Aneta Kosińska
email: aneta.kosinska@chemia.uni.lodz.pl

David Virieux
email: david.virieux@enscm.fr

Jean-Luc Pirat
email: jean-luc.pirat@enscm.fr

Kamila Czarnecka
email: kamila.czarnecka@umed.lodz.pl

Malgorzata Girek
email: malgorzata.girek@umed.lodz.pl

Pawel Szymański
email: pawel.szymanski@umed.lodz.pl

Slawomir Wojtulewski

Saranya Vasudevan
email: saranya@cbmm.lodz.pl

Arkadiusz Chworos
email: achworos@cbmm.lodz.pl

Bogna Rudolf
email: bogna.rudolf@chemia.uni.lodz.pl

Ana María Díez-Pascual

Aneta Kosi Ńska

Kamila Czarnec

Małgorzata Gire

Paweł Szyma Ński

Sławomir Wojtulewski

Synthesis and Biological Studies of Novel Aminophosphonates and Their Metal Carbonyl Complexes (Fe, Ru)

Keywords: aminophosphonates, metallocarbonyl complexes, serine esterase inhibitors

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In recent years, considerable efforts have been devoted to finding an effective treatment for Alzheimer's disease, which is the leading cause of dementia. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two clinically significant serine esterases, are involved in neurodegenerative processes related to this pathology. Interestingly, inhibition of these enzymes helps to increase cognitive functions, and for this reason, designing and synthesizing potent inhibitors of AChE and BuChE are of great interest [START_REF] Makhaeva | Kinetics and mechanism of inhibition of serine esterases by fluorinated aminophosphonates[END_REF][START_REF] Lejczak | Inhibition of aminopeptidases by aminophosphonates[END_REF][START_REF] Makhaeva | Kinetics and mechanism of inhibition of serine esterases by fluorinated carbethoxy 1-aminophosphonates[END_REF][START_REF] Turcheniuk | Recent advances in the synthesis of fluorinated aminophosphonates and aminophosphonic acids[END_REF][START_REF] Pawelczap | Synthesis of phosphono dipeptides, inhibitors of cathepsin C. Phosphorus Sulfur Silicon[END_REF][START_REF] Bernhard | Mechanism of enzyme inhibition by phosphate esters[END_REF].

Several organophosphorus compounds (OP) such as esters, thiol esters, acid halides, or anhydrides of pentavalent phosphorus-containing acids (phosphates and phosphonates) are known to inhibit serine esterases [START_REF] Makhaeva | Kinetics and mechanism of inhibition of serine esterases by fluorinated aminophosphonates[END_REF][START_REF] Eto | Organophosphorus Pesticides: Organic and Biological Chemistry[END_REF][START_REF] Thompson | Pesticide Toxicology and International Regulation[END_REF]. The general structure of such inhibitors is R 1 R 2 P(O)X, where R 1 and R 2 substituents are usually simple alkyl or aryl groups that can be connected to phosphorus directly or through an intervening -O-, -S-or -NH-linkage [START_REF] Makhaeva | Kinetics and mechanism of inhibition of serine esterases by fluorinated aminophosphonates[END_REF]. Among thoroughly studied OP serine esterase inhibitors we can lineup DFP, diisopropyl Inhibition of serine esterases by OP generally involves the phosphorylation of the serine residue in the active site. This inhibition is based on the nucleophilic attack of the hydroxyl group of the serine residue on the phosphorus atom of phosphonate forming a covalent bond between the serine and the phosphonate compound. Phosphorylation of the serine results in immediate loss of the leaving group at the phosphorus atom [START_REF] Powers | Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases[END_REF].

