N-Methyl Costaricine and Costaricine, Two Potent Butyrylcholinesterase Inhibitors from Alseodaphne pendulifolia Gamb.

Studies have been conducted over the last decade to identify secondary metabolites from plants, in particular those from the class of alkaloids, for the development of new anti-Alzheimer’s disease (AD) drugs. The genus Alseodaphne, comprising a wide range of alkaloids, is a promising source for the discovery of new cholinesterase inhibitors, the first-line treatment for AD. With regard to this, a phytochemical investigation of the dichloromethane extract of the bark of A. pendulifolia Gamb. was conducted. Repeated column chromatography and preparative thin-layer chromatography led to the isolation of a new bisbenzylisoquinoline alkaloid, N-methyl costaricine (1), together with costaricine (2), hernagine (3), N-methyl hernagine (4), corydine (5), and oxohernagine (6). Their structures were elucidated by the 1D- and 2D-NMR techniques and LCMS-IT-TOF analysis. Compounds 1 and 2 were more-potent BChE inhibitors than galantamine with IC50 values of 3.51 ± 0.80 µM and 2.90 ± 0.56 µM, respectively. The Lineweaver–Burk plots of compounds 1 and 2 indicated they were mixed-mode inhibitors. Compounds 1 and 2 have the potential to be employed as lead compounds for the development of new drugs or medicinal supplements to treat AD.


Introduction
Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease that gradually causes cognitive decline and memory loss. ACh is a cholinergic neurotransmitter in the central nervous system (CNS), which relays information in the form of an electrical impulse from one neuron to another during neurotransmission. AD disrupts this communication network in the brain through the hydrolysis and inactivation of ACh by the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes, in turn terminating the signal transmission. The inhibition of the activities of AChE and BChE, which subsequently increases the ACh concentration, has been the primary step in the treatment of AD [1][2][3][4][5][6][7]. 2 of 11 The cholinesterase inhibitors approved by the Food and Drug Administration (FDA) to treat patients with mild to moderate AD include donepezil, galantamine, and rivastigmine. Though frequently used, the capacity of these drugs to cross the blood-brain barrier (BBB) is limited. Furthermore, these drugs have also shown various dose-associated side-effects, particularly at higher doses. Thus, there is still a need for new cholinesterase inhibitors with lower toxicity and higher BBB penetration [1,8].
Alkaloids and non-alkaloids have both been explored for their potential as cholinesterase inhibitors. Among these two classes of compounds, natural and semi-synthetic alkaloids have been found to be more promising candidates owing to their complex nitrogencontaining structures. It has been reported that one of the binding sites of AChE involves the interaction with the positively charged nitrogen. Hence, alkaloids serve as a template for the development of potent and effective new cholinesterase inhibitors [9,10].
Alseodaphne pendulifolia Gamb. is one out of eight species within the genus Alseodaphne that has been studied for its chemical constituents. It has been reported to yield isoquinolinetype alkaloids such as aporphine, oxoaporphine, and bisbenzylisoquinoline [15]. However, till present, there has only been two reports on the compounds which have been isolated and characterized from this species [16,17]. Hence, exploring the chemical constituents of A. pendulifolia and their biological activities is valuable.
In an attempt to further identify new and potent AChE or BChE or dual-cholinesterase inhibitors with either an isoquinoline or indole backbone, our group explored the alkaloidal content of the bark of A. pendulifolia and assessed their enzyme inhibitory activities.

