Letter pubs.acs.org/OrgLett
Lycoplanine A, a C16N Lycopodium Alkaloid with a 6/9/5 Tricyclic Skeleton from Lycopodium complanatum Zhi-Jun Zhang,†,§,∥ Yin Nian,‡,∥ Qin-Feng Zhu,†,§ Xiao-Nian Li,† Jia Su,† Xing-De Wu,† Jian Yang,*,‡ and Qin-Shi Zhao*,† †
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ Key Laboratory of Bioactive Peptides of Yunnan Province/Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, People’s Republic of China § University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *
ABSTRACT: Lycoplanine A (1), a Lycopodium alkaloid with a 6/9/5 tricyclic ring skeleton fused with the γ-lactone ring and featuring an unusual 1-oxa-6-azaspiro[4.4]nonane moiety and an unprecedented 3-azabicyclo[6.3.1]dodecane unit, was isolated from the club moss Lycopodium complanatum. The structure and absolute configuration of 1 were identified by a combination of NMR spectroscopic analysis and single-crystal X-ray diffraction. Biological studies showed that 1 is a potent Cav3.1 T-type calcium channel (TTCC) inhibitor with an IC50 value of 6.06 μM. Lycopodium alkaloids (LAs) have attracted much scientific attention because of their important biological activities related to acetylcholinesterase (AChE) and unique structural characteristics. To date, more than 300 LAs have been reported. Of these, huperzine A, a highly specific and potent inhibitor of AChE,1 has already been marketed as a new drug to treat Alzheimer’s disease (AD) in China and as a dietary supplement in the USA.2 Lycopodium complanatum (L.) Holub (syn.: Diphasiastrum complanatum) is a traditional Chinese herbal medicine for the treatment of arthritic pain, quadriplegia, and contusion.3,4 Previous investigations of this species led to reports of a large number of LAs with fascinating strutures, such as lyconadin A,5 complanadine A,6 lycopladine A,7 and lycopladine H.8 In 2013, our group also reported a unique LA with a C15N skeleton, lycospidine A, from this species collected from Yunnan Province in China.9 Since different ecological environments may cause differences in the secondary metabolites,10 we continued to explore structurally novel LAs of L. complanatum collected from Guizhou Province in China. As a result, a highly modified LA, namely, lycoplanine A (1), was isolated and identified. Compound 1 possesses a 6/9/5 tricyclic ring skeleton that is fused with the γ-lactone ring and constructed from an unusual 1-oxa-6-azaspiro[4.4]nonane moiety and an unprecedented 3-azabicyclo[6.3.1]dodecane unit (Figure 1). Although LAs have diverse heterocyclic frameworks, such as C11N, C15N, C16N, C16N2, C22N2, and C27N3 types, their bioactivities are mainly related to cholinesterase.11−13 Thus, it is of interest to expand screening targets for LAs. Therefore, Ttype calcium channels (TTCCs, Cav3.1−3.3), attractive © 2017 American Chemical Society
Figure 1. Structure of 1.
therapeutic targets in modern drug development dealing with neuropathic pain, absence epilepsy, insomnia, and Parkinson’s disease,14 were used to evaluate compounds 1 and 2 in the present study. Significantly, compound 1 exhibited noticeable inhibition of Cav3.1 TTCC peak current with an IC50 value of 6.06 μM. Unfortunately, the known compound 2 showed no inhibitory activity. Reported herein are the isolation, structure elucidation, proposed biosynthetic pathway, and Cav3.1 TTCC inhibition of lycoplanine A (1). The whole plant of L. complanatum was collected from Guizhou Province and identified by Prof. Xiao Cheng of Kunming Institute of Botany, Chinese Academy of Sciences (voucher no. 2013-9-15). The air-dried and powdered sample (30 kg) was extracted with 60% EtOH (24 h × 3) at room temperature, and the extract was partitioned between EtOAc and 1.0‰ HCl/H2O. Then water-soluble materials, which were Received: July 27, 2017 Published: August 22, 2017 4668
DOI: 10.1021/acs.orglett.7b02293 Org. Lett. 2017, 19, 4668−4671
Letter
Organic Letters
2). Its 1H−1H COSY spectrum revealed the presence of three fragments: a, −CH2−CH2−CH2− (for C-1/C-2/C-3); b,
adjusted to pH 10 with saturated aqueous Na2CO3, were extracted with CHCl3 to give an alkaloidal extract (60 g). The alkaloidal extract was subjected to medium-pressure liquid chromatography (MPLC) over RP-18 gel and eluted with MeOH/H2O (1:9−1:0) to yield five fractions, A−E. Fraction B (10 g) was chromatographed over repeated silica gel columns (CHCl3/MeOH) to yield compound 1 (3 mg). Fraction E (9 g) was further purified by silica gel column chromatography (CHCl3/MeOH) using Sephadex LH-20 to afford lycopladine H (2) (5 mg). Lycoplanine A (1), colorless needles (MeOH), had the molecular formula of C16H21NO3 as determined by the HR-EIMS peak at m/z 275.1515 (calcd 275.1521), corresponding to seven degrees of unsaturation. The IR spectrum showed absorptions for carbonyl groups (1703 and 1633 cm−1). Its 13C and DEPT spectrum exhibited 16 carbon signals (Table 1),
Figure 2. Selected 2D NMR correlations of 1.
