Wortmannine F and G, two new pyranones from Talaromyces wortmannii LGT-4, the endophytic fungus of Tripterygium wilfordii

Phytochemistry Letters 29 (2019) 115–118

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Wortmannine F and G, two new pyranones from Talaromyces wortmannii LGT-4, the endophytic fungus of Tripterygium wilfordii

T

Jun-Wen Zhaoa,b, Zhong-Duo Yanga, , Shuan-Yan Zhouc, Li-Jun Yanga, Jian-Hui Suna, ⁎ Xiao-Jun Yaoc, Zong-Mei Shua, Shuo Lia, ⁎

a

School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, PR China The Provincial Education Key Laboratory of Screening, Evaluation and Advanced Processing of Traditional Chinese Medicine and Tibetan Medicine, School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, PR China c Department of Chemistry, Lanzhou University, Lanzhou 730000, PR China b

ARTICLE INFO

ABSTRACT

Keywords: Wortmannine Talaromyces wortmannii Endophytic fungus Phosphoinositide 3-kinase inhibitors

Two new pyranones, Wortmannine F (1) and G (2), were isolated from Talaromyces wortmannii LGT-4, which is an endophytic fungus from Tripterygium Wilfordii. Their structures were elucidated on the basis of NMR and ESIMS spectral data, as well as chemical calculation. Additional known compounds (4–8) were also isolated. The isolates were tested for monoamine oxidase (MAO), acetylcholinesterase (AChE) and phosphoinositide 3-kinase α (PI3Kα) inhibitory activities. Compounds 1, 2 showed potent PI3Kα inhibitory activity with IC50 of 25 and 5 μM, respectively.

1. Introduction Endophytic fungi which have coevolved for millions of years with their eukaryotic hosts are relatively unstudied and potential sources of novel natural products for exploitation in medicine, agriculture, and industry (Strobel and Daisy, 2003; Bérdy, 2012). The effects of environmental changes and culture conditions on gene expression will be helpful for optimizing secondary metabolite production by endophytic fungi under laboratory conditions (Wani et al., 2016). Moreover, we can obtain secondary metabolites which have unique structures and a wide range of biological activities, such as antitumour, antimicrobial and antituberculosis activities (Sun et al., 2004), by changing the culture conditions. During our ongoing work on endophytic fungi, a fungal strain LGT-4 (GenBank Accession no. KF850714) was isolated from the traditional Chinese medicinal plant Tripterygium Wilfordii. Investigation on the chemical constituents of this fungus showed that the list of compounds in the fungus varied with the culture medium. By changing the fermentation medium, including potato-dextrose broth culture medium, corn culture medium, rice culture medium and sweet potato culture medium, we have obtained a series of novel compounds including secovironolide (Ding et al., 2015), wortmannine A–D (Fu et al., 2016a), deacetylisowortmin A and B (Fu et al., 2016b), wortmannolol (Zhi

et al., 2016) and wortmannine E (Sun et al., 2017). In this paper, we report another two new compounds named wortmannine F (1) and G (2) (Fig. 1), which were obtained from the organic extract of King’s B culture medium. Monoamine oxidase (MAO) is a potential target for new drugs of antidepressants, anxiolytics, and anti-Parkinson's disease (Herraiz et al., 2010). Acetylcholinesterase (AChE) plays a significant role in the termination of nerve impulse transmission, which is a useful target for the drugs of Alzheimer's disease (Houghton et al., 2006). Phosphoinositide 3-kinases (PI3Ks) are potential targets for anti-tumour drugs (Stephens et al., 2005). To obtain natural monoamine oxidase, acetylcholinesterase and PI3K inhibitors, we tested the inhibitory activities of all isolated compounds against these three enzymes. Compounds 1, 2 only showed potent activity against PI3K α. 2. Results and discussion Wortmannine F (1) was isolated as a white amorphous powder and exhibited the molecular formula C16H28O2 (3 degrees of unsaturation) by HRESIMS (m/z 275.1979 [M + Na]+, calcd. for C16H28O2Na, 275.1982). The IR spectrum showed a band at νmax = 1720 cm−1 indicative of the carbonyl group functionality. The 1H and 13C NMR spectra of 1 (Table 1) showed signals for three methyl groups, eight

⁎ Corresponding authors at: School of Life Science and Engineering, Lanzhou University of Technology, 287 Langongping Road, Qilihe District, Lanzhou 730050, PR China. E-mail addresses: [email protected] (Z.-D. Yang), [email protected] (S. Li).

https://doi.org/10.1016/j.phytol.2018.11.023 Received 12 October 2018; Received in revised form 22 November 2018; Accepted 27 November 2018 1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

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Fig. 1. The structures of compounds 1-2.

