Chinese Chemical Letters  2018, Vol. 29 Issue (3): 535-537   PDF    
Asperterzine, a symmetric aromatized derivative of epipolythiodioxopiperazine, from the endophytic fungus Aspergillus terreus PR-P-2
Jiao Baia,b, Feng Guoa,b, Rui Wanga,b, Gang Chena,b, Zhanlin Lia,b, Meili Shaoa,b, Chunmei Xuea,b, Huiming Huaa,b,1    
a Key Laboratory of Structure-Based Drug Design and Discovery(Shenyang Pharmaceutical University), Ministry of Education, Shenyang 110016, China;
b School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
Abstract: A new epipolythiodioxopiperazine (ETP), asperterzine (1), along with two known analogs, bisdethiobis (methylthio)-acetylaranotin (2) and bisdethiobis(methylthio)-acetylapoaranotin (3), was isolated from the plant endophytic fungus Aspergillus terreus PR-P-2. The structure elucidation of 1 was accomplished by a combination of spectral methods and electronic circular dichroism (ECD) spectrum. Asperterzine (1) was a symmetric aromatized ETP found as a natural product for the first time. Compounds 2 and 3 showed strong cytotoxicity against HL-60 cell line. The putative biosynthetic pathway of 1 was also detailed in the text.
Key words: Asperterzine     Epipolythiodioxopiperazine     Plant endophytic fungus     Aspergillus terreus     Cytotoxicity    

Epipolythiodiketopiperazines (ETPs) are a unique class of fungal metabolites originating from diketopiperazines (DKPs) characterized by a di-or polysulfide bridges, generally exhibiting potent biological activity [1]. Nearly twenty distinct families have been found and characterized of ETPs since the seminal discovery of gliotoxin in 1936 [2]. Especially, epidithiodioxopiperazines including aranotins [3], hyalodendrins [4], gliotoxins [5], emestrins [6], epicorazines [7], and emethallicins [8], etc., isolated from several genera of Aspergillus, Penicillium, Hyalodendron, Emericella, Podospora, Epicoccum, showed extensive bioactivities such as antiviral [3], antibacterial [3, 9], cytotoxic [10, 11], antifungal [12], and histamine inhibitory activities [13]. As one of diverse resources, the filamentous fungus Aspergillus terreus can produce acetylaranotin and related natural products [2]. During our searching for new bioactive metabolites derived from endophytic fungi, a fungus Aspergillus terreus PR-P-2 was obtained from Camellia sinensis var. Assamica (Mast.) Kitam [14]. Recently, we isolated a new symmetric aromatized derivative of ETP, asperterzine (1), along with its two known analogs, bisdethiodis(methylthio)-acetylaranotin (2) [15] and bisdethiodis(methylthio)-acetylapoaranotin (3) [16] from the fermented culture of the strain PR-P-2 (Fig. 1). Herein, we report the isolation and structure elucidation of the new compound. The cytotoxic activities of compounds 1–3 against HL-60 (human promyelocytic leukemia cells) cell line were also evaluated.

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Fig. 1. The structures of compounds 1–3

The strain PR-P-2 was isolated from Camellia sinensis var. assamica (Mast.) Kitam. of Puer city in Yunnan, China, in September 2012. It was identified as Aspergillus terreus (GenBank accession no. KP260560) and has been deposited in School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University.

