Microporenic Acids A–G, Biofilm Inhibitors, and ... - ACS Publications

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Microporenic Acids A−G, Biofilm Inhibitors, and Antimicrobial Agents from the Basidiomycete Microporus Species Clara Chepkirui,† Kamila T. Yuyama,∥ Lucy A. Wanga,⊥ Cony Decock,‡ Josphat C. Matasyoh,§ Wolf-Rainer Abraham,∥ and Marc Stadler*,† †

Department of Microbial Drugs, Helmholtz Centre for Infection Research; and German Centre for Infection Research (DZIF), Partner Site Hannover/Braunschweig, Inhoffenstrasse 7, 38124 Braunschweig, Germany ‡ Mycothéque de l’ Universite Catholique de Louvain (BCCM/MUCL), Place Croix du Sud 3, B-1348 Louvain-la-Neuve, Belgium § Department of Chemistry, Faculty of Sciences, Egerton University, P.O. Box 536, 20115, Njoro, Kenya ⊥ Department of Biochemistry, Faculty of Sciences, Egerton University, P.O. Box 536, 20115, Njoro, Kenya ∥ Department of Chemical Microbiology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany S Supporting Information *

ABSTRACT: The need for effective compounds to combat antimicrobial resistance and biofilms which play important roles in human infections continues to pose a major health challenge. Seven previously undescribed acyclic diterpenes linked to isocitric acid by an ether linkage, microporenic acids A−G (1−7), were isolated from the cultures of a hitherto undescribed species of the genus Microporus (Polyporales, Basidiomycota) originating from Kenya’s Kakamega forest. Microporenic acids D and E (4 and 5) showed antimicrobial activity against a panel of Gram positive bacteria and a yeast, Candida tenuis. Moreover, microporenic acids A and B (1 and 2) demonstrated dose-dependent inhibition of biofilm formation by Staphylococcus aureus. Compound 1 further showed significant activity against Candida albicans and Staphylococcus aureus preformed biofilms.

I

Fungi are one of the most diverse groups of organisms and represent an important source of novel bioactive compounds.5 However, the secondary metabolites of many fungal species remain unstudied. The Basidiomycota particularly represent a relatively neglected source for promising bioactive natural products.6 From our continuous search for novel antimicrobial agents from Kenya’s tropical basidiomycetes, we have reported several bioactive compounds.7−9 Herein we report the isolation, structure elucidation, and biological activity of microporenic acid A−G (1−7), seven new diterpenes with an attached isocitric acid moiety from cultures of a Microporus species (Polyporales). These compounds are the first metabolites to be reported from this genus.

nfections that are associated with antimicrobial resistance continue to pose a major challenge in public health, resulting in high rates of morbidity and mortality, increased length of hospitalization, and higher healthcare costs.1 Biofilms also play important roles in human infections. The National Institute of Health reported that all infections (including those that involve biomaterials) are associated with biofilms.2 Biofilm formation confers to individual bacteria the ability to collaborate and to adapt to a range of harsh environmental conditions and evade predation by phagocytic microbes.3 Most Candida infections are biofilm related; they pose a significant health burden with the Center for Disease Control (CDC) estimating that approximately 7% of infants, 31% of patients suffering from AIDS, and 20% of the cancer patients undergoing chemotherapy develop oral candidiasis.4 On the other hand, CDC reports that bacterial biofilms are involved in 65% of the human bacterial infections. CDC also reports that despite this fact, there are currently no biofilm inhibitors approved for clinical use. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 7, 2017

A

DOI: 10.1021/acs.jnatprod.7b00764 J. Nat. Prod. XXXX, XXX, XXX−XXX

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with 7 degrees of unsaturation from the HRMS data. 1H NMR exhibited five singlets for methyl groups at ∂ 1.58 (H3-18), 1.60 (H3-26), 1.62 (H3-27), 1.66 (H3-17), 1.68(H3-25). Doublets of doublets occurring at ∂ 4.23 and 4.10 suggested a methylene attached to oxygen while the peak at ∂ 4.26 gave an indication of a hydroxy methine in the molecule. HMBC correlations between H3-17/H3-18 to C-15/C-16, H3-25 to C-3/C-4/C-5, H3-26 to C-7/C-8/C-9, and H3-27 to C-11/C-12/C-13 were observed. In the correlation spectroscopy (COSY) data correlations of H2-2 to H-3, H2-6 to H2-5/ H-7, H2-10 to H2-9/H-11, and H2-14 to H2-13/H-15 were recorded. These heteronuclear multiple bond correlation (HMBC) and COSY correlations suggested that one part of the molecule consisted of a chain with four isoprene units. On the other hand, HMBC correlations of H-19 to C-20/C-21/C23/C-24, H-20 to C-19/C-21/C-22/C-23/C-24, and H2-21 to C-19/C-20/C-22/C-23 established the tricarboxylic acid moiety, the second part of the molecule. This part of the molecule is presumably derived from isocitric acid. Cross peaks in the COSY spectra between of H-20 and H-19/H2-21 were observed. HMBC correlation of H-19 to C-2 confirmed the linkage of the two parts of the molecule via the oxygen atom. The upfield shifts of C-25 (∂16.5), C-26 (∂ 16.2), and C-27 (∂ 16.1) showed that all these olefinic bonds had an E configuration. To determine the absolute stereochemistry at C-19 and C-20 compound 1 was treated with both S and R phenylglycine methyl ester (PGME). The difference in chemical shifts (Δ∂) between R and S-PGME amides were used to assign the stereochemistry at the C-19 and C-20 for β-substituted carboxylic acid.10 Protons H-19 and H-20 gave negative values for Δ∂ = ∂(R-PMGE amide) − ∂ (S-PGME amide), i.e., −0.06 and −0.23, respectively. This indicated that the two carboxylic acid substituents at C-19 and C-20 were oriented to the same side of the plane (Figure 2). This was further supported by the cross peaks observed in the ROESY spectra between H-19 and H-20/Hb-21 (∂ 2.59). Therefore, the absolute stereochemistry for 1 was assigned as 19R and 20S. Terpenes with isocitric acid moieties have been reported before from Ganoderma neojaponicum, Cryptoporus volvatus, and Polyporus arcularius.11−13 Compounds 2−7 were found to possess the same tricarboxylic acid moiety as in 1 with the difference being the substitutions along the chain. Nuclear Overhauser effect

