Four New Chromones from the Endophytic Fungus Phomopsis asparagi DHS-48 Isolated from the Chinese Mangrove Plant Rhizophora mangle

Four new chromones, phomochromenones D–G (1–4), along with four known analogues, diaporchromone A (5), diaporchromanone C (6), diaporchromanone D (7), and phomochromenone C (8), were isolated from the culture of Phomopsis asparagi DHS-48 from Chinese mangrove Rhizophora mangle. Their structures were elucidated on the basis of comprehensive spectroscopic analysis. The absolute configurations of 1 and 4 were assigned on the basis of experimental and calculated electronic circular dichroism (ECD) data, and those of enantiomers 2 and 3 were determined by a modified Mosher’s method and basic hydrolysis. To the best of our knowledge, phomochromenones D–F (1–4) possessing a 3-substituted-chroman-4-one skeleton are rarely found in natural sources. Diaporchromone A (5) showed moderate to weak immunosuppressive activity against T and/or B lymphocyte cells with IC50 of 34 μM and 117 μM.


Introduction
Marine-derived fungi are morphologically and physiologically adapted to harsh environmental stresses, such as high salinity, high temperature, extreme tides, oxygen pressure, high humidity, and light and air limitations, which have increasingly attracted the attention of both pharmaceutical and natural product chemists in recent decades [1,2]. Fungi colonized in mangrove forests, which comprise the second largest ecological group of marine fungi, have especially adapted their metabolic mechanisms to the unique properties of the marine environment via the generation of a large variety of structurally unprecedented and biologically interesting metabolites of pharmaceutical importance [3][4][5]. One fungal genus which is especially productive with regard to the accumulation of a diverse array of mostly bioactive compounds is Phomopsis. Chemical investigation of this fungal genus has resulted in the discovery of over 70 potentially bioactive secondary metabolites, such as subintestinal vessel plexus (SIV) accelerator phomopsis-H76 A [6], cytotoxic phomopchalasins B and C [7], mycoepoxydiene [8], dicerandrols [9], antibiotic phomoxanthone A [10], phomodiol [11], phompsichalasin [12], antimicrotubule phomosidin [13], and antiinflammatory phomol [14]. As part of our ongoing investigation on bioactive metabolites from mangrove endophytic fungi [15][16][17][18][19], Phomopsis asparagi DHS-48 was isolated from a fresh root of the mangrove plant Rhizophora mangle. Four new chromones (1)(2)(3)(4), and five known compounds, including diaporchromone A (5) [20], diaporchromanone C (6) [20], diaporchromanone D (7) [20], and phomochromenone C (8) [21] (Figure 1) were isolated from the EtOAc extract of P. asparagi after fermentation on a solid rice medium containing sea salt. Herein, we report the isolation, structural elucidation, and exploration on the biological activities of compounds 1-8.

