Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Fusicoccane-Derived Diterpenoids from Alternaria brassicicola: Investigation of the Structure−Stability Relationship and Discovery of an IKKβ Inhibitor Zhengxi Hu,†,§ Weiguang Sun,†,§ Fengli Li,†,§ Jiankun Guan,‡,§ Yuanyuan Lu,‡ Junjun Liu,† Ying Tang,‡ Guang Du,‡ Yongbo Xue,† Zengwei Luo,† Jianping Wang,*,† Hucheng Zhu,*,† and Yonghui Zhang*,† †
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, and Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China
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‡
S Supporting Information *
ABSTRACT: The structure−stability relationships of 1−6 were investigated to show that 1 converted to 5 via a kinetic, solution-mediated autoxidation. In addition, alterbrassicene A (7), a fusicoccane-derived diterpenoid bearing an unprecedented 5/9/4-fused carbocyclic skeleton with a rare fused 2cyclobuten-1-one motif, was characterized from Alternaria brassicicola. Its absolute structure was elucidated by spectroscopic analyses and quantum chemical calculations. Compound 7 was the first fusicoccane derivative acting as a potent IKKβ inhibitor in the NF-κB signaling pathway.
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ubiquitous eukaryotic adapter proteins that participate in the modulation of the cell cycle, signaling transduction, protein transportation and apoptosis, and their ability to potently activate the plasma membrane H+-ATPase, giving them remarkable phytohormone-like properties. Studies on the mechanism of action, chemical synthesis, and biosynthesis of these intriguing molecules have been continuously published in recent years.6 Notably, brassicicene C, which is a fusicoccane originally misassigned to have a unique C12 methyl functionality, drew much attention from the biosynthetic community to identify the gene clusters involved.6e−g However, the long road toward the identification of fusicoccanes with classical dicyclopenta[a,d]cyclooctane skeletons seemed to end in 2016. Inspired by the computational predictions and biosynthetic logic-based strategies, we reassigned a class of architecturally complex structures (brassicicenes C−H, J, and K) to unprecedented bridgehead double-bond-containing tricyclo[9.2.1.03,7]tetradecane skeletons.7 These new findings pushed the studies on fusicoccanes to new heights. The fateful misdiagnosis of these fusicoccanes were attributed to their lack of two-dimensional nuclear magnetic resonance (2D NMR) data, thus exploring chemical evidence of the original structures seemed significant, challenging, and urgent. To address this problem, we attempted to recreate the original liquid cultivation conditions. As a result, the originally misassigned brassicicenes C, F, G, H, J, and K (1−6; see Figure
he evolutionarily conserved nuclear factor-κB (NF-κB) signaling pathways play key roles in inflammatory and immune responses by regulating the transcription of numerous target genes.1 In the canonical pathway, NF-κB dimers are sequestered in the cytoplasm through binding to inhibitor of NF-κB (IκB) proteins, which mask their nuclear localization signals. When cells are stimulated by various NF-κB inducers, including cytokines such as tumor necrosis factor alpha (TNFα) or interleukin 1β (IL-1β), growth factors and microbial infections, IκB proteins are rapidly phosphorylated by an active IκB kinase (IKK) complex and subsequently undergo proteasomal degradation, which liberates free NF-κB dimers that can enter the nucleus to promote gene transcription.2 Aberrant activation of the NF-κB signaling pathway is known to be involved in many human diseases, including autoimmune and chronic inflammatory diseases, which render IKKs potentially important therapeutic targets, because of their central roles in the canonical NF-κB pathway.3 Recently, there has been a concerted effort to identify small-molecule inhibitors of IKKα/β, and some of them have shown promising inhibitory effects in various experimental animal models.4 Nevertheless, because of the current lack of clinical data, there is still an urgent need for the development of novel IKKα/β inhibitors with high efficacy and low toxicity. Fusicoccanes, which are a promising but underexploited family of terpenoids sharing a unique dicyclopenta[a,d]cyclooctane skeleton, have attracted, and continue to attract, much attention from the scientific community, because of their surprising biological functions; the functions of interest are their ability to target 14−3−3 proteins,5 which are a class of © XXXX American Chemical Society
Received: July 9, 2018
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DOI: 10.1021/acs.orglett.8b02137 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
the “observable” or “unstable” ranges are indicative of possible errors in the structural assignment.8 In our study, the OS energies of these fusicoccanes, as well as their parent alkenes, are all predicted to be in the “isolable” range (see Table S4 in the Supporting Information), suggesting that these bridgehead double-bond-containing bicyclo[6.2.1]undecane carbon skeletons are intrinsically stable, and this is unlike what is seen in the stabilities of fusicoccanes 1−6 and is somewhat influenced by the functional groups present; however, these functional groups cannot increase the OS energies of the NPs from the “isolable” to “observable” or “unstable” ranges.8b Despite 1 being predicted to be in the “isolable” range, it still decomposes to 5, indicating that this reaction is probably facilitated by a solution-mediated kinetic effect. Moreover, a novel fusicoccane-derived diterpenoid, alterbrassicene A (7), and the biogenetically related intermediates 8−10 were also discovered (see Figure 3). Compound 7 was
1) were reisolated and recharacterized. Importantly, the detailed 2D NMR analysis of the compounds, as well as the
Figure 1. (a) Revised structures of brassicicenes C (1), F (2), G (3), H (4), J (5), and K (6) from the reisolated compounds. (b) X-ray structure of 5, showing the absolute configuration.
