Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Amichalasines A−C: Three Cytochalasan Heterotrimers from Aspergillus micronesiensis PG‑1 Zhaodi Wu,† Qingyi Tong,† Xiaotian Zhang, Peng Zhou, Chong Dai, Jianping Wang, Chunmei Chen,* 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, China
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S Supporting Information *
ABSTRACT: Amichalasines A−C (1−3), which represent a new type of cytochalasan heterotrimers, were isolated from Aspergillus micronesiensis PG-1. Compounds 1 and 2 possess an undecacyclic 5/6/11/5/5/6/6/5/ 11/6/5 ring system, and 3 has an additional furan ring with a dodecacyclic 5/6/11/5/5/6/6/5/5/11/6/5 ring system. 1 and 2 exhibited potent cytotoxic activities through apoptosis induction mediated by caspase-3 activation and PARP degradation, and their IC50 values against HL60 cells were 1.71 and 3.74 μM, respectively.
C
uncharacterized enzymes responsible for these extraordinary reactions that are waiting to be discovered.14 In our ongoing search for bioactive cytochalasans, three novel cytochalasan heterotrimers (1−3) and two known compounds (4 and 5)8,15 (Figure 1) were isolated from Aspergillus micronesiensis PG-1, an endophytic fungus derived from the root of Phyllanthus glaucus. Amichalasines A (1) and B (2) possess a highly complex structure with an undecacyclic 5/6/11/5/5/6/6/5/11/6/5 ring system, and amichalasine C (3) possesses an additional furan ring with a dodecacyclic 5/6/11/5/5/6/6/5/5/11/6/5 ring system. Asperchalasine A (4),8 the cytochalasan heterotrimer, was also obtained in this work. Compounds 1− 3 were evaluated for their cytotoxicities against six cancer cell lines and a normal cell line. Compounds 1 and 2 exhibited much stronger cytotoxicities than most of the previously reported cytochalasans with the IC50 values of 1.71 and 3.74 μM, respectively (against HL60 cell lines), compound 2 was twice as potent as the model compounds, cytochalasins B and D.16−20 Mechanistically, 1 and 2 exerted their effects through apoptosis induction mediated by caspase-3 activation and PARP degradation. Herein, we describe the isolation, structural elucidation, and biological evaluation of compounds 1−3. Amichalasine A (1) was obtained as a white, amorphous powder. Its molecular formula, C57H72N2O12, was deduced from the positive HRESIMS ion peak at m/z 999.4971 [M + Na]+ (calcd for C57H72N2O12Na, 999.4983). The 1H NMR and HSQC spectra revealed the presence of 11 methyl groups
ytochalasans are a group of fungal metabolites normally characterized by a tricyclic ring system with a highly substituted perhydro-isoindolone moiety.1 Since the first discovery of cytochalasins A and B in 1966,2 nearly 400 cytochalasans have been isolated and characterized. Concomitantly, investigations related to cytochalasans, such as total synthesis of cytochalasins B and D,3,4 biosynthesis elucidation and modification of chaetoglobosin A,5,6 and mechanism study of chaetoglobosin A against chronic lymphocytic leukemia cells,7 have flourished in the past decades. Notably, our recent discovery of merocytochalasans, which are generated by the fusion of one or two cytochalasans with one or two epicoccine units, is a significant breakthrough in the field of cytochalasan research.8−11 The highly functionalized structures resulted in merocytochalasans with promising antitumor activities. For example, asperflavipine A showed moderate cytotoxicity and induced apoptosis in Jurkat, NB4, and HL60 cell lines,10 and asperchalasine A caused a significant G1-phase cell cycle arrest effect in cancer but not normal cells, highlighting its potential as a selective cell cycle regulator against cancer cells.8 Hence, merocytochalasans have attracted substantial interest from organic synthetic and biosynthetic communities. Recently, based on our proposed biosynthetic pathways of merocytochalasans, Deng’s group completed the total synthesis of asperchalasine A,12 and at the same time, Tang’s group reported a different strategy for the total synthesis of the same compound through a biomimetic Diels−Alder reaction followed by an oxidative cycloaddition.13 In addition, determining the biosynthetic pathways of merocytochalasans has attracted great attention from biosynthetic chemists; they believe that there are many © XXXX American Chemical Society
Received: December 20, 2018
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DOI: 10.1021/acs.orglett.8b04066 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Figure 4. Experimental ECD spectra of 1−3.
Scheme 1. Proposed Biogenetic Pathways of 1−4
Figure 1. Structures of compounds 1−5.
Figure 2. Key 1H−1H COSY and HMBC correlations and fusion pattern of units A−C of 1.
Figure 3. X-ray structure of 1. Figure 5. Dose−response viability curves of HL60 after treatment with 2 and cytochalasins B and D for 48 h. Data represent the mean ± SEM of three independent experiments.
