from d-glucose. A recent report suggested the use of electronic circular dichroism (ECD) to verify such assignments in similar, but somewhat simpler cases than structure 2.¹⁹ Despite the somewhat variable NMR signal behavior of 2 in solution, we explored application of this method. Like the simpler literature examples (which lack the second, substituted C-1′−C7′ aromatic ring unit) and the calculated spectra published for truncated versions thereof, the ECD curve for 2 showed a positive Cotton effect near 300 nm and a negative trend below 250 nm. The data for 1 were a bit more complex, but showed similar trends in these regions. Although not considered definitive, these similarities provided support for the proposed d-glucose-type absolute configuration for the C-glycoside unit in 2 and, by analogy, in 1.

Compounds 1 and 2 are new depsides each containing two polyketide-derived alkyl-substituted aromatic rings connected by an ester linkage, with one of the rings also bearing a C-glucosyl unit. In the case of 1, a third biosynthetic unit is also incorporated. Other fungal depsides containing alkyl side-chains have reportedly exhibited anti-inflammatory^20,21 and antitumor activities.²² Depsides are most commonly found in lichens and plants, although their production by nonlichenized ascomycetes has been reported occasionally. The presence of a sugar substituent is uncommon in depsides, but glucose, galactose, and glucuronic acid derivatives are found in metabolites such as TPI-1 to TPI-5,²³ exophilic acid,²⁴ aquastatin,²⁵ CRM646-A/B,²⁶ and KS-501/KS-502.²⁷ However, in these precedents, the carbohydrate is bound by an O-glycosyl linkage to carbon C-4. C-Glycosylated compounds are well known from plants and to some degree from bacterial sources, but are not common as fungal products. The only other fungal C-glycosylated depside described to date is stromemycin, a natural product reported from an unidentified fungus (DSM 12038),¹⁷ Aspergillus stellatus,²⁸ and Penicillium pinophilum²⁹ (although the structure is not correctly drawn in the latter reference, which shows a methoxy group in place of the acid group). Stromemycin is the closest literature analogue to compound 2, having C9 diene-containing side-chains rather than C7 alkyl groups and lacking the cyclohexenone unit present in 1. Compound 2 could also be considered a 3-C-glucosyl derivative of de-O-methylprasinic acid.³⁰ A methyl ester of the hydrolysis product of stromemycin called carnemycin A (i.e., having only the C-glycosylated aromatic ring unit) has also been reported as a product of a marine isolate of Aspergillus carneus.³¹ An additional report of carnemycins and several closely related metabolites from another marine-derived Aspergillus sp. isolate has also recently appeared.^{19 1}H and ¹³C NMR shift assignments for relevant portions of all of these compounds match very well with those of 1 and 2, further supporting the structure assignments.

Compounds 1 and 2 are assembled from three to four different biosynthetic units, with the core aromatic units of 1 and 2 likely having a straightforward heptaketide origin. Attachment of glucose to the aromatic core presumably involves a C-glycosyl transferase enzymatic step. The hydroxylated cyclohexenone unit in 1 could originate from nucleophilic epoxide ring opening of epoxydon, a cytotoxic tetraketide fungal metabolite derived from the 6-methylsalicylic acid (6-MSA) pathway.^32–35

Compounds 1 and 2 were produced by all four strains of P. arenicola (Table 1), indicating that they are potential metabolite markers for the species. Interestingly, medium I, prepared with nitrate as the primary nitrogen source, was the only medium among the five tested that yielded compound 1, while 2 was produced under all conditions. The nitrogen source and its bioavailability can be a potent regulator of secondary metabolism in fungi and can play a role in differential metabolite expression.^36,37 Nitrogen availability is very important for regulation of 6-methylsalicylic acid synthase,³⁸ and inorganic nitrogen sources stimulate the production of patulin, an end-product of the 6-MSA pathway in several Penicillium species,^39,40 which is consistent with the observation of 1 in the only medium with nitrate as nitrogen source. This observation also reinforces the strategic benefit of testing strains under culture conditions that vary in nutritional composition and physical environment in order to expand the expression of secondary metabolism.^41,42

Compounds 1 and 2 were assayed against bacterial and fungal human pathogens. However, assays against Staphylococcus aureus, Candida albicans, and Cryptococcus neoformans at 100 μg/mL did not show any growth inhibition. The compounds were also evaluated for in vitro cytotoxicity against three human cancer cell lines by assessing cell viability after 48 h of incubation (Figure 2). The results showed that compound 1 decreased cell viability in all three cell lines in a dose-dependent manner (IC₅₀ values: 7.3, 6.0, and 9.7 μM in HCT-116, IMR-32, and BT-474 cells, respectively), while 2 did not cause significant cytotoxicity toward these cell lines at levels up to 30 μM. The potency of compound 1 in the tested cancer cell lines compared favorably with that of 5-fluorouracil (5-FU; IC₅₀ values: 6.5 and 5.7 μM in HCT-116 and IMR-32 cells, respectively), a chemotherapeutic agent commonly used for the treatment of breast, colorectal, and pancreatic cancer.^43,44

Figure 2.