RESULTS AND DISCUSSION The culture of Microporus sp. strain MUCL 55563 was fermented in shake flasks and its extracts subjected to extensive chromatographic studies as described in the Experimental Section, resulting in the isolation and identification of seven new metabolites and one known compound. Compound 1 was isolated as white oil from the mycelia (Figure 1). The molecular formula was deduced as C26H40O7

Figure 1. Chemical structures of compounds 1−8.

Figure 2. COSY, HMBC, and rotating-frame Overhauser effect spectroscopy (ROESY) correlations of 1. B

DOI: 10.1021/acs.jnatprod.7b00764 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. Antimicrobial and Cytotoxic Activities of Compounds 1−8a test strains

a

1

2

positive controls

3

4

5

6

7

8

Bacteria Bacillus subtilis DSM 10 Micrococcus luteus DSM 1790 Staphylococcus aureus DSM 346 Escherichia coli DSM 498

MIC (μg/mL) 300 300 37.5 37.5 300 300 − −

− 75 − −

37.5 18.75 75 −

18.8 9.4 150 −

− n.t − −

− 300 − −

300 75 − −

2.3 8.3 1 2.3

ciprofloxacin oxytetracycline oxytetracycline ciprofloxacin

Fungi Mucor plumbeus MUCL 49355 Candida tenuis MUCL 29982 Pichia anomala DSM 6766

75 300 150

− − −

75 37.5 −

75 37.5 −

− − −

300 − −

75 − −

9.4 16.7 16.7

nystatin nystatin nystatin

Cell lines KB 3.1 HeLa L929

Cytotoxicity IC50 (μM) 6 − − − − −

− −

− −

− −

− −

− −

0.22 1.4

epothilon B epothilon B

150 − −

−, not active; nt, not tested. Starting concentration for antimicrobial assay and cytotoxicity assay were 300 and 37 μg/mL, respectively.

showed that 5 was similar to 1 with one isoprene unit missing. Compound 5 has been reported before as a product of chemical synthesis in a patent application,14 but to the best of our knowledge it has never before been isolated from a natural source. No NMR data were reported, but the chemical structure and the MS data match our own findings. Compound 6, isolated as a yellow oil from the supernatant crude extract, had a molecular formula C26H42O9 and 6 levels of unsaturation established from the HRMS data. The 1D and 2D NMR data of 6 indicated a closely related structure to that of 1 with the difference being on the substituents at C-11 and C-12. In the 13 C NMR spectra, signals from two of the olefinic carbons were missing and instead two new signals were observed at ∂ 77.9 (C-11) and ∂ 74.5 (C-12). The methyl group proton H3-27 which was slightly shifted upfield (∂ 1.10) exhibited HMBC to C-11/C-12/C-13. H-11 showed correlations to C-9/C-10/C-12/C-13/C-27 in the HMBC. The hydroxy groups were attached to C-11 (∂ 77.9) and C-12 (∂ 74.5) based on their chemical shifts. Cross peaks between H-11 and H2-10 in the COSY spectra were observed. To assign the relative stereochemistry at C-11 and C-12, Jresolved HMBC was used to determine the heteronuclear longrange coupling constants.15 A small coupling constant (3JC−H) of 2.09 Hz between H-11 and C-27 was measured. Furthermore, 3JC−H recorded between H-11 and C-13 was 2.10 Hz indicating that the conformation of C-27 and C-13 was gauche relative to H-11. This assignment was further supported by the strong ROESY correlations between H3-27 and H-11. A network of ROESY correlations between H-11 to H2-10a/H29b/H2-13b/H2-14b was also observed. The absolute configuration of 6 at C-11 and 12 was determined using Mosher’s method.16 The (S) and (R)-MTPA esters were obtained by treatment of 6 with (R)-(−) and (S)-(+) MTPA chloride, respectively. The difference in chemical shifts (Δ∂SR) between S- and R-MTPA esters led to the assignment of the absolute configuration as 11S and 12R (Table 1 Supporting Information). Compound 7 was isolated as a yellow oil from both the supernatant and the mycelial crude extracts. From the HR mass spectrum its molecular formula was deduced as C26H42O9 with six degrees of unsaturation. In the 13 C NMR spectra of 7, signals for two of the olefinic carbons at C-15 and C-16 found in compound 1 were replaced by two signals for oxygenated carbons, observed at ∂ 78.4 (C-15) and ∂ 72.9 (C-16). HMBC

