Triterpene-Based Carboxamides Act as Good Inhibitors of Butyrylcholinesterase

A set of overall 40 carboxamides was prepared from five different natural occurring triterpenoids including oleanolic, ursolic, maslinic, betulinic, and platanic acid. All of which were derived from ethylene diamine holding an additional substituent connected to the ethylene diamine group. These derivatives were evaluated regarding their inhibitory activity of the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) employing Ellman’s assay. We further determined the type of inhibition and inhibition constants. Carboxamides derived from platanic acid have been shown to be potent and selective BChE inhibitors. Especially the mixed-type inhibitor (3β)-N-(2-pyrrolidin-1-ylethyl)-3-acetyloxy-20-oxo-30-norlupan-28-amide (35) showed a remarkably low Ki of 0.07 ± 0.01 µM (Ki′ = 2.38 ± 0.48 µM) for the inhibition of BChE.


Introduction
It is now more than a century since Alois Alzheimer, a German physician, described a new disease of the brain [1], being today one of the most threatening plagues for the elderly and one of the greatest challenges for chemists and biologists. The eponymous disease is today one of the greatest scourges of humanity. Alzheimer's disease (AD), the most common cause of dementia, is a neurodegenerative disorder, causing the progressive loss of cognitive functions and memory. As a result of demographic changes, the number of AD patients is steadily rising. This disease is characterized by an increasing decline in acetylcholine (ACh, neurotransmitter) levels in the cholinergic system [2]. A common strategy for the management of AD is to develop inhibitors that suppress the degradation of ACh caused by hydrolases acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE,EC 3.1.1.8).
Ursolic acid (UA), oleanolic acid (OA), maslinic acid (MA), betulinic acid (BA), and platanic acid (PA) are naturally occurring pentacyclic triterpenoids (Figure 1), which are widely distributed in various plants. Triterpenes and their derivatives represent a group of pharmacologically interesting substances showing a variety of biological activities such as antitumor [3], antiviral, and anti-HIV [4,5], antibacterial [6,7], and antifungal [8] properties. Pentacyclic triterpenoic acids have already been tested for cholinergic activities with moderate anti-cholinesterase activity [9,10]. Cyclic terpene derivatives, which act as inhibitors of cholinesterases, have been our research focus for a long time. Results from our group have demonstrated that structural modifications of the terpenoid backbone have a high impact onto the inhibitory potential for cholinesterases (ChEs) [11][12][13]. Exceptionally good ChE In patients, BChE activity increases with progression of AD while the level of AChE remains constant [16]. Therefore, selective BChE inhibitors represent legitimate therapeutic options to improve the deficit in the neurotransmitter ACh. Deduced from the results of our previous studies, UA, OA, MA, BA and PA carboxamides holding a terminal amino moiety were synthesized from the parent pentacyclic triterpenoic acids. Altogether, 40 different compounds were synthesized and screened for their ChE inhibitory activity using Ellman's assay.

Biology
In this study, carboxamides with different triterpenoic backbones (ursolic, oleanolic, maslinic, betulinic, and platanic acids), and various amine residues (Scheme 1), were investigated. All compounds 11-50 (except those being not soluble under the conditions of the assay) were subjected to Ellman's assays to determine their inhibition rates and constants (K i for competitive inhibition and K i for uncompetitive inhibition) for the cholinesterases AChE and BChE. Galantamine hydrobromide (GH), one of the gold standard drugs for treating AD symptoms, was used for comparison. In general, pre-screening the 37 triterpenoic acid amides using AChE (electric eel) and BChE (equine serum) identified seventeen compounds as potential inhibitors with inhibition rates equal or even better than GH. The results are summarized in Figures 2 and 3 (details are found in the Supplementary Part,  Table S1).
Molecules 2019, 24 FOR PEER REVIEW 3 GH . The results are summarized in Figures 2 and 3 (details are found in the supplementary part,  Table 1).

