Letter pubs.acs.org/OrgLett
Rhodomollacetals A−C, PTP1B Inhibitory Diterpenoids with a 2,3:5,6Di-seco-grayanane Skeleton from the Leaves of Rhododendron molle Junfei Zhou, Na Sun, Hanqi Zhang, Guijuan Zheng, Junjun Liu, 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: Three novel diterpenoids with an unprecedented 2,3:5,6-di-seco-grayanane carbon skeleton, rhodomollacetals A−C (1−3), are isolated from the leaves of Rhododendron molle. Their structures are elucidated by comprehensive spectroscopic techniques and single-crystal X-ray diffraction. Rhodomollacetal A (1) possesses a novel cis/cis/cis/cis-fused 6/6/6/6/5 pentacyclic ring system, featuring an unprecedented 11,13,18-trioxa-pentacyclo [8.7.1.15,8.02,8.012,17]nonadecane scaffold. Compounds 2 and 3 have a rare 4-oxatricyclo[7.2.1.01,6]dodecane moiety and a 2,3dihydro-4H-pyran-4-one unit. Compounds 1−3 showed moderate PTP1B inhibitory activities, and their molecular dockings were investigated.
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rayanane diterpenoids featuring a unique 5/7/6/5 tetracyclic carbon skeleton exclusively exist in the Ericaceae plants and exhibit a broad spectrum of significant bioactivities, such as sodium channel antagonistic, antinociceptive, anticancer, and antiviral activities.1 Due to their complex, highly oxygenated, polycyclic structures, and significant bioactivities, the total synthesis of grayanane diterpenoids, such as grayanotoxin III2 and pierisformoside C,3 have attracted considerable attention from the community of organic synthesis chemists. Rhododendron molle is famous for the abundance of grayanane and related diterpenoids. Up to now, nearly 70 diterpenoids, belonging to grayanane,4 kalmane,5 seco-kalmane,6 3,4-seco-grayanane,7 1,10:2,3-di-seco-grayanane,8 C-nor-D-homograyanane,9 and D-homograyanane10 carbon skeletons, respectively, have been reported from the flowers, fruits, and roots of R. molle. Protein tyrosine phosphatase 1B (PTP1B) has been proven to be an important therapeutic target for type II diabetes and obesity because of its pivotal role as a negative modulator in the insulin signal pathway.11 In a previous search for novel PTP1B inhibitors from Ericaceae plants, rhodomollanol A, a novel diterpenoid with an unprecedented 5/7/5/5 tetracyclic rhodomollane diterpene carbon skeleton from R. molle, showed PTP1B inhibitory activity.12 This exciting result inspired us to further search for more potent PTP1B inhibitory diterpenoids from the leaves of R. molle, leading to the isolation of three novel diterpenoids with an unprecedented 2,3:5,6-di-seco-grayanane carbon skeleton, rhodomollacetals A−C (1−3) (Figure 1), as well as their plausible biogenetic precursor rhodojaponin VI13 (4). Compound 1 possesses a novel cis/cis/cis/cis-fused 6/6/6/ 6/5 pentacyclic system, featuring an unprecedented 11,13,18trioxa-pentacyclo[8.7.1.15,8.02,8.012,17]nonadecane scaffold, and 2 and 3 are a pair of C-6 epimers, bearing a rare 4-oxatricyclo© 2017 American Chemical Society
Figure 1. Structures of rhodomollacetals A−C (1−3).
[7.2.1.01,6]dodecane moiety and a 2,3-dihydro-4H-pyran-4-one unit. Herein, we report the isolation, structure elucidation, PTP1B inhibitory activity, and molecular docking of compounds 1−4. Rhodomollacetal A (1) was obtained as colorless prisms, mp 167−168 °C. The molecular formula of 1 was determined as C21H32O7 by the HRESIMS ion at m/z 419.2051 [M + Na]+ (calcd for C21H32O7Na, 419.2046) and 13C NMR data, suggesting six degrees of unsaturation. The IR spectrum suggested the presence of hydroxy (3408 cm−1) and ketone (1704 cm−1) groups in 1. The 1H NMR spectrum of 1 (Table S1) exhibited resonances for four methyl singlets at δH 1.37 (H3-20), 1.33 (H3-17), 1.23 (H3-19), and 1.04 (H3-18), an oxygenated methyl singlet at δH 3.43 (3-OCH3), an oxygenated methine at δH 4.11 (H-14), and three acetals at δH 5.62 (d, H-2), 5.47 (d, H-6), and 4.57 (s, H-3). The 13C NMR spectrum showed a total of 21 carbon resonances, assignable by DEPT and HSQC spectra to be four methyls, an oxygenated methyl, four methylenes, seven methines (one oxygenated at δC 79.9, and three acetals at δC 91.3, 94.7, and 106.3), and five quaternary carbons (two oxygenated at Received: August 24, 2017 Published: September 27, 2017 5352
DOI: 10.1021/acs.orglett.7b02633 Org. Lett. 2017, 19, 5352−5355
Letter
Organic Letters δC 80.6 and 76.8, and one ketone carbonyl at δC 210.9). A ketone carbonyl accounts for one degree of unsaturation, and the rest of the five degrees of unsaturation required the existence of a pentacyclic system in 1. The planar structure of compound 1 (Figure 2) was deduced from 1H−1H COSY, HSQC, and HMBC spectra analyses.
