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Aug 21, 2013 - Pseudallescheria boydii is a ubiquitous filamentous fungus that is distributed widely in soil, decaying vegetation, and even polluted w...
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Polyketides from the Littoral Plant Associated Fungus Pseudallescheria boydii Ya-Chih Chang,† Tzu-Shing Deng,‡ Ka-Lai Pang,⊥ Che-Jen Hsiao,§ Yi-Ying Chen,∥ Shye-Jye Tang,▽ and Tzong-Huei Lee*,∥ †

School of Pharmacy, Taipei Medical University, Taipei, Taiwan 110 Department of Agronomy, National Chung Hsing University, Taipei, Taiwan 402 ⊥ Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan 202 § School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan 110 ∥ Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan 110 ▽ Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan 202 ‡

S Supporting Information *

ABSTRACT: Four previously unreported chemical entities, boydone A (1), boydone B (2), botryorhodine F (3), and botryorhodine G (4), along with five known compounds, fusidilactone A (5), (R)-(−)-mevalonolactone (6), (R)(−)-lactic acid (7), ovalicin (8), and botryorhodine C (9), were isolated from the ethyl acetate extracts of the fermented broths of the fungal strain Pseudallescheria boydii NTOU2362. The structures of 1−9 were characterized on the basis of their spectroscopic data analyses. The absolute configurations of 1 and 2 were established by comparison with the literature and the modified Mosher’s method. The growth inhibitory activities of 1−9 against the A549 non-small-cell lung cancer cell line were evaluated, and 2 and 8 exhibited moderate to potent bioactivities with GI50 values of 41.3 and 4.1 μM, respectively, in comparison with fluorouracil (GI50 = 3.6 μM).

Pseudallescheria boydii is a ubiquitous filamentous fungus that is distributed widely in soil, decaying vegetation, and even polluted waters.1,2 Previously, Allescheria boydii, Petriellidium boydii, Monosporium ampiospermum, and Scedosporium apiospermum were reported as synonyms of P. boydii,3 and among these P. boydii and S. apiospermum were classified as the sexual reproductive stage and the asexual reproductive stage of the same species, respectively; however they were recently reported to belong to two different species.4 P. boydii causes a wide range of infections that effect practically all the organs of the human body in immunocompetent and immunocompromised patients.5−7 One alkaloid, pseurotin A,8 and a series of cyclic peptides, pseudacyclins A−E,4 have been described from P. boydii. However, it was predicted that many other bioactive principles remained to be discovered from this fungus to account for its dominance in the environment and its high level of pathogenicity toward humans. During our preliminary screening, ethyl acetate extracts of the fermented broths of P. boydii NTOU2362 isolated from the leaves of the littoral plant Sesuvium portulacastrum at a concentration of 100 μg/mL exhibited potent cytotoxicity against RAW264.7 mouse macrophage cells. We investigated the chemical constituents of this fungal strain, and this work resulted in the isolation and identification of four new polyketides, 1−4 (Figure 1), together with five known compounds, 5−9. Their growth inhibitory activities against the A549 non-small-cell lung cancer cell line were also assessed. © XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. Chemical structures of 1−4.

From the ethyl acetate extracts of the fermented broths of P. boydii NTOU2362, nine compounds including four new Received: March 6, 2013

A

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Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1−4 [δ in ppm, mult. (J in Hz)] 1 no.

13 a,c

C

1 2 3 4 5 6 7 8

201.7 s 60.4 s 196.6 s 142.6 s 149.2 s 101.7d s 153.6 d 51.8 t

9 10 11 12 13 14 15 16

61.9 t 22.7d q 32.9d t 9.5 q 59.4d q 34.6d d 19.0d q 28.2d t

17

13.0 q

OH-5 NH

2 1

He

13 a,c

7.49 d (11.8) 3.46 m

206.7 s 50.7 s 75.8 d 178.8 s 123.9 s 29.7 d 17.9 q 27.0 t

3.79 t (5.1) 1.25 s 1.85 m 0.71 t (7.4) 3.63 s −h 1.26 d (7.1) 1.66 m 1.74 m 0.89 t (7.4)

