Synthesis of a Novel Series of Phosphonate-Functionalized 1,2,3-Triazoles as Potential Candidates for Allosteric Modulation of α 7 Nicotinic Acetylcholine Receptors

: Nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels formed by the assembly of five subunits. Receptor activity could be subjected to both positive and negative modulation at allosteric sites by endogenous neurotransmitters as well as synthetic ligands such as steroids, bivalent cations, alcohols, and a range of drugs. The subtype of α 7 nAChR has been considered a potential therapeutic target for Alzheimer’s disease, schizophrenia and other neurological and psychiatric disorders. In this work we present the synthesis of a novel series of phosphonate-functionalized 1,4-disubstituted 1,2,3-triazoles with potential activity over α 7 AChR. These compounds were synthetized through the copper-catalyzed Huisgen 1,3-dipolar cycloaddition of organic azides and alkynes. Copper nanoparticles (CuNPs) immobilized on different supports were prepared using the CuCl 2 -Li-DTBB reducing system previously reported by our group.


Introduction
Nicotinic acetylcholine receptors (nAChRs) are transmembrane receptors that are activated by the neurotransmitter acetylcholine. These receptors are distributed throughout the central and peripheral nervous systems and play important roles in several (patho)physiological processes [1]. Seventeen subunits have been identified in vertebrates (α1-10, β1-4, γ, δ and ε), which co-assemble to form a diverse family of receptor subtypes. In particular, the human 7 nAChR has been identified as a potential target for therapeutic drug discovery and has been implicated in a number of neurological and psychiatric disorders [2]. Considering the fact that α7 nAChR undergoes very rapid desensitisation in response to agonist activation, several positive allosteric modulators (PAMs) were synthesized and evaluated. α7 nAChR PAMs are classified in two types: those that do not change the AChR desensitization properties are called type I whereas those that decrease the level of desensitization are labeled as type II.
The identified α7 PAMs have considerable diversity, ranging from proteins to small molecules. Even the small molecule PAMs vary significantly in structure, as well as in properties and probably mechanisms of the modulation. Nitrogen-containing heterocycles such as indoles, isoxazoles, thiazoles, pyridines and pyrroles are largely present in both type I and type II PAMs [3].
Frequently, isosteric replacement of this heterocycles, or amide linkers, with triazole rings allows the efficient generation of small libraries of ligands for nAChR via one-pot copper(I)-catalyzed azidealkyne cycloaddition. In this way, several triazole-containing agonists of nAChR were synthesized [4]. 1,2,4-triazoles are also present in structures acting over nicotinic receptors. Derivatives of 4BP-TQS, a potent allosteric agonist of α7 nAChRs, showed properties of type I or II PAMs [5].
Our group developed methodologies to synthesize triazoles. Copper nanoparticles (CuNPs) can be obtained through the reducing system CuCl2·2H2O, lithium metal, and a catalytic amount of 4,4'di-tert-butylbiphenyl (DTBB) in tetrahydrofuran (THF) at room temperature. We showed that these unsupported copper nanoparticles (10 mol%) effectively catalysed the 1,3-dipolar cycloaddition of organic azides and terminal alkynes in the presence of triethylamine at 65 °C in THF. Moreover, these CuNPs can be supported over activated carbon and perform the multicomponent synthesis of 1,2,3triazoles from organic halides in water, allowing the recycling and reuse of the catalyst [6].
In the other hand phosphonates and related compounds exhibit a wide range of applications in medicinal chemistry. For example, some of these compounds are potent inhibitors of human acetylcholinesterase [7]. Thus, in this work we present the synthesis of a novel series of phosphonatefunctionalized 1,4-disubstituted 1,2,3-triazoles.

Synthetic Procedures
Reagents employed were analytical grade. THF and CH2Cl2 were freshly distilled over sodium/benzophenone or CaH2 respectively. Moisture-sensitive reactions were conducted under inert atmosphere of dry nitrogen using oven dried glassware. Reactions were monitored by analytical thin layer chromatography (TLC) performed on Merck silica gel 60 F254 plates and visualized under ultraviolet (UV) light and/or using phosphomolybdic acid or p-anisaldehyde solutions. Purification of crude materials was performed by flash chromatography on silica gel (200-400 mesh, Merck).

General Procedure for Azide-Alkyne Cycloaddition Employing CuNPs/C in Water
NaN3 (1.2 eq.), the organic halide (1 eq.), and the alkyne (1 eq.) were added to a suspension of CuNPs/C (5 mol% Cu) in H2O. The reaction mixture was warmed to 70 °C overnight. Then, the mixture was diluted with water and extracted with EtOAc. The collected organic phases were dried with anhydrous Na2SO4, dried and purified by flash chromatography.

General Procedure for Azide-Alkyne Cycloaddition Employing CuNPs/C in THF
Organic azide (1 eq.), alkyne (1 eq.) and triethyl amine (1 eq.) were added to a suspension of CuNPs/C (5 mol% Cu) in THF. The reaction mixture was heated under reflux overnight. Then, THF was removed and the mixture was washed with water, followed by extractions with EtOAc. The collected organic phases were dried with anhydrous Na2SO4, dried, and purified by flash chromatography.

Results and Discussion
The 1,4-disubstitued triazole derivatives synthesized in this work are shown in Figure 1. We have successively synthesized two series of derivatives, taking into account the preliminary results in their evaluation as α7 nAChRs PAMs. In the first series, structure activity relationship strategies (SAR) were employed and we observed that compounds bearing a phosphonate moiety presented the desired activity. Thus, we continued synthesizing phosphonate derivatives in order to enhance the activity.

Synthesis of 1,2,3-Triazoles Using Supported Copper Nanoparticles
We found that CuNPs supported over activated carbon were able to promote the 1,3-dipolar cycloaddition between 1-azidometylnaphtalene, formed from the corresponding chloride in the reaction media, and propargyl alcohol in water at 70 °C. Afterwards, as shown in Scheme 1, compound 1 was transformed in the corresponding bromide, which was subjected to the Michaelis-Becker reaction with different hydrogen phosphonates, obtaining dialkyl phosphonates esters 3a-c. Finally, phosphonic acid 4 was obtained through the hydrolysis of compound 3a in the presence of trymethyl sylil bromide (TMSBr). Scheme 1. Synthesis of 1,4-disubstituted 1,2,3-triazole series I.

Structure Activity Relationship (SAR) Studies. Synthesis of Homologs of Phosphonate-Triazoles and Invertion of Triazole Geometry
Considering that in preliminary studies of the first series of compounds dimethyl phosphonate ester 3a exerted higher activity as α7 nAChRs PAM, we decided to synthesize homologs of this compound. We made variations in the distances between the groups' naphthalene-triazole and phosphonate-triazole, leading to derivatives 5 and 6. In both cases CuNPs/C were used as catalyst for the cycloaddition reaction. 1-azidonaphtalene was obtained via diazonium salt in excellent yields. In this case, dimethyl phosphonates esters were obtained via the Arbuzov reaction with higher yields than the former method.

Conclusions
The synthesis of a novel series of phosphonate-functionalized 1,4-disubstituted 1,2,3-triazoles was achieved through the methodologies developed by our group, consisting in the preparation of copper-based catalysts in the form of supported nanoparticles. Preliminary studies on their activity as α7 nAChR PAMs show that those with phosphonate functionality, especially dimethyl phosphonate esters, present the desired activity.

Conflicts of Interest:
The authors declare no conflict of interest.