Natural and Semisynthetic Tigliane Diterpenoids with New Carbon

Letter pubs.acs.org/OrgLett

Natural and Semisynthetic Tigliane Diterpenoids with New Carbon Skeletons from Euphorbia dracunculoides as a Wnt Signaling Pathway Inhibitor Li Wang,†,§,⊥ Jing Yang,†,⊥ Ling-Mei Kong,† Jun Deng,† Zijun Xiong,†,§ Jianping Huang,† Jianying Luo,† Yijun Yan,† Yikao Hu,‡ Xiao-Nian Li,† Yan Li,† Yong Zhao,*,‡ and Sheng-Xiong Huang*,† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, and Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China ‡ College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650500, China § University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: Euphordraculoates A (1) and B (2), featuring tigliane diterpenoids with two new carbon skeletons, were characterized as metabolites of Euphorbia dracunculoides and semisynthetic products, respectively. Their structures were determined by spectroscopic analyses and X-ray crystallography. The respective biosynthetic and chemical formation mechanisms for 1 and 2 from a known tigliane 3 was proposed. The detailed decarbonization mechanism from 3 to 2 was further explored by 18O-labeling experiment. Compound 2 could inhibit Wnt pathway in a dose- and time-dependent manner.

T

igliane diterpenoids, which were reported mainly from Euphorbiaceae and Thymelaeaceae families,1 have a unique architecture of a 5/7/6/3-tetracyclic ring system.1,2 Tiglianes were reported to show a wide variety of biological activities, including proinflammatory activity, cocarcinogenic and tumor-promotive activities, cell differentiation, anticancer and antiviral activities, and etc.1−4 Some tiglianes, which could serve as protein kinase C (PKC) modulators, attracted the most attention because the activating capacity of PKCs was responsible for diverse biological activities,5 especially for anticancer and anti-HIV.6,7 Currently, the most progressed compound in this class of natural products is phorbol 12myristate 13-acetate, which is currently in phase II clinical trials for treatment of acute myeloid leukemia.1 For their fascinating structures and striking bioactivities, this class of molecules has attracted great interest as challenging targets to chemists for total synthesis and biosynthesis over the past 20 years.8 As part of natural products discovery efforts from Traditional Chinese Medicine plants and associated endophytes in China,9 euphordraculoate A (1) (Figure 1), an unprecedented diterpenoid lactone, which exhibits a novel 6/6/3-fused ring system with a 2-methyl-2-cyclopentenone moiety, was isolated from Euphorbia dracunculoides. Initially, we intended to realize the biomimetic conversion of 1 from a known tigliane precursor 3,9a,10 which was isolated as one of the main metabolites in E. dracunculoides. However, the desired transformation failed; an unexpected product 2 with an unprecedented 5/5/6/3-fused tetracyclic ring core (Figure 1) © 2017 American Chemical Society

Figure 1. Chemical structures of compounds 1−3.

was yielded from 3 with the aid of NaH in dry THF. In this paper, the isolation, structural elucidation, plausible biosynthetic pathway of 1, and formation mechanism of 2 from 3, as well as the Wnt signaling pathway inhibitory activity, are described. Euphordraculoate A (1) was obtained as colorless gum. The molecular formula of 1, C31H36O7, which indicates 14 degrees of unsaturation, was deduced from the HRESIMS data (m/z 543.2354 [M + Na]+, calcd 543.2353). The 13C NMR spectrum (Supporting Information, Table S1) of 1 revealed 31 carbon resonances, composed of seven methyls, one methylene, 13 methines, and ten quaternary carbons. By combining the 1H and 13C NMR data of 1 (Table S1), one benzoyloxy group Received: June 15, 2017 Published: July 13, 2017 3911

