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Rhodomollanol A, a Highly Oxygenated Diterpenoid with a 5/7/5/5 Tetracyclic Carbon Skeleton from the Leaves of Rhododendron molle Junfei Zhou, Guanqun Zhan, Hanqi Zhang, Qihua Zhang, Ying Li, Yongbo Xue, and Guangmin Yao* Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China S Supporting Information *

ABSTRACT: A novel diterpenoid with an unprecedented carbon skeleton, rhodomollanol A (1), and a new grayanane diterpenoid, rhodomollein XXXI (2), were isolated from the leaves of Rhododendron molle. Their structures were elucidated using comprehensive spectroscopic methods and single-crystal X-ray diffraction. Compound 1 possesses a unique cis/trans/ trans/cis/cis-fused 3/5/7/5/5/5 hexacyclic ring system featuring a rare 7-oxabicyclo[4.2.1]nonane core decorated with three cyclopentane units. The plausible biosynthetic pathway for 1 was proposed. Compound 1 exhibited moderate PTP1B inhibitory activity.

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ricaceae plants are famous not only for their beautiful flowers but also for their structurally intriguing and bioactive diterpenoids components.1 The total synthesis of Ericaceae diterpenoids such as grayanotoxin III, kalmanol, and pierisformoside C has been attempted due to their complex, highly oxygenated, polycyclic carbon skeleton structures and significant bioactivities.2 Rhododendron molle G. Don, a deciduous shrub, is a well-known traditional Chinese medicine used to treat rheumatism, bruises, asthma, pain, psoriasis, and scabies.3 Previous phytochemical studies on R. molle have only focused on the flowers,4 roots,5 and fruits,6 and a total of 67 diterpenoids, belonging to seven carbon skeletons, grayanane,4a kalmane,4b seco-kalmane,4c 3,4-seco-grayanane,4d 1,10:2,3-disecograyanane,5a C-nor-D-homograyanane,6a and D-homograyanane,6b have been isolated. Some of them have showed significant anticancer,4d antiviral,5a,6b antinociceptive,5b immunomodulatory,5c and sodium channel antagonistic6c activities. However, there are no reports on the leaves of R. molle. Expecting the other plant parts to be chemically distinct from the previously well-studied flowers, fruits, and roots, the leaves of R. molle were investigated for the first time, leading to the isolation of a highly oxygenated diterpenoid with an unprecedented carbon skeleton, rhodomollanol A (1), and a new grayanane diterpenoid, rhodomollein XXXI (2) (Figure 1), as well as their

related biogenetic precursor, rhodojaponin III (3). Rhodomollanol A (1) possesses an unprecedented 5/7/5/5 tetracyclic diterpene carbon skeleton featuring a rare 7-oxabicyclo[4.2.1]nonane core fused with three cyclopentane moieties. In this paper, we report the isolation, structure elucidation, plausible biosynthetic pathways, and PTP1B inhibitory activities of 1−3. The leaves of R. molle collected at Qichun, Hubei Province of China, were powdered and extracted with 95% EtOH at room temperature. The extract was suspended in water and extracted successively with petroleum ether, CHCl3, EtOAc, and n-BuOH. The CHCl3 extract was fractionated by silica gel column chromatography (CC) and then purified by repeated reversedphase (RP) C18 silica gel and Sephadex LH-20 as well as RP C18 HPLC to yield three diterpenoids (1−3). Rhodomollanol A (1), [α]D20 −44.9 (c 0.1, MeOH), was obtained as a colorless prism, mp 187−188 °C (MeOH). The molecular formula of 1 was determined to be C20H28O5 by the quasimolecular ion at m/z 371.1825 [M + Na]+ (calcd for C20H28O5Na, 371.1834) in the HRESIMS and 13C NMR data, indicating seven degrees of unsaturation. The 1H NMR spectrum (Table 1) of 1 showed resonances attributed to an olefinic proton at δH 5.49 (s, H-15), five oxygenated methines at δH 3.82 (d, J = 3.2, 0.6 Hz, H-2), 3.17 (dd, J = 3.2 Hz, H-3), 3.86 (dd, J = 10.6, 5.8 Hz, H-6), 4.20 (ddd, J = 12.0, 7.8, 5.6 Hz, H-12), and 4.53 (s, H-14), as well as four methyls at δH 1.88 (s, H3-17), 1.10 (s, H318), 1.29 (s, H3-19), and 1.44 (s, H3-20). The 13C NMR and DEPT spectra (Table 1) indicated the presence of four methyls, two methylenes, nine methines (five oxygenated and one olefinic), and five quaternary carbons (two oxygenated and one olefinic). A double bond accounts for one degree of unsaturation,

Figure 1. Structures of compounds 1 and 2.

