Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Cytochathiazines A−C: Three Merocytochalasans with a 2H‑1,4Thiazine Functionality from Coculture of Chaetomium globosum and Aspergillus flavipes Wenjing Wang, Fanrong Zeng, Qiong Bie, Chong Dai, Chunmei Chen, Qingyi Tong, Junjun Liu, Jianping Wang, Yuan Zhou, Hucheng Zhu,* and Yonghui Zhang*
Downloaded via UNIV OF TEXAS AT EL PASO on October 16, 2018 at 22:45:47 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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 S Supporting Information *
ABSTRACT: Cytochathiazines A−C (1−3), which represent a new type of merocytochalasan, were isolated from coculture of Chaetomium globosum and Aspergillus flavipes. Compounds 1−3 are the first natural products featuring an unprecedented 2H-1,4-thiazine functionality. Plausible biosynthetic pathways for 1−3 with a chaetoglobosin and a dipeptide as the main constitutional units were proposed. Compound 2 induced apoptosis in leukemia cells through the activation of caspase-3 and the degradation of poly ADP-ribose polymerase.
C
ytochalasans are a series of well-known fungi metabolites featuring multiple ring systems that are generated via PKS-NRPS hybrid pathways.1 Changing the amino acid residues and polyketides involved in the biosynthesis results in cytochalasans with various skeletons, and these differences cause these compounds to have a wide variety of biological activities.1,2 Therefore, cytochalasans have attracted substantial attention from natural products chemists, synthetic chemists, biosynthetic chemists, and pharmacologists.3 Over the past few decades, more than 300 cytochalasans with diverse structures have been isolated and characterized.4 Our previous investigations on Aspergillus flavipes revealed new dimensions to the diversity of the cytochalasan family known as merocytochalasans, which arise from the dimerization of one or more cytochalasans with one or more epicoccine units, resulting in intriguing structures.4,5 Total syntheses of asperchalasine A, a representative of merocytochalasans, were reported by two groups of synthetic chemists at almost the same time.3i,j Coculture is a convenient and effective approach for activating silent biosynthetic genes and exploring the structural diversity of microbial secondary metabolites.6 Hence, this technique was used in our studies on cytochalasans of Chaetomium globosum and A. f lavipes and led to the isolation of a series of functionalized cytochalasans.3c In our further studies on the metabolites of the coculture of C. globosum and A. f lavipes, a new type of merocytochalasan (cytochathiazines A−C, 1−3), with unprecedented 2H-1,4-thiazine functionalities, was isolated and characterized (Figure 1). Natural products containing thiazine rings are rare, and most of them are derived from Streptomyces sp. and marine organisms, and only three examples have been isolated from fungi (Table S1). © XXXX American Chemical Society
Figure 1. Structures of compounds 1−3.
Cytochathiazines A−C (1−3), formed by the fusion of a dipeptide and a cytochalasan, are the first examples of natural products with a 2H-1,4-thiazine ring. Biological research indicated that compound 2 could induce apoptosis in leukemia cells through the activation of caspase-3 and the degradation of PARP. Herein, we report the details for the isolation, structural Received: September 14, 2018
A
DOI: 10.1021/acs.orglett.8b02942 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters elucidation, biological evaluation, and plausible biogenetic pathways of 1−3. Cytochathiazine A (1) was obtained as a pale-yellow powder. Its molecular formula, C38H42N4O7S, with 20 degrees of unsaturation, was deduced from the pseudo-molecular ion peak at m/z 721.2689 [M + Na]+ (calcd for C38H42N4O7SNa, 721.2672) in its HRESIMS spectrum. The 1H NMR (Table S2) and HSQC data of 1 indicated the presence of a 3substituted indolyl unit based on the characteristic signals at δH 7.47 (1H, d, J = 7.9 Hz), 7.38 (1H, d, J = 8.0 Hz), 7.19 (1H, brs), 7.09 (1H, t, J = 7.3 Hz), and 7.00 (1H, t, J = 7.0 Hz); a terminal double bond with two protons at δH 4.90 (1H, brs) and 5.17 (1H, brs); a methoxy group at δH 3.64 (3H, s); and three methyl groups at δH 1.79, 1.04, and 0.81. The 13C NMR data of 1 (Table S2) displayed 38 carbon signals, including 2 carbonyls [δC 194.3 (C-19) and 206.6 (C-23)], 3 amide/ester carbonyls [δC 173.8 (C-1), 161.8 (C-5″), and 170.1 (C-8″)], 17 olefinic/aromatic carbons, 1 sp3 quaternary carbon [61.2 (C-9)], 4 sp3 methylene groups, 7 sp3 methine groups including an oxygenated methine [δC 68.4 (C-7)], a methoxy group, and 3 methyl groups. The aforementioned data, along with our previous investigations on C. globosum and A. f lavipes, indicated that 1 was likely a Chae-type cytochalasan.3c,7 The cytochalasan moiety (fragment A) of 1, which is similar to chaetoglobosin Fex,8 was elucidated by detailed analyses of the 1H−1H COSY and HMBC spectra (Figure 2). However,
Figure 3. Four candidates of the structure of compound 1.
