Letter Cite This: Org. Lett. 2018, 20, 5506−5509
pubs.acs.org/OrgLett
Belamchinanes A−D from Belamcanda chinensis: Triterpenoids with an Unprecedented Carbon Skeleton and Their Activity against AgeRelated Renal Fibrosis Ying-Ying Song,‡,||,⊥ Jin-Hua Miao,§,⊥ Fu-Ying Qin,†,‡,|| Yong-Ming Yan,† Jing Yang,‡ Da-Peng Qin,† Fan-Fan Hou,*,§ Li-Li Zhou,*,§ and Yong-Xian Cheng*,†
Org. Lett. 2018.20:5506-5509. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 09/08/18. For personal use only.
†
Guangdong Key Laboratory for Genome Stability & Disease Prevention, School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen 518060, PR China ‡ State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China § State Key Laboratory of Organ Failure Research, Guangdong Provincial Institute of Nephrology, Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China || University of Chinese Academy of Sciences, Beijing 100049, PR China S Supporting Information *
ABSTRACT: Belamchinanes A−D (1−4), four triterpenoids with an unprecedented skeleton, were isolated from the seeds of Belamcanda chinensis and fully characterized. The structures of belamchinanes feature a 4/6/6/6/5 polycyclic system in which a fourmembered carbocyclic ring bridges the C-1 and C-11 positions of a classical triterpenoid framework. A plausible pathway for the biosynthesis of 1−4 is proposed. Biological studies reveal that 1−4 dose-dependently protect age-related renal fibrosis in vitro.
T
he Iridaceae family contains an estimated 1700 species of plants, many of which are known to have ornamental value and some are used for medicinal purposes. A member of the latter group, Iris pallasii, has yielded an extract that contains the well-known irisquinone and benquinone derivatives, which have been developed as radiosensitizing agents for use in the treatment of cancer.1 In addition, the rhizomes of the plant Belamcanda chinensis (Iridaceae) have been used as a traditional Chinese medicine for the treatment of throat disorders.2 Isoflavonoids, iridal-type triterpenoids, quinones, and stilbenes have been isolated from this plant.3 Although the seeds of B. chinensis have not been used medicinally, they are documented to contain dibenzofurans4 in addition to the substances mentioned above. In our recent effort on searching active compounds from natural origins, we embarked on a study of B. chinensis seeds that resulted in the isolation of four triterpenoids, belamchinanes A−D (1−4) (Figure 1), which possess a new carbon skeleton featuring a 4/6/6/6/5 polycyclic system, i.e., a pentacyclic triterpenoid skeleton containing an A−B ring bridging four-membered ring. Herein, we describe the isolation, structure characterization, and renal protection evaluation of these substances. Belamchinanes A−D (1−4) were isolated from the seeds of B. chinensis by using the procedure described in the Supporting Information (SI). Belamchinane A (1),5 isolated as needles, was found to have a molecular formula of C31H46O5 by analysis of its HRESIMS, 13C NMR, and DEPT spectra, indicating nine degrees of unsaturation. The 1H NMR spectrum of 1 contains six methyl © 2018 American Chemical Society
Figure 1. Structures of 1−4.
singlets, including 1 methoxy (δH 3.62, δC 51.9), 2 methyl doublets, and 2 olefinic proton resonances. Analysis of 13C NMR and DEPT spectra (Table S1) reveals that 1 contains 31 carbons attributed to 8 methyl (1 methoxy), 7 methylene (1 olefinic), 6 methine (1 oxygendated) and 10 quaternary carbons (1 ketocarbonyl, 1 ester carbonyl, 3 olefinic, and 5 aliphatic with 1 being oxygenated). The data suggest that 1 is an arborane-6 or a fernane-type7 triterpenoid. The structural construction of 1 was aided by the results of 2D NMR experiments. The 1H−1H COSY spectrum of 1 displays Received: August 3, 2018 Published: August 28, 2018 5506
DOI: 10.1021/acs.orglett.8b02490 Org. Lett. 2018, 20, 5506−5509
Letter
Organic Letters
calculations were carried out on 1 at the B3LYP/6-31G(d,p) level (Figure 3). The results provide a weighted ECD spectrum
interactions of H-18/H-19/H-20/H-21/H-22/H3-29 (H3-30), in connection with HMBC correlations of H3-29/, C-21, C-22, C-30, H3-30/C-21, C-22, C-29 (Figure 2), H-18/C-17, C-21,
Figure 3. Calculated and experimental ECDs of 1.
of the enantiomer (1S,5S,7R,10R,11R,13S,14R,17R,18R,21R)-1 that agrees well with that determined experimentally. Fortunately, a solution of 1 in cyclohexane (1 mL) containing one drop of isopropyl alcohol afforded crystals, which were subjected to X-ray crystallographic analysis using Cu Kα radiation. The results (Figure 4) verify the proposed structural and stereochemical assignments of 1.