Results and Discussion
Phomochromenone D (1), a white amorphous powder, has the molecular formula C 16 4 Hz, and δ H 7.10 (d, J = 2. 4 Hz,, one methine at δ H 4.25 (m, H-2 ), one methylene at δ H 2.92 (dd, J = 14.0, 8.0 Hz, H a -1 ) and 2.60 (dd, J = 14.0, 5.1 Hz, H b -1 ), and two methyl at δ H 2.02 (s, H 3 -9) and δ H 1.29 (d, J = 6. 3 Hz,, and two methoxy at δ H 3.917 (s, H 3 -11) and δ H 3.924 (s, H 3 -12),. The 13 C NMR and DEPT spectra showed 16 carbon resonances corresponding to two sp 2 methine (δ C 114.52 and 102.3), ten sp 2 quaternary (δ C 165. 1, 164.8, 159.5, 135.9, 119.1, 114.49, one carbonyl at δ C 171.4, one conjugated carbonyl at δ C 178.4), one oxygenated methine (δ C 67.1), two methoxy (δ C 57.0, 53.5), one methylene (δ C 42.6), and two methyl (δ C 23.7, 10.3) carbons. The HMBC correlation ( Figure 2) from H-6 to C-5, C-7, and C-10, and from H-8 to C-7, C-4a and C-8a indicated the presence of the chromone moiety. Moreover, HMBC correlations from H 3 -9 to C-2, C-3 and C-4; and from H 2 -1 to C-2 and C-3, suggested that a 2-hydroxypropyl group was attached to C-2 of the chromone core. The absolute configuration of C-2 of 1 was determined by the comparison of its experimental and time-dependent density functional theory (TDDFT)-calculated electronic circular dichroism spectrum. The experimental ECD spectrum (CH 3 OH) for 2 S-1 matched well with the calculated spectrum ( Figure 3), which confirmed the unambiguous assignment of the absolute configuration of 1 as S, and the trivial name, phomochromenone D, was assigned.    Phomochromenones E (2) and F (3) were isolated as a mixture of two enantiomers and shared the same NMR data, 1 H-1 H COSY and HMBC spectra. Based on the HR-ESIMS ion detected at m/z 321.0989 (calcd. for [M−H] − , 321.0974), the mixture (2/3) had the same molecular formula of C16H18O7 (i.e., differing from that of 1 by an additional hydroxyl group). The 1 H and 13 C NMR spectrum clearly indicated that this hydroxyl group was attached at C-6. Supporting evidence for this assignment was obtained from the downfield chemical shifts of C-8 (δC 138.2, s) and the absence of the proton signal of H-6 in 2/3 (δH 7.10, d, J = 2.4 Hz for H-8 and δC 102.3, d for C-8 in 1, respectively). Moreover, the presence of a hydroxyl group at C-8 was corroborated by the observed HMBC correlation from H-6 (δH 7.10, d, J = 2.4 Hz) to C-8, C-7 (δC 151.4), C-5 (δC 123.2), C-4a (δC 115.4), and C-10 (δC 172.4). However, the antipode rotation and ECD were detected, which suggested the mixture was not an optically pure compound. Since the chiral phase HPLC (CHI-RALPAK IC) did not afford the separation of these two enantiomers, a modified Mosher's experiment was performed to obtain its MPA esters. The products of Mosher's reactions were subsequently analyzed by UPLC-ESI-MS through a RP-C18 chromatography column to afford two pairs of diastereomeric esters (  Phomochromenones E (2) and F (3) were isolated as a mixture of two enantiomers and shared the same NMR data, 1 H-1 H COSY and HMBC spectra. Based on the HR-ESIMS ion detected at m/z 321.0989 (calcd. for [M−H] − , 321.0974), the mixture (2/3) had the same molecular formula of C 16 H 18 O 7 (i.e., differing from that of 1 by an additional hydroxyl group). The 1 H and 13 C NMR spectrum clearly indicated that this hydroxyl group was attached at C-6. Supporting evidence for this assignment was obtained from the downfield chemical shifts of C-8 (δ C 138.2, s) and the absence of the proton signal of H-6 in 2/3 (δ H 7.10, d, J = 2.4 Hz for H-8 and δ C 102.3, d for C-8 in 1, respectively). Moreover, the presence of a hydroxyl group at C-8 was corroborated by the observed HMBC correlation from H-6 (δ H 7.10, d, J = 2.4 Hz) to C-8, C-7 (δ C 151.4), C-5 (δ C 123.2), C-4a (δ C 115.4), and C-10 (δ C 172.4). However, the antipode rotation and ECD were detected, which suggested the mixture was not an optically pure compound. Since the chiral phase HPLC (CHIRALPAK IC) did not afford the separation of these two enantiomers, a modified Mosher's experiment was performed to obtain its MPA esters. The products of Mosher's reactions were subsequently analyzed by UPLC-ESI-MS through a RP-C 18 H3-14). The 13 C NMR and DEPT spectra showed 20 carbon signals, including a keto group, an ester carbonyl group, eight olefinic carbon signals (including four oxygenated carbons), three oxy-methines, one methylene, two methoxy group, and three methyl group. Comparison of the NMR data of 4 with those of phomochromenone B [21], previously isolated from endophytic fungus Phomopsis sp. HNY29-2B derived from mangrove plant Acanthus ilicifolius Linn, revealed that both compounds differed with regard to the nature of the side chain at C-1, where the hydroxyl group of the latter was replaced by the 3-hydroxybutan-2yloxyl group of 3. This was confirmed through 1 H-1 H COSY correlations of H3-4′/ H-1′/ H- Phomochromenone G (4) was obtained as a white amorphous powder and had a molecular formula of C 20 5.73(s, H-9), δ H 4.40 (m, H-11), δ H 3.60 (m, H-1 ), and δ H 3.54 (m, H-2 ); one methylene at δ H 2.77 (dd, J = 17.9, 3.7 Hz, H a -10) and 2.68 (dd,J = 17.9,10.7 Hz,; three methyl at δ H 1.37 (d, J = 6. 2 Hz,, δ H 1.13 (d, J = 6. 0 Hz,, and δ H 1.19 (d, J = 6. 0 Hz,; two methoxy groups at δ H 3.96(s, H 3 -15) and δ H 3.87(s, H 3 -14). The 13 C NMR and DEPT spectra showed 20 carbon signals, including a keto group, an ester carbonyl group, eight olefinic carbon signals (including four oxygenated carbons), three oxy-methines, one methylene, two methoxy group, and three methyl group. Comparison of the NMR data of 4 with those of phomochromenone B [21], previously isolated from endophytic fungus Phomopsis sp. HNY29-2B derived from mangrove plant Acanthus ilicifolius Linn, revealed that both compounds differed with regard to the nature of the side chain at C-1, where the hydroxyl group of the latter was replaced by the 3-hydroxybutan-2-yloxyl group of Figure 2) from H-9 to C-11(δ C 63.9), C-4(δ C 176.8), C-2(δ C 166.7), and C-1 (δ C 67.1). The relative configuration of 4 was based on the NOESY correlations as indicated in Figure 2. The NOESY correlations of H-9 to H-11, H 3 -1 and H-3 ; H-11 to H 3 -1 ; and H 3 -4 to H-2 indicated that H-9, H-11, H 3 -1 , and H 3 -4 were on the opposite side of the H 3 -12, H-2 , and H 3 -4 . The absolute configuration of 4 were also determined by comparing experimental and calculated electronic circular dichroism (ECD) spectra for the truncated model (9R, 12S, 2 R, 3 S)-4 and the truncated model (9S, 12R, 2 S, 3 R)-4 using time-dependent density functional theory (TDDFT). The theoretical spectrum of 4 showed an excellent fit with the experimental plot recorded in MeOH (Figure 3), which supported the absolute configuration to be 9R, 12S, 2 R, 3 S. Thus, the structure of 4 was determined and named phomochromenone F.

