Cysteine Residue Containing Merocytochalasans and 17,18-seco

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Cysteine Residue Containing Merocytochalasans and 17,18-secoAspochalasins from Aspergillus micronesiensis Zhaodi Wu, Xiaotian Zhang, Weaam Hasan Al Anbari, Qun Zhou, Peng Zhou, Mi Zhang, Fanrong Zeng, Chunmei Chen, Qingyi Tong, Jianping Wang, Hucheng Zhu,* and Yonghui Zhang* Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China

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S Supporting Information *

ABSTRACT: Two cysteine residue containing merocytochalasans (cyschalasins A and B, 1 and 2) and two 17,18-secoaspochalasins (secochalasins A and B, 3 and 4) were isolated from the endophytic fungus Aspergillus micronesiensis. Cyschalasins A and B represent a new type of merocytochalasan featuring the fusion of an aspochalasin with a modified cysteine residue. Secochalasins A and B are the first 17,18seco-aspochalasins to be reported and represent a previously undescribed carbon skeleton. Plausible biosynthetic pathways of 1−4 were proposed. Compounds 1 and 2 were cytotoxic and active against Gram-positive bacteria.

C

sulfur are rare in nature, and there are only a few examples including our previously reported 2H-1,4-thiazine ring containing merocytochalasans.10 Plausible biosynthetic pathways of 1−4 are discussed, and cysteine is proposed as the key building block in the biosynthesis of 1 and 2. Compounds 1 and 2 exerted cytotoxic activities, and their IC50 values against five human cancer cell lines (HL60, A549, Hep3B, MCF-7, and SW480) ranged from 3.0 to 19.9 μM. Compounds 1 and 2 were active against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans. The MIC50 and MIC90 values against MRSA of 1 were 17.5 and 28.4 μg/mL, respectively, and those of compound 2 were 10.6 and 14.7 μg/mL, respectively. Herein, we reported the isolation, structural elucidation, and biological evaluation of these metabolites. Compound 1 was obtained as a white, amorphous powder. Its molecular formula was determined to be C28H42N2O5S by HRESIMS and 13C NMR data. The 1H NMR data (Table 1) displayed signals for two olefinic protons [δH 6.27 (1H, brd, J = 10.6 Hz) and 5.27 (1H, brs)] and six methyl groups [δH 1.93 (3H, s), 1.78 (3H, s), 1.32 (3H, brd, J = 1.1 Hz), 1.26 (3H, d, J = 6.9 Hz), and 0.92 (6H, d, J = 6.5 Hz)]. The 13C NMR with DEPT spectra showed a total of 28 carbon resonances assigned to two ketone carbonyls (δC 204.6 and 202.4), two amide carbonyls (δC 176.8 and 173.2), four olefinic carbons (δC 142.0, 139.7, 126.3, and 124.5), and 20 sp3 carbons [a quaternary carbon (δC 65.9), seven methines (δC 73.2, 54.1, 52.8, 45.9, 45.3, 37.2, and 25.8), six methylenes (δC 50.0, 42.8, 40.2, 39.3, 29.9, and 28.7), and six methyls (δC 24.2, 22.5, 21.9,

ytochalasans are fungal polyketide synthase−nonribosomal peptide synthetase (PKS-NRPS) hybrid metabolites, which can be attributed to the cytochalasin, chaetoglobosin, aspochalasin, pyrichalasin, and alachalasin classes, according to the amino acid involved in their biosynthesis.1 Cytochalasans are cytotoxic due to their capping actin filaments ability, while some cytochalasans also exhibit anti-HIV, anti-inflammatory, and phytotoxic effects.2 Approximately 400 cytochalasans with various carbon skeletons have been discovered from fungi, and the structural diversity and biological activity of cytochalasans have drawn considerable attention from chemists and pharmacologists. In a previous investigation, we isolated a series of cytochalasan heterodimers, heterotrimers, and heterotetramers from Aspergillus f lavipes.3−6 The total syntheses of some of the merocytochalasans have been reported.7,8 Recently, much progress has been reported in the study of cytochalasans. For example, three cytochalasan heterodimers formed by combining chaetoglobosin and aureonitol derivatives have been discovered from Chaetomium globosum,9 three merocytochalasan heterodimers containing a 2H-1,4-thiazine ring generated by the fusion of a dipeptide with the chaetoglobosin unit were discovered from the coculture of C. globosum and A. f lavipes,10 and two cytochalasans with a tetracyclic skeleton were reported from Xylaria curta E10.11 In our continuing investigations into cytochalasans, two cysteine residue containing merocytochalasans (cyschalasins A and B, 1 and 2) and two 17,18-seco-aspochalasins (secochalasins A and B, 3 and 4) were discovered from Aspergillus micronesiensis. The cysteine residue was connected to C-20 of the aspochalasin moiety via a sulfur atom in compounds 1 and 2. To the best of our knowledge, cytochalasans containing © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 7, 2019