Considering the chemical shifts of C-20 (δC 151.5) and the methine (δC 119.5) in the thiazine ring, which were located at the α and β positions of the carbonyl C-19, respectively, structure b is less likely than a. Although these analyses suggested a is the most likely structure for these structurally unique compounds, more solid evidence was necessary to verify this structure. Compound 1 possesses 8 stereocenters, and all of them are located in fragment A. Therefore, the relative stereochemistry of 1 could be deduced by a NOESY experiment, even though the whole planar structure of 1 had not been fully determined. The NOESY correlations (Figure 4) of H-5/H-8, H-3/CH3-
Figure 2. Key HMBC and COSY correlations of 1.
C-20 and C-21 of fragment A were connected to unexpected substructures via a single bond and double bond, respectively. In addition, fragment B, a side chain containing amide and ester groups, was determined by the HMBC correlations from CH3-9″ to C-8″, from H2-7″ to C-8″ and C-5″, and from NH6″ to C-5″ (Figure 2). With fragments A and B deciphered, two sp2 carbons (δC 119.5 and 133.9) as well as a sulfur atom and a nitrogen atom remained to be assigned to satisfy the molecular formula. Therefore, considering the remaining three degrees of unsaturation, fragments A and B were proposed to be connected via an unprecedented thiazine ring. On the basis of the 2D NMR spectra and the chemical shift of C-21, the sulfur atom should be connected to C-21 directly, which leads to four potential candidates, a−d (Figure 3), with 1,2-thiazine, 1,3-thiazine, or 1,4-thiazine functionalities. Further examination of the HMBC spectrum of 1 revealed a key HMBC correlation from the unassigned sp2 methine proton (δH 7.27) to the amide carbonyl (δC 161.8) in fragment B, which excluded candidates c and d and indicated that only candidates a and b should be further considered (Figure 3). Comprehensive investigation of the 2D NMR spectra of 1, especially the 1H−1H COSY and HMBC spectra, revealed that both candidates satisfied the observed correlations, which were not sufficient for distinguishing a and b (Figure 3).
Figure 4. Key NOESY correlations of 1 shown in fragment A.
11, H-4/H-10, H-8/H-14, H-14/H-16, H-7/H-13, and H-16/ CH3-25 indicated that the core structure of 1 is identical to that of chaetoglobosin Fex.8 In addition, the NOESY correlations of H-13/H-15b, H-15b/H-17, H-13/H-22a, H17/H-22a, and H-4/H-22b revealed the α-orientation of H22a (2.88, dd, J = 19.7, 11.3 Hz) and the β-orientation of H22b (1.23, dd, J = 19.7, 2.2 Hz). Therefore, the NOESY correlation of H-21/H-22b and the large coupling constant (11.3 Hz) between H-21 and H-22a suggested that H-21 was β-orientation. To confirm the structure of 1, the 13C NMR chemical shifts for both a and b were calculated; the calculated 13C NMR data of a were in good agreement with the experimental data, and a higher correlation coefficient was obtained for a (0.9977) than for b (0.9929) (Figure S2). In addition, the average absolute B
DOI: 10.1021/acs.orglett.8b02942 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
consistent with that of 1, as determined by the contiguous NOESY correlations of H-10b/H-22b, H-13/H-22a, H-17/H22a, and H-21/H-22b. Comparison of experimental ECD spectra of 2 and 1 was used to establish the absolute configuration of 2. The ECD curves of 2 and 1 were almost identical, which allowed us to assign the absolute configuration of 2 as 3S,4R,7S,8R,9R,16S,21R (Figure 7).
deviation of a (2.25 ppm) was substantially lower than that of b (3.26 ppm) (Figure 5). To obtain a single-crystal of 1 to
Figure 5. Calculated NMR shifts of a and b.
confidently determine its structure and absolute configuration, a number of attempts were made in various solvents, and finally, crystallization was achieved in ethanol−water (20:1). The X-ray diffraction experiment (Figure 6) using Cu Kα radiation unambiguously determined the whole structure and absolute configuration of 1 as 3S,4R,5S,7S,8R,9R,16S,21R (Flack parameter 0.073 (4)).
Figure 7. Experimental ECD spectra of 1−3 in MeOH.
