Cascarinoids A–C, a Class of Diterpenoid Alkaloids with Unpredicted

Letter Cite This: Org. Lett. 2018, 20, 228−231

pubs.acs.org/OrgLett

Cascarinoids A−C, a Class of Diterpenoid Alkaloids with Unpredicted Conformations from Croton cascarilloides Xin-Hua Gao,†,‡,⊥ Yan-Sheng Xu,§,⊥ Yao-Yue Fan,† Li-She Gan,∥ Jian-Ping Zuo,*,†,§ and Jian-Min Yue*,† †

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People’s Republic of China ‡ University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, People’s Republic of China § Laboratory of Immunology and Virology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, People’s Republic of China ∥ Institute of Modern Chinese Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China S Supporting Information *

ABSTRACT: Cascarinoids A−C (1−3), a new class of diterpenoid alkaloids with unpredicted conformations, were isolated and structurally characterized from Croton cascarilloides. It was demonstrated that the dispersion interaction might be one of the main contributors that stabilized the folded conformations for compounds 1−3. Compounds 2 and 3 showed moderate immunosuppressive activity against T and/or B lymphocyte cells.

T

para-disubstituted benzene group. The 13C NMR data (Table S1) with the aid of DEPT and HSQC experiments resolved 28 carbon resonances including those from a para-disubstituted benzene, two double bonds, and two carbonyls (δC 210.2 and 170.7). The aforementioned functionalities accounted eight out of the 14 DBEs in the molecule, and the remaining ones thus required the attendance of six additional rings in compound 1.

he plants of the Croton genus (Euphorbiaceae) are distributed widely in the tropical and subtropical areas of the world.1 Many plants of this genus have long been used as folk medicine to treat diseases such as cancer, diabetes, hyperlipidemia, hypertension, and malaria.2 Chemical investigations on the Croton plants led to the isolation of diverse compound classes, for instance, diterpenoids,3 polyphenols,4 alkaloids,5 and flavonoids.6 C. cascarilloides, a shrub plant, grows mainly in the region of southern Asia.1 Previous chemical studies on the plants of C. cascarilloides collected from Okinawa, Japan, afforded a number of crotofolane diterpenoids.3a−d In our continuous search for structurally interesting and bioactive important components from the Croton species,7 three compounds, cascarinoids A−C (1−3) representing the first example of crotofolane diterpenoid alkaloids, were isolated from the twigs and leaves of C. cascarilloides, which is native to the Yunnan Province of China. Their structures were established by spectroscopic data, single-crystal X-ray crystallography, and electronic circular dichroism (ECD) data. Presented herein are the isolation, structural determination, and biological evaluation of these diterpenoid alkaloids. Cascarinoid A (1), colorless crystals, was assigned a molecular formula C28H31NO5 on the basis of the (+)-HRESIMS protonated molecular ion peak at m/z 462.2285 [M + H]+ (calcd 462.2280) with 14 double-bond equivalents (DBEs). The IR spectrum implied the presence of hydroxy (3431 cm−1), keto (1745 cm−1), and amide (1653 cm−1) functionalities. Analysis of the 1H NMR data (Table S1) revealed the presence of two singlet methyls (δH 1.05 and 2.03, s, each 3H), one doublet methyl (δH 1.04, d, J = 7.10 Hz), an exocyclic double bond (δH 4.97 and 5.18), and a characteristic © 2017 American Chemical Society

The framework of diterpenoid alkaloid 1 was constructed by analysis of its 2D NMR spectra. First, six spin-coupling segments a−f as depicted in bold bonds were identified by the 1H−1H COSY correlations (Figure 1A). These fragments, the quaternary carbons and the heteroatoms were then put together by interpretation of the HMBC spectrum (Figure 1A), in which the multiple HMBC correlation networks of H3-20/C5, C-6, and C-7; H3-19/C-1; H3-17/C-8, C-15, and C-16; HReceived: November 19, 2017 Published: December 18, 2017 228

DOI: 10.1021/acs.orglett.7b03592 Org. Lett. 2018, 20, 228−231

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Figure 1. Key 2D NMR correlations for cascarinoid A (1). Figure 2. X-ray structures of 1 and 2.