spectroscopy (NOESY), COSY, and HMBC correlations for all the other compounds isolated for this part of the molecule were in agreement with those of compound 1. Considering the fact that the compounds were obtained as congeners, they are likely to be products of the same biosynthetic pathway, and therefore similar R and S configuration at C-19 and C-20, respectively, can be assumed. Compound 2 was isolated as a yellow oil from the mycelia. The HR mass spectrum revealed the molecular formula to be C27H42O7 with seven degrees of unsaturation. Analysis of the 1 H and 13C NMR data for 2 revealed a structure closely related to that of 1 with the difference being an additional methoxy group signal occurring at ∂ 52.0 in the 13C NMR data. The methoxy group protons (∂ 3.70) showed HMBC correlations to C-22 (∂ 172.2). Compound 3 with the molecular formula C26H38O9 deduced from HRMS data was isolated from both the supernatant and the mycelial extracts as a yellow oil. The 1D and 2D NMR data for 3 suggested a similar structure as 1 with the difference being at C-17 (∂ 168.9) assigned as a carboxylic acid moiety based on the chemical shift and the molecular formula. This assignment was confirmed by the HMBC correlations of H-15 and H3-18 to this C-17. The upfield shift of C-18 (∂ 12.6) suggests that this proton is trans to H-15, hence the olefinic bond between C-15 and C-16 has an E configuration. Compound 4 was isolated from the supernatant and mycelia as a yellow oil with the molecular formula C26H40O8 obtained from the HR mass spectrum. The 1D and 2D NMR data for compound 4 revealed a closely related structure to 1 with the difference being at one end of the chain (C-17). In place of one of the methyl group attached to C-16 in 1 an oxygenated methylene resonating at ∂ 68.4 was attached. The oxygenated methylene protons ∂ 3.91 (H-17) exhibited HMBC correlations to C-15/C-16/C-18. Further, long-range COSY correlations were observed between H-18 and H-15/H-17. Compound 5 with a molecular formula of C21H32O7 and six degrees of unsaturation deduced from the HR mass spectrum was isolated from both the supernatant and the mycelial extracts as a yellow oil. Peaks occurring at ∂ 1.59 (H3-13), 1.62 (H3-22), 1.65 (H3-14), and 1.69 (H3-21) were observed in the 1 H NMR spectrum, revealing four methyl groups. Further, the doublet of doublets at ∂ 4.11 and ∂ 4.25 (H2-2) for oxygenated methylene and a doublet at ∂ 4.26 for oxygenated methine were also present in the 1H NMR. The 1D and 2D NMR data C

DOI: 10.1021/acs.jnatprod.7b00764 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. Biofilm and Preformed Biofilm Inhibition Activity of 1 and 2a 1 test organisms

MIC (μg/mL)

Staphylococcus aureus DSM 1104

>256

Candida albicans DSM 11225

64

MIC (μg/mL)

49% (256 μg mL−1) 37% (128 μg mL−1) 1.5% (64 μg mL−1) 72% (16 μg mL−1) 52% (8 μg mL−1)

>256

biofilm inhibition % 86% (256 μg mL−1) 54% (64 μg mL−1) 28% (16 μg mL−1) −

coprinuslactone

2 preformed biofilm inhibition

−

biofilm inhibition %

biofilm inhibition %

86% (256 μg mL−1) 38% (128 μg mL−1) 11% (64 μg mL−1) −

99% (150 μg mL−1) 92% (75 μg mL−1) 85% (37 μg mL−1) −

Reference: nystatin, antifungal, MIC 16.7 μg/mL; oxytetracycline, antibacterial, MIC 1 μg/mL; −, not tested. Coprinuslactone,23 MIC 150 μg mL−1; no activity against preformed biofilms. a