The fungus PR-P-2 was cultured on a rotary shaker (180 rpm) at 28 ℃ for 7 days in liquid medium (containing mannitol 2%, D-glucose 2%, yeast extract 0.5%, peptone 1%, KH2PO4 0.05%, MgSO4 0.03%, corn syrup 0.1%). After 7 days, the fermented culture (65 L) was filtered to be separated into the broth and the mycelia. The former was concentrated to 15 L and extracted three times with EtOAc, while the latter was extracted three times with 80% aqueous acetone by the ultraphonic method. The acetone solution was evaporated under vacuum to afford an aqueous solution, which was then extracted three times with EtOAc. The crude EtOAc extract (62.4 g) of the broth was subjected to silica gel column chromatography (CC) using gradient elution with CH2Cl2–CH3OH (from 100:0 to 0:100) to give six fractions (Fr. B1–B6). Fr. B3 was further separated by ODS CC with CH3OH-H2O (from 5:95 to 100:0) to give four fractions (Fr. B3.1–B3.4). Fr. B3.4 was purified by semipreparative HPLC (CH3OH:H2O = 70:30) to afford compound 3 (2.9 mg). Fr. B5 was subjected to silica gel CC with petroleum etheracetone (from 100:0 to 0:100) to obtain five fractions (Fr. B5.1– B5.5) and Fr. B5.4 was separated by semi-preparative HPLC (CH3OH:H2O = 65:35) afford 2 (22.2 mg). The crude EtOAc extract (40.0 g) of the mycelia was fractionated by silica gel CC using gradient elution with petroleum ether–acetone (from 100:0 to 0:100) to give four fractions (Fr. M1–M4). Fr. M3 was subjected to Sephadex LH-20 CC (CH3OH) and purified by semi-preparative HPLC (CH3OH:H2O = 60:40) to afford 1 (5.6 mg) (Fig. 1).

Asperterzine (1), isolated as a colorless needle crystal, had the molecular formula C18H14N2O2S2 determined by HR-ESI-MS at m/z 355.0568 [M+H]+ (calcd. 355.0569), implying the presence of 13 degrees of unsaturation. The IR absorption bands at 3064 (C-H of sp2 hybridization), 2919 (C-H of sp3 hybridization), 2348 (S-H), 1692 (carbonyl), 1587, 1466, 1447 (benzene) cm-1 and the UV absorption maximum at 242 and 283 nm revealed the existence of mercapto, carbonyl and benzene ring. The 1H NMR spectrum (Table 1) of compound 1 showed the signals of a mercapto proton at δH 9.70 (s, 1H), an ortho-disubstituted benzene at δH 7.24 (d, 1H, J = 7.4 Hz), 7.19 (d, 1H, J = 7.6 Hz), 7.17 (t, 1H, J = 7.6 Hz), 7.09 (t, 1H, J = 7.4 Hz), and a methylene at δH 3.99 (d, 1H, J = 16.4 Hz), 3.55 (d, 1H, J = 16.4 Hz). The 13C NMR spectrum (Table 1) exhibited only nine carbon signals classified into a carbonyl carbon (δC 167.1), six aromatic carbons (δC 137.6, 137.2, 127.6, 125.1, 124.4 and 121.3), and two aliphatic carbons (δC 74.0 and 45.1). The NMR spectral data combined with the molecular formula allowed us to assume that compound 1 should be a highly symmetrical molecule. The HMBC spectrum of 1 revealed the correlations of H-5 with C-7 and C-9, H-6 with C-4 and C-8, H-7 with C-5 and C-9, H-8 with C-4 and C-6, confirming the presence of the benzene ring. Moreover, both H-3a and H-3b correlated with C-9, C-5, C-1, C-2, suggesting that the methylene connected to C-4 of the benzene ring and C-2. In addition, the mercapto group was located at C-2 by the HMBC correlations of S-H with C-1, C-2, C-3. Meanwhile, a nitrogen atom was simultaneously connected to C-2 and C-9 according to the chemical shifts of C-2 (δ 74.0) and C-9 (δ 137.6). Thus, one-half structure of 1 was established as shown in Fig. 2. All the aforementioned evidences corroborated that compound 1 was a diketopiperazine condensed from two molecules of 2-(mercapto)-1H-indole-2, 3-dihydro-2-carboxylic acid.