DOI: 10.1021/acs.orglett.7b01813 Org. Lett. 2017, 19, 3911−3914

Letter

Organic Letters (OBz) (δH 8.02 × 2, 7.57, 7.46 × 2; δC 130.4, 129.8 × 2, 128.6 × 2, 133.1, 166.0) and one isobutyryl (OiBu) (δH 1.07, 1.08, 2.45; δC 18.8, 18.9, 34.3, 175.8) were obviously presented in 1.10,11 As a result, the remaining 20 carbons should be from the parent nucleus with eight degrees of unsaturation. Moreover, among the carbon signals of the core skeleton of 1, four olefinic carbons (δC 160.3, CH; 141.2, C; 127.7, C; 141.6, CH) and two carbonyls (δC 206.9 and 163.5) were unambiguous. Therefore, 1 was speculated to be a diterpenoid with a tetracyclic core skeleton. A known tigliane (3),9a,10 which shared some similar 13C NMR data with 1, was also isolated from this plant. A careful comparison of the NMR data between 1 and 3 (Table S1) reached a conclusion that 1 also possesses the following subunits: a methylated α, β-unsaturated ketone moiety (C-1, C2, C-3, and C-16), a gem-dimethylcyclopropane unit (C-9, C10, C-11, C-18, C-19) and a six-membered ring substituted by a benzoyl and an isobutyryl. Meanwhile, a new upfield shifted carbonyl carbon (δC 163.5) and a methylene (δC 37.9) appeared in 1 instead of two methines in 3. Moreover, the 13 C NMR signals of C-6, C-7, C-8, C-15 in 3 presented severe variations in 1. Consequently, 1 was inferred to be different from 3 in the seven-membered ring. The HMBC correlations from H-1 to C-2, C-3, C-4, C-15, and C-16; from H-4a to C-1, C-2, and C-3; and from H-4b to C-3 and C-15, along with the spin system at H-1/H-15/H2-4 revealed by COSY correlations, demonstrated the presence of a 2-methyl-2-cyclopentenone moiety in 1 (Figure 2). Without

dimethylcyclopropane unit in 1 was supposed to be cis-fused like tiglianes,2 and H-9 and 11-OiBu were all α-oriented. The ROESY correlations of H-9/Me-19, H-8/Me-18, H-8/H-15, H8/H-13, H-12/Me-20 allowed the H-8, cyclopentenone moiety at C-14 and the benzoyloxy group at C-12 to be β-oriented, while Me-20 was to be α-oriented. The absolute configuration of 1 was determined by comparing experimental and calculated ECD spectra predicted by time-dependent density-functional theory. The result showed that the experimental and calculated ECD spectra of the compound 1 were in good agreement (Figure 3, left). Thus, the absolute configuration of compound 1 was assigned as 8S, 9R, 11R, 12R, 13R, 14S, and 15S.

Figure 3. Calculated and experimental ECD spectra of compounds 1 (left) and 2 (right).

The above analyses certify euphordraculoate A (1) as a diterpenoid with new carbon skeleton architecture. The cooccurrence of 1 with a known tigliane 3 within the same plant raises the possibility that the new carbon skeleton results from a further modification of 3. Herein, we propose a possible biosynthetic route for 1 (Scheme 1, dashed path) in order to Scheme 1. Plausible Biosynthetic Pathway of 1 (dashed) and the Proposed Formation Mechanism of 2 from 3 (Solid)

Figure 2. Key 2D correlations of 1 and the X-ray structure of 2.

doubt, the long-range HMBC couplings H-15/C-14 and H-8/ C-15 verified that this 2-methyl-2-cyclopentenone moiety should be connected to C-14 through a single bond formed by C-14 and C-15. In addition, on the basis of the observation of the HMBC correlations of H-7/C-8, H-7/C-17, H-8/C-6, and H-8/C-7, and the COSY cross peak of H-7/H-8, a propenyl was linked to the six-membered ring at C-8. Besides, only two correlations from H-7 and H3-17 to the upfield carbonyl at δC 163.5 were detected in the HMBC spectrum of 1. It was inferred that this carbonyl was linked to the above propenyl moiety at C-6 in a mode of δ-lactone for the reason that the chemical shift of this carbonyl δC 163.5 was consistent with the typical 13C NMR values of α,β-unsaturated δ-lactone.12 Accordingly, the last unidentified ring (α, β-unsaturated δlactone moiety) and the only unassigned oxygen atom between C-14 and C-5 in 1, as well as the two Dalton molecular weight variation between 1 (MW: 520) and 3 (MW: 522), were all accounted for. Thus, the planar structure of 1 was established. The relative configuration of 1 was elucidated by ROESY experiment (Figure 2). The six-membered ring and the gem-

trigger further studies to its biomimetically total chemical synthesis. First, a retro-aldol reaction would occur in 3 and opens the seven-membered ring at C-4 and C-5, leaving the carbon C-5 in a form of aldehyde subsequently. Then, the aldehyde intermediate is further oxygenated to be a carboxylic acid, which could undergo an intramolecular esterification with the hydroxyl at C-14 later on. In this way, the structure of 1 is produced. 3912

DOI: 10.1021/acs.orglett.7b01813 Org. Lett. 2017, 19, 3911−3914

Letter

Organic Letters

Scheme 2. Chemical Synthesis of 18O-Labeled 5 from Inhoffen Lythgoe Diol under 18O2 Atmosphere

