Emestrins: Anti-Cryptococcus Epipolythiodioxopiperazines from

Eleven emestrin-type epipolythiodioxopiperazines, including four new compounds, emestrins H–K (1–4), were isolated from the crude extracts of two ...
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Emestrins: Anti-Cryptococcus Epipolythiodioxopiperazines from Podospora australis Yan Li,† Qun Yue,† Nicole M. Krausert,‡ Zhiqiang An,† James B. Gloer,‡ and Gerald F. Bills*,† †

Texas Therapeutic Institute, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77054, United States ‡ Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States S Supporting Information *

ABSTRACT: Eleven emestrin-type epipolythiodioxopiperazines, including four new compounds, emestrins H−K (1−4), were isolated from the crude extracts of two strains of the coprophilous fungus Podospora australis. The structures of 1−4 were established primarily by analysis of NMR data, and the absolute configuration of C-6 in 1 was independently assigned using the modified Mosher method. Four of the known emestrins obtained (emestrins C−E and MPC1001C) were found to selectively inhibit the growth of Cryptococcus neoformans. These results also represent the first report of chemistry from any strain of P. australis.

erations, we hypothesized that screening with a Cryptococcuscentric approach would reveal antifungal molecules not detected by Candida- or Aspergillus-based phenotypic assays. Moreover, these prospects could be enhanced by targeting types of fungi that have rarely been included in screening programs to date, such as coprophilous fungi, which are fungi adapted to complete their life cycles through dispersal and growth in animal dung.4 In the course of exploring fermentation extracts of rarely studied coprophilous fungi for natural products with activity against C. neoformans, two different strains of Podospora australis (Ascomycota, Lasiosphaeriaceae) were isolated from horse (TTI-0248) and rabbit (TTI-0313) dung from Texas (Figure S1). Methyl ethyl ketone (MEK) extracts of solidsubstrate fermentations of TTI-0248 and TTI-0313 strongly inhibited the growth of C. neoformans H99 in agar inhibition assays, but negligibly affected growth of C. albicans ATCC 10231 (Figure 1) or Staphylococcus aureus ATCC 43300 (not shown). Comparison of the HPLC-MS profiles of the two extracts indicated that they contained a substantially similar array of metabolites. Fractionation of these extracts led to the isolation of 11 emestrin-type epipolythiodioxopiperazine metabolites, including four new compounds, which we named

Cryptococcus species are among the most common causes of invasive fungal infections, affecting one million people each year and resulting in >600 000 deaths worldwide.1,2 Cryptococcus species, especially C. neoformans, most often cause disease in people with compromised immune function. Roughly a third of all HIV/AIDS-associated deaths are due to cryptococcal disease, exceeding the number due to tuberculosis.3 Current treatments employ a limited number of antifungal agents (amphotericin B, flucytosine, fluconazole), with no new therapies introduced in the past few decades. These options remain unsatisfactory because of their toxicity, their inability to eradicate the fungus, and the emergence of drug resistance. Even when treated, Cryptococcus infections are further complicated by high recurrence rates and the need for longterm suppressive therapy. Natural products from fungi have been critical to the development of antifungal agents, e.g., the echinocandins. However, efforts to discover new antifungal natural products generally have been highly biased toward seeking agents for C. albicans and Aspergillus species, both of which belong to the Ascomycota, while Cryptococcus species belong to the Tremellales of the Basidomycota and differ substantially from ascomycetes in regard to ecology, epidemiology, life cycle, and genomic structure. In fact, agents used in treating Cryptococcus infections, such as amphotericin, azoles, and flucytosine, are all nonexclusive to Cryptococcus. On the basis of these consid© XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 30, 2016

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DOI: 10.1021/acs.jnatprod.6b00498 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Anti-Cryptococcus and anti-Candida assay results of the crude extracts (10 μg/well) from TTI-0248 and TTI-0313: (1) crude extract from TTI-0248 (wheat medium); (2) crude extract from TTI-0248 (supermalt medium); (3) crude extract from TTI-0313 (wheat medium); (4) crude extract from TTI-0313 (supermalt medium); (5) amphotericin B (2.5 μg/well).

emestrins H−K (1−4), and seven known emestrins: secoemestrin D (5), 5 emestrin C (6; MPC1001), 6,7 MPC1001C (7),6 emestrin D (8; MPC1001D),6,7 emestrin E (9),7 MPC1001F (10),6 and MPC1001H (11).6 Herein, we report on the identification of the producing organisms, their fermentation, the isolation, structure determination, and antibiotic activity of these new and known emestrins and, in particular, their selective antifungal activity toward C. neoformans.