C

12.4 q 22.3 q 26.0 t 9.5 q 57.1 q

3 Hf

13 b,c

1

C

4.49 s

2.47 m 1.07 d (7.1) 1.45 m 1.55 m 0.76 t (7.4) 1.09 s 1.58 m 0.92 t (7.4) 4.07 s

197.9 s 124.9 s 153.8 s 85.4 d 77.9 s 78.6 d 21.4 q 8.6 q 164.0 s 112.7 s 157.5 s 116.8 s 160.4 s 114.2 d 141.5 d 20.2 q 51.5 t

4 1

Hg

5.39 d (1.2) 3.77 s 1.54 s 1.69 d (1.2)

6.56 s 2.27 s 4.55 d (11.2) 4.57 d (11.2) 5.53 s

13 b,c

C

140.4 s 113.5 s 135.3 s 142.5 s 114.7 s 143.0 s 9.6 q 9.6 q

1

Hg

2.27 s 2.09 s

163.6 s 112.0 s 160.4 s 116.6 s 161.3 s 114.9 d 140.8 s 20.8 q

2.30 s

52.2 t

4.70 s

6.60 s

11.03 brd (11.8)

a

Measured in CDCl3 (125 MHz). bMeasured in DMSO-d6 (125 MHz). cMultiplicities were obtained from DEPT experiment. dPeaks broadening at 298 K and can be overcome in CD3OD at 333 K. eMeasured in CDCl3 (800 MHz). fMeasured in CDCl3 (500 MHz). gMeasured in DMSO-d6 (500 MHz). hSignal not found at 298 K, but observed at δH 2.78 and 3.04 when measured in CD3OD at 193 and 333 K, respectively.

-11; H3-13/C-4; H3-15/C-5, -14, and -16; H-7/C-1, -5, and -8) of 1 (Figure 2). The double-bond equivalence of 1 was calculated to

polyketides (1−4) were identified. The structures of the known compounds were determined to be fusidilactone A (5),9 (R)(−)-mevalonolactone (6),10 (R)-(−)-lactic acid (7),11 ovalicin (8),12 and botryorhodine C (9).13 Compound 1 was obtained as a yellow oil, and its estimated molecular formula, C17H27NO4, was further deduced by the pseudomolecular ion [M + H]+ (m/z = 310.2012, calcd for C17H28NO4 [M + H]+, 310.2018) on the positive mode of HRESIMS as well as 13C NMR coupled with DEPT spectra. The IR absorptions at 3430, 1670, and 1607 cm−1 indicated the presence of a hydroxy, a conjugated ketone carbonyl, and a conjugated double bond, respectively. Seventeen carbon resonances observed in the 13C NMR spectrum coupled with the DEPT interpretations of 1 were attributable to five methyls at δC 59.4 (q, C-13), 22.7 (q, C-10), 19.0 (q, C-15), 13.0 (q, C-17), and 9.5 (q, C-12), four methylenes at δC 61.9 (t, C-9), 51.8 (t, C8), 32.9 (t, C-11), and 28.2 (t, C-16), two methines at δC 153.6 (d, C-7) and 34.6 (d, C-14), and six quaternary carbons at δC 201.7 (s, C-1), 196.6 (s, C-3), 149.2 (s, C-5), 142.6 (s, C-4), 101.7 (s, C-6), and 60.4 (s, C-2) (Table 1). The 1H NMR spectrum exhibited signals for five methyl resonances at δH 3.63 (s, H3-13), 1.26 (d, J = 7.1 Hz, H3-15), 1.25 (s, H3-10), 0.89 (t, J = 7.4 Hz, H3-17), and 0.71 (t, J = 7.4 Hz, H3-12), four methylene resonances at δH 3.79 (t, J = 5.1 Hz, H2-9), 3.46 (m, H2-8), 1.85 (m, H2-11), and 1.74, 1.66 (each 1H, m, H2-16), two methine resonances at δH 7.49 (d, J = 11.8 Hz, H-7) and 2.78 (m, H-14), and a quite lower field −NH signal at δH 11.03 (brd, J = 11.8 Hz) (Table 1). A preliminary linear skeleton bearing four branches was deduced to be C-3−C-2 (−branch)−C-1−C-6 (−branch)− C-5 (−branch)−C-4 (−branch) from complete interpretations of key cross-peaks in the COSY spectrum (H-7/−NH; −NH/ H2-8; H2-8/H2-9; H2-11/H3-12; H2-16/H3-17) together with key cross-peaks in the HMBC spectrum (H3-10/C-1, -2, -3, and