In vitro toxicity assay against HCT-116 (colorectal carcinoma), IMR-32 (neuroblastoma), and BT-474 (ductal carcinoma) cells for (a) arenicolin A (1); (b) arenicolin B (2); and (c) 5-fluorouracil (positive control).

EXPERIMENTAL SECTION

General Experimental Procedures.

Optical rotations were measured on a Rudolph Research Autopol III automatic polarimeter. UV data were obtained using a Varian Cary III UV spectropho-tometer. ECD data were collected with a Jasco model J-815 spectropolarimeter. NMR data were collected on a Bruker 500 MHz NMR instrument equipped with a 5 mm triple resonance cryoprobe at 298 K, with CD₃OD as solvent and tetramethylsilane as internal standard (δ_H/δ_C = 0). HRESIMS data were recorded using a Waters Q-TOF Premier mass spectrometer. Semipreparative HPLC separations and HPLC-MS data were acquired on an Agilent 1260 system equipped with a diode array detector (DAD) and coupled to an Agilent 6120 single quadrupole mass spectrometer (MS), with a binary solvent system consisting of 0.1% aqueous formic acid (solvent A) and 0.1% formic acid in MeCN (solvent B).

Strains and Culture Conditions.

Four strains of Penicillium arenicola (Table 1) were obtained from the USDA National Regional Research Laboratory (NRRL) culture collection and were grown in five different media. These media were as follows: medium I⁴ (glucose 20 g, casein peptone 5 g, NaNO₃ 3.0 g, K₂HPO₄ 1.0 g, KCl 0.5 g, MgSO₄·7H₂O 0.5 g, FeSO₄·7H₂O 10 mg per 1000 mL of deionized H₂O); medium IV⁴⁵ (meat peptone 30.5 g, soybean meal 15.5 g, dextrin 2.0 g, blackstrap molasses (Brer Rabbit) 10.5 g, Na₂HPO₄ 4.5 g, MgSO₄·7H₂O 5.5 g, FeSO₄·7H₂O 0.10 g, ZnSO₄·7H₂O 4.55 mg, cottonseed oil 40 mL per 1000 mL of deionized H₂O); medium VI⁴⁶ (glycerol 10.0 g, peptone 8.6 g, soybean meal 40.0 g, cottonseed oil 20.0 g, mannitol 97.3 g, l-ornithine 5.3 g, l-proline 5.0 g, CaCl₂ 5 g, K₂HPO₄ 8.4 g, MgSO₄·7H₂O 5.0 g, CuSO₄·5H₂O 0.6 g, MnSO₄·H₂O 0.1 g, FeSO₄·7H₂O 0.05 g per 1000 mL of deionized H₂O); medium TG 106⁴⁷ (d-mannitol 100.0 g, NZ amine 33.0 g, yeast extract 10.0 g, (NH₄)₂SO₄ 5.0 g, KH₂PO₄ 9.0 g per 1000 mL of deionized H₂O), and medium SMY (Bacto neopeptone 10.0 g, maltose 40.0 g, yeast extract 10.0 g per 1000 mL of deionized H₂O).

For the seed culture stage, six agar discs from 3-week-old YM agar (malt extract 10.0 g, yeast extract 2.0 g, agar 20.0 g, 1000 mL of deionized H₂O) cultures were inoculated into medium SMY with 0.4% agar in 50 mL aliquots in 250 mL flasks. Seed cultures were grown at 24 °C, 220 rpm for 4 days. For the production cultures, 1.0 mL aliquots of the seed growth were transferred to flasks with 50 mL of medium I, medium IV, medium IV, medium TG 106, and medium SMY, respectively, and kept at 24 °C, 220 rpm for 12 days.

Extraction and LC-MS Analysis.

Each fermentation was extracted by the addition of an equal volume of MeOH followed by shaking for 2 h. The aqueous phase was filtered, a 5.0 mL aliquot removed for LC-MS analysis, and the remaining extract evaporated to dryness under vacuum. The aliquot was evaporated to dryness under gentle heating in an air stream, resuspended in 0.5 mL of MeOH, filtered through 0.2 μm regenerated cellulose membrane, and analyzed by HPLC-MS (Ace Equivalence 5 C₁₈, 4.6 × 150 mm, 5 μm, 10–100% B for 28 min, 1.0 mL/min). The chromatographic profiles were monitored by wavelength scanning from 190 to 400 nm and by positive and negative ESIMS from m/z 160 to 1500.

