Anticholinesterase activity of β-carboline-1,3,5-triazine hybrids

Abstract

The β-carboline-1,3,5-triazine hydrochlorides 8-13 were evaluated in vitro against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The analysed compounds were selective to BuChE, with IC₅₀ values in the range from 1.0-18.8 µM being obtained. The N-{2-[(4,6-dihydrazinyl-1,3,5-triazin-2-yl)amino]ethyl}-1-phenyl-β-carboline-3-carboxamide (12) was the most potent compound and kinetic studies indicate that it acts as a competitive inhibitor of BuChE. Molecular docking studies show that 12 strongly interacts with the residues of His438 (residue of the catalytic triad) and Trp82 (residue of catalytic anionic site), confirming that this compound competes with the same binding site of the butyrylthiocholine.

Keywords:
β-carboline. 1,3,5-triazine. Acetylcholinesterase. Butyrylcholinesterase

INTRODUCTION

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder (Larson, Kukull, Katzman, 1992), characterised by cognitive impairment, which is associated by means of cholinergic hypothesis, to the loss cholinergic of neurons and a decrease in levels of cholinergic neurotransmission (Francis et al., 1999; Pinto, Lanctôt, Herrmann, 2011). The hydrolysis of the neurotransmitter acetylcholine (ACh) into choline and acetic acid, a reaction catalysed by enzymes of the cholinesterase family, is necessary to allow a cholinergic neuron to return to its resting state after activation (Čolović et al., 2013). The acetylcholinesterase (AChE) predominates in the healthy brain, while the butyrylcholinesterase (BuChE) is considered to play a minor role in the regulation of synaptic ACh levels (Silva et al., 2014). In fact, most of the current drugs used for AD treatment are based on this hypothesis, acting mainly by the inhibition of AChE (Silva et al., 2014; Čolović et al., 2013). However, studies have shown that as AD progresses, the BuChE activity progressively increases, while AChE activity remains unchanged or gradually decreases. When this occurs, the BuChE assumes function of metabolize the ACh in the synapse (Darvesh, Hopkins, Geula, 2003; Anand, Singh, 2013; Li et al., 2017). Thus, also inhibiting BuChE, cognitive improvements associated with the current cholinesterase inhibitors can be obtained. As a result, both AChE and BuChE can be considered as therapeutic targets in Alzheimer's disease treatment (Darvesh, Hopkins, Geula, 2003; Anand, Singh, 2013; Li et al., 2017).

The treatment of AD usually is performed with cholinesterase inhibitors (donepezil, rivastigmine and galantamine), which enhance cholinergic signalling in the central nervous system and cognitive symptoms. However, these drugs do not prevent the AD progression, and unfortunately there is still no cure for this disease (Silva et al., 2014; Pinto, Lanctôt, Herrmann, 2011; Čolović et al., 2013).

In recent years, several works have been carried out with the aim of obtaining new inhibitors for AChE and BuChE, for the treatment of AD (Anand, Singh, 2013; Li et al., 2017). In this context, several classes of heterocyclic compounds, including those containing the 1,3,5-triazine and β-carboline nucleus, were described as potential anticholinesterase inhibitors (Veloso et al., 2013; Jameel et al., 2017; Maqbool et al., 2016; Rook et al., 2010; Jin-Shuai et al., 2014; Horton et al., 2017).

Studies have demonstrated that 1,3,5-triazine derivatives were found to act as multi-target anti-Alzheimer agents (Veloso et al., 2013; Jameel et al., 2017; Maqbool et al., 2016; Trifunović et al., 2017). Trisubstituted triazines (I, Figure 1), for example, inhibited important targets associated with AD, such as AChE, BuChE and Aβ aggregation (Veloso et al., 2013). The triazine-triazolopyrimidine hybrid II (Figure 1) showed the inhibition of AChE (IC₅₀ = 0.065 µM) and BuChE (IC₅₀ = 1.88 µM) similar to donepezil (IC₅₀ = 0.047 µM for AChE and 2.72 µM for BuChE), which is a potential candidate for anti-Alzheimer’s drug (Jameel et al., 2017). Also, cyanopyridine-triazine hybrids inhibit AChE and BuChE, and can reduce neuronal death induced by H₂O₂-mediated oxidative stress and Aβ _1-42 induced cytotoxicity (Maqbool et al., 2016).