Figure 2. Key COSY, HMBC, and NOESY correlations of 1.

be five, including two ketone groups (C-1 and C-3) and two double bonds (Δ4 and Δ6), together with a ring. The cyclized structure and location of each branch of 1 were further corroborated by the NOESY spectrum, in which key correlations of H3-12/H3-13, H3-13/H3-17, H3-15/H-7, and H-7/H2-8 were observed (Figure 2). The high NMR data compatibility of C-2and C-14-carrying moieties in 1 and its related analogue similin B, a C15 hexacyclic polyketide isolated previously from the coprophilous fungus Sporormiella similis,14 as well as their consistent levorotations ([α]31D −43.8 and [α]31D −14.6, respectively) supported the relative configurations of C-2 and C-14 of 1 to be R* and S*, respectively. The structure of 1 was assigned as shown in Figure 1 and was named boydone A. The molecular formula for 2, C13H22O3, was determined by 13 C NMR and HRESIMS data. Its IR absorption bands at 3400, 1738, and 1661 cm−1 indicated the presence of a hydroxyl, a carbonyl, and a double bond, respectively. The 1H and 13C NMR spectra of 2 were similar to those of 1, suggesting a similar B

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-12, and -13, and H2-17/C-11, -12, and -13 in the HMBC spectrum and 13C−13C connectivity of C-15/C-14, C-14/C-13, C-13/C-12, C-12/C-17, and C-10/C-11 in the 2D INADEQUATE spectrum established the gross structure of 3 (Figure 4). The relative stereochemistry of C-4, -5, and -6 was

skeleton in part. Their major differences involved the lack of the C-6−NH−C-7−C-8−C-9−OH branch and the ketone group at C-3 in 1 substituted by a carbinol functionality [δC 75.8 (d); δH 4.49 (s)] in 2 (Table 1). Key cross-peaks of H3-12/H2-11, H3-9/ H2-8, H2-8/H-6, and H3-7/H-6 in the COSY spectrum along with key cross-peaks of H3-10/C-1, -2, -3, and -11 and H-6/C-1, -4, -5, -7, and -8 in the HMBC spectrum of 2 (Figure 3) suggested

Figure 4. Key HMBC, INADEQUATE, and NOESY correlations of 3. Figure 3. Key COSY, HMBC, and NOESY correlations of 2.

determined to be R*, R*, and S*, respectively, by distinctive mutual correlations of H3-7/H-6, H3-7/H-4, and H-6/H-4 (Figure 4). Accordingly, 3 was assigned as shown in Figure 1 and was named botryorhodine F. Compound 4 had the molecular formula C17H16O7 as determined by negative ion mode HRESIMS and 13C NMR interpretation. When comparing the 13C and 1H NMR data of 4 with those of 3 (Table 1), it is obvious that 4 had the same number of carbon atoms and shared a partial structure (ring B, C9−C-17) with 3. The other eight signals in the 13C NMR of 4 [δC 143.0 (s, C-6), 142.5 (s, C-4), 140.4 (s, C-1), 135.3 (s, C-3), 114.7 (s, C-5), 113.5 (s, C-2), 9.6 (q, C-7), and 9.6 (q, C-8)] as well as their corresponding signals in the 1H NMR of 4 [δH 2.27 (s, H3-7) and 2.09 (H3-8)] suggested that the residual C8 part of 4 was a dimethyl-bearing aromatic moiety (A ring) possessing four oxygenated carbons (Table 1). The NMR data of the A ring in 4 were similar to those of botryorhodine C (9) except that the H-6 [δH 6.46 (s); δC 112.7 (d)] in 9 was replaced by OH-6 [δC 143.0 (s)] in 4. HMBC correlations of H3-8/C-1, -2, and -3 and H3-7/C-4, -5, and -6 together with spatial mutual correlations of H3-7 and H2-17 in the NOESY spectrum (Figure 5) allowed the final determination of 4 as shown in Figure 1, and it was named botryorhodine G.

