Antimicrobial metabolites from the plant endophytic fungus Penicillium sp.

Antimicrobial metabolites from the plant endophytic fungus Penicillium sp.

Fitoterapia 116 (2017) 72–76 Contents lists available at ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote Antimicrobial m...

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Fitoterapia 116 (2017) 72–76

Contents lists available at ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Antimicrobial metabolites from the plant endophytic fungus Penicillium sp. Ming-Hua Yang 1, Tian-Xiao Li 1, Ying Wang, Rui-Huan Liu, Jun Luo, Ling-Yi Kong ⁎ State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China

a r t i c l e

i n f o

Article history: Received 25 August 2016 Received in revised form 9 November 2016 Accepted 10 November 2016 Available online 19 November 2016 Chemical compounds studied in this article: (R)-MTPA-Cl (PubChem CID: 3080792) (S)-MTPA-Cl (PubChem CID: 2724611) 4-Dimethylaminopyridine (PubChem CID: 14284) Ethyl ethanoate (PubChem CID: 8857) Pyridine (PubChem CID: 1049)

a b s t r a c t Five rare dichloro aromatic polyketides (1–5) were obtained from an endophytic fungus Penicillium sp., along with five known metabolites (6–10). Their structures were elucidated by extensive spectroscopic analysis, Mosher methods, as well as [Rh2(OCOCF3)4]-induced electronic circular dichroism (ECD) experiments. Compounds 2– 4 and 6 structurally involved acyclic 1.3-diols, the uneasy configuration determinations of which were well carried out by double-derivation NMR methods. Compounds 1–10 were evaluated for their antibacterial and antifungal activities against five strains of human pathogenic microorganisms. Helvolic acid (7) showed potent inhibitory effects against Staphylococcus aureus and Pseudomonas aeruginosa with MIC (minimum inhibitory concentration) values of 5.8 and 4.6 μg/mL, respectively. © 2016 Elsevier B.V. All rights reserved.

Keywords: Penicillium sp. Phenolic metabolites Dichlorine Antimicrobial

1. Introduction Penicillium genus is well-known for its antibiotic metabolite penicillin, which is still widely used in clinic since its discovery in 1920s. Several decades later, the bioactive metabolites of this fungal genus are still attractive. These metabolites, diverse in structural types like polyketides [1], tetramic acid derivatives [2], and alkaloids [3], remain potential sources of antitumor and antimicrobial prodrugs [3–5]. In our continuous search for bioactive fungal metabolites [6–8], a Penicillium strain that harbored in the tubers of Pinellia ternata aroused our attention. Its EtOAc extract exhibited obvious antibacterial activities against Staphylococcus aureus and Pseudomonas aeruginosa with MIC (minimum inhibitory concentration) values of 287 and 232 μg/mL, respectively. Thus, the chemical investigations of this extract were carried out and led to the isolation of seven dichloro aromatic polyketides (1–5, 6, and 9), helvolic acid (7), the sulfur-containing diketopiperazine (8), and trypacidin A (10) (Fig. 1). All the isolated compounds were reevaluated for their antimicrobial activities against a panel of human

⁎ Corresponding author. E-mail address: [email protected] (L.-Y. Kong). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.fitote.2016.11.008 0367-326X/© 2016 Elsevier B.V. All rights reserved.

pathogenic microorganisms. Helvolic acid (7) was found to be the most effective one, especially against S. aureus and P. aeruginosa (MIC = 5.8 and 4.6 μg/mL). Herein, the isolation, structural elucidation, and biological activities of 1–10 are described.

2. Experimental 2.1. General experimental procedures ECD spectra were recorded on a JASCO 810 spectrometer. The optical rotations were measured on a JASCO P-1020 polarimeter. UV spectra were scanned by a Shimadzu UV-2450 spectrophotometer and IR spectra were recorded in KBr discs on a Bruker Tensor 27 spectrometer. Standard 1D and 2D NMR spectra were performed on the Bruker Avance III NMR instrument (1H, 500 MHz and 13C, 125 MHz) and chemical shifts were referenced by the solvent peaks (δH 3.31, δC 49.1 for CD3OD and δH 7.26, δC 77.2 for CDCl3). HRESIMS was accomplished with an Agilent 6520B UPLC-Q-TOF mass spectrometer. HPLC was carried out with an Agilent 1100 system equipped with G1314 UV detector and ZORBAX SB-C18 column (9.4 × 250 mm). Preparative HPLC was performed on a Shimadzu LC-10A system with Shim-pack RP-C18 column (20 × 200 mm) and Shimadzu SPD-20A detector at the flow rate of

M.-H. Yang et al. / Fitoterapia 116 (2017) 72–76

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Fig. 1. Structures of compounds 1–10.

