Anthraquinone derivatives from a coral associated fungus Stemphylium lycopersici

Abstract Two new anthraquinone derivatives, alterporriol Y (1) and macrosporin 2-O-α-D-glucopyranoside (2), together with five known analogues (3-7) were isolated from the fungus Stemphylium lycopersici associated with the gorgonian coral Dichotella gemmacea collected from the South China Sea. Their structures were determined on the basis of detailed spectroscopic analysis and comparison with reported data. The absolute configurations were determined by the ECD method. In an in vitro cytotoxic assay, compound 3 and 4 showed potent effects against HCT-116 and MCF-7 cell lines. Compound 4 also exhibited cytotoxicity toward Huh7 stem cell-like cells. Graphical Abstract


Introduction
Chemistry of gorgonian coral Dichotella gemmacea is featured by briarane diterpenes which have been reported to exhibit cytotoxicity and antifouling effect (Li et al. 2011. Chemical investigation of microorganisms associated with D. gemmacea started from 2010 when five new sesquiterpenes were isolated from the fungus Aspergillus sp. (Wei et al. 2010). Subsequent investigations of D. gemmacea associated fungi involved Aspergillu sp., Cladosporium sp., Eutypella sp., and Cochliobolus lunatus, offering different types of metabolites, such as polyketides, sesquiterpenes, alkaloids (Shao et al. 2011a(Shao et al. , 2011bLiao et al. 2017).
As part of our ongoing search for bioactive compounds from marine invertebrates and associated fungi (Xu et al. 2014;Sun et al. 2016Sun et al. , 2017Li et al. 2018), a strain of Stemphylium lycopersici was isolated from D. gemmacea collected from the South China Sea. This fungus was recognized as a kind of plant pathogenic fungi, and there has been no report of its chemistry. Metabolites from the genus of Stemphylium were reported to include anthraquinones, chromones, a-pyrones, and phytotoxins (Barash et al. 1975;Manulis et al. 1984;Liu et al. 2014;Zhou et al. 2014a;Liu et al. 2015). Anthraquinone derivatives were the main constituents (Zheng et al. 2012;Zhou et al. 2014b;Liu et al. 2015). Of particular interest the fungi of this genus were recently found to produce dimers or trimers of anthraquinone (Zheng et al. 2012). The metabolites displayed antiviral, cytotoxic, antioxidative, and antifungal activities (Zheng et al. 2012;Liu et al. 2014;Zhou et al. 2014b;Liu et al. 2015;Li, Xue, et al. 2017).