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correlations of H-15 to C-13/C-16 and H3-17/H3-18 to C-15/ C-16 observed confirmed these assignments. COSY correlations were observed between H2-14 to H2-13/H-15 in addition to those observed for 1. In the ROESY spectra, cross peaks were observed between H-15 and H3-17/H3-18/H2-14b/H213a. Derivatization of 7 with both (R)- (−) and (S)-(+) MTPA chloride allowed the absolute configuration assignment of the stereocenter at C-15 as S. Protons H-15 and H3-17/18 gave +0.02 and −0.02, respectively, for Δ∂ = ∂ (S-MTPA ester) − ∂ (R-MTPA ester). The known compound 8, which has been previously reported from the basidiomycete Pholiota adiposa, was purified as white solid and characterized as 15-Hydroxy-6α, 12-epoxy7β, 10αH, 11βH-spiroax-4-ene (8) by comparison of the NMR data with those reported in the literature.17 It has recently also been obtained from a Tyromyces species (Polyporaceae) collected in China.18 All compounds were tested for their antimicrobial activities against various organisms (Table 1). Compounds 4 and 5 showed moderate activities against Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus, whereas 1−5 had moderate activity against M. luteus. Compounds 4 and 5 further exhibited antifungal activity against Candida tenuis and Mucor plumbeus. Compound 1 demonstrated weak cytotoxicity activity against KB3.1 cell lines. The other compounds had no activity against KB3.1 and L929 cell lines at concentrations of 37 μg/mL. When compound 1 was tested against C. tenuis, we observed that 1 was fungistatic (it did not kill the cells but stopped growth) and also dispersed biofilm formed by the yeast. Therefore, all compounds isolated showing antimicrobial activities except 4 and 5 were subjected to the biofilm inhibition assay using Candida albicans and S. aureus; compound 1 and 2 displayed activity in this assay. Compound 1 exhibited significant activity against preformed C. albicans biofilm and moderate activity against S. aureus preformed biofilms (Table 2). 1 and 2 demonstrated moderate dosedependent activity in the S. aureus biofilm formation. Biofilm formation is an important factor associated with drug resistance.19,20 Compounds with the ability to inhibit the formation of biofilms are thought to evade the rapid evolution of resistance, since unlike antibiotics they do not pose a fatal threat to microorganisms.21 There is also an urgent need for compounds dispersing biofilms, including pathogenic fungi like C. albicans, but not many molecules with such activities are known to science yet.22 Therefore, compounds like 1 hold considerable promise in designing bioactive lead molecules that could function as adjunctive agents in combination therapy with antibiotics.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined with a PerkinElmer 241 spectrometer. UV spectra were recorded with a Shimadzu UV−vis spectrophotometer UV-2450. NMR spectra were recorded with Bruker Ascend 700 spectrometer with 5 mm TXI cryoprobe (1H 700 MHz, 13C 175 MHz) and Bruker AV II-500 (1H 500 MHz, 13C 120 MHz) spectrometers. HR-ESI-MS mass spectra were recorded with an Agilent 1200 series HPLC-UV system (column 2.1 mm × 50 mm, 1.7 μm, C18 Acquity UPLC BEH (Waters); solvent A, H2O + 0.1% formic acid; solvent B, AcCN + 0.1% formic acid; gradient, 5% B for 0.5 min increasing to 100% B in 19.5 min and then maintaining 100% B for 5 min; flow rate 0.6 mL min−1; UV−vis detection 200−600 nm combined with ESI-TOF-MS (Maxis, Bruker) (scan range 100−2500 m/z, capillary voltage 4500 V, dry temperature 200 °C). Fungal Material. The fungal strain Microporus sp. (Polyporaceae) was obtained from a specimen collected from the Kakamega equatorial rainforest, located in the western part of Kenya (0° 17′ 3.19″ N 34° 45′ 8.24″ E) by C. Decock and J. C. Matasyoh, February 17, 2015. The dried herbarium specimen and culture are deposited at MUCL, Louvain-la-Neuve, Belgium as MUCL 55563. The fungus was assigned to the genus Microporus by morphological studies and partial sequencing of the rDNA operon (5.8S gene region, the internal transcribed spacer ITS1 and ITS2). These sequence data are deposited in GenBank with accession number MG687373. According to preliminary results, it represents a new species, which will be described in a later taxonomic paper. Genomic DNA Miniprep kit (Bio Basic Canada Inc., Markham, Ontario, Canada). A Precellys 24 homogenizer (Bertin Technologies, France) was used for cell disruption at a speed of 6000 rpm for 2 × 40 s. The DNA regions were amplified with primers ITS 1f and NL4. Fermentation and Extraction. A well grown culture grown on an YMG agar plate was cut into small pieces using a cork borer (7 mm) and five pieces per flask were inoculated in a batch of 25 500 mL Erlenmeyer flasks containing 200 mL of the medium (for details on the composition of the medium see the Supporting Information). The cultures were incubated at 23 °C on a rotary shaker (140 rpm). The growth of the fungus was monitored by constantly checking the amount of free glucose (using Bayer Diastix Harnzuckerstreifen). The fermentation was terminated 5 days after glucose depletion. The mycelia and supernatant were separated via vacuum filtration. The mycelia were extracted with 4 × 500 mL of acetone in ultrasonic bath at 40 °C for 30 min and the solvent was evaporated in vacuo (40 °C). The remaining aqueous residue was diluted with the same amounts of ethyl acetate and water and extracted three times. The extracts were combined, dried over anhydrous sodium sulfate, and evaporated to dryness in vacuo (40 °C). After filtration using a RP solid phase cartridge (Strata-X 33 μm, Polymeric Reversed Phase; Phenomenex, Aschaffenburg, Germany) 800 mg of crude extract was obtained. The supernatant was mixed with 100 g of adsorbent resin (Amberlite XAD-16 N) and incubated on a shaker for 5 h. The Amberlite resin was then filtered and eluted with 4 × 500 mL of acetone. The resulting acetone extract was evaporated and the remaining aqueous phase subjected to the same procedure as the D

DOI: 10.1021/acs.jnatprod.7b00764 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 3. NMR Data (1H 500 MHz, 13C 125 MHz) in Acetone-d6 for 1 and in CDCl3 for 2 and (1H 700 MHz, 13C 175 MHz, Acetone-d6) for 3 and 4 1

2

position

δC, type

δH (J in Hz)

2

67.7, CH2

3 4 5

121.3, CH 141.9, C 40.4, CH2

4.24, dd (11.8, 6.4) 4.11, dd, (11.8, 7.5) 5.36, m a

2.04−2.07, m 1.98−2.02, m a 12.08−2.14, m

δC, type 67.5, CH2 118.9, CH 143.2, C 39.7, CH2

a

6

27.2, CH2

7 8 9

125.2, CH 135.6, C 40.5, CH2

a

5.10−5.16, m

a

2.04−2.07, m 1.98−2.02, m a 12.08−2.14, m

26.3, CH2 123.6, CH 135.0, C 39.6, CH2

27.4, CH2

11 12 13

124.9, CH 136.0, C 40.6, CH2

a

3.10−5.16, m

a

2.04−2.07, m 1.98−2.02, m a 12.08−2.14, m

124.4, CH 135.6, C 39.8, CH2

a

a

14

27.5, CH2

15 16 17 18 19 20 21

125.3, CH 131.7, C 25.9, CH3 17.8, CH3 77.7, CH 44.9, CH 32.6, CH2

22 23 24 25 26 27

172.0, C 172.2, C 173.3, C 16.5, CH3 16.2, CH3 16.1, CH3

a

3.10−5.16, m

1.66, 1.58, 4.26, 3.43, 2.82, 2.59,

s s d (4.1) m dd (17.2, 9.5) dd, (17.2, 4.6)