X-ray crystallographic structures of 3 and 5, explicitly support our previously proposed structural revisions. The force-field-based method, which utilized olefin strain (OS) energy to classify the parent bridgehead olefins as “isolable” (OS ≤ 17 kcal/mol), “observable” (17 ≤ OS ≤ 21 kcal/mol), and “unstable” (OS ≥ 21 kcal/mol), has been recognized as a diagnostic tool for estimating the stabilities of complex natural products (NPs) with bridgehead olefins.8 By using this reliable method, fusicoccanes 1−6 were initially predicted to be “isolable”7 and were thus expected to be stable at room temperature.8a However, despite repeated highperformance liquid chromatography (HPLC) purifications using methanol−H2O, pure 1 could not be obtained. After standing for several weeks under atmospheric conditions, methanol solutions of 1 afforded crystals suitable for X-ray crystallography, and they were explicitly identified as hydroperoxide 5. Unexpectedly, when the HPLC eluent was acetonitrile−H2O, pure 1 was obtained, and it provided clean NMR data. These data indicated that different solvents might influence the stability of 1. Then, solutions of pure 1 were prepared in different solvents (1 mg/mL for each), allowed to stand for 3 days under atmospheric conditions, and then subjected to HPLC analysis. The chromatograms showed that 1 was slowly oxidized to 5 in all solutions, but the transformation was much slower in acetonitrile−H2O, which explained why a rapid HPLC isolation using this solution as the mobile phase could provide pure 1. Surprisingly, when neat 1 was stored in a closed bottle, after four months, HPLC analysis showed that 1 was still pure. These results (see Figure S3 in the Supporting Information) reveal that 1 slowly converted to 5 via a kinetic, solution-mediated autoxidation; the mechanism (Figure 2) of this oxidation likely involves a
Figure 3. Structures of compounds 7−10 and originally misassigned structure of brassicicene C.
uniquely defined by an unprecedented 5/9/4-fused carbon skeleton bearing an extremely rare fused 2-cyclobuten-1-one motif; and 8 was the first example of a bridgehead C10−C11 double-bond-containing NP with a bicyclo[6.2.1]undecane skeleton. Using a force-field-based method, the new bridgehead double-bond-containing NPs 8 and 9 were predicted to be “isolable” (see Table S4). Alterbrassicene A (7) was found to have a molecular formula of C21H30O5, based on the (+)-HRESIMS ion at m/z 385.1998 [M + Na]+, corresponding to seven degrees of unsaturation. Apart from three degrees of unsaturation occupied by the four olefinic carbons (δC 128.8, 150.6, 172.8, and 149.0) and one carbonyl carbon (δC 194.2), a tetracyclic diterpenoid should exist in 7, since its planar structure was determined via thorough evaluation of its 2D NMR spectra (see Figure 4). Analysis of the NOESY spectrum (Figure 4), including the crosspeaks of H-4β/H2-16/Me-17/H-8, H-4α/H-6/Me-19, H-
Figure 2. Proposed mechanism of the autoxidation from 1 to 5.