13/C-1, C-11, C-12, and C-14; H-5/C-4 and C-14; H-7 and H9/C-8; and H-3/C-4; H2-18/C-11and C-12 allowed the establishment of the down-left part of the molecule, a tricyclic ring system with appendages that feature the carbon scaffold of crotofolane-type diterpenoids.8 The up-right part was identified as a 4-hydroxyphenylethyl amino moiety by the NMR data and the HMBC correlations from H-2′ to C-3′ and C-4′ (or C-8′), which was then connected with the C-9 and C-16 via the nitrogen atom to furnish an α, β-unsaturated γ-lactam ring by the key HMBC correlations from H-1′ to C-9 and C-16. The attendances of 4,14- and 5,6-epoxides were suggested by the strongly shielded chemical shifts of C-4, C-5, C-6, and C-14 to satisfy the requirements for the remaining two DBEs. The planar structure of 1 was thus defined as an unprecedented crotofolane-type diterpene alkaloid. The relative configuration of 1 was established by NOESY correlations (Figure 1B) in which the correlations of H-13 with H-11β, H-9, and H3-20; H-5 with H-3β and H3-20; H3-19 with H-3β indicated that these protons were on the same side of the molecule and were randomly assigned to be β-oriented. The NOESY correlations of H-7/H-18a and H-11α/H-18b thus suggested that H-7 was α-oriented. The orientation of 4,14epoxide was left unassigned due to the lack of the available NOESY correlations. Fortunately, the suitable quality crystals were obtained from the recrystallization of 1 in methanol at ambient temperature, which allowed a successful performance of X-ray crystallography study using Cu Kα radiation (λ = 1.54178 Å). Analysis of the X-ray data (Figure 2) not only completed the assignment of the relative configuration for 1 but also unambiguously determined its absolute configuration as 2R,4S,5S,6S,7S,9S,13S,14R on the basis of the perfect Flack parameter [−0.09(5)].9 Cascarinoid B (2) was obtained as colorless crystals. It possessed the same molecular formula C28H31NO5 with compound 1 as established by the (+)-HRESIMS ion peak at m/z 462.2283 [M + H]+ (calcd 462.2280). The 13C NMR spectrum with DEPT experiments showed the presence of two double bonds, two carbonyls, and one phenyl group, which accounted for eight DBEs and required the attendance of six additional rings in compound 2. The severely shielded chemical shifts assignable to an oxygenated methine carbon at δ 64.9 and

three oxygenated quaternary carbons at δ 56.4, 62.9, and 68.4 suggested the presence of a trisubstituted and a tetrasubstituted epoxide moieties in compound 2 (Table S1). Further examination of the 2D NMR spectra of 2, especially the HMBC data (Figures S2 and S17), indicated that it shared the same planar structure with compound 1, and this is consistent with their similar 1D NMR patterns. The major changes of the chemical shifts for the protons (H-3α, H-3β, H-7, and H3-19) and carbons (C-1, C-2, C-3, C-4, C-5, C-6, C-7, C-12, C-13, C18, and C-19) of two stereoisomers 1 and 2 occurred mainly in the A and B rings, indicating that the stereochemistries of the A and/or B ring of two compounds are different. In the NOESY spectrum of 2 (Figure 3), the mutual correlations of H-9 with H-13 and H-10β; H-13 with H3-20;

Figure 3. Selected NOESY correlations for 2 and 3.

and H-5 with H-3β and H3-20 indicated that these protons were on the same side of the molecular plane and were βdirected. The NOESY correlation networks of H-10α with H18b; H-18a with H-7 and H3-19; and H3-19 with H-3α suggested that they were α-oriented. The stereochemistry of the 4,14-epoxide was uncertain due to the lack of reliable NOESY correlations. To complete the structural assignment for 229