Figure 2. Key 2D NMR correlations of 1 and 2.
and H-21/C-17, C-18. The findings indicate the presence of an E ring in 1 possessing an isopropyl group at C-21. Moreover, observations of 1H−1H COSY correlations of H-15/H-16, accompanied by HMBC correlations of H3-28/C-16, C-17, C18, C-21; H3-27/C-12, C-13, C-14, C-18; H3-26/C-8, C-13, C14, C-15, H-15/C-13, C-14, H-16/C-14, H-18, indicate that 1 contains a D ring fused to the E ring. Additional HMBC correlations of H3-27, H-18/C-12 (δC 216.2) and H3-26, H-15/ C-8 (δC 139.9) suggest the presence of a ketone moiety at C-12 and a double bond involving C-8. 1H−1H COSY correlations of H-5/H-6/H-7 (δH 4.52) and HMBC correlations of H-6, H-7/ C-8, H-7/C-9 (δC 140.8), H-5, H3-25/C-9, C-10 suggest the existence of a B ring in 1 with a Δ8(9) double bond and a hydroxyl group at C-7. The existence of cross peaks in the HMBC spectrum associated with H3-24, Ha-23 (δH 4.86), Hb-23 (δH 4.65)/C4, C-5, H-5/C-4, C-23 (δC 112.1), and C-24 suggest that 1 is a 3,4-seco-triterpenoid, a conclusion that is in accord with a 1 H−1H COSY correlation of H-1/H-2 and HMBC correlations of H-1, H-2, OCH3/C-3 (δC 172.2). Lastly, HMBC correlations of H-1/C-5, C-9, C-10, C-11 (δC 83.7), C-12, C-25 indicate the presence of a four-membered A ring bridging the B- and C-rings via C-1 and C-11. The chemical shift of C-11 indicates that it is also bonded to oxygen. As a consequence, the planar structure of 1 was deduced to be that shown in Figure 1, which is comprised of 3,4-seco-8-ferna-4(23),9(11)-dien-3-oic acid8 possessing a strained four-membered ring. The relative configurations at the stereogenic centers in 1 were elucidated using ROESY experiments. ROESY cross-peaks of H-22, H3-27/H3-28, H-2/H-5, and H3-27 (Figure 2) indicate that they are vicinal and all have α-orientations. In particular, the observation of an H-2/H3-27 interaction suggests the presence of a β-disposed OH group at C-11. Likewise, ROESY correlations of H-18/H-21, H3-26; H3-25/H3-24, H3-26, H-7/ H3-26 show that, in a manner that is consistent with reported observations,9 H-7, H-18, H-21, H3-25, and H3-26 are spacially vicinal and are oriented in the β-direction. In this way, the relative configurations at all the stereogenic centers in 1 were determined to be 1S,5S,7R,10R,11R,13S,14R,17R,18R,21R. To gain additional evidence for the above assignment of relative stereochemistry and for establishing the absolute configurations at the stereogenic centers, electronic circular dichroism (ECD)
Figure 4. X-ray structure of 1.