Received: June 19, 2017 Published: July 13, 2017

© 2017 American Chemical Society

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DOI: 10.1021/acs.orglett.7b01863 Org. Lett. 2017, 19, 3935−3938

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Organic Letters Table 1. 1H (400 MHz) and 13C NMR (100 MHz) Spectroscopic Data for 1 in CD3OD 1 no.

δH (J, Hz)

1α 2α 3α 4 5 6α 7α 7β 8 9β 10 11α 11β 12α 12β 13 14 15 16 17 18 19 20

2.29, d (0.6) 3.82, dd (3.2, 0.6) 3.17, d (3.2)

3.86, dd (10.6, 5.8) 1.87, dd (13.2, 5.8) 2.05, dd (13.2, 10.6) 3.46, dd (12.0, 6.4) 1.72, ddd (12.0, 6.4, 5.6) 1.63, q (12.0)

2 δC

δH (J, Hz)

δC

55.9 59.5 64.7 48.4 82.6 73.9 43.1

2.33, d (0.6) 3.79, dd (3.0, 0.6) 3.18, d (3.0)

54.8 60.8 65.3 48.7 81.1 73.6 40.5

61.7 49.0 88.2 35.4 74.8

4.20, ddd (12.0, 7.8, 5.6) 2.46, d (7.8) 4.53, s 5.49, s 1.88, s 1.29, s 1.10, s 1.44, s

64.4 88.4 128.0 148.3 18.1 20.7 20.6 22.4

3.86, dd (9.2, 6.4) 1.88, dd (13.8, 6.4) 1.85, dd (13.8, 9.2) 1.62, d (6.8) 1.66, m 1.53, m 1.99, m 1.41, m 2.44, t (2.4) 4.16, s 4.90, q (1.6) 1.71, d (1.6) 1.25, s 1.15, s 1.43, s

55.4 50.0 79.4 23.1 24.8 57.0 80.2 132.1 138.7 15.4 20.3 21.5 30.6

(δC 88.2) and C-14 (δC 88.4) were obviously shifted downfield compared to a typical hydroxylated quaternary carbon and hydroxylated methine, respectively; thus, an oxygen bridge must exist between C-10 and C-14. The above deductions were supported by the molecular formula and resulting degrees of saturation of 1. Consequently, the planar structure of rhodomollanol A (1) was established to be a 3/5/7/5/5/5 hexacyclic skeleton (Figure 2). The relative configuration of rhodomollanol A (1) was determined by the coupling constants and NOESY data analyses (Figure 3). The α-orientation of H-1 was randomly assigned in 1.

and the remaining six degrees of unsaturation suggested the presence of a hexacyclic system in 1. As shown in Figure 2, 1H−1H COSY and HSQC data suggested the presence of four partial structures in 1, (a) C-1/C-

Figure 2. Selected 1H−1H COSY and HMBC correlations of rhodomollanol A (1).

2/C-3, (b) C-6/C-7, (c) C-9/C-11/C-12/C-13, and (d) C-14/ C-15. The HMBC data were used to construct the planar structure of 1 from these four partial structures. HMBC correlations from two gem-dimethyls (H3-18/H3-19) to C-3/ C-4/C-5 and correlations from H-3 to C-4 and C-5 suggested the direct connections of C-3, C-5, C-18, and C-19 toward C-4. The connection of C-1 and C-6 through C-5 was confirmed by the HMBC correlations from H-1 to C-5/C-6, H-6 to C-1/C-5, from H2-7 to C-5. HMBC correlations from the methyl singlet H3-20 to C-1/C-9/C-10 established the connections of C-1, C-9, and C-20 to the oxygenated quaternary carbon C-10. HMBC correlations from H2-7 to C-8/C-9/C-13/C-14, H-9 to C-7/C8/C-14, H-13 to C-7/C-8/C-9/C-14, and from H-14 to C-7/C13 defined the connections of C-7, C-9, C-13, and C-14 to C-8. The connections of C-13/C-15/C-17 to quaternary carbon C-16 were indicated by the HMBC correlations of methyl singlet H317 to C-13/C-15/C-16. The chemical shifts of C-2 (δC 59.5) and C-3 (δC 64.7) suggested the presence of a 2,3-epoxy group.4a In addition, C-10

Figure 3. Selected NOESY correlations of rhodomollanol A (1).