that the gross structure of 2 with a pentacyclic ring was a C13 analogue of 1. Due to the biogenetic relationship between 1 and 2 as well as high NMR data compatibility of C-2- and C-6carrying moieties in 2 and its related compound similin A, a C13 pentacyclic polyketide of fungal origin,14 it is proposed that 2 adopted relative configurations of C-2 and C-6 consistent with their corresponding carbons in 1 and similin A. In the NOESY spectrum of 2, mutual correlations of H3-10/H-3 and H-3/H3-13 were observed (Figure 3), while no cross-peaks of H2-11/H-3 or H3-12/H-3 appeared. The relative configuration of C-3 was thus deduced to be R*. Compound 2 was further treated with (S)(+)- and (R)-(−)-MTPACl to afford the (R)- and (S)-MTPA esters, respectively, by following the modified Mosher’s method.15 The Δδ values of H3-13 and -12 obtained from the 1 H chemical shifts of (R)- and (S)-MTPA esters were −0.16 and +0.02, respectively. Unambiguously, the absolute configurations of C-2, -3, and -6 were corroborated to be S, R, and S, respectively. Accordingly, the structure of 2 was determined to be as shown in Figure 1 and was named boydone B. Compound 3 was obtained as one colorless crystal, and the pseudomolecular ion [M − H]− (m/z = 349.0915, calcd for C17H17O8 [M − H]−, 349.0923) in the negative mode of HRESIMS along with 13C NMR assignments established its molecular formula to be C17H18O8. In the IR spectrum of 3, the absorption bands at 3276, 1698, and 1600 collocated with 1446 cm−1 indicated the presence of a hydroxy, a conjugated ketone carbonyl, and an aromatic functionality, respectively. The 13C NMR of 3 coupled with a DEPT experiment showed that there were 17 resonances including three methyls [δC 21.4 (q, C-7), 20.2 (q, C-16), and 8.6 (q, C-8)], one methylene [δC 51.5 (t, C17)], four methines [δC 141.5 (d, C-15), 114.2 (d, C-14), 85.4 (d, C-4), and 78.6 (d, C-6)], and nine quaternary carbons [δC 197.9 (s, C-1), 164.0 (s, C-9), 160.4 (s, C-13), 157.5 (s, C-11), 153.8 (s, C-3), 124.9 (s, C-2), 116.8 (s, C-12), 112.7 (s, C-10), and 77.9 (s, C-5)] (Table 1). The 1H NMR data of 3 accompanied by the HSQC spectrum revealed three methyls [δH 2.27 (s, H3-16), 1.69 (d, J = 1.2 Hz, H3-8), and 1.54 (s, H3-7)], a geminal mutually coupled methylene [δH 4.57 and 4.55 (each 1 H, d, J = 11.2 Hz, H2-17)], two carbinoyl protons [δH 5.39 (d, J = 1.2 Hz, H-4) and 3.77 (s, H-6)], an olefinic proton [δH 6.56 (s, H-14)], and a hydroxy group [δH 5.53 (s, OH-5)] (Table 1). Conspicuous cross-peaks of H3-7/C-4, -5, and -6, H3-8/C-1, -2, and -3, H3-16/ C-10, -14, and -15, H-4/C-3, -5, and -11, H-6/C-1, H-14/C-10,

Figure 5. Key HMBC and NOESY correlations of 4.