Table 1. NMR Data (500 MHz, CDCl3) of 1 position

δC

1 3 4 5a 6 7 8 10 10a 11

165.6 68.5 167.2 64.9 72.3 110.9 137.7 137.4 107.3 38.9

11a 1′ 2′/6′ 3′/5′ 4′ 7′

68.4 133.2 130.7 128.8 128.1 46.3

SCH3-3 SCH3-11a NH-2 OH-6

14.3 14.7

δH (J in Hz)

4.57, 4.64, 4.93, 6.19, 6.36,

HMBCa

d (7.7) dd (7.7, 1.8) dd (8.2, 1.8) dd (8.2, 2.4) t (2.4)

6, 10, 10a, 11a 5a, 7, 8 5a, 6, 8 6, 7, 10 5a, 8, 10a, 11

2.62, d (15.0) 1.87, dt (15.0, 2.4)

5a, 10, 10a, 11a 1, 10, 10a, 11a

7.19, 7.31, 7.31, 3.64, 3.03, 2.25, 2.44, 6.27, 5.13,

2′/6′, 4′, 7′

dd (7.7, 1.7) m m d (13.5) d (13.5) s s s brs

3, 4, 1′, 2′/6′ 3, 4, 1′, 2′/6′ 3 11a 1, 3

a

HMBC correlations are from proton(s) stated to the indicated carbon.

10 and C-10a and from H-8 to C-6 and C-10 and comparison with data for known emestrins enabled assignment of a 5a,6dihydrooxepine, whereas those from H-10 to C-5a, C-10a, and C-11 and from H2-11 to C-1, C-5a, C-10, and C-11a along with the chemical shifts of C-5a and C-11a indicated that the 5a,6dihydrooxepine was fused to the ETP core through a pyrrolidine ring. Correlations from H2-7′ to C-3, C-4, C-1′ and C-2′/C-6′ indicated that C-7′ is attached to both the C-3 and the monosubstituted benzene ring. Correlations from the S-methyl signals for SCH3-3 and SCH3-11a to C-3 and C-11a, respectively, located the two S-methyl groups at C-3 and C-11a. Considering the chemical shifts of C-6 (δC 72.3) and the unsaturation requirement for 1 located the remaining hydroxy group (OH-6) at C-6. Collectively, these data permitted assignment of the planar structure of 1. The 7.7 Hz coupling between H-5a and H-6 in 1 suggested their trans-relationship, by analogy to other members of this class.6−8 Independent establishment of the relative configuration at other positions was complicated by the shortage of

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RESULTS AND DISCUSSION Emestrin H (1) was assigned the molecular formula C20H22N2O4S2 (11 degrees of unsaturation) on the basis of HRESIMS. Its 1H and 13C NMR spectra showed resonances for two exchangeable protons (δH 6.27 and 5.13), two S-methyl groups, two methylenes, two methines, including one oxymethine, one monosubstituted benzene ring, four olefinic carbons (three of which are protonated), two sp3 quaternary carbons, and two amide/carboxyl carbons (δC 167.2 and 165.6). Analysis of these data (Table 1) suggested the presence of the epipolythiodioxopiperazine (ETP) moiety characteristic of emestrins,6,7 but because the data did not clearly match those of any of the known members of the class, detailed 2D NMR analysis was undertaken. 1H−1H COSY data showed the presence of an isolated spin system corresponding to the C-5a− C-6−C-7−C-8 unit in 1. HMBC correlations from H-5a to CB

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structural features nearly identical to those found in 2, except that the S-methyl signals were absent in 3, indicating that a disulfide bond was formed between C-3 and C-11a to satisfy the unsaturation requirement of 3. Therefore, the planar structure of 3 was assigned as shown. Analysis of 1H−1H coupling constants for 3 and comparison of NMR data with other analogues obtained suggested the same relative configuration found in other related compounds, such as secoemestrin C1.7 The molecular formula of emestrin K (4) was determined to be C29H30N2O6S2 (16 unsaturations) on the basis of HRESIMS analysis, indicating the presence of one less oxygen atom than the formula of 2. Analysis of its 1H and 13C NMR data (Table 2) as well as COSY and HMBC correlations revealed structural features matching those of 2, except that the signals for the C5a−C-10a fragment differed significantly. On the basis of 1 H−1H COSY correlations and couplings observed for relevant protons (e.g., 5 Hz coupling between H-8 and H-10), C-8 was directly connected to C-10, extending the continuous spin system from H-5a−H-10 and suggesting the presence of a 1,3cyclohexadiene unit in place of the oxepin, which was further supported by an HMBC correlation from H-8 to C-10a. On the basis of these data, the planar structure of 4 was assigned as shown. Since 4 was presumed to be a precursor of 6−9, which were also isolated in the current work, the absolute configuration of 4 was proposed to be analogous to those of the other emestrins. Although they lack the macrocyclic architecture of 6−9 and the diphenyl ether unit of 10 and 11, the clear biogenetic relationships suggest that all of the compounds have the same absolute configuration at relevant centers. ECD data collected for compounds 1−4 support this conclusion. The sign of the Cotton effect (CE) at 230−270 nm for epidithiodioxopiperazines reportedly depends on the disulfide bridge orientation.7,9 Among the new analogues, only 3 has an intact disulfide bridge, but all of the compounds showed negative CEs between 220 and 240 nm and positive CEs between 260 and 300 nm, which are analogous to data for emestrin,8 emestrin C (6),6 and other representative epidithiodioxopiperazines.8 The specific rotation of a sample of 6, which was the major known emestrin analogue isolated in this work, was virtually identical with the literature value ([α]20D +118 (c 0.09, MeOH); lit. [α]20D +117 (c 0.3, MeOH)), further supporting assignment of the same absolute configuration. On the basis of the biogenetic analogy and the similarity of ECD curves of all of these compounds, the absolute configurations of 1−4 were assigned to be analogous to that of 6 (3R, 5aS, 6S, 11aR). The known compounds 5−11 isolated from the crude extracts were identified as secoemestrin D, emestrin C (also named MPC1001), MPC1001C, emestrin D, emestrin E, MPC1001F, and MPC1001H, respectively, by comparison of their NMR and MS data with those previously reported.5−7 Compounds 1−11 were tested for antifungal activity against C. neoformans H99 and C. albicans ATCC 10231 (Table 3). Emestrin C (6) and MPC1001C (7), containing both macrocyclic skeletons and disulfide bridges across a dioxopiperazine, showed significant activity against C. neoformans H99, with MIC values of 0.8 and 1.6 μg/mL, respectively, while the positive control amphotericin B showed a minimum inhibitory concentration (MIC) value of 0.8 μg/mL. The trisulfur-bridged (emestrin D) and tetrasulfur-bridged (emestrin E) compounds showed weaker anti-Cryptococcus activity, with MIC values of 6.4 and 25.6 μg/mL, respectively, while the other macrocycle