The small coupling constant J = 0.6 Hz between H-1α and H-2 indicated that their dihedral angle is about 90, thus, H-2 was determined to be α-orientated.4a The smaller coupling constant J = 3.2 Hz between H-2α and H-3 suggested the α-orientation of H-3.4a The cross peak of H-1α and H-6 in the NOESY spectrum revealed the α-orientation of H-6. The NOESY correlations between H-6α and H-14 established the orientation of H-14. The NOESY correlations between H-6α and H-7α, and H-7β and H9 suggested that H-9 was β-oriented. The β-orientation of H-12 3936

DOI: 10.1021/acs.orglett.7b01863 Org. Lett. 2017, 19, 3935−3938

Letter

Organic Letters

ing a rare 7-oxabicyclo[4.2.1]nonane core and three cyclopentane units. The biosynthetic pathway of rhodomollanol A (1) could be traced back to the grayanane diterpenoid rhodojaponin III (3).4a As illustrated in Scheme 1, the loss of H2O in 3 could

was determined by NOESY correlation between H-9β and H-12. Moreover, the NOESY correlations between H-13 and H2-7 confirmed the β-orientation of H-13. Finally, the structure of rhodomollanol A (1) was confirmed by single-crystal X-ray diffraction with Cu Kα radiation (Figure 4). Rhodomollanol A (1) possesses a unique cis/trans/trans/cis/

Scheme 1. Proposed Biosynthetic Pathway of 1

Figure 4. X-ray crystal structure of rhodomollanol A (1).

cis-fused 3/5/7/5/5/5 hexacyclic ring system composed of a rare 7-oxabicyclo[4.2.1]nonane core and three cyclopentane units. The resulting Flack parameter −0.04(19)7 assigned the absolute configuration of 1 as 1R,2S,3R,5R,6R,8S,9R,10R,12S,13R,14S. Rhodomollein XXXI (2) was isolated as a colorless prism, mp 235−236 °C. On the basis of the [M + Na]+ peak ion at m/z 373.1990 in HRESIMS and 13C NMR, the molecular formula of compound 2 was established as C 20 H 30 O 5 (calcd for C20H30O5Na, 373.1991), exhibiting six degrees of unsaturation. The 1H and 13C NMR data (Table 1) of compound 2 resembled those of rhodojaponin III (3),4a except for the presence of a trisubstituted double bond (δH 4.90, q, J = 1.6 Hz, H-15; δC 132.1, C-15; 138.7, C-16) in 2, replacing a methylene (δH 1.79, d, J = 15.0 Hz, H-15β; 1.95, d, J = 15.0 Hz, H-15α; δC 59.8, C-15) and an oxygenated quaternary carbon (δC 81.4, C-16) in 3. Thus, compound 2 should be a dehydration product of 3. In the HMBC spectrum, the correlations from H3-17 (δH 1.71, d, J = 1.6 Hz) to C-13/C-15/C-16 revealed the double bond was located at C-15 and C-16. The structure of compound 2 was finally determined to be 2β,3β-epoxy-5β,6β,10α,14β-tetrahydroxygrayan-15(16)ene by single-crystal X-ray diffraction (Figure 5), and the absolute configuration was assigned as 1S,2S,3R,5R,6R,8S,9R,10R,13S,14R by the resulting Flack parameter −0.0(1).7 Rhodomollanol A (1) is a highly oxygenated diterpenoid with an unprecedented 5/7/5/5 tetracyclic carbon skeleton, possess-

produce the new grayanane diterpenoid 2. Compound 3 and its isomer 4 could be formed by the addition of H2O to 2 under the Markovnikov rule and anti-Markovnikov rule, respectively. Then, a carbocation center is formed at C-15 in 6 by the protonation of 15-OH in 4 and then loss of H2O in 5. The migration of the 12,13-bond to C-15 could be accomplished by an enzymatic Wagner−Meerwein rearrangement8 in 6, which is the key step to form a new carbon−carbon bond between C-12 and C-15 in 7. Consequently, the carbocation center created at C-13 in intermediate 7 could be neutralized by the formation of a new oxygen cation bridge between C-13 and C-14. Then, 10-OH attacks C-14 in 8 to trigger a nucleophilic addition reaction to form a new oxygen cation bridge between C-10 and C-14 in 9. Finally, rhodomollanol A (1) is formed by the successive deprotonation, dehydration, and oxidation of 9. Rhodomollanol A (1) represents a new 5/7/5/5 tetracyclic diterpene carbon skeleton, and the name “rhodomollane” is suggested for this new skeleton type. The new rhodomollane skeleton is considered to be formed by the migration of the 12,13-bond to C-15 in grayanane rather than the migration of the 7,8-bond to C-14 in kalmane, based on the β-orientation of H-13 (the biogenetic label is H-15) in 1 (Scheme 2). Actually, H-13 is α-oriented in grayanane and kalmane (formed by the migration Scheme 2. Biogenetic Relationships of the Rhodomollane, Grayanane, and Kalmane Diterpene Carbon Skeletons