10 mL/min. ODS, silica gel, and sephadex LH-20 were employed for the column chromatography (CC). Precoated silica gel GF254 plates (Qingdao Marine Chemical, Co., Ltd., China) were used for thin layer chromatography (TLC).

MeOH). Fr. 5 (1.5 g) was chromatographed over silica gel CC using CH2Cl2–MeOH (20:1 to 1:1, v/v) and further purified by preparative HPLC to give 9 (3.2 mg, tR 24.8 min) and 10 (5.0 mg, tR 33.0 min) using MeOH–H2O (50:50) as the mobile phase.

2.2. Fungal material 2.4. Spectroscopic data The title endophyte was isolated from the tubers of P. ternata that were collected from the suburb of Nanjing, Jiangsu, P. R. China, in October 2012. It was identified as Penicillium sp. by internal transcribed spacer (ITS) region and 18S rDNA sequence analysis (99% sequence homology to Penicillium sp. MZKI P-265, GenBank Accession no. EU069420.1). The fungal was cultured on PDA (potato 200 g/L, dextrose 20 g/L, and agar 18 g/L) at 28 °C for 5 days. Then the agars were cut into small pieces, which were transferred aseptically into a 1000 mL Erlenmeyer flask (containing 300 mL of potato dextrose broth). The flask was incubated on a rotary shaker at 150 rpm and 28 °C for 7 days to prepare seed culture. The seed culture was then added into ten 500 mL Erlenmeyer flask (each containing 80 g of rice and 120 mL of sterile water) to cultivate at 28 °C for 30 days. 2.3. Extraction and isolation The fermented culture was extracted with EtOAc three times, and the EtOAc solvent was removed under vacuum to afford a crude extract (6.8 g). The extract was separated into six fractions (Fr. 1–Fr. 6) upon silica gel CC by TLC analysis, using a gradient elution of petroleum ether– EtOAc (40:1, 20:1, 5:1, 2:1, 1:2, v/v). Fr. 4 (2.1 g) was applied to ODS CC with a step gradient elution of MeOH–H2O (20:80 to 80:20, v/v) to give five subfractions (Fr. 4.1–Fr. 4.5). Fraction Fr. 4.3 (0.5 g) was chromatographed over Sephadex LH-20 CC and further purified by preparative HPLC using acetonitrile–H2O (35:65) to yield 2 (5.9 mg, tR 25.2 min) and 3 (3.2 mg, tR 26.5 min). Fr. 4.2 (0.3 g) was processed in the same way with acetonitrile–H2O (25:75) to afford 4 (4.4 mg, tR 30.4 min) and 6 (15.6 mg, tR 32.0 min), respectively. Separation of Fr. 3 (0.4 g) was achieved by preparative HPLC with MeOH–H2O (60:40), giving 5 (9.6 mg, tR 28.3 min) and 7 (7.0 mg, tR 31.5 min). Compound 1 (2.6 mg, tR 21.4 min) was obtained from Fr. 3 by preparative HPLC using MeOH–H2O (50:50), and so was 8 (18.5 mg, tR 18.9 min, 70%

(+)-(2R)-3′-methoxyl citreovirone (1): colorless oil; [α]25 D + 0.7 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 193 (3.53), 204 (4.09), 280 (2.96) nm; ECD (2 × 10− 4, MeOH) λmax (Δε) 365 (+ 3.67), 426 (−2.92) nm; IR (KBr) νmax 3443, 2920, 2850, 2355, 1632, 1468, 1384, 1124, 1025, 517 cm−1; ESIMS, m/z 307 [M + H]+; HRESIMS, m/z 307.0499 [M + H]+ (C13H17O4Cl2, calcd for 307.0498); 1H and 13C NMR data (CDCl3) see Table 1. (+)-(2R,4S)-3′-methoxyl citreochlorol (2): colorless needles; [α]25 D + 4.6 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 206 (4.13), 274 (2.86), 280 (2.86) nm; IR (KBr) νmax 3436, 2922, 2850, 2375, 2309, 1601, 1465, 1352, 1205, 1151, 1070, 833, 779 cm−1; ESIMS, m/z 309 [M + H]+; HRESIMS, m/z 331.0475 [M + Na]+ (C13H18NaO4Cl2, calcd for 331.0474); 1H and 13C NMR data (CDCl3) see Table 1. (−)-(2S,4R)-3′-methoxyl citreochlorol (3): colorless needles; [α]25 D − 2.3 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 195 (3.41), 206 (4.14), 274 (2.91), 280 (2.87) nm; IR (KBr) νmax 3428, 2919, 2849, 2375, 2347, 1599, 1458, 1362, 1205, 1087, 827, 777 cm−1; ESIMS, m/z 309 [M + H]+; HRESIMS, m/z 331.0473 [M + Na]+ (C13H18NaO4Cl2, calcd for 331.0474); 1H and 13C NMR data (CDCl3) see Table 1. (−)-Citreochlorol (4): pale brown needles; [α]25 D − 2.1 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 194 (3.20), 205 (3.91), 275 (2.64), 280 (2.63) nm; IR (KBr) νmax 3441, 2920, 2850, 2353, 1600, 1384, 1155, 853, 517 cm− 1; ESIMS, m/z 317 [M + Na]+; HRESIMS, m/z 317.0321 [M + Na]+ (C12H16NaO4Cl2, calcd for 317.0318); 1H and 13C NMR data (CD3OD) see Table 1. (−)-(3S,10R)-dichlorodiaportal (5): brownish solid; [α]25 D − 12.9 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 215 (4.58), 257 (4.21), 299 (3.97) nm; IR (KBr) νmax 3444, 2923, 2851, 2375, 2347, 2318, 1748, 1629, 1510, 1436, 1385, 1161, 1060, 840, 782 cm− 1; ESIMS, m/z 337 [M + H]+; HRESIMS, m/z 335.0097 [M − H]− (C13H13O6Cl2, calcd for 335.0095); 1H and 13C NMR data (CDCl3) see Table 2.