All the pure isolated compounds (1−9) were evaluated for their growth inhibitory activities against the A549 lung cancer cell line. Compounds 2 and 8 exhibited growth inhibitory activities against the A549 non-small-cell lung cancer cell line, and their GI50 values were calculated to be 41.3 and 4.1 μM, respectively. Under the same condition, the GI50 value of fluorouracil was 3.6 μM. C

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310; ESIMS negative mode m/z [M − H]−, 308; HREIMS m/z 310.2012 (calcd for C17H28NO4 [M + H]+, 310.2018). Boydone B (2): yellow oil; [α]24D −19.4 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 254 (4.0); IR (ZnSe) νmax 3400, 2958, 2919, 1738, 1661, 1455, 1326, 1079, 1030, 929 cm−1; 13C and 1H NMR data, see Table 1; ESIMS m/z [M + H]+, 227; HRESIMS m/z 227.1640 (calcd for C13H23O3 [M + H]+, 227.1647). Botryorhodine F (3): colorless crystal; [α]24D +6.5 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 266 (3.9), 219 (4.2); IR (ZnSe) νmax 3276, 2933, 1698, 1600, 1446, 1265, 1123, 1077, 1021, 853 cm−1; 13C and 1H NMR data, see Table 1; ESIMS negative mode m/z [M − H]−, 349; HREIMS m/z 349.0915 (calcd for C17H17O8 [M − H]−, 349.0923). Botryorhodine G (4): amorphous, white solid; UV (MeOH) λmax (log ε) 269 (3.7); IR (ZnSe) νmax 3253, 1698, 1607, 1465, 1284, 1142, 1077 cm−1; 13C and 1H NMR data, see Table 1; ESIMS negative mode m/z [M − H]−, 331; HRESIMS m/z 331.0816 (calcd for C17H15O7 [M − H]−, 331.0818). Growth Inhibitory Assay. By following the methods reported previously,16 A549 cells were seeded in 96-well plates in medium with 5% FBS. After 24 h, cells were fixed with 10% CCl3COOH to represent cell population at the zero time of drug addition. After additional incubation of vehicle (0.1% DMSO) or 1−9 for 48 h, cells were fixed with 10% CCl3COOH, and SRB was added to stain cells. Unbound SRB was washed out with 1% AcOH, and SRB-bound cells were solubilized with 10 mM Trizma base. The absorbance was read at 515 nm. Using the following absorbance measurements, such as time zero (T0), control growth (C), and cell growth in the presence of compound (Tx), the percentage growth was calculated at each of the compound concentration levels. Percentage growth inhibition was calculated as 100 − [(Tx − T0)/(C − T0)] × 100. Growth inhibition of 50% (GI50) is determined at the drug concentration that results in 50% reduction of total protein increase in control cells during the compound incubation.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-2000 polarimeter (Tokyo, Japan). 1H, 13C NMR, DEPT, COSY, NOESY, HSQC, HMBC, and INADEQUATE were acquired on a Bruker DRX-500 SB and AVIII-800 spectrometer (Ettlingen, Germany). Low-resolution and high-resolution mass spectra were obtained using a VG Platform electrospray ESI/MS (VG, England) and high-definition mass spectrometry system with an ESI interface and a TOF analyzer (Waters Corp., Manchester, UK), respectively. IR spectra were recorded on a JASCO FT/IR 4100 spectrometer (Tokyo, Japan). Sephadex LH-20 (Amersham Biosciences, Filial Sverige, Sweden) and Diaion HP20 (Mitsubishi Chemical, Tokyo, Japan) were used for open column chromatography. TLC was performed using silica gel 60 F254 plates (200 μm, Merck). A reflective index detector (Bischoff, Leonberg, Germany) was used in HPLC purification. Fermentation of Pseudallescheria boydii NTOU2362. P. boydii NTOU2362 was isolated from the leaves of the littoral plant Sesuvium portulacastrum collected at Chiayi County, Taiwan, and was identified by one of us (K.L.P.). This fungal strain was deposited at the Institute of Marine Biology, National Ocean University, Keelung, Taiwan. The mycelium of P. boydii was inoculated into 5 L serum bottles, each containing 30 g of dextrose, 6 g of peptone, 