Screening of Extracts for Cholinesterase Inhibitory Activities
Preliminary screening of the dichloromethane and methanol extracts of the bark of A. pendulifolia at a concentration of 100 µg/mL revealed that the dichloromethane extract (47.01 ± 1.81% and 95.36 ± 0.67%, respectively) was more effective in inhibiting the activities of AChE and BChE compared to the methanol (<10% and 28.41 ± 4.2%, respectively) extract. Therefore, the dichloromethane extract was further investigated with the intention of identifying the compound(s) that were responsible for giving rise to the AChE and BChE inhibitory activities.
The UV spectrum of compound 1 exhibited characteristic absorption peaks of a bisbenzylisoquinoline moiety at λmax 223 and 282 nm [26,27]. The IR spectrum revealed Compound 1 (Figure 1) was isolated as an optically active yellow amorphous powder. The positive LCMS-IT-TOF analysis, which exhibited a pseudo-molecular ion [M + H] + at m/z 597.2866 ( Figure S1) (calcd. for C 36 H 41 N 2 O 6 597.2965), enabled a molecular formula of C 36 H 40 N 2 O 6 with 18 degrees of unsaturation to be proposed. The high molecular mass and considering the fact that bisbenzylisoquinoline-type alkaloids have been characterized in the genus Alseodaphne, it was reasonable to postulate the possibility of compound 1 being such a compound [23][24][25].
The paired signals in the 13 C NMR spectrum implied that the structure of compound 1 was not symmetrical in nature. Therefore, the structure of compound 1 was proposed to be constructed from two partial structures, 1a and 1b. Partial structures 1a and 1b each comprised three substructures, rings A-C and rings A'-C' (Figure 1).
A closer look at the structures of compounds 1-5 provided further insight as to how the cholinesterase enzyme inhibitory activities of these alkaloids might have been influenced by the chemical groups in their respective structures. Although compound 1 is almost identical in structure to compound 2, the presence of a methyl group bonded to the nitrogen atom in ring B could have slightly suppressed its AChE and BChE inhibitory activities in comparison to compound 2 in which the methyl group was absent (Figure 1). The BChE inhibiting potential of compounds 3-5 though considered to be moderately active, exhibited varying degrees of potency. Although compounds 3-5 each possessed an aporphine nucleus with a 1,2,10,11-tetra substituted biphenyl system, it is evident that the methyl group bonded to the nitrogen atom in ring B of compounds 4 and 5 and the hydroxyl group bonded to position C-1 of ring A in compound 5 could have caused the decrease in their respective BChE inhibitory activities compared to the activity of compound 3 in which the methyl group was absent and a methoxyl group occupied position C-1 instead (Figure 1).
With regard to the selectivity of the compounds (Table 2), compounds 1 and 2 were found to be BChE selective inhibitors in contrast to galantamine, which is an AChE selective inhibitor.

Enzyme Kinetic Study
Kinetic studies were subsequently carried out on compounds 1 and 2, which actively inhibited the BChE, in order to determine their mode of inhibition. As illustrated in the Lineweaver-Burk plot analyses (Figure 3), compounds 1 and 2 both displayed mixed-mode inhibition against BChE as indicated by their data lines, which either intersected in the first (for compound 1) or second (for compound 2) quadrants. This type of inhibitor is able to bind to the active site of the enzyme, as well as at different sites of the enzyme (allosteric site) due to the allosteric effect [40,41]. The inhibition constants, K i , were derived from the secondary plots for compounds 1 (0.67 µM) and 2 (0.05 µM), implying that compound 2 has a 13-fold higher affinity to BChE compared to compound 1. a Data presented as mean ± SD (n = 3). b Selectivity for AChE is defined as IC50 (BChE)/IC50 (AChE). c Selectivity for BChE is defined as IC50 (AChE)/IC50 (BChE). NT = not tested.
A closer look at the structures of compounds 1-5 provided further insight as to how the cholinesterase enzyme inhibitory activities of these alkaloids might have been influenced by the chemical groups in their respective structures. Although compound 1 is almost identical in structure to compound 2, the presence of a methyl group bonded to the nitrogen atom in ring B could have slightly suppressed its AChE and BChE inhibitory activities in comparison to compound 2 in which the methyl group was absent (Figure 1). The BChE inhibiting potential of compounds 3-5 though considered to be moderately active, exhibited varying degrees of potency. Although compounds 3-5 each possessed an aporphine nucleus with a 1,2,10,11-tetra substituted biphenyl system, it is evident that the methyl group bonded to the nitrogen atom in ring B of compounds 4 and 5 and the hydroxyl group bonded to position C-1 of ring A in compound 5 could have caused the decrease in their respective BChE inhibitory activities compared to the activity of compound 3 in which the methyl group was absent and a methoxyl group occupied position C-1 instead (Figure 1).
With regard to the selectivity of the compounds (Table 2), compounds 1 and 2 were found to be BChE selective inhibitors in contrast to galantamine, which is an AChE selective inhibitor.