DOI: 10.1021/acs.orglett.7b03592 Org. Lett. 2018, 20, 228−231

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Organic Letters compound 2, we steered our attention to single-crystal X-ray crystallography, and suitable quality crystals were then obtained in acetonitrile at room temperature after many recrystallization solvents and conditions were tried. The relative configuration of 2 was finally established by the X-ray crystallography study (Figure 2), but its absolute stereochemistry was undetermined due to the relatively poor Flack parameter [−0.4(2)] for compound 2.9 The (+)-HRESIMS analysis of cascarinoid C (3) gave a molecular formula C28H31NO6 based on the protonated ion peak at m/z 478.2217 [M + H]+ (calcd 478.2230), which is 16 mass units more than that of compound 2 and suggestive of an oxygenated derivative of 2. Comparison of its 1D NMR data (Table S1) with those of compound 2 showed that the obvious changes of the chemical shifts were appeared around the C-9 at the C and D rings, indicating that the C-9 (δC 93.2) of compound 3 was oxygenated to form a hemiketal amine. This deduction was corroborated by the 2D NMR spectra, in particular the key correlations from H-7, H2-11, and H2-1′ to C9 in the HMBC spectrum (Figures S2 and S33). The relative configuration of compound 3 was fixed to be identical with that of 2 by the coupling constants of the key protons and the NOESY correlations (Figure 3). The mutual NOESY correlations of H-13/OH-9 and H3-20 and H-5/H-3β and H3-20 indicated that these protons were on the same side of the molecular plane and were assigned to be β-directed. The NOESY correlation networks of H-18a/H-7 and H3-19, H3-19/ H-3α, and H-10α/H-18b suggested that they were α-oriented. The chemical shifts of the carbons in the rings A and B (especially C-1, C-2, C-3, C-6, C-13, and C-14) of 3 were very similar to those of 2, indicating that the 4,14-epoxide ring was also β-directed. The tendencies of ECD curves of compounds 2 and 3 at the range ca. 200−270 nm (Figure 4) were similar to that of

Figure 5. Newman projections along the C-1′/C-2′ bond for the observed (A) and predicted (B) conformations of compound 1 (Dit stands for the diterpenoid core and Ph stands for the benzene ring).

conformers for compound 1 were analyzed by a 1D potential energy surface scanning (PES) method on the dihedral angle of C(3′)−C(2′)−C(1′)−N(1) with and without dispersion correction at the HF/STO-3G and B3LYP/6-31G level in the Gaussian 09 software package, respectively. The relaxed PES scans were performed in internal redundant coordinates with 36 steps of 10 degrees increasing in the dihedral angle, in which the relative energy levels of the extended conformers in each scan were set as 0 kcal/mol. The results showed that the dispersion interactions led to the decreased tendency of relative energy ΔE as the extended conformers rotated to both directions (Figure 6). The lowest relative energy was appeared

Figure 6. 1D PES scan on the dihedral angle of C(3′)−C(2′)−C(1′)− N(1) of 1 with (red dotted lines) and without (black dotted lines) dispersion correction at the HF/STO-3G level (A) and B3LYP/6-31G level (B).

at a dihedral angle of C(3′)−C(2′)−C(1′)−N(1) about −52°, which matched the favored folded conformation for the solid state of compound 1 as indicated by X-ray diffraction. The aforementioned analysis suggested that the dispersion interaction might be one of the main driving forces that stabilized the folded conformations for compounds 1−3.12 Immunosuppressive agents have been widely used in clinical practice for organ transplant and other immunologicalassociated ailments.13 Natural products have been an important source of immunosuppressive agents and have attracted broad interest from the organic chemistry community.14 Compounds 1−3 were evaluated for immunosuppressive activity against the proliferation of T and B lymphocyte cells in vitro with cyclosporin A (CsA) as the positive control.14a,15 The test results (Table 1; Figure S1) showed that compounds 2 and 3 exhibited moderate activities against the ConA-induced proliferation of T lymphocyte cells and/or LPS-induced proliferation of B lymphocyte cells with IC50 values ranging from 6.14 to 16.27 μM. The results indicated that the presence

Figure 4. CD spectra of 1−3.

compound 1. The similar tendency of ECD curves and the cooccurrence with compound 1 as well as the biogenetic consideration allowed the assignment of the absolute configurations of compounds 2 and 3 as depicted.10 The biosynthetic origin of this compound class could be traced back to crotofolane diterpenoids, which underwent aminolysis and oxidative modifications to produce compounds 1−3, respectively (see details in Scheme S1). It is thought-provoking that compounds 1−3 took unpredicted conformations (Figure 5) both in the solid state and solution, in which the benzene ring was folded with the α,β-unsaturated γ-lactam (conformer A) instead of the predicted far away arrangement (conformer B).11 To seek the inherent driving forces for these unpredicted conformations, the conformational conversions of the folded and extended 230