series of processes as follows:9 the loss of a hydrogen atom (H11) of 1 could provide a key radical a, which then underwent radical addition to O2, thus forming a peroxide radical b. Afterward, the peroxide radical b abstracted a hydrogen atom to generate 5. To the best of our knowledge, all known bridgehead doublebond-containing NPs are “isolable”, while OS energies within
Figure 4. Selected 1H−1H COSY (blue bold lines), HMBC (red solid arrows), and NOESY (red dashed arrows) correlations of 7. B
DOI: 10.1021/acs.orglett.8b02137 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
rearrangement10 of ii could generate carbocation iii with its unprecedented 5/9/4-fused core skeleton, which continued through oxidization and hemiketal reactions to form 7. In our anti-inflammatory screenings of compounds from the NPs library,11 the effects of all the isolates on the production of NO, as well as the pro-inflammatory cytokines TNF-α and IL1β in a mouse macrophage cell line RAW264.7 stimulated by lipopolysaccharide (LPS), were evaluated. Among the isolates evaluated (see Figure S13 in the Supporting Information), 7 showed a significant inhibitory effect at 10 μM and markedly decrease these indices in a dose-dependence manner; meanwhile, inhibited gene expressions of TNF-α, IL-1β, IL-6, and CD86, as well as elevated gene expression of CD206, were also observed after treatment with 7 (see Figure S14 in the Supporting Information). These results suggest that 7 has potential as an in vitro anti-inflammatory agent, which inspired us to investigate the underlying mechanism. Several inflammatory therapeutic targets, including IKKα, IKKβ, inducible nitric oxide synthase (iNOS), sirtuin 2 (SIRT2), vascular cell adhesion molecule (VCAM), intercellular cell adhesion molecule (ICAM), and Janus kinase 1 (JAK1), etc., were explored in a virtual screening. The total score (see Table S6 in the Supporting Information) predicted that 7 was most likely to exhibit an IKKβ inhibitory effect. The computational docking technique was then applied to elucidate the proposed mechanism. As shown in Figure 6A, 7 binds to
7/H-9α, and H-9β/Me-18, indicated that HO-3 and H-6 were on the same face and α-orientated, while Me-17, Me-18, and the oxygen bridge C8−O−C12 were β-oriented. Thus, the relative configuration of 7 was elucidated. The 13C NMR chemical shifts of 7 were calculated at the B3LYP/6-311++G(d,p) level with the PCM in MeOH, and the results agreed well with the experimental data (R2 = 0.9986; see Figure 5), supporting our proposed relative
Figure 5. (a) Linear correlation between the experimental and calculated 13C NMR chemical shifts for 7. (b) Experimental ECD curve of 7, together with the calculated ECD spectra of 7 and ent-7 after optimization at the B3LYP/6-311++G** level of theory.
configuration. Furthermore, the electronic circular dichroism (ECD) spectra (Figure 5) of 7 and ent-7 were computed via a time-dependent density functional theory method. A good qualitative agreement between the experimental and predicted ECD spectra of 7 confirmed its absolute configuration to be 3R, 6S, 7R, 8S, 11R, and 12S. Architecturally, metabolite 7, with an unprecedented 5/9/4fused carbon skeleton, did not adhere to the biogenetic isoprene rule, because of sequential unexpected rearrangements of the classic dicyclopenta[a,d]cyclooctane skeleton, which greatly diversified the structure of the fusicoccanes. A plausible biosynthetic pathway (see Scheme 1) could be traced Scheme 1. Plausible Biosynthetic Pathway for 7
Figure 6. Compound 7 is a novel IKKβ inhibitor: (A) representative view of a simulated binding complex (the left side shows the global view of the IKKβ structure, and the right side shows an expanded view of 7 in the binding site); (B) IKKβ inhibitory effects of 7 and BMS345541 at a series of concentrations.
the IKKβ protein presumably at the hinge loop connecting the N and C lobes, a selective inhibitor binding region that recognizes the adenine in ATP. In addition, 7 might form favorable hydrogen bonding interactions with residues including Arg31 and Leu21. To prove this hypothesis, an in vitro enzyme-base experiment was performed (Figure 6B); indeed, 7 exhibited a potent inhibitory effect on IKKβ (IC50 = 2.48 μM), which was comparable to that of the well-known IKKβ inhibitor BMS-345541 (IC50 = 0.97 μM).12 To further validate the effect of 7 binding to the IKKβ protein, the microscale thermophoresis (MST) was used.13 The Kd values of 7 (15.36 μM) and BMS-345541 (11.43 μM) (see Figure S15 in the Supporting Information) were consistent with enzyme inhibitory effects and confirmed the specific binding of 7 to IKKβ. We then evaluated the mechanism of 7 on LPS-induced NFκB activation. Phosphorylation of IκB induced by activated IKK is a key step in the activation of the canonical NF-κB pathway. As expected (Figures 7A and 7B), phosphorylated IκBα was detected in RAW264.7 cells after exposure to LPS or
back to brassicicene B (10), which originated from geranylgeranyl pyrophosphate (GGPP) via a series of enzyme-catalyzed reactions.6d Protonation and dehydration of 10 at C12 could furnish carbocation i, which then underwent C1−C12 ring closure and C1−C11 ring opening to afford vital carbocation intermediate ii, thus leading to the formation of the unusual tricyclo[9.2.1.03,7]tetradecane derivatives 1−6, 8, and 9. Alternatively, a Wagner−Meerwein C
DOI: 10.1021/acs.orglett.8b02137 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
of this class of fusicoccanes featuring unusual bridgehead double-bond-containing tricyclo[9.2.1.03,7]tetradecane skeletons was explored, and 1 was found to slowly convert to 5 via a kinetically driven autoxidation in solution. Notably, the discovery of 7 greatly diversifies the diterpenoid skeleton of architecturally complex fusicoccanes from classic dicyclopenta[a,d]cyclooctane to bridgehead double-bond-containing tricyclo[9.2.1.03,7]tetradecane, and now to a 5/9/4-fused skeleton decorated with a rare fused 2-cyclobuten-1-one motif. In addition, with the aid of in silico target screening and cell and animal studies, 7 was found to be the first fusicoccane derivative acting as a potent IKKβ inhibitor of the NF-κB signaling pathway, highlighting its potential as a lead compound for the development of new anti-inflammatoryrelated therapeutic agents.