Figure 2.
Percentage of inhibition of selected carboxamides and galantamine hydrobromide (GH as standard), final concentration of the inhibitor 10 µM, determined by Ellman's assay using acetylcholinesterase (green in the front) and butyrylcholinesterase (purple in the back). The carboxamides showed a considerably higher inhibition for BChE than for AChE. With the exception of 17 (88.61 ± 0.22%) and 49 (82.72 ± 0.09%), none of the tested compounds showed any notable activity for AChE. Further kinetic studies (see supplementary part, Table 2) showed (3β)-N-(2-piperidin-1-ylethyl)- 3-hydroxy-lup-20 (29)-en- 28-amide (49) as a good mixed-type AChE inhibitor with inhibition constants: Ki = 1.00 ± 0.09 µM and Ki' = 1.42 ± 0.08 µM. The results of the screening experiments suggest that many of the synthesized carboxylic acid amides inhibit BChE activity to a high degree. Fifteen derivatives showed promising inhibitory rates, five of which seemed to inhibit BChE almost completely (88.37-94.60% inhibition rate). The evaluation of the Dixon [17], , and  plots showed that all active compounds were mixed-type   Table 1).
To explain the results from the biological testing, some molecular modeling studies were performed. Table 3 summarizes the results for the most favored docking position of each ligand. From these results, nice correlations between the gold fitness values (the higher the value the better the predicted affinity between ligand enzyme), as well as the interaction energies, with the experimental K i values were established.  Figure 4 displays details of the docking arrangements of ligands with BChE. All ligands fit into the binding pocket-preferentially based on hydrophobic interactions ( Figure 5) with the side chains of residues W79, W228, L283, Y329, and F326. The highest affinity was observed for 35 ( Figure 4a). Thus a hydrogen bond of the acetyl group of 35 with the phenolic hydroxyl group of Y79 stabilizes the interaction with BChE. This hydrogen bond, however, is not present in compounds 31 and 34. The modifications in ring E of 31 (six-membered ring) compared to compounds 34 and 35 (five-membered rings) slightly changed the docking arrangement (cf. Figure 4d) and caused reduced hydrophobic interactions in particular with W79.

General
All chemicals, reagents and technical equipment were purchased in Germany unless otherwise stated. NMR spectra were recorded using the Varian spectrometers Gemini 2000 or Unity 500 (δ given in ppm, J in Hz; typical experiments: H-H-COSY, HMBC, HSQC, NOESY), MS spectra were taken on a Finnigan MAT LCQ 7000 (electrospray, voltage 4.1 kV, sheath gas nitrogen) instrument. The optical rotations were measured on a Perkin-Elmer polarimeter at 20 • C; TLC was performed on silica gel (Merck 5554, detection with cerium molybdate reagent) melting points were uncorrected (Leica hot stage microscope or BÜCHI Melting Point M-565) and elemental analyses were performed on a Foss-Heraeus Vario EL (CHNS) unit. IR spectra were recorded on a Perkin Elmer FT-IR spectrometer Spectrum 1000 or on a Perkin-Elmer Spectrum Two (UATR Two Unit). The solvents were dried according to usual procedures. The purity of the compounds was determined by HPLC and found to be >96%. Ursolic (1), betulinic (4), and platanic acid (5) were obtained from Betulinines (Stříbrná Skalice, Czech Republic), oleanolic acid (2) was purchased from Carbone Scientific (London, UK) and maslinic acid (3) was synthesized as previously described [20,21].

Biology
A TECAN SpectraFluorPlus working in the kinetic mode and measuring the absorbance at λ = 415 nm was used for the enzymatic studies. Acetylcholinesterase (from Electrophorus electricus), 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) and acetylthiocholine iodide were purchased from Sigma. Butyrylcholinesterase (from equine serum) was purchased from Fluka. Preparation of the solutions, assay procedure, as well as molecular modeling conditions can be found in the Supplementary Materials.

Conclusions
In this study, 40 triterpenoic acid amides , with different triterpenoic backbones and various amine residues were synthesized and subjected to Ellman's assay to determine their potential as inhibitors of cholinesterases. Furthermore, some enzyme kinetic studies were performed. Thus, systematic variation of the amine substituent led to analogs possessing the same or even better BChE-inhibiting properties as standard galantamine hydrobromide. Outstanding derivatives were 2-pyrrolidin-1-ylethyl substituted compounds 34 (from BA), 36 (from UA), and 37 (from OA), showing K i values of 0.39 ± 0.04 µM, 0.47 ± 0.08 µM, and 0.55 ± 0.02 µM, respectively. Furthermore, Ellman's assay revealed several platanic acid compounds as excellent inhibitors of BChE. Particularly, 40, 45, and 50 were great inhibitors, showing inhibition rates even in the nanomolar range. The most active compound in the test was a hybrid holding a platanic acid backbone and a pyrrolidinyl residue. For (3β)-N-(2-pyrrolidin-1-ylethyl)- 3-acetyloxy-20-oxo-30-norlupan-28-amide (35), inhibition constants K i = 0.07 ± 0.01 µM and K i = 2.38 ± 0.48 µM have been determined. The results obtained in the biological assay can be explained by appropriate molecular modeling calculations. All active compounds were mixed-type BChE inhibitors with a dominating competitive part (K i < K i ). The best inhibitor for acetylcholinesterase was a betulinic acid derived piperidinyl derivative (49), acting as a mixed-type inhibitor showing K i = 1.00 ± 0.09 µM and K i = 1.42 ± 0.08 µM, respectively.