Isolation.

The dried extracts from the above fermentations were pooled and dissolved in H₂O/MeOH (1:1 v/v), and most of the MeOH was removed under vacuum. The remaining aqueous sample was extracted with an equal volume of methyl ethyl ketone (MEK). After 2 h, the organic phase was separated and evaporated under vacuum. The resulting sample (830 mg) was dissolved in a mixture of MeCN and H₂O (1:1 v/v) and fractionated with a Grace Reveleris X2 flash chromatography system using a Reveleris C₁₈ RP 12 g cartridge (10–100% ACN over 16 min, flow rate 30 mL/min), using UV and ELSD detection, resulting in 16 fractions.

Fraction 8 was dried, dissolved in ACN/H₂O (1:1 v/v), and further purified by semipreparative HPLC (Agilent Zorbax SB-C₁₈ column; 5 μm; 9.4 × 250 mm; gradient of 30–60% B in 20 min, 4.0 mL/min), yielding 7.2 mg of 1. Fraction 9 was further purified by semipreparative HPLC (Agilent Zorbax SB-C₁₈ column; 5 μm; 9.4 × 250 mm; gradient of 50–100% B for 20 min (solvent A, 0.1% formic acid in H₂O; solvent B, 0.1% formic acid in MeCN, 4.0 mL/min)), yielding 2.1 mg of 2.

In an effort to obtain more material and to provide representative results for a single fungal culture in a single medium, strain NRRL 31507 was cultured in medium I (four 1 L Erlenmeyer flasks with 250 mL of medium per flask). Production of 1 and 2 was monitored by LCMS. Two of the cultures were extracted as before after 12 days, and the remaining two after 15 days, as the titers appeared to be decreasing. After concentration under vacuum, extraction with MEK, flash chromatography of the resulting combined, dried extracts, and semipreparative HPLC using the protocols above afforded an additional 6 mg of 2 and 1 mg of 1.

Arenicolin A (1):

off-white solid; [α]_D −29 (c 0.11, MeOH); UV (MeOH) λ_max (log ε) 221 (4.3), 272 (4.1), 308 (3.8); ECD (MeOH, 4.8 mM) λ_max (Δε) 225 (−15), 240 (−56), 262 (+20), 272 (+2.5), 275 (−3.5), 311 (+8.2) nm; 1D and 2D NMR data (CD₃OD), Table 2; HRESIMS m/z 803.3495 [M – H]⁻ (calcd for [C₄₁H₅₆O₁₆ – H], m/z 803.3490).

Arenicolin B (2):

off-white solid; [α]_D +16 (c 0.19, MeOH); UV (MeOH) λ_max (log ε) 220 (4.4), 273 (4.1), 307 (3.9); ECD (MeOH, 7.4 mM) λ_max (Δε) 217 (−16), 227 (−4.6), 250 (−2.7), 311 (+4.3) nm; 1D and 2D NMR data (CD₃OD), Table 3; HRESIMS m/z 647.3064 [M − H] ⁻ (calcd for [C₃₄H₄₈O₁₂ − H], 647.3068).

Antimicrobial Assay.

Samples of 1 and 2 were dissolved in DMSO at 0.1 mg/mL, and 10 μL of each solution was applied to a 4 mm well aspirated from a plate of YM agar seeded with an overnight culture of Staphylococcus aureus (ATCC 43300), Candida albicans (ATCC 10231), or Cryptococcus neoformans H99. Plates were incubated at 37 °C and examined after 24–48 h for zones of inhibition.⁴⁸

Cytotoxicity Assay.

The cytotoxicities of compounds 1 and 2 were determined on HCT-116, IMR-32, and BT-474 cancer cell lines as previously described,⁴⁹ with minor modifications. Briefly, 2000 cells/well were plated in 96-well half-area white microplates. After 2 h of preincubation, serial dilutions of 1 and 2 were added (0.3 through 30.0 μM) and treatment continued for 48 h. Cells were similarly treated with 5-FU and fresh medium as positive and negative control, respectively. Each concentration was evaluated in triplicate. The resulting cell viability was measured using CellTiter-Glo (Promega) according to the manufacturer’s protocol and the Synergy HTX Multi-Mode (BioTek). Data were analyzed with GraphPad Prism software using the logistic nonlinear regression model.