Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
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Aureochaeglobosins A−C, Three [4 + 2] Adducts of Chaetoglobosin and Aureonitol Derivatives from Chaetomium globosum Ming-Hua Yang,† Mei-Ling Gu,† Chao Han, Xiao-Jiang Guo, Guo-Ping Yin, Pei Yu, and Ling-Yi Kong* Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China S Supporting Information *
ABSTRACT: Aureochaeglobosins A−C (1−3), three novel [4 + 2] cycloaddition heterodimers of chaetoglobosin and aureonitol derivatives, were obtained from the culture of endophytic fungus Chaetomium globosum, representing the first adduct examples of chaetoglobosins. Their structures were elucidated by extensive spectroscopic analyses, single-crystal X-ray diffraction, and a modified Mosher’s method. Compounds 2 and 3 showed significant cytotoxicities against human MDA-MB-231 cancer cells with IC50 values of 7.6 and 10.8 μM, respectively.
C
to screen out a C. globosum strain in our searching for cytotoxic fungal metabolites. However, a following HPLC−MS analysis revealed metabolites possessing a chaetoglobosins-like UV absorption and more importantly, a chaetoglobosins-unlike mass-to-charge ratio larger than 700 (Figure S1). A targeted isolation was subsequently fulfilled to afford three novel cytochalasans, aureochaeglobosins A−C (1−3), in which an aureonitol derivative fused to the chaetoglobosin moiety via [4 + 2] cycloaddition (Figure 1). To the best of our knowledge, aureochaeglobosins A−C are the first adduct examples occurring in chaetoglobosins.
ytochalasans are a large group of fungal metabolites characterized by a perhydro-isoindolone moiety to which a typical 9−15-membered ring is fused.1 They are hybrid products produced through the combined action of polyketide synthases and nonribosomal peptide synthetases.2 On the basis of the amino acids involved in the biosynthesis of isoindolone structure, cytochalasans could be divided into five subgroups, which are cytochalasins, pyrichalasins, chaetoglobosins, aspochalasins and alachalasins.1 Although cytochalasans are best known to inhibit cytokinesis by capping actin filaments, they are found with a wide range of other biological functions, including cytotoxic,3 antiviral,4 antimicrobial,5 immunosuppressive,6 and so on. Cytochalasans also play an important role in fungal virulence and are related with fungus−plant symbiosis.7 Therefore, considerable attention from chemists, botanists, and pharmacologists has been given to cytochalasins.1,8 To date, more than 300 cytochalasans have been reported from various fungi origins such as Chaetomium, Aspergillus, and Periconia species.8 Most isolates feature in the above five types of carbon skeletons with differences at functional groups. The macrocyclic ring structure sometimes could undergo different intramolecular reactions to produce varied skeletons such as armochaeglobine A from Chaetomium globosum,9a phomopchalasin A from Phomopsis sp.,9b and cytoglobosin A from Chaetomium globosum.9c Epicochalasines10a and asperchalasines10b with intricate polycyclic structures are hybrid products of aspochalasins and epicoccines, and are first samples of cytochalasan adduct which enriched the structure variety of cytochalasans. However, since those complex products were all metabolized by the same strain source, Aspergillus f lavipes,10 and no other adducts were reported afterward, we cannot tell whether or not it is a unique case. Chaetomium globosum, a well-known member of the Chaetomium genus, is a major source of chaetoglobosins. Different chaetoglobosins are being revealed from C. globosum ever since the first isolation in 1973.11 Chaetoglobosins are widely reported for their cytotoxicity; thus, it was not a surprise © XXXX American Chemical Society
Figure 1. Structures of compounds 1−3. Received: April 19, 2018
A
DOI: 10.1021/acs.orglett.8b01243 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
