Fungal ABC Transporter-Associated Activity of Isoflavonoids from the

Article pubs.acs.org/jnp

Fungal ABC Transporter-Associated Activity of Isoflavonoids from the Root Extract of Dalea formosa Gil Belofsky,*,† Marcin Kolaczkowski,*,‡ Earle Adams,§ John Schreiber,† Victoria Eisenberg,† Christina M. Coleman,⊥ Yike Zou,⊥ and Daneel Ferreira⊥ †

Department of Chemistry, Central Washington University, Ellensburg, Washington 98926, United States Department of Biophysics, Wrocław Medical University, Wrocław, Poland PL-50-368 § Department of Chemistry, The University of Montana, Missoula, Montana 59812, United States ⊥ Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States ‡

S Supporting Information *

ABSTRACT: New potential treatments for disseminated fungal infections are needed, especially for infections caused by the commonly drug-resistant pathogens Candida albicans and C. glabrata. These pathogens cause systemic candidiasis, a significant cause of mortality in immune-compromised patients. ABC transporters of the pleiotropic drug resistance subfamily, such as Cdr1p of C. albicans, play an important role in antifungal resistance and are potential bioassay targets for antifungal therapies against drug-resistant pathogens. We observed strong antifungal growth inhibitory activity in the methanol extract of Dalea formosa roots. This extract afforded six new isoflavonoids, sedonans A−F (1−6), a new but-2-enolide, 4′-O-methylpuerol A (7), and the new pterocarpan entsandwicensin (8). The structures and absolute configurations of these compounds were assigned using spectroscopic and chiroptical techniques. The direct antifungal activity of 1 against C. glabrata (MIC = 20 μM) was higher than that of fluconazole. Sedonans A−F and ent-sandwicensin were also active against Saccharomyces cerevisiae strains that express differing ABC transporter-associated resistance mechanisms but differed in their susceptibility to Cdr1p-mediated detoxification. A sedonan A (1)/ent-sandwicensin (8) combination exhibited synergistic growth inhibition. The results demonstrate that multiple crude extract compounds are differentially affected by efflux-mediated resistance and are collectively responsible for the observed bioactivity. lants produce numerous “secondary” metabolites that play important roles in the establishment of plant−fungal interactions. Increasing evidence suggests that cellular transport via proteins of the ABC transporter superfamily plays a key role in this process. Members of this ubiquitous group have been observed to play a role in the translocation of secondary metabolites not only between different plant organs1 but also to their external environment, thereby affecting the microbial communities in their immediate surroundings.2−4 These external effects range from the signaling necessary for the establishment of symbiotic interactions to toxicity that helps to eliminate pathogens. A number of phytoalexins, produced in increased amounts upon pathogen exposure, have been shown to exhibit direct antifungal activity5,6 and may serve a protective function against infection.7,8 In turn, fungi have been shown to respond to plant phytoalexins by inducing the expression of multidrug resistance (MDR) ABC transporters. Plant−fungal chemical interactions may therefore play a role in pathogenesis and fungal resistance development.9 Cytotoxic compounds of plant origin have been found to be substrates for multiple eukaryotic ABC transporters, such as

P

© XXXX American Chemical Society and American Society of Pharmacognosy

human P-glycoprotein (P-gp). Induced overexpression of P-gp leads to chemotherapy resistance of cancer cells.10 The best characterized functional homologue of P-gp found in fungi is Pdr5p of Saccharomyces cerevisiae. It shares common substrates and inhibitors with P-gp.11 PDR5 and its homologous genes SNQ2 and YOR1 are the major targets of transcriptional activation by the master regulators of yeast pleiotropic drug resistance (PDR), Pdr1p and Pdr3p.12 These major members of the pleiotropic drug resistance network of genes have been shown to mediate resistance to many structurally varied compounds, including most classes of prescribed antifungals and plant natural product crude extracts or purified compounds.13−15 This broad type of resistance to small molecules increases the likelihood of success of the yeast S. cerevisiae on fruits and other plant materials that are often rich in defensive secondary metabolites. Similar efflux-mediated resistance mechanisms operate in pathogenic yeast of the genus Candida,16 the most frequent Received: January 25, 2013

A

dx.doi.org/10.1021/np4000763 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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determined by NMR and HRMS techniques, and the absolute configurations of compounds 1−7 were examined by electronic circular dichroism. Details of the isolation, structural elucidation, and differential antifungal activities of these metabolites are reported herein.

causal agents of human fungal infections in patients with compromised immune function.17 Cdr1p is the major multidrug transporter of C. albicans and shares extensive sequence identity and functional similarity to Pdr5p of S. cerevisiae.13,14,18−20 Transient or chronic induction of Cdr1p expression leads to increased resistance to antifungal drugs, especially to the azoles, a mainstay of antifungal therapy.21 Of further interest is C. glabrata, the second most frequent pathogenic yeast in immunocompromised patients, which is intrinsically less susceptible to azoles than C. albicans.17 Synergistic and other interactions between multiple components are widely thought to be responsible for the observed activity of crude plant extracts.22 Prior work that sheds light on possible synergistic mechanisms involving multidrug transporters includes the potentiating effect of 5-Omethylhydnocarpin on the antibiotic activity of berberine against Staphylococcus aureus. Termed an “efflux pump” inhibitor, 5-O-methylhydnocarpin effectively inhibited the MDR pump NorA-mediated efflux of berberine.23 Similar “efflux pump” inhibitors have also been shown to act as ABC transport inhibitors in eukaryotic cells.15,24,25 Such agents may work in combination with antimicrobials to overcome certain MDR resistance mechanisms that are dependent on overexpression of ABC transporters. The broad substrate specificity of many biosynthetic enzymes often results in a series of structurally related biochemicals with similar biological functions.26 Fungal MDR mechanisms mediated by ABC transporters may have coevolved accordingly to expel compounds with diverse chemotypes such as defensive phytochemicals. Such complex plant−fungal interactions represent a relatively unexplored area of interest, and a better understanding of such interactions could facilitate the identification of new antifungal agents and the development of alternative treatment methods less susceptible to resistance development. Use of the well-characterized PDR network of the model eukaryote S. cerevisiae enabled us to probe antifungal activity that is dependent on the function of the major yeast effectors of MDR. Dalea formosa Torr (Fabaceae), the striking “featherplume Dalea”, is a woody shrub native to the Southwestern United States and Northern Mexico. A recent phylogenetic study places Dalea in the tribe Amorpheae, along with the closely related “daleoid” genera Marina and Psorothamnus.27 Members of the genus Dalea have been the subject of prior chemical investigations that have yielded a variety of biologically active phenolic compounds.28−33 One prior chemical study of D. formosa focused on essential oil composition.34 The leaves of the plant were used in Native American traditional medicine, as a cathartic by the Jemez, and as an emetic and strengthener by the Keres.35 The twigs and leaves were used by Pueblo and Apache for analgesic purposes.36 The plant was used by Hopi for influenza and other respiratory infections.36 In preliminary testing, a crude extract of aerial portions of D. formosa was found to exhibit antifungal activity, prompting further study.14 From a re-collection of plant material, the methanolic extract of the roots was found to exhibit strong activity against wild-type and model fungal strains expressing different aspects of effluxmediated resistance mechanisms. Fractionation of the root extract of D. formosa by silica gel VLC, Sephadex LH-20, and gradient chromatography over silica gel afforded the six new isoflavonoids sedonans A−F (1− 6), a new but-2-enolide, 4′-O-methylpuerol A (7), and the new pterocarpan ent-sandwicensin (8). Their structures were