H NMR signals near the stereocenters and an associated lack of relevant correlations in NOESY data. However, as noted above, several known emestrins were also obtained, and the close similarities among these compounds enabled assignments to be made. The absolute configuration of the major known analogue isolated from both strains, emestrin C (6), has been demonstrated by chemical conversion from emestrin,7 whose configuration has been established by X-ray crystallography and ECD analysis.9 The identity of the sample obtained in the present work with emestrin C was established by comparison of 1 H NMR, 13C NMR, MS, and [α]D data with literature values.6 Given the clear biosynthetic relationship, the similarities in 1H and 13C NMR data at relevant positions, and ECD comparisons with 6 and other compounds isolated (see below), the absolute configuration of 1 is proposed to be analogous to that of 6, as shown. To help verify this stereochemical assignment, the absolute configuration at C-6 in 1 was independently assigned using the modified Mosher method. Treatment of 1 with (S)- and (R)-αmethoxy-α-trifluoromethylphenylacetic acid (MTPA) Cl afforded the R- (1a) and S-MTPA (1b) esters, respectively. The differences in chemical shift values (Δδ = δS − δR) for 1b and 1a were recorded and utilized to assign the 6S configuration (Figure 2).10,11

Figure 2. Δδ values (in ppm; = δS − δR) obtained for (S)- and (R)MTPA esters 1b and 1a.

Emestrin I (2) was isolated as a white, amorphous solid with a molecular formula of C29H30N2O7S2 (16 degrees of unsaturation), established by HRESIMS. The 1H and 13C NMR spectra of 2 showed resonances similar to those of 1 (Table 2), except for the presence of an N-methyl group (δH/ δC 3.27/30.0) and a 1″,3″,4″-trisubstituted benzene bearing a carboxylic carbon (C-7″) substituent, a phenolic OH group, and an O-methyl group (Table 2). Chemical shift values, the coupling pattern, and HMBC correlations from aromatic protons H-2″ and H-6″ to C-7″, from the O-methyl group to C-4″, and from the phenolic OH group to C-2″, C-3″, and C4″ indicated the substitution pattern shown for this unit in 2. In addition, HMBC correlations from the N-methyl proton signal to C-1 and C-3 located this methyl group at N-2. The absence of other exchangeable proton signals and a weak HMBC correlation from H-6 to C-7″ together with the significant downfield shift of the H-6 oxymethine signal (δH 5.98 in 2; δH 4.64 in 1) indicated that the 1″,3″,4″-trisubstituted benzoyl group acylates the C-6 oxygen atom. Therefore, the planar structure of emestrin I was assigned as shown in 2. The relative configuration of 2 was deduced as shown by analogy to 1, and this assignment was supported by the nearly identical ECD spectra recorded for the two compounds.6 The molecular formula of emestrin J (3) was determined to be C27H24N2O7S2 (17 degrees of unsaturation) by HRESIMS, which was 30 mass units (C2H6) less than that of 2. Analysis of its 1H NMR, 13C NMR (Table 2), and HMBC data revealed C

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Table 2. NMR Data (500 MHz, CDCl3) of 2−4 2 position

δC

1 3 4 5a 6 7 8 10 10a 11

165.6 76.1 164.3 61.5 72.3 106.2 139.4 137.0 109.4 39.3

11a 1′ 2′/6′ 3′/5′ 4′ 7′

69.0 134.1 130.0 128.7 127.9 42.2

1″ 2″ 3″ 4″ 5″ 6″ 7″ CH3-2 SCH3-3 SCH3-11a OCH3-4″ OH-3″

122.9 116.1 145.3 150.7 110.0 123.6 165.9 30.0 13.2 15.1 56.0

3

δH mult. (J in Hz)

5.09, 5.98, 4.86, 6.32, 6.42,

d (8.2) dd (8.2, 2.1) dd (8.2, 2.1) dd (8.2, 2.4) t (2.4)

