Commiphoratones A and B, Two Sesquiterpene Dimers from Resina

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Commiphoratones A and B, Two Sesquiterpene Dimers from Resina Commiphora Jia-Wang Liu,†,‡,∥,# Ying Liu,†,# Yong-Ming Yan,† Jing Yang,‡ Xi-Feng Lu,*,† and Yong-Xian Cheng*,†,§ †

Guangdong Key Laboratory for Genome Stability & Disease Prevention, School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen 518060, China ‡ State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China § Henan University of Chinese Medicine, Zhengzhou 450008, China ∥ University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: Two sesquiterpene dimers, commiphoratones A (1) and B (2), were isolated from Resina Commiphora. Their structures were elucidated by spectroscopic, computational, and crystallographic methods. Compounds 1 and 2 represent an unusual pattern of dimerization between two types of sesquiterpenes. Moreover, compound 1 has a saddle shape. The plausible biosynthetic pathway for 1 and 2 is presented. Bioassay showed that 1 and 2 significantly block lipid metabolism in a concentration-dependent manner.

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lants biosynthesize resins for different purposes. Terpenoids are the main components of resins that are utilized for chemical defense.1 Much attention has been given to structurally and biologically intriguing terpenoids exemplified by bolivianine,2 gochnatiolides,3 and (+)-absinthin.4 In recent years, research in our laboratory has focused on natural products present in plant resins. This effort led to the isolation of GQ5 and sesquiterpenoids with new carbon skeletons from Toxicodendron vernicifluum, which have interesting kidney protection abilities. 5 Moreover, we found that Resina Commiphora is a natural resin secreted from the genus Commiphora (Burseraceae) such as C. myrrh. This resin has been used as a Chinese medicine for the treatment of blood stagnation.6 Thus far, more than 300 terpenoids have been isolated from Commiphora species.6 Likewise, we identified a number of terpenoids from Resina Commiphora that are renoprotective, as well as those that have novel skeletons.7 In a recent investigation of Resina Commiphora described below, we characterized two new sesquiterpene dimers, commiphoratones A (1) and B (2), the first of which contains a unique 6/6/5/5/6(5)/6 heptacyclic architecture (Figure 1). The biological activities of these substances toward regulation of lipid metabolism were also assessed. Commiphoratone A (1),8 colorless needles (MeOH), possesses the molecular formula C30H32O6, as determined by evaluating its HRESIMS, 13C NMR, and DEPT spectra. The 1H NMR spectrum of 1 contains 3 methyl singlets, 3 methyl doublets, and 1 aromatic proton. Analysis of the 13C NMR and DEPT spectra (Table S1) reveals that 30 carbons in 1 are associated with 6 methyl, 4 methylene, 6 methine (1 aromatic, 5 aliphatic with 2 oxygenated), and 14 quaternary carbons (2 © XXXX American Chemical Society

Figure 1. Structures of 1 and 2.

Figure 2. Key 2D NMR correlations of 1.

ketones, 1 carbonyl, 9 olefinic, and 2 aliphatic with 1 being oxygenated). The spectroscopic data, along with a consideration of the chemical profile of the genus Commiphora, Received: February 15, 2018

A

DOI: 10.1021/acs.orglett.8b00561 Org. Lett. XXXX, XXX, XXX−XXX

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Scheme 1. Plausible Pathway for the Biogenesis of 1 and 2

Figure 3. X-ray structure of 1. Displacement ellipsoids are drawn at the 30% probability level.

Figure 4. Key 2D NMR correlations of 2.

