Phenols and diketopiperazines isolated from Antarctic-derived fungi, Penicillium citreonigrum SP-6

Phytochemistry Letters 27 (2018) 114–118

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Phenols and diketopiperazines isolated from Antarctic-derived fungi, Penicillium citreonigrum SP-6

T

Jia-ning Huanga,1, Qingbo Zoub,1, Jun Chena, Si-han Xua, Dan Luoa, Feng-guo Zhanga, ⁎ Yuan-yuan Lua, a b

Department of Marine Pharmacy, China Pharmaceutical University, Nanjing 210009, China Department of Pharmacy, Daqing Oil-field General Hospital, Daqing, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Penicillium citreonigrum Antitumor activities Phenols Diketopiperazines

One diketopiperazine (1) and three phenols (3, 5 and 6) were isolated from static culture of Antarctic fungus, Penicillium citreonigrum SP-6, as well as their structures were determined by NMR and CD spectroscopic methods. Additionally, two known compounds, cyclo-(L-Trp-L-Phe) (2) and (-)-(3S, 10R)-dichlorodiaportal (4), were isolated and identified. Antitumor biological studies demonstrated that compounds 1 and 3 showed weak inhibitions against HCT116 cancer cell lines with IC50 values of 26.7 and 46.3 μM, respectively, comparing to cisdichlorodiamine platinum.

1. Introduction

2. Results and discussion

The genus Penicillium is a well-known producer of natural compounds including indole alkaloids (Li, 2010), sesquiterpenoid (Vansteelandt et al., 2013), polyketides (Ren et al., 2006; Kossuga et al., 2013) and hydroisocoumarins (Qi et al., 2013). These compounds have displayed various biological activities such as cytotoxic (Li, 2010; Vansteelandt et al., 2013), antibacterial (Qi et al., 2013), and antifouling (Ren et al., 2006). Nowadays, many research groups focus on Antarctic fungus due to its new and interesting source of microbes as well as many bioactive compounds isolated from these fungus (Wang et al., 2017; Figueroa et al., 2015; Zhou et al., 2015; Wu et al., 2013; Wang et al., 2016; Lin et al., 2014; Wu et al., 2012; Wang et al., 2015). Therefore, we focused on the exploration of secondary metabolites produced by Antarctic-derived fungi (Li et al., 2018). In this study, Penicillium citreonigrum SP-6, which was isolated from soil around the China Great Wall Station, was selected for further research due to its cytotoxic activities. Totally, one diketopiperazine (1) and three phenols (3, 5 and 6) together with two known compounds, cyclo-(L-Trp-L-Phe) (2) and (-)-(3S, 10R)- dichlorodiaportal (4), were isolated from Penicillium citreonigrum SP-6 (see Fig. 1). This study elucidated the isolation procedure, structural identification and anticancer activities of the compounds from this fungus.

Compound 1, white amorphous power, showed its positive HRESIMS ions at m/z 366.1451 (calcd for C20H20N3O4+, 366.1448). The 1H NMR spectrum of 1 (Table 1) afforded one mono-substituted aromatic ring (δH 7.01 (2H, d, J = 7.5 Hz, H-19, 23), 7.05 (2H, d, J = 7.5 Hz, H-20, 22), 6.86 (1H, t, J = 7.5 Hz, H-21)) and one orthodisubstituted ring (δH 7.05 (1H, d, J = 7.2 Hz, H-4), 6.75(1H, t, J = 7.2 Hz, H-5), 7.15 (1H, t, J = 7.2 Hz, H-6) and 6.71 (1H, d, J = 7.2, H-7)). Combined HSQC spectrum, the other hydrogen nuclear signals were assigned as two methylene signals (δH 2.15 (1H, H-10α), 1.43 (1H, H-10β), δc 36.8 (C-10); δH 2.98 (1H, H-17α), 2.81 (1H, H-17β), δc 40.1 (C-17), respectively), two N-substituted methine units (δH 4.38(1H, H11), δc 56.4 (C-11); δH 4.30 (1H, H-14), δc 56.9 (C-14), respectively) and one methine signal (δH 5.28(1H, s, H-2), δc 101.8 (C-2)). Through the comparison of the NMR data of 1 with Cyclo-(L-Trp-L-Phe) (Kahina et al., 2015) indicated that the highly similarity of their structures, except for forming a 1, 2-dioxetane unit between C-2 (δc 101.8) and C-3 (δc 76.6) in 1. The stereochemistry of compound 1 was determined by biosynthesis pathway and theoretical ECD (electronic circular dichroism) methods. The 11S and 14S of 1 could be deducted by its biosynthesis pathway because it might be peroxidated from compound 2, Cyclo-(L-Trp-LPhe). The stereochemistry of 1 had been performed as 2R, 3R, 11S and 14S which were further evidenced by comparing CD experimental with

⁎

Corresponding author. E-mail address: [email protected] (Y.-y. Lu). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.phytol.2018.07.013 Received 23 April 2018; Received in revised form 29 June 2018; Accepted 13 July 2018 1874-3900/ © 2018 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.