A

DOI: 10.1021/acs.jnatprod.9b00016 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. 1H−1H COSY and key HMBC correlations of 1−4.

20.0, 15.2, and 13.9)]. The 1H and 13C NMR data (Table 1) suggested that 1 was an aspochalasin-based compound. The HMBC correlations (Figure 1) from H3-11 to C-4, C-5, and CTable 1. 1H and

13

C NMR Data of 1−4a 1

no.

δC

1 3 4 5 6 7 8 9 10a 10b 11 12 13 14 15a 15b 16a 16b 17 18 19a 19b 20a 20b 21 22 23 24 25 1′a 1′b 2′a 2′b 3′ 4′

176.8 52.8 54.1 37.2 142.0 126.3 45.9 65.9 50.0 13.9 20.0 124.5 139.7 39.3 28.7 73.2 204.6 42.8 45.3 202.4 25.8 24.2 21.9 15.2 29.9 40.2 173.2 22.5

6, from H3-12 to C-5, C-6, and C-7, from H3-25 to C-13, C-14, and C-15, and from H-19 to C-17, C-18, C-20, and C-21, together with the 1H−1H COSY correlations of H-3 with H-4

δH (J, Hz) 3.15 ddd (9.8, 4.1, 2.8) 2.55 m 2.52 m 5.27 brs 2.91 brd (10.6) 1.46 1.21 1.26 1.78 6.27

m m d (6.9) s brd (10.6)

2.15 2.15 2.43 1.52 4.20

m m m m dd (10.1, 1.0)

3.10 dd (12.1, 3.0) 2.98 t (12.1) 5.05 dd (12.1, 3.0)

1.66 0.92 0.92 1.32 2.59 2.51 3.32 3.32

m d (6.5) d (6.5) brd (1.1) m m m m

1.93 s

2 δC 176.7 52.8 54.1 37.2 142.0 126.3 45.8 65.8 49.9 13.9 20.1 124.5 139.7 39.3 28.6 73.1 204.7 42.9 45.8 202.6 25.8 24.1 21.8 15.2 35.0

3

δH (J, Hz)

δC 177.4 53.5 53.8 36.0 140.6 127.6 40.4 67.9 49.7

3.15 m 2.56 m 2.54 m 5.27 brs 2.91 m 1.53 1.21 1.26 1.78 6.29

m m d (6.9) s brd (10.6)

2.15 2.15 2.44 1.53 4.19

m m m m dd (10.2, 1.0)

14.6 20.3 125.5 136.2 35.9 33.7 175.5 176.6 28.9

3.19 dd (12.1, 2.9) 2.93 t (12.1) 5.07 dd (12.1, 2.9)

71.6

1.67 0.92 0.92 1.32 2.87 2.75 4.36

174.3 52.8

3.75 s

36.7 209.7 26.1 24.2 21.7 16.2 52.0

m d (6.5) d (6.5) brd (1.0) dd (13.4, 4.4) dd (13.4, 6.5) dd (6.5, 4.4)

δH (J, Hz) 3.18 dt (10.0, 3.6) 2.54 m 2.53 m 5.32 brs 3.50 brd (10.0) 1.46 1.28 1.21 1.77 5.86

ddd (14.0, 10.0, 4.1) m d (6.8) s brd (10.0)