Table 1 1 H (600 M) and

13

CNMR (150 M) data of compounds 1-2.a

1 in CDCl3

2 in CD3OD

No 1

1

H –

13

C 166.5

No 1

2 3 4 5 6

– 6.64 (d, J = 6.3 Hz) 2.31 (m) 4.36 (m) 1.49 (m) 1.77 (m) 1.49 (m) 1.29 (m) 1.29 (m) 1.29 (m) 1.29 (m) 1.29 (m) 1.49 (m) 1.29 (m) 1.29 (m) 0.87 (t, J = 7.3 Hz) 0.99 (d, J = 7.1 Hz) 1.90 (s)

127.1 145.9 32.5 80.4 32.0

3 4 4a 5 6 7 8 8a 9

7 8 9 10 11 12 13 14 15 16

Mcdonald, 2006) and a nonyl unit (see δH and δC data from H-C-6 to HC-14 in Table 1). The presence of these two partial units was also supported by 2D-NMR correlations (Fig. 2). The HMBC correlations between H-6/C-4 indicated that the nonyl unit was linked with the dihydropyran-2-one unit at C-5. The observed NOESY correlation between H-4/H-5 showed that the relative configuration of H-4 and H-5 was cis-. Molecular calculation studies were executed to determine the absolute configurations at C-5 and C-4. The calculated [α]D value of the (4R, 5S) isomer (-111.0, while the calculated [α]D value of the (4S, 5R) isomer was -35.0) was close to the measured value of 1 (-162.0), which revealed that the absolute configurations of C-4 and C-5 were R and S, respectively. Wortmannine G (2) was isolated as a white amorphous powder and exhibited the molecular formula C12H14O4 (6 degrees of unsaturation) by HRESIMS (m/z 245.0786[M + Na]+, calcd. for C12H14O4Na, 245.0784). The IR spectrum showed bands at νmax = 3396, 1689 cm−1 indicative of carbonyl and hydroxyl functionalities. The 1H and 13C NMR spectra of 1 (Table 1) showed signals for one methyl group, three methylenes, three methines and five quaternary carbons including three sp2 quaternary carbons, one sp3 quaternary carbon and one carbonyl carbon. The 1H and 13C NMR data of 2 were very similar with those of the known compound penicisochroman K (3) (Bunbamrung et al., 2014), which showed that 2 was an analogue of 3. In the 1H and 13C NMR spectra of 2, there was an additional propyl (δH: 2.02 (ddd, J = 13.7, 11.7, 4.7 Hz), 1.76 (ddd, J = 13.7, 11.8, 4.7 Hz), 1.47 (m), 1.35 (m), 0.89 (t, J = 6.4 Hz); δC: 38.1, 16.2, 13.9) instead of a methyl in 3 which suggested there was a propyl at C-3 in 2. The presence of the propyl was also confirmed by 1H-1HCOSY correlations (Fig. 2) between H-9/H-10, H-10/H-11. The HMBC correlations (Fig. 2) between H-9/C4 and H-9/C-3 further showed that the propyl was located at C-3. The specific rotation ([α]D20 +4.0) of 2 suggested that the absolute

25.4 29.4 29.6 29.6 29.7 31.6

10 11

1

H 5.05 4.88 – – – 7.47 7.18 6.97 – – 2.02 1.76 1.47 1.35 0.89

13

(d, J = 16.3 Hz) (d, J = 16.3 Hz)

(d, J = 7.8 Hz) (t, J = 7.9 Hz) (d, J = 8.0 Hz) (ddd, J = 13.7, 11.7, 4.7 Hz) (ddd, J = 13.7, 11.8, 4.7 Hz) (m) (m) (t, J = 6.4 Hz)

C 57.5 96.5 192.4 129.0 117.7 127.6 119.6 152.3 128.8 38.1 16.2 13.9

22.8 14.6 11.5 17.1

a Assignments were aided by a combination of 1H-1H COSY, HMQC and HMBC experiments.

methylenes, three methines and two quaternary carbons including one sp2 quaternary carbon and one ester carbonyl carbon. The 1H and 13C NMR data of 1 suggested that 1 had a 3,5-dimethyl-5,6-dihydropyran-2one unit (see δH and δC data from H-C-1 to H-C-5 in Table 1) (Chen and

Fig. 2. 1H–1H COSY, HMBC and NOESY of 1 and 2.