A detailed analysis of the HRESIMS, 13C NMR, and DEPT spectra and 1D and 2D NMR data of belamchinane B (2)10 shows that it has the same molecular formula and planar structural architecture as those of 1. Thus, we anticipated that 1 and 2 are stereoisomers. Indeed, observations of ROESY correlations (Figure 2) of H-1/H-5, H3-27; H-2/H3-25, 11-OH; H3-26/H-7, H-18; and H-5/7-OH, and H3-28/H3-27, H-22 indicate that 2 is a C-1 epimer of 1. This finding, together with the results of the ECD calculations (SI), enables assignment of the absolute configuration of 2 as 1R,5S,7R,10R,11R,13S,14R,17R,18R,21R. Analysis of the HRESIMS, 13C NMR, and DEPT spectra of belamchinane C (3)11 shows that it has the molecular formula of C31H46O6 and 9 degrees of unsaturation. The NMR data of 3 closely resemble those of 1, differing in that 3 has an additional hydroxyl group attached to C-19. This conclusion is supported by the observation of H-18/H-19 (δH 4.22)/H-20/H-21/H-22/ H3-29 (H3-30) in the 1H−1H COSY spectrum (Figure S1). In addition, ROESY correlations of H-19/H-22, H3-27, H3-28; H327/H-2, H3-28; and H-2/H-5, H3-27 suggest that H-2, H-5, H19, H-22, H3-27, H3-28 all have the same α-orientation. Likewise, correlations of H-18/H-21, H3-26; H-1/11-OH, H325; and H-7/H3-26 show that H-1, H-7, H-18, H-21, 11-OH, H3-25, and H3-26 are β-disposed. These findings along with those coming from analysis of the calculated and experimental ECD spectra (SI) show that the absolute configurations at the 5507
DOI: 10.1021/acs.orglett.8b02490 Org. Lett. 2018, 20, 5506−5509
Letter
Organic Letters
kidneys displaying renal fibrosis have many of the clinical characteristics of premature aging or accelerated senescence.17 These observations suggest that aging is an independent risk factor in the progression of CKD.16,18 Thus, an urgent need exists for pharmaceutical agents to treat age-related renal fibrosis and consequently decrease the morbidity and mortality of CKD. An investigation was designed to assess the potential agerelated renal fibrosis inhibition properties of 1−4. For this purpose, human proximal tubular (HKC-8) cells were treated with D-galactose (D-gal), an inducer of accelerated aging.19 As is seen in Figure 5A−D, administration of D-gal induces the
stereogenic centers in 3 are 1S,5S,7R,10R,11R,13S,14R,17R,18R,19S,21R. As determined by using HRESIMS, belamchinane D (4)12 has the molecular formula of C31H44O6. Analysis of its 13C NMR and DEPT spectra suggests that 4 contains 10 degrees of unsaturation. Moreover, the NMR data of 4 are similar to those of 3, except for the fact that C-7 of 3 is a ketone moiety in 4. The 1H−1H COSY correlation of H-5/H-6 and HMBC correlations of H-5, H-6/C-7 (δC 196.8) support this conclusion. The presence of an α,β-unsaturated ketone group in 4 is evidenced by the presence of an absorption at 254 nm in the UV spectrum. Information about the stereochemistry of 4 came from observations of ROESY correlations of H3-28/H-19, H-22, H3-27, H-2/H-5, H3-27, which suggest that H-2, H-5, H19, H-22, H3-27, H3-28 all have the same α-orientation. Likewise, correlations of H-18/19-OH, H-21, H3-26; H-1/11OH, H3-25, show that H-1, H-18, H-21, 11-OH, 19-OH, H3-25 and H3-26 are β-oriented. Finally, the absolute configuration of 4 was elucidated to be 1S,5S,10R,11R,13S,14S,17R,18R,21R utilizing the same method as that applied to 1−3. Belamchinanes A−D (1−4) have structures that resemble those of fernane-type triterpenoids, which are widely found in the Marattiaceae,9 Rubiaceae,13 and Physciaceae14 families as well as the genus Lonicera.15 However, to the best of our knowledge, triterpenoids such as 1−4, which possess a 3,4-seco ring, are relatively rare. Thus far, only plants in the Euphorbiaceae 8 family have been reported to contain triterpenoids of this type, and in contrast to fernane-type triterpenoids containing 3,4-seco rings, those lacking a C-3−C-4 bond and containing a C-1−C-11 bridge are unprecedented. With this in mind, a plausible pathway for biosynthesis of terpenoids 1−4 is proposed (Scheme 1). The route starts with
Figure 5. Western blot analysis showing that 1−4 block D-gal-mediated age-related renal fibrosis. HKC-8 cells were treated with D-gal (10 mg/ mL) for 60 h in the absence or presence of different concentrations of 1−4. Cell lysates were probed with antibodies against p16 and fibronectin.