2.30 2.30 2.44 2.44

t t t t

(7.5) (7.5) (7.5) (7.5)

2.56 2.43 2.87 2.87

m m m m

1.68 0.93 0.92 1.59 3.65

m d (6.6) d (6.6) brd (1.1) s

4 δC 177.4 53.5 53.9 35.9 140.5 127.6 40.6 67.9 49.7 14.6 20.2 125.2 136.5 36.1 34.1 177.5 174.9 28.5 36.5 209.7 26.1 24.2 21.7 16.3 52.1

δH (J, Hz) 3.19 dt (9.8, 3.5) 2.51 m 2.51 m 5.34 brs 3.48 brd (10.0) 1.44 1.29 1.22 1.77 5.88

ddd (13.9, 9.8, 4.2) ddd (13.9, 9.8, 3.5) d (6.6) s brd (10.0)

2.30 2.30 2.39 2.39

m m m m

2.61 2.46 2.91 2.91

m m m m

1.69 0.94 0.93 1.58 3.65

m d (6.6) d (6.6) brd (1.0) s

a

400 MHz for 1H and 100 MHz for 13C, measured in CD3OD. B

DOI: 10.1021/acs.jnatprod.9b00016 J. Nat. Prod. XXXX, XXX, XXX−XXX

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and H-10, of H-8 with H-7 and H-13, of H-16 with H-15 and H-17, and of H-19 with H-20 indicated that 1 possessed an aspochalasin unit similar to flavichalasine M.12 However, the chemical shift of C-20 (CH) differed greatly (δC 45.3 for 1 and 74.4 for flavichalasine M). Except for the 24 carbons assigned to the aspochalasin moiety, there were four additional carbons including an amide carbonyl (δC 173.2, C-3′), two methylenes (δC 40.2, C-2′; and 29.9, C-1′), and a methyl (δC 22.5, C-4′). The HMBC correlations (Figure 1) from H-20 to C-1′, from H-1′ to C-2′ and C-20, from H-2′ to C-1′ and C-3′, and from H 3 -4′ to C-3′, together with the chemical formula, C28H42N2O5S, revealed that an N-acetyl-cysteamine chain was connected to C-20 via an S atom in compound 1. Thus, the planar structure of 1 was determined. The NOESY correlations from H-13 to H-17 and from H-13 to H-20 revealed their cofacial and α-orientations (Figure 2).

Figure 3. ΔδH values (in ppm) for the MTPA esters of 2.

Figure 2. Key NOESY correlations of 1 and 2.

Compound 2 was assigned a molecular formula of C28H41NO7S, based on positive HRESIMS. The overall NMR data of 2 were similar to those of 1, and the differences were that the C-2′ methylene (δC 40.2) in 1 changed to an oxygenated methine (δC 71.6) in 2 and the C-3′ amide carbonyl (δC 173.2, C-3′; 22.5, C-4′) in 1 changed to an ester carbonyl (δC 174.3, C-3′; 52.8, C-4′) in 2. The HMBC correlations (Figure 1) from H-20 to C-1′, from H-1′ to C-2′, C-3′, and C-20, and from H3-4′ to C-3 constructed the structure of the modified cysteine chain, as well as the fusion pattern through a C-20−S−C-1′ linkage in compound 2. The NOESY correlations from H-13 to H-17 and H-20 revealed that H-17 and H-20 were α-oriented (Figure 2); however, there was no evidence to determine the relative configuration of 2′-OH. In order to the determine the absolute configuration of C-2′, (S)- and (R)-MTPA esters (2a and 2b) were prepared.13 The 1H NMR Δδ values (Δδ = δ2a − δ2b) revealed the S-configuration of C-2′ (Figure 3).14 The absolute configurations of the aspochalasin moiety of compounds 1 and 2 were determined by comparing their electronic circular dichroism (ECD) spectra with that of aspochalasin P (Figure 4), whose absolute configuration was confirmed by X-ray diffraction analysis in our previous study.12 Compound 3 was obtained as a colorless gum, and its chemical formula was determined to be C25H37NO6, based on positive HRESIMS. The 1H NMR spectrum revealed the presence of two olefinic protons [δH 5.86 (brd, 1H, J = 10.0 Hz) and 5.32 (brs, 1H)], five methyls [δH 1.77 (3H, s), 1.59 (3H, brd, J = 1.1 Hz), 1.21 (3H, d, J = 6.8 Hz), 0.93 (3H, d, J = 6.6 Hz), and 0.92 (3H, d, J = 6.6 Hz)], and a methoxyl [δH 3.65 (3H, s)]. These data together with the 13C NMR spectrum, which displayed 25 carbon signals, suggested that 3