■

RESULTS AND DISCUSSION The HRESIMS, 13C NMR (Table 1), and DEPT data for compound 1 indicated a molecular formula of C21H20O6. The 13 C NMR spectrum in acetone-d6 exhibited a duplication of signals for at least six carbons, likely due to restricted rotation about the C-3−C-1′ bond. A 2:1 mixture of CDCl3−methanold4 was found to coalesce these signals into single resonances. Methine (δH 4.12; Table 1) and oxymethylene (δH 4.34 and 4.12) proton signals of the C-ring spin system correlated to carbons at δC 70.9 and 47.8 in the HSQC spectrum, a pattern characteristic of an isoflavanone core structure. The 13C and DEPT data indicated the presence of 10 quaternary carbons, including a ketocarbonyl at δC 198.2 and five oxygenated sp2 carbons between δC 151 and 168. These data suggested a substituted isoflavanone structure bearing a methoxy group (δC 56.0), and its formula suggested the additional presence of a prenyl group. The overall structural connectivity was established by HSQC and HMBC spectroscopic data (Figures S6 and S7, Supporting Information) and by comparison with known compounds.31,37 HMBC correlations verified the position of cyclization of the C-3′ prenyl group. The assignment of C-4′ (δC 155.8) was derived from its correlations with the O-methyl protons and H-1″. Also, H-1″ and H-3 both correlated to C-2′ (δC 151.9), indicating this to be the point of attachment of the C-3″ oxygen. When taken in conjunction B



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Table 1. NMR Data for Isoflavones 1−3 1 (2:1 CDCl3−methanol-d4) δ Ca

δHb (mult.; JHH)

δ Ca

2α 2β 3 4 5 5-OH 6 7 8 9 10 1′ 2′ 3′ 4′ 4′-OCH3 5′ 6′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴ 3‴

70.9

4.50 (br t; 11.1) 4.34 (dd; 11.1, 5.7) 4.12 (dd; 11.1, 5.7)

73.2

47.8 198.2 164.9

δHb (mult.; JHH) 4.73 (d; 11.2) 4.36 (d; 11.2)

74.4 195.2 161.6

3 (acetone-d6) δCa 71.0 48.1 198.7 162.7c

11.74 (s) 97.0c 167.2 95.9c 164.3 103.4 115.5 151.9 111.4 155.8 56.0 103.7 130.8 117.2 129.4 77.2 28.4 27.9

5.94 (d; 2.1) 5.90 (d; 2.1)

3.78 6.40 6.88 6.60 5.52

(s) (d; (d; (d; (d;

8.5) 8.5) 10.0) 10.0)

1.31 (s) 1.30 (s)

4‴ 5‴ a

2 (CDCl3)

position

107.9 165.4 96.0 161.2 101.9 115.1 156.2c 107.5 156.8c

5.98 (s)

6.45 (s)

124.4 124.6 21.4 121.2 136.1 26.0 18.1 39.9 148.0 113.8

6.89 (s) 3.37 (d; 7.2) 5.23 (br t; 7.2) 1.77 (s) 1.82 (s) 6.08 5.29 5.25 1.24

27.1 26.9

(dd; 17.7, 10.5) (d; 17.7) trans (d; 10.5) cis (s)

δHb (mult.; JHH) 4.60 (t; 10.8) 4.41 (dd; 5.4, 10.8) 4.20 (dd; 5.4, 10.8)

12.68 (s) 109.1 164.7 95.1 162.2c 103.5 113.3 155.2 105.0 156.7 126.2 130.0 21.8 123.8 131.2 26.0 17.9 40.7 149.1 110.4 27.5 27.5

6.02 (s)

6.44 (s)

7.00 (s) 3.25 (d; 7.1) 5.24 (br t; 7.1) 1.63 (s) 1.74 (s) 6.22 4.96 4.90 1.41

(dd; 10.7, 17.6) (dd; 1.2, 17.6) trans (dd; 1.2, 10.7) cis (s)

75 MHz. b400 MHz. cAssignments may be interchanged.

correlations between H2-1″ and H-2‴ and the A- and B-ring carbons, respectively. The core A- and C-ring arrangement was supported by its characteristic 13C NMR pattern and by the sharp singlet (δH 11.74) representing the hydrogen-bonded hydroxy proton. This hydroxy proton and H2-1″ both correlated to C-5 (δC 161.6) in the HMBC spectrum, thus facilitating its differentiation from C-9 (δC 161.2). The C-3 absolute configuration of compound 2 was defined by comparison of experimental ECD data (Figure 1) with reported data.41,42 These authors assigned the absolute configuration of a series of 3-hydroxyisoflavanones by comparison of the ECD data with those of isoflavanones, i.e., the CEs of the n → π* and π → π* electronic transitions. Thus, based on the presence of a positive CE for the n → π* transition and a negative CE for the π → π* transition in the respective 320−350 and 260−310 nm regions of the experimental ECD spectrum, the C-3 absolute configuration of compound 2 was assigned as S.38 It should be emphasized that the orientation of the B-ring is the same for (3R)isoflavanones and (3S)-hydroxyisoflavanones due to a change in the Cahn-Ingold−Prelog (CIP) preferences when going from −H to −OH at C-3. Indeed, erroneous assignment of CIP preferences combined with misinterpretation of experimental ECD data has led to the incorrect assignment of the absolute configuration for the 3-hydroxyisoflavanones phyllanone A42 and the sophoronols41 when experimental ECD data indicated (3S)- and (3R)-configurations, respectively. Such errors in configurational assignment combined with questions regarding

with the aromatic AB-spin system comprised of H-5′ and H-6′ (δH 6.40, 6.88, respectively; J5′,6′ = 8.5 Hz), these data defined the 3-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl) substituent. The remainder of the structure was readily determined by HSQC and HMBC analyses. Electronic circular dichroism (ECD) was used to define the absolute configuration at C-3 in compound 1. The ECD spectrum showed a broad, low-amplitude, local minimum around 320 nm and lacked distinct Cotton effects (CEs) in the 200−240, 260−300, and 320−352 nm regions expected for ECD spectra of enantiopure or enantio-enriched isoflavanones.38 Racemization of isoflavanones during the process of extraction and isolation is fairly common,39 due to the lability of the C-3 hydrogen. The specific rotation of +6.0 indicated a slight excess of the dextrorotatory enantiomer, but generally supported the conclusions drawn from the ECD data. Thus, compound 1 comprised a racemic mixture of the new isoflavanone 5,7-dihydroxy-3-(5-methoxy-2,2-dimethyl-2Hchromen-8-yl)chroman-4-one, named sedonan A. The molecular formula, C25H28O7, of compound 2 (sedonan B) was corroborated by HRESIMS. TOF positive mode analysis revealed a significant ion at m/z 301/302 that likely resulted from the fragmentation of two prenyl groups. The presence and position of the C-3 hydroxy group was evident from HMBC correlations (Figure S11, Supporting Information) from H2-2 and H-6′ (δH 6.89) to C-3 (δC 74.4) and by analogy to sedonan C (3) and known compounds.31,40 The location of the two prenyl groups was evident from HMBC C