2.69, d (15.0) 1.64, dt (15.0, 2.4)

7.11, 7.29, 7.29, 3.65, 3.12,

dd (7.6, 1.6) m m d (13.5) d (13.5)

7.74, d (2.0)

6.96, d (8.5) 7.81, dd (8.5, 2.0) 3.27, 1.94, 2.16, 3.99, 5.88,

s s s s s

δC 165.4 78.6 162.0 62.9 70.6 105.8 141.1 139.3 113.4 36.4

5.37, 5.87, 4.71, 6.35, 6.70,

7.26, 7.11, 7.19, 4.15, 3.16,

123.1 116.4 145.2 150.6 109.8 123.7 166.3 29.4

6.92, d (8.5) 7.74, dd (8.4, 2.0) 3.02, s

3.99, s 5.74, s

Table 3. Antifungal Activities of 6−9a

a

C. albicans ATCC 10231

0.8 1.6 6.4 25.6 0.8

6.4 >25.6 >25.6 >25.6 1.6

δC 166.1 76.1 164.3 65.3 75.9 128.3 125.4 119.4 134.7 39.1 73.0 134.3 130.0 128.6 127.8 42.5 123.7 115.9 145.3 150.5 110.0 123.1 166.1 30.1 13.1 15.1 56.03

δH mult. (J in Hz)

5.11, 6.42, 5.76, 5.97, 5.82,

d (14.0) d (14.0) d (10.0) dd (10.0, 5.0) dd (5.0, 1.8)

2.66, d (15.5) 1.46, dd (15.5, 1.8)

7.07, 7.24, 7.24, 3.54, 3.06,

dd (7.6, 1.6) m m d (13.5) d (13.5)

7.73, d (2.0)

6.94, d (8.4) 7.76, dd (8.4, 2.0) 3.28, 2.21, 2.26, 3.99, 5.74,

s s s s s

dioxopiperazine moieties, but the substituents at C-6 and N-2 differ. Emestrin J (3) is the C-4″ methyl ether of secoemestrin C1,7 while emestrin K (4) is a new member of the emestrin class having the bis(methylthio)dioxopiperazine unit, but differing from the known analogues6 by incorporation of a 1,3-cyclohexadiene unit instead of the dihydrooxepin. Biosynthetically, emestrins 1−11 are likely formed from two Lphenylalanine units by a peptide cyclization pathway similar to that of gliotoxin, which is encoded by the gli gene cluster in Aspergillus f umigatus.13−17 However, biosynthesis of emestrins would require additional ring-expansion and macrocyclization steps (Scheme 1). The first emestrin-type compound was isolated in 1986 from mycelial acetone extracts of Aspergillus striatus (=Emericella striata).18 Since then, more than 10 distinct subtypes of these fungal metabolites have been characterized, and some antifungal activity against Trichophyton, Penicillium, and Fusarium species has been observed.5−7,19−21 The most noteworthy of these compounds is emestrin C (6; MPC1001), which was isolated from Cladorrhinum sp. as an antiproliferative agent possessing strong activity against the human prostate cancer cell line DU145 (IC50 = 9.3 nM)6,7 and has been the subject of extensive synthetic studies.22−25 Herein, we report that the macrocycle ring-containing emestrins, especially emestrin C, strongly inhibit the growth of C. neoformans H99 (MIC = 0.8 μg/mL), but only moderately affect the growth of C. albicans ATCC 10231 (MIC = 6.4 μg/ mL). Mechanistic studies of the effects of emestrins on rat liver

MIC (μg/mL) C. neoformans H99

d (7.5) t (7.5) t (7.5) d (18.4) d (18.4)

7.71, d (2.0)

56.1

compound

dd (8.7, 1.6) dt (8.7, 1.6) dd (8.7, 1.6) dd (8.2, 2.1) d (2.1)

3.70, s

73.4 133.9 129.9 128.4 127.4 35.4

6 7 8 9 amphotericin B

4

δH mult. (J in Hz)

Compounds 1−5, 10, and 11 were inactive at 256 μg/mL.

ring-opened compounds did not visibly affect growth of Cryptococcus at 256 μg/mL, indicating that the macrocyclic ring and polysulfide structures are critical for the antiCryptococcus activity. Only 6 displayed modest activity against C. albicans ATCC 10231, with an MIC value 6.4 μg/mL, while the positive control amphotericin B exhibited an MIC value of 1.6 μg/mL in this assay. Consistent with the lack of bacterial growth inhibition by the crude extracts, compounds 1−11 had no noticeable effect on growth of the Gram-positive bacterium Staphylococcus aureus ATCC 43300 or the Gram-negative bacterium Pseudomonas aeruginosa ATCC 10145 at 512 μg/mL. Natural products have been reported from only a limited number of Podospora species to date,4 and this is the first report of the chemistry from P. australis. The new compounds emestrin H (1) and emestrin I (2) are closely related to asteroxepin, a metabolite from Aspergillus terreus.12 All three compounds contain both dihydrooxepin and bis(methylthio)D

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Scheme 1. Proposed Biosynthetic Route to 1−11

mitochondria26 led to the hypothesis that the antifungal and cytotoxic activities of emestrins were due to the inhibition of ATP synthesis in mitochondria, causing an uncoupling of oxidative phosphorylation and depression of respiration. Since ATP synthesis and mitochondrial function are conserved features of fungi and other eukaryotes, other factors, such as efflux, cell permeability, or effects on secondary targets, may explain the differential sensitivity between C. albicans and C. neoformans.