Figure 4. Experimental ECD spectra of 1−4 and aspochalasin P.

was also an aspochalasin derivative. Analysis of the 2D NMR spectra of 3 indicated that the 11-membered macrocycle was opened between C-17 and C-18, resulting in a novel 17,18seco-aspochalasin skeleton. The assignment of C-17 (δC 175.5, ester carbonyl) and C-18 (δC 176.6, carboxyl) was based on the HMBC correlations from H-15, H-16, and H3-1′ to C-17 and from H-19 and H-20 to C-18 (Figure 1). Therefore, the structure of 3 was determined. Compound 4 had the same molecular formula as 3, and the NMR data of 4 were similar to those of 3. However, in the structure of 4, C-17 was a carboxyl, and C-18 was an ester carbonyl, which was evidenced by the HMBC correlations from H-15 and H-16 to C-17 (δC 177.5) and from H-19, H-20, and H3-1′ to C-18 (δC 174.9) (Figure 1). Thus, the structure of 4 was determined. Since the experimental ECD spectra of 3 and 4 agreed well with that of aspochalasin P (Figure 4), the absolute configuration of 3 and 4 was determined to be 3S,4R,5S,8S,9S. Compounds 1 and 2 represent a new type of merocytochalasan featuring the fusion of an aspochalasin with a modified cysteine residue. Aspochalasin B and cysteine were proposed as biosynthetic precursors (Scheme 1). Aspochalasin B combines with cysteine via a 1,4-addition to form the intermediate i. Intermediate i is transformed into intermediates ii and iii through acylation and deamination reactions, respectively. Subsequently, ii undergoes a decarboxylation reaction to form compound 1, and iii undergoes a methylation reaction to generate compound 2. In addition, compounds 3 and 4 are C

DOI: 10.1021/acs.jnatprod.9b00016 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Scheme 1. Plausible Biosynthetic Pathways of 1 and 2

Table 2. Cytotoxicity against Five Human Cancer Cells of 1 and 2 (IC50, μM) compound