This was confirmed through 1 H-1 H COSY correlations of H 3 -4 / H-1 / H-2 / H 3 -3 and HMBC correlations (
Our primary application of immunosuppressive activity screening indicated that a crude extract of P. asparagi DHS-48 showed strong inhibitory of splenic lymphocyte growth with IC 50 of 6 µg/mL. An immunosuppressive assay showed that compound 5 exhibited moderate to weak inhibitory activity against ConA-induced T and LPS-induced B murine splenic lymphocytes in vitro with IC 50 values of 34 and 117 µM, respectively (Table 2, Figure 5), whereas the other investigated compounds showed no obvious inhibitory effect. The cytotoxicity of 5 was tested in splenocyte cultures for 72 h using the tetrazolium salt-based CCK-8 assay. The results showed that it inhibited splenic lymphocyte growth with relatively lower toxicity (IC 50 47µM), at which the survival of normal splenic cells was slightly influenced in comparison with that of CsA (IC 50 11 µM). The results showed that compound 5 with a 1,3,4,10-tetrahydropyrano [4,3-b]chromene nucleus displayed significant immunosuppressive activity compared to compounds (1-3, 6, 7) with a chromone nucleus and compound 8 with a 10H-chromeno [3,2-c]pyridine nucleus. The additional 11-OH group in 5 is essential for its stimulated splenic lymphocyte inhibitory compared to compound 4. Table 2. Immunosuppressive activities of isolated compounds 1-8.

