3 Fused Schinortriterpenoids

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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Schincalactones A and B, Two 5/5/6/11/3 Fused Schinortriterpenoids with a 13-Membered Carbon Ring System from Schisandra incarnata Jian Song,† Ming Zhou,† Jia Zhou, Jing-Jing Liang, Xiao-Gang Peng, Junjun Liu, and Han-Li Ruan* School of Pharmacy, Tongji Medical College, Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Huazhong University of Science and Technology, Wuhan 430030, P.R. China S Supporting Information *

ABSTRACT: Two novel schinortriterpenoids (SNTs), schincalactones A (1) and B (2), featuring a unique 5/5/6/11/3 ring system, together with schincalide B (3), were isolated from Schisandra incarnata. Their structures were elucidated by detailed spectroscopic analysis, and the absolute configurations of 1 and 3 were confirmed by single-crystal X-ray diffraction. Compounds 1 and 2 possess a 13membered carbon ring and are the first examples in the SNT family. Plausible biosynthetic pathways of 1−3 were postulated.

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he Schisandraceae family, placed under the tribe Magnoliales in the Engler system, is taxonomically composed of the genera Schisandra and Kadsura. Among the 66 species of this family in the world, 27 (19 Schisandra and 8 Kadsura) are distributed throughout China.1 Since the 1970s, much attention has been focused on this family for their diverse application in folk medicine with tonic effects and sedative, antiasthenic, antiaging, and heptaprotective properties. Phytochemical investigations on Schisandraceae plants resulted in the characterization of a variety of secondary metabolites, with the main components being lignans2 and triterpenoids.3 The most intriguing triterpenoids from this family are schinortriterpenoids (SNTs), which represent a special class of highly oxygenated and rearranged nortriterpenoids with C26−C29 frameworks. Their fascinating structures and various biological activities3b,4 have attracted the interest of both natural product and synthetic chemists over the past decade.5 Recently, a lot of excellent work on the total synthesis of SNTs (e.g., schilancidilactones A and B,6 lancifodilactone G,7 rubriflordilactone B,8 (+)-19-dehydroxyl arisandilactone A,9 rubriflordilactone A,10 schilancitrilactones B and C,11 and propindilactone G12) has made this compound class a hot topic in related scientific communities.5,13 Our previous phytochemical studies on Schisandra incarnata afforded one tricyclo[5.2.1.01,6]decane-bridged system14 and five 1oxaspiro[6.6]tridecane motif15 SNTs. In our continuing search for structurally interesting and bioactive important components from this species, schincalactones A (1) and B (2), two unique SNTs featuring a bicyclo[11.1.0]tetradecane skeleton, together with schincalide B (3), the C-23 epimer of schincalide A, were isolated (Figure 1). Although up to 226 SNTs3,14−16 with diverse structures have been isolated so far, 1 and 2 represent the first examples of hexacyclic SNTs featuring a unique 13-membered carbon ring. Previously, a number of triterpenoids were reported to have potent immunosuppressive activities.17 Our recent investigations on S. incarnata revealed that some SNTs have immunosup© XXXX American Chemical Society

Figure 1. Structures of schincalactones A (1), B (2), and schincalide B (3).

pressive potentiality. Immunosuppressants (e.g., ciclosporin, tacrolimus (FK506), everolimus, and rapamycin) have been used in clinical practice for organ transplant and other immunologically related ailments for decades. Some immunosuppressive drugs may cause serious side effects, such as dyslipidemia, hyperglycemia, lipodystrophy, liver and renal injury, and increased susceptibility to infection. New immunosuppressants with high efficiency and less adverse effects still remain to be explored by scientists, with natural products being an important source.18 Compounds 1−3 were tested for their in vitro immunosuppressive activity and exhibited inhibition activities against the proliferation of T and B lymphocyte cells of different degrees. Herein, we report the isolation, structural determination, hypothetical biogenetic pathways, and immunosuppressive activities of these SNTs. Schincalactone A (1), colorless platelet crystals, has a molecular formula of C29H36O10 as determined by HRESIMS Received: March 19, 2018