HL60

A549

Hep3B

MCF-7

SW480

1 2 DDPa

9.3 ± 0.2 3.0 ± 0.0 2.1 ± 0.1

>20 16.7 ± 0.9 7.2 ± 0.3

19.9 ± 0.6 8.2 ± 0.1 17.6 ± 0.4

16.1 ± 0.7 17.1 ± 0.2 15.0 ± 0.4

10.1 ± 0.3 13.6 ± 1.1 14.3 ± 0.1

a

DDP was used as the positive control. spectra were measured on Shimadzu UV1750, Bruker Vertex 70 FTIR, and Jasco J-810 spectrometers, respectively. High-resolution electrospray ionization mass spectroscopy was obtained using a Thermo Fisher LC-LTQ-Orbitrap XL spectrometer. NMR spectra were recorded on a Bruker AM-400 spectrometer. Chemical shifts were referenced to the residual CD3OD (δH 3.31/δC 49.0) signals. Silica gel (100−200 mesh and 200−300 mesh), ODS (50 μm, YMC, Japan), and Sephadex LH-20 were used for column chromatography (CC). Semipreparative high-performance liquid chromatography (HPLC) was performed using a Dionex Ultimate 3000 HPLC (Dionex, Sunnyvale, CA, USA) with a UV detector and a Welch Ultimate XB-C18 (10 × 250 mm, 5 μm) column. Thin-layer chromatography (TLC) was performed on precoated silica gel GF254 plates. Fungal Material. The fungus Aspergillus micronesiensis was isolated from the root of the traditional Chinese medicinal plant Phyllanthus glaucus, collected from LuShan Mountain, Jiangxi Province, China (2015). Its sequence data have been submitted to the DDBJ/EMBL/GenBank database with the accession no. MH938722. A voucher sample (ZYH20150717) has been preserved in the culture collection center of Tongji Medical College, Huazhong University of Science and Technology. Fermentation and Isolation. The strain was cultured on potato dextrose agar (PDA) for 5 days to prepare the seed culture. Agar plugs were inoculated into 150 sterilized Erlenmeyer flasks (1 L), each containing 200 g of rice and 200 mL of water. All flasks were incubated at 25 °C for 5 weeks. The fermented rice substrate was extracted seven times with 95% aqueous EtOH at room temperature, and the solvent was evaporated under vacuum. The residue was then partitioned with EtOAc to afford the EtOAc fraction (approximately 120 g). The EtOAc extract was subjected to silica gel CC eluting with petroleum ether−EtOAc (20:1−5:1) to remove the majority of the lipids and steroids and then eluting with CH2Cl2−MeOH (100:1− 0:1) to furnish eight fractions (Fr. A−H). Fraction C (15.0 g) was separated on ODS CC (gradient elution with MeOH−H2O, 50:50−100:0) to afford 10 subfractions (Fr. C1− C10). Fr. C3 (eluted with MeOH−H2O, 70:30) was chromatographed on Sephadex LH-20 (MeOH−CH2Cl2, 1:1) and then a silica gel column to give three subfractions (C3.1−C3.3). C3.1 was

proposed to be generated from aspochalasin B via oxidization and methylation reactions. Cytochalasans are able to cap actin filaments and subsequently inhibit cytokinesis, which affects vital cellular processes including cell adhesion, motility, signaling, and cytokinesis.2 This specific property endows cytochalasans with potent cytotoxic activity. Compounds 1−4 were initially evaluated for their in vitro cytotoxicity against five human cancer cell lines (HL60, A549, Hep3B, MCF-7, and SW480) (Table 2). Compounds 1 and 2 showed moderate activities against these cancer cells, and the IC50 values ranged from 3.0 to 19.9 μM, while compounds 3 and 4 were inactive. Compounds 1−4 were also evaluated for their antimicrobial effects against five types of bacteria (Escherichia coli, Staphylococcus aureus, MRSA, Salmonella enterica, Pseudomonas aeruginosa) and a fungus (Candida albicans). As a result, compounds 1 and 2 showed activities against Gram-positive bacteria and fungus (Table 3); notably, 1 and 2 were active Table 3. Antimicrobial Activity of 1 and 2 (MIC, μg/mL) compound 1 2

MIC50 MIC90 MIC50 MIC90

Staphylococcus aureus 40.8 58.5 14.5 27.0

± ± ± ±

5.0 1.5 0.4 0.2

MRSA

Candida albicans

± ± ± ±

43.3 ± 1.5 89.9 ± 1.1 94.7 ± 1.3 >100

17.5 28.4 10.6 14.7

0.3 0.1 0.1 0.0

against MRSA with MIC50 values of 17.5 and 10.6 μg/mL and MIC90 values of 28.4 and 14.7 μg/mL, respectively. This is the first time cytochalasans with anti-MRSA activity have been reported.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation values were obtained using a PerkinElmer PE-341 polarimeter. UV, IR, and ECD D