Figure 5. Calculated and experimental ECDs of 2 (red and green calculated at the B3LYP-PCM/6-31G(d,p)//B3LYP/6-31G(d,p) level in CH3OH; blue, experimental in CH3OH).

prompted us to speculate that 1 is a dimeric cadinane-type sesquiterpene. The structural assignment of 1 was aided by the results of 2D NMR experiments. The observation of 1H−1H COSY correlations of H-3/H-4/H-5, H-4/H3-15, H-3′/H-4′/H-5′, and H-4′/H3-15′, accompanied by HMBC correlations of H-3/ C-2 (δC 198.0), H-4/C-2, C-6, H-5/C-6, H3-15/C-3, C-4, C-5, and of H-3′/C-2′ (δC 195.7), C-1′, H-5′/C-1′, C-2′, C-6′, H315′/C-3′, C-4′, C-5′ (Figure 2), indicates that 1 contains two symmetric, terminal subunits (rings A and F). Coupling of these rings with the remaining carbon signals led to construction of two cadinane-type sesquiterpene components (blue line and red line in 1 in Figure 1). The existence of HMBC correlations of H-5/C-6, C-7, H3-14/C-1, C-2, C-10, C-9 and H-9/C-10, C-6, C-7, C-8 in the blue part and consideration of the aromatic natures of C-1 and C-6−C-10 suggest the presence of an aromatic ring B. Likewise, HMBC correlations of H3-13/C-11, C-12, C-7; H-12/C-11, C-8 show that rings B and C merge via C-8−O−C-12. In this way, the blue structural fragment is an 11,12-disubstituted derivative of

Figure 6. HepG2 cells were treated with 5, 10, and 20 μM of 1 (A) or 2 (B) for 48 h followed by MTT assay. HMGCR, SQLE, and LDLR mRNA levels of 1 (C) or 2 (D) were expressed relative to those of controls by Q-PCR. HMGCR, SQLE, and LDLR protein expressions of 1 (E) or 2 (F) were analyzed by using Western blot. Data are expressed as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 vs control.

myrrhone, which was isolated previously from C. myrrha.9 In an analogous manner, the fusion of E and F rings via a Δ1(6) double bond is supported by HMBC correlations of H-5′/C-6′, C-1′, C-7′, H-10′/C-1′, C-2′, C-6′, C-8′, H-9′/C-10′, C-1′, C7′, C-8′, H3-14′/C-10′, C-9′, C-1′ and the 1H−1H COSY correlations of H-9′/H-10′/H3-14′. Furthermore, the HMBC cross-peaks of H3-13′/C-11′ (δC 123.1), C-12′ (δC 171.7), C-7′ B