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of 2 and the known compound dalversinol A, which differs from 3 with regard to the position of the A-ring prenyl group.31 The point of attachment of this group was determined to be C6 (δC 109.1) by the presence of a HMBC correlation from H2″. The latter signal also correlated to C-5 and C-7 (δC 162.7 and 164.7, respectively). Key correlations from the C-5 hydrogen-bonded hydroxy proton (δH 12.68) to C-5, C-6 (δC 109.1) and C-10 (δC 103.5) further verified the position of the A-ring prenyl group. The remainder of the structure of 3 was supported by observed HMBC correlations (Figure S16, Supporting Information) and agreed with the assignments for 2 and known compounds.31,40,43 The ABC spin system of the heterocyclic protons was consistent with their chemical shifts, observed coupling constants (e.g., the smaller coupling constant between H-2β and H-3, Table 1), and a comparison with literature examples.17 As with compound 1, the ECD spectrum of compound 3 lacked the significant CEs of enantiopure or enantio-enriched isoflavanones; this observation was further supported by the absence of optical activity ([α]D = 0). Compound 3 was therefore determined to be a racemic mixture of the new isoflavanone 3-[2,4-dihydroxy-5-(2-methylbut-3-en2-yl)phenyl]-5,7-dihydroxy-6-(3-methylbut-2-en-1-yl)chroman4-one and named sedonan C.37 This substitution pattern is identical to that of a flavanone, 2′,4′-dihydroxy-5′-(1‴,1‴dimethylallyl)-6-prenylpinocembrin (6PP), isolated from Dalea elegans.44 HRESIMS of compound 4 indicated a molecular formula of C21H22O5, and the 1H NMR spectrum exhibited a characteristic spin pattern for H2-2, H-3, and H2-4, reminiscent of an

Figure 1. Experimental ECD spectrum of sedonan B (2).

the direct applicability of the empirical rules for isoflavanones to the 3-hydroxyisoflavanones initiated further investigation of the configurational assignment for this compound class using computationally simulated ECD spectra, the results of which will be discussed in a separate publication. Thus, structure 2 of the new 3-hydroxyisoflavanone (S)-3-[2,4,-dihydroxy-5-(2methylbut-3-en-2-yl)phenyl]-3,5,7-trihydroxy-6-(3-methylbut2-enyl)chroman-4-one (sedonan B) was assigned as shown. HRESIMS analysis of compound 3 indicated a molecular formula of C25H28O6. Spectroscopic data for 3 resembled those Table 2. NMR Data for Isoflavans 4−6 in CDCl3 4 position

δC

2α 2β 3 4α 4β 5 6 7 7-OH 8 8-OH 9 10 1′ 2′ 2′-OCH3 3′ 4′ 4′-OCH3 5′ 6′ 1″ 2″ 3″

70.5

4″ 5″ a

a

32.1 30.1 120.1 107.9 142.4

5

δH (mult.; JHH)

δC

(ddd; 10.1, 3.3, 1.8) (dd; 10.1, 10.1) (m) (ddd; 15.7, 5.0, 1.1) (dd; 15.7, 10.8) (d; 8.4) (d; 8.4)

70.6

b

4.40 4.08 3.55 2.87 3.01 6.58 6.53

a

6

δH (mult.; JHH)

δC

(ddd; 10.2, 3.3, 1.8) (dd; 10.2, 10.2) (m) (ddd; 15.7, 5.0, 1.2) (dd; 15.7, 11.0) (d; 8.4) (d; 8.4)

70.6

b

4.38 4.08 3.57 2.86 3.03 6.56 6.51

32.3 30.3 120.0c 107.8 142.3

a

32.1 30.1 120.1 107.9 142.3c

δHb (mult.; JHH) 4.43 4.07 3.56 2.91 3.02 6.57 6.51

(br d; 10.0) (dd; 10.0, 10.0) (m) (dd; 15.8, 5.2) (dd; 15.8, 10.4) (d; 8.4) (d; 8.4)

3.81 6.47 6.94 3.47 5.24

(s) (d; 8.5) (d; 8.5) (d; 7.0) (br t; 7.0)

5.15 (s) 131.8

131.7

131.7 5.30 (s)

142.1 115.0 121.1 151.4 110.8 154.4 55.8 103.0 127.1 117.2 129.1 76.2 28.0c 27.9c

3.82 6.40 6.91 6.70 5.60

(s) (d; (d; (d; (d;

8.7) 8.7) 10.0) 10.0)

1.45 (s) 1.42 (s)

142.0c 115.0 120.6 154.0

142.0 115.1 120.1c 156.8c 55.7c 96.8 158.1c 55.6c 128.6 126.3 40.3 148.3 109.9

3.85 (s) 6.50 (s) 3.84 (s) 7.01 (s) 6.15 4.94 4.93 1.41 1.41

27.5c 27.4c

(dd; 17.2, 10.9) (d; 10.9) (d; 17.2) (s) (s)

114.8 156.7 56.0 103.2 125.4 22.7 121.7 136.2 26.1 18.1

1.79 (s) 1.86 (s)

75 MHz. b400 MHz. cAssignments may be interchanged. D



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isoflavan ring system. Three pairs of sp2 hydrogens were ocoupled, and their positions established through HMBC correlations, in particular from H-1″ to C-2′ and C-4′ of the B-ring and from H-2″ to C-3′. A key NOESY correlation between H-5′ and OCH 3 -4′ lent support to HMBC correlations (Figure S21, Supporting Information) that led to placement of the methoxy group at C-4′. HMBC correlations from H-6′ to C-3 and H2-2 to C-1′ confirmed the B- to C-ring linkage. Interestingly, the C-5 and C-1′ signals at δC 120.1 and 121.1, respectively, were indistinguishable by HMBC, but were resolved using HSQC-TOCSY. The overall structure and NMR assignments for compound 4 were consistent with data reported for similar compounds,40 and the assignments for H-2α and H-2β (Table 2) were consistent with a recent detailed report describing the C-ring conformation of the isoflavan glabridin.45 The absolute configuration of compound 4 was defined by comparison of its experimental ECD data with those of known isoflavans.38,46 The ECD spectrum of 4 (Figure S1, Supporting Information) exhibited a low-amplitude positive CE at 227 nm in the 1La transition region (220−260 nm) and a low-amplitude negative CE at 292 nm in the 1Lb transition region (260−320 nm). The broadening and bathochromically shifted locations of these electronic transitions, relative to those for compounds 5 and 6 (see below), can be attributed to the presence of the additional double bond conjugated to the aromatic B-ring. The presence of negative and positive CEs for the 1Lb and 1La bands, respectively, reflects M-helicity for the heterocyclic Cring, hence necessitating the (3S)-absolute configuration in order for the B-ring to attain a conformationally preferred αequatorial orientation.38 Compound 4 was therefore defined as the new isoflavan (S)-3-(5-methoxy-2,2-dimethyl-2H-chromen8-yl)chroman-7,8-diol and named sedonan D. HRESIMS of compound 5 indicated a molecular formula of C22H26O5, and 1H, 13C, and DEPT NMR data exhibited characteristics of an isoflavan structure, with close similarities to 4 in the assignments for the A- and C-rings. The substitution of the A-ring was established by two- and three-bond correlations from the OH-7 and OH-8 protons to their points of attachment and nearby carbons, as well as to the o-coupled H-5 and H-6 (J = 8.4 Hz). The spectroscopic data of the B-ring and the prenyl substituent were similar to that of compound 3, with the two m-hydroxy groups in 3 being replaced by O-methyl groups in 5. The locations of the two methoxy groups were confirmed by HMBC correlations (Figure S25, Supporting Information). Compound 5 had an experimental ECD spectrum (Figure S2, Supporting Information) distinct from that of compound 4, with a low-amplitude negative CE at 224 nm in the 1La transition region and a low-amplitude positive CE at 280 nm in the 1Lb transition region. Such CEs reflect P-helicity and indicate the (3R)-absolute configuration.38 Compound 5 was thus defined as the new isoflavan (R)-3-[2,4-dimethoxy-5-(2methylbut-3-en-2-yl)phenyl]chroman-7,8-diol and named sedonan E. HRESIMS of compound 6 indicated a molecular formula of C21H24O5, and its NMR spectroscopic data showed similarities to compounds 4 and 5. The locations of the o-coupled aromatic protons H-5/H-6 (J = 8.4 Hz) and H-5′/H-6′ (J = 8.5 Hz) were supported by HMBC data (Figure S29, Supporting Information). A key correlation from H-6′ to C-3 confirmed the connectivity between the B- and C-rings. Two additional hydrogens in the molecular formula of 6, compared with that of 4, were accounted for by the acyclic C-3′ prenyl group and the