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1047−1054. (c) Kawakami, S.; Toyoda, H.; Harinantenaina, L.; Matsunami, K.; Otsuka, H.; Shinzato, T.; Takeda, Y.; Kawahata, M.; Yamaguchi, K. Chem. Pharm. Bull. 2013, 61, 411−418. (d) Kawakami, S.; Matsunami, K.; Otsuka, H.; Shinzato, T.; Takeda, Y.; Kawahata, M.; Yamaguchi, K. Tetrahedron Lett. 2010, 51, 4320−4322. (e) Ramos, F.; Takaishi, Y.; Kashiwada, Y.; Osorio, C.; Duque, C.; Acuña, R.; Fujimoto, Y. Phytochemistry 2008, 69, 2406−2410. (f) Adelekan, A. M.; Prozesky, E. A.; Hussein, A. A.; Ureña, L. D.; van Rooyen, P. H.; Liles, D. C.; Meyer, J. J. M.; Rodríguez, B. J. Nat. Prod. 2008, 71, 1919−1922. (4) Cai, Y.; Evans, F. J.; Roberts, M. F.; Phillipson, J. D.; Zenk, M. H.; Gleba, Y. Y. Phytochemistry 1991, 30, 2033−2040. (5) (a) Ravanelli, N.; Santos, K. P.; Motta, L. B.; Lago, J. H. G.; Furlan, C. M. S. Afr. J. Bot. 2016, 102, 153−156. (b) Burnell, R.; Chapelle, A.; Bird, P. J. Nat. Prod. 1981, 44, 238−238. (6) (a) Zou, G. A.; Su, Z. H.; Zhang, H. W.; Wang, Y.; Yang, J. S.; Zou, Z. M. Molecules 2010, 15, 1097−1102. (b) Guerrero, M. F.; Puebla, P.; Carrón, R.; Martín, M. L.; Román, L. S. J. Pharm. Pharmacol. 2002, 54, 1373−1378. (7) (a) Zhang, D. D.; Zhou, B.; Yu, J. H.; Xu, C. H.; Ding, J.; Zhang, H.; Yue, J. M. Tetrahedron 2015, 71, 9638−9644. (b) Liu, C. P.; Xu, J. B.; Zhao, J. X.; Xu, C. H.; Dong, L.; Ding, J.; Yue, J. M. J. Nat. Prod. 2014, 77, 1013−1020. (c) Wang, G. C.; Zhang, H.; Liu, H. B.; Yue, J. M. Org. Lett. 2013, 15, 4880−4883. (8) (a) Chavez, K.; Compagnoneb, R.; Riina, R.; Briceño, A.; Gonzalez, T.; Squitieri, E.; Landaeta, C.; Soscrún, H.; Suárez, A. Nat. Prod. Commun. 2013, 8, 1679−1682. (b) Jogia, M. K.; Andersen, R. J.; Parkanyi, L.; Clardy, J.; Dublin, H. T.; Sinclair, A. R. E. J. Org. Chem. 1989, 54, 1654−1657. (c) Chan, W. R.; Prince, E. C.; Manchand, P. S.; Springer, J. P.; Clardy, J. J. Am. Chem. Soc. 1975, 97, 4437−4439. (9) Flack, H. D. Acta Crystallogr., Sect. A: Found. Crystallogr. 1983, 39, 876−881. (10) (a) Uchida, I.; Kuriyama, K. Tetrahedron Lett. 1974, 15, 3761− 3764. (b) Beecham, A. F. Tetrahedron 1972, 28, 5543−5554. (11) (a) Takahashi, O.; Kohno, Y.; Nishio, M. Chem. Rev. 2010, 110, 6049−6076. (b) Gardner, R. R.; Christianson, L. A.; Gellman, S. H. J. Am. Chem. Soc. 1997, 119, 5041−5042. (c) Burgstahler, A. W.; Ziffer, H.; Weiss, U. J. Am. Chem. Soc. 1961, 83, 4660−4661. (12) (a) Hwang, J.; Dial, B. E.; Li, P.; Kozik, M. E.; Smith, M. D.; Shimizu, K. D. Chem. Sci. 2015, 6, 4358−4364. (b) Kasavajhala, K.; Bikkina, S.; Patil, I.; MacKerell, A. D.; Priyakumar, U. D. J. Phys. Chem. B 2015, 119, 3755−3761. (c) Duan, G.; Smith, V. H., Jr.; Weaver, D. F. J. Phys. Chem. A 2000, 104, 4521−4532. (13) (a) Kahan, B. D. Nat. Rev. Immunol. 2003, 3, 831−838. (b) Meier-Kriesche, H. U.; Li, S.; Gruessner, R. W. G.; Fung, J. J.; Bustami, R. T.; Barr, M. L.; Leichtman, A. B. Am. J. Transplant. 2006, 6, 1111−1131. (c) Hong, J. C.; Kahan, B. D. Semin. Nephrol. 2000, 20, 108−125. (14) (a) Fan, Y. Y.; Gan, L. S.; Liu, H. C.; Li, H.; Xu, C. H.; Zuo, J. P.; Ding, J.; Yue, J. M. Org. Lett. 2017, 19, 4580−4583. (b) Fan, Y. Y.; Zhang, H.; Zhou, Y.; Liu, H. B.; Tang, W.; Zhou, B.; Zuo, J. P.; Yue, J. M. J. Am. Chem. Soc. 2015, 137, 138−141. (c) Wang, Y.; Liu, Q. F.; Xue, J.-J.; Zhou, Y.; Yu, H. C.; Yang, S. P.; Zhang, B.; Zuo, J. P.; Li, Y.; Yue, J. M. Org. Lett. 2014, 16, 2062−2065. (d) Zhu, Y.; Zhang, Q.; Li, S.; Lin, Q.; Fu, P.; Zhang, G.; Zhang, H.; Shi, R.; Zhu, W.; Zhang, C. J. Am. Chem. Soc. 2013, 135, 18750−18753. (e) Chooi, Y. H.; Fang, J.; Liu, H.; Filler, S. G.; Wang, P.; Tang, Y. Org. Lett. 2013, 15, 780−783. (f) Zhang, B.; Wang, Y.; Yang, S. P.; Zhou, Y.; Wu, W. B.; Tang, W.; Zuo, J. P.; Li, Y.; Yue, J. M. J. Am. Chem. Soc. 2012, 134, 20605− 20608. (g) Wu, X.; Stockdill, J. L.; Wang, P.; Danishefsky, S. J. J. Am. Chem. Soc. 2010, 132, 4098−4100. (15) Wan, J.; Zhu, Y. N.; Feng, J. Q.; Chen, H. J.; Zhang, R. J.; Ni, J.; Chen, Z. H.; Hou, L. F.; Liu, Q. F.; Zhang, J.; Yang, L.; Tang, W.; Yang, Y. F.; Nan, F. J.; Zhao, W. M.; Zuo, J. P. Int. Immunopharmacol. 2008, 8, 1248−1256.