A

DOI: 10.1021/acs.orglett.8b00889 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters ([M + Na]+ m/z 567.2199, calcd 567.2206) and 13C NMR data, requiring 12 double bond equivalents (DBEs). The IR spectrum suggested the presence of hydroxyl (3405 cm−1) and carbonyl (1758 and 1612 cm−1) groups. The 1H NMR data (Table S3) of 1 displayed typical resonances for one secondary methyl at δH 1.00 (d, J = 6.4 Hz, H3-21) and four tertiary methyls at δH 1.07 (s, H3-18), 1.21 (s, H3-29), 1.37 (s, H3-30), 2.03 (s, H3-27), three oxygenated methines at δH 4.30 (br s, H-14), 4.64 (m, H-7), 4.78 (d, J = 6.0 Hz, H-1), and two olefinic protons at δH 5.43 (d, J = 11.4 Hz, H-22), 7.58 (s, H-24). The 13C NMR spectrum of 1 exhibited 29 carbon resonances, assignable with the aid of DEPT and HSQC spectra to 10 quaternary carbons (two olefinic, three oxygenated, two ester carbonyl, and two ketone carbonyl carbons), nine methines (two olefinic and three oxygenated methines), five methylenes, and five methyls (Table S3 and Figures S11−S13). The aforementioned functionalities accounted for 6 out of the 12 DBEs in the structure of 1, and the rest required the presence of six additional rings for 1. The planar structure of 1 was established by assembling three partial structures I−III from the 2D NMR spectroscopic data. Comparison of the 1D NMR data of 1 (Table S3) with those of preschisanartanin P16b suggested similar substructure I for both compounds, which was further corroborated by 2D NMR correlations (Figure 2a). An α-methyl α,β-unsaturated γ-lactone

65.9), C-9 (δC 210.5), C-10 (δC 95.0), and C-19 (δC 96.7), as well as the requirement of two remaining DBEs. Herein, substructure III was constructed (Figure 2a), which could be further confirmed by single-crystal X-ray crystallographic analysis. Furthermore, HMBC correlations of H-5/C-6, C-10, and H-6/ C-10 showed the connection of unit III and I through the carbon bond between C-5 and C-10, whereas partial structures III and II were connected together through a linkage of C-17−C-20, as seen by the 1H−1H COSY association of H-17/H-20 and HMBC correlation of H-20/C-17. Therefore, the planar structure of compound 1 was assigned, as shown in Figure 2b. The relative configuration of 1 was determined by NOESY spectrum (Figure S16). Biogenetically, H-5 was assigned to be αorientated. The correlations of H-5/H3-30; H-1, H-6β/H3-29; H-6β, H-8β/H-7; H-8α/H-14 in the NOESY spectrum revealed that H-1, H-6β, and H-7 were β-orientated and H-14 was αorientated. The NOESY cross-peaks of H-17, H3-18/H-16 suggested that they shared the same orientation. The chemical shifts of H-22 (δH 5.43, d, J = 11.4 Hz) and H-24 (δH 7.58, s) in the 1H NMR spectrum, along with the appearance of a NOESY cross-peak of H-20/H-24 and the absence of the NOESY correlation between H-22 and H-24, suggested the E-geometry of Δ22 in 1.19 Experimental electronic circular dichroism (ECD) spectrum of 1 exhibited a positive Cotton effect around 275 nm, suggesting that the absolute configuration at C-20 should be S (Figure 3).20 However, the relative configurations of C-10 and C-

Figure 2. (a) Key HMBC and 1H−1H COSY correlations of units I−III. (b) Key HMBC and 1H−1H COSY correlations of 1.