1.68, s 1.60, s 1.62, s

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

67.7, CH2

4.25, dd (11.8, 6.5) 4.11, dd (11.8, 7.3) 5.36, m

67.7, CH2

4.24, dd (11.6, 6.5) 4.11, dd (11.6, 7.5) 5.36, m

a

2.03−2.07, 1.96−2.00, a 2.07−2.11, a 1.96−2.00, a 5.03, m

m m m m

a

2.03−2.07, 1.96−2.00, a 2.07−2.11, a 1.96−2.00, a 5.03, m

m m m m

a

m m m m

a

26.6, CH2

2.03−2.07, 1.96−2.00, a 2.07−2.11, a 1.96−2.00, a 5.03, m a

26.7, CH2 124.2, CH 131.3, C 25.7, CH3 17.7, CH3 75.7, CH 43.9, CH 31.3, CH2 172.2, C 175.6, C 175.8, C 16.5, CH3 16.2, CH3 16.1, CH3 52.0, OCH3

4

4.16, dd (11.9, 6.7) 4.04, dd (11.9, 7.8) 5.30, m

a

a

10

3

1.69, 1.60, 4.20, 3.59, 2.80, 2.63,

1.55, 1.59, 1.62, 3.70,

121.3, CH 141.9, C 40.4, CH2

a

2.06−2.07, m 1.99−2.02, m a 2.13−2.14, m

121.3, CH 141.9, C 40.3, CH2

a

27.4, CH2 125.0, CH 135.9, C 40.3, CH2

5.16, m a

2.06−2.07, m 1.99−2.02, m a 2.13−2.14, m

125.9, CH 134.8, C 39.0, CH2

5.18, m 2.12−2.16, m

2.04−2.08, m 1.98−2.02, m 2.12−2.16, m a

27.2, CH2 125.0, CH 135.9, C 40.5, CH2

a

27.2, CH2

a

5.15, m a

2.04−2.08, m 1.98−2.02, m 2.12−2.16, m a

27.1, CH2 125.2, CH 135.5, C 40.4, CH2

5.16, m a

2.04−2.08, m 1.98−2.02, m 2.12−2.16, m a

s s d (3.4) m dd (17.2, 9.1) dd (17.2, 5.9)

27.9, CH2

2.31, q (7.3)

27.9, CH2

142.9, CH 128.5, C 169.3, C 12.6, CH3 77.7, CH 44.9, CH 32.6, CH2

6.77, m

124.9, CH 136.3, C 68.4, CH2 13.3, CH3 77.7, CH 44.9, CH 32.6, CH2

172.1, C 172.3, C 173.3, C 16.7, CH3 16.1, CH3 16.2, CH3

s s s s

1.80, 4.26, 3.43, 2.82, 2.60,

s d (4.1) m dd (17.1, 9.5) dd, (17.1, 4.6)

1.69, s 1.62, s 1.64, s

172.1, C 172.3, C 173.3, C 16.6, CH3 16.2, CH3 16.1, CH3

5.38, m 3.91, s 1.62, brs 4.2, d (3.4) 3.43, m 2.82, dd (17.2, 9.5) 2.60, dd, (17.2, 4.5)

1.68, s 1.62, brs 1.62, brs

Signals overlapped with other signals.

acetone−water phase of the mycelia to afford 600 mg of dark brown solid. Isolation. The mycelia and supernatant crude extracts were fractionated using preparative reverse phase HPLC (PLC 2020, Gilson, Middleton). VP Nucleodur 100-5 C18 ec column (250 mm × 40 mm, 7 μm, Macherey-Nagel) used as a stationary phase. Deionized water (Milli-Q, Millipore, Schwalbach, Germany) (solvent A) and acetonitrile (solvent B) were used as the mobile phase. Elution gradient used was 5−100% solvent B in 60 min and thereafter isocratic conditions at 100% solvent B for 5 min. UV detection was carried out at 210, 254, and 350 nm. A total of 10 fractions were collected according to the observed peaks (F-1−F-10) for the supernatant and four (F-1−F-4) for the mycelial extract. Fraction F-4 from the mycelial culture was purified by reverse phase LC (solvent A/solvent B), elution gradient 70−90% solvent B for 25 min followed by gradient shift from 90 to 100% in 3 min, and finally isocratic condition at 100% solvent B for 5 min with preparative (Kromasil) 250 mm × 20 mm, 7 μm C18 column as stationary phase to give compound 1 (60 mg) and compound 2 (10 mg). Unless stated otherwise, the same column was used for purification of the other fractions. Fractions F-1 and F-2 from mycelium and F-6 and F-7 were combined as they contained same compounds. These fractions were also purified by reverse phase LC (solvent A/solvent B), elution gradient 40−65% solvent B for 30 min, followed by a gradient shift from 65 to 100% in 5 min, and finally