Figure 3. X-ray crystal structure of compound 1.
group in 2. The 1H NMR spectrum of 2 (Table S1) displayed resonances for an olefinic proton at δH 7.55 (s, H-2), two acetals at δH 4.83 (s, H-3) and δH 4.83 (dd, H-6), two oxygenated methyls at δH 3.50 (s, 3-OCH3) and 3.42 (s, 6-OCH3), and four methyl singlets at δH 1.32 (H3-17), 1.00 (H3-18), 1.12 (H3-19), 1.56 (H3-20). The 13C NMR and DEPT spectra (Table S1) indicated the existence of four methyls, two oxygenated methyls, four methylenes, six methines (one oxygenated, two acetals, and one olefinic), and six quaternary carbons (two oxygenated, one olefinic, and one ketone carbonyl). A double bond and a carbonyl account for two degrees of unsaturation, and the remaining four degrees of unsaturation indicated the presence of four rings in 2. As shown in Figure 4, 1H−1H COSY and HSQC spectra revealed the presence of two partial structures in 2: (a) C(6)H−
Figure 2. 1H−1H COSY, key HMBC, and NOESY correlations of compound 1. 1
H−1H COSY spectrum revealed the presence of three partial structures: (a) C(1)H−C(2)H, (b) C(6)H−C(7)H2, and (c) C(9)H−C(11)H 2 −C(12)H 2 −C(13)H−C(14)H. In the HMBC spectrum of 1 (Figure 2), the cross peaks from H3-18/ H3-19 to C-3, C-4, and C-5, from H-1 to C-4, from H-2 and 3OCH3 to C-3, and from H-3 to C-2, as well as the chemical shifts of C-2 (δC 91.3) and C-3 (δC 106.3), established the A-ring of a 2methoxy-3,3-dimethyl-pyran-4-one in 1. HMBC correlations of H3-20 to C-1, C-9, and C-10, of H-6 to C-2 and C-10, and of H-2 to C-6, as well as the chemical shift of C-6 (δC 94.7), constructed the partial structure B-ring. The partial structure C-ring was determined by the HMBC correlations from H-7 to C-8 and C-9 and from H-6 to C-8 and C-10. Thus, the 2,9-dioxabicyclo [3.3.1]nonane scaffold of rings B and C was established. In the same way, the connections of rings D and E were established by the HMBC correlations of H3-17 to C-13, C-16, and C-15, of H15 to C-8, C-9, and C-14, H-14 to C-8, C-9, and C-15, and H-9 to C-14 and C-15, which are the same for the typical grayanane diterpenoids. The relative configuration of compound 1 was confirmed by NOESY spectrum (Figure 2). H-9 was randomly assigned as the β-orientation, which is the same as that in the grayanane diterpenoids. NOESY correlations between H-9β and H-1, and between H-1 and H-2 indicated that H-1 and H-2 were βoriented. The α-orientation of H-3 was deduced from the NOESY correlation between H-2β and 3-OCH3. The NOESY correlations between H-9 and one of the H-15 and between H-14 and H3-20 suggested the α-orientation of H-14 and H3-20. NOESY correlations between H-9β and H-7β and between H-7α and H-6 suggested that H-6 is α-oriented. Finally, the structure of 1 was confirmed by single-crystal X-ray diffraction with Cu Kα radiation (Figure 3), and the absolute configuration of 1 was determined to be 1S,2S,3R,6R,8R,9R,10R,13R,14R,16R by the resulting Flack parameter of −0.12(11).14 Rhodomollacetal B (2), [α]20D +7.8 (c 0.1, MeOH), was obtained as colorless prisms, mp 180−181 °C. Its molecular formula was established as C22H34O7 by the [M + Na]+ ion at m/ z 433.2220 (calcd for C22H34O7Na, 433.2220) in the HRESIMS and 13C NMR data, indicating six degrees of unsaturation. The UV absorption at 265 (4.02) nm and the IR absorptions at 1679 and 1618 cm−1 indicated the presence of a conjugated carbonyl
Figure 4. 1H−1H COSY, key HMBC, and NOESY correlations of compound 2.