DOI: 10.1021/acs.orglett.8b00561 Org. Lett. XXXX, XXX, XXX−XXX

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sesquiterpenoid parts in 2 are connected via a CH2 bridge between C-10 and C-4′, and C-9−C-5′ to form an unusual core comprising five-membered rings. This conclusion is supported by 1H−1H COSY correlation of H-9/H-5′ and the HMBC cross-peaks of H2-11′/C-10, C-1, C-9, C-14, C-4′, C-3′, C-5′. The relative configurations of the stereogenic centers in 2 were determined by using ROESY correlations. As shown in Figure 4, the “triangle cycle” interactions between H-1′/H-5′, H-7′, and H-5′/H-7′ indicate that these protons are vicinal. Furthermore, ROESY correlations of H-9/H-6′, H3-15′, H-6′/ H3-15′, Hb-2′, and H3-15′/Hb-2′ suggest that H-6′ and H3-15′ have the same spatial orientation as that of H-9. Likewise, H-1, H-4, H3-14, and H3-16 each has the same orientation as does H-9 with support of correlations of H-9/H-1, H3-14, H3-14/H1, H-6′, H-1/H-4, H3-16/H-1, H-4, and H3-14. In addition, interpretation of the cross-peaks of H-5′/H3-15, H-5, H3-15/H2, H-5, H-2/H-5, H2-11′/H-2, and H-5 demonstrates that H-2, H-5, and H3-15 are located on the same face of ring A and have the same orientation as that of H-5′. Finally, H-5′ and H-9 were assigned as trans based on the JH‑5′,H‑9 value of 11.4 Hz, as well as the simultaneously observed ROESY interactions of H-5′/ H3-15, H-5, H-9/H-1, H3-14, H-6′, and H3-15′. The relative configurations of the stereocenters in 2 follow from this. Subsequent electronic circular dichroism (ECD) calculations show that the calculated weighted ECD spectrum of 1R,2S,4S,5R,9R,10R,1′S,5′R,6′S,7′R,10′S-2 coincides well with that of the measured one (Figure 5), enabling the absolute configuration of 2 to be finally assigned. Note that the presence of ring D makes 2 a hexacyclic structure with 11 successive chiral centers. In this effort, we characterized the new natural products 1 and 2 as dimeric sesquiterpenoids. The presence and structural context of ring D gives 1 a unique saddle-like architecture. In addition, the unique presence of a D ring in 2 allows it to have a 5(5)/7/5/6/6 ring system. To explore these issues, we proposed the plausible pathway for the biogenesis of 1 and 2 shown in Scheme 1. In the route, the universal sesquiterpenoid progenitor farnesyl pyrophosphate is the precursor of the three cadinane-type intermediates i, ii, and iv and one guaiane-type intermediate iii. Key steps in the sequence involving aldol condensation13 of the two similar cadinane sesquiterpenoids i and ii, along with sequential esterification and condensation, produce 1. For compound 2, the assembly of an intriguing ring D might result from two-step Michael addition reactions. In detail, intermediates v and vi could be generated from iii and iv, respectively. The intermolecular Michael addition between intermediates v and vi could lead to the formation of C-10−C11′. Similarly, intermediate vii would give rise to viii by the formation of C-9−C-5′ via an intramolecular Michael addition reaction. Intermediate viii would finally generate 2 by undergoing reactions such as oxidation, esterification, and reduction. Disturbances of lipid metabolism are caused by many disorders, including diabetes, fatigue, and atherosclerosis.14 As a result, the development of natural substances that target regulation of lipid metabolism and avoid adverse effects of currently marketed, lipid-lowering drugs is important. Within this context, a study was conducted to determine if the substances isolated from Resina Commiphora regulate lipid metabolism. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) and squalene epoxidase (SQLE) are enzymes that promote two rate-limiting steps in cholesterol biosynthesis and, as a result, that serve as pharmacological targets to lower plasma