moiety was interpreted by the key HMBC and 1H−1H COSY correlations shown in Figure 2a. The sequence of −CH(C-22)− CH(C-20)−CH3 (C-21) was deduced by 1H−1H COSY correlations of H-20/H3-21, H-22, and HMBC correlations of H-20, H3-21/C-22, and H-20/C-21. The unit of −CH(C-22)− CH(C-20)−CH3(C-21) and the α-methyl α,β-unsaturated γlactone moiety were connected by an olefinic bond between C22 and C-23, which was confirmed by the HMBC correlations of H-20 and H-22/C-23. Thus, substructure II was established (Figure 2a). The HMBC associations of H-16, H-17, H-18/C-13 and H-16, H-17/C-18, coupled with the 1H−1H COSY resonance of H-16/H-17, indicated the presence of a cyclopropane ring. The 1H−1H COSY cross-peaks of H-5/H2-6/H-7/ H2-8/H-14 and H2-11/H2-12 and HMBC interactions of H-5/ C-6, C-10; H-6α/C-5, C-7, C-8, C-10; H-6β/C-5, C-7, C-8; H8α/C-7; H-8β/C-14; H-11/C-9, C-12, C-13; H-12α/C-11, C18; H-12β/C-16, H-17/C-15, established the structural unit of −C(C-10)−CH(C-5)−CH 2 (C-6)−CH(C-7)−CH 2 (C-8)− CH(C-14)−C(C-15)−CH(C-16)−C(C-13)−CH 2 (C-12)− CH2(C-11)−C(C-9)−. The structural units of −C(C-10)− C(C-19)−O−CH(C-7)− and −C(C-19)−C(C-9)− were proposed in consideration of the chemical shifts of C-7 (δC

Figure 3. Selected NOESY correlations of compound 1.

19 were still unassigned due to the lack of available NOESY correlations. Fortunately, the suitable quality crystals of 1 were obtained, allowing a successful X-ray crystallography study by Cu Kα radiation. The perfect Flack parameter21 of −0.02(8) not only confirmed the elucidated planar structure and relative configuration for 1 but also unambiguously determined its absolute configuration as 1R,5S,7R,10R,13R,14S,16S,17R,19R,20S (CCDC 1830622, Figure 4). Schincalactone B (2) was isolated as a white powder. On the basis of the [M + Na]+ peak ion at m/z 583.2150 (calcd for C29H36O11Na, 583.2155) in HRESIMS and 13C NMR data, the molecular formula of 2 was established as C29H36O11, which has 16 mass units more than that of compound 1. Careful comparison of the 1D NMR data of 2 with those of 1 suggested that the structure of 2 was quite similar to 1. The main difference was found to exist around the C-2 at ring A, where the methene (δC 37.4, t) in 1 was replaced by an oxygenated methine (δC 76.8, B

DOI: 10.1021/acs.orglett.8b00889 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

SNTs.3b Based on this hypothesis, a pathway (Scheme 1) for 1 and 2 was proposed. Preschisanartanin P16b was assumed to be a Scheme 1. Hypothetical Biosynthesis of Schincalactones A (1) and B (2)

Figure 4. X-ray ORTEP drawing of compound 1.

d) in 2, which indicated that a hydroxyl should be located at C-2. This deduction was corroborated by the correlation from H-1 to C-2 in the HMBC spectrum (Figure S25). The relative configuration of 2 was fixed to be identical to that of 1 by the NOESY spectrum (Figure S27). The NOESY correlations of H5, H3-30/H-2 implied that H-2 was α-oriented (Figure S3). Comparison of the experimental ECD spectrum of 2 with that of 1 showed identical tendency curves, with the biogenetic consideration allowing the assignment of the absolute configuration of 2, as depicted in Figure 1. Schincalide B (3) was obtained as colorless needles. It possessed the same molecular formula C29H32O9 as the previously reported schincalide A,14 as established by its HRESIMS ion peak at m/z 547.1887 ([M + Na]+, calcd for 547.1944). The UV spectrum showed a λmax at 202 nm in MeOH. Extensive analysis of the 1D and 2D NMR spectral data revealed that 3 shared the same planar structure with schincalide A. The proposed structure of 3 was fully interpreted by its HSQC, 1 H−1H COSY, and HMBC spectra (Figures S36−S38), and its absolute configuration was finally deduced as 1R,2R,5S,9R,10R,12S,13R,17S,20R,22R,23R by X-ray diffraction (CCDC 1830624, Figure 5) with a Flack parameter21 of 0.0(2). The structure of 3 was established as the C-23 epimer of schincalide A, named schincalide B. The biogenesis of the SNTs has attracted great interest because of their highly oxygenated and multicyclic frameworks. Recently, a biosynthetic pathway hypothesized that the SNTs were derived from 3,4:9,10-disecocycloartane by decarboxylation at C-28. Following this, a sequence of oxidations and skeletal rearrangements resulted in the formation of various types of