C(7)H2 and (b) C(9)H−C(11)H 2−C(12)H2 −C(13)H− C(14)H. HMBC correlations from H3-18/H3-19 to C-1, C-4, and C-5, from H-2 to C-1, C-3, and C-5, and from 3-OCH3 to C3, as well as the chemical shift of C-3 (δC 110.1) and H-3 (δH 4.83), constructed the A-ring of a 2-methoxy-3,3-dimethyl-2,3dihydro-4H-pyran-4-one unit in 2. The B-ring of the 2-methoxy6-methyl pyrane unit was determined by the HMBC correlations from H3-20 to C-9 and C-10, from H-6 to C-8 and C-10, from H2-7 to C-8 and C-9, and from 6-OCH3 to C-6. Similarly, rings C and D are the same as those in grayanane diterpenoids and are confirmed by the HMBC correlations of H3-17 to C-13, C-16, and C-15, of H-15 to C-8, C-9, and C-14, of H-14 to C-8, C-9, and C-15, and of H-9 to C-14 and C-15. Similar to that in compound 1, H-9 in 2 was randomly assigned to be β-oriented. The α-orientation of H-6 was deduced from the NOESY correlations between H-9β and H-7β and between H-7α and H-6. NOESY correlations between H-14 and H3-20 and between H-14 and H-6 indicated that H-6 and H3-20 are αoriented. The absolute configuration of 2 (Figure 5) was determined to be 3S,6S,8R,9R,10R,13R,14R,16R by the Flack parameter of 0.1(1)14 and the Hooft parameter of 0.05(6)15 5353
DOI: 10.1021/acs.orglett.7b02633 Org. Lett. 2017, 19, 5352−5355
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Organic Letters
for their PTP1B inhibitory activities in vitro. Results (Table 1 and Figure S2) indicated that 1−3 exhibited moderate PTP1B Table 1. PTP1B Inhibitory Activities of Compounds 1−4 compd
inhibition (%)a
IC50 (μΜ)b
1 2 3 4 oleanolic acidc
67.5 78.6 82.4 15.4 99.2
42.42 ± 1.40 19.88 ± 1.11 16.66 ± 0.40 >200 4.71 ± 0.16
a The preliminary screening concentration was 200 μΜ. bValues are expressed as the means ± SD, n = 3. cOleanolic acid was used as the positive control.
Figure 5. X-ray crystal structure of compound 2.
resulting from single-crystal X-ray diffraction with Cu Kα radiation. Rhodomollacetal C (3), [α]20D −13.7 (c 0.1, MeOH) , a white powder, has the same molecular formula as 2, which was confirmed by the [M + Na]+ ion at m/z 433.2207 (calcd for C22H34O7Na, 433.2220) and 13C NMR data. The NMR data of 3 were very similar to those of 2, except that H-6 (δH 4.77, dd) in 3 shifted upfield more than that (δH 4.83, dd) in 2, while C-6 (δC 101.6) in 3 shifted downfield more than that (δC 98.0) in 2. Thus, 3 should be a 6-epimer of 2. The coupling constants of H-6 (J = 3.9 and 1.5 Hz) in 3 were totally different from that (J = 10.0 and 2.2 Hz) in 2, supporting the above deduction. The absolute configuration of 3 was the same as that of 2, ignoring C-6, based on their similar ECD spectra. Rhodomollacetals A−C (1−3) possess an unprecedented 2,3:5,6-di-seco-grayanane carbon skeleton, and their biosynthetic pathways could be traced back to the coisolated grayanane diterpenoid rhodojaponin VI (4)13 in this study. As shown in Scheme 1, a key tetra-carbonyl intermediate 5 is formed by the
inhibitory activities with IC50 values of 42.42 ± 1.40, 19.88 ± 1.11, and 16.66 ± 0.40 μM, respectively, and 4 did not show significant PTP1B inhibitory activity (IC50 > 200 μM). A preliminary structure−activity relationship (SAR) study revealed that the 4-oxatricyclo[7.2.1.01,6]dodecane and 2-methoxy-2,3dihydro-4H-pyran-4-one units may be essential for the PTP1B inhibitory activity, while the orientation of 6-OCH3 in 2 and 3 does not significantly affect the PTP1B inhibitory activity. To further investigate their SAR and modes of action, the binding modes of compounds 1−4 with PTP1B were obtained by molecular docking. Results (Figures 6, S3−S6) revealed that
Scheme 1. Proposed Biosynthetic Pathways for 1−3
Figure 6. Low-energy binding conformations of 1−4 (A−D) bound to PTP1B enzyme generated by virtual ligand docking. Dotted green and orange lines indicate hydrogen bonds and σ−π interactions, respectively.
