Monoterpene Indole Alkaloids with Cav3.1 T-Type Calcium Channel Inhibitory Activity from Catharanthus roseus

Catharanthus roseus is a well-known traditional herbal medicine for the treatment of cancer, hypertension, scald, and sore in China. Phytochemical investigation on the twigs and leaves of this species led to the isolation of two new monoterpene indole alkaloids, catharanosines A (1) and B (2), and six known analogues (3–8). Structures of 1 and 2 were established by 1H-, 13C- and 2D-NMR, and HREIMS data. The absolute configuration of 1 was confirmed by single-crystal X-ray diffraction analysis. Compound 2 represented an unprecedented aspidosperma-type alkaloid with a 2-piperidinyl moiety at C-10. Compounds 6–8 exhibited remarkable Cav3.1 low voltage-gated calcium channel (LVGCC) inhibitory activity with IC50 values of 11.83 ± 1.02, 14.3 ± 1.20, and 14.54 ± 0.99 μM, respectively.


Introduction
Monoterpene indole alkaloids (MIAs) are one of the largest natural product families constructed from indole and monoterpene moieties, and commonly found in Apocynaceae, Rubiaceae, and Loganiaceae families [1]. To date, more than 3000 MIAs have been reported, many of which have been found to exhibited important pharmaceutical effects [2,3]. Representative MIAs such as, reserpine, vinblastine/vincristine, and quinine are used clinically for the treatment of hypertension, cancer, and malaria, respectively [4]. In light of their diverse and complex structures and high druggability, MIAs have attracted great interest from chemical and pharmacological communities and have been a potent resource for new drug discovery.
Catharanthus roseus (L.) G. Don (Apocynaceae), a tropical perennial subshrub, is a well-known traditional herbal medicine for treating cancer, hypertension, scald, and sore in China [5]. Early phytochemical studies on this plant have led to the isolation of an array of MIAs, including the well-known anticancer drugs vinblastine and vincristine [6]. The discovery of these two drugs has been regarded as one of the most important developments in both natural product chemistry and the clinical treatment of cancer during the 1960s to 1980s [7][8][9][10]. In recent years, some new and bioactive MIAs were still reported from this plant [11][12][13][14][15][16][17]. In our continuing search for structurally unique and pharmaceutically interesting MIAs from medicinal plants [18][19][20][21][22], a phytochemical study of the twigs and leaves of C. roseus was undertaken and led to the identification of two new MIAs, catharanosines A (1) and B (2), and six known analogues ( Figure 1). Compound 2 was found to represent an unprecedented aspidosperma-type alkaloid with a piperidine moiety at C-10. Due to the limited amount of 1 and 2, only compounds 3-8 were screened for their inhibitory activity on Ca v 3.1 low voltage-gated calcium channel (LVGCC), an important therapeutic target for cardiovascular disease [23]. Herein, the isolation, structure determination, and bioactivities of compounds 1-8 are described.
FOR PEER REVIEW 2 of 11 from this plant [11][12][13][14][15][16][17]. In our continuing search for structurally unique and pharmaceutically interesting MIAs from medicinal plants [18][19][20][21][22], a phytochemical study of the twigs and leaves of C. roseus was undertaken and led to the identification of two new MIAs, catharanosines A (1) and B (2), and six known analogues ( Figure 1). Compound 2 was found to represent an unprecedented aspidosperma-type alkaloid with a piperidine moiety at C-10. Due to the limited amount of 1 and 2, only compounds 3-8 were screened for their inhibitory activity on Cav3.1 low voltage-gated calcium channel (LVGCC), an important therapeutic target for cardiovascular disease [23]. Herein, the isolation, structure determination, and bioactivities of compounds 1-8 are described.

Biological Activity
Due to the traditional use of C. roseus for treating hypertension in China, compounds 3-8 were evaluated for the effects on Cav3.1 low-voltage-gated calcium channel, which plays an important role in the regulation of cardiovascular disease. At a concentration of 50 μM, compounds 6-8 showed strong inhibitions on Cav3.1 ( Figure 5), while compounds 3-5 exhibited weak activity with inhibition rate of less than 50%. Then, compounds 6-8 were further evaluated for their dose-dependent relationships on Cav3.1 at a concentration range from 1.6 to 50.0 μM. The results showed that compounds 6-8 dose-dependently inhibited on Cav3.1 with IC50 values of 11.83 ± 1.02, 14.30 ± 1.20, and 14.54 ± 0.99 μM, as compared to mibefradil, an inhibitor of T-type VGCC, with IC50 value of 3.09 ± 0.41 μM ( Figure 6). These results indicated that compounds 6-8 were important antihypertensive active components of C. roseus.

Biological Activity
Due to the traditional use of C. roseus for treating hypertension in China, compounds

Biological Activity
Due to the traditional use of C. roseus for treating hypertension in C 3-8 were evaluated for the effects on Cav3.1 low-voltage-gated calcium plays an important role in the regulation of cardiovascular disease. At a 50 μM, compounds 6-8 showed strong inhibitions on Cav3.1 (Figure 5), w 3-5 exhibited weak activity with inhibition rate of less than 50%. Then were further evaluated for their dose-dependent relationships on Cav3.1 range from 1.6 to 50.0 μM. The results showed that compounds 6-8 d inhibited on Cav3.1 with IC50 values of 11.83 ± 1.02, 14.30 ± 1.20, and 14 compared to mibefradil, an inhibitor of T-type VGCC, with IC50 value (Figure 6). These results indicated that compounds 6-8 were important active components of C. roseus.