precursor, and the pathway involved oxidative cleavage and dehydration followed by oxidation and ring closure to form 1 and 2. Additionally, a plausible biosynthetic pathway of 3 was also postulated, which was similar to that of schincalide A (Scheme S2).14 Compounds 1−3 were tested for their in vitro immunosuppressive activities against the proliferation of T and B lymphocytes. As a result, 1 and 3 showed activities against the proliferation of T cells, with IC50 values of 50.91 and 65.91 μM (positive control CsA = 0.022 μM) and the proliferation of B cells with IC50 values of 36.84 and 38.86 μM (MMF = 9.42 μM), respectively (Table S1). The effects of 1−3 on the level of mouse IgG1 of the spleen lymphocyte cell supernatant were also investigated. The secretions of IgG1 were significantly inhibited by 1−3 at an equal concentration of 12.5 μg/mL (Figure S1). To further investigate the mechanisms of the immunosuppressive activities of 1−3, several selected therapeutic targets including FK506 binding proteins (FKBPs) were implemented in the AutoDock software. Taking the binding energy and distance (Table S2) into consideration, the interactions between the targets and the molecules were assessed. The calculated results predicted that FKBPs exhibited good binding affinity with compound 1 (binding energy = −8.4 kcal/mol and distance = 0.65 Å, as shown in Table S2). Tacrolimus, characterized by a 23membered macrolide lactone, could reduce peptidylprolyl isomerase activity by binding to FKBP12 to create a FKBP12− FK506 complex, which could inhibit T-lymphocyte signal transduction.22 As observed from the results of the molecular docking (Figure 6), FK506 and FKBP12 exhibited strong hydrogen bond interactions (e.g., FK506 to Tyr82) and a

Figure 5. X-ray ORTEP drawing of compound 3.