isocratic condition at 100% solvent B for 5 min to give 3 mg of 6 and 5.2 mg of 7. Compounds 3 (3.2 mg), 4 (3 mg), and 8 (4.2 mg) (5.2 mg) were obtained from fraction F-8 of the supernatant and F-3a of the mycelia by reverse phase LC (solvent A/solvent B), elution gradient 60−70% solvent B for 25 min, followed by a gradient change from 70 to 100% in 5 min, and thereafter isocratic conditions at 100% solvent B for 5 min. Compound 5 (3.3 mg) was obtained from another chromatographic separation of the combined fractions F-3 (mycelial extract) and F-9 (supernatant extract) using the same method as for the above fractions (elution gradient 65%−75% solvent B in 25 min, 75%−100% solvent B in 5 min, and similar isocratic condition at 100% solvent B). Antimicrobial Assay. The antifungal and antibacterial activities (minimum inhibition concentrations, MIC) were determined in serial dilution assays against Candida albicans DSM 1665, Candida tenuis MUCL29982, Mucor plumbeus MUCL49355, Pichia anomala DSM 6766, Bacillus subtilis DSM10, Escherichia coli DSM498, Micrococcus luteus DSM 1790, and Staphylococcus aureus DSM 346 as described previously.24 The assays were carried out in 96-well microtiter plates in YMG medium for filamentous fungi and yeasts and EBS for bacteria in triplicate. The starting concentration was 300 μg/mL. Cytotoxicity Assays. In vitro cytotoxicity (IC50) of compounds 1− 5 and 7−9 was determined against mouse fibroblast L929 and HeLa (KB-3.1) cell lines as previously described.25 E

DOI: 10.1021/acs.jnatprod.7b00764 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 4. NMR Data (1H 700 MHz, 13C 175 MHz, Acetone-d6) for 5 and 6 and (1H 500 MHz, 13C 125 MHz, Acetone-d6) for 7 5

6

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

2

67.7, CH2

67.7, CH2

121.4, CH 141.8, C 40.4, CH2

4.24, dd (11.8, 6.5) 4.11, dd (11.8, 6.5) 5.35, m

67.6, CH2

3 4 5

4.25, dd (11.8, 6.5) 4.11, dd (11.8, 7.5) 5.36, m

4.24, dd (11.6, 6.4) 4.12, dd (11.6, 6.4) 5.35, m

a

2.13−2.15, 2.07−2.09, a 2.13−2.15, a 2.07−2.09, 5.15, m

m m m m

a

m m m m

a

6

27.1, CH2

7 8 9

124.9, CH 136.0, C 40.5, CH2

2.07−2.09, 1.98−2.00, a 2.07−2.09, a 1.98−2.00, 5.10, m a

a

7

position

10

27.5, CH2

11 12 13

125.3, CH 131.7, C 25.9 CH3

14

121.4, CH 141.8, C 40.4, CH2

2.17−2.20, 2.03−2.06, a 2.17−2.20, a 2.03−2.06, 5.18, m a

26.9, CH2 124.6, CH 136.4, C 37.8, CH2

1.65, s

17.8, CH3

1.59, s

22.8, CH2

15 16 17

77.7, CH 44.9, CH 32.7, CH2

4.26, 3.42, 2.82, 2.59,

126.3, CH 131.3, C 25.9, CH3

18 19 20 21

172.1, C 172.4, C 173.2, C 16.6, CH3

1.69, s

22 23 24 25 26 27

16.2, CH3

1.62, s

17.8, 77.7, 44.9, 32.6,

m m m m

2.30, m a 2.03−2.06, m 1.72, m 1.38, m 3.32, dd, (10.5, 1.5)

30.4, CH2 77.9, CH 74.5, C 38.6, CH2

d (3.9) m dd, (16.9, 9.2) dd, (16.9, 4.5)

a

1.58, m 1.40, m 2.17−2.20, m 5.12, m 1.65, s

CH3 CH CH CH2

1.60, 4.26, 3.42, 2.83, 2.59,

172.1, C 172.3, C 173.3, C 16.6, CH3 16.3, CH3 23.0, CH3

s d (4.1) m dd, (17.2, 9.5) dd, (17.2, 4.7)

1.68, s 1.62, s 1.10, s

121.4, CH 141.9, C 40.4, CH2

a

2.05−2.08, m 1.98−2.02, m a 2.10−2.16, m a

27.4, CH2 124.9, CH 135.9, C 40.6, CH2

5.16, m a

2.05−2.08, m 1.98−2.02, m a 2.10−2.16, m a

27.0, CH2 125.1, CH 136.0, C 37.7, CH2 27.5, CH2 78.4, CH 72.9, C 25.0, CH3 25.0, 77.5, 44.8, 32.6,

CH3 CH CH CH2

172.1, C 172.2, C 173.3, C 16.5, CH3 16.2, CH3 16.1, CH3

5.16, m 2.25, m a 1.98−2.02, m 1.66, m 1.32, m 3.26, dd, (10.5, 1.7) 1.11, s 1.11, 4.26, 3.44, 2.83, 2.59,

s d (3.9) m dd, (17.2, 9.5) dd, (17.2, 4.4)