C-2′ hydroxy proton. The ECD spectrum of compound 6 (Figure S3, Supporting Information) was similar to that of compound 4, with low-intensity positive and negative CEs at 218 and 286 nm in the respective 1La and 1Lb transition regions, thus indicating a (3S)-absolute configuration.38 Compound 6 was therefore defined as the new isoflavan (S)-3-[2-hydroxy-4methoxy-3-(3-methylbut-2-en-1-yl)phenyl]chroman-7,8-diol and named sedonan F. The HRESIMS data for compound 7 indicated a molecular formula of C18H16O5. Its 1H and 13C NMR data showed that it belongs to a different structural class than compounds 1−6. The 1H NMR data indicated an aromatic ring exhibiting an ABC spin pattern and a p-disubstituted benzylic ring with an AA′BB′ spin pattern. These conclusions were supported by 1D and 2D NMR spectroscopic data (Figures S30 and S33, Supporting Information) and by comparison with the closely related known compound puerol A. 47−49 Key HMBC correlations facilitated confirmation of the likely α-hydroxydihydrochalcone-derived47 core of the molecule. The three rings were linked by correlations from H-6″, H-3, and H2-5a to C-4. NOESY correlations supporting this arrangement were observed between H-6″ and both H-3 and H-5. The benzylic H2-5a correlated to both H-5 and H-2′/H-6′. A NOESY correlation between OCH3-4′ and H-3′/H-5′ confirmed the position of the methoxy group. The unambiguous assignment of the overall structure of the new compound 4′-Omethylpuerol A (7), as shown, is in alignment with previously proposed structural determinations and revisions.47,48 The chiral 5-substituted 2(5H)-furanone structural moiety in 7 is a common motif of several classes of biologically active terrestrial and marine natural products.50−52 Much effort has been devoted to the definition of the C-5 absolute configuration within this class of compounds using ECD data, especially by application of the simple helicity correlation models developed by Uchida and Kuriyami53 and Gawronski and associates.54 According to these empirical models, the C-5 absolute configuration is correlated to the sign of the CEs corresponding to the n → π* and π → π* electronic transitions, as shown in Figure 2. Gawronski and Feringa

Figure 2. Correlation of the n → π* and π → π* Cotton effects with the C-5 absolute configuration of but-3-enolides.

proposed that the CE resulting from the π → π* transition is preferred for the assignment of absolute configuration, as the sign and amplitude of the n → π* CE can be significantly affected by the electronic nature of the C-5 substituent.54 However, this work does not take into account the possible effects on the ECD spectrum when π-electron-rich C-4 substituents, such as the aryl substituent of compound 7, are present and conjugated to the core 2(5H)-furanone moiety. For compound 7, it is suspected that the presence of such extended conjugation results in a bathochromic shift for the electronic transitions of the 2(5H)-furanone unit, with the addition of electronic transitions corresponding to the aryl substituent. Accordingly, in the ECD spectrum of compound 7 (Figure 3), the negative CEs at 325 and 285 nm were assigned E



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diminishes the induction of their expression upon xenobiotic exposure. This approach allowed recognition of substrates of these major fungal ABC MDR transporters. It also allowed for monitoring of possible involvement of other resistance genes regulated by Pdr1p and Pdr3p. A complementary approach was based on verification of the effect of the hyperactive PDR1-3 mutant allele expressed in the triple knockout of PDR5, SNQ2, and YOR1. This strain served also as the host for heterologous overproduction and analysis of the interactions of the crude extract and its components with Cdr1p of C. albicans. Its increased expression in a strain devoid of major endogenous MDR transporters enhanced the sensitivity of detection during the search of potential multidrug efflux pump inhibitors.13,14 The crude D. formosa root extract showed inhibitory activity against the tested wild-type strains of the three yeast species, with greater inhibition of C. glabrata and S. cerevisiae (MIC = 30 μg/mL) than of C. albicans (MIC = 60 μg/mL). Inactivation of PDR1 and PDR3, as well as PDR5, SNQ2, and YOR1, resulted in a marked increase in activity against S. cerevisiae (MIC = 3.8 and 7.6 μg/mL respectively), indicating the presence of growth inhibitory substrates of Pdr5p, Snq2p, and Yor1p in the crude extract. The lack of an MIC increase in the hyperactive gain-of-function PDR1−3 mutant, bearing deletions in PDR5, SNQ2, and YOR1, as compared to the isogenic strain with the wild-type PDR1 allele, indicated that other PDR1 and PDR3 activated genes do not make a significant contribution to fungal resistance to the crude extract. However, a large increase in the MIC was observed upon overproduction of Cdr1p, demonstrating that the presence of this single ABC multidrug transporter of C. albicans was sufficient to reduce the growth inhibition effects of the crude extract (MIC = 244 μg/mL). No increase in specific activity against C. albicans was observed in chromatographic fractions until purified samples of 1, 3, 5, and 6 became available. In the case of the most active compound, sedonan A (1), the growth inhibitory activity against C. albicans (MIC = 15 μg/mL) and C. glabrata (MIC = 7.6 μg/mL) increased only 4-fold as compared to the crude extract, while a 2-fold increase was observed with 3, 5, and 6 against strains of both species. Considering the large increase in purity from the crude extract, these data suggested that loss of combined effects for components within the crude mixture could be responsible for the discrepancy between enhanced purity and the resulting specific activity. The activity of some of the purified compounds against both Candida species did not increase and was equal to that of the crude extract. These included sedonan D (4) (C. glabrata MIC = 30.5 μg/mL, C. albicans MIC = 61 μg/mL) and ent-sandwicensin (8) (C. glabrata MIC = 30.5 μg/mL). In contrast, the activity of sedonan B (2) and 4′-O-methylpuerol A (7) was decreased (C. glabrata MIC = 122 and 244 μg/mL, respectively) relative to the activity of the crude extract. Both 2 and 7 were inactive against C. albicans, as was ent-sandwicensin (8), which was in agreement with a prior report for sandwicensin.56 It has been generally observed that C. glabrata is more sensitive to plant natural products than to the synthesized and frequently prescribed drug fluconazole, to which C. albicans is more sensitive.14,15 The exact amounts of 1−8 in the crude extract were uncertain due to the complexity of the mixtures. Isolated yields suggested that 1 and 2 were major components at approximately 0.4% and 1.2% by weight, while 3−8 ranged from 0.04% to 0.21%. In other terms, sedonan A (1) was present in this extract at approximately 3900 ppm, while its

Figure 3. Experimental ECD spectrum of 4′-O-methylpuerol A (7).