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M.-H. Yang et al. / Fitoterapia 116 (2017) 72–76

Table 1 1 H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data of 1–4. Position 1 2 3

75.1 72.7 43.2 206.8 51.2

3.68 (s)

1′ 2′ 3′ 4′ 5′ 6′ 3′-OCH3 5′-OCH3 b

δC

5.79 (d, 4.0) 4.37 (dt, 4.0, 7.6, 11.8) 2.87 (dd, 7.6, 17.7) 2.95 (dd, 4.4, 17.7)

4 5

a

1a δH (multi, J in Hz)

135.4 107.8 161.4 99.6 161.4 107.8 55.6 55.6

6.34 (t, 2.1) 6.39 (t, 2.1) 6.35 (t, 2.1) 3.78 (s) 3.78 (s)

2a δH (multi, J in Hz) 5.72 (d, 4.0) 4.17 (m) 1.80 (m) 2.04 (m) 4.12 (m) 2.69 (dd, 4.4, 13.5) 2.82 (dd, 8.5, 13.5)

6.36 (br s) 6.36 (br s) 3.78 (s) 3.78 (s)

72.4 44.9

5.75 (d, 4.4) 4.25 (m) 1.88 (m) 1.89 (m) 4.17 (m) 2.67 (dd, 8.9, 13.4) 2.81 (dd, 4.1, 13.4) 6.37 (br s) 6.36 (br s) 6.37 (br s) 3.78 (s) 3.78 (s)

δC 76.8 73.9 38.6 69.5 44.8 140.2 107.6 161.4 99.1 161.4 107.6 55.6 55.6

4b δH (multi, J in Hz) 5.91 (d, 3.4) 4.04 (m) 1.75 (m) 1.91 (m) 4.00 (m) 2.66 (dd, 8.9, 13.4) 2.68 (dd, 4.1, 13.4)

δC 76.2 74.0 38.4 70.0 43.6

6.31 (t, 2.1)

140.7 108.7 158.0 99.0 160.9 106.2

3.73 (s)

54.2

6.29 (t, 2.11) 6.22 (t, 2.1)

Recorded in CDCl3. Recorded in CD3OD.

A sample of 2 (0.8 mg) was dissolved in freshly distilled dry pyridine (150 μL), and several dry crystals of dimethylaminopyridine were added. Treated 2 with (R)-(−)-MTPA-Cl to prepare the (S)-MTPA ester (2a) at 60 °C for 10 h. The reaction was quenched with 1 mL of methanol. Then the solvent was removed and the residue was purified by preparative HPLC. Similarly, a sample of 2 (0.8 mg) was treated with (S)-(+)-MTPA-Cl to afford the (R)-MTPA ester 2b. The (S)- and (R)-MTPA esters of 3–6 were prepared in the same manner. 1H NMR signals of these MTPA esters (3a, 3b, 4a, 4b, 5a, 5b, 6a, and 6b) were assigned unambiguously as below. Data for 2a: (1H NMR, 500 MHz, CDCl3) δ 7.552–7.260 (10H, aromatic), 6.359 (1H, br t, H-4′), 6.333 (2H, br t, H-2′/6′), 5.774 (1H, d, J = 2.8 Hz, H-1), 5.372 (1H, m, H-2), 5.353 (1H, m, H-4), 3.761 (6H, s, 3′/ 5′-OCH3), 3.545 (3H, s, MTPA-OCH3), 3.409 (3H, s, MTPA-OCH3), 2.982 (1H, dd, J = 4.6, 14.0 Hz, H-5a), 2.901 (1H, dd, J = 8.2, 13.8 Hz, H-5b), 2.271 (1H, m, H-3a), 2.260 (1H, m, H-3b); ESIMS positive m/z 741 [M + H]+. Data for 2b: (1H NMR, 500 MHz, CDCl3) δ 7.548–7.362 (10H, aromatic), 6.314 (1H, br t, H-4′), 6.234 (2H, br t, H-2′/6′), 5.935 (1H, d, J = 3.1 Hz, H-1), 5.358 (1H, m, H-2), 5.258 (1H, m, H-4), 3.710 (6H, s, 3′/ 5′-OCH3), 3.612 (3H, s, MTPA-OCH3), 3.421 (3H, s, MTPA-OCH3), 2.757