General
Optical rotations were measured with a Horiba SEPA-300 polarimeter (Horiba, Tokyo, Japan). Melting point was recorded on an X-4 micro melting point apparatus (Beijing Second Optical Instrument Factory, Beijing, China). IR spectra were obtained by a Tensor 27 spectrophotometer (Bruker, Karlsruhe, Germany) with KBr pellets. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer (Shimadzu, Kyoto, Japan). 1D and 2D spectra were run on a Bruker AM-400 or an Avance III 600 spectrometer (Bruker, Karlsruhe, Germany) with TMS as the internal standard. Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. EIMS were recorded on a Waters Autospec Premier P776 spectrometer (Waters Corporation, Milford, MA, USA). Column chromatography (CC) was performed using silica gel (200-300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China) and MCI gel (75-150 mm; Mitsubishi Chemical Corporation, Tokyo, Japan). Fractions were monitored by TLC (GF254, Qingdao Marine Chemical Co., Ltd., Qingdao, China), and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH. All solvents were distilled prior to use.

General
Optical rotations were measured with a Horiba SEPA-300 polarimeter (Horiba, Tokyo, Japan). Melting point was recorded on an X-4 micro melting point apparatus (Beijing Second Optical Instrument Factory, Beijing, China). IR spectra were obtained by a Tensor 27 spectrophotometer (Bruker, Karlsruhe, Germany) with KBr pellets. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer (Shimadzu, Kyoto, Japan). 1D and 2D spectra were run on a Bruker AM-400 or an Avance III 600 spectrometer (Bruker, Karlsruhe, Germany) with TMS as the internal standard. Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. EIMS were recorded on a Waters Autospec Premier P776 spectrometer (Waters Corporation, Milford, MA, USA). Column chromatography (CC) was performed using silica gel (200-300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China) and MCI gel (75-150 mm; Mitsubishi Chemical Corporation, Tokyo, Japan). Fractions were monitored by TLC (GF254, Qingdao Marine Chemical Co., Ltd., Qingdao, China), and spots were visualized by heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH. All solvents were distilled prior to use.

Plant Material
The whole plants of C. roseus were purchased from the Herb Material Market of Juhuacun, Kunming, Yunnan Province, P. R. China, in June 2011, and identified by Prof. Xiao Cheng, Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (20110620C) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and Isolation
The air-dried whole plants of C. roseus (60 kg) were powdered and extracted with methanol (4 × 200 L) under reflux. The methanol was evaporated under reduced pressure to produce a residue, which was dissolved in hot water and adjusted to pH 2 with 0.5% HCl and then extracted with ethyl acetate (50 L × 3). The water-soluble portion was adjusted to pH 9.0 with sat. Na 2 CO 3 and partitioned with CHCl 3 to yield the total crude alkaloids (200 g), which were chromatographed over silica gel column using a step-gradient eluting with CHCl 3 -Me 2 CO (1:0-0:1) to obtain five fractions A-E. Fraction B was applied to MCI gel column eluted with MeOH-H 2 O (40%-100%) to give subfractions B1-B4. Fraction B1 was subjected to silica gel CC (CHCl 3 -MeOH, 10:1) to obtain compounds 6 (15 mg), 7 (15 mg), and 8 (12 mg). Fraction B2 was subjected to silica gel CC (CHCl 3 -MeOH, 10:1) to obtain compounds 3 (13 mg) and 4 (11 mg). Fraction C was subjected to silica gel CC (CHCl 3 -MeOH, 9:1) to obtain compound 5 (5 mg (8). Crystallographic data for compound 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition numbers: CCDC 2106217). Copies of these data can be obtained free of charge via www.ccdc.cam.ac.uk.

Ca v 3.1 T-Type Calcium Channel Inhibitory Activity Assay
HEK293T cells purchased from ATCC were cultured at 37 • C with 5% CO 2 in Dulbecco's modified Eagle medium with glucose, L-glutamine, pyruvate, 10% FBS, and 1% Pen-Strep. Cells were seeded at low density onto 24-well plates 24 h before transfection. Adherent cells were transfected using Lipofectamine 2000 reagent (Invitrogen) with 300 ng Ca v 3.1 cDNA and recorded after 48 h. Whole-cell voltage-clamp recordings were performed at room temperature (24 • C). The peak currents of Ca v 3.1 were elicited by 150 ms depolarization from a holding potential of −100 mV to −40 mV at 4 s intervals. Borosil-icate glass micropipettes were pulled to produce a resistance of 4-6 MΩ and filled with intracellular recording solution containing 130 mM CsCl, 2 mM MgCl 2 , 10 mM EGTA, 5 mM Na-ATP, 10 mM HEPES (pH 7.2 with CsOH). The extracellular recording solution was composed of 145 mM CsCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 10 mM glucose, 10 mM HEPES (pH 7.4 with CsOH). The current trace of Ca v 3.1 in different states was analyzed by the Clampfit 10.6. Data were processed using the software Graphpad Prism 8.0.