Figure 5. X-ray crystal structure of rhodomollein XXXI (2). 3937

DOI: 10.1021/acs.orglett.7b01863 Org. Lett. 2017, 19, 3935−3938

Letter

Organic Letters of the 8,9-bond to C-14 in grayanane)8b diterpenoids. The migration of the 7,8-bond to C-14 in kalmane will produce a 13epi-rhodomollane. The protein tyrosine phosphatase 1B (PTP1B) has been recognized as a potential target for the treatment of type 2 diabetes and obesity.9 Since grayanane diterpenoids have been reported to possess PTP1B inhibitory activities,10 compounds 1−3 were evaluated for their PTP1B inhibitory activities in vitro.10 Preliminary screening results (Table 2) at the

ORCID

Yongbo Xue: 0000-0001-9133-6439 Guangmin Yao: 0000-0002-8893-8743 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by Fundamental Research Funds for the Central Universities (HUST: 2016YXMS148) and the National Natural Science Foundation of China (81001368 and 31170323). We thank the Analytical and Testing Center at Huazhong University of Science and Technology for spectroscopic data collection.

Table 2. PTP1B Inhibitory Activities of Compounds 1−3 compd

inhibitiona (%)

IC50b (μM)

1 2 3 oleanolic acidc

87.4 31.5 13.4 101.3

24.32 ± 0.56 >200 >200 4.54 ± 0.25

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(1) Li, Y.; Liu, Y. B.; Yu, S. S. Phytochem. Rev. 2013, 12, 305. (2) (a) Kan, T.; Hosokawa, S.; Nara, S.; Oikawa, M.; Ito, S.; Matsuda, F.; Shirahama, H. J. Org. Chem. 1994, 59, 5532. (b) Borrelly, S.; Paquette, L. A. J. Am. Chem. Soc. 1996, 118, 727. (c) Chow, S.; Kreß, C.; Albæk, N.; Jessen, C.; Williams, C. M. Org. Lett. 2011, 13, 5286. (3) State Administration of Traditional Chinese Medicine. Zhong Hua Ben Cao; Shanghai Science and Technology Publishing Company: Shanghai, 1999; Vol. 6, pp 5266. (4) (a) Klocke, J. A.; Hu, M. Y.; Chiu, S.; Kubo, I. Phytochemistry 1991, 30, 1797. (b) Chen, S. N.; Zhang, H. P.; Wang, L. Q.; Bao, G. H.; Qin, G. W. J. Nat. Prod. 2004, 67, 1903. (c) Zhou, S. Z.; Yao, S.; Tang, C. P.; Ke, C. Q.; Li, L.; Lin, G.; Ye, Y. J. Nat. Prod. 2014, 77, 1185. (d) Wang, S. J.; Lin, S.; Zhu, C. G.; Yang, Y. C.; Li, S.; Zhang, J. J.; Chen, X. G.; Shi, J. G. Org. Lett. 2010, 12, 1560. (e) Zhong, G. H.; Hu, M. Y.; Wei, X. Y.; Weng, Q. F.; Xie, J. J.; Liu, J. X.; Wang, W. X. J. Nat. Prod. 2005, 68, 924. (f) Zhang, Z. R.; Zhong, J. D.; Li, H. M.; Li, H. Z.; Li, R. T.; Deng, X. L. J. Asian Nat. Prod. Res. 2012, 14, 764. (g) Chen, S. N.; Bao, G. H.; Wang, L. Q.; Qin, G. W. Zhongguo Tianran Yaowu 2013, 11, 525. (h) Zhou, S. Z.; Tang, C. P.; Ke, C. Q.; Yao, S.; Lin, G.; Ye, Y. Chin. Chem. Lett. 2017, 28, 1205. (5) (a) Li, Y.; Liu, Y. B.; Zhang, J. J.; Li, Y. H.; Jiang, J. D.; Yu, S. S.; Ma, S. G.; Qu, J.; Lv, H. N. Org. Lett. 2013, 15, 3074. (b) Li, Y.; Liu, Y. B.; Zhang, J. J.; Liu, Y.; Ma, S. G.; Qu, J.; Lv, H. N.; Yu, S. S. J. Nat. Prod. 2015, 78, 2887. (c) Bao, G. H.; Wang, L. Q.; Cheng, K. F.; Feng, Y. H.; Li, X. Y.; Qin, G. W. Planta Med. 2003, 69, 434. (d) Bao, G. H.; Wang, L. Q.; Cheng, K. F.; Qin, G. W. Chin. Chem. Lett. 2002, 13, 955. (e) Zhi, X.; Xiao, L.; Liang, S.; Yi, F.; Ruan, K. F. Chem. Nat. Compd. 2013, 49, 454. (6) (a) Li, Y.; Liu, Y. B.; Liu, Y. L.; Wang, C.; Wu, L. Q.; Li, L.; Ma, S. G.; Qu, J.; Yu, S. S. Org. Lett. 2014, 16, 4320. (b) Li, Y.; Liu, Y. B.; Yan, H. M.; Liu, Y. L.; Li, Y. H.; Lv, H. N.; Ma, S. G.; Qu, J.; Yu, S. S. Sci. Rep. 2016, 6, 36752. (c) Li, C. J.; Liu, H.; Wang, L. Q.; Jin, M. W.; Chen, S. N.; Bao, G. H.; Qin, G. W. Acta. Chim. Sinica 2003, 61, 1153. (d) Li, C. J.; Wang, L. Q.; Chen, S. N.; Qin, G. W. J. Nat. Prod. 2000, 63, 1214. (7) Flack, H. D.; Bernardinelli, G. Chirality 2008, 20, 681. (8) (a) Dewick, P. M. In Medicinal Natural Products: A Biosynthetic Approach, 3rd ed.; John Wiley & Sons, Ltd.: Chichester, 2009; pp 15. (b) Zhang, M. K.; Zhu, Y.; Zhan, G. Q.; Shu, P. H.; Sa, R. J.; Lei, L.; Xiang, M.; Xue, Y. B.; Luo, Z. W.; Wan, Q.; Yao, G. M.; Zhang, Y. H. Org. Lett. 2013, 15, 3094. (9) Shi, L.; Yu, H. P.; Zhou, Y. Y.; Du, J. P.; Shen, Q.; Li, J. Y.; Li, J. Acta Pharmacol. Sin. 2008, 29, 278. (10) Liu, C. C.; Lei, C.; Zhong, Y.; Gao, L. X.; Li, J. Y.; Yu, M. H.; Li, J.; Hou, A. J. Tetrahedron 2014, 70, 4317.