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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.orglett.8b02137. Experimental procedures, 1D and 2D NMR, HRESIMS, UV, and IR spectra, and detailed quantum chemical calculations (PDF)
Figure 7. IKKβ-NF-κB inflammation signal was inhibited by compound 7. RAW264.7 cells were preincubated with the indicated concentrations of 7 for 2 h before stimulation with (A) 1 μg/mL LPS or (B) 20 ng/mL TNF-α. The cells were harvested after 2 h, and the total cell lysates were tested via Western blot experiments for the occurrence of IκBα phosphorylation. DMSO and BMS-345541 were used as the negative and positive controls, respectively. (C) NF-κB activation detected by nuclear translocation. RAW264.7 cells were treated with LPS (1 μg/mL) in the absence or presence of 7 (5 and 10 μM) for 2 h. Then, NF-κB p65 nuclear translocation was investigated by staining with an anti-p65 subunit antibody (red) and DAPI (blue). (D) Hemotoxylin and eosin (H&E) staining of mouse liver and lung sections, showing the spleen immunohistochemical staining results of IκBα and p-IκBα. Less sinusoidal cell loss, tissue destruction, and erythrocyte influx were observed after treatment with 7 or BMS-345541.
Accession Codes
CCDC 1571928, 1571929, 1813586, and 1813587 contain 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]. uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (J. Wang). *E-mail:
[email protected] (H. Zhu). *E-mail:
[email protected] (Y. Zhang).
TNF-α. Strikingly, compared with pretreatment with BMS345541, pretreatment with 5 or 10 μM of 7 could also effectively block the phosphorylation of IκBα, resulting in a similar inhibitory effect against several stimulants that induced NF-κB activation. Moreover, addition of 7 abolished LPSinduced nuclear translocation of p65 (Figure 7C). Taken together, these results confirm that 7 acted as a potent IKKβ inhibitor of the NF-κB pathway induced by diverse stimuli in vitro. Encouraged by its promising IKKβ inhibitory potency, we attempted to evaluate the effect of 7 on LPS-triggered inflammatory responses in vivo. As shown in Figure 7D, tissue destruction and wide hemorrhagic necrosis from liver and lung tissues were highly induced in mice challenged with LPS, compared with the control mice, and the pathological change was greatly attenuated in mice pretreated with different concentrations of 7. Meanwhile, 7 also inhibited IκBα phosphorylation in spleen tissue induced by LPS injection (Figure 7D). Together, the in vitro and in vivo results reveal that 7 could tightly bind IKKβ, leading to the inactivation of the NF-κB signaling pathways and providing remarkable antiinflammatory activity. In summary, the current work provides chemical evidence for our previously proposed structural revisions. Based on this foundation, the relationship between structures and stabilities
ORCID
Zhengxi Hu: 0000-0002-1247-5615 Junjun Liu: 0000-0001-9953-8633 Yongbo Xue: 0000-0001-9133-6439 Yonghui Zhang: 0000-0002-7222-2142 Author Contributions §
Z.H., W.S., F.L., and J.G. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This project was financially supported by the Program for Changjiang Scholars of Ministry of Education of the People’s Republic of China (No. T2016088), the National Science Fund for Distinguished Young Scholars (No. 81725021), the Innovative Research Groups of the National Natural Science Foundation of China (No. 81721005), the National Natural Science Foundation of China (Nos. 31370372, 21702067 and 81602986), and the China Postdoctoral Science Foundation Funded Project (Nos. 2017M610479 and 2018T110777). D
DOI: 10.1021/acs.orglett.8b02137 Org. Lett. XXXX, XXX, XXX−XXX
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REFERENCES
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DOI: 10.1021/acs.orglett.8b02137 Org. Lett. XXXX, XXX, XXX−XXX
