Novel Conjugates of Tacrine with 1,2,4,-Thiadiazole as Highly Effective Cholinesterase Inhibitors, Blockers of NMDA Receptors, and Antioxidants
Abstract—Conjugates of tacrine with 1,2,4-thiadiazole derivatives were synthesized for the first time. Their esterase profile and effects on the key NMDA receptor-binding sites as well as antioxidant activity were inves- tigated. The obtained compounds effectively inhibited cholinesterases (with a predominant effect on butyr- ylcholinesterase), simultaneously blocked two NMDA receptor-binding sites (allosteric and intrachannel sites, and exhibited a high radical-scavenging activity. Our study shows that the obtained compounds are promising to design drugs for the treatment of Alzheimer’s disease and other multifactorial neurodegenerative diseases.
Alzheimer’s disease (AD) is a neurodegenerative disease currently posing a serious social and health problem due to the progressive increase in the number of patients and the lack of effective therapy. Despite the huge efforts in search for new effective agents for the AD treatment, no new drug has appeared on the pharmaceutical market in the past 11 years [1, 2].The progression of AD is associated with signifi- cant impairment of many neurotransmitter systems. The cholinergic and glutamatergic systems are affected most severely [3, 4]. Modern AD therapy includes the use of cholinesterase inhibitors (Aricept, Exelon, and Galantamine) and the NMDA receptor antagonist memantine. The combination of anticho- linesterase drugs with memantine is the most effective. This approach to AD treatment is based on the syner- gistic effect of cholinesterase inhibitors and NMDA receptor modulators for improving memory and cog- nitive functions [5].One of the main factors adversely affecting the functioning of brain neurons is oxidative stress. In this regard, conferring an additional antioxidant function to cholinesterase inhibitors and NMDA receptor blockers is a promising direction in the development of effective pharmacotherapy of AD [6].Taking into account the multifactorial nature of AD [1, 2], it is of interest to design drugs with a com- bined effect, which could simultaneously affect several targets during the disease progression (in particular, the enzymes of the cholinesterase group and NMDA receptors) and would exhibit antioxidant properties [2, 6, 7].In this study, we propose to use tacrine, a potent inhibitor of acetylcholinesterase (AChE) and butyryl- cholinesterase (BChE), as the main anticholinesterase pharmacophore. The high affinity of the tacrine frag- ment for the catalytic site of AChE is widely used to create hybrid compounds, conjugates with a wide range of pharmacological activity [8].
Although tacrine was the first drug approved for the AD treatment, its practical application has been limited by its hepatotoxicity. However, it was later demonstrated that the hepatotoxicity of tacrine can be reduced in the presence of free-radical scavengers. As a result, a new direction in the search of multifunc- tional drugs for AD therapy has appeared: the devel- opment of tacrine conjugates conferred with addi- tional antioxidant properties. Recent examples of such molecules are lipocrine, tacrine–melatonin hybrids, derivatives of tacrine with 8-hydroxyquinoline, etc., which can also protect cells from oxidative stress [8].We propose to use the 1,2,4-thiadiazole scaffold as a second pharmacophore. 1,2,4-Thiadiazole deriva- tives are of great interest due to their wide-range bio- logical activity [9, 10]. This class of compoundsincludes AChE inhibitors, antioxidants, and ligands of glutamate receptors of the CNS. Drug Tideglusib, a 1,2,4-thiadiazolidine derivative, was tested in phase II of clinical trials in patients with AD.Our recent study [10] of the biological activity of 3- substituted 5-anilino-1,2,4-thiadiazoles containing various substituents at position 3 of the thiadiazole ring showed their inhibitory activity and selectivity for BChE and a high radical-scavenging activity.The purpose of this work to perform a directed syn- thesis of tacrine conjugates with derivatives of 1,2,4- thiadiazoles and to study their esterase profile, the effect on the key binding sites of NMDA receptor, and the antioxidant activity.Target compounds were synthesized by the method developed by us earlier [11] using the reaction of 5- anilino-3-(2-oxopropyl)-1,2,4-thiadiazole (2) withdiamine (1) (Scheme 1).1H NMR spectra were recorded with a Bruker DPX-200 spectrometer (Bruker, Germany); chemical shifts are given on a -scale relative to Me4Si.
Melting points were determined on a Boetius heating stage without correction (Table 1).To evaluate the esterase profile of compounds, we studied their inhibitory activity against human eryth- rocyte AChE (EC 3.1.1.7), equine serum BChE (EC 3.1.1.8), and a structurally related to cholinesterases, por- cine liver carboxylesterase (CaE, EC 3.1.1.1). The enzymes were purchased from Sigma-Aldrich (United States). The activity of AChE and BChE was deter- mined by the method of Ellman ( = 412 nm) in 100 mM phosphate buffer (pH 7.5) at 25C using 1 mM acetylthiocholine or 1 mM butyryl thiocholine as a substrate; the procedure is described in detail in [7]. The activity of CaE was determined spectrophoto- metrically ( = 405 nm) by the release of 4-nitrophe- nol in 100 mM phosphate buffer (pH 8.0) at 25C using 1 mM 4-nitrophenyl acetate as a substrate; theprocedure is described in detail in [7]. Measurements were performed in a FLUOStar Optima microplate reader (LabTech, Germany). Compounds were dis- solved in DMSO, the incubation mixture contained 2% solvent. The inhibitory activity of the compounds was characterized by IC50 values. The range of concen-tration of the test compounds was 1 × 10–12–1 × 10–4 M. Each experiment was performed in triplicate. IC50 val- ues were calculated using the Origin 6.1 software for Windows (OriginLab, United States). The results of this series of experiments are summarized in Table 2.The binding of compounds to different modulatory regions of NMDA receptors was studied by the radi- oligand method. The ability of compounds to interact with the entire pool of NMDA receptors was evaluated by the competitive binding with [3H]-MK-801 (dizo- cilpine) [12].