tentatively to the n → π* and π → π* electronic transitions of the 2(5H)-furanone moiety, respectively, and the positive CE at 251 nm to transitions of the C-4 aryl substituent. In terms of the correlation model (Figure 2), the negative CE at 285 nm, as the π → π* electronic transition, would therefore be preferred for the assignment of configuration and would indicate Mhelicity and (5R)-absolute configuration. Such an assignment was supported by a specific rotation of −43 in MeOH, consistent with the negative and positive values reported for a series of synthetic (5R)- and (5S)-4-phenyl-2(5H)-furanones, respectively.55 The structure of compound 7, 4′-O-methylpuerol A, has therefore been tentatively assigned as (R)-4-(2,4dihydroxyphenyl)-5-(4-methoxybenzyl)furan-2(5H)-one. The influence of extended conjugation on the n → π* and π → π* electronic transitions of the 2(5H)-furanone class of compounds and the effects of such conjugation on the interpretation of the ECD spectra, in terms of C-5 absolute configuration, will be described elsewhere. The NMR data of compound 8 (Figures S34−37, Supporting Information) was identical to those of the known pterocarpan sandwicensin,37,56 with the molecular formula, C21H22O4, confirmed by HRESIMS. The (6aS, 11aS) absolute configuration of 8 was inferred from its large, positive specific rotation, which was opposite in sign but similar in magnitude to the reported value.37,56 Therefore, compound 8 was identified as the new compound ent-sandwicensin. The antifungal activities of the crude extract, chromatographic fractions, and purified new compounds A−F (1−6), 4′O-methylpuerol A (7), and ent-sandwicensin (8) were monitored by growth assays with selected yeast species, including C. albicans, C. glabrata, and several strains of S. cerevisiae (see Experimental Section). The wild-type and model strains used for the assays included those with deletions in the major transporters and regulators of yeast MDR and other strains modified to overexpress the major endogenous MDR pumps (Pdr5p, Snq2p, Yor1p) and the major MDR transporter Cdr1p of C. albicans. These S. cerevisiae strains, showing different levels of expression of resistance mechanisms affecting cell permeability to toxic insults, offered the advantage of increased sensitivity of detection of direct antifungal activity. This was achieved with deletions in the three major MDR transporter encoding genes, PDR5, SNQ2, and YOR1, and genes encoding their major transcriptional activators Pdr1p and Pdr3p. The double deletion in PDR1 and PDR3 reduces the expression of PDR5, SNQ2, YOR1, and other target genes and F



Article

MIC toward C. glabrata was in the range of 15 ppm, suggesting that these metabolites, collectively, are present at levels that may afford an effective chemical defense. The activity profile of the crude extract against different strains suggested involvement of the major components of the yeast pleiotropic drug resistance network of genes in the detoxification of D. formosa compounds. This possibility was evaluated using S. cerevisiae strains that possessed increased sensitivity to known antifungal compounds. The wild-type S. cerevisiae strain showed a similar trend to that observed with Candida spp., exhibiting a minimal increase in specific activity of pure compounds as compared to crude fractions. Purified compounds 1−6 and 8 possessed increased antifungal activity, relative to the wild-type strain, against the double knockout of PDR1 and PDR3, as well as the triple knockout of PDR5, SNQ2, and YOR1. This effect was seen irrespective of the presence or absence of the hyperactivating PDR1−3 mutation (Table 3). Additionally, changes in specific activity of crude

Figure 4. Fold of direct growth inhibitory activity change. (A) In response to deletions of genes encoding major activators of pleiotropic drug resistance, PDR1 and PDR3 (denoted Δ13), and major multidrug transporters, PDR5, SNQ2, and YOR1 (denoted Δ125), in relation to the wild-type S. cerevisiae strain. (B) After purification of 1−8 (indicated at the bottom), in relation to the activity of the crude extract, evaluated in the wild-type S. cerevisiae strain (wt) and strains bearing deletions in the major pleiotropic drug resistance genes. Changes were calculated on the basis of MIC determinations from the growth-based microdilution assay performed as described in the Experimental Section.

Table 3. Minimum Inhibitory Concentrations (MIC) [μg/ mL] of the Crude D. formosa Root Extract and Compounds (1−6 and 8) against S. cerevisiae Strainsa strain compound

wt

Δ13

Δ125

1−3Δ125

1−3Δ125 CDR1

crude extract 1 2 3 4 5 6 8

30.5 7.6 61 15.2 30.5 15.2 15.2 30.5

3.8 1.9 15.2 0.95 15.2 3.8 3.8 3.8

7.6 1.9 3.8 0.95 30.5 3.8 3.8 1.9

7.6 1.9 3.8 0.95 30.5 3.8 3.8 1.9

244 244 244 >244 244 61 30.5 >244

Figure 5. Fold increase in resistance to 1−8 upon heterologous Cdr1p overproduction in the S. cerevisiae host strain bearing deletions in major endogenous multidrug transporters encoding genes PDR5, SNQ2, and YOR1. Changes were calculated on the basis of MIC determinations from the growth-based microdilution assay performed with the Cdr1p overproducing MKCDR1h in relation to the expression host AK100, as described in the Experimental Section.

a wt, wild type; Δ13, double knockout of the major activators of yeast pleiotropic drug resistance, PDR1 and PDR3; Δ125, triple knockout of major multidrug ABC transporters, PDR5, SNQ2, and YOR1. The presence of the hyperactive PDR1−3 allele (1−3) and the heterologously overproduced Cdr1p gene (CDR1) is marked.

fractions against these hypersensitive mutants resulting from subsequent purification steps were of similar magnitude to those observed with the wild-type S. cerevisiae strain (Figure 4B) and resembled those found with C. glabrata, not exceeding 4-fold. These results suggested that synergy between the components of the crude extract may account for its high activity relative to the isolated compounds and that this synergy is irrespective of the involvement of the analyzed members of the yeast pleiotropic drug resistance network, including the heterologously overproduced Cdr1p, in the detoxification of the isolated compounds (Figures 4A and 5). This is in line with the successive analyses of the abilities of extracts, fractions, and purified compounds that did not reveal significant potentiation of Cdr1p-mediated ketoconazole resistance. The present results, identifying 3 among substrates of Cdr1p, shed new light on the possible interactions of the previously identified 6PP,44 which differed by the relative position of Band C-rings, with yeast MDR transporters. As would be expected for a putative efflux pump substrate, 6PP did not effectively inhibit the transport of Cdr1p substrate rhodamine 6G, exerting its effect only at high concentrations, otherwise toxic to yeast cells. This toxic effect of 6PP, related to its mitochondrial mechanism of action, was proposed to

contribute to the synergistic effect on growth inhibition observed with fluconazole at lower concentrations.57 For insight into the effects of compound combinations, a two-dimensional checkerboard assay was performed with sedonan A (1) and ent-sandwicensin (8). These compounds were chosen based on their strong antifungal activities and their pronounced structural differences, which likely correlate to their potential ability to affect different intracellular targets. Calculations of the corresponding fractional inhibitory concentration (FIC) and FICindex values were used to evaluate possible synergistic effects.58,59 Measurements with the wildtype S. cerevisiae strain revealed that the MIC of 1 alone, 21 μM, decreased to 2.6 μM in the presence of 8, and the MIC of 8 alone, 84 μM, decreased to 5.4 μM in the presence of 1. A similar reduction was observed with the more sensitive double PDR1 and PDR3 knockout strain: the MIC of 1 alone, 5.2 μM, decreased to 1.3 μM in the presence of 8, whereas the MIC of 8 alone, 10.4 μM, decreased to 1.3 μM in the presence of 1. The corresponding FICindex values of 0.19 (wild-type strain) and 0.37 (regulatory mutant strain) indicated synergistic interactions of the compounds against both strains.60 G