FIGURE 1
Structures of 1,3,5-triazine (I and II) and β-carboline (III, IV and Va,b) derivatives with anticholinesterase activity, of DYRK1A inhibitor VI and of β-carboline-1,3,5-triazine hybrid VII.

Studies focusing on the properties of (-carbolines concerning neurodegenerative diseases have also been intensified in recent years, and several researches have highlighted these alkaloids as a new class of anti-Alzheimer agents. β-Carboline derivatives showed activities in neurological disorders associated with AD, acting as potent inhibitors of AChE and BuChE (Rook et al., 2010; Jin-Shuai et al., 2014; Horton et al., 2017; Torres et al., 2012), dual specificity tyrosine phosphorylation regulated kinase-1A (DYRKA) (Drung et al., 2014; Rüben et al., 2015) and monoamine oxidase (MAO) (Santillo et al., 2014). The bivalent β-carboline derivative III (Figure 1) showed potent anticholinesterase activity, displaying AChE inhibition (IC₅₀ = 0.5 nM) higher than the reference drug tacrine (IC₅₀ = 45 nM), and approximately the same activity as that of tacrine for BuChE (IC₅₀ ≅ 5 nM) (Rook et al., 2010). On the other hand, the harmane (IV, Figure 1) and its β-carbolinium derivatives Va and Vb (Figure 1) exhibited greater selectivity towards BuChE over AChE. The compounds Va and Vb (IC₅₀ = 0.23 and 0.637 µM) were more active to BuChE than physostigmine (IC₅₀ = 3.7 µM) making them suitable prototypes in the search for anti-Alzheimer drugs (Torres et al., 2012). Also, β-carbolines with an extended aromatic ring system were highly active and selective for BuChE, and it was found that over 60% of the studied compounds showed a better inhibitory activity of BuChE than the drug galantamine (Horton et al., 2017).

In our previous work, we investigated the properties of (-carbolines related to neurodegenerative diseases, which led us to identify compound VI (Figure 1) as a potent DYRK1A and MAO-A inhibitor (Drung et al., 2014). By continuing our research, and taking in account the related proprieties of β-carbolines and 1,3,5-triazines, in this work we evaluated the anticholinesterase activity of β-carboline-1,3,5-triazine hybrids VII (Figure 1) against AChE and BuChE. Additionally, kinetic and molecular docking studies were carried out for the most potent compound, aiming to evaluate its inhibition mode against BuChE.

MATERIAL AND METHODS

Synthesis of β-carboline-1,3,5-triazine hydrochlorides (8-13)

The β-carboline-1,3,5-triazine hybrids were synthesised as described for Baréa et al. (2018). The hydrochloride salts 8-13 were prepared from the treatment of β-carboline-1,3,5-triazine hybrids (1 mmol) with hydrochloric acid (12 M) in methanol, at room temperature for 4 h. Compounds 8-13 were obtained in yields in the range from 50-80%. Elemental analysis for compound 12, calculated for C₂₃H₂₃N₁₁O.5HCl: C 42.38, H 4.33, N 23.64, found: C 43.26, H 4.76, N 20.20.

In vitro assays

In vitro inhibition studies on AChE and BuChE

AChE (from electrophorus electricus, type VI-S, lyophilised powder, lot 041M7009V), BuChE (from equine serum, lyophilised powder, lot SLBB2114V). 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman’s reagent), acetylthiocholine iodide, and S-butyrylthiocholine iodide were purchased from Sigma Aldrich. Absorbance measurements were taken using a Molecular Devices FlexStation 3 Microplate Reader with Softmax Pro 5.3 software.