Table 2 1 H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data of 5 and 6.

Position 1 3 4 4a 5 6 7 8 8a 9 10 11 3-OH 6-OCH3 8-OH 10-OH b

76.5 75.8 37.5

139.8 107.7 161.4 99.1 161.4 107.7 55.6 55.6

6.36 (br s)

2.5. Preparation of the (S)- and (R)-MTPA ester derivatives

a

3a δH (multi, J in Hz)

δC

5a δH (multi, J in Hz)

3.10 (s) 6.31 (d, 1.5) 6.38 (d, 1.5)

2.18 (dd, 3.0, 14.0) 2.37 (dd, 11.0, 14.0) 4.86 (m) 5.68 (d, 3.4) 3.59 (br s) 3.90 (s) 11.05 (s) 6.16 (br s)

Recorded in CDCl3. Recorded in CD3OD.

δC

Position

168.7 103.1 39.5 138.6 107.6 166.4 99.9 164.8 101.4 40.0

1 2 3

72.9 75.6 55.8

4 5 1′ 2′ 3′ 4′ 5′ 6′ 5′-OCH3

6b δH (multi, J in Hz) 5.96 (d, 3.4) 4.21 (m) 1.79 (m) 1.81 (m) 4.11 (m) 2.72 (dd, 6.1, 13.4) 2.80 (dd, 7.1, 13.4) 6.37 (br t) 6.30 (br t) 6.39 (br t) 3.81 (s)