Article

Central Washington University, and has been deposited in the herbarium of the same department. Roots and aerial portions were separated, and the material was air-dried for several days and stored in a −20 °C freezer prior to extraction. Extraction and Isolation. D. formosa roots (air-dried ∼72 h; 344 g) were extracted with MeOH (4 L) by a combination of soaking (larger pieces) and processing in a Waring blender (smaller pieces) to provide, after evaporation under reduced pressure, 15.8 g of crude combined extract. This material was preadsorbed in MeOH solution onto ∼10 g of silica gel, the solvent removed under vacuum, and the resulting powder subjected to VLC over a prepacked column bed, 10 cm (i.d.) × 5 cm (h), of TLC-grade silica gel (Selecto Scientific). The column was eluted using a stepwise gradient of solvents beginning with hexanes (1 L) and continuing with mixtures (500 mL each) of EtOAc in hexanes (20, 40, 60, 80, and 100%), followed by mixtures of MeOH in CH2Cl2 (2, 5, 8, 10, and 30%). The three fractions that eluted with 20−60% EtOAc were combined on the basis of TLC analysis, and the solvents were evaporated. A portion (2.5 g) of this residue was further fractionated by Sephadex LH-20 (Sigma) CC (2.5 cm × 58 cm) eluting with 1 L of 3:1:1 (v/v) hexanes−toluene−MeOH at a flow rate of 0.3−0.5 mL/min, and collecting ∼8 mL fractions. Materials of similar composition as determined by TLC were pooled, resulting in 52 fractions. Fractions 20 and 42 from this column comprised compounds 4 (sedonan D; 32 mg) and 2 (sedonan B; 191 mg), respectively. Fractions 17 and 18 from the Sephadex LH-20 column were combined (170 mg) and dissolved in CH2Cl2 and, upon gradual addition of hexanes, compound 1 (sedonan A; 62 mg) precipitated. The remaining supernatant (112 mg) was further purified over silica gel (2.5 cm × 13 cm; 60−200 mesh, ∼20 mL/min) using a continuous linear gradient of EtOAc (0−15%) in hexanes. Fractions from this column that eluted with 12−15% EtOAc afforded compound 5 (sedonan E; 21 mg). Fractions 33 and 34 from the Sephadex LH-20 column were combined (128 mg) and further purified in a similar manner to 5, but using a continuous linear gradient of EtOAc (0− 35%) in hexanes. One fraction, eluting with 18−20% EtOAc, afforded compound 6 (sedonan F; 13 mg). The combined fractions 35−37 (191 mg) from the Sephadex LH-20 column were further purified over silica gel in three successive stages using these same gradient methods (0−50% EtOAc in hexanes, 0−5% MeOH in CH2Cl2, and finally 10−40% of a 1:1 EtOAc−CH2Cl2 mixture in hexanes) to yield compound 3 (sedonan C; 33 mg). Fractions 11 (203 mg) and 39 (40 mg) from the Sephadex LH-20 column were further fractionated using silica gel chromatography (1.5 cm × 9 cm; 60−200 mesh) with step gradients of MeOH in CH2Cl2 (0−2%) and EtOAc in hexanes (0−100%), respectively. These separations resulted in compound 8 (ent-sandwicensin; 6.5 mg) and compound 7 (4′-O-methylpuerol A; 7.5 mg). Susceptibility Testing. Inhibition of yeast growth was measured using a microdilution assay in 96-well plates. Samples were added to liquid growth medium as DMSO stock solutions, and serial dilutions were prepared before inoculation. An identical solvent amount (not exceeding 1.2%) was added to control cultures and served as a negative control, which had no effect on growth at the applied concentrations. Serial dilutions of trifluoperazine, ketoconazole, and fluconazole served as a positive control for growth inhibition and as controls for possible variations between the different runs of the assay. Growth was monitored by measuring the optical density at 550 nm (OD550) in a microplate reader as well as by visual inspection at 24 and 48 h. The lowest concentration of compound that inhibited growth by at least 80%, compared to the drug-free control, was used as the MIC breakpoint. The MIC values reported represent the values observed in three independent measurements in which the error did not exceed the window determined by the serial dilution factor. Growth assays with Candida albicans and C. glabrata (strains CCM8320 and CCM8270, respectively, from the Czech Collection of Microorganisms) were performed according to the CLSI M27-A procedure, using MOPS (morpholinepropanesulfonic acid)-buffered (pH 7) RPMI 1640 medium.65 Assays with Saccharomyces cerevisiae were performed in a similar way, as described previously.14,66

Since the double knockout of PDR1 and PDR3 exhibited a markedly decreased level of expression of the major drug efflux pumps and a decreased ability to induce them upon xenobiotic exposure,61,62 the observed synergy toward this strain therefore suggests activity toward varied intracellular targets rather than MDR efflux inhibition. The structural differences of 1 and 8, combined with their inability to potentiate Cdr1p-mediated ketoconazole resistance, support the hypothesis that these compounds act against other, as yet unidentified, intracellular targets. If drug-induced upregulation of efflux pumps, a process dependent on the major transcriptional activators of yeast pleiotropic drug resistance,62 was elicited in the described system, the combination of these two agents would be expected to result in either the loss of synergy or antagonism in the growth inhibition assay.63 This result, however, was not observed, as tests in both wild-type and knockout strains resulted in similar synergistic effects. The results described herein demonstrate that synergistic interactions between multiple growth inhibitory compounds operate in plants. This, together with the generation of closely related structural derivatives less prone to multidrug efflux, enhances the success of plants in overcoming the major fungal resistance efflux mechanisms. This knowledge may contribute to a better understanding of how plant−fungal interactions are maintained and may help guide the development of more effective ways to control fungal pathogens in both clinical and agricultural settings.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting point determinations were made on an Electrothermal Mel-Temp, model 1001. Optical rotations were recorded on a Perkin-Elmer 341 polarimeter (Na lamp, 589 nm). UV spectra were recorded on an HP-Agilent 8453 photodiode array instrument. ECD spectra were obtained at room temperature using either an OLIS DSM 20 CD spectrophotometer or a JASCO J-815 CD spectrometer with a 1 cm path length cylindrical quartz cuvette and high-purity MeOH (Fisher Scientific). Spectra for each compound were obtained at multiple concentrations in order to determine the concentration that gave the highest quality data (see Table S1). To correct for small amounts of noise, a “digital filter” smoothing algorithm64 as provided by the OLIS GlobalWorks software was applied to all raw OLIS data, producing the curves shown in Figures 3 and S2 (Supporting Information), and a binomial smoothing algorithm with 10 passes, as provided with the JASCO Spectra Manager Ver. 1.54 software, was applied to all raw JASCO data, producing the curves shown in Figures 1 and S1 and S3 (Supporting Information). IR spectra were recorded on a Nicolet Protégé 460 spectrometer. NMR spectra (CWU) were obtained on a Bruker Avance 400 MHz system with Topspin 1.3 software. 2D NMR spectra (Montana) were performed on Varian VNMRS systems: at 500 MHz using inverse and broadband autotunable probes and at 600 MHz using a triple resonance, HCN and salt-tolerant 13C-enhanced cryoprobe. HRESIMS were run on a Waters Q-TOF Premier quadrupole time-of-flight hybrid mass spectrometer. A Waters Acquity UPLC was used to inject samples into 1:1 MeCN−H2O using flow injection analysis (100 μL/min) with no column. Positive electrospray ionization was used to generate [M + H]+ or [M + Na]+ ions. TLC plates (EMD Chemicals, Inc.; silica gel 60, F254) were eluted with 9:1 CH2Cl2−MeOH or mixtures of hexanes−EtOAc and visualized with UV (254 nm) and the spray reagent vanillin/H2SO4 (1 g/100 mL w/ v) after gentle heating. Plant Material. Whole plants of D. formosa were collected by G.B. on April 16, 2009, 1−200 m from the roadside on Yavapai County Rd. 78, 0.8 mile from Hwy. 179 near Sedona, AZ, GPS coordinates N 34°44.245′, W 111°47.257′, alt. 3392 ft. A voucher specimen was authenticated by Dr. Tom Cottrell, Department of Biological Sciences, H