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from rabbit dung) in TTI’s culture collection at the Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA. Both strains were identified as P. australis based on the combination of hairy ascomata, four-spored asci, large, apiculate, aseptate, ellipsoidal ascospores with two apical whip-like appendages, and a minute apiculate pedicel (Figure S1). We also observed that both strains formed a Cladorrhinum-like conidial state on the aerial mycelium (Figure S1). To the best of our knowledge, this is the first report of a conidial state for P. australis. To corroborate the morphological identifications, we analyzed their approximate phylogenetic relationships based on the internal transcribed spacer (ITS) and large subunit (LSU) rDNA sequences. DNA extraction from cultured mycelium and DNA cloning and sequencing protocols were conducted as previously described.27 The DNA sequences have been accessioned in GenBank as KX015764 (TTI0248, ITS + LSU) and KX015765 (TTI-0313, ITS + LSU). Recent attempts at phylogenetic reconstructions of the Lasiosphaericeae have demonstrated that the family is paraphyletic.28 Podospora and allied genera Arnium, Cercophora, Schizothecium, and Immerisella formed several polyphyletic lineages within the Sordariales, and the genera and species are interspersed within these sublineages, therefore calling into question family and generic concepts within the group.28 Sequences of the large subunit of the rDNA of TTI-0248 and TTI0313 were intercalated within a previous alignment of LSU sequences from coprophilous Lasiosphaeriaceae and other species of the Sordariales.28 A maximum likelihood analysis of these sequences executed in Mega 6.029 resulted in a phylogenetic tree (Figure S2) with topology similar to that previously reported. The sequences of TTI-0248 and TTI-0313 grouped tightly with two other sequences from P. australis isolated from bison dung in the USA (Figure S2). Fermentation. For each cultured strain, agar plugs from YMA cultures were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of SMYA seed medium (1% Bacto neopeptone, 4% maltose, 1% yeast extract, and 0.4% agar). Seed media were incubated for 5 days with agitation at 220 rpm. A scaled-up fermentation was then carried out in 10 Erlenmeyer flasks (500 mL) each containing 30 g of wheat. A 50 mL amount of a base solution (0.2% yeast autolysate, 1% sodium tartrate, 0.1% KH2PO4, 0.1% MgSO4·7H2O, and 0.005% FeSO4·7H2O in deionized H2O) was added to each flask, and the contents were soaked overnight before autoclaving at 20 psi for 20 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the seed culture, mixed with the wheat, and standing incubated at 23 °C for 14 days.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were acquired using a Rudolph Research AUTOPOL III automatic polarimeter. UV data were obtained for solutions in MeOH with a Varian Cary III UV/vis spectrophotometer. ECD data were recorded with an Olis Cary-17 spectrophotometer (1 cm cell) using MeOH as solvent. All 1H, 13C, APT, and 2D NMR (COSY, HSQC, HMBC, NOESY) data were collected using a Bruker AVANCE-500 MHz spectrometer with a 5 mm triple-resonance cryoprobe at 298 K using solvent signals (CDCl3; δH/δC, 7.26/77.2) as references. The HMQC and HMBC experiments were optimized for 145 and 8 Hz, respectively. 13C NMR multiplicities were established by APT experiments and were consistent with the position assignments in Tables 1 and 2. ESIMS data were recorded on an Agilent 6120 single quadrupole LC−MS using positive ion electrospray ionization. Highresolution mass spectra were acquired with a Waters Q-Tof Premier instrument. Fungal Strains and Their Identification. Strains of Podospora australis were isolated from horse dung collected along Airport Road, Houston, Harris County, Texas, and white-tailed rabbit dung collected along US Route 90, near Brackettville, Kinney County, Texas. Briefly, dung samples were rehydrated with deionized water and incubated at room temperature in deep-dish Petri plates lined with moist filter paper until ascomata formed on the dung surface. Ascospores and asci were dissected from mature ascomata and transferred to cornmealdextrose agar supplemented with 50 μg/mL chlortetracycline and streptomycin sulfate. Agar plates with dissected ascospores and asci were sealed and incubated overnight at 45 °C to promote ascospore germination. Germinated ascospores were then transferred to YMA medium (1% maltose extract, 0.2% yeast extract, and 2% agar) to establish axenic cultures. The two isolates were assigned the accession numbers TTI-0248 (isolate from horse dung) and TTI-0313 (isolate E