(δC 155.8) and consideration of the chemical shift of C-8′ (δC 96.0) and the framework of cadinane-type sesquiterpenes support the presence of ring G and another characteristic of an 8′,10′-disubstituted cadinane-type sesquiterpene.10 Thus, the presence of two sesquiterpene units was clarified, and their connection through a furan motif (ring D) was demonstrated by key HMBC interactions of H3-13/C-8′, H-12/C-8′, C-9′, H9′/C-8′, C-11, and C-12. ROESY experiments were used to assign the relative configurations of the stereocenters in 1. ROESY correlations of H3-13/H-12, H-9′, and H-12/H-9′ (Figure 2) indicate that H3-13, H-12, and H-9′ are vicinally disposed. Likewise, ROESY correlations of H3-13′/H3-14′ and H3-15′ show the orientations of two methyl groups relative to rings E and F, and that of H-4/ H3-13 reveals the orientation of H3-15. An additional ROESY correlation of H-9′/H3-14′ suggests that H-9′ and H3-14′ have a close spatial location. Although it is difficult to assign the configuration of C-8′, the observed ROESY correlations of H313′/H3-15, H3-14′, H3-15′, H-10′/H-12, H-9, H-4′/H3-14, H-9, H-9/Ha-3′, Hb-5′ indicate that only one orientation is possible for C-8′. It should be noted that the ROESY observations show that 1 has an intriguing saddle-shaped structure, which is caused by the same orientations of the two bulky ring systems that surround ring D. Finally, the assigned structure of 1 was unambiguously determined by using single-crystal X-ray diffraction analysis with Cu Kα radiation (Figure 3). This analysis also enables assignment of the absolute configurations of the stereocenters in 1 as 4R,11R,12R,4′R,8′R,9′S,10′R. Commiphoratone B (2),11 a white amorphous powder, has the molecular formula of C31H44O4. The 1H NMR spectrum contains 7 methyl groups (4 singlets, 3 doublets). Apart from an evident methoxy group (δH 3.21, δC 56.2), the remaining 30 carbons are ascribed to 6 methyl, 5 methylene, 12 methine (10 aliphatic with 1 oxygenated and 2 olefinic), and 7 quaternary carbons (1 ketone, 2 aliphatic with 1 oxygenated, and 4 olefinic with 1 oxygenated). These spectroscopic characteristics suggest that 2 is composed of two types of sesquiterpenes, classified as a guaiane-type (in blue) and cadinane-type (in red) (Figure 1). The looplike 1H−1H COSY correlations (Figure 4) of H-1/H2/H-3/H-4/H-5/H-1 and H-4/H3-15, aided by the HMBC correlation (Figure 4) of H3-16/C-2 (δC 84.2), show the presence in 2 of a five-membered ring A containing a methoxy group at C-2. Evidence that rings A and B are fused at C-1 and C-5 arises from the HMBC cross-peaks of H-1/C-6, C-9, C-10, C-14 and H-5/C-6, C-10. A diagnostic proton resonance at δH 6.99 and HMBC cross-peaks of H-12/C-11, C-13, C-7, C-8 (δC 162.6), H3-13/C-11, C-7, C-12 (δC 138.8) suggest the presence of a furan (ring C). In addition, fusion between the sevenmembered ring B with ring C is deduced from HMBC correlations of H-9/C-7, C-8, C-10, C-1 and H3-14/C-1, C-10, C-9. These data suggest a guaiane-type sesquiterpene structure whose signals are the same as those of myrrhterpenoid I.12 Similarly, the other sesquiterpene fragment was deduced to have a cadinane-type structure. The successive 1H−1H COSY correlations of H-1′/H-6′/H-7′/H-8′/H-9′, H-7′/H-12′/H13′, H-12′/H-14′, together with the HMBC cross-peaks of H3-15′/C-1′, C-9′, C-10′ (δC 72.8), H-12′/C-6′, C-7′, C-8′, indicate the existence of ring F and the presence of an isopropyl group attached to C-7′ and a hydroxyl bonded to C-10′. The remaining 1H−1H COSY correlations of H-3′/H-2′/H-1′ and H-5′/H-6′ led to assignment of fusion between rings F and E and the presence of a bicyclic cadinane motif. Finally, two C

DOI: 10.1021/acs.orglett.8b00561 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters cholesterol.15 Inhibiting hepatic HMGCR upregulates lowdensity lipoprotein receptor (LDLR) expression, as a consequence of a limited intracellular cholesterol supply. Statins, the most successful and widely used cholesterollowering drugs, utilize this mechanism to accelerate plasma LDL clearance.16 Interestingly, we observed that 1 and 2 potently reduce HMGCR and SQLE transcript and protein levels in a dose-dependent manner (Figure 6). Additionally, 1 increases LDLR transcript and protein levels in a dosedependent manner, whereas 2 is less capable of inducing LDLR expression. These findings suggest that 1 and 2 display behaviors that are similar to that of statins. Moreover, at the doses tested, 1 and 2 do not affect the viability of hepatic cells, excluding the possibility that their cytotoxicity induces changes in HMGCR, SQLE, and LDLR transcript and protein levels. Therefore, the findings suggest that 1 and 2 may be useful in treating cardiovascular diseases, such as hypercholesterolemia and atherosclerosis, by regulating cholesterol metabolism. However, the exact molecular mechanism of this regulation needs to be elucidated.