Both enzymes AChE and BuChE include a catalytic triad in their active site, like that used by serine proteases such as α-chymotrypsin. The essential features of the catalytic site (i.e., a catalytic triad of Ser-His-Glu, an oxyanion hole, a π cation binding site, and an acyl-binding pocket) are the same in both targeted proteins, AChE, and BuChE. According to theoretical and experimental studies inhibitors can interact with either or both of AChE's binding sites, peripheral anionic site (PAS), and catalytic triad [START_REF] Pang | Novel and viable acetylcholinesterase target site for developing effective and environmentally safe insecticides[END_REF][START_REF] Ekström | Crystal structures of acetylcholinesterase in complex with HI-6, Ortho-7 and obidoxime: Structural basis for differences in the ability to reactivate tabun conjugates[END_REF]. A PAS is a binding site for allosteric modulators and PAS inhibitors at the protein surface where it is situated at the edge of the active-site gorge [START_REF] Johnson | Inhibitors tethered near the acetylcholinesterase active site serve as molecular rulers of the peripheral and acylation sites[END_REF]. PAS contains Tyr72, Tyr124, Trp286, Tyr341, and Asp74 aromatic residues [START_REF] Bourne | Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site[END_REF]. The PAS-recognized AChE inhibitors or those that interact with both the PAS and the catalytic site may have a dual pharmacological impact. This combination is thought to improve cholinergic neurotransmission while decreasing AChE's pro-aggregating activity, making it a potentially effective novel treatment for AD [START_REF] Cacabelos | Pharmacogenomics and therapeutic prospects in Alzheimer's disease[END_REF][START_REF] Bhattacharjee | Discovery of non-oxime reactivators using an in silico pharmacophore model of oxime reactivators of OP-inhibited acetylcholinesterase[END_REF]. The active triad is the major focus for the regulated reduction of AChE in patients with acetylcholine deficiency, such as those with Alzheimer's disease or myasthenia gravis, which is described by varying muscle weakness and exhaustion as a result of a loss of receptors. On the other hand, organophosphorus (OP) substances such as soman, tabun, sarin, and VX which are known as nerve agents, are also directly bound at the active triad of the AChE enzyme. In addition, some organophosphorus compounds may also have interactions with the peripheral anionic site [START_REF] Barak | Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite[END_REF][START_REF] Radić | Peripheral site ligands accelerate inhibition of acetylcholinesterase by neutral organophosphates[END_REF][START_REF] Čadež | Assessment of four organophosphorus pesticides as inhibitors of human acetylcholinesterase and butyrylcholinesterase[END_REF]. Organophosphorus compounds block the activity of AChE by attaching a phosphorus conjugate covalently to the catalytic serine residue.

The acyl-binding pocket, responsible for the difference in substrate specificity between the two enzymes, is larger in BuChE [START_REF] Nicolet | Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products[END_REF][START_REF] Harel | Conversion of acetylcholinesterase to butyrylcholinesterase: Modeling and mutagenesis[END_REF][START_REF] Saxena | Differences in active site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase[END_REF][START_REF] Nachon | Crystal structures of human cholinesterases in complex with huprine W and tacrine: Elements of specificity for anti-Alzheimer's drugs targeting acetyl-and butyrylcholinesterase[END_REF][START_REF] Radić | Three distinct domains in the cholinesterase molecule confer selectivity for acetyl-and butyrylcholinesterase inhibitors[END_REF]. For example, tetraisopropyl pyrophosphoramide (iso-OMPA) is a selective BuChE inhibitor, where increased steric constraints in the acyl-binding pocket prevent interactions with AChE. The various inhibitors of these two enzymes also bind to different amino acids of the cholinesterase active site and are well characterized. The tricyclic acridine compound, tacrine, and the phenothiazine, ethopropazine, bind to the anionic site [START_REF] Johnson | Cholinesterase-like catalytic antibodies: Reaction with substrates and inhibitors[END_REF].

Makhaeva et al. reported new fluorinated aminophosphonates that irreversibly inhibit four serine esterases: AChE, BuChE, carboxylesterase, and neuropathy target esterase, a phospholipase that deacetylates intracellular phosphatidylcholine to produce glycerophosphocholine (Scheme 1) [START_REF] Makhaeva | Kinetics and mechanism of inhibition of serine esterases by fluorinated aminophosphonates[END_REF]. The presence of the two trifluoromethyl groups increased the electrophilic character of phosphonate which has a higher rate of reaction with nucleophiles. Inhibition of serine esterases by OP generally involves the phosphorylation of the serine residue in the active site. This inhibition is based on the nucleophilic attack of the hydroxyl group of the serine residue on the phosphorus atom of phosphonate forming a covalent bond between the serine and the phosphonate compound. Phosphorylation of the serine results in immediate loss of the leaving group at the phosphorus atom [START_REF] Powers | Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases[END_REF].