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HRESIMS m/z 605.1394 [M + Na]+ (calcd for C29H30N2O7S2Na, 605.1392). Emestrin J (3): white powder; [α]25D −49 (c 0.040, MeOH); UV (MeOH) λmax (log ε) 207 (4.29), 260 (3.73), 294 (3.29) nm; ECD (c 50 μM, MeOH) λmax (Δε) 206 (+8), 232 (−15), 266 (+7) nm; 1H and 13C NMR data see Table 2; HMBC data (CDCl3, 500 MHz) H-5a → C-6, 10a, 11a; H-6 → C-5a, 7, 8, 7″; H-7 → C-5a, 8; H-8 → C-6, 7, 10; H-10 → C-5a, 8, 10a; H2-11 → C-1, 10, 10a, 11a; H-2′/6′ → C-4′, 2′/6′, 7′; H-3′/5′ → C-1′, 3′/5′; H-4′ → C-2′/6′; H2-7′ → C-3, 4, 1′, 2′/6′; H-2″ → C-1″, 3″, 4″, 6″, 7″; H-5″ → C-1″, 3″, 4″, 6″; H-6″ → C-2″, 4″; 2-CH3 → C-1, 3; 4-OCH3 → C-4; 3″−OH → C-2″, 3″, 4″; HRESIMS m/z 575.0918 [M + Na]+ (calcd for C27H24N2O7S2Na, 575.0923). Emestrin K (4): white powder; [α]25D −6 (c 0.087, MeOH); UV (MeOH) λmax (log ε) 208 (4.52), 260 (4.08), 292 (3.74) nm; ECD (c 29 μM, MeOH) λmax (Δε) 209 (−15), 221 (−29), 274 (+11), 286 (+9), 292 (+10) nm; 1H and 13C NMR data see Table 2; HMBC data (CDCl3, 500 MHz) H-5a → C-6; H-6 → C-5a, 7, 8; H-7 → C-5a, 10; H-8 → C-6, 10, 10a; H-10 → C-5a, 8; H2-11 → C-5a, 10, 10a, 11a; H2′/6′ → C-4′, 2′/6′, 7′; H-3′/5′ → C-1′, 3′/5′; H-4′ → C-2′/6′; H2-7′ → C-3, 4, 1′, 2′/6′; H-2″ → C-1″, 3″, 4″, 6″, 7″; H-5″ → C-1″, 3″, 4″, 6″; H-6″ → C-2″, 4″, 7″; 2-CH3 → C-1, 3; 3-SCH3 → C-3; 11a-SCH3 → C-11a; 4-OCH3 → C-4; 3″-OH → C-2″, 3″, 4″; HRESIMS m/z 589.1450 [M + Na]+ (calcd for C29H30N2O6S2Na, 589.1443). Secoemestrin D (5), emestrin C (6), MPC1001C (7), emestrin D (8), emestrin E (9), MPC1001F (10), and MPC1001H (11): 1H NMR, 13 C NMR, and MS data were consistent with literature values.5−7 Antifungal Assays. In vitro antifungal activity was measured according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations.30 The MIC was determined by means of the serial dilution method in 96-well plates with YM (1% maltose extract, 0.2% yeast extract, and 0.4% agar) as the test medium. Amphotericin B was used as the positive control. Test compounds were dissolved in DMSO and serially diluted in growth medium. Visual end point and the optical density readings of microplate wells were measured relative to positive and negative controls. The strains were incubated at 25 °C, and the MICs were determined at 24 h for C. albicans ATCC 10231 and at 48 h for C. neoformans H99. Viability was determined with the aid of a plate reader using PrestoBlue resazurin dye (Life Technologies) as the viability indicator. The spectrophotometric MIC value was defined as the lowest concentration of a test compound that resulted in a culture with a density equal to 100% inhibition when compared to the growth of the untreated control.