Table 1. Immunosuppressive Tests of 1−3 ConA-induced T-cell proliferation

LPS-induced B-cell proliferation

compd

CC50(μM)

IC50(μM)

SIa

IC50(μM)

SIa

1 2 3

− 40.41 11.11

− − 6.14

− − 1.81

− 16.27 10.29

− 2.48 1.08

a

SI is determined as the ratio of the concentration of the compound that reduced cell viability to 50% (CC50) to the concentration of the compound needed to inhibit the proliferation by 50% relative to the control value (IC50). “−” stands for inactive.

of the hemiketal amine group at C-9 is crucial for the immunosuppressive activity of this compound class.

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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.7b03592. Tabulated NMR data of 1−3; experimental procedures; bioassay; X-ray crystallographic data for 1 and 2; and raw spectroscopic data including IR, MS, and NMR spectra for compounds 1−3 (PDF) Accession Codes

CCDC 1586400−1586401 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 Authors

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

Jian-Min Yue: 0000-0002-4053-4870 Author Contributions ⊥

X.-H.G. and Y.-S.X. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation (Nos. 21532007, U1302222) of the People’s Republic of China. We thank Prof. Y.-K. Xu of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences (CAS), for the identification of the plant material.

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REFERENCES

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DOI: 10.1021/acs.orglett.7b03592 Org. Lett. 2018, 20, 228−231