1.68, s 1.62, s 1.60, s

Signals overlapped with other signals. further incubated for 24 h at 37 °C. All experiments were made in triplicate with two repetitions. The compounds’ abilities to disperse the preformed biofilms were evaluated using the MTT reduction assay according to ref 29 and staining with crystal violet as described26 for C. albicans and S. aureus, respectively. Methanol was used as a negative control. Microporenic Acid A (1). White oil; [α]25 D + 26° (c 0.001, MeOH); UV (MeOH) λmax(log ε) 204 (4.23); ESIMS m/z 951(20) [2M + Na] + , 587 (22) [M + Na]+, 465(3) [M + H]+, 447 (0.5) [M + H − H2O]+; HRMS m/z 465.2824 [M + H]+ (calcd for C26H41O7, 465.2852). NMR data see Tables 3 and 4. Microporenic Acid B (2). Yellow oil; [α]25 D + 33° (c 0.001, MeOH); UV (MeOH) λmax(log ε) 204 (4.03); ESIMS m/z 979 (100) [2M + Na] +, 501 (84) [M + Na]+, 478 (3) [M + H]+, 461 (0.2) [M + H − H2O]+; HRMS m/z 479.3002 [M + H]+ (calcd for C27H43O7, 479.3008); NMR data see Tables 3 and 4. Microporenic Acid C (3). Yellow oil; [α]25 D + 13° (c 0.001, MeOH); UV (MeOH) λmax(log ε) 205 (4.37); ESIMS m/z 1011 (100) [2M + Na]+, 517 (49) [M + Na]+, 495 (1) [M + H]+, 477 (1) [M + H − H2O]+; HRMS m/z 495.2590 [M + H]+ (calcd for C26H39O9, 495.2594); NMR data see Tables 3 and 4. Microporenic Acid D (4). Yellow oil; [α]25 D + 19° (c 0.001, MeOH); UV (MeOH) λmax(log ε) 204 (4.26); ESIMS m/z 983 (100) [2M + Na] +, 503 (70) [M + Na]+, 481 (1) [M + H]+, 463 (10) [M + H −

Biofilm Inhibition Assay. Staphylococcus aureus was adjusted to match the turbidity of a 0.5 McFarland standard and incubated in 96 well tissue microtiter plates (TPP, Switzerland) in CASO with 4% glucose broth together with serial diluted compounds and incubated for 24 h at 37 °C. The biofilm inhibition activity of the test compounds was evaluated by crystal violet staining following the protocol of O’Toole.26 In brief, the supernatant was discarded, the biofilm stained with crystal violet for 15 min, washed three times, the dye in the biofilm was extracted with diluted acetic acid, and the absorbance of this extract was finally quantified in a plate reader at 550 nm. Standard deviations of two repeats with three parallel experiments each were 10% or less. Methanol was used as a negative control. Evaluation of the antimicrobial activities of the tested compounds (MIC, bactericidal/fungicidal vs bacteriostatic/fungistatic effects) was carried out as described recently.27,28 The previously reported coprinuslactone23 was used as a standard. Preformed Biofilm Inhibition Assay. C. albicans and S. aureus cultures were adjusted to match the turbidity of a 0.5 McFarland standard. C. albicans was incubated in 96 well non-tissue microtiter plates (FalconMicro Test) for 4 h in SB broth at 37 °C. S. aureus was incubated in 96 well tissue microtiter plates (TPP, Switzerland) for 24 h in CASO with 4% glucose broth. The supernatant was removed from the wells, and 150 μL of the respective media (fresh) was added together with serial diluted compounds into the wells. The plates were F

DOI: 10.1021/acs.jnatprod.7b00764 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

H2O]+; HRMS m/z 481.2788 [M + H]+ (calcd for C26H41O8+, 481.2801); NMR data see Tables 3 and 4. Microporenic Acid E (5). Yellow oil; [α]25 D + 20° (c 0.001, MeOH); UV (MeOH) λmax(log ε) 204 (4.39); ESIMS m/z 419 (100) [M + Na]+, 397 (0.3) [M + H]+. HRMS m/z 397.2211 [M + H]+ (calcd for C21H33O7+, 397.2226). NMR data see Tables 3 and 4. Microporenic Acid F (6). Yellow oil; [α]25 D + 22° (c 0.001, MeOH); UV (MeOH) λmax(log ε) 203 (4.05); ESIMS m/z 1019 (100) [2M + Na]+, 997 (8) [2M + H]+, 521 (82 [M + Na]+, 499 (3) [M + H]+, 481 (10) [M + H − H2O]+; HRMS m/z 499.2882 [M + H]+ (calcd for C26H43O9+, 499.2907). NMR data see Tables 3 and 4. Microporenic Acid G (7). Yellow oil; [α]25 D + 13° (c 0.001, MeOH); UV (MeOH) λmax(log ε) 204 (3.95); ESIMS m/z 1019(100) [2M + Na] +, 521 (46) [M + Na]+, 499 (0.3) [M + H]+, 481 (3) [M+H− H2O]+; HRMS m/z 499.2879 [M + H]+ (calcd. for C26H43O9+, 499.2907). NMR data see Tables 3 and 4.