upregulation of cellular p16, a typical senescence-related protein marker.20 Age-related fibrosis is also reflected in upregulation of the matrix protein fibronectin.21 Western blot analysis shows that D-gal promotes overproduction of fibronectin in HKC-8 cells (Figure 5A−D). We next determined the effects of 1−4 on cell viability by MTT assay. The results show that 1−4 in the range of concentrations used (SI) do not have cytotoxic effects on HKC-8 cells. Next, we assessed the effects of these substances on D-gal-induced tubular cell senescence. Inspection of the gels (Figure 5) demonstrates that D-gal-induced cellular senescence is inhibited by 1−4 dose-dependently. It has been shown that mitochondrial functions gradually decrease following the development of aging.22 Hence, damage of mitochondrial function could reflect the extents of aging. To elucidate more thoroughly the effects of 1−4 on age-related renal fibrosis, the protective role these substances play on mitochondrial function was determined. We first examined the phosphorylation of peroxisome proliferator activated receptor gamma coactivatior-1-α (PGC-1α), a master regulator of mitochondrial biogenesis,23 can be seen (Figure 6), D-gal treatment clearly decreases the expression of PGC-1α in HKC-8 cells and, in a consistent manner, it reduces expression of mitochondria transcription factor A (TFAM), a transcriptional factor regulated by PGC-1α that controls mitochondrial DNA.22 Importantly, addition of 1−4 reverses D-gal-induced expression of phospho-PGC-1α and TFAM, suggesting that these substances protect against mitochondrial damage. The observation that 1−4 have protective effects on mitochondrial function at concentrations as low as 0.5−1 μM suggests that mitochondrial protection might be the mechanism for their antiaging efficacy.
Scheme 1. Plausible Pathway for the Biogenesis of 1−4
multistep transformation of the universal triterpenoid precursor, 2,3-oxidosqualene (A), to the fernane-type intermediate (B). The 3,4-seco fernane-type intermediate (D−F) would then form through a complex sequence likely involving oxidative A-ring cleavage. Finally, the four-membered ring present in 1−4 could arise by an intramolecular Michael addition reaction of the 3,4seco fernane. In the next phase of these studies, we explored the biological properties of belamchinanes A−D. Chronic kidney disease (CKD) has become a worldwide health problem, and renal fibrosis, the final common pathological feature for various forms of this disease, has a very high prevalence in the elderly as compared to the younger population.16 Moreover, diseased 5508
DOI: 10.1021/acs.orglett.8b02490 Org. Lett. 2018, 20, 5506−5509
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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.8b02490. NMR data, NMR and MS spectra of 1−4, isolation procedures, ECD calculations, crystallographic data of 1, and bioassay methods (PDF) Accession Codes
CCDC 1857194 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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REFERENCES
(1) Fu, L. W.; Li, X. B.; Liang, Y. J.; Feng, H. L.; Zhang, Y. M.; Pan, Q. C. Chem. Pharm. Bull. 2001, 17, 234−235. (2) Chinese Pharmacopoeia Committee. Chin. Pharmacopoeia 2010, 1, 267−268. (3) Yang, Y.; Chen, J. J.; Zhang, D. Y.; Dong, X. F.; Zhao, C. Q. Youji Huaxue 2013, 33, 1244−1253. (4) Seki, K.; Haga, K.; Kaneko, R. Phytochemistry 1995, 38, 703−709. (5) Belamchinane A (1): colorless needles (cyclohexane); [α]D23 +18.7 (c 0.38, MeOH); UV (MeOH) λmax (log ε) 207 (3.58) nm; CD (MeOH) Δε222 +2.66, Δε246 −0.48, Δε317 +0.80; ESIMS (positive) m/ z 521 [M + Na]+; HRESIMS m/z 521.3246 [M + Na]+,a clcd for C31H46NaO5 521.3243; 1H and 13C NMR data, see Table S1. (6) Fan, J. T.; Kuang, B.; Zeng, G. Z.; Zhao, S. M.; Ji, J.; Zhang, Y. M.; Tan, N. H. J. Nat. Prod. 