Cajanusflavanols A–C, Three Pairs of Flavonostilbene Enantiomers

Letter Cite This: Org. Lett. 2018, 20, 876−879

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Cajanusflavanols A−C, Three Pairs of Flavonostilbene Enantiomers from Cajanus cajan Qi-Fang He,†,‡,§ Zhen-Long Wu,†,‡,§ Xiao-Jun Huang,†,‡ Yuan-Lin Zhong,† Man-Mei Li,‡ Ren-Wang Jiang,†,‡ Yao-Lan Li,†,‡ Wen-Cai Ye,*,†,‡ and Ying Wang*,†,‡ †

Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China ‡ Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM & New Drugs Research, Jinan University, Guangzhou 510632, People’s Republic of China S Supporting Information *

ABSTRACT: Three pairs of new flavonostilbene enantiomers, cajanusflavanols A−C (1−3), along with their putative biogenetic precursors 4−6, were isolated from Cajanus cajan. Compound 1 possesses an unprecedented carbon skeleton featuring a unique highly functionalized cyclopenta[1,2,3-de]isobenzopyran-1-one tricyclic core. Compounds 2 and 3 are the first examples of methylene-unit-linked flavonostilbenes. Their structures with absolute configurations were elucidated by spectroscopic analyses, X-ray diffraction, and computational calculations. Compounds 1 and 2 exhibited significant in vitro anti-inflammatory activities. continuing studies, three pairs of new flavonostilbene enantiomers, cajanusflavanols A−C (1−3) (Figure 1), together with their putative biosynthetic precursors [cajanolactone A (4), pinostrobin (5), and 3-methoxy-5-hydroxystilbene (6)], were

F

lavonostilbenes are a small but growing group of natural products that features a stilbene moiety coupled with a flavonoid unit through various linkage patterns.1 To date, about 33 flavonostilbenes have been isolated from plants of genera Polygonum (Polygonaceae),1,2 Sophora (Leguminosae),3−6 Picea (Pinaceae),7 Gnetum (Gnetaceae),8,9 and Struthiola (Thymelaeaceae).10 Of particular note, due to the activities of the conjugated double bond in the characteristic 1,2-diphenylethylene backbone of stilbene, in most cases, the stilbene and flavonoid subunits of the reported flavonostilbenes were coupled through single C−C bond directly, or via a dihydropyran or a dihydrofuran ring, between the olefinic carbons in stilbenes and the aromatic ring in flavonoids.2,11,12 Recently, a complex flavonostilbene with an unprecedented rearranged flavanol skeleton fused to stilbene via a hexahydrocyclopenta[c]furan moiety was isolated from the rhizomes of Polygonum cuspidatum, suggesting the potential structural diversity and complexity of this class of natural products.2 Apart from their unique structural features, some members of flavonostilbenes exhibited pronounced anti-inflammatory,1,4 antioxidant,8 antibacterial,3,13 and antitumor activities,5 as well as reversal activity on multidrug resistance (MDR).6 The leaves of Cajanus cajan (Linn.) Millsp. (Leguminosae) were used as folk medicine in China mainly for the treatment of diabetes14 and avascular necrosis of the femoral head.15 As a part of our ongoing program for exploring structurally unique natural products with interesting bioactivities from medicinal plants distributed in southern China, our group had reported the isolation of a pair of enantiomeric stilbene dimers with a unique coupling pattern from the leaves of the title plant.16 In our © 2018 American Chemical Society

Figure 1. Chemical structures of (+)-1/(−)-1, (+)-2/(−)-2, and (+)-3/ (−)-3. Received: January 2, 2018 Published: January 22, 2018 876