from H-8 to C-9 and C-1. The chemical shifts of C-6 and C-7 were of typical epoxy structure,12 and accordingly revealed the 6,7-epoxide structure. Moreover, the 1H−1H COSY cross-peaks of H-8/H-13/H-14/H2-15/H-16/H-17/H3-24, and the HMBC correlations from H-8 to C-13, C-14 and C-23, from H3-24 to C-15, C-16 and C-17, and from H-19 to C-17, C-18 and C-20 established the similar 13-membered macrocyclic ring to chaetoglobosin A.11a Rather than the Δ21(22) double bond in chaetoglobosin A, the C-21 and C-22 in 1 were determined as two methines, which had mutual 1H−1H COSY correlations and HMBC correlations with C-23 and C-20. Aside from those of chaetoglobosin moiety, the remained 13 carbon signals were attributed to a moiety of aureonitol derivative on the basis of 1 H−1H COSY correlations shown in Figure 2 and the key
Herein, we present the isolation, structure elucidation, and bioactivity evaluation, as well as plausible biogenetic pathway of 1−3. Aureochaeglobosin A (1) was isolated as colorless crystals. The HRESIMS ion peak at m/z 757.3825 ([M + Na]+) revealed its molecular formula C45H54N2O7 with 20 degrees of unsaturation. The 1H NMR data (Table 1) showed three Table 1. 1H (600 MHz) and 13C NMR (150 MHz) NMR Data of 1 in CDCl3 δH
no. 1 3 4 5 6 7 8 9 10a 10b 11 12 13 14 15a 15b 16 17
3.70 m 2.39 m 1.94 m 2.76 d (4.3) 2.50 dd (5.7, 4.3) 2.70 m 2.80 d (12.5) 1.20 d (6.9) 1.12 s 6.18 dd (15.5, 5.7) 5.55 m 1.90 m 2.31 brd (13.0) 2.60 m 5.46 d (10.0)
18 19 20 21 22 23 24 25
4.87 d (6.0) 3.98 brs 3.25 brd (7.2) 1.08 d (6.1) 1.42 s
δC 174.5 52.7 48.5 37.1 56.7 62.8 44.1 64.9 34.3
33.0 139.2 131.3
10′′
81.5 212.4 39.8 38.8 211.0 21.3 11.1
11′′ 12′′ 13′′a 13′′b 2-NH 1′-NH 19-OH
13.2 19.5 128.4 134.1 41.6
δH
no. 2′ 3′ 4′ 5′ 6′ 7′ 1′a 3′a 1′′a 1′′b 2′′ 3′′ 4′′ 5′′ 6′′ 7′′ 8′′ 9′′
7.24 s 7.52 7.14 7.21 7.38
d (7.8) dd (7.8, 7.3) dd (8.0,7.3) d (8.0)
2.07 brd (16.5) 2.64 m 5.87 overlap 5.87 overlap 2.36 m 4.09 dd (8.9, 6.1) 3.86 t (6.1) 2.67 m 5.36 dd (14.4, 8.4) 6.02 dd (14.4, 10.7) 5.97 dd (13.4, 10.7) 5.62 dt (13.4, 6.5) 1.72 d (6.5) 3.67 m 4.04 t (8.1) 7.20 brs 8.83 brs 3.61 d (6.0)
δC 124.7 110.7 118.2 119.7 122.2 111.6 136.6 127.3 26.1 126.2 123.3 41.1 84.7 81.8 53.2 127.9 133.3 131.0
Figure 2. Key 1H−1H COSY and HMBC correlations of 1. 129.6 18.2 70.3
HMBC correlations from H2-13′′ to C-5′′, C-7′′ and C-8′′.13 More importantly, the 1H−1H COSY cross-peaks of H2-1′′/H22/H-21/H-4′′, along with the HMBC correlations from H21′′ and H-4′′ to C-21 and C-22, unambiguously established the connections of two moieties across C-21/C-4′′ and C-22/C1′′. Thus, the planar structure of 1 was established. The relative stereochemistry was mainly determined by ROESY correlations (Figure 3). ROESY cross-peaks from H-5 to H-4/H-8, from H3-12 to H-7/H3-11/H-3, and from H3-11 to H-3 indicated that H-4, H-5 and H-8 are on the same side of cyclohexane ring, and that H-3, H-7, H3-11, and H3-12 are approached in space. The E-geometry of the 13,17-diene was
secondary methyls at δH 1.08 (d, J = 6.1 Hz), 1.20 (d, J = 6.9 Hz), and 1.72 (d, J = 6.5 Hz); two tertiary methyls at δH 1.12 (s) and 1.42 (s); nine olefinic hydrogen protons at δH 5.36− 6.18; one single-substituted indolyl with four coupled aromatic protons at δH 7.14 (dd, J = 7.8, 7.3 Hz), δH 7.21 (dd, J = 8.0, 7.3 Hz), δH 7.38 (d, J = 8.0 Hz), δH 7.52 (d, J = 7.8 Hz), a singlet proton at δH 7.24 and two broad NH singlets at δH 7.20 and 8.83. The 13C NMR together with the HSQC spectra indicated 45 carbons ascribed to two carbonyls, an amide carbonyl, nine double bonds, six oxygenated carbons (including a quaternary, four methines, and a methylene), 13 aliphatic carbons, and five methyls. Comparing the NMR data with that of reported from C. globosum,11 1 was inferred as a chaetoglobosin adduct. The chaetoglobosin moiety was further established by analyzing 2D NMR data. Its characteristic 10-(indol-3-yl) group was determined by the 1H−1H COSY cross-peaks of H4′/H-5′/H-6′/H-7′ and the HMBC correlations from H-4′ to C-5′ and C-3′, from H-2′ to C-3′, and from H2-10 to C-3′ and C-3. Analysis of 1H−1H COSY spectrum then determined the spin system of C-11/C-5/C-4/C-3/C-10, which further constructed the perhydro-isoindolone core according to the HMBC correlations mainly from H-3 to C-1 and C-9, from H311 to C-4, C-5 and C-6, from H3-12 to C-5, C-6 and C-7, and