The preliminary screening concentration was 200 μM. bValues are expressed as the means ± SD, n = 3. cOleanolic acid was used as the positive control. a

concentration of 200 μM showed that compound 1 exhibited strong PTP1B inhibitory activity with an inhibition rate of 87.4%, while compounds 2 and 3 showed moderate and weak PTP1B inhibitory activities with inhibition rates of 31.5% and 13.4%, respectively. Further screening studies at five different concentrations (Figure S1) revealed that 1 exhibited moderate inhibitory activity with an IC50 value of 24.32 ± 0.56 μM. Compound 2 possessing a double bond at C-15 and C-16 showed stronger PTP1B inhibitory activities than 3 without a double bond. Thus, Δ15(16) double bond will increase the PTP1B inhibitory activity, which is consistent with the reported conclusion by Hou group.10 Compound 1 with a new rhodomollane diterpene carbon skeleton showed stronger PTP1B protein inhibitory activity than compounds 2 and 3, suggesting that rhodomollane diterpenoids may possess stronger PTP1B protein inhibitory activity than grayanane diterpenoids. In summary, the leaves of R. molle were investigated for the first time, leading to the isolation of two new (1 and 2) and a known (3) diterpenoids. Rhodomollanol A (1) possesses an unprecedented 5/7/5/5 tetracyclic diterpene carbon skeleton, featuring a rare 7-oxabicyclo[4.2.1]nonane core fused with three cyclopentane units. Rhodomollanol A (1) enriched the chemical diversity of Ericaceae diterpenoids. In addition to the fascinating structural complexity, rhodomollanol A (1) exhibited moderate PTP1B inhibitory activity. This finding provides a new structural class to design PTP1B inhibitors.

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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.7b01863. Experimental procedures; concentration−inhibition ratio curves of rhodomollanol A (1) and oleanolic acid; 1D and 2D NMR spectra; HRESIMS of 1 and 2 (PDF) X-ray crystallographic data for 1 (CCDC 1556790) (CIF) X-ray crystallographic data for 2 (CCDC 1556791) (CIF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 3938

DOI: 10.1021/acs.orglett.7b01863 Org. Lett. 2017, 19, 3935−3938