As mentioned above, a biomimetic chemical transformation from 3 to 1 was planned via the key retro-aldol reaction. Retroaldol reactions were attempted with different bases including NaH, DBU, LDA, and LHMDS; however, none of them could generate the proposed intermediate 1a from 3. Fortunately, main product 2 was yielded from 3 under the condition of NaH in dry THF. Intriguingly, product 2 has a molecular formula of C30H36O7 determined by the HRESIMS data (m/z 531.2357 [M + Na]+, calcd 531.2353), which has one carbon atom less than that of the starting material 3. In order to solve the unforeseen structure of 2, detailed NMR experiments toward 2 were carried out (Figure S1). As in the case of compound 1, a methylated α, β-unsaturated ketone subunit, a six-membered ring, a gem-dimethylcyclopropane modification fused at C-9 and C-11, and a benzoyl and an isobutyryl esterifications at C12 and C-11, respectively, were all definitive in 2 from the NMR data (Table S1). The COSY spin system H-15/H-4/H6/H-8, along with the HMBC cross peaks H-15/C-14 and H8/C-14, evidenced a pentacyclic subunit in 2 instead of a sevenmembered ring in 3 (Figure S1). The HMBC correlations from OH-14 to C-14, C-15, and C-1 permitted a hydroxyl substituted at C-14. Similarly, HMBC correlations H-6/C-7 and H3-17/C-7 implied an acetyl linked to C-6. Consequently, product 2 was deduced to be rearranged tigiliane occupying unprecedented 5/5/6/3-fused carbon rings. To further confirm this unique structure, a single crystal X-ray analysis of 2 was carried out (Figure 2). The X-ray data13 corroborated the structure of 2 elucidated by NMR data. The absolute configuration of 2 was then determined in the same manner as that of 1 by comparing experimental and calculated ECD spectra (Figure 3, right). As shown in Scheme 1 (solid arrows), the formation mechanism of 2 from 3 was devised. Initially, opening of the seven-membered ring of 3 at C-4 and C-5 via retro-aldol reaction with the aid of NaH yields the proposed intermediate 1a containing an α,β-unsaturated aldehyde moiety. Second, an intramolecular Michael addition occurs to generate the 5/5/6/ 3-fused core skeleton intermediate 2a. Finally, a carbon in the intermediate 2a is taken off presumably through a 1,2cycloaddition between oxygen and the double bond of the enolate moiety in the intermediate 2a and subsequent C−C bond cleavage of 2b (Scheme 1, solid path). There are several papers reporting a similar decarbonization reaction to generate ketone using the aldehyde as material in the literature.14 As no literature proved the detailed mechanism of the above-mentioned oxidative addition and C−C bond cleavage steps, an 18O-labeling experiment toward this type of reaction was designed. Due to the amount limit of compound 3 and the difficulty to capture the enolate intermediate after Michael addition, we did not use 3 to conduct further experiments directly. Inspired by the literature reports,14 a model compound of the reaction intermediate, aldehyde 4, which can be obtained from inhoffen lythgoe diol with the aid of Dess-Martin Periodinane, was used instead. We treated 4 with NaH under an oxygen atmosphere to give product 5 as a pair of isomers (Scheme 2). Then the oxidative C−C bond cleavage of aldehyde from 4 was conducted using 18O2 as the oxidant. As a result, the 18O-labeled compound 5a was obtained (HREIMS: m/z 196.1350 M+, calcd for C12H1816O18O, 196.1349). This experiment not only supports the decarbonization mechanism reported decades ago14c but also suggests that the proposed intermediate 2a containing an aldehyde moiety is reasonable in Scheme 1.

Aberrant activation of the Wnt/β-catenin signaling pathway is involved in the development and progression of various cancers. Antagonists of Wnt/β-catenin signaling might be very potent if developed into effective antitumor agents.15 For compounds 1−3, the Wnt pathway inhibitory activities were conducted in HEK293 cells stably transfected with Wnt3a, Renilla, and SuperTopflash luciferase (ST-Luc),16 which was named HEK293W. Only 2 exhibited potent and selective inhibitory activity. As shown in Figure 4A, the incubation of

Figure 4. Compound 2 inhibits Wnt pathway. (A) HEK293W cells with DMSO, 1.5625, 3.125, 6.25, 12.5, 25 μM 2 for 24 h. (B) Western blot analysis of Wnt signaling downstream target proteins in HEK293W and HT29 cells. (C) Western blot analysis of β-catenin and phospho-β-catenin (Ser33, Ser37, and Thr41) in HEK293W cells treated with 2. (D) Western blot analysis of β-catenin and phospho-βcatenin (Ser33, Ser37 and Thr41) in HEK293W cells treated with increasing time of 2.