Extraction and Isolation. The wheat cultures of TTI-000248 were combined and extracted repeatedly with MEK (3 × 1.0 L), and the organic solvent was evaporated to dryness under vacuum to afford a crude extract (5.0 g), which was fractionated by silica gel VLC using n-hexane−EtOAc gradient elution. The fraction (300 mg) eluted with 50% EtOAc was separated further by Sephadex LH-20 column chromatography using 1:1 CH2Cl2−MeOH as the eluent, and resulting subfractions were further purified by RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 44−53% MeCN in H2O with 0.1% formic acid over 55 min; 2 mL/min) to afford 1 (2.0 mg, tR 23.0 min), 2 (4.0 mg, tR 44.5 min), and 5 (2.5 mg, tR 49.5 min). The silica column fraction eluted with 70% EtOAc (490 mg) was separated again by Sephadex LH-20 column chromatography eluting with 1:1 CH2Cl2−MeOH, and the resulting subfractions were combined and further purified by RP HPLC (50% MeCN in H2O with 0.1% formic acid for 40 min) to afford 10 (5.5 mg, tR 17.4 min), 11 (4.1 mg, tR 19.1 min), 8 (2.6 mg, tR 21.6 min), 4 (5.1 mg, tR 23.9 min), 9 (1.5 mg, tR 25.4 min), 6 (12.6 mg, tR 30.9 min), and 2 (6.1 mg, tR 36.7 min). Similarly, the wheat cultures of TTI-000313 were combined and extracted repeatedly with MEK (3 × 1.0 L), and the organic solvent was evaporated to dryness under vacuum to afford a crude extract (3.0 g), which was fractionated on a silica gel column (20−100% CH2Cl2 in n-hexane over 20 min; 40 mL/min) using a Grace Reveleris X2 flash chromatography system. Fractions (550 mg) eluted with 60%, 70%, and 80% CH2Cl2 were combined and fractionated by Sephadex LH-20 column chromatography using 1:1 CH2Cl2−MeOH as the eluent. The resulting subfractions were further purified by RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 50% MeCN in H2O with 0.1% formic acid over 50 min; 2 mL/min) to afford 6 (14.5 mg, tR 30.9 min), 3 (3.5 mg, tR 35.6 min), 2 (8.5 mg, tR 36.7 min), 5 (6.2 mg, tR 39.3 min), and 7 (4.5 mg, tR 48.5 min). Fractions (95 mg) eluted with 85% and 90% CH2Cl2 were combined and further purified by RP HPLC (using the same gradient as in purification of 6) to afford 8 (8.2 mg, tR 21.6 min) and 9 (8.9 mg, tR 25.4 min). Emestrin H (1): white powder; [α]25D −97 (c 0.071, MeOH); UV (MeOH) λmax (log ε) 208 (4.22), 307 (3.34) nm; ECD (c 53 μM, MeOH) λmax (Δε) 203 (+2), 227 (−23), 300 (+4) nm; 1H NMR, 13C NMR, and HMBC data see Table 1; HRESIMS m/z 441.0922 [M + Na]+ (calcd for C20H22N2O4S2Na, 441.0919). Preparation of (R)- (1a) and (S)-MTPA (1b) Esters. A solution of 1 (1.0 mg, 0.005 mmol) in CHCl3 was transferred to an NMR tube, and the solvent was then removed under vacuum. Pyridine-d5 (0.5 mL) and (S)-MTPACl (4.5 μL, 0.025 mmol) were added into the NMR tube, and all contents were mixed thoroughly by shaking the NMR tube. The reaction was performed at room temperature, and the solution was allowed to stand for 24 h. 1H NMR data for the resulting R-MTPA ester (1a) were obtained without purification: 1H NMR data for key signals (pyridine-d5, 500 MHz) δ 6.57 (1H, s, H-10), 6.50 (1H, dd, J = 8.3, 2.3 Hz, H-8), 5.70 (1H, d, J = 8.3 Hz, H-6), 5.48 (1H, d, J = 8.3 Hz, H-7), 4.98 (1H, d, J = 8.3, Hz, H-5a), 2.30 (3H, s, 11aSCH3), 2.28 (3H, s, 3-SCH3). In a similar fashion, another sample of 1 (1.0 mg, 0.005 mmol), (R)MTPACl (4.5 μL, 0.025 mmol), and pyridine-d5 (0.5 mL) were allowed to react in an NMR tube at ambient temperature for 24 h and processed as described above for 1a to afford 1b: 1H NMR (pyridined5, 500 MHz) δ 6.57 (1H, s, H-10), 6.46 (1H, dd, J = 8.0, 2.3 Hz, H8), 5.74 (1H, d, J = 8.0 Hz, H-6), 5.42 (1H, t, J = 8.0 Hz, H-7), 5.16 (1H, d, J = 8.0 Hz, H-5a), 2.55 (3H, s, 11a-SCH3), 2.31 (3H, s, 3SCH3). Emestrin I (2): white powder; [α]25D +6 (c 0.045, MeOH); UV (MeOH) λmax (log ε) 208 (4.39), 261 (3.81), 296 (3.39) nm; ECD (c 38 μM, MeOH) λmax (Δε) 211 (−1), 223 (−12), 228 (−11), 232 (−11), 295 (+3) nm; 1H and 13C NMR data see Table 2; HMBC data (CDCl3, 500 MHz) H-5a → C-6, 10a; H-6 → C-5a, 7; H-7 → C-5a, 6, 8; H-8 → C-6, 7, 10; H-10 → C-5a, 8, 10a, 11; H2-11 → C-1, 5a, 10, 10a, 11a; H-2′/6′ → C-4′, 2′/6′, 7′; H-3′/5′ → C-1′, 3′/5′; H-4′ → C-2′/6′; H2-7′ → C-3, 4, 1′, 2′/6′; H-2″ → C-1″, 3″, 4″, 6″, 7″; H-5″ → C-1″, 3″, 4″, 6″; H-6″ → C-2″, 4″, 7″; 2-CH3 → C-1, 3; 3-SCH3 → C-3; 11a-SCH3 → C-11a; 4-OCH3 → C-4; 3″−OH → C-2″, 3″, 4″;

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00498. 1 H NMR, 13C NMR, and ECD spectra of 1−4 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the University of Texas Health Science Center at Houston new faculty start-up funds and the Kay and Ben Fortson Professorship to G.B.

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REFERENCES

(1) May, R. C.; Stone, N. R.; Wiesner, D. L.; Bicanic, T.; Nielsen, K. Nat. Rev. Microbiol. 2016, 14, 106−117.