δC 78.3 74.1 40.0 69.9 46.0 142.3 110.1 159.5 100.3 162.3 107.6 55.6

(1H, dd, J = 7.0, 14.0 Hz, H-5a), 2.631 (1H, dd, J = 6.2, 14.0 Hz, H-5b), 2.242 (1H, m, H-3a), 2.118 (1H, m, H-3b); ESIMS positive m/z 741 [M + H]+. Data for 3a: (1H NMR, 500 MHz, CDCl3) δ 7.524–7.391 (10H, aromatic), 6.332 (1H, br t, H-4′), 6.237 (2H, br t, H-2′/6′), 5.844 (1H, d, J = 2.4 Hz, H-1), 5.251 (1H, m, H-2), 5.153 (1H, m, H-4), 3.714 (6H, s, 3′/ 5′-OCH3), 3.464 (6H, s, MTPA-OCH3), 2.994 (1H, dd, J = 6.5, 13.2 Hz, H-5a), 2.760 (1H, dd, J = 6.9, 14.4 Hz, H-5b), 2.191 (1H, m, H-3a), 2.106 (1H, m, H-3b); ESIMS positive m/z 741 [M + H]+. Data for 3b: (1H NMR, 500 MHz, CDCl3) δ 7.548–7.380 (10H, aromatic), 6.329 (1H, br t, H-4′), 6.263 (2H, br t, H-2′/6′), 5.837 (1H, d, J = 3.1 Hz, H-1), 5.304 (1H, m, H-2), 5.226 (1H, m, H-4), 3.709 (6H, s, 3′/ 5′-OCH3), 3.596 (3H, MTPA-OCH3), 3.480 (3H, MTPA-OCH3), 3.029 (1H, dd, J = 6.1, 13.8 Hz, H-5a), 2.705 (1H, dd, J = 7.2, 13.8 Hz, H-5b), 2.263 (1H, m, H-3a), 2.152 (1H, m, H-3b); ESIMS positive m/z 741 [M + H]+. Data for 4a: (1H NMR, 500 MHz, CDCl3) δ 7.612–7.290 (15H, aromatic), 6.633 (1H, br t, H-2′), 6.579 (1H, br t, H-4′), 6.562 (1H, br t, H-6′), 5.785 (1H, d, J = 3.5 Hz, H-1), 5.345 (1H, m, H-2), 5.329 (1H, m, H-4), 3.715 (3H, s, 5′-OCH3), 3.643 (3H, s, MTPA-OCH3), 3.527 (3H, s, MTPAOCH3), 3.371 (3H, s, MTPA-OCH3), 3.019 (1H, dd, J = 4.6, 14.2 Hz, H5a), 2.907 (1H, dd, J = 8.6, 14.2 Hz, H-5b), 2.265 (1H, m, H-3a), 2.252 (1H, m, H-3b); ESIMS positive m/z 960 [M + NH4]+. Data for 4b: (1H NMR, 500 MHz, CDCl3) δ 7.639–7.335 (15H, aromatic), 6.531 (1H, br t, H-2′), 6.519 (1H, br t, H-4′), 6.472 (1H, br t, H-6′), 5.954 (1H, d, J = 3.2 Hz, H-1), 5.356 (1H, m, H-2), 5.221 (1H, m, H-4), 3.720 (3H, s, 5′-OCH3), 3.667 (3H, s, MTPA-OCH3), 3.604 (3H, s, MTPAOCH3), 3.392 (3H, s, MTPA-OCH3), 2.721 (1H, dd, J = 7.2, 14.1 Hz, H5a), 2.649 (1H, dd, J = 5.7, 14.1 Hz, H-5b), 2.240 (1H, m, H-3a), 2.144 (1H, m, H-3b); ESIMS positive m/z 960 [M + NH4]+. Data for 5a: (1H NMR, 500 MHz, CDCl3) δ 7.816–7.291 (10H, aromatic), 6.631 (1H, br d, H-7), 6.615 (1H, br d, H-5), 6.236 (1H, s, 3-OH), 6.078 (1H, d, J = 3.0 Hz, H-11), 5.693 (1H, m, H-10), 3.896 (3H, s, 6OCH3), 3.775 (3H, s, MTPA-OCH3), 3.541 (3H, s, MTPA-OCH3), 3.413 (2H, s, H-4), 3.224 (1H, dd, J = 3.7, 15.4 Hz, H-9a), 3.034 (1H, dd, J = 9.9, 15.2 Hz, H-9b); ESIMS positive m/z 769 [M + H]+. Data for 5b: (1H NMR, 500 MHz, CDCl3) δ 7.811–7.094 (10H, aromatic), 6.597 (1H, d, J = 2.3 Hz, H-7), 6.491 (1H, d, J = 2.3 Hz, H-5), 6.090 (1H, d, J = 3.2 Hz, H-11), 6.030 (1H, br s, 3-OH), 5.717 (1H, m, H-10), 3.892 (3H, s, 6-OCH3), 3.796 (3H, s, MTPA-OCH3), 3.646 (3H, br s, MTPA-OCH3), 3.579 (2H, s, H-4), 3.128 (1H, dd, J = 3.4, 15.5 Hz, H-9), 2.955 (1H, dd, J = 9.8, 15.4 Hz, H-9); ESIMS positive m/z 769 [M + H]+. Data for 6a: (1H NMR, 500 MHz, CDCl3) δ 7.617–7.385 (15H, aromatic), 6.559 (2H, br t, H-2′/6′), 6.511 (1H, br t, H-4′), 5.851 (1H, d, J = 3.1 Hz, H-1), 5.275 (1H, m, H-2), 5.194 (1H, m, H-4), 3.728 (3H, s, 5′OCH3), 3.655 (3H, s, MTPA-OCH3), 3.532 (3H, s, MTPA-OCH3), 3.446