DOI: 10.1021/acs.orglett.8b00010 Org. Lett. 2018, 20, 876−879

Letter

Organic Letters isolated from the plant. Structurally, compound 1 possesses an unprecedented carbon skeleton with a unique densely functionalized cyclopenta[1,2,3-de]isobenzopyran-1-one tricyclic core (rings A−C). Compounds 2 and 3 represent the first examples of flavonostilbenes featuring an unusual linkage pattern, in which the stilbene and flavonoid substructures were connected via a methylene group. In this Letter, we report the isolation, structural elucidation, hypothetical biogenetic pathway, and in vitro anti-inflammatory activities of 1−3. Cajanusflavanol A (1) was yielded as purple red block-shaped crystals. The molecular formula of 1 was determined to be C37H34O9 based on a quasi-molecular ion at m/z 645.2097 [M + Na]+ (calcd for C37H34O9Na, 645.2095) in its HR-ESI-MS. The UV spectrum of 1 exhibited absorption maxima at 207 and 289 nm. The IR spectrum implied the existence of hydroxyl (3451 cm−1) and carbonyl (1636 cm−1) groups, as well as aromatic rings (1487 and 1448 cm−1). The 1H and 13C NMR spectra of 1 displayed the signals for a ketone carbonyl (δC 196.9), an ester carbonyl (δC 168.4), two methoxyls [δH 3.93 and 3.63 (each 3H, s); δC 56.4 and 55.8], two methyls [δH 0.76 and 0.75 (each 3H, s); δC 27.6 and 25.9], as well as two monosubstituted benzene rings and two pentasubstituted benzene rings. Comprehensive analysis of 1H−1H COSY, HSQC, and HMBC spectra of 1 resulted in the unambiguous assignment of its 1H and 13C NMR signals as shown in Table 1.

Figure 2. Key 1H−1H COSY, HMBC, and NOESY correlations of 1.

(δC 87.8) and upfield shift of the carbonyl carbon C-15 (δC 168.4) could result in the establishment of the lactone ring B, which fused to the benzene ring A. Besides, the HMBC correlations between H-7 and C-18, as well as between H-17 and C-8 indicated that the C-17 of the isopentyl side chain was connected with C-7 via C−C bond to form a five-membered ring C, which fused to both rings A and B sharing the same joined carbon C-1 and established the substructure 1a (Figure 2). The HMBC cross peaks between H-2′ and C-4′/C-9′/C-2″/C-6″, between H-3′a and C-10′, between H-3′b and C-1″, between H6′ and C-8′/C-10′, between 5′−OH (δH 12.24) and C-5′, as well as between 7′-OMe (δH 3.63) and C-7′ suggested the presence of a flavanone unit 1b (Figure 2). A comparison of the NMR data of 1b with those of pinostrobin (5), a coexistence flavanone isolated by us in presented study, further confirmed the structure of the fragment 1b. Furthermore, the HMBC correlations between H16 and C-7′/C-9′ and between H-17 and C-8′ indicated that the substructures 1a and 1b were connected through C-16−C-8′ bond. Therefore, the planar structure of 1 was established as shown in Figure 2. In the NOESY spectrum of 1, the cross peaks between H3-19 and H-7/H-16 suggested that these protons were situated at the same side of ring C. Meanwhile, the NOE correlations between H-8 and H-17 indicated that the two protons were cofacial and located at another side of ring C (Figure 2). Finally, the structure and relative stereochemistry of 1 were completely established by a single-crystal X-ray diffraction experiment (Figure 3). Notably, according to the X-ray diffraction structure of 1, the steric interactions between the bulk substituents vicinal to the C-16− C-8′ bond indicated the existence of an axial chirality in the molecule. Besides, the existence of a centrosymmetric space

Table 1. NMR Data for 1 (in CDCl3, J in Hz)a no. 1 2 3 4 5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 a

δH

6.31 (s)

3.26 (dd, 11.4, 7.2) 3.97 (d, 11.4) 7.23 (d, 6.5) 7.35 7.35 7.35 7.23 (d, 6.5) 4.80 (d, 6.8) 2.63 (t, 7.0) 0.75 (s) 0.76 (s)

δC

no.

147.9 99.5 161.8 97.6 161.8

3-OH 5-OMe 1′ 2′ 3′

121.7 48.1

4′ 5′

87.8 137.6 127.2 128.7 129.2 128.7 127.2 168.4 41.2 60.0 72.5 25.9 27.6

6′ 7′ 8′ 9′ 10′ 1″ 2″ 3″ 4″ 5″ 6″ 5′-OH 7′-OMe

δH 9.70 (s) 3.63 (s) 4.68 (dd, 14.1, 2.4) a. 2.69 (dd, 17.3, 2.4) b. 3.40 (dd, 17.3, 14.1)

δC 55.8 80.3 41.0

196.9 163.2 6.17 (s)

7.08 7.35 7.35 7.35 7.08 12.24 (s) 3.93 (s)

92.9 164.5 112.5 159.8 103.6 136.0 127.7 128.9 130.6 128.9 127.7 56.4

Overlapped signals were reported without designating multiplicity.