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(2) Desnos-Ollivier, M.; Patel, S.; Raoux-Barbot, D.; Heitman, J.; Dromer, F. mBio 2015, 6, e00311. (3) Beardsley, J.; Wolbers, M.; Kibengo, F. M.; Ggayi, A. B.; Kamali, A.; Cuc, N. T.; Binh, T. Q.; Chau, N. V.; Farrar, J.; Merson, L.; Phuong, L.; Thwaites, G.; Van Kinh, N.; Thuy, P. T.; Chierakul, W.; Siriboon, S.; Thiansukhon, E.; Onsanit, S.; Supphamongkholchaikul, W.; Chan, A. K.; Heyderman, R.; Mwinjiwa, E.; van Oosterhout, J. J.; Imran, D.; Basri, H.; Mayxay, M.; Dance, D.; Phimmasone, P.; Rattanavong, S.; Lalloo, D. G.; Day, J. N.; CryptoDex, I. N. Engl. J. Med. 2016, 374, 542−554. (4) Bills, G. F.; Gloer, J. B.; An, Z. Q. Curr. Opin. Microbiol. 2013, 16, 549−565. (5) Xu, Y. M.; Espinosa-Artiles, P.; Liu, M. X.; Arnold, A. E.; Gunatilaka, A. A. J. Nat. Prod. 2013, 76, 2330−2336. (6) Onodera, H.; Hasegawa, A.; Tsumagari, N.; Nakai, R.; Ogawa, T.; Kanda, Y. Org. Lett. 2004, 6, 4101−4104. (7) Herath, K. B.; Jayasuriya, H.; Ondeyka, J. G.; Polishook, J. D.; Bills, G. F.; Dombrowski, A. W.; Cabello, A.; Vicario, P. P.; Zweerink, H.; Guan, Z.; Singh, S. B. J. Antibiot. 2005, 58, 686−694. (8) Seya, H.; Nakajima, S.; Kawai, K.; Udagawa, S. J. Chem. Soc., Chem. Commun. 1985, 657−658. (9) Seya, H.; Nozawa, K.; Nakajima, S.; Kawai, K. I.; Udagawa, S. I. J. Chem. Soc., Perkin Trans. 1 1986, 109−115. (10) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512−519. (11) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092−4096. (12) Kirby, G.; Robins, D.; Stark, W. J. Chem. Res., Synop 1986, 302− 303. (13) Dolan, S. K.; O’Keeffe, G.; Jones, G. W.; Doyle, S. Trends Microbiol. 2015, 23, 419−428. (14) Scharf, D. H.; Chankhamjon, P.; Scherlach, K.; Heinekamp, T.; Willing, K.; Brakhage, A. A.; Hertweck, C. Angew. Chem., Int. Ed. 2013, 52, 11092−11095. (15) Scharf, D. H.; Remme, N.; Habel, A.; Chankhamjon, P.; Scherlach, K.; Heinekamp, T.; Hortschansky, P.; Brakhage, A. A.; Hertweck, C. J. Am. Chem. Soc. 2011, 133, 12322−12325. (16) Forseth, R. R.; Fox, E. M.; Chung, D.; Howlett, B. J.; Keller, N. P.; Schroeder, F. C. J. Am. Chem. Soc. 2011, 133, 9678−9681. (17) Davis, C.; Carberry, S.; Schrettl, M.; Singh, I.; Stephens, J. C.; Barry, S. M.; Kavanagh, K.; Challis, G. L.; Brougham, D.; Doyle, S. Chem. Biol. 2011, 18, 542−552. (18) Seya, H.; Nozawa, K.; Udagawa, S.; Nakajima, S.; Kawai, K. Chem. Pharm. Bull. 1986, 34, 2411−2416. (19) Herath, H. M.; Jacob, M.; Wilson, A. D.; Abbas, H. K.; Nanayakkara, N. P. Nat. Prod. Res. 2013, 27, 1562−1568. (20) Tsumagari, N.; Nakai, R.; Onodera, H.; Hasegawa, A.; Rahayu, E. S.; Ando, K.; Yamashita, Y. J. Antibiot. 2004, 57, 532−534. (21) Terao, K.; Ito, E.; Kawai, K.; Nozawa, K.; Udagawa, S. Mycopathologia 1990, 112, 71−79. (22) Schuber, P. T.; Williams, R. M. Heterocycles 2012, 84, 1193− 1207. (23) Schuber, P. T., Jr.; Williams, R. M. Tetrahedron Lett. 2012, 53, 380−382. (24) Peng, J.; Clive, D. L. J. Org. Chem. 2009, 74, 513−519. (25) Peng, J.; Clive, D. L. Org. Lett. 2007, 9, 2939−2941. (26) Kawai, K.; Ishizaki, K.; Nakamaru, T.; Hisada, K.; Nozawa, Y.; Kawai, K. I. Mycotoxin Res. 1989, 5, 2−8. (27) Jayanetti, D. R.; Yue, Q.; Bills, G. F.; Gloer, J. B. J. Nat. Prod. 2015, 78, 396−401. (28) Kruys, A.; Huhndorf, S. M.; Miller, A. N. Fungal Divers. 2015, 70, 101−113. (29) Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. Mol. Biol. Evol. 2013, 30, mst197. (30) National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts. Clinical and Laboratory Standards Institute. Approved standard M27-A3, 3rd ed.; Wayne, PA, 2008.

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