M.-H. Yang et al. / Fitoterapia 116 (2017) 72–76

(3H, s, MTPA-OCH3), 3.027 (1H, dd, J = 6.4, 14.3 Hz, H-5a), 2.781 (1H, dd, J = 6.0, 14.3 Hz, H-5b), 2.273 (1H, m, H-3a), 2.131 (1H, m, H-3b); ESIMS positive m/z 960 [M + NH4]+. Data for 6b: (1H NMR, 500 MHz, CDCl3) δ 7.558–7.374 (15H, aromatic), 6.575 (1H, br t, H-2′), 6.543 (1H, br t, H-4′), 6.494 (1H, br t, H-6′), 5.848 (1H, d, J = 2.9 Hz, H-1), 5.207 (1H, m, H-2), 5.138 (1H, m, H-4), 3.738 (3H, s, 5′-OCH3), 3.655 (3H, s, MTPA-OCH3), 3.600 (3H, s, MTPAOCH3), 3.432 (3H, s, MTPA-OCH3), 2.984 (1H, dd, J = 7.8, 14.9 Hz, H5a), 2.813 (1H, dd, J = 6.5, 14.8 Hz, H-5b), 2.172 (1H, m, H-3a), 2.100 (1H, m, H-3b); ESIMS positive m/z 960 [M + NH4]+. 2.6. Antimicrobial assay Broth microdilution method was used for determining the MIC of a pure compound according to the antimicrobial susceptibility testing standards of National Center for Clinical Laboratory Standards (NCCLS). The assays were carried out using sterile 96-well microtiter plates in triplicate [6]. The test samples were first dissolved in DMSO, then the solutions were diluted with broth by a serial 2-fold method (DMSO b 1%). Fluconazole was used as the positive control for antifungal, amoxicillin for Gram-positive bacteria, and streptomycin for Gramnegative bacteria assays. Control wells were only incubated with medium to ensure the sterility. MIC value was defined as the drug concentration that caused 50% reduction of the microbial growth, which was obtained by the optical density measurement after incubation for 24 h. 3. Results and discussion Compound 1 was obtained as colorless oil. Its molecular formula C13H16O4Cl2 was deduced on the basis of the positive HRESIMS ion at m/z 307.0499 [M + H]+ (calcd for C13H17O4Cl2, 307.0498). IR spectrum suggested the presence of hydroxy (3443 cm−1) and keto (1632 cm−1) groups. In its 1D NMR spectrums (Table 1), three meta-coupled aromatic protons (δH 6.39, 6.35, and 6.34), two equative methoxyls (both at δH 3.78), along with the symmetric aromatic carbon signals (δC 161.4 × 2, 135.4, 107.8 × 2, 99.6), indicated the presence of a symmetrical trisubstituted phenyl unit. The remaining 1D NMR data combined with HSQC signals were attributed as two methylenes, a hydroxy methine, a dichloromethyl, and a carbonyl. HMBC correlations (Fig. 2) from H-3 to C-1/C-2/C-4 and from H-5 to C-3/C-4, revealed the presence of 1,1dichloro-2-hydroxy-4-pentanone side chain. And the HMBC correlations from H-5 to C-1′ (δC 135.4) and from H-2′ (δH 6.34) to C-5 suggested the 5–1′ connection of the above side chain with the benzene ring. Therefore, 1 was identified as an analogue of citreovirone (9) [9], namely 3′-methoxyl citreovirone. Absolute configuration of the C-2 secondary alcohol was determined by [Rh2(OCOCF3)4]-induced ECD experiment. In the induced ECD spectrum (Supporting information Fig. S25), the significant positive Cotton effect at 350 nm indicated the bS configuration, corresponding to the 2R absolute configuration according to the bulkiness rule [10–12].

Fig. 2. HMBC correlations of compounds 1–3 and 5.