HEK293W cells with 2 resulted in a dose-dependent decrease in Wnt3a induced ST-Luc transcription. Meanwhile, the exposure of HEK293W cells to increased concentrations of 2 for 24 h obviously suppressed the expression of Wnt target genes Axin 2, c-myc, cyclin D1, and survivin (Figure 4B, left). Consistently, a similar effect was confirmed in HT29 colorectal cancer cells with constitutively activated Wnt signaling (Figure 4B, right), whereas β-catenin is an important component of the canonical Wnt signaling pathway that is phosphorylated by the destruction complex and then ubiquitinated and degraded through the proteasome pathway.17 Thus, the respective protein levels of total and phosphorylated β-catenin were investigated. Promotion of phosphorylation and degradation of β-catenin were observed in a time- and dose-dependent manner in HEK293W cells incubated with 2 (Figure 4C−D). For the reason that Wnt pathway is closely associated with cell proliferation, the growth inhibitory effect using MTS assay in colorectal cancer cells with abberant Wnt signaling showed that 2 inhibited all the tested cells, Caco2, HT29, and HCT116 with IC50 values of 10.97, 31.78, and 42.58 μM, respectively. Taken together, our study indicates that 2 could be included as a new 3913

DOI: 10.1021/acs.orglett.7b01813 Org. Lett. 2017, 19, 3911−3914

Letter

Organic Letters

(5) (a) Brooks, G.; Morrice, N. A.; Ellis, C.; Aitken, A.; Evans, A. T.; Evans, F. J. Toxicon 1987, 25, 1229. (b) Wada, R.; Suto, Y.; Kanai, M.; Shibasaki, M. J. Am. Chem. Soc. 2002, 124, 10658. (c) Roux, P. P.; Ballif, B. A.; Anjum, R.; Gygi, S. P.; Blenis, J. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 13489. (6) El-Mekkawy, S.; Meselhy, M. R.; Nakamura, N.; Hattori, M.; Kawahata, T.; Otake, T. Phytochemistry 2000, 53, 457. (7) McKernan, L. N.; Momjian, D.; Kulkosky, J. Adv. Virol. 2012, 2012, 805347. (8) (a) Kawamura, S.; Chu, H.; Felding, J.; Baran, P. S. Nature 2016, 532, 90. (b) Wender, P. A.; Kee, J.-M.; Warrington, J. M. Science 2008, 320, 649. (c) Ovaska, T. V.; Reisman, S. E.; Flynn, M. A. Org. Lett. 2001, 3, 115. (d) Lee, K.; Cha, J. K. J. Am. Chem. Soc. 2001, 123, 5590. (e) Luo, D.; Callari, R.; Hamberger, B.; Wubshet, S. G.; Nielsen, M. T.; Andersen-Ranberg, J.; Hallström, B. M.; Cozzi, F.; Heider, H.; Møller, B. L.; Staerk, D.; Hamberger, B. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E5082. (9) (a) Wang, L.; Ma, Y.-T.; Sun, Q.-Y.; Li, X.-N.; Yan, Y.; Yang, J.; Yang, F.-M.; Liu, F.-Y.; Zang, Z.; Wu, X.-H.; Huang, S.-X.; Zhao, Y. Tetrahedron 2015, 71, 5484. (b) Yan, Y.; Ma, Y.-T.; Yang, J.; Horsman, G. P.; Luo, D.; Ji, X.; Huang, S.-X. Org. Lett. 2016, 18, 1254. (c) Yan, Y.; Yang, J.; Yu, Z.; Yu, M.; Ma, Y.-T.; Wang, L.; Su, C.; Horsman, G. P.; Huang, S.-X. Nat. Commun. 2016, 7, 13083. (10) Duarte, N.; Lage, H.; Ferreira, M.-J. U. Planta Med. 2008, 74, 61. (11) Aljančić, I. S.; Pešić, M.; Milosavljević, S. M.; Todorović, N. M.; Jadranin, M.; Milosavljević, G.; Povrenović, D.; Banković, J.; Tanić, N.; Marković, I. D.; Ruždijić, S.; Vajs, V. E.; Tešević, V. V. J. Nat. Prod. 2011, 74, 1613. (12) (a) Yang, J.-H.; Pu, J.-X.; Wen, J.; Li, X.-N.; He, F.; Xue, Y.-B.; Wang, Y.-Y.; Li, Y.; Xiao, W.-L.; Sun, H.-D. J. Nat. Prod. 2010, 73, 12. (b) Wang, W.; Xu, Z.; Yang, M.; Liu, R.; Wang, W.; Liu, P.; Guo, D. Magn. Reson. Chem. 2007, 45, 522. (13) Crystallographic data for compound 2 have been deposited with the Cambridge Crystallographic Data Centre (CCDC 1531928). These data can be obtained free of charge via https://www.ccdc.cam. ac.uk. (14) (a) Chen, B.; Kawai, M.; Wu-Wong, J. R. Bioorg. Med. Chem. Lett. 2013, 23, 5949. (b) Posner, G. H.; Crawford, K.; Siu-Caldera, M.L.; Reddy, G. S.; Sarabia, S. F.; Feldman, D.; van Etten, E.; Mathieu, C.; Gennaro, L.; Vouros, P.; Peleg, S.; Dolan, P. M.; Kensler, T. W. J. Med. Chem. 2000, 43, 3581. (c) Huber, J. E. Tetrahedron Lett. 1968, 9, 3271. (15) Anastas, J. N.; Moon, R. T. Nat. Rev. Cancer 2013, 13, 11. (16) Kong, L.-M.; Feng, T.; Wang, Y.-Y.; Li, X.-Y.; Ye, Z.-N.; An, T.; Qing, C.; Luo, X.-D.; Li, Y. Oncotarget 2016, 7, 10203. (17) Klaus, A.; Birchmeier, W. Nat. Rev. Cancer 2008, 8, 387. (18) Johnston, C. W.; Skinnider, M. A.; Dejong, C. A.; Rees, P. N.; Chen, G. M.; Walker, C. G.; French, S.; Brown, E. D.; Bérdy, J.; Liu, D. Y.; Magarvey, N. A. Nat. Chem. Biol. 2016, 12, 233.