3 g of yeast extract (Becton, Dickinson and Company, Sparks, MD, USA), and 3 L of deionized water. The fermentation was conducted with aeration at 25−30 °C for 30 days. Extraction and Isolation. The filtered fermented broths (60 L) of P. boydii NTOU2362 were partitioned three times with 30 L of recycled ethyl acetate, then concentrated in vacuum to dryness (3.9 g). Subsequently, the crude extracts were redissolved in 20 mL of MeOH, then applied onto a Sephadex LH-20 column (2.8 cm i.d. × 68 cm) eluted by MeOH with a flow rate of 2.2 mL/min. Each fraction (24 mL) was checked for its compositions by TLC using EtOAc− hexane−AcOH (19:1:1, v/v/v) for development, and vanillin−sulfuric acid was used in the detection of compounds with similar skeletons. All the fractions were combined into seven portions, I−VII. Portion I (fr. 10, 11) was combined and rechromatographed on a Diaion HP20 column (1.5 cm i.d. × 24 cm) eluted by aqueous MeOH in stepwise gradient mode with a flow rate of 5.0 mL/min. Each subfraction (150 mL) collected was checked for its composition by the same TLC system and combined into six subportions, I-I−I-VI. Subportion I-III (eluted by 40% MeOH) was purified by HPLC on a semipreparative reversedphase column (Thermo Hyperail HS C18, 10 × 250 mm, Bellefonte, PA, USA) with MeOH−H2O (9:11, v/v) as eluent, 2 mL/min, which gave 9 (30.8 mg, tR = 18.9 min). The subportion I-IV (eluted by 60% MeOH) was purified by the same HPLC column with MeCN−H2O (1:1, v/v) as eluent, 2 mL/min, affording 8 (6.2 mg, tR = 29.6 min). Subportion I-V (eluted by 80% MeOH) was purified by the same HPLC column with MeOH−H2O (3:2, v/v) as eluent, 2 mL/min, affording 7 (68.4 mg, tR = 30.2 min). Portion II (fr. 12−14) was combined and rechromatographed on the same Diaion HP20 column eluted by aqueous MeOH in stepwise gradient mode to afford five subportions, II-I−II-V. Subportion II-IV (eluted by 75% MeOH) was purified by a Phenomenex Luna PFP HPLC column (Torrance, CA, USA) with MeCN−H2O (9:11, v/v) as eluent, 2 mL/min, which resulted in the isolation of 6 (29.7 mg, tR = 24.7 min). Portion IV (fr. 17−19) was combined and rechromatographed on the same Diaion HP20 column system to afford five subportions, IV-I− IV-V. Subportion IV-II (eluted by 25% MeOH) was purified by a Phenomenex Luna PFP HPLC column with MeCN−H2O (3:17, v/v) as eluent, 2 mL/min, which gave 4 (4.4 mg, tR = 10.3 min) and 5 (6.9 mg, tR = 17.4 min). Subportion IV-III (eluted by 50% MeOH) was purified by a Phenomenex Luna PFP HPLC column with MeCN−H2O (2:3, v/ v) as eluent, 2 mL/min, to afford 1 (10.6 mg, tR = 21.2 min). Subportion IV-IV (eluted by 75% MeOH) was purified by a Phenomenex Luna PFP HPLC column with MeCN−H2O (13:7, v/v) as eluent, 2 mL/min, producing 2 (25.7 mg, tR = 22.9 min) and 3 (9.9 mg, tR = 30.3 min). Boydone A (1): yellow oil; [α]31D −43.8 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 348 (4.3), 216 (4.1) nm; IR (ZnSe) νmax 3430, 2966, 2930, 2874, 1660, 1607, 1554, 1442, 1294, 1212, 1060 cm−1; 13C and 1H NMR data, see Table 1; ESIMS positive mode m/z [M + H]+,

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of the new compounds 1−4. This material is available free of charge via the Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +886 2 27361661, ext. 6156. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. S.-H. Wang of the Instrumentation Center of Taipei Medical University and Ms. S.-L. Huang of the Instrumentation Center of the College of Science, National Taiwan University, for the general NMR data acquisition and Dr. C.-F. Chang of Genomics Research Center, Academia Sinica, for the 2D INADEQUATE data acquisition.

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REFERENCES

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