Figure 6. Binding poses of FK506 and 1 bound to FKBP12. C

DOI: 10.1021/acs.orglett.8b00889 Org. Lett. XXXX, XXX, XXX−XXX

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(2) Ward, R. S. Nat. Prod. Rep. 1999, 16, 75. (3) (a) Xiao, W. L.; Li, R. T.; Huang, S. X.; Pu, J. X.; Sun, H. D. Nat. Prod. Rep. 2008, 25, 871. (b) Shi, Y. M.; Xiao, W. L.; Pu, J. X.; Sun, H. D. Nat. Prod. Rep. 2015, 32, 367. (4) (a) Li, R. T.; Xiang, W.; Li, S. H.; Lin, Z. W.; Sun, H. D. J. Nat. Prod. 2004, 67, 94. (b) Lei, C.; Pu, J. X.; Huang, S. X.; Chen, J. J.; Liu, J. P.; Yang, L. B.; Ma, Y. B.; Xiao, W. L.; Li, X. N.; Sun, H. D. Tetrahedron 2009, 65, 164. (5) Li, X.; Cheong, P. H. Y.; Carter, R. G. Angew. Chem., Int. Ed. 2017, 56, 1704. (6) Wang, H.; Zhang, X.; Tang, P. Chem. Sci. 2017, 8, 7246. (7) Liu, D. D.; Sun, T. W.; Wang, K. Y.; Lu, Y.; Zhang, S. L.; Li, Y. H.; Jiang, Y. L.; Chen, J. H.; Yang, Z. J. Am. Chem. Soc. 2017, 139, 5732. (8) Yang, P.; Yao, M.; Li, J.; Li, Y.; Li, A. Angew. Chem., Int. Ed. 2016, 55, 6964. (9) Han, Y. X.; Jiang, Y. L.; Li, Y.; Yu, H. X.; Tong, B. Q.; Niu, Z.; Zhou, S. J.; Liu, S.; Lan, Y.; Chen, J. H.; Yang, Z. Nat. Commun. 2017, 8, 14233. (10) Li, J.; Yang, P.; Yao, M.; Deng, J.; Li, A. J. Am. Chem. Soc. 2014, 136, 16477. (11) Wang, L.; Wang, H.; Li, Y.; Tang, P. Angew. Chem., Int. Ed. 2015, 54, 5732. (12) You, L.; Liang, X. T.; Xu, L. M.; Wang, Y. F.; Zhang, J. J.; Su, Q.; Li, Y. H.; Zhang, B.; Yang, S. L.; Chen, J. H.; Yang, Z. J. Am. Chem. Soc. 2015, 137, 10120. (13) (a) Goh, S. S.; Chaubet, G.; Gockel, B.; Cordonnier, M. C. A.; Baars, H.; Phillips, A. W.; Anderson, E. A. Angew. Chem., Int. Ed. 2015, 54, 12618. (b) Chaubet, G.; Goh, S. S.; Mohammad, M.; Gockel, B.; Cordonnier, M. C. A.; Baars, H.; Phillips, A. W.; Anderson, E. A. Chem. Eur. J. 2017, 23, 14080. (c) Xiao, Q.; Ren, W. W.; Chen, Z. X.; Sun, T. W.; Li, Y.; Ye, Q. D.; Gong, J. X.; Meng, F. K.; You, L.; Liu, Y. F.; Zhao, M. Z.; Xu, L. M.; Shan, Z. H.; Shi, Y.; Tang, Y. F.; Chen, J. H.; Yang, Z. Angew. Chem., Int. Ed. 2011, 50, 7373. (14) Zhou, M.; Liu, Y.; Song, J.; Peng, X. G.; Cheng, Q.; Cao, H.; Xiang, M.; Ruan, H. Org. Lett. 2016, 18, 4558. (15) Song, J.; Liu, Y.; Zhou, M.; Cao, H.; Peng, X. G.; Liang, J. J.; Zhao, X. Y.; Xiang, M.; Ruan, H. L. Org. Lett. 2017, 19, 1196. (16) (a) Shi, Y. M.; Cai, S. L.; Li, X. N.; Liu, M.; Shang, S. Z.; Du, X.; Xiao, W. L.; Pu, J. X.; Sun, H. D. Org. Lett. 2016, 18, 100. (b) Liu, Y.; Tian, T.; Yu, H. Y.; Zhou, M.; Ruan, H. L. Fitoterapia 2017, 118, 38. (c) Li, F.; Zhang, T.; Sun, H.; Gu, H.; Wang, H.; Su, X.; Li, C.; Li, B.; Chen, R.; Kang, J. Molecules 2017, 22, 1931. (d) Wang, X.; Fronczek, R. F.; Chen, J.; Liu, J.; Ferreira, D.; Li, S.; Hamann, T. M. Molecules 2017, 22, 65. (e) Shi, Y. M.; Hu, K.; Pescitelli, G.; Liu, M.; Li, X. N.; Du, X.; Xiao, W. L.; Sun, H. D.; Puno, P. T. Org. Lett. 2018, 20, 1500. (f) Liu, M.; Luo, Y. Q.; Wang, W. G.; Shi, Y. M.; Wu, H. Y.; Du, X.; Pu, J. X.; Sun, H. D. Nat. Prod. Commun. 2015, 10, 2045. (g) Yeon, J. H.; Cheng, L.; He, Q. Q.; Kong, L. Y. Chin. J. Nat. Med. 2014, 12, 782. (h) Minh, P. T. H.; Lam, D. T.; Tien, N. Q.; Tuan, N. N.; Nhung, V. P.; Van Hai, N.; Van Kiem, P.; Nhiem, N. X.; Van Minh, C.; Ju, P. S.; Hyun, K. S. Nat. Prod. Commun. 2014, 9, 373. (17) (a) Duan, H.; Takaishi, Y.; Momota, H.; Ohmoto, Y.; Taki, T.; Jia, Y.; Li, D. Phytochemistry 2000, 53, 805. (b) Alanazi, A. M.; Al-Omar, M. A.; Abdulla, M. M.; Amr, A. E. G. E. Int. J. Biol. Macromol. 2013, 58, 245. (18) (a) Clardy, J.; Walsh, C. Nature 2004, 432, 829. (b) Li, J. W. H.; Vederas, J. C. Science 2009, 325, 161. (19) Shi, Y. M.; Yang, J.; Xu, L.; Li, X. N.; Shang, S. Z.; Cao, P.; Xiao, W. L.; Sun, H. D. Org. Lett. 2014, 16, 1370. (20) Wang, J. R.; Kurtan, T.; Mandi, A.; Guo, Y. W. Eur. J. Org. Chem. 2012, 2012, 5471. (21) Flack, H. D.; Bernardinelli, G. Acta Crystallogr., Sect. A: Found. Crystallogr. 1999, 55, 908. (22) Van Duyne, G. D.; Standaert, R. F.; Karplus, P. A.; Schreiber, S. L.; Clardy, J. Science 1991, 252, 839.