DOI: 10.1021/acs.jnatprod.9b00016 J. Nat. Prod. XXXX, XXX, XXX−XXX

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19a, H-1′a), 3.14 (1H, m, H-3), 3.05 (1H, m, H-19b), 3.02 (1H, m, H-8), 2.96 (1H, m, H-4), 2.74 (1H, dd, J = 13.6, 3.9 Hz, H-1′b), 2.54 (1H, m, H-5), 2.24 (1H, m, H-16a), 2.15 (2H, m, H-15a,b), 1.79 (3H, s, H3-12), 1.69 (1H, m, H-22), 1.62 (1H, m, H-16b), 1.44 (1H, m, H-10a), 1.35 (3H, brd, J = 1.4 Hz, H3-25), 1.29 (3H, m, H3-11), 1.21 (1H, m, H-10b), 0.89 (3H, d, J = 6.6 Hz, H3-23), 0.86 (3H, d, J = 6.6 Hz, H3-24); ESIMS m/z 990 [M + Na]+. Cytotoxic and Antimicrobial Assay. Five human cancer cell lines (HL60, A549, Hep3B, MCF-7, and SW480), five types of bacteria (Escherichia coli, Staphylococcus aureus, MRSA, Salmonella enterica, and Pseudomonas aeruginosa), and a fungus (Candida albicans) were used in the biological assay, according to the previously described method.15

separated on ODS CC (gradient elution with MeOH−H2O, 55:45− 80:20) to afford five subfractions (C3.1.1−C3.1.5). C3.1.4 was purified by semipreparative HPLC (MeOH−H2O, 72:28, v = 2.0 mL/ min) to afford compound 3 (13.8 mg, tR = 32.5 min). Fr. C4 (eluted with MeOH−H2O, 80:20) was chromatographed on Sephadex LH-20 (MeOH−CH2Cl2, 1:1) and then using silica gel to give seven subfractions (C4.1−C4.7). C4.3 was separated on ODS CC (gradient elution with MeOH−H2O, 60:40−80:20) to afford three subfractions (C4.3.1−C4.3.3). C4.3.1 was purified by semipreparative HPLC (MeOH−H2O, 72:28, v = 2.0 mL/min) to afford compound 4 (10.5 mg, tR = 33.7 min). C4.4 was separated on ODS CC (gradient elution with MeOH−H2O, 60:40−80:20) to afford four subfractions (C4.4.1−C4.4.4). C4.4.2 was purified by semipreparative HPLC (MeOH−H2O, 80:20, v = 2.0 mL/min) to give compound 1 (4.7 mg, tR = 19.0 min). Fraction D (20.0 g) was separated on ODS CC (gradient elution with MeOH−H2O, 50:50−100:0) to afford nine subfractions (Fr. D1−D9). Fr. D4 (eluted with MeOH−H2O, 70:30) was chromatographed on Sephadex LH-20 (MeOH−CH2Cl2, 1:1) and then using silica gel to give three subfractions (D4.1−D4.3). D4.3 was separated on ODS CC (gradient elution with MeOH−H2O, 60:40−80:20) to afford five subfractions (D4.3.1−D4.3.5). D4.3.4 was purified by semipreparative HPLC (MeCN−H2O, 66:34, v = 2.0 mL/min) to afford compound 2 (3.8 mg, tR = 31.5 min). Cyschalasin A (1): white, amorphous powder; [α]20D +63.6 (c 1.0, MeOH); ECD (MeOH) λ (Δε) 228 (+3.9), 302 (+7.4) nm; UV (MeOH) λmax (log ε) 203 (4.29) nm; IR νmax 3434, 2957, 2926, 1685, 1656, 1551, 1440, 1383, 1300, 1047, 1020 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 541.2683 [M + Na]+ (calcd for C28H42N2O5SNa, 541.2707). Cyschalasin B (2): white, amorphous powder; [α]20D +80.3 (c 0.4, MeOH); ECD (MeOH) λ (Δε) 234 (+2.9), 302 (+6.9) nm; UV (MeOH) λmax (log ε) 203 (4.23) nm; IR νmax 3435, 2948, 2853, 1687, 1631, 1504, 1439, 1384, 1217, 1092, 1022 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 558.2488 [M + Na]+ (calcd for C28H41NO7SNa, 558.2501). Secochalasin A (3): colorless gum; [α]20D −28.1 (c 1.4, MeOH); ECD (MeOH) λ (Δε) 213 (+5.0); UV (MeOH) λmax (log ε) 203 (4.17) nm; IR νmax 3436, 2958, 1709, 1639, 1557, 1439, 1385, 1208, 1171, 1028 