Table 1
1H (600 MHz) and 13C (75 MHz) NMR spectroscopic data of asperterzine (1) in DMSO-d6

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Fig. 2. The key HMBC (H→C) correlations of one-half structure of asperterzine (1)

The absolute configuration of 1 was elucidated by comparing its experimental ECD curve with the ECD spectra calculated for all options (Fig. 3). There are two possible isomers of 1 (2S, 2'S-1 and 2R, 2'R-1) and for each isomer an optimized conformation was calculated using DFT at the B3LYP/6-31G* (d, p) in the GAUSSIAN 09 program to generate its ECD property. Since the ECD spectrum of 1 showing the Cotton effects at 217 nm (Δe +16.29), 256 nm (Δε -2.22) and 276 nm (Δε +2.93) was matched well with that of the isomer 2S, 2'S-1, it allowed us to unambiguously deduce the stereochemistry of 1 with the absolute configuration of 2S, 2'S. Therefore, asperterzine (1) was determined to be (6aS, 13aS)-6a, 13a-dimercapto-6a, 7, 13a, 14-tetrahydro-6H, 13H-pyrazino[1, 2-a:4, 5-a']diindole-6, 13-dione.

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Fig. 3. Calculated and experimental ECD spectra for asperterzine (1)

The known compounds 2 and 3 were identified as bisdethiobis (methylthio)-acetylaranotin [15] and bisdethiobis(methylthio) acetylapoaranotin [16] by comparing their measured spectroscopic data with those reported in the literatures, respectively.

Wang and coworkers have studied the biosynthesis of 2 and 3 using target gene-deletion and defined a cluster of nine genes that is required for acetylaranotin biosynthesis [2, 15]. According to this finding, the biosynthetic pathway of 1 was similarly proposed in Scheme 1. Condensation and cyclization of two molecules of phenylalanine would generate diketopiperazine 4. Bishydroxylation of 4, followed by glutathione (GSH) conjugation, then produced dithiol 7. Epoxidation of 7 gave intermediate 8, which could undergo nucleophilic attack of the amide nitrogen to afford 9. Dehydration 9 would afford aromatic dithiol diketopiperazine 1.

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Scheme 1. Plausible biosynthetic pathway of asperterzine (1)

The cytotoxic activities of 1–3 against HL-60 (human promyelocytic leukemia cells) cell line were evaluated. Compounds 2 and 3 exhibited potent inhibitory effects with IC50 values of 9.34 and 16.30 mmol/L, while compound 1 showed no cytotoxic activity with IC50 value of >100 (5-FU: IC50 = 2.80 mmol/L) toward HL-60 cells. It was found that the activity appeared to decrease with the aromaticity of the compounds.

All natural epidithiodioxopiperazines discovered to date contain (or are derived from) at least one aromatic amino acid (tyrosine-and/or phenylalanine-derived and tryptophan-derived). The subgroup of epidithiodioxopiperazines derived from phenylalanine and/or tyrosine-is characterized by the presence of at least one seven membered dihydrooxepine ring or dihydrobenzene ring, and includes the aranotins, emethallicins, and emestrins [2]. The completely symmetric aromatized derivative, such as asperterzine (1), is found in the natural ETPs for the first time.

In this paper, the structure of asperterzine (1) was elucidated by a combination of spectral methods (UV, IR, HR-ESI-MS, and NMR) and ECD spectrum. The putative biosynthetic pathway of compound 1 was also proposed. The bioassay for the compounds showed that compounds 2 and 3 had strong cytotoxic activity against HL-60 cells.

The general experimental procedures, physiochemical data and bioassay for the compounds were provided in Supplementary data, along with a listing of UV, IR, ESI-MS, 1D and 2D NMR, ECD spectra for compound 1.

Acknowledgments

This work was supported by the Fund of Natural Science of Liaoning Province (No. 2015020730). It was also supported by Program for Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team in University. We gratefully acknowledge Mr. Yi Sha and Mrs. Wen Li, Department of Analytical Testing Center, Shenyang Pharmaceutical University, for measurements of the NMR data.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2017.09.006.

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