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configuration at position 3 was S, compared to that of the similar structured penicisochroman K ([α]D20 –3.49) (Bunbamrung et al., 2014). Four known compounds were identified as cyclo (L-Leucyl-L-Prolyl) (4) (Yan et al., 2004), cyclo [L-(4-hydroxyprolinyl)-D-leucine] (5) (Jr et al., 1998), cis-cyclo (L-Ala-L-Pro) (6) (Stark and Hofmann, 2005), tryptamine (7) (Mourshid et al., 2016) and cyclo (D-Pro-L-Leu) (8) (Kumar et al., 2013) by comparing their physical data (MS, 1H and 13C NMR) with those reported in the literature. The inhibitory activities of the isolated compounds 1–2, 4-8 against monoamine oxidase (MAO), acetylcholinesterase (AChE) and phosphoinositide 3-kinase α (PI3Kα) were tested. Only compounds 1, 2 showed potent PI3Kα inhibitory activity with IC50 of 25 and 5 μM, respectively, when the IC50 value of GDC 0941 as a positive drug was 4.2 nM. However, the other compounds (4-8) did not show any inhibitory activities against the above three enzymes. Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that play key regulatory roles in cell proliferation, survival, and cell translation (Stephens et al., 2005). It has been proved that PI3Ks are potential targets for chemical therapy, especially PI3K α as one of the most important targets of anti-tumour drugs (Stephens et al., 2005). Up to now, many PI3K inhibitors have been found, including wortmannin derivatives, thienopyrimidine derivatives, quinazolone derivatives, pyridofuropyrimidines, imidazopyridine analogues, midazoquinoline analogues, etc. (Wu et al., 2014). However, not too many natural PI3K inhibitors were found (Wu et al., 2014). To obtain natural PI3K inhibitors, we tested the PI3K α inhibitory activities of 1-2 and found that they were potent natural PI3K α inhibitors.

fractions (Fr. 3–1 to 3–6). Fr. 3–2 (54 mg) was subjected to semi-preparative HPLC purification (Gemini-NX, 5 μm, C18, 110 A, 250 × 10.0 mm, flow rate of 1 mL/min, UV 254 nm) eluting with 58% MeOH–H2O to yield compound 1 (tR = 45 min, 5 mg). Fr. 3–3 (34 mg) was also subjected to semi-preparative HPLC purification (Gemini-NX, 5 μ, C18, 110 A, 250 × 10.0 mm, flow rate of 1 mL/min, UV 254 nm) eluting with 42% MeOH–H2O to yield compound 2 (tR = 50.6 min, 3 mg). Fr. 4 (82 mg) was separated on a silica gel column eluted with a gradient of petroleum ether–acetone (40:1, 30:1, 10:1, 5:1, 1:1) to yield compound 4 (11 mg) and Fr. 4-3. Fr. 4-3 was separated on a silica gel column eluted with a gradient of petroleum ether-acetone (20:1, 10:1, 8:1, 5:1, 1:1) to yield compound 5 (6 mg). Fr. 2 (64 mg) was subjected to semi-preparative HPLC purification (Gemini-NX, 5 μ, C18, 110 A, 250 × 10.0 mm, flow rate of 1 mL/min, UV 210 nm) eluting with 35% MeOH–H2O to yield compounds 6 (tR = 35 min, 5 mg) and 7 (tR = 42 min, 7 mg). Fr. 1 (78 mg) was separated on a silica gel column eluted with a gradient of petroleum chloroform-methanol (10:1, 8:1, 5:1, 3:1,1:1) to yield compound 8 (5 mg). 3.4.1. Wortmannine F (1) White amorphous powder; [α]D20 -162.0 (c 40 mM, CHCl3); IR (KBr): νmax 1720 cm−1; 1 H and 13C NMR spectroscopic data, see Table 1; HRESIMS 275.1979 [M + Na]+ (calcd. for C16H28O2Na, 275.1982).