hydrophobic binding pocket between FK506 and Phe36 and Ile99, while just one hydrogen bond interaction between compound 1 and Tyr82 and a hydrophobic binding pocket between 1 and Phe36 and Phe99 were found. Compound 1 possessed less binding pocket sites and smaller molecule size compared to that of tacrolimus, which helps us justify that the moderate immunosuppressive activity of 1 and FKBP12 is very likely a target of 1. The above speculation remains to be validated by biophysical assays in further research. In summary, schincalactones A (1) and B (2), featuring a 5/5/ 6/11/3 ring system and possessing a unique 13-membered carbon ring in their structures, are the first examples in the SNT family. A biosynthetic pathway of 1 and 2 was proposed. Our bioassays provide further evidence that SNTs have potent immunosuppressive activities. The discovery of 1 and 2 enriches the diversity of the SNT family and may attract increased interest from the biosynthetic, synthetic, and pharmacological communities for further investigation.

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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.8b00889. Experimental details and full NMR, HRESIMS, ECD, UV, and IR spectra of 1−3 (PDF) Accession Codes

CCDC 1830622 and 1830624 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], 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

Junjun Liu: 0000-0001-9953-8633 Han-Li Ruan: 0000-0003-0882-1009 Author Contributions †

J.S. and M.Z. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by the Natural Science Foundation of China (Nos. 31770380, 31270394, and 21572073) and the Fundamental Research Funds for the Central Universities (No. 2016YXMS150). We are grateful to Professor Shuming Li from Philipps University of Marburg for his helpful suggestions on the biogenetic pathway, and Mr. Xianggao Meng from Central China Normal University for X-ray analysis. Thanks are also given to the staff at the Analytical and Testing Center of Huazhong University of Science and Technology for collecting the spectroscopic data.

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REFERENCES

(1) An Editorial Committee of Flora of China. Flora of China; Science Press and Missouri Botanical Garden Press: Beijing, 2008; Vol. 7, p 39. D

DOI: 10.1021/acs.orglett.8b00889 Org. Lett. XXXX, XXX, XXX−XXX