Compound
Cytotoxicity a IC 50 (µM) b  (Figure 3), which supported the absolute configuration to be 9R, 12S, 2′R, 3′S. Thus, the structure of 4 was determined and named phomochromenone F. Our primary application of immunosuppressive activity screening indicated that a crude extract of P. asparagi DHS-48 showed strong inhibitory of splenic lymphocyte growth with IC50 of 6 μg/mL. An immunosuppressive assay showed that compound 5 exhibited moderate to weak inhibitory activity against ConA-induced T and LPS-induced B murine splenic lymphocytes in vitro with IC50 values of 34 and 117 μM, respectively (Table 2, Figure 5), whereas the other investigated compounds showed no obvious inhibitory effect. The cytotoxicity of 5 was tested in splenocyte cultures for 72 h using the tetrazolium salt-based CCK-8 assay. The results showed that it inhibited splenic lymphocyte growth with relatively lower toxicity (IC50 47μM), at which the survival of normal splenic cells was slightly influenced in comparison with that of CsA (IC50 11 μM). The results showed that compound 5 with a 1, 3,4,10-tetrahydropyrano[4,3-b]chromene nucleus displayed significant immunosuppressive activity compared to compounds (1-3, 6, 7) with a chromone nucleus and compound 8 with a 10H-chromeno [3,2-c]pyridine nucleus. The additional 11-OH group in 5 is essential for its stimulated splenic lymphocyte inhibitory compared to compound 4.  Table 2. Immunosuppressive activities of isolated compounds 1-8.

General Procedures
Specific rotations were obtained on a WYA-2S digital Abbe refractometer (Shanghai Physico-optical Instrument Factory, Shanghai, China). UV spectra were determined using a Shimadzu UV-2401 PC spectrophotometer (Shimadzu Corporation, Tokyo, Japan), while CD spectra were measured on a JASCO J-715 spectra polarimeter (Japan Spectroscopic, Tokyo, Japan). 1 H, 13 C and 2D NMR spectra were recorded on a Bruker AV 400 NMR spectrometer using TMS as an internal standard. High-resolution ESI-MS were performed on an LTQ Orbitrap XL instrument (Thermo Fisher Scientific, Bremen, Germany) using peak matching. TLC and column chromatography (CC) were carried out over silica gel (200-400 mesh, Qingdao Marine Chemical Inc., Qingdao, China), or a Sephadex-LH-20 Merck,Darmstadt,Germany), respectively. UPLC analysis (Waters Corporation, Milford, MA, USA) was recorded using a Waters system equipped in ESI mode on an Acquity UPLC H-Class connected to an SQ Detector 2 mass spectrometer using a BEH RP C 18 column (2.1 × 50 mm, 1.7 µm, 0.5 mL/min). Semi-preparative HPLC was performed using a Waters equipped with a 2998 PDA detector (Waters Corporation, Milford, MA, USA) and a RP C 18 column (YMC-Pack ODS-A, 10 × 250 mm, 5 µm, 3 mL/min).

Fungal Material
Endophytic fungus Phomopsis asparagi was isolated with PDA medium from the fresh root of the mangrove plant Rhizophora mangle, collected in October 2015 in Dong Zhai Gang-Mangrove Garden on Hainan Island, China. The fungus (strain no.DHS-8) was identified using a molecular biological protocol by DNA amplification and sequencing of the ITS region (GenBank Accession no.MT126606) [22]. A voucher strain was deposited at one of the authors' laboratories (J.X.).

Immunosuppressive Assay
Compounds 1-8 were evaluated for immunosuppressive activity against the proliferation of concanavalin A (ConA)-induced T and lipopolysaccharide (LPS)-induced B murine splenic lymphocyte in vitro using a CCK-8 method according to previously reported methods [24]. Cyclosporine A was used as a positive control.
Author Contributions: J.X. conceived and designed the experiments; C.W. and Z.F. isolated the metabolites; C.S. elucidated structures; X.Z. performed the bioactivity assays; J.X. and C.W. wrote the paper. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement:
All experiments conducted on animal material were approved by the Ethics Committee of the Hainan University (HNUAUCC-2021-00082, Feb. 19th, 2021, and all methods used were compliant with the regulations of the Ethics Committee.