1−4 were posed in similar catalytic/pocket domains of the PTP1B, including the Asp181 and Phe182 residues of the WPD loop, Cys215 residue of the catalytic site, Ser216, Ala217, Gly218, Ile219, Gly220, and Arg221 residues of the secondary phosphated-binding loop (P-loop), and Tyr46, Asp48, Lys120, Gln262, and Gln266 residues of the hydrophobic pocket site, and 1−4 can be stabilized by the simultaneous establishment of multiple hydrogen bonds and van der Waals contacts in the pocket site (Figures S3−S6). Interestingly, H-9β in 1, H-15β in 2, and H-9β and H-15β in 3 have strong σ−π interactions with the benzene ring of the Phe182 residue in the catalytic WPD loop, which plays an important role to stabilize the binding with the ligands.18 This indicates that 1−3 may reduce the mobility of the
enzyme (such as cytochrome P45016)-mediated oxidization7,8 of two sets of the adjacent dihydroxy groups in 4, and then, a hemiacetal 6 is produced by the attack of the 10-OH to the 6carbonyl group in 5. In path A, the new 6-OH attacks the 2carbonyl group to generate an acetal intermediate 7, followed by the formation of a hemiacetal intermediate 8. Finally, rhodomollacetal A (1) is produced from path A, while 2 and 3 are generated from the similar path B. LC−HRMS analysis of the crude extract of R. molle revealed that 1−3 are natural products (Figure S1). Since grayanane diterpenoids have been reported to possess PTP1B inhibitory activities,17 compounds 1−4 were evaluated 5354
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(9) 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. (10) 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. (11) Tamrakar, A. K.; Maurya, C. K.; Rai, A. K. Expert Opin. Ther. Pat. 2014, 24, 1101. (12) Zhou, J.; Zhan, G.; Zhang, H.; Zhang, Q.; Li, Y.; Xue, Y.; Yao, G. Org. Lett. 2017, 19, 3935. (13) 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. (14) Parsons, S.; Flack, H.; Wagner, T. Acta Crystallogr., Sect. B: Struct. Sci., Cryst. Eng. Mater. 2013, B69, 249. (15) Hooft, R. W. W.; Straver, L. H.; Spek, A. L. J. Appl. Crystallogr. 2010, 43, 665. (16) Hrycay, E. G.; Bandiera, S. M. Arch. Biochem. Biophys. 2012, 522, 71. (17) 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. (18) Feldhammer, M.; Uetani, N.; Miranda, D.; Tremblay, M. L. Crit. Rev. Biochem. Mol. Biol. 2013, 48, 430. (19) Popov, D. Biochem. Biophys. Res. Commun. 2011, 410, 377. (20) Yip, S. C.; Saha, S.; Chernoff, J. Trends Biochem. Sci. 2010, 35, 442.
WPD loop to a more rigid conformation, which maintains the WPD loop in an open conformation to prevent substrate binding.19 More importantly, 14-OH in 2 and 3 exhibit strong hydrogen bonds with the Ser216 residue closed to the highaffinity catalytic site Cys215 of PTP1B enzyme, which could attack the phosphate group though nucleophilic reaction to release phenol substrate.20 The hydrogen bond interaction with Ser216 could imply stronger binding affinity with the active site Cys215 and increase the PTP1B inhibitory activity of 2 and 3. However, 4 neither shows strong σ−π interaction with the Phe182 residue of the WPD loop nor shows strong hydrogen bonds with the Ser216 residue. Maybe, that is why 4 did not show significant PTP1B inhibitory activity (IC50 > 200 μM). Therefore, the Phe182 and Ser216 residues play an important role in recognizing the ligands. This evidence provides useful clues to develop novel PTP1B inhibitors based on 2,3:5,6-di-secograyanane diterpenoids.
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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.7b02633. Detailed experimental procedures, NMR spectroscopic data, HRESIMS, UV, IR, and NMR spectra for 1−3, binding energies and docking models for 1−4, and 1D NMR spectra for 4 (PDF) X-ray crystallographic data for 1 (CCDC 1567619) (CIF) and 2 (CCDC 1567620) (CIF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Junjun Liu: 0000-0001-9953-8633 Guangmin Yao: 0000-0002-8893-8743 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Analysis and Measurement Centre at HUST for the IR and ECD analyses. This work was supported financially by the Fundamental Research Funds for the Central Universities (HUST: 2016YXMS148) and the National Natural Science Foundation of China (81001368 and 31170323).
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REFERENCES
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DOI: 10.1021/acs.orglett.7b02633 Org. Lett. 2017, 19, 5352−5355