Figure 3. Key ROESY correlations of 1. B
DOI: 10.1021/acs.orglett.8b01243 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters deduced from the coupling constant (J13,14 = 15.5 Hz) and the ROESY correlations of H-8/H-14 and H3-25/H-16. Simultaneously, the ROESY correlations of H-8/H-1′′a and H-14/H16 oriented these protons on the same side, while the ROESY correlations of H-13/H-15a, H-15a/H-17, H-17/H-19, and H19/H-21 indicated their opposite side to H-8. The ROESY correlations were also found between H-4′′, H-22 and H-1′′a, yet none of them was observed NOE effect with H-21 which, instead, was correlated with H-5′′, H-19, and H-17. H-4′′ and H-22 were accordingly assigned on the opposite side to H-21 of newly formed cyclohexene ring. Additionally, the ROESY correlation of H-7′′/H-5′′ revealed their same orientations, while the ROESY correlations from H-6′′ to H-8′′, H-4′′, and H-3′′ settled the opposite orientation of H-6′′. An X-ray diffraction experiment (Figure 4) finally confirmed the planar
Figure 5. Δδ values between S-MTPA and R-MTPA esters of 2 and 3.
situation. The assignment of all chiral centers was consequently fulfilled and was the same as for 1. The molecular formula of aureochaeglobosin C (3) was determined as C45H54N2O7 by HRESIMS ion peak at m/z 757.3823 ([M + Na]+). Its NMR data (Table S1) were almost superimposable with those of 1 and 2, indicating that 3 was another adduct of chaetoglobosin and aureonitol derivatives. The Δ6(12) terminal double bond in 3 was revealed by HMBC correlations from two singlets of H2-12 at δH 5.05 and 5.24 to C-5 (δC 38.5), C-6 (δC 147.2), and C-7 (δC 70.6). The rest of the structure, including relative configuration was verified identical to those of 1 through carefully analyzing 1H−1H COSY, HMBC, and ROESY correlations (Figure S3). The modified Mosher’s method was also conducted to determine absolute configuration (Figure 5). Unlike the data of 2, the ΔδSR values of the proton signals adjacent to C-7 and C-6′′ significantly revealed the 7S and 6′′S configurations of 3. Thus, the absolute configuration of 3 was determined as 3S, 4R, 5S, 7S, 8R, 9R, 16S, 19R, 21S, 22S, 4′′R, 5′′R, 6′′S and 7′′R. The [4 + 2] cycloaddition reaction between two moieties is the key step in biosynthesis of 1−3. As proposed for [3 + 2] cycloaddition products of cytochalasan, asperchalasine A, and epicochalasines A and B, free-radical and Diels−Alder reactions are both possible hypotheses. Considering the reported isolation of aureonitol precursor with stable s-cis diene,13d we propose that Diels−Alder reactions are a more probable mechanism involved in the biosynthetic pathways of 1−3. We doubted whether the dimerization would occur spontaneously in fermentation. After all, the dienophile precursors, chaetoglobosins with Δ21(22) double bond, had been widely and early isolated from the Chaetomium genus. The electrophilic 20,23dicarbonyl structure could also make the dienophile more reactive under mild conditions. Trying to discover clues of the above problem, HPLC−MS guided isolations were used to obtain precursor analogues of 1−3 for testing the dimeric reaction in tube. The exact cytochalasan precursor of 1, chaetoglobosin A,11a was obtained, but there was no aureonitol analogues detected under MS and UV analysis. Several diene substitutes (furan, pyrrole and 2,3-dimethyl-1,3-butadiene) were therefore employed for attempting Diels−Alder reactions under different conditions. However, when different Lewis acids were used under multiple thermal conditions (see the Supporting Information for details), no adducts was found in TLC or HPLC detection. The negative results led us to consider the dimerization as an enzyme-catalyzed reaction, especially when noticing that 1−3 are the same stereospecific products, while self-construction is usually resulted in nonstereospecific outcomes.14 Nevertheless, direct evidence is needed before any firm conclusion can be drawn.