3. Experimental

3.4.2. Wortmannine G (2) White amorphous powder; [α]D20+4.0° (c 4 mM, CHCl3); IR(KBr): νmax 3396, 1689 cm−1; 1 H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z: 245.0786 [M + Na]+, (calcd. for C12H14O4Na, 245.0784).

3.1. General experimental procedures

3.5. Bioassays

The 1H and 13C NMR spectra were obtained on Bruker 600 MHz spectrometers, using TMS as an internal standard. The IR spectrum was recorded on a Perkin-Elmer Paragon spectrophotometer. Separation and purification were performed by a semi-preparative HPLC system (consisted of a Jasco PU-2086 Plus, UV-2075 Plus detector and GeminiNX ODS column (5 μm, φ 250 × 10 mm, Phenomenex Co. Ltd.)). The silica gel (200–300 mesh) for column chromatography and silica GF254 for TLC were supplied by Qingdao Marine Chemical Inc., China. The macroporous resin was from Cangzhou Bon Adsorber Technology Co., Ltd. (Cangzhou, China). All other organic solvents were of analytical grade.

3.5.1. Monoamine oxidase assay The procedure for testing the monoamine oxidase (MAO) inhibiting activity was the same as that reported in our previous paper (Zhi et al., 2014). 3.5.2. Acetylcholinesterase assay The procedure for testing the AChE inhibiting activity was the same as that reported in our previous paper (Yang et al., 2012). 3.5.3. PI3Kα biochemical assay The PI3Kα activity assay was conducted using the ADP-Glo Kinase assay (Han et al., 2015). Briefly, the ADP-Glo™ kinase detection kit was from Promega. PI3Kα kinase and the substrate PIP2/PS were from Thermo Fisher Scientific. All assays were performed in a black 384-well plate at room temperature. The kinase buffer contained 50 mM Hepes (pH 7.5), 3 mM MgCl2, 100 mM NaCl, 1 mM EGTA, 0.03% CHAPS and 2 mM DTT. The PI3Kα kinase mixture was prepared by diluting PI3Kα in the kinase buffer to 0.9 ng/μL. The ATP/substrate mixture contained 10 μM PIP2/PS and 50 μM ATP. The compounds for testing were diluted in DMSO to obtain different concentrations. 2 μL of the diluted compound and 4 μL of the ATP/substrate mixture were added to individual wells of 384-well assay plates. The reactions were started by adding 4 μL of PI3Kα kinase mixture per well. The assay plates were covered, and the reactions were allowed to proceed for 1 h, after which 10 μL of Kinase Glo™ reagent per well was added. The plates were incubated for 40 min, and then 20 μL of kinase detection reagent per well was added. The plates then were equilibrated in the dark for 25 min, after which luminescence was measured. The percentage of inhibition was calculated based on the following equation: %inhibition = [1 – (RLUcompound – RLUmin)/(RLUmax–RLUmin)] × 100, where RLUcompound is the luminescence reading at a given compound concentration, RLUmin is the luminescence reading at the highest concentration (10 mM) of the positive control (compound GDC0941) to completely inhibit PI3Kα

3.2. Isolation and identification of the fungal strain The strain LGT-4 was isolated from the Chinese herb medicine Tripterygium wilfordii and was identified as T. wortmannii based on both the morphology on PDA and analysis of the DNA sequences of the ITS15.8S-ITS2 ribosomal DNA gene region (GenBank Accession no. KF850714) (Fu et al., 2016a). 3.3. Fermentation and extraction The strain LGT-4 was cultured in King’s B Medium (Siddiqui et al., 2005) for 20 d at 28 °C on a 50 L fermenter. The fermenting broth was filtered, and then, the filtrate was extracted with EtOAc (50 L × 2). 3.4. Isolation The crude extract (3 g) was chromatographed on a macroporous resin column with a step gradient of EtOH-H2O (20, 40, 60, 80 and 100% EtOH) to yield five fractions (Fr. 1–5). Fr. 3 was further separated using silica gel column chromatography with a step gradient of petroleum ether-acetone (30:1, 20:1, 8:1, 5:1, 2:1 and 1:1) to yield six 117

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kinase activity, and RLUmax is the luminescence reading in the absence of a compound.

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