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Fig. 1. Structures of the isolated compounds 1-6 from Penicillium citreonigrum. Table 1 NMR spectrum data of compound 1 in CD3OD. No.

13 a

1

HMBCc

2 3 4 5 6 7 8 9 10

101.8 76.6 124.6 120.6 131.7 111.3 132.2 149.5 36.8

5.28(1H,s)

C3,C9,C10

7.05 6.75 7.15 6.71

C3,C6,C9 C6,C8 C4,C9 C4,C5,C8

11 13 14 16 17

56.4 168.5 56.9 164.0 40.1

18 19, 23 20, 22 21 a b c

C

136.7 131.7 130.0 128.4

Hb (J Hz)

Table 2 NMR Spectrum data of compound 3 in CD3OD.

(1H, (1H, (1H, (1H,

d, 7.2) t, 7.2) t, 7.2) d, 7.2)

α 2.15 (1H, dd, 13.8, 5.5) β 1.43 (1H, dd, 13.8, 8.3) 4.38 (1H, dd, 8.3, 5.5)

C3,C10,C13,C16

4.30 (1H, t, 4.9)

C13,C16,C17,C18

α 2.98(1H, dd, 13.9, 4.9) β 2.81 (1H, dd, 13.9, 4.9)

C13,C14,C18,C19,C23

7.01(2H, d, 7.5) 7.05(2H, d, 7.5) 6.86 (1H, t, 7.5)

C17,C21 C18,C21 C19,C23

13 a

C

C2,C3,C5,C11,C16

1 3 4

171.7 81.3 33.2

4a 5 6 7 8 8a 9 10 11

131.1 119.0 122.9 145.9 151.6 109.7 30.4 31.1 176.6

a b c

1

Hb (J Hz)

Key HMBCc

4.63 (1H, m) α 2.95 (1H, dd, 3.0, 16.0) β 2.87(1H, dd, 11.0, 16.0)

C4 C3, C4a, C5, C7, C8a, C9

6.64 (1H, d, 8.0) 7.00 (1H, d, 8.0)

C1, C4, C6, C7, C8, C8a C4a, C7, C8

2.55 (2H, m) 2.07 (2H, m)

C3, C10, C11 C3, C4, C9, C11

125 MHz for 13C NMR. 500 MHz for 1H NMR. 500 MHz for 2D NMR.

elucidated by HMBC spectrum. For example, the C-7 was especially assigned as δC 145.9 by strong correlation with H-6 and relatively weak correlation with H-5, respectively, due to 2JH-C and 4JH-C coupling between the hetero-nuclear. In addition, the 7,8-dihydroxy of aromatic ring were further evidenced by comparing 13 C NMR data of C-7 (δC 145.9) and C-8 (δC 151.6) with the reported NMR data of 7,8-dihydroxy and 5,8-dihydroxy isocoumarin (Hussain et al., 2009), in which the chemical shifts of C-7 and C-8 appeared at δC 145.2 and δC 155.0 in 7,8dihydroxy isocoumarin, while δC 143.5(C-7) and 146.2 (C-8) were shown in 5,8-dihydroxy isocoumarin (Qi et al., 2013), respectively. The absolute configuration 3R of 3 was determined by comparison of experimental with theoretical ECD (electronic circular dichroism, shown in Fig. S2). Thus, the structure of compound 3 was elucidated as 3R-(7, 8-dihydroxy- 1-oxoisochroman-3-yl) propanoic acid. Compound 5, white amorphous power, afforded the molecular formula C13H18N2O4 by the positive HRESIMS data, m/z 267.1340 [M +H]+ (calcd for C13H19N2O4, 267.1339). The 1H NMR spectrum of compound 5 (Table 3) demonstrated three aromatic signals (δH 7.48 (1H, s), 7.39 (1H, d, J = 8.0 Hz) and 6.87 (1H, d, J = 8.0 Hz)), one oxygenated methyl (δH 3.94 (3H, s)), three methylene units (δH 3.43 (2H, t, J = 6.5 Hz), 3.29 (2H, t, J = 6.5 Hz), 1.81 (2H, t, J = 6.5 Hz)) and one methyl signal (δH 1.99 (3H, s)). The 13C NMR and HSQC spectrum (Table 3) showed total 13 carbon signals, including two carboxylic carbon signals (δC 173.6 and 170.1), six aromatic carbon signals (δC 151.5, 149.0, 127.0, 122.1, 116.1 and 112.2), one oxygenated