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The molecular formula C13H18O4Cl2 of 2 was assigned by the positive ion at m/z 331.0475 [M + Na]+ (calcd for C13H18NaO4Cl2, 331.0474). Analysis of its NMR data (Table 1) with those of 1 indicated their structural similarity. However, the presence of additional hydroxy methine (δH 4.12, 1H, m; δC 72.4) suggested the reduction of 4-carbonyl, which was further confirmed by the HMBC correlations (Fig. 2). Yet, the hydroxylation gave typical 1.3-diols for 2, the absolute configurations of which were quite uneasy to determine. Therefore, the double-derivation NMR method [13,14] were introduced and treatment of 2 with (R)- and (S)-MTPA-Cl gave (S)- and (R)-MTPA esters 2a and 2b, respectively. According to the anti-1.3-type A mode, the positive ΔδH(S–R) values of H-2, H-3, and H-4 (Fig. 3) established the 2R and 4S absolute configuration for 2. Compound 3 had the same molecular formula as 2. Its NMR data resembled those of 2 but slightly differed at C-2 and C-4, suggesting that 3 was a stereoisomer of 2. Similar to the case of 2, the 2S and 4R absolute configuration of 3 was assigned by the negative ΔδH(S–R) values of H-2, H-3, and H-4 (Fig. 3) on the basis of anti-1,3-type B mode [13]. Compounds 4 and 6, with the same molecular formula of C12H16O4Cl2, had the identical planar structure to citreochlorol [15] as demonstrated by their NMR data (Tables 1 and 2). The optical value 30 ([α]25 D − 2.1) of 4 was negative while citreochlorol ([α]D + 2.4) and 6 25 ([α]D + 4.7) were positive [15], implying that 4 was a stereoisomer of citreochlorol. To determine the absolute configurations of these 1,3diols in both 4 and 6, the above NMR method was performed likewise. The 1H NMR signals of (S)- and (R)-MTPA esters were attributed and the ΔδH(S−R) values were calculated (Fig. 3), giving 2R,4R and 2R,4S absolute configurations for 4 and 6, respectively. The [M − H]− ion at m/z 335.0097 gave the molecular formula C13H14O6Cl2 for 5, which showed six indices of hydrogen deficiency. The tetrasubstituted benzene ring was revealed by a pair of metacoupled aromatic protons (δH 6.38, 6.31, each 1H, d, J = 1.5 Hz), a phenolic hydroxy (δH 11.05, 1H, s), as well as an aromatic methoxyl (δH 3.90, 3H, s). In combination with its HSQC spectrum, the other signals could be assigned as a singlet methylene (δH 3.10, 2H, s; δC 39.5), a coupled methylene (δH 2.37, 1H, dd, J = 11.0, 14.0 Hz; δH 2.18, 1H, dd, J = 3.0, 14.0 Hz; δC 40.0), an oxygenated methine (δH 4.86, 1H, m; δC 72.9), a dichloromethine (δH 5.68, 1H, d, J = 3.4 Hz; δC 75.6), an ester carbonyl (δC 168.7), and an acetal carbon (δC 103.1). Thorough comparison of the NMR data with those of dichlorodiaportin [16] indicated their structural similarity, except for the distinguishing C-3 and C-4. In particular, the olefinic bond (Δ3(4)) in dichlorodiaportin was oxidized to tertiary alcohol in 5, as confirmed by its HMBC correlations (Fig. 2). Hence, the planar structure of 5 was established. The absolute configuration of 10-secondary alcohol was demonstrated by Mosher method [17] and the positive ΔδH(S−R) value of H-9 and the negative H-11 revealed the 10R configuration. In addition, to determine the absolute configuration of 3-tertiary alcohol, [Rh2(OCOCF3)4]-induced ECD experiment was applied to 5a. The positive sign of the E band at around 350 nm (Supporting Information Fig. S26) suggested the 3S absolute configuration [10,11]. Thus, the structure of dichlorodiaportal (5) was well confirmed. Five known compounds were identified as (+)-citreochlorol (6) [15], helvolic acid (7) [18], cis-bis-(methylthio)-silvatin (8) [19], citreovirone (9) [9], and trypacidin A (10) [20] by comparison of their spectroscopic data with those in the literature. All the isolated compounds (1−10) were evaluated for their antimicrobial activities against a panel of pathogens including Gram-positive bacteria (S. aureus ATCC 25923 and B. subtilis ATCC 6633), Gram-negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 9027), as well as the fungal strain Candida albicans ATCC 24433. Among them, 7 was active to all the four test bacteria, showing potent inhibition of S. aureus and P. aeruginosa (MIC = 5.8 and 4.6 μg/mL) as well as mild effect against B. subtilis and E. coli (MIC = 42.2 and 75.0 μg/mL). Compounds 1 and 9 were found to have moderate antibacterial abilities against E. coli and S. aureus (MIC = 62.6 and 76.6 μg/mL).

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Fig. 3. ΔδH(S−R) values (measured in CDCl3) for the MTPA esters of 2–6.

Whereas their derivatives 2–6 with hydroxylated C-4 were inactive (MIC N 100.0 μg/mL), suggesting that the C-4 carbonyl might be important for their antimicrobial abilities. Compounds 8 and 10 exhibited antibacterial ability against S. aureus with MIC values of 43.4 and 76.0 μg/mL and 10 also displayed effect against B. subtilis (MIC = 54.1 μg/mL). However, no antifungal activities against C. albicans were found for 1–10. Conflict of interest The authors declared no conflict of interest. Acknowledgements This research work was funded by the National Natural Science Foundation of China (81503218), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the youth fund project of basic research program of Jiangsu Province (Natural Science Foundation, BK20130651), and the Fundamental Research Funds for the Central Universities (2016ZZD010). Appendix A. Supplementary data HRESIMS, 1D and 2D NMR spectra of compounds 1–6 are available as Supporting information. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j. fitote.2016.11.008. References [1] Z.J. Lin, T.J. Zhu, Y.C. Fang, Q.Q. Gu, W.M. Zhu, Polyketides from Penicillium sp JP-1, an endophytic fungus associated with the mangrove plant Aegiceras corniculatum, Phytochemistry 69 (2008) 1273–1278. [2] Z.J. Lin, Z.Y. Lu, T.J. Zhu, Y.C. Fang, Q.Q. Gu, W.M. Zhu, Penicillenols from Penicillium sp. GQ-7, an endophytic fungus associated with Aegiceras corniculatum, Chem. Pharm. Bull. 56 (2008) 217–221. [3] H.M. Ge, Y. Shen, C.H. Zhu, S.H. Tan, H. Ding, Y.C. Song, R.X. Tan, Penicidones A–C, three cytotoxic alkaloidal metabolites of an endophytic Penicillium sp, Phytochemistry 69 (2008) 571–576.