Cytochathiazine C (3) had the same molecular formula (C38H42N4O7S) as compounds 1 and 2. The 1H and 13C NMR data (Table S2) of 3 closely resembled those of 1 and 2, indicating that they were structurally similar, except for the lack of a C=C double bond and the concomitant occurrence of an sp3 quaternary carbon (δC 56.7). These data in conjunction with the HMBC correlations from H3-12 to C-5 (δC 36.1), C-6 (δC 56.7), and C-7 (δC 60.2) revealed the presence of an epoxide ring at C-6 and C-7. Therefore, the planar structure of 3 was elucidated, as shown. On the basis of the observed key correlations between H-3/CH3-11, H-3/CH3-12, H-7/H-13, and H-5/H-8 in the NOESY spectrum (Figure S4), CH3-11, CH3-12, and H-7 were cofacial and α-oriented. In addition, the remaining chiral centers of compound 3 were determined to be the same as those in 2 and 1. The ECD curves of 3 and 1 were identical, allowing the absolute configuration of 3 (Figure 7). Cytochathiazines A−C have been identified as a new type of merocytochalasan that features a rare 2H-1,4-thiazine ring. Two possible biogenetic pathways (pathways A and B) are proposed in Scheme 1, and further evaluation of those pathways may be of interest to biosynthetic chemists. In pathway A, dipeptide derivative b was formed by the reaction of cysteine and glycine followed by an esterification. After that, b fused with a molecular of cytochalasan via a Michael addition to generate the C−S bond of intermediate c at C-21, and subsequently, c underwent condensation along with the elimination of water to generate Schiff base d, which was then oxidized to form the formation of 2H-1,4-thiazine functionality, generating compound 3. Further isomerization of 3 could produce compounds 1 and 2. In pathway B, the reactions were similar to those in pathway A, but they occurred in a different order. Cysteine fused with the cytochalasan moiety instead of forming the dipeptide first, and the peptide bond between the cysteine and the glycine was formed as the second step. Finally, compound 3 was formed via an additional esterification reaction. Compounds 1−3 were evaluated for their cytotoxicities against six human cancer cell lines (SW480, NB4, HL-60, A549, MCF-7, and SMMC-7721).3c,9 Among them, compound 2 showed moderate antiproliferative activities against NB4 and HL-60 cells with IC50 values of 9.6 and 12.5 μM (Table S4),
Figure 6. X-ray ORTEP drawing of compound 1.
Cytochathiazine B (2) was also isolated as a pale-yellow powder and possessed the same molecular formula (C38H42N4O7S) as compound 1 as indicated by the HRESIMS ion peak at m/z 721.2672. The 1H and 13C NMR data (Table S2) of 2 were similar to those of 1, except for the absence of the terminal double bond in 1 and the appearance of two olefinic carbons (δ C 125.5 and 133.7). The HMBC correlations from H-3 to C-5 (δC 125.5), CH3-12 to C-5, CH3-11 to C-6 (δC 133.7), and H-8 to C-6 revealed that the terminal double bond in 1 was replaced by an endocyclic double bond between C-5 and C-6 in 2. The 2H-1,4-thiazine unit was deduced to be the same as that of 1 by comparison of their 1D NMR spectra and analyses of its 1H−1H COSY and HMBC spectra in the same manner as described for 1 (Figure S3). Accordingly, the planar structure of 2 was deduced, and it was determined to be a structural isomer of 1. The relative configuration of 2 was determined by detailed evaluation of the NOESY spectrum (Figure S3). The NOESY correlations of H7/H-13, H-13/H-15b, and H-15b/H-17 indicated the αorientation of these protons. The β-orientation of H-4, H-8, H14, H-16 and CH3-25 were suggested by the NOESY correlations of H-4/H-10, H-4/H-8, H-8/H-14, H-14/H-16, and H-16/CH3-25. The relative configuration of C-21 was C
DOI: 10.1021/acs.orglett.8b02942 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Scheme 1. Proposed Biosynthetic Pathways of 1
compounds enriches the structural diversity of merocytochalasans, and their total synthesis and biosynthesis may attract the interest of synthetic chemists and biosynthetic chemists.
respectively. Moreover, the apoptosis induction effects of compound 2 against NB4 and HL-60 cells were assessed. As shown in Figure 8, compound 2 induced moderate apoptosis of NB4 and HL-60 cells by activation of caspase-3 and degradation of PARP.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b02942. Experimental details and copies of 1D and 2D NMR spectra; experimental details for X-ray analysis of 1 (PDF) Accession Codes
CCDC 1827757 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected].
Figure 8. Cytotoxicity and apoptosis induction effects of compound 2. (A) Inhibition rates of 2 against NB4 and HL60 cell lines were evaluated by a CCK-8 kit after 48 h of treatment. Values are the mean ± SD of three independent experiments. IC50 values were calculated by SPSS. (B) After treatment with DMSO (