125 MHz for 13C NMR. 500 MHz for 1H NMR. 500 MHz for 2D NMR.

theoretical ECD data (Fig. S1). Above all, the structure of compound 1 enlightened 2R, 3R- dioxeto-Cyclo-(L-Trp-L-Phe). Compound 3, known as white amorphous powder, was identified to have the molecular formula C12H12O6 on the basis of positive HRESIMS data, m/z 275.0531 [M + Na]+ (calcd for C12H13O6Na+, 275.0526). The 1H NMR spectrum (Table 2) of 3 showed the presence of three methylene units (δH 2.95 (1H, dd, J1 = 3.0 Hz, J2 = 16.0 Hz, H-4α), 2.87 (1H, dd, J1 = 11.0 Hz, J2 = 16.0 Hz, H-4β), 2.55 (2H, m, H-9) and 2.07 (2H, m, H-10)), one oxygenated methine (δH 4.63(1H, m, H-3)), two aromatic hydrogen signals with AB coupling system (δH 6.64 (1H, d, J = 8.0, H-5) and 7.00 (1H, d, J = 8.0, H-6)). The 13C NMR and HSQC spectrum demonstrated 12 carbon signals, including six nonprotonated carbons, three methines and three methylene units. An aromatic ring was deduced in the molecule by the AB coupling system of 1H NMR and the 13 C NMR data δC 131.1 (C-4a), 119.0 (C-5), 122.9 (C-6), 145.9 (C-7), 151.6 (C-8) and 109.7(C-8a). Furthermore, H-4 correlated to C-3, C-4a, C-5, C-7, C-8a and C-9 in HMBC spectrum (Table 2 and Fig. 2) basically indicated that the skeleton of compound 3 is dihydro-isocoumarin (Rukachaisirikul et al., 2009; Hussain et al., 2009). The completed assignments of 1H and 13C NMR data were well 115

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Fig. 2. Key HMBC correlations of compounds 1, 3, 5 and 6.

correlated to C-2 and C-4 in HMBC spectrum, meanwhile the C-4 chemical shift was moved to low field comparing to C-2 for the hydroxylation of C-4, the C-4 was ultimately assigned at δC 149.0 according to the above two evidences. The 3-acetamidopropyl unit was established by H-9, H-10 and H-12 correlated to C-11 in the HMBC spectrum. Finally, the two units could be combined together due to H-3, H-7 and H-8 correlated to C-1 in HMBC spectrum, respectively. Thus, the structure of 5 was demonstrated as N-(3-acetamidopropyl)- 4-hydroxy3-methoxybenzamide. Compound 6, white amorphous power, gave its positive HRESIMS ions at m/z 267.1341 (calcd for C13H19N2O4+, 267.1340). Considering the similarity between compounds 5 and 6 (Table 3), we speculated the methoxyl group might be substituted at C-5 in compound 6, which also coincidence with these NMR signals of 3-hydroxy-4-methoxybenzoic acid (Lee et al., 2001). The conclusion was further validated by the HMBC correlation from 5-OCH3 (δH 3.94) to C-5 (δC 152.2) (Fig. 2). Above all, the structure of compound 6 was demonstrated as N-(3acetamidopropyl)-3-hydroxy-4-methoxybenzamide. Compounds 2 and 4 were identified as cyclo-(L-Trp-L-Phe) (He et al., 2015) and (-)-(3S, 10R)-dichlorodiaportal (Yang et al., 2017) by spectroscopic analyses and comparisons with literature data, respectively. All the compounds were tested for their cytotoxic activities against Human colon cancer cell line (HCT116), Human breast cancer cell line (MCF7), Human hepatocellular carcinoma cell line (HepG2), Human lung adenocarcinoma cell line (A549) and Normal human liver cell (L02). These results were shown in Table 4. Compounds 1 and 3 showed weak inhibition against HCT116 cancer cell lines with IC50 values of 26.7 and 46.3 μM, respectively, comparing to cis-dichlorodiamine platinum. Considering the cytotoxic activities of the two compounds (1 and 2), the 1, 2-dioxetane radical group in compound 1 is likely to elevate the cytotoxic activities of the diketopiperazine.