Enzyme Kinetic Study
Kinetic studies were subsequently carried out on compounds 1 and 2, which actively inhibited the BChE, in order to determine their mode of inhibition. As illustrated in the Lineweaver-Burk plot analyses (Figure 3), compounds 1 and 2 both displayed mixedmode inhibition against BChE as indicated by their data lines, which either intersected in the first (for compound 1) or second (for compound 2) quadrants. This type of inhibitor is able to bind to the active site of the enzyme, as well as at different sites of the enzyme (allosteric site) due to the allosteric effect [40,41]. The inhibition constants, Ki, were derived from the secondary plots for compounds 1 (0.67 µM) and 2 (0.05 µM), implying that compound 2 has a 13-fold higher affinity to BChE compared to compound 1.

General Experimental Procedures
Analytical and preparative TLC was carried out on Merck 60 F254 silica gel plates (absorbent thickness: 0.25 and 0.50 mm, respectively) (Merck, Darmstadt, Germany). Column chromatography (CC) was performed using silica gel 60 (70-230 mesh, ASTM) (Merck, Germany). All solvents were of analytical-grade and were distilled prior to use. IR spectra were recorded using a Perkin-Elmer Spectrum 400 FT-IR Spectrometer. NMR spectra were acquired in CDCl 3 with tetramethylsilane as an internal standard (Merck, Germany) using either a JOEL ECX 500 MHz NMR Spectrometer or a BRUKER Advance III 600 NMR Spectrometer (BRUKER, Billerica, MA, USA). LCMS-IT-TOF spectra were obtained using an Agilent 6530 Accurate-Mass Q-TOF LC/MS system (Agilent, Santa Clara, CA, USA). UV spectra were recorded using a Shimadzu UV-250 UV-Vis Spectrophotometer (Shimadzu, Tokyo, Janpan). A Jasco P-1020 polarimeter was used to record the optical rotation (JASCO, Hachioji, Tokyo, Japan).

Plant Material
A. pendulifolia was collected from the Sungai Tekam Reserve Forest, Jerantut, Pahang, Malaysia. The plant was identified by a botanist, Mr. Teo Leong Eng, and a voucher specimen (KL5732) has been deposited with the University of Malaya herbarium.

In Vitro Cholinesterase Enzyme Inhibitory Activity
The cholinesterase inhibitory activities of the extracts and fompounds 1-6 were performed as previously described [42]. AChE from Electrophorus electricus and BChE from equine serum were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). An amount of 0.1 M of sodium phosphate buffer (pH 8) was added to a 96-well microplate followed by the sample and 1U of the AChE or BChE. Then, 10 mM of 5,5 -dithiobis(2nitrobenzoic acid) (DTNB) was added followed by 14 mM of acetylthiocholine iodide or S-butyrylthiocholine chloride. The final concentration of DMSO was 1%. Galanthamine was used as the standard for validation and result comparison. The absorbance was measured using a Tecan Infinite 200 Pro Microplate Spectrometer at 412 nm for 30 min.

Enzyme Kinetic Studies
The enzyme kinetic studies of compounds 1 and 2 against BChE were performed in a similar manner to the in vitro BChE inhibition assay. The enzyme inhibition kinetic was carried out in the presence of three concentrations of the inhibitors, compounds 1 and 2, at various concentrations of the substrate, S-butyrylthiocholine chloride by constructing a Lineweaver-Burk plot (reciprocal plots of 1/V versus 1/[S]). The inhibition constant (Ki) was estimated from the secondary plot of the Lineweaver-Burk plot [14].

Conclusions
N-methyl costaricine (1), a new bisbenzylisoquinoline alkaloid, along with costaricine (2), hernagine (3), N-methyl hernagine (4), corydine (5), and oxohernagine (6) were isolated and characterized from the bark of A. pendulifolia. The AChE level decreases significantly and the ratio of BChE to AChE changes dramatically in the cortical regions with AD progression [43,44]. Hence, a BChE inhibitor could have better curative effects for AD. In this study, compounds 1 and 2 displayed potent BChE inhibitory activity. Therefore, the potency of compounds 1 and 2 in the current investigation suggested that further studies can be carried out to evaluate their amyloid-β-inhibition potential in vitro, and these alkaloids could be employed as lead compounds for the development of new drugs or medicinal supplements to treat AD.