Results and discussion
Alterporriol Y (1) was obtained as a red amorphous powder. Its molecular formula of C 32 H 30 O 12 was determined by HRESIMS, required eighteen degrees of unsaturation. The IR spectrum of 1 demonstrated signals for hydroxyl groups (3392 cm À1 , 3182 cm À1 ) and carbonyl groups (1715 cm À1 , 1643 cm À1  Table S1). The 13 C NMR and DEPT spectra exhibited sixteen carbon signals, including ten sp 2 carbon atoms (2 C ¼ O, 7 C ¼ C, 1 C ¼ CH) and 6 sp 3 carbon atoms (1 CO, 1 CHO, 2 CH 2 , 1 CH 3 , 1 OCH 3 ) (Table S1), taking account six degrees of unsaturation. The established molecule formula and the presence of only 16 signals in the 13 C NMR spectrum indicated 1 to be a symmetrical tetrahydroanthraquinone dimer. The NMR data of 1 were almost identical to those of co-isolated altersolanol B (3) (Yagi et al. 1993). However, the aromatic methine at C-8 (d C 106.8, CH; d H 6.97, 1H) in 3 was replaced by a quaternary carbon (d C 122.6) in 1, indicating 1 to be a C-8 À C-8' dimeric derivative of 3. This dimeric linkage was supported by the similar chemical shift of C-8 in 1 as that in alterporriol N (Zheng et al. 2012), a 8,8'-linked dimer analogue obtained from soft coralderived Alternaria sp. fungus. This established planar structure was further confirmed by the 2 D NMR analysis as shown in Figure S1. The relative configuration of 1 was proven the same as that of 3 based on the NOE experiment ( Figure S2). The equatorial orientation of Me-11 was indicated by its NOE effects with both with H 2 -1. The NOE cross peaks of H-3 with Me-11 and H-1b suggested a b orientation of these protons. The relative configuration of (2S Ã ,3R Ã )-1 was then determined.
Alterporriol Y (1) is an 8,8'-linked anthraquinone homodimer with chiral nonracemic monomeric units and axial chirality due to the hindered rotation along the biaryl axis. The absolute configuration of the co-isolated monomeric unit, altersolanol B (3), has been recently determined by single crystal X-ray diffraction analysis as (2S,3R) (Li, Chen, et al. 2017). The ECD spectra of axially chiral biaryls are governed by the (aR) or (aS) axial chirality and the central chirality elements has only minor influence on the ECD transitions (Debbab et al. 2012). In the ECD spectrum of 1 ( Figure  S3), two positive Cotton effects (CEs) were observed at 306 and 272 nm and negative CEs appeared at 252 and 227 nm, which showed mirror image relationship with the ECD spectra of alterporriol N and D having (aS) axial chirality and close similarity with that of (aR)-alterporriol E (Debbab et al. 2012;Kanamaru et al. 2012). Thus the absolute configuration of alterporriol Y (1) was determined as (aR,2S,3R,2'S,3'R).
Macrosporin 2-O-a-D-glucopyranoside (2) was afforded as a yellow amorphous powder with the molecular formula C 22 H 22 O 10 as determined by HRESIMS data. The presence of hydroxyl groups (3393 cm À1 , 3362 cm À1 , 3285 cm À1 ) and carbonyl groups (1666 cm À1 , 1644 cm À1 ) were concluded by analysis the IR spectrum of 2. The 1 H NMR and 13 C NMR spectrum including the coupling constant of the glucose moiety (Table  S1) of 2 was almost identical to that of macrosporin 2-O-(6'-acetyl)-a-D-glucopyranoside (Zhou et al. 2014b), with the difference recognized being the absence of an acetoxy group at C-6', with the chemical shifts of C-5' and C-6' moved downfield and upfield respectively. This conclusion was supported by the similar positive rotation of 2 (½a 25 D þ 106) as that of macrosporin 2-O-(6'-acetyl)-a-D-glucopyranoside (½a 25 D þ 120) (Zhou et al. 2014b). Thus, the structure of compound 2 was the deacetylation of macrosporin 2-O-(6'-acetyl)-a-D-glucopyranoside.

General experimental procedures
Optical rotations were recorded using an Autopol VI polarimeter while ECD spectra was measured by a JASCO-715 spectropolarimeter. Varian Cary 300 Bio UV-Visible and Bruker Vertex 70v FT-IR spectrophotometers were applied to record UV and IR spectra, respectively. NMR spectra were acquired by employing a Bruker DRX 600 spectrometer at 300 K. The chemical shifts were referenced by the residual DMSO signal (d H 2.50 ppm; d C 39.5 ppm) or C 5 D 5 N signal (d H 8.74, 7.58, 7.22; d C 150.3, 135.9, 123.9). The HRMS data was obtained by using an Agilent Q-TOF Micro mass spectrometer. The purification of compounds was conducted on an Agilent 1100 HPLC system, which was equipped with a YMC Pack ODS-A column (250 Â 10 mm, 5 lm). Open column chromatography (CC) was carried out on commercial silica gel (200-300, 300 À 400 mesh, Huanghai, Yantai). Glass sheets with pre-coated silica gel (HSGF-254, Huanghai, Yantai) were used to carry out TLC analysis. Spots on TLC were observed by spraying with 10% anisaldehyde in H 2 SO 4 , followed by heating.

Fungal material
A white filamentous fungal strain was isolated from the internal tissues of gorgonian coral D. gemmacea, which was collected from the South China Sea in Oct of 2015, and was identified as S. lycopersici by analysis of internally transcribed spacer (ITS) region (GenBank accession number MH158517). A voucher strain of this fungus (internal strain No. ZH-1IV) was kept in the laboratory of Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University.

Biological assay
The cytotoxicity effects of the compounds 1-6 were evaluated against HCT-116 human colon cancer cells, MCF-7 human breast cancer cells, and Huh7 cancer stem cell-like cells using CCK-8 (Cell Counting Kit-8) method. Adriamycin was used as the positive control, with IC 50 values of 5.4 lM, 6.2 lM and 15.4 lM for HCT-116, MCF-7 and Huh7 cancer stem cell-like cells, respectively.

Conclusions
In conclusion, two new anthraquinone derivatives, alterporriol Y (1) and macrosporin 2-O-a-D-glucopyranoside (2) were isolated from the fungus S. lycopersici associated with the gorgonian coral D. gemmacea. This is the first report of chemical investigation of S. lycopersici. The interesting growth inhibitory activity against cancer stem cell of 4 may hold as a basis for further attempt of investigations for such cluster of metabolites.