Table 3 NMR spectrum data of compounds 5 and 6 in CD3OD. No.

5

6

13 a

C

1 2 3 4 5 6

170.1 127.0 112.2 149.0 151.5 116.1

7

122.1

8

38.4

9

30.5

10

38.1

11 12 4-OCH3 5-OCH3

173.6 22.7 56.4 　

a b c

1

Key HMBCc

13 a

(1H, d,

　 　 C1, C5, C7 　 　 C2, C4

170.2 128.6 112.2 147.8 152.2 115.5

(1H, d,

C1, C3, C5

120.5

(2H, t,

C1, C9, C10 C10, C11

38.3

C8, C9, C11 　 C11 C4 　

38.1

b

H (J Hz)

7.48 (1H,s)

6.87 8.0) 7.39 8.0) 3.43 6.5) 1.81 6.5) 3.29 6.5)

(2H, t, (2H, t,

1.99 (3H, s) 3.94 (3H, s) 　

C

30.5

1

Hb (J Hz)

Key HMBCc 　

7.48 (1H,s)

C1, C5, C7

7.01 8.0) 7.37 8.0) 3.42 6.5) 1.80 6.5) 3.29 6.5)

(1H, d,

C2, C4

(1H, d,

C1, C3, C5

(2H, t,

C1, C9, C10 C10, C11

(2H, t, (2H, t,

C8, C9, C11

173.7 22.7

1.99 (3H, s)

C11

56.6

3.94 (3H, s)

C5

125 MHz for 13C NMR. 500 MHz for 1H NMR. 500 MHz for 2D NMR.

methyl carbon signal (δC 56.4), three methylene units (δC 38.4, 38.1 and 30.5) and a methyl signal (δC 22.7). The aromatic signals were identical to these of brynamide B (Sahranavard et al., 2010), which indicated that a 4-hydroxy-3-methxy benzamide exist in the structure. The 4-hydroxy-3-methxy benzamide unit was elucidated by HMBC correlations (Fig. 2), including H-3, H-5 correlated to C-7 and 4-OCH3 (δH 3.94) correlated to C-4 (δC 149.0), respectively. In addition H-6 Table 4 Cytotoxicity dataa for metabolites 1–6 (μM). Compounds

HCT116

1 2 3 4 5 6 CDDPb

26.7 ± 60.2 ± > 100 57.3 ± > 100 > 100 14.6 ±

a b

9.3 11.3 12.4

2.6

HepG2

MCF7

A549

L02

> > > > > > 7.9

> 100 > 100 > 100 > 100 > 100 > 100 12.2 ± 2.4

> 100 > 100 43.0 ± 8.3 79.1 ± 12.4 > 100 > 100 18.1 ± 5.1

> 100 > 100 > 100 > 100 > 100 > 100 10.1 ± 1.1

100 100 100 100 100 100 ± 1.5

Data represented as mean ± SD (standard deviation) (n = 3). cis-dichlorodiamine platinum. 116

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3. Experimental

C13H19N2O4+, 267.1341).

3.1. General

3.4. Cytotoxic assays

All NMR spectra were recorded at 500 MHz (1H) and 125 MHz (13C) with a Bruker DRX-500 spectrometers, and TMS used as an internal reference. Si gel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., China), Octadecylsilanized silica gel (ODS) (40–63 μm, YMC Co. Ltd., Japan) and Sephadex LH-20 (GE Healthcare Co. Ltd., USA) were used for column chromatography. HPLC and Semi-preparative HPLC were performed on a Jasco PU-2086 and UV-2075 system, using Shim-pack ODS-C18 column (5 μm, Ф4.6 × 250 mm, SHIMADZU CO. Ltd., Japan) and YMC ODS-C18column (10 μm, Ф20 × 250 mm, YMC CO. Ltd., Japan), respectively. ESI-MS and HRESI-MS experiments were recorded on Mariner ESI-TOF spectrometer, respectively.