−CH2−CH2−CH2− (for C-9/C-10/C-11), and c, −CH2− CH−CH2−CH(CH3)− (for C-6/C-7/C-8/C-15/C-16), as shown in Figure 2. In the HMBC spectrum, correlations from H-15 and H-6 to the tetrasubstituted olefinic carbons at δC 162.1 (C-5) and δC 135.8 (C-14), together with the existence of the −CH2CHCH2−CH(CH3)− spin system, indicated the presence of a six-membered ring, A, as shown in Figure 2. In addition, HMBC cross-peaks from H-15 to the carbonyl carbon at δC 173.9 (C-13) and from H-6 to the oxygen-bearing carbon at δC 109.8 (C-4) suggested the existence of a five-membered α,β-unsaturated γ-lactone ring, D, which is fused to ring A at C-5 and C-14. Further analyses of HMBC associations of H-8 and H-11 to C-12 revealed that ring A and fragment b are linked by the ketone group at δC 215.5 (C-12). Similarly, fragment a is attached to ring D through C-4 on the basis of the HMBC correlations of H-3 and H-2 to C-4. To fulfill the molecular formula and unsaturation requirement, a nitrogen atom still needed to be assigned and two remaining degrees of unsaturation needed to be constructed. In the HMBC spectrum, a correlation between H-1 and C-9 was observed, which suggested their connection through the nitrogen atom. In addition, HMBC cross-peaks from H-9 and H-1 to C-4 further indicated the linkage between C-4 and the nitrogen atom and established rings B and C as shown in Figure 2. Thereby, the planar structure of 1 was established. Compound 1 possesses a 6/9/5 tricyclic ring skeleton fused with the γlactone ring, a structure that has not been found in any other LAs. Besides, rings A and B show an unprecedented 3azabicyclo[6.3.1]dodecane unit, while rings C and D exhibit an unusual 1-oxa-6-azaspiro[4.4]nonane moiety. The relative configuration of 1 was established by a ROESY experiment (Figure 2). The cross-peaks of H-16 and H-6a revealed that these protons have the same orientation. Besides, the H-11a/H-7, H-11a/H-6b, H-9a/H-6b, and H-3a/H6a correlations suggested the conformations of rings A, B, C, and D as shown. To confirm the structure and determine the absolute configurations of C-4, C-7, and C-15, 1 was crystallized from MeOH to afford a crystal of the hexagonal space group P65 with unit cell dimensions a = 12.9639(5) Å, b = 12.9639(5) Å, and c = 14.8451(6) Å, which was analyzed by X-ray crystallography. On the basis of the presence of three oxygen atoms in the molecule, the final refinement of the Cu Kα data resulted in a Flack parameter of 0.02(5), a final R1 (I > 2σ(I)) of 0.0343, and an wR(F2) (I > 2σ(I)) of 0.0864. These allowed the unambiguous assignment of not only the unusual 1-oxa-6azaspiro[4.4]nonane moiety and the unprecedented 3azabicyclo[6.3.1]dodecane moiety but also the 4S,7R,15R
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR data for 1 in CD3OD (δ in ppm, J in Hz) δH
no. 1a 1b 2a 2b 3a 3b 4 5 6a 6b 7 8a 8b 9a 9b 10a 10b 11a 11b 12 13 14 15 16
3.45 2.89 2.06 2.00 2.34 2.16
3.10 2.51 2.94 2.58 1.36 2.28 2.00 1.68 1.68 3.21 1.88
(td, 15.7, 4.5) (dd, 15.7, 8.4) (m) (overlapped) (ddd, 10.2, 6.6, 3.0) (m)
(dt, 16.1, 3.0) (ddd, 16.1, 4.4, 3.0) (m) (m) (ddd, 13.6, 8.5, 4.0) (dt, 15.6, 3.2) (overlapped) (overlapped) (overlapped) (m) (dt, 14.4, 4.2)
2.71 (m) 1.20 (d, 7.1)
δC 54.2 22.9 34.0 109.8 162.1 27.0 50.2 33.2 43.6 25.6 33.9 215.5 173.9 135.8 26.5 20.1
HMBC (1H−13C) 2, 3, 1, 1, 1, 1,
3, 4, 3, 3, 2, 2,
4 9 4 4 4 4
5, 8, 12, 14 4, 5, 7, 8, 14 7, 14, 15 7, 12, 14, 15, 16 1, 4, 10, 11 1, 4, 10, 11 9, 11, 12 9, 11, 12 10, 12 9, 12
5, 8, 14, 16 8, 14, 15
attributed to five quaternary carbons, two tertiary carbons, eight methylenes, and one methyl group. For the quaternary carbons, the sp3 quaternary carbon (δC 109.8) was ascribed to carbon C4 bearing both an oxygen atom and a nitrogen atom. Three sp2 quaternary carbons were assigned to an α,β-unsaturated γlactone moiety formed by C-5 (δC 162.1), C-14 (δC 135.8), and C-13 (δC 173.9). In addition, another sp2 quaternary carbon was attributable to the ketone group (δC 215.5). In 1H and HSQC spectra, eight methylenes (δH 1.36−3.45, each 2H), two methines (δH 2.71 and 2.94, each 1H), and one methyl (δH 1.20, 3H) were observed, consistent with the functional units shown by 13C and DEPT spectroscopic data. The aforementioned functionalities accounted for four degrees of unsaturation, thus requiring the existence of three additional rings in the molecule. The planar structure of 1 was determined by interpretation of 2D NMR spectra, especially the HMBC spectrum (Figure 4669
DOI: 10.1021/acs.orglett.7b02293 Org. Lett. 2017, 19, 4668−4671
Letter
Organic Letters absolute configuration, as shown in Figure 3. Crystallographic data of 1 have been deposited at the Cambridge Crystallographic Data Centre (CCDC) (deposition no. 1560538).