The analysis of the competitive binding with [3H]-ifenprodil was used to determine the ability of the compounds to interact with the NMDA recep- tor type comprising NR2B subunits [13]. The com- pounds were tested in vitro in a concentration range of 1 × 10–9–1 × 10–4 M. The results of these experiments are summarized in Table 3.The antioxidant activity of the compounds was assessed in the ABTS assay [14]. The compounds were incubated with the ABTS•+ radical at 30C for 1 h in the dark. The compounds were tested in a concentra- tion range of 1 × 10–6–1 × 10–4 M. The degree of ABTS•+ discoloration was measured at a wavelength of 734 nm in a xMark™ microplate reader (BioRad, United States). Trolox was used as a standard antioxi- dant. All measurements were performed in triplicate. The radical-scavenging activity of the compounds is represented in TEAC units (Trolox equivalent antiox- idant capacity) as the ratio between the slopes obtained from the linear correlation for concentra- tions of Trolox and the antiradical compounds withabsorbance. For all of the studied tacrine conjugates, we also calculated IC50 values (the antioxidant con- centration in M at which the concentration of ABTS•+ decreased by 50%). Calculations were per- formed using the Origin 6.1 software for Windows (OriginLab Corp., United States). The results of this series of experiments are shown in Table 3.The results were statistically processed using the GraphPad Prism software v. 6.05 for Windows (GraphPad Software, United States).As a result of kinetic studies, we determined the inhibitory activity of compounds 3a–3d against AChE, BChE, and CaE. Carboxylesterase hydrolyzes many drugs containing ester, amide, or carbamate groups, and the inhibition of this enzyme by anticho- linesterase compounds used for AD treatment may cause adverse drug interactions. The obtained IC50 values characterizing the esterase profile of the studied compounds 3a–3d are shown in Table 2.
The analysis of the esterase profile of com- pounds 3a–3d (Table 2) showed that all the studied conjugates of tacrine with 1,2,4-thiadiazole effectively inhibited AChE and BChE, exhibiting a higher activ- ity against BChE. The IC50 values of these com- pounds were in the range of 0.07–0.1 M, which is only slightly inferior to the activity of tacrine. Similarly to tacrine, the synthesized conjugates showed a selec- tivity towards BChE as compared to AChE; however, they also inhibited CaE.In a healthy brain, acetylcholine is hydrolyzed pri- marily by AChE, whereas BChE plays an auxiliary role. However, with the AD progression, the activity of AChE decreases, whereas the activity of BChE gradu- ally increases [15]. In this regard, the role of BChE as a therapeutic target for compensation for the choliner- gic deficiency observed in AD increases [3, 15]. The high activity of conjugates 3a–3d as inhibitors of both AChE and BChE and their higher selectivity for BChE compared to AChE and CaE (Table 2) is of interest for designing drugs for effective and safer treatment of AD.The use of NMDA receptor antagonists for the treatment of AD and other neurodegenerative diseases is currently restricted by memantine, a low-affinity blocker of the intrachannel binding site of MK-801, which shows a good binding kinetics to NMDA recep- tor [5]. Data presented in Table 3 show that the studied conjugates more efficiently interact with the binding site of MK-801 as compared to tacrine but slightly inferior to memantine. However, they acquire the ability to block the binding site of ifenprodil, which is absent in both original pharmacophores.Today, the number of known NMDA receptor blockers that are able to simultaneously block the binding sites of MK-801 and ifenprodil is very small.
Therefore, the fact that the synthesized compounds exhibit the properties of blockers of the allosteric modulatory site of NMDA receptors (the binding siteof ifenprodil) and simultaneously acquire the proper- ties of blockers of the intrachannel (MK-801-specific) binding site is an important result that can enhance the therapeutic potency of these compounds for the treat- ment of AD, stroke, or depressions as compared to the monoligand drugs.The study of the antioxidant properties of the con- jugates of tacrine with 1,2,4-thiadiazole in the test with the model radical ABTS•+ showed that com- pounds 3a–3d exhibit a high radical-scavenging activ- ity, which is higher than the activity of the standard antioxidant Trolox (Table 3). Similarly to Trolox, all the compounds exhibited a high initial ABTS•+-bind- ing rate, and the maximum binding of the radical was reached as soon as 10 min after the start of the reac- tion. The results show that the introduction of the thiadiazole moiety confers the synthesized tacrine conjugates with the antioxidant properties.Thus, in this study we found novel multifunctional compounds, conjugates of tacrine with 1,2,4-thiadi- azole derivatives, which effectively inhibit cholinester- ases with a primary effect on BChE, simultaneously block two binding sites of NMDA receptor (allosteric and intrachannel sites), and exhibit a high radical- scavenging activity. The results of this study allow these compounds to be considered as promising can- didates to design drugs for therapy of AD and other neurodegenerative diseases.