Analysis of the 1H−1H COSY spectrum of 1 revealed the existence of four spin-coupling systems as drawn with blue bold lines in Figure 2. In the HMBC spectrum, correlations between H-4 and C-2/C-6, between H-7 and C-6/C-9, between H-8 and C-1/C-10/C-14, between H-16 and C-1/C-5/C-18, between H17 and C-19/C-20, between 3-OH (δH 9.70) and C-3, as well as between 5-OMe (δH 3.63) and C-5 suggested the existence of a stilbene moiety with an isopentyl side chain. Moreover, the obvious downfield shift of the sp3-hybridized tertiary carbon C-8

Figure 3. X-ray ORTEP drawing of 1. 877

DOI: 10.1021/acs.orglett.8b00010 Org. Lett. 2018, 20, 876−879

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Organic Letters

flavonostilbene. Detailed analysis of its 1H−1H COSY and HMBC spectra allowed the assembly of the similar structural fragments (3a, 3b, and methylene, Figure 4) to 2. Different from 2, the NMR spectra assigned to the flavanone moiety (3b) in 3 showed the signals due to a para-substituted benzene ring instead of the signals corresponding to a monosubstituted benzene ring in 2b. A comparison of the NMR data assigned to 3b with those of sakuranetin further confirmed the structure of 3b.17 In addition, the HMBC correlations between H-1‴ and C-3/C-5/ C-7′/C-9′ were observed, revealing that the CH2 unit bridged fragments 3a and 3b via C-4 and C-8′ positions, respectively. Thus, the gross structure of 3 was established as shown in Figure 4. Because of the barely measurable optical rotation values of 2 and 3, they were both presumed to be racemic mixtures. Subsequently, the chiral HPLC separation of 2 and 3 afforded two pairs of enantiomers, (+)-2 and (−)-2 as well as (+)-3 and (−)-3, appropriately in a ratio of 1:1, respectively. The absolute configurations of the resolved enantiomers were also determined by their experimental ECD spectra coupled with ECD calculation using the similar TDDFT method. Both of the experimental ECD spectra of (+)-2 and (+)-3 exhibited positive Cotton effect at 290 nm (π → π* absorption band), which was consistent with that of the flavanone with 2R configuration.18 Furthermore, the predicted ECD spectra of 2′R-2 and 2′R-3 revealed good agreement with the experimental ones of (+)-2 and (+)-3, respectively (Figures S2 and S3). Therefore, the absolute configurations of (+)-2 and (+)-3 were both established as 2′R. On the contrary, both of the experimental and calculated ECD spectra of (−)-2 and (−)-3 displayed similar signal intensity but opposite curve with those of (+)-2 and (+)-3, respectively, indicating the absolute configurations of (−)-2 and (−)-3 were 2′S. Compound 1 represents the first example of flavonostilbene whose stilbene and flavonoid substructures were linked through the isopentenyl side chain of the stilbene moiety and the aromatic ring of the flavonoid unit. Based on the findings of key biogenetic precursors in the plant, a hypothetical biosynthetic pathway of 1 was proposed as shown in Scheme 1. As a precursor,

group P21/c in its crystal structure, combined with its barely measurable optical rotation value, suggested that 1 was a racemic mixture. Subsequently, 1 was separated into two enantiomers, (+)-1 and (−)-1, by HPLC using a chiral column. The absolute configurations of the two enantiomers of 1 were further determined by comparison of their experimental ECD spectra with those predicted by time-dependent density functional theory (TDDFT) calculation at CAM-B3LYP/6-31+G(d) level (see the Supporting Information). As a result, the calculated ECD curves of 7S,8S,16R,16M,17R,2′S-1 and 7R,8R,16S,16P,17S,2′R-1 displayed good agreement with the experimental ones of (+)-1 and (−)-1 (Figure S1), respectively, which could establish their absolute configurations. The molecular formula of cajanusflavanol B (2) was deduced to be C32H28O6 on the basis of its HR-ESI-MS at m/z 507.1811 [M − H]− (calcd for C32H27O6, 507.1813). The UV absorption maxima at 208 and 293 nm as well as the IR absorption bands at 3424, 1629, 1593, and 1448 cm−1 suggested the presence of functional groups including hydroxyl, carbonyl, and aromatic ring in 2. Analysis of the 1H and 13C NMR data of 2 indicated the presence of a carbonyl, four benzene rings, a vinyl group, an oxygenated methine, two methylenes, and two methoxyls. The above spectral data suggested that 2 was also a flavonostilbene derivative. Based on the analysis of 1D and 2D NMR spectra, the 1 H and 13C NMR data of 2 were assigned and listed in Table S1 (see the Supporting Information). The correlations in its 1H−1H COSY spectrum established four spin systems in 2 (Figure 4). In the HMBC spectrum,