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(7) Chepkirui, C.; Richter, C.; Matasyoh, J. C.; Stadler, M. Phytochemistry 2016, 132, 95−101. (8) Mudalungu, C. M.; Richter, C.; Wittstein, K.; Abdalla, A. M.; Matasyoh, J. C.; Stadler, M.; Süssmuth, R. D. J. Nat. Prod. 2016, 79, 894−898. (9) Chepkirui, C.; Matasyoh, J. C.; Decock, C.; Stadler, M. Phytochem. Lett. 2017, 20, 106−110. (10) Yabuuchi, T.; Kusumi, T. J. Org. Chem. 2000, 65, 397−404. (11) Cabrera, G. M.; Roberti, M. J.; Wright, J. E.; Seldes, A. M. Phytochemistry 2002, 61, 189−193. (12) Asakawa, Y.; Hashimoto, T.; Mizuno, Y.; Tori, M.; Fukazawa, Y. Phytochemistry 1992, 31, 579−592. (13) Wu, W.; Zhao, F.; Ding, R.; Bao, L.; Gao, H.; Lu, J.; Yao, X.; Zhang, X.; Liu, H. Chem. Biodiversity 2011, 8, 1529−1538. (14) Kobayashi, T.; Tamura, K.; Yoshida, M.; Koga, H. Tricarboxylic acid derivative having squalene synthetase inhibitor activity. PCT International Application WO 9504025 A1, February 9, 1995. (15) Furihata, K.; Seto, H. Tetrahedron Lett. 1999, 40, 6271−6275. (16) Hoye, T. R.; Jeffrey, C. S.; Shao, F. Nat. Protoc. 2007, 2, 2451− 2458. (17) Liu, D. B.; Jia, R.; Wang, F.; Liu, J. Z. Naturforsch. 2008, 63b, 111−113. (18) Guo, H.; Feng, T.; Li, Z. H.; Liu, J. K. Yao Xue Xue Bao 2014, 49, 1578−1581. (19) Finkel, J. S.; Mitchell, A. P. Nat. Rev. Microbiol. 2011, 9, 109− 118. (20) Uppuluri, P.; Srinivasan, A.; Ramasubramanian, A.; Lopez-Ribot, J. L. Antimicrob. Agents Chemother. 2011, 55, 3591−3593. (21) Shareck, J.; Belhumeur, P. Eukaryotic Cell 2011, 10, 1004−1012. (22) Estrela, A. B.; Abraham, W.-R. Agriculture 2016, 6, 37−63. (23) De Carvalho, M. P.; Gulotta, G.; do Amaral, M. W.; Lünsdorf, H.; Sasse, F.; Abraham, W.-R. Environ. Microbiol. 2016, 18, 4254− 4264. (24) Noumeur, S. R.; Helaly, S. E.; Jansen, R.; Gereke, M.; Stradal, T. E. B.; Harzallah, D.; Stadler, M. J. Nat. Prod. 2017, 80, 1531−1540. (25) Sandargo, B.; Thongbai, B.; Stadler, M.; Surup, F. J. Nat. Prod. 2018, 81, 286−291. (26) O’Toole, G. A. J. Visualized Exp. 2011, 47, 2437−2437. (27) Yuyama, K. T.; Neves, T. S. P.; Memória, M. T.; Tartuci, I. T.; Abraham, W.-R. AIMS Microbiology. 2017, 3, 50−60. (28) Yuyama, K.; Chepkirui, C.; Wendt, L.; Fortkamp, D.; Stadler, M.; Abraham, W.-R. Microorganisms 2017, 5, 80. (29) Brambilla, L. Z. S.; Endo, E. H.; Cortez, D. A. G.; Filho, B. P. D. Rev. Bras. Farmacogn. 2017, 27, 112−117.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00764. Experimental procedures, 1D and 2D NMR data, LCMS data, and ITS-DNA sequence of the producing organism (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 531 6181-4240. Fax: +49 531 6181 9499. E-mail: [email protected]. ORCID

Marc Stadler: 0000-0002-7284-8671 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to W. Collisi for conducting the bioassays, C. Kakoschke and C. Schwager for recording NMR and HPLCMS data, and to Dr. Frank Surup for valuable scientific discussions. Financial support by the ASAFEM Project (Grant No. IC-070) under the ERAfrica Programme to J.C.M. and M.S. and a personal Ph.D. stipend from the German Academic Exchange Service (DAAD), the Kenya National Council for Science and Technology (NACOSTI), and the Science Without Border (Ciências sem Fronteiras)” Program of ́ Superior Coordenaçaõ de Aperfeiçoamento de Pessoal de Nivel (CAPES) and the Conselho Nacional de Desenvolvimento ́ Cientifico e Tecnológico (CNPq) from Brazil to C.C. and K.T.Y. are gratefully acknowledged.

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REFERENCES

(1) Neidell, M. J.; Cohen, B.; Furuya, Y.; Hill, J.; Jeon, C. Y.; Glied, S.; Larson, E. L. Clin. Infect. Dis. 2012, 55, 807−815. (2) National Institute of Health. Research on Microbial Biofilms (PA03−047). December 20, 2002, http://grants.nih.gov/grants/guide/pafiles/PA-03-047.html (accessed on June 16, 2017). (3) Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Nat. Rev. Microbiol. 2004, 2, 95−108. (4) Centre for Disease Control and Prevention. http://www.cdc. gov/fungal/diseases/candidiasis/thrush/statistics.html (retrieved on June 16, 2017). (5) Bills, G. F.; Gloer, J. B. Microbiology spectrum 2016, 4(6). (6) Karwehl, S.; Stadler, M. Curr. Top. Microbiol. Immunol. 2016, 398, 303−338. G

DOI: 10.1021/acs.jnatprod.7b00764 J. Nat. Prod. XXXX, XXX, XXX−XXX