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Sedonan D (4): off-white solid; mp 75−82 °C; [α]20D +23 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 226 (4.09), 279 (3.71) nm; ECD (c 0.002, MeOH) λmax (θ); 210 (−5.9 × 103), 227 (4.8 × 103), 249 (1.8 × 103), 292 (−1.6 × 103) nm; IR (film on NaCl) νmax 3439 (br OH), 2973, 2928, 1637, 1595, 1492, 1280, 1246, 1204, 1095 cm−1; 1H and 13C NMR data, see Table 2; HMBC correlations (CDCl3) H-2α → C-3, 4, 9, 1′; H-2β → C-3, 4, 9; H2-4 → C-2, 3, 5, 9, 10; H-5 → C4, 7, 8*, 9; H-6 → C-7, 8, 10; OCH3-4′ → C-4′; H-5′ → C-1′, 2′*, 3′, 4′, 1″*; H-6′ → C-3, 2′, 3′*, 4′; H-1″ → C-2′, 3′, 4′, 3″; H-2″ → C-3′, 3″, 4″, 5″; H3-4″ → C-1″*, 2″, 3″, 5″; H3-5″ → C-1″*, 2″, 3″, 4″ (* indicates weak four-bond correlation); HRESIMS found m/z 355.1556 [M + H]+, calcd for C21H23O5, 355.1545. Sedonan E (5): white solid; mp 64−70 °C; [α]20D +22 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 209 (3.78), 236 (3.53), 281 (3.49) nm; ECD (c 0.0022, MeOH) λmax (θ); 206 (−9.6 × 104), 224 (−7.6 × 103), 241 (3.3 × 103), 280 (2.8 × 103) nm; IR (film on NaCl) νmax 3440 (br OH), 2958, 2924, 1633, 1612, 1505, 1479, 1463, 1289, 1202, 1086, 1024 cm−1; 1H and 13C NMR data, see Table 2; HMBC correlations (CDCl3) H-2α→ C-4, 9; H-2β → C-3, 4; H2-4 → C-2, 3, 9, 10; H-5 → C-4, 7, 8*, 9; H-6 → C-7, 8, 10; OH-7 → C-6, 7, 8; OH8 → C-7, 8, 9; OCH3-2′ → C-2′; H-3′ → C-3*, 1′, 5′, 4′, 2′, 1″*; OCH3-4′ → C-4′; H-6′ → C-3, 5′, 4′, 3′*,2′, 1″; H-2″ → C-5′, 1″, 4″, 5″; H2-3″ → C-1″, 2″; H3-4″ → C-5′, 1″, 2″, 5″; H3-5″ → C-5′, 1″, 2″, 4″ (* indicates weak four-bond correlation); HRESIMS found m/z 371.1854 [M + H]+, calcd for C22H27O5, 371.1858. Sedonan F (6): yellow oil; [α]20D +8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (4.69), 228 (sh) (4.35), 276 (3.75) nm; ECD (c 0.002, MeOH) λmax (θ); 205 (−1.3 × 103), 218 (1.2 × 104), 250 (2.6 × 103), 286 (−1.4 × 103) nm; IR (film on NaCl) νmax 3411 (br OH), 2923, 1709, 1610, 1494, 1479, 1464, 1440, 1362, 1275, 1222, 1089 cm−1; 1H and 13C NMR data, see Table 2; HMBC correlations (CDCl3) H2-2 → C-3, 4, 9; H2-4 → C-2, 3, 5, 9, 10; H-5 → C-4, 7, 9; H-6 → C-8, 10; OCH3-4′ → C-4′; H-5′ → C-1′, 3′, 4′; H-6′ → C-3, 2′, 4′; H-1″ → C-2′, 3′, 4′, 2″, 3″; H-2″ → C-1″, 4″, 5″; H-4″ → C-2″, 3″, 5″; H-5″ → C-2″, 3″, 4″; HRESIMS found m/z 357.1705 [M + H]+, calcd. for C21H25O5 357.1702. (−)-4′-O-Methylpuerol A (7): yellow solid; mp >180 °C (dec); [α]20D −43 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 201 (4.80), 218 (4.68), 243 (sh) (4.38), 284 (4.38), 325 (4.42) nm; ECD (c 0.0025, MeOH) λmax (θ); 203 (−1.4 × 104), 213 (8.7 × 103), 231 (−5.5 × 103), 251 (1.3 × 104), 285 (−1.8 × 104), 325 (−1.3 × 104) nm; IR (film on NaCl) νmax 3219 (br OH), 2921, 1700, 1606, 1516, 1448, 1301, 1244, 1173, 1105, 1030 cm−1; 1H NMR (acetone-d6, 400 MHz) δ 7.42 (1H, d, J = 8.6 Hz, H-6″), 7.03 (2H, d, J = 8.6 Hz, H-2′, H-6′), 6.78 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.60 (1H, d, J = 2.3 Hz, H-3″), 6.53 (1H, dd, J = 8.6, 2.3 Hz, H-5″), 6.18 (1H, d, J = 1.2 Hz, H-3), 5.87 (1H, ddd, J = 6.7, 3.2, 1.2 Hz, H-5), 3.74 (3H, s, OCH3), 3.27 (1H, dd, J = 14.5, 3.2 Hz, H-5a), 2.78 (1H, dd, J = 14.5, 6.7 Hz, H-5a); 13 C NMR data (acetone-d6, 100 MHz) δ 173.6 (C, C-2), 165.7 (C, C4), 162.2 (C, C-4″), 159.6 (C, C-4′), 158.8 (C, C-2″), 132.1 (CH, C6″), 131.6 (CH, C-2′, C-6′), 129.2 (C, C-1′), 114.3 (CH, C-3′, C-5′), 113.7 (CH, C-3), 110.8 (C, C-1″), 109.2 (CH, C-5″), 104.1 (CH, C3″), 83.9 (CH, C-5), 55.5 (CH3, OCH3), 39.8 (CH2, C-4a); HMBC correlations (acetone-d6) H-3 → C-2, 4, 5, 1″; H2-5a → C-4, 5, 1′, 2′; H-2′ → C-4a, 3′, 4′, 6′; H-3′ → C-1′, 4′, 5′; OCH3-4′ → C-4′; H-5′ → C-1′, 3′, 4′; H-6′ → C-5a, 2′, 4′, 5′; H-3″ → C-1″, 2″, 4″, 5″; H-5″ → C-1″, 2″*, 3″, 4″; H-6″ → C-4, 2″, 4″ (* indicates weak four-bond correlation); HRESIMS found m/z 313.1074 [M + H]+, calcd for C18H16O5, 313.1076. ent-Sandwicensin (8): off-white solid; mp 76−90 °C (lit. 88.5−89.5 °C);55 [α]20D +72 (c 0.1, CHCl3); IR and 1H and 13C NMR were consistent with published values for (6aR,11aR)-sandwicensin.37,55 The molecular structure of 8 was confirmed by HSQC and HMBC NMR spectroscopy; HRESIMS found m/z 339.1597 [M + H]+, calcd for C21H23O4, 339.1596.