[δH 1.88 (3H, s), 1.75 (6H, s), 1.48 (3H, s), 1.27 (3H, s), 1.26 (3H, d, J = 7.2 Hz), 1.17 (3H, d, J = 7.2 Hz), 0.98 (6H, d, J = 6.5 Hz), 0.96 (3H, d, J = 6.5 Hz), and 0.91 (3H, d, J = 6.5 Hz)] and four olefinic protons [δH 6.32 (1H, d, J = 9.6 Hz, H13′), 6.03 (1H, d, J = 10.9 Hz, H-13), 5.35 (1H, brs, H-7), and 5.23 (1H, brs, H-7′)]. The 13C NMR with DEPT spectra displayed 57 carbons including 5 carbonyls, 2 amide carbonyls, 10 olefinic carbons (6 quaternary carbons and 4 methines),
and 40 sp3 carbons (5 quaternary carbons, 18 methines, 6 methylenes, and 11 methyls) (Table S1, Supporting Information). The 1H and 13C NMR data suggested that 1 is a cytochalasan heterotrimer containing two cytochalasans and B
DOI: 10.1021/acs.orglett.8b04066 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Table 1. IC50 (μM, 48 h) Values of 1−3 against Six Cancer Cell Lines and a Normal Cell Line 1 2 3
HL60
U87MG
MDA-MB-231
A549
Hep3B
SW480
NCM460
BSIa
3.74 1.71 18.02
8.71 5.64 35.84
8.42 5.44 16.92
6.40 3.36 25.65
7.86 3.11 26.21
9.91 3.47 23.15
10.78 6.19 27.24
2.88 3.62 1.61
a
BSI for the Biggest Selective Index, BSI = IC50 value of NCM460/the smallest IC50 value of the cancer cell lines.
to be β-oriented.10 Thus, only two chiral centers, C-2″ and C4″, remained to be determined. Considering the rigid ring system and steric constraints, the two cytochalasan monomers should be on the opposite sides of the epicoccine moiety forming a sandwich-type structure; therefore, the configurations at C-2″ and C-4″ are self-explanatory. However, further evidence was necessary to verify the structure of 1. Fortunately, after numerous attempts in different solvents at different temperatures, a small crystal suitable for single-crystal X-ray diffraction was finally obtained from a mixed solvent system of EtOH−CH2Cl2−H2O (approximately 3:1:0.1) (Figure 3), and this analysis confirmed the planar structure of 1 as well as its absolute configuration (3S,4R,5S,8S,9S,17R,18S,19S,20S, 3′S,4′R,5′S,8′S,9′S,19′S,20′S,1″R,2″R,3″S,4″R,8″S). The 1H and 13C NMR data of 2 closely resembled those of 1 (Table S1, Supporting Information), and the only difference was that the carbonyl at C-17′ (δC 200.9) in 1 was reduced to a methylene (δC 39.7) in 2, which was confirmed by the 1H−1H COSY correlation of H-16′/H-17′. The absolute configuration of 2 was determined to be identical with that of 1 based on their similar ECD spectra (Figure 4). Compound 3 had the same molecular formula as 1, and its NMR data were similar to those of 1 (Table S2, Supporting Information). Detailed analyses of its 2D NMR data revealed that the C-18 oxygenated methine (δC 83.7) in 1 was oxidized to a hemiketal (δC 105.0) in 3, and the C-18′ carbonyl (δC 196.5) in 1 was reduced to an oxygenated methane (δC 87.9) in 3. Moreover, to satisfy the molecular formula of 3, C-18′ was suggested to be connected to C-3″ via an oxygen atom forming an additional furan ring. This structural assignment was further supported by the obvious downfield chemical shift of C-3″ (δC 111.7). Thus, the gross structure of 3 was established. The relative configurations at the chiral centers of 3 are the same as those of 1, as evidenced by their similar ROESY correlations. Additionally, the ROESY correlation between H17 and H-20 of 3 suggested that 18-OH was β-oriented, the obvious ROESY correlation of H-18′/H-19′ together with the absence of an H-18′/H-20′ ROESY correlation indicated that H-18′ and H-19′ were cofacial. Thus, the relative configuration of 3 was determined. Since the experimental ECD spectrum of 3 was similar to that of 1 (Figure 4), the absolute configuration of 3 was determined. In our previous study, the biogenetic pathways of epicochalasines A and B were proposed involving Diels− Alder and [3 + 2] cycloaddition reactions.9 Subsequently, three biosynthetic intermediate derivatives (aspergilasines B−D) were isolated, which supported our biosynthetic hypothesis.10 Therefore, we proposed that compounds 1−4 were also formed through Diels−Alder and [3 + 2] cycloaddition reactions as shown in Scheme 1. The coisolated aspochalasin D (5) was supposed as the key block; it suffers oxidization/ reduction reactions to form the cytochalasan monomers of 1− 3. Compound 5 undergoes a Diels−Alder reaction with the oxidized epicoccine (i) to yield the intermediate a, similarly,
Figure 6. Apoptosis induction effects of compounds 1 and 2 against cancer cell lines. Cells were exposed to vehicle control (DMSO,