Anticholinesterase activities of β-carboline-1,3,5-triazine hydrochlorides against AChE and BuChE were evaluated according to Ellman’s modified method (Ellman et al., 1961). Stock solutions of the tested compounds 8-13 were prepared in milli-Q water. The tests were performed in polystyrene 96-well plate, with 125 µL of DTNB (0.5 mM), 50 µL of buffer solution of phosphate (pH 8), 25 µL of sample solution at different concentrations and without the inhibitor (control), and 25 µL of enzyme solution of AChE (0.23 U mL^-1 prepared in buffer solution of phosphate) or BuChE (0.23 U mL^-1 prepared in buffer solution of phosphate) were added to each well. The plate was incubated at 30°C for 15 minutes under stirring, and then absorbance was measured at a wavelength of 412 nm. After this time, 25 µL of substrate (acetylthiocholine or butyrylthiocholine, 5 mM, prepared in milli-Q water) was added to each well. The plate was kept at 30°C, under stirring, and the absorbance was measured again at the same wavelength after 4 minutes. The tests were performed in triplicate.

The rates of reactions were calculated using appropriate software (Origin 6.1). The inhibition percentages were calculated by comparing of control reaction rate with the sample reaction rate using Eq.1:

$\%\text{inhibition}=\left(\left(\text{control reaction rate-sample reaction rate}\right)/\text{control reaction rate}\right)\times 100$ (1)

The inhibition curve was obtained by plotting an inhibition percentage graph versus the logarithm of the inhibitor concentration.

Kinetic analysis of BuChE inhibition

Kinetic studies were carried out by Ellman’s modified method (Ellman et al., 1961) for compound 12, using a 0.23 U mL^-1 solution of BuChE from equine. The test was performed without the inhibitor, in 0.3 and 3.0 µM concentrations of inhibitor 12 for BuChE. Butyrylthiocoline iodine was used as substrate of the reaction in the following final concentrations: 0.05, 0.125, 0.50, 0.75, 1.0 and 2.0 µM. The absorbance was measured in 10 s for 6 min. The obtained data were used to create substrate-velocity curves which were transformed using the Origin 6.1 program into Linerweaver-Burk plots.

Molecular Modelling

The crystal structure of BuChE complexed with butyrylcholinesterase (code ID: 1P0P) (Nicolet et al., 2003) was obtained from the Protein Data Bank and the N-{2-[(4,6-dihydrazinyl-1,3,5-triazin-2-yl)amino]ethyl}-1-phenyl-β-carboline-3-carboxamide (12) structure was drawn using the Marvin Sketch Version 14.8.25 ChemAxon. The molecular docking studies were performed using the AutoDock Vina program (Trott, Olson, 2010) implemented at the interface PyRx 0.9 (Wolf, 2009) using default parameters. For each PDB file, some molecules of water and other ligands (except butyrylcholinesterase) were removed. The box dimensions were set at 25 × 22 × 22 Å and the center of the grid box was placed at coordinates x = 133.7, y = 115.1, z = 41.0.

Re-docking simulations were performed to validate the parameters that had been chosen and were repeated four times which gave a RMSD values below 0.5 Å. The best results were submitted to energy minimisation with the NAMD2 program (Phillips et al., 2005). The force field adopted for proteins was CHARMM C35b2-C36a2, and for the ligands, they were generated in the same format as the SwissParam server (Zoete et al., 2011). The results were shown with the CCP4 Molecular Graphics software (McNicholas et al., 2011).

RESULTS AND DISCUSSION

Chemistry

The β-carboline-1,3,5-triazine hybrids 8-13 (Scheme 1) were synthesised as described for Baréa et al. (2018). Briefly, the β-carboline intermediate 1, obtained from L-tryptophan commercial, was subjected to reaction with cyanuric chloride in the absence or presence of different amines, under basic medium. The hydrochloride salts were prepared from the treatment of compounds 8-13 with hydrochloric acid in methanol (Scheme 1).

SCHEME 1
Synthesis of compounds 8-13. Reagents and conditions: (a) Cyanuric chloride, NaOH (1M), H₂O, CH₃CN, THF, 0ºC, 1 h. (b) 1) Cyanuric chloride, NaOH (1M), H₂O, CH₃CN, THF, 0ºC, 1h; 2) Amine (cyclohexylamine for 9; 1-methylpiperazine for 10; benzylamine for 11; hydrate hydrazine for 12; isopropylamine for 13), 70°C, 48 h; (c) MeOH, HCl 12 M, r,t., 4 h.