Figure 4. X-ray crystallographic structure of 1.
structure and relative configuration of 1 and also unambiguously determined the absolute configuration to be 3S, 4R, 5S, 6R, 7S, 8R, 9R, 16S, 19R, 21S, 22S, 4′′R, 5′′R, 6′′S and 7′′R (Flack parameter 0.04 (7)). Aureochaeglobosin B (2) had the same molecular formula of 1, and its 1D NMR spectra were also very similar to those of 1. Detailed analysis of NMR data (Table S1) revealed that 1 and 2 were only different at the perhydro-isoindolone core. The H311 (δH 1.73) and H3-12 (δH 1.53) in 2 were low-field shifted and appeared as singlets, which along with the existence of olefinic carbons at δC 125.6 and 134.2, suggested the Δ5(6) double bond in 2. The C-7 was substituted with a hydroxyl due to the shifts to low field of H-7 (δH 3.80) and C-7 (δC 67.7). Further support for the proposed transformation was provided by the HMBC correlations, mainly from H3-11 to C-4/C-6, H312 to C-5/C-7, and H-7 to C-5/C-8/C-9. Meanwhile, ROESY correlations between H-7 and H-13 assigned H-7 an opposite orientation to H-8. Other relative configurations in 2 were deduced identical to 1 by elucidating their similar ROESY correlations (Figure S2). The absolute stereochemistry was established by the modified Mosher’s method. The R and S trisMTPA esters of the alcohol at C-7, C-19, and C-6′′ were prepared, and subsequent 1H NMR analysis of the Δδ values for two esters were calculated, as shown in Figure 5. The 7S configuration was unambiguously determined by Mosher’s method. Possibly due to the interference from MTPA esters at C-19, both positive and negative ΔδSR values coexisted for one side of the C-6′′ stereocenter. Nevertheless, the 6′′S configuration indicated by the overall ΔδSR trend was in accordance with the deduction from NOE effects under the 7S C
DOI: 10.1021/acs.orglett.8b01243 Org. Lett. XXXX, XXX, XXX−XXX
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Compounds 1−3 were tested for their cytotoxic activities against three human cancer cell lines (MDA-MB-231, U20S and HepG2). The results showed that 2 and 3 exhibited significant cytotoxic activities against MDA-MB-231 breast cancer cells. The IC50 values were 7.6 ± 0.5 and 10.8 ± 0.64 μM for 2 and 3 respectively, while the positive control (doxorubicin) had an IC50 value of 1.0 ± 0.10 μM.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b01243. Detailed experimental procedures, full spectroscopic data (NMR, MS, UV, and IR) of compounds 1, 2 and 3 (PDF) Accession Codes
CCDC 1574956 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 data_
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Ling-Yi Kong: 0000-0001-9712-2618 Author Contributions †
M.H.Y. and M.L.G. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research work was funded by the National Natural Science Foundation of China (81602985), the Program for Changjiang Scholars and the Innovative Research Team in University (IRT_15R63), the 111 Program of Introducing Talents of Discipline to Universities (B18056), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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DOI: 10.1021/acs.orglett.8b01243 Org. Lett. XXXX, XXX, XXX−XXX