Human colon cancer cell line (HCT116), Human breast cancer cell line (MCF7), Human hepatocellular carcinoma cell line (HepG2), Human lung adenocarcinoma cell line (A549) and Normal human liver cell (L02) cells were purchased from the Cell Bank of the Shanghai Institute of Cell Biology. HCT116 and A549 cells were cultured in RPMI 1640 medium (Gibco/BRL, Grand Island, New York, USA), 10% fetal FBS (v/v), penicillin (100 units/mL) and streptomycin (100 mg/L). HepG2 and MCF7 were cultured in DMEM medium (Gibco/BRL, Grand Island, New York, USA) added with 10% fetal bovine serum (FBS, Gibco/BRL, v/v), penicillin (100 units/mL) and streptomycin (100 mg/ L). All the Cells were cultured at 37 °C in an atmosphere of 5% CO2. Compounds were dissolved in DMSO as a 100 mmol/L stock solution for the treatment of cells. Compounds 1–6 were added in 96-well plates at different doses (0 to 50 μM) to treat the Cells (4 × 104 per well) for 48 h. Cell viabilities were analyzed as following: MTT (20 μL, 5 mg/mL, SunshineBio, Nanjing, Jiangsu, China) was added to each well, and the plates were kept at 37 °C in 5% CO2 for 4 h. The optical densities (OD) of the samples were obtained at 570 nm on automatic plate reader (Bio-Rad, Hercules, California, USA). The positive controls, cis-dichlorodiamine platinum (CDDP), were purchased from Sigma (China).

3.2. Fungal material The fungus, Penicillium citreonigrum SP-6 (GeneBank number: MH482861), was isolated from a marine sediment sample, which was collected from the site in the Antarctic Great Wall Station (62. 22 °S, 58. 96 °W). The strain (Deposit number: SP00006) is preserved at the Department Marine Pharmacy, China Pharmaceutical University. 3.3. Fermentation, extraction and isolation

Conflict of interest

For chemical investigation, the fungal was statically fermented for 45 days at 28 °C on rice medium (10 kg). After fermentation, the rice medium was extracted with ethanol (20 L) three times. The ethanol solution was evaporated under reduced pressure to afford a crude methanol extract, which was then suspended in water and extracted by CHCl3 (200 mL × 3). The CHCl3 fraction (44.3 g) was further separated by silica gel column chromatography, gradient eluted with CH3OH-CHCl3 (1/40, 1/ 15, 1/13 and 1/5, v/v), affording four fractions Fr. 1 to Fr. 4. Compound 3 was purified from Fr.1 (156.6 mg) by Semi-preparative HPLC eluted with MeOH-H2O (45/55, 0.1% TFA, v/v). Fr.2 (228.6 mg) was purified by ODS column chromatography to afford 1 (6.8 mg) and 2 (10.2 mg). Compounds 5 (3 mg, tR = 29 min) and 6 (20.1 mg, tR = 29 min) were further purified by Semi-preparative HPLC eluted with MeCN-H2O (10/90, v/v) from fraction 3 (243.6 mg). Compound 4 was isolated from fraction 5 (132.1 mg) by Sephadex LH-20 chromatography and eluted with methanol.

Authors declare no conflict of interest. Acknowledgements We gratefully acknowledge the financial support from The Project Program of National Nature Science Foundation of China for Young Scientists (No. 81001395), the Priority Academic Program Development of Jiangsu Higher Education Institution and Qing Lan Project. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2018.07.013. References

3.3.1. Compound 1 White amorphous power; M. P. 185–187 °C; [α]D 25= +45.7 (c 1.0 CH3OH) ; UV (CH3OH) λmax (log ε) 205 (2.79), 242 (2.01), 297 (1.53) nm; 1H and 13C NMR data (CD3OD), see Table 1; (+)-HRESIMS m/z 366.1451 (calcd for C20H20N3O3+, 366.1488).

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3.3.2. Compound 3 White amorphous power; M. P. 179–180 °C; [α]D 25= -34.1 (c 1.0 CH3OH) ; UV (CH3OH) λmax (log ε) 223 (3.00), 257 (2.60), 332 (2.36) nm; 1H and 13C NMR data (CD3OD), see Table 2; (+)-HRESIMS m/z 275.0531 [M + Na]+ (calcd for C12H13O6Na+, 253.0526). 3.3.3. Compound 5 White amorphous power; M. P. 225–227 °C; UV (CH3OH) λmax (log ε) 209 (3.07), 258 (2.65), 289 (2.40) nm; 1H and 13C NMR data (CD3OD), see Table 3; (+)-HRESIMS 267.1340 [M+H]+ (calcd for C13H19N2O4, 267.1339). 3.3.4. Compound 6 White amorphous power; M. P. 220–221 °C; UV (CH3OH) λmax (log ε) 208 (2.90), 255 (2.49), 291 (2.32) nm; 1H and 13C NMR data (CD3OD), see Table 3; (+)-HRESIMS m/z 267.1339 (calcd for 117

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