Anticholinesterase activity

The anticholinesterase activities of β-carboline-1,3,5-triazine hydrochlorides 8-13 and donepezil (reference compound) were evaluated according to the Ellman method (Ellman et al., 1961). Firstly, the compounds 8-13 were evaluated in vitro at concentrations of 10 µM and 100 µM against AChE and BuChE and their percentages of inhibition were determined (Table I). All compounds showed less than 50% or no inhibition for AChE, and their IC₅₀ (50% Inhibitory Concentration) values were not determined for this enzyme. On the other hand, all hybrids inhibited the BuChE at a concentration of 100 µM, showing inhibition percentage in the range from 67.3-91.9%, and most of them also inhibited this enzyme at concentration of 10 µM (40.5-82.7%). Thus, the IC₅₀ values were determined for compounds that inhibited more than 40% of this enzyme, at a concentration of 10 µM (Table I). The obtained IC₅₀ values ranged from 1.0-18.8 µM, with derivative 12, containing the hydrazinyl group at 6- and 4-positions of 1,3,5-triazine ring, the most active among the tested compounds for BuChE.

TABLE I
Inhibition percentages of compounds 8-13 against AChE and BuChE and theirs IC₅₀ values for BuChE

		% inhibition AChE		% inhibition BuChE		BuChE IC50 (µM)
Comp.	R	10 µM	100 µM	10 µM	100 µM
8	Cl	NI	6.5 ± 3.4	44.9 ± 5.4	80.9 ± 2.2	10.2 ± 0.3
9		NI	0.5 ± 0.1	NI	67.3 ± 9.9	N.D.
10		NI	45.9 ± 2.8	40.5 ± 2.9	83.4 ± 1.6	12.0 ± 1.4
11		NI	NI	49.3 ± 5.4	83.0 ± 2.2	18.8 ± 3.8
12	NHNH2	7.6 ± 0.2	29.2 ± 6.5	82.7 ± 0.3	91.9 ± 5.5	1.0 ± 0.1
13		9.7 ± 3.3	17.1 ± 1.5	71.6 ± 3.5	80.1 ± 6.2	5.8 ± 4.1

• NI = No Inhibition. ND = Not Determined. Donepezil was used as positive control (IC_{50 AChE} = 10.8 ± 3.0 nM; IC_{50 BuChE} = 2.9 ± 0.5 μM).

In summary, our results show that the analysed β-carboline derivatives were selective to BuChE. This selectivity can be explained based on the volume of the BuChE active site gorge, which is ∼200 ų larger than the AChE gorge (Saxena et al., 1997; Johnson, Moore, 2012; Masson, Carletti, Nachon, 2009), allowing the accommodation of tested β-carboline-1,3,5-triazine hybrids. Moreover, other compounds of the β-carboline class with a flexible linker also exhibited selectivity for BuChE (Zhao et al., 2018). The authors explain that folded molecules are suitable for the relatively spherical and large cavity of BuChE, but not stretched and slender enough to fit the narrow gorge of AChE (Zhao et al., 2018). Therefore, the presence of the flexible N-aminoethyl-carboxamide group between the β-carboline and 1,3,5-triazine moieties in 8-13 probably corroborated the obtained selectivity.

Enzyme kinetics

Due to the potent activity observed for compound N-{2-[(4,6-dihydrazinyl-1,3,5-triazin-2-yl)amino]ethyl}-1-phenyl-β-carboline-3-carboxamide (12), this compound was submitted to kinetics studies to investigate its type of BuChE inhibition. The kinetics studies were performed using the modified Ellman’s method (Ellman et al., 1961). To assess the kinetic parameters, we measured the initial rate of enzyme activity at different concentrations of substrate butyrylthiocholine (0.05 to 2.0 mM) in the absence and presence of the compound 12. Lineweaver-Burk plots (Figure 2) were generated by plotting the reciprocal of the initial rate (1/v₀) against the reciprocal of substrate concentrations (1/[S]) for the different concentrations of 12, resulting from the substrate-velocity curves for BuChE.