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(3) Li, C.; Dian, L. Y.; Zhang, W. D.; Lei, X. G. J. Am. Chem. Soc. 2012, 134, 12414−12417. (4) Zhang, W. H.; Luo, S. J.; Fang, F.; Chen, Q. S.; Hu, H. W.; Jia, X. S.; Zhai, H. B. J. Am. Chem. Soc. 2005, 127, 18−19. (5) Ai, J.; Nie, J.; He, J. B.; Guo, Q.; Li, M.; Lei, Y.; Liu, Y. H.; Zhou, Z. M.; Zhu, F. X.; Liang, M.; Cheng, Y. X.; Hou, F. F. J. Am. Soc. Nephrol. 2015, 26, 1827−1838. (6) Shen, T.; Li, G. H.; Wang, X. N.; Lou, H. X. J. Ethnopharmacol. 2012, 142, 319−330. (7) Dong, L.; Cheng, L. Z.; Yan, Y. M.; Wang, S. M.; Cheng, Y. X. Org. Lett. 2017, 19, 286−289. (8) Commiphoratone A (1): colorless needles (MeOH); [α]D23 +151.1 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 278 (3.95), 237 (3.97) nm; CD (MeOH) Δε300 + 28.44, Δε258 − 14.30, Δε223 − 20.67; ESIMS (positive) m/z 511 [M + Na]+; HRESIMS m/z 511.2092 [M + Na]+ (calcd for C30H32NaO6 511.2097); for 1H and 13 C NMR data, see Table S1. (9) Zhu, N. Q.; Sheng, S. Q.; Sang, S. M.; Rosen, R. T.; Ho, C. T. Flavour Fragrance J. 2003, 18, 282−285. (10) Shen, T.; Wan, W. Z.; Wang, X. N.; Yuan, H. Q.; Ji, M.; Lou, H. X. Helv. Chim. Acta 2009, 92, 645−652. (11) Commiphoratone B (2): white powders (MeOH); [α]D24 +58.5 (c 0.22, MeOH); UV (MeOH) λmax (log ε) 280 (3.29), 195 (3.72); CD (MeOH) Δε280 + 3.37, Δε211 − 10.82, Δε195 − 12.90; ESIMS (positive) m/z 503 [M + Na]+; HRESIMS m/z 503.3138 [M + Na]+ (calcd for C31H44NaO4 503.3137); for 1H and 13C NMR data, see Table S1. (12) Xu, J.; Guo, Y. Q.; Li, Y. S.; Zhao, P.; Liu, C. Z.; Ma, Y. G.; Gao, J.; Hou, W. B.; Zhang, T. J. Planta Med. 2011, 77, 2023−2028. (13) Yuan, T.; Zhu, R. X.; Yang, S. P.; Zhang, H.; Zhang, C. R.; Yue, J. M. Org. Lett. 2012, 14, 3198−3201. (14) Nicolson, G. L. J. Cell. Biochem. 2007, 100, 1352−1369. (15) Puleston, D. J.; Villa, M.; Pearce, E. L. Cell Metab. 2017, 26, 131−141. (16) Goldstein, J. L.; Brown, M. S. Arterioscler., Thromb., Vasc. Biol. 2009, 29, 431−438.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b00561. NMR data, NMR and MS spectra of 1 and 2, detailed isolation procedures, crystallographic data of 1, ECD calculations of 2, bioassay methods (PDF) Accession Codes

CCDC 1817676 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 Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jia-Wang Liu: 0000-0003-4009-0541 Yong-Xian Cheng: 0000-0002-1343-0806 Author Contributions #

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

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Science Fund for Distinguished Young Scholars (81525026) and National Key Research and Development Program of China (2017YFA0503900).

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REFERENCES

(1) Plant Resins: Chemistry, Evolution, Ecology, And Ethnobotany; Langenheim, J. H., Ed.; Timber Press: Portland, Cambridge, 2003. (2) Yuan, C. C.; Du, B.; Yang, L.; Liu, B. J. Am. Chem. Soc. 2013, 135, 9291−9294. D

DOI: 10.1021/acs.orglett.8b00561 Org. Lett. XXXX, XXX, XXX−XXX