2011, 74, 2069−2080. (7) Tanaka, R.; Matsunaga, S. Phytochemistry 1989, 28, 3149−3154. (8) Tanaka, R.; Ida, T.; Kita, S. Phytochemistry 1996, 41, 1163−1168. (9) Chen, C. R.; Liao, Y. W.; Wu, H. T.; Shih, W. L.; Teng, C. Y.; Yang, S. Z.; Hernanden, C. E.; Chang, C. I. Chem. Pharm. Bull. 2010, 58, 408− 411. (10) Belamchinane B (2): white solids (MeOH); [α]D23 +74.0 (c 0.37, MeOH); UV (MeOH) λmax (log ε) 205 (3.96); CD (MeOH) Δε205 +4.95, Δε223 +2.65, Δε244 −1.13, Δε329 +3.29; ESIMS (positive) m/z 521 [M + Na]+; HRESIMS m/z 521.3237 [M + Na]+, calcd for C31H46NaO5 521.3243; 1H and 13C NMR data, see Table S1. (11) Belamchinane C (3): white solids (MeOH); [α]D23 +26.5 (c 0.49, MeOH); UV (MeOH) λmax (log ε) 204 (3.62) nm; CD (MeOH) Δε205 +2.06, Δε244 −0.90, Δε322 +1.38; ESIMS (positive) m/z 537 [M + Na]+; HRESIMS m/z 537.3188 [M + Na]+, calcd for C31H46NaO6 537.3192; 1H and 13C NMR data, see Table S2. (12) Belamchinane D (4): white solids (MeOH); [α]D22 +10.9 (c 0.31, MeOH); UV (MeOH) λmax (log ε) 254 (3.12), 203 (3.12) nm; CD (MeOH) Δε211 +0.87, Δε251 −1.06, Δε319 +1.62; ESIMS (positive) m/z 535 [M + Na]+; HRESIMS m/z 535.3030 [M + Na]+, calcd for C31H44NaO6 535.3036; 1H and 13C NMR data, see Table S2. (13) Liou, M. J.; Wu, T. S. J. Nat. Prod. 2002, 65, 1283−1287. (14) Maier, M. S.; Rosso, M. L.; Fazio, A. T.; Adler, M. T.; Bertoni, M. D. J. Nat. Prod. 2009, 72, 1902−1904. (15) Kikuchi, M.; Kawarada, N.; Yaoita, Y. Chem. Pharm. Bull. 1999, 47, 663−666. (16) Minutolo, R.; Borrelli, S.; De Nicola, L. Am. J. Kidney Dis. 2015, 66, 184−186. (17) Stenvinkel, P.; Larsson, T. E. Am. J. Kidney Dis. 2013, 62, 339− 351. (18) Sturmlechner, I.; Durik, M.; Sieben, C. J.; Baker, D. J.; van Deursen, J. M. Nat. Rev. Nephrol. 2017, 13, 77−89. (19) Chang, Y. M.; Chang, H. H.; Kuo, W. W.; Lin, H. J.; Yeh, Y. L.; Padma Viswanadha, V.; Tsai, C. C.; Chen, R. J.; Chang, H. N.; Huang, C. Y. Int. J. Mol. Sci. 2016, 17, 466. (20) Baker, D. J.; Childs, B. G.; Durik, M.; Wijers, M. E.; Sieben, C. J.; Zhong, J.; Saltness, R. A.; Jeganathan, K. B.; Verzosa, G. C.; Pezeshki, A.; Khazaie, K.; Miller, J. D.; van Deursen, J. M. Nature 2016, 530, 184− 189. (21) Luo, C. W.; Zhou, S.; Zhou, Z. M.; Liu, Y. H.; Yang, L.; Liu, J. F.; Zhang, Y. F.; Li, H. Y.; Liu, Y. H.; Hou, F. F.; Zhou, L. L. J. Am. Soc. Nephrol. 2018, 29, 1238−1256. (22) Gomes, A. P.; Price, N. L.; Ling, A. J.; Moslehi, J. J.; Montgomery, M. K.; Rajman, L.; White, J. P.; Teodoro, J. S.; Wrann, C. D.; Hubbard, B. P.; Mercken, E. M.; Palmeira, C. M.; de Cabo, R.; Rolo, A. P.; Turner, N.; Bell, E. L.; Sinclair, D. A. Cell 2013, 155, 1624−1638. (23) Hickey, F. B.; Corcoran, J. B.; Docherty, N. G.; Griffin, B.; Bhreathnach, U.; Furlong, F.; Martin, F.; Godson, C.; Murphy, M. J. Am. Soc. Nephrol. 2011, 22, 1475−1485.
Figure 6. Western blot analysis showing that 1−4 protect against D-galmediated mitochondrial damage. HKC-8 cells were treated with D-gal (10 mg/mL) for 60 h in the absence or presence of different concentrations of 1−4. Cell lysates were immunoblotted with antibodies against PGC-1α and TFAM.
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AUTHOR INFORMATION
Corresponding Authors
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[email protected]. ORCID
Yong-Xian Cheng: 0000-0002-1343-0806 Author Contributions ⊥
Y.-Y.S. and J.-H.M 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), the National Key Research and Development Program of China (2017YFA0503900), and the National Natural Science Foundation of China (81722011, 81521003, 81570620). We thank Dr. L.H. Jiang at Instrumental Analysis Center of Shenzhen University (Xili Campus) for NMR data collection. We thank the Institute of Traditional Chinese Medicine & Natural Products, Jinan University for assistance in ECD analyses. 5509
DOI: 10.1021/acs.orglett.8b02490 Org. Lett. 2018, 20, 5506−5509