Figure 4. Inhibitory effect of lycoplanine A (1) on Cav3.1. (A) Representative Cav3.1 peak current traces elicited by 150 ms depolarization to −40 mV at 3 s intervals from a holding potential (HP) of −100 mV in the absence (control) and presence of different concentrations of 1. Zero current is indicated by dashed lines. (B) Dose−response relationships of 1 for Cav3.1 at an HP of −100 mV. Data points represent mean ± SEM of four measurements. The solid curve represents a fit to the Hill equation.
Figure 3. X-ray crystal structure of 1.
A plausible biogenetic pathway for 1 is proposed as shown in Scheme 1. Lycopladine H (2) was a novel alkaloid with a fused
performed on mock-transfected cells, and 1 showed no detrimental effect. Compounds 1 and 2 were also tested for β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory activity16 and for AChE inhibitory activities using the Ellman method as reported previously (huperzine A as positive control, IC50 = 0.03 μM).17 Unfortunately, they showed no inhibitory activity (IC50 > 100 μM) after two repeated experiments. In summary, we have described in this work the isolation and evaluation of a new type of LA, lycoplanine A (1), that potently inhibit Cav3.1 TTCC. This molecule has a novel chemical structure, possessing a 6/9/5 tricyclic ring skeleton fused with the γ-lactone ring, and differs markedly from known Cav3.1 inhibitors. In addition, the Cav3.1 inhibitory effect of 1 is in line with the traditional use of L. complanatum to cure arthritic pain, which provides exciting opportunities to discover more potent TTCC inhibitors from this species.
Scheme 1. Plausible Biogenetic Pathway for 1
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tetracyclic skeleton that was isolated in 2009 from L. complanatum,8 the same plant as in this study. Since compound 2 possesses the same 6/12 fused ring system as 1 (the red and blue sticks shown in Scheme 1), we propose that 2 is probably the precursor of 1. Consequently, compound 2 undergoes oxidative cleavage at C-12/C-13 to produce key 6/9/6 tricyclic intermediate i. This intermediate is converted to keto amine ii through a retro-Michael addition reaction, which is followed by a cyclization reaction between N and C-4 to produce carbinol amine iii. Then iii undergoes intramolecular esterification to give 1. As noted above, the effects of LAs on Cav3.1 TTCC have not been revealed. Therefore, we initiated such a study. Cav3.1 was expressed heterologously in HEK293 cells, and the inhibitory effect of 1 on Cav3.1 peak currents was examined by whole-cell patch clamp. Dose−response relationships were obtained for 1, which exhibited notable inhibition at 5, 10, and 30 μM (Figure 4 and Table S1). The IC50 value for 1 was 6.06 μM, with a Hill coefficient of 2.9. Mibefradil, a classic TTCC inhibitor15 that was once clinically used for the treatment of hypertension, inhibited Cav3.1 with an IC50 value of 1.32 μM and a Hill coefficient of 1.9 under our experimental conditions (Figure S12). Interestingly, the apparent potency of compound 1 is weaker than that of mibefradil. However, it shows significantly stronger cooperative binding to Cav3.1 TTCC than mibefradil does. This phenomenon may be due to the unique mechanism of action of compound 1 and deserves further study. In addition, to exclude the possibility that the Cav3.1 inhibition of 1 was related to cellular toxicity, the same experiments were
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b02293. 1D and 2D NMR, IR, UV, ESI-MS, and HR-ESI-MS data; detailed experimental procedures, X-ray crystallographic data; and Cav3.1 TTCC inhibition data (PDF) X-ray crystallographic data for 1 (CIF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Yin Nian: 0000-0002-9916-064X Qin-Shi Zhao: 0000-0002-1249-2917 Author Contributions ∥
Z.-J.Z. and Y.N. 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 (U0932602 and U1502223), the 4670
DOI: 10.1021/acs.orglett.7b02293 Org. Lett. 2017, 19, 4668−4671
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Organic Letters National Basic Research Program of China (973 Program) (2011CB915503), the Foundation of the Chinese Academy of Sciences (ZSTH-019), and the Yunnan Major Science and Technology Project (2015ZJ002).
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