Scheme 1. Hypothetical Biosynthetic Pathway for 1 Figure 4. Key 1H−1H COSY and HMBC correlations of 2 and 3.

correlations between H-7 and C-2/C-6/C-9, between H-8 and C-1/C-10/C-14, and between 3-OMe (δH 3.39) and C-3 established a stilbene motif 2a. The structure of 2a was further confirmed by comparing its NMR data with those of the coisolated known stilbene 3-methoxy-5-hydroxystilbene (6). Besides, the HMBC correlations between H-2′ and C-4′/C-9′/ C-2″/C-6″, between H-3′a and C-10′/C-1″, between H-6′ and C-8′/C-10′, and between 7′-OMe (δH 3.74) and C-7′ assembled a flavanone moiety 2b, which was identical to 1b. Moreover, the HMBC correlations between H-1‴ and C-1/C-3/C-7′/C-9′ indicated that 2a and 2b were connected through C-2 and C-8′ via the remaining unassigned CH2 unit. Therefore, the planar structure of 2 was established. The molecular formula of cajanusflavanol C (3) was established as C32H28O7 by its HR-ESI-MS at m/z 523.1762 [M − H]− (calcd for C32H27O7, 523.1762). Similar to 2, the 1H and 13C NMR spectra of 3 also showed the characteristic signals for stilbene and flavanone moieties, as well as an additional methylene group, indicating that 3 was also a methylene linked

cajanolactone A (4) first underwent epoxidation and dehydration reactions to give intermediate A. Through deprotonation and dispersion, the resonance-stabilized free radicals B and C could be obtained.16 Radical C might subsequently involve a key intramolecular radical addition reaction to produce intermediate D with the 5/6/6-fused tricyclic framework.19 After the intermolecular nucleophilic addition and reduction between the intermediate D and flavonoid 5,20 the novel flavonostilbene 878