The following isogenic strains characterized by a common genetic background (MATa, ura3-52, trp1Δ63, leu2Δ1, his3Δ200, GAL2+) and by different levels of expression of endogenous and heterologous major multidrug resistance genes were used: FY1679-28C, wild type; FY1679/TDEC, knockout of PDR1 and PDR3 genes encoding major transcriptional activators of yeast pleiotropic drug resistance (pdr1Δ2::TRP1, pdr3-Δ::HIS3);67 FYAK 26/8-10B1, triple knockout of PDR5, SNQ2, and YOR1 genes encoding major multidrug ABC transporters (pdr5-Δ1::hisG, snq2-Δ1::hisG, yor1-1::hisG);13 AK100, triple knockout of PDR5, SNQ2, and YOR1 in the hyperactive gain-offunction mutant PDR1−3 (PDR1−3, pdr5-Δ4::rep500, snq2-Δ1::hisG, yor1-1::hisG); and MKCDR1h, Cdr1p overproducer in the AK100 host (PDR1−3, pdr5-Δ4::CDR1h, snq2-Δ1::hisG, yor1-1::hisG).66 Direct antifungal activity was first measured in crude extracts, and expanded susceptibility testing, as described herein, was used in the initial two rounds of purification by VLC and Sephadex LH-20 chromatography. The effect of analyzed samples on Cdr1p-mediated azole resistance was evaluated with the MKCDR1h overproducing strain using a similar growth-based assay. In this case, the medium was supplemented with a subinhibitory concentration of ketoconazole, as described previously, to determine if tested samples were able to potentiate the growth inhibitory effect of the azole.14 The growth inhibitory effects of the combinations of 1 and 8 were determined by a microdilution checkerboard procedure.58 The fractional inhibitory concentration index (FICindex) was defined following the equation FICindex = FICA + FICB = (MICA comb/ MICA alone) + (MICB comb/MICB alone), where MICA alone and MICB alone are the MICs of compounds acting alone and MICA comb and MICB comb are MICs of compounds A and B in combination. Compound interactions were defined as synergistic if the FICindex was ≤0.5, indifferent if the FICindex was >0.5 and ≤4.0, and antagonistic if the FICindex was >4, according to criteria described previously.59,60 Sedonan A (1): white solid; mp >85 °C (dec) (CH2Cl2−hexanes); [α]20D +6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (4.39), 280 (4.28) nm; IR (film on NaCl) νmax 3321 (br OH), 2973, 1638, 1595, 1492, 1285, 1249, 1160, 1124, 1097 cm−1; 1H and 13C NMR data, see Table 1; HMBC correlations (2:1 CDCl3−methanol-d4) H2-2 → C-3, 4, 9, 1′; H-3 → C-2, 1′, 2′, 6′; H-6 → C-5, 7, 8, 10; H-8 → C-6, 7, 9, 10; OCH3-4′ → C-4′; H-5′ → C-1′, 2′*, 3′, 4′; H-6′ → C-3, 2′, 3′*, 4′; H-1″ → C-2′, 4′, 3″; H-2″ → C-3′, 3″; H3-4″ → C-1″*, 2″, 3″, 5″; H3-5″ → C-1″*, 2″, 3″, 4″ (* indicates weak four-bond correlation); HRESIMS found m/z 369.1345 [M + H]+, calcd for C21H21O6, 369.1338. Sedonan B (2): pale yellow oil; [α]20D −19 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.68), 228 (sh) (4.33), 292 (4.21), 341 (sh) (3.53) nm; ECD (c 0.0026, MeOH) λmax (θ); 217 (1.6 × 104), 237 (−9.6 × 103), 255 (0), 298 (−9.2 × 103), 335 (5.2 × 103), 368 (−886) nm; IR (film on NaCl) νmax 3394 (br OH), 2968, 1634, 1501, 1446, 1377, 1281, 1156, 1096, 1078 cm−1; 1H and 13C NMR data, see Table 1; HMBC correlations (CDCl3) H2-2 → C-3, 4, 9, 1′; OH-5 → C-5, 6, 10; H-8 → C-6, 7, 8, 9, 10; H-3′ → C-3*, 1′, 2′, 4′, 5′, 1‴*; H6′ → C-3, 2′, 3′*, 4′, 5′, 1‴; H2-1″ → C-5, 6, 7, 2″, 3″, 4″*; H-2″ → C-6, 1″, 4″, 5″; H3-4″ → C-2″, 3″, 5″; H3-5″ → C-2″, 3″, 4″; H-2‴ → C-5′, 1‴, 4‴, 5‴; H2-3‴ → C-1‴, 2‴; H3-4‴ → C-5′, 1‴, 2‴; H3-5‴ → C-5′, 1‴, 2‴ (* indicates weak four-bond correlation); HRESIMS found m/z 463.1742 [M + Na]+, calcd for C25H28O7Na, 463.1733. Sedonan C (3): yellow solid; mp 98−107 °C; [α]20D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 237 (3.84), 289 (4.16) nm; IR (film on NaCl) νmax 3360 (br OH), 2967, 2929, 1637, 1601, 1505, 1444, 1382, 1281, 1183 cm−1; 1H and 13C NMR data, see Table 1; HMBC correlations (acetone-d6) H2-2 → C-3, 4, 9, 1′; H-3 → C-2, 4, 1′, 2′, 6′; OH-5 → C-5, 6, 10; H-8 → C-4*, 6, 7, 9, 10; H-3′ → C-1′, 2′, 4′, 5′, 1‴*; H-6′ → C-3, 2′, 3′*, 4′, 1‴; H2-1″ → C-5, 6, 7, 2″, 3″, 4″*; H-2″ → C-4″, 5″; H-4″ → C-2″, 3″, 5″; H-5″ → C-2″, 3″, 4″; H2‴ → C-5′, 1‴,4‴,5‴; H2-3‴ → C-1‴, 2‴; H-4‴ → C-4′*, 5′, 6′*, 1‴, 2‴, 3‴*, 5‴; H-5‴ → C-4′*, 5′, 6′*, 1‴, 2‴, 3‴*, 4‴ (* indicates weak four-bond correlation); HRESIMS found m/z 425.1961 [M + H]+, calcd for C25H29O6, 425.1964. I



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ASSOCIATED CONTENT

S Supporting Information *

ECD spectra of compounds 4−6. Concentrations examined to obtain spectra for compounds 1−7. 1H, 13C, HSQC, and HMBC spectra of compounds 1−8 and NOESY spectra of compounds 2 and 3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(G. Belofsky) Tel: 509-963-2882. Fax: 509-963-1050. E-mail: [email protected]. (M. Kolaczkowski) Tel: (48) 71 7841403. Fax: (48) 71 7840088. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the University of Iowa High Resolution Mass Spectrometry Facility, operating under a NIH shared instrument grant (S10-RR023384-01), for discounted services. We gratefully acknowledge Central Washington University, College of the Sciences, for a Faculty Summer Research Grant and for G.B.’s start-up funding. M.K. was supported in part by funds from Wrocław Medical University. We thank the USDA Agricultural Research Service Specific Cooperative Agreement No. 58-64-2-0009 for partial financial support. We also would like to thank the Mississippi College Department of Chemistry and Biochemistry, Clinton, MS, especially Dr. D. Rosado, for use of the JASCO J-815 spectrometer.

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