chemotype of Wnt signaling inhibitor and has the potential to be an anticancer drug lead for further development. In summary, our discovery of euphordraculoate A (1) from E. dracunculoides has expanded the diterpenoids skeleton of the structurally complex tiglianes to possess a 6/6/3-fused core system decorated with a 2-methyl-2-cyclopentenone moiety. Our efforts to biomimetically synthesize 1 from a proposed tigliane precursor 3 failed, but, surprisingly, led to a Wnt signaling pathway inhibitor 2, which contains another new scaffold with 5/5/6/3-fused carbon rings. The conversion from 3 to 2 and detailed decarbonization mechanism study not only provide potential synthetic utility in the context of 5/5/6-fused carbon rings containing complex natural products but also validates the long-standing proposal14c that the decarbonization mechanism from 2-methyl aldehyde to ketone under strong base conditions involves a 1,2-cycloaddition of the singlet molecular oxygen to the double bond of the enolate and a subsequent C−C bond cleavage. To the best of our knowledge, this is the first indication that tiglianes or their derivatives could inhibit the Wnt signaling pathway highlighting the natural products chemists’ suspicion that novel natural product scaffolds with specific activity often have a high likelihood of acting on novel targets.18

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01813. Detailed experimental procedures, spectroscopic data, ECD calculations of 1 and 2, and NMR and MS spectra for 1, 2, 4, 5a, 5b, and 18O-labled 5a (PDF) X-ray data for 2 (CIF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.-X.H). *E-mail: [email protected] (Y.Z.). ORCID

Sheng-Xiong Huang: 0000-0002-3616-8556 Author Contributions ⊥

L.W. and J.Y. contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No. 81522044 to S.-X.H.; No. 21162044 to Y.Z.), Applied Basic Research Foundation of Yunnan Province (No. 2013HA022 to S.-X.H.), and Foundation from Chinese Academy of Sciences (QYZDBSSW-SMC051 to S.-X.H.).

■

REFERENCES

(1) Wang, H.-B.; Wang, X.-Y.; Liu, L.-P.; Qin, G.-W.; Kang, T.-G. Chem. Rev. 2015, 115, 2975. (2) Vasas, A.; Hohmann, J. Chem. Rev. 2014, 114, 8579. (3) Shi, Q.-W.; Su, X.-H.; Kiyota, H. Chem. Rev. 2008, 108, 4295. (4) Durán-Peña, M. J.; Botubol Ares, J. M.; Collado, I. G.; Hernández-Galán, R. Nat. Prod. Rep. 2014, 31, 940. 3914

DOI: 10.1021/acs.orglett.7b01813 Org. Lett. 2017, 19, 3911−3914