[4] J. Qi, C.L. Shao, Z.Y. Li, L.S. Gan, X.M. Fu, W.T. Bian, H.Y. Zhao, C.Y. Wang, Isocoumarin derivatives and benzofurans from a sponge-derived Penicillium sp. fungus, J. Nat. Prod. 76 (2013) 571–579. [5] C. Intaraudom, N. Boonyuen, R. Suvannakad, P. Rachtawee, P. Pittayakhajonwut, Penicolinates A–E from endophytic Penicillium sp. BCC16054, Tetrahedron Lett. 54 (2013) 744–748. [6] Y. Wang, M.H. Yang, X.B. Wang, T.X. Li, L.Y. Kong, Bioactive metabolites from the endophytic fungus Alternaria alternate, Fitoterapia 99 (2014) 153–158. [7] Y. Liu, M.H. Yang, X.B. Wang, T.X. Li, L.Y. Kong, Caryophyllene sesquiterpenoids from the endophytic fungus Pestalotiopsis sp. Fitoterapia 109 (2016) 119–124. [8] T.X. Li, X.B. Wang, J. Luo, M.H. Yang, L.Y. Kong, Antioxidant sordariol dimers from Sordaria macrospora and the absolute configuration determinations of their two simultaneous linear 1,2-diols, Tetrahedron Lett. 57 (2016) 2754–2757. [9] Y. Shizuri, M. Nagahama, S. Yamamura, K. Kawai, N. Kawai, H. Furukawa, Isolation and structures of citreovirenone and citreovirone, Chem. Lett. 22 (1986) 1129–1132. [10] J. Frelek, W.J. Szczepek, [Rh2(OCOCF3)4] as an auxiliary chromophore in chiroptical studies on steroidal alcohols, Tetrahedron Asymmetry 10 (1999) 1507–1520. [11] M. Gerards, G. Snatzke, Circular dichroism, XCIII determination of the absolute configuration of alcohols, olefins, epoxides, and ethers from the CD of their in situ complexes with [Rh2(O2CCF3)4], Tetrahedron Asymmetry 1 (1990) 221–236. [12] J. He, Y. Shen, J.S. Jiang, Y.N. Yang, Z.M. Feng, P.C. Zhang, S.P. Yuan, Q. Hou, New polyacetylene glucosides from the florets of Carthamus tinctorius and their weak anti-inflammatory activities, Carbohydr. Res. 346 (2011) 1903–1908. [13] J.M. Seco, E. Quinoa, R. Riguera, Assignment of the absolute configuration of polyfunctional compounds by NMR using chiral derivatizing agents, Chem. Rev. 112 (2012) 4603–4641. [14] F. Freire, J.M. Seco, E. Quiñoá, R. Riguera, Determining the absolute stereochemistry of secondary/secondary diols by 1H NMR: basis and applications, J. Org. Chem. 70 (2005) 3778–3790. [15] S. Lai, Y. Shizuri, S. Yamamura, K. Kawai, H. Furukawa, Three new phenolic metabolites from Penicillium species, Heterocycles 32 (1991) 297–305. [16] T.O. Larsen, J. Breinholt, Dichlorodiaportin, diaportinol, and diaportinic acid: three novel isocoumarins from Penicillium nalgiovense, J. Nat. Prod. 62 (1999) 1182–1184. [17] J.A. Dale, H.S. Mosher, Nuclear magnetic resonance enantiomer regents. Configurational correlations via nuclear magnetic resonance chemical shifts of diastereomeric mandelate, O-methylmandelate, and alpha-methoxy-alphatrifluoromethylphenylacetate (MTPA) esters, J. Am. Chem. Soc. 95 (1973) 512–519. [18] H. Fujimoto, E. Negishi, K. Yamaguchi, N. Nishi, M. Yamazaki, Isolation of new tremorgenic metabolites from an Ascomycete, Corynascus setosus, Chem. Pharm. Bull. 44 (1996) 1843–1848. [19] R.J. Capon, M. Stewart, R. Ratnayake, E. Lacey, J.H. Gill, Citromycetins and bilains A– C: new aromatic polyketides and diketopiperazines from Australian marine-derived and terrestrial Penicillium spp. J. Nat. Prod. 70 (2007) 1746–1752. [20] E.A. Pinheiro, J.M. Carvalho, D.C. dos Santos, A.O. Feitosa, P.S. Marinho, G.M.S. Guilhon, L.S. Santos, A.L. de Souza, A.M. Marinho, Chemical constituents of Aspergillus sp. EJC08 isolated as endophyte from Bauhinia guianensis and their antimicrobial activity, An. Acad. Bras. Cienc. 85 (2013) 1247–1253.