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 470.2517 [M + Na]+ (calcd for C25H37NO6Na, 470.2519). Secochalasin B (4): colorless gum; [α]20D −26.6 (c 1.5, MeOH); ECD (MeOH) λ (Δε) 213 (+5.0); UV (MeOH) λmax (log ε) 203 (4.14) nm; IR νmax 3434, 2952, 2922, 1703, 1638, 1553, 1505, 1439, 1383, 1212, 1173, 1028 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 470.2552 [M + Na]+ (calcd for C25H37NO6Na, 470.2519). Preparation of (S)- and (R)-MTPA Esters (2a and 2b) of 2. A mixture of compound 2 (0.5 mg), (R)-MTPACl (10.0 μL), 4(dimethylamino)pyridine (5.0 mg), and triethylamine (2 μL) in CH2Cl2 (500 μL) was stirred at room temperature for 5 h. After evaporation of solvent, the residue was purified by silica gel column chromatography (petroleum ether−EtOAc, 5:1) to prepare (S)MTPA ester (2a, 0.3 mg). The (R)-MTPA ester (2b, 0.4 mg) was prepared similarly. 2a: colorless gum; 1H NMR (CD3OD) δ 7.36−7.64 (overlap, aromatic protons), 6.35 (1H, d, J = 10.7 Hz, H-13), 5.30 (1H, brs, H7), 5.17 (1H, dd, J = 10.6, 3.4 Hz, H-20), 4.28 (1H, m, H-17), 4.22 (1H, dd, J = 5.6, 2.1 Hz, H-2′), 3.73 (3H, s, H3-4′), 3.20 (2H, m, H19a, H-1′a), 3.14 (1H, m, H-3), 2.97 (1H, m, H-8), 2.93 (1H, m, H19b), 2.88 (1H, m, H-1′b), 2.55 (1H, m, H-4), 2.50 (1H, m, H-5), 2.32 (1H, m, H-16a), 2.20 (2H, m, H-15a,b), 1.79 (3H, s, H3-12), 1.67 (1H, m, H-22), 1.62 (1H, m, H-16b), 1.47 (1H, m, H-10a), 1.34 (3H, brs, H3-25), 1.29 (3H, m, H3-11), 1.20 (1H, m, H-10b), 0.91 (3H, d, J = 6.6 Hz, H3-23), 0.87 (3H, d, J = 6.6 Hz, H3-24); ESIMS m/z 990 [M + Na]+. 2b: colorless gum; 1H NMR (CD3OD) δ 7.35−7.65 (overlap, aromatic protons), 6.34 (1H, d, J = 10.7 Hz, H-13), 5.31 (1H, brs, H7), 5.16 (1H, dd, J = 10.6, 3.4 Hz, H-20), 4.28 (1H, m, H-17), 4.22 (1H, dd, J = 5.8, 2.0 Hz, H-2′), 3.77 (3H, s, H3-4′), 3.20 (2H, m, H-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00016. 1

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H NMR spectra of 2a and 2b; HRESIMS, UV, IR, and NMR spectra of 1−4 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (H. Zhu). *Tel: 86-27-83692892. E-mail: [email protected]. (Y. Zhang). ORCID

Yonghui Zhang: 0000-0002-7222-2142 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Program for Changjiang Scholars of Ministry of Education of the People’s Republic of China (T2016088); the National Natural Science Foundation for Distinguished Young Scholars (81725021); the National Natural Science Foundation of China (21772048, 81573316, 21602068, and 81773597); the Innovative Research Groups of the National Natural Science Foundation of China (81721005); the National Science and Technology Project of China (2018ZX09201001-001-003); Natural Science Foundation of Hubei Province of China (2018CFA076 and 2018CFA027); the Academic Frontier Youth Team of HUST; and the Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College (HUST). We thank the Analytical and Testing Center at Huazhong University of Science and Technology for IR, UV, and ECD analyses.

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DOI: 10.1021/acs.jnatprod.9b00016 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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DOI: 10.1021/acs.jnatprod.9b00016 J. Nat. Prod. XXXX, XXX, XXX−XXX