DOI: 10.1021/acs.orglett.8b00010 Org. Lett. 2018, 20, 876−879

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756−760. (b) Asakawa, Y. Bull. Chem. Soc. Jpn. 1970, 43, 2223−2229. (c) Asakawa, Y. Bull. Chem. Soc. Jpn. 1971, 44, 2761−2766. (2) Li, F.; Zhan, Z.; Liu, F.; Yang, Y.; Li, L.; Feng, Z.; Jiang, J.; Zhang, P. Org. Lett. 2013, 15, 674−677. (3) Wan, C. X.; Luo, J. G.; Ren, X. P.; Kong, L. Y. Phytochemistry 2015, 116, 290−297. (4) Kwon, J.; Basnet, S.; Lee, J. W.; Seo, E. K.; Tsevegsuren, N.; Hwang, B. Y.; Lee, D. Bioorg. Med. Chem. Lett. 2015, 25, 3314−3318. (5) Shour, S.; Iranshahy, M.; Pham, N.; Quinn, R. J.; Iranshahi, M. Nat. Prod. Res. 2017, 31, 1270−1276. (6) Ni, K.; Yang, L.; Wan, C.; Xia, Y.; Kong, L. Nat. Prod. Res. 2014, 28, 2195−2198. (7) Wada, S.; Yasui, Y.; Hitomi, T.; Tanaka, R. J. Nat. Prod. 2007, 70, 1605−1610. (8) Iliya, I.; Tanaka, T.; Ali, Z.; Iinuma, M.; Furusawa, M.; Nakaya, K.; Shirataki, Y.; Murata, J.; Darnaedi, D.; Matsuura, N.; Ubukata, M. Heterocycles 2003, 60, 159−166. (9) Ali, F.; Assanta, M. A.; Robert, C. J. Med. Food 2011, 14, 1289− 1297. (10) Ayers, S.; Zink, D. L.; Brand, R.; Pretorius, S.; Stevenson, D.; Singh, S. B. Nat. Prod. Commun. 2008, 3, 189−192. (11) Shen, T.; Wang, X. N.; Lou, H. X. Nat. Prod. Rep. 2009, 26, 916− 935. (12) Keylor, M. H.; Matsuura, B. S.; Stephenson, C. R. J. Chem. Rev. 2015, 115, 8976−9027. (13) Silva, L. N.; Zimmer, K. R.; Macedo, A. J.; Trentin, D. S. Chem. Rev. 2016, 116, 9162−9236. (14) Grover, J. K.; Yadav, S.; Vats, V. J. Ethnopharmacol. 2002, 81, 81− 100. (15) Yuan, H. PCT Int. Appl. WO 2000078324 A1 20001228, 2000. (16) Li, X. L.; Zhao, B. X.; Huang, X. J.; Zhang, D. M.; Jiang, R. W.; Li, Y. J.; Jian, Y. Q.; Wang, Y.; Li, Y. L.; Ye, W. C. Org. Lett. 2014, 16, 224− 227. (17) Liu, Y. L.; Ho, D. K.; Cassady, J. M.; Cook, V. M.; Baird, W. M. J. Nat. Prod. 1992, 55, 357−363. (18) Slade, D.; Ferreira, D.; Marais, J. P. J. Phytochemistry 2005, 66, 2177−2215. (19) Cichewicz, R. H.; Kouzi, S. A. Stud. Nat. Prod. Chem. 2002, 26, 507−579. (20) Cao, J. Q.; Huang, X. J.; Li, Y. T.; Wang, Y.; Wang, L.; Jiang, R. W.; Ye, W. C. Org. Lett. 2016, 18, 120−123. (21) Zraunig, A.; Pacher, T.; Brecker, L.; Greger, H. Phytochem. Lett. 2014, 9, 33−36. (22) Massaro, C. F.; Katouli, M.; Grkovic, T.; Vu, H.; Quinn, R. J.; Heard, T. A.; Carvalho, C.; Manley-Harris, M.; Wallace, H. M.; Brooks, P. Fitoterapia 2014, 95, 247−257. (23) Wollenweber, E.; Wehde, R.; Dörr, M.; Lang, G.; Stevens, J. F. Phytochemistry 2000, 55, 965−970. (24) Garo, E.; Hu, J. F.; Goering, M.; Hough, G.; O’Neil-Johnson, M.; Eldridge, G. J. Nat. Prod. 2007, 70, 968−973.

skeleton was formed and subsequently yielded 1. Compounds 2 and 3 are the first examples of methylene-linked flavonostilbenes, in which the stilbene and flavonoid substructures were connected via an additional CH2 unit. Interestingly, to the best of our knowledge, neither aromatic ring methylation stilbene nor flavonoid had ever been reported from plants of family Leguminosae.21−24 Actually, stilbenes characterized by Cmethylation on the aromatic ring are very rare in the plant kingdom.21 Although the reasonable flavanone (5) and stilbene (6) precursors were both isolated in our present study, however, the biosynthesis of 2 and 3 might undergo an unusual route, which was of great interest and needed to be addressed by further studies. Compounds 1−3 were evaluated for their abilities to inhibit lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells. As a result (Table S3), 1 and 2 displayed favorable inhibitory effects on NO production with IC50 values of 13.62 ± 0.49 and 17.52 ± 0.66 μM, respectively, whereas 3 exerted a moderate suppression of NO secretion and weak inhibitory activity against the proliferation of cells.

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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.8b00010. Detailed descriptions of the experimental procedure; UV, IR, MS, and NMR spectra for compounds 1−6; ECD calculations for 1−3 (PDF) Accession Codes

CCDC 1814247 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

Man-Mei Li: 0000-0003-2423-3119 Ren-Wang Jiang: 0000-0002-2163-1683 Wen-Cai Ye: 0000-0002-2810-1001 Author Contributions §

Q.-F.H. and Z.-L.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 81473117, 81622045, 81630095, and U1401225) and the Science and Technology Planning Project of Guangdong Province (No. 2016B030301004).

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REFERENCES

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DOI: 10.1021/acs.orglett.8b00010 Org. Lett. 2018, 20, 876−879