FIGURE 2
Lineweaver-Burk plot for the inhibition of BuChE with different butyrylthiocholine concentrations (0.05 to 2.0 mM) in the absence and presence of compound 12 at concentrations of 0.3 and 3.0 µM.

Graphical analysis of Lineweaver-Burk plots (Figure 2) and the kinetic parameters of BuChE activity showed a practically unchanged V _max value (V _max = 0.11, 0.10 and 0.11 µMs^-1 in the absence and presence of 0.3 and 3.0 µM of 12, respectively) and an increasing K _m value (K _m = 0.28, 0.30 and 1.58 mM in the absence and presence of 0.3 and 3.0 µM of 12, respectively) with increasing inhibitor concentrations, i.e. increasing slopes and the same intercepts on the y-axis (-1/V _max). This pattern indicates a competitive type of inhibition (Copeland, 2000). It is shown that compound 12 and substrate (butyrylthiocholine) compete for the same active site, i.e. the inhibitor interacts with the same binding site as the substrate.

For hybrid 12, the dissociation constant (K_i) value obtained was 0.55 µM while the K_m value of butyrylthiocholine iodide for BuChE was 0.28 mM, which shows that the binding capacity of 12 with BuChE is approximately 509-fold that of the substrate. In addition, the hybrid 12 exhibited a K_i value similar to derivative Vb (Figure 1, K_i = 0,64 µM for BuChE) (Torres et al., 2012) and showed a binding capacity that was approximately 164-fold greater than that of harmane (IV, Figure 1, K_i = 90 µM for BuChE) (Torres et al., 2012).

Molecular modelling studies

The molecular docking calculations were performed using AutoDockVina program (Trott, Olson, 2010) implemented at the interface PyRx 0.9 (Wolf, 2009). Compound 12 was docked in the active site of BuChE (PDB: 1P0P) (Nicolet et al., 2003) derived from the complex of the enzymes with butyrylcholinesterase obtained from the Protein Data Bank (PDB). The best docked poses, i.e. the lowest energy conformer in the most populated cluster of conformers, were subjected to energy minimisation by NAMD (Phillips et al., 2005) program and analysed to explain interactions between ligands and the target enzyme. Figure 3 shows that compound 12 is oriented in active site gorge of BuChE. The hydrogen atom of protonated triazine moieties interacts with the carboxylate oxygen atom of Gly115 via an H-bond. The oxygen atom of the carbonyl group forms an H-bond with OH group of Thr120 and NH group also forms an H-bond with the carboxylate oxygen atom of Gly116. The protonated nitrogen of hydrazine moieties interacts with Tyr440 and Trp82 by π-cation interaction. Both hydrogen atoms of the protonated hydrazine moieties are therefore likely to form an H-bond with the carboxylate oxygen atoms of His438 and Gly439 and the other protonated hydrazine moieties also interact with Glu197 by H-bond interactions. This strong interaction with His438 (residue of the catalytic triad) (Nicolet et al., 2003) and the π-cation interaction with Trp82 (residue of catalytic anionic site) (Nicolet et al., 2003) confirm that the compound competes with the same binding site of the butyrylthiocholine.

FIGURE 3
Binding mode of 12 and BuChE. The compound is rendered in green ball-and-stick models, and the residues are rendered in grey coloured sticks.

CONCLUSION

In conclusion, we evaluated the anticholinesterase activity of the β-carboline-1,3,5-triazine hybrids against AChE and BuChE. All of the compounds showed significant activity and selectivity for BuChE. The kinetics and molecular docking studies for the most active hybrid 12 indicate that this compound inhibited BuChE via a competitive type of inhibition.

ACKNOWLEDGMENTS

This work was supported by Fundação Araucária, PR, Brazil (Sarragiotto MH, No. 2/2017 Prot. 47223 FA/UEM). We thank CAPES for fellowship (Baréa P) and Complexo de Centrais de Apoio à Pesquisa of Universidade Estadual de Maringá (COMCAP-UEM) for the facilities.

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Publication Dates

• Publication in this collection
  15 July 2022
• Date of issue
  2022

History

• Received
  20 Nov 2019
• Accepted
  31 Mar 2020