Both enzymes AChE and BuChE include a catalytic triad in their active site, like that used by serine proteases such as α-chymotrypsin. The essential features of the catalytic site (i.e., a catalytic triad of Ser-His-Glu, an oxyanion hole, a π cation binding site, and an acyl-binding pocket) are the same in both targeted proteins, AChE, and BuChE. According to theoretical and experimental studies inhibitors can interact with either or both of AChE's binding sites, peripheral anionic site (PAS), and catalytic triad [START_REF] Pang | Novel and viable acetylcholinesterase target site for developing effective and environmentally safe insecticides[END_REF][START_REF] Ekström | Crystal structures of acetylcholinesterase in complex with HI-6, Ortho-7 and obidoxime: Structural basis for differences in the ability to reactivate tabun conjugates[END_REF]. A PAS is a binding site for allosteric modulators and PAS inhibitors at the protein surface where it is situated at the edge of the active-site gorge [START_REF] Johnson | Inhibitors tethered near the acetylcholinesterase active site serve as molecular rulers of the peripheral and acylation sites[END_REF]. PAS contains Tyr72, Tyr124, Trp286, Tyr341, and Asp74 aromatic residues [START_REF] Bourne | Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site[END_REF]. The PAS-recognized AChE inhibitors or those that interact with both the PAS and the catalytic site may have a dual pharmacological impact. This combination is thought to improve cholinergic neurotransmission while decreasing AChE's pro-aggregating activity, making it a potentially effective novel treatment for AD [START_REF] Cacabelos | Pharmacogenomics and therapeutic prospects in Alzheimer's disease[END_REF][START_REF] Bhattacharjee | Discovery of non-oxime reactivators using an in silico pharmacophore model of oxime reactivators of OP-inhibited acetylcholinesterase[END_REF]. The active triad is the major focus for the regulated reduction of AChE in patients with acetylcholine deficiency, such as those with Alzheimer's disease or myasthenia gravis, which is described by varying muscle weakness and exhaustion as a result of a loss of receptors. On the other hand, organophosphorus (OP) substances such as soman, tabun, sarin, and VX which are known as nerve agents, are also directly bound at the active triad of the AChE enzyme. In addition, some organophosphorus compounds may also have interactions with the peripheral anionic site [START_REF] Barak | Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite[END_REF][START_REF] Radić | Peripheral site ligands accelerate inhibition of acetylcholinesterase by neutral organophosphates[END_REF][START_REF] Čadež | Assessment of four organophosphorus pesticides as inhibitors of human acetylcholinesterase and butyrylcholinesterase[END_REF]. Organophosphorus compounds block the activity of AChE by attaching a phosphorus conjugate covalently to the catalytic serine residue.

The acyl-binding pocket, responsible for the difference in substrate specificity between the two enzymes, is larger in BuChE [START_REF] Nicolet | Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products[END_REF][START_REF] Harel | Conversion of acetylcholinesterase to butyrylcholinesterase: Modeling and mutagenesis[END_REF][START_REF] Saxena | Differences in active site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase[END_REF][START_REF] Nachon | Crystal structures of human cholinesterases in complex with huprine W and tacrine: Elements of specificity for anti-Alzheimer's drugs targeting acetyl-and butyrylcholinesterase[END_REF][START_REF] Radić | Three distinct domains in the cholinesterase molecule confer selectivity for acetyl-and butyrylcholinesterase inhibitors[END_REF]. For example, tetraisopropyl pyrophosphoramide (iso-OMPA) is a selective BuChE inhibitor, where increased steric constraints in the acyl-binding pocket prevent interactions with AChE. The various inhibitors of these two enzymes also bind to different amino acids of the cholinesterase active site and are well characterized. The tricyclic acridine compound, tacrine, and the phenothiazine, ethopropazine, bind to the anionic site [START_REF] Johnson | Cholinesterase-like catalytic antibodies: Reaction with substrates and inhibitors[END_REF].

Makhaeva et al. reported new fluorinated aminophosphonates that irreversibly inhibit four serine esterases: AChE, BuChE, carboxylesterase, and neuropathy target esterase, a phospholipase that deacetylates intracellular phosphatidylcholine to produce glycerophosphocholine (Scheme 1) [START_REF] Makhaeva | Kinetics and mechanism of inhibition of serine esterases by fluorinated aminophosphonates[END_REF]. The presence of the two trifluoromethyl groups increased the electrophilic character of phosphonate which has a higher rate of reaction with nucleophiles. Scheme 1. Proposed mode of inhibition of a serine esterase (Enz-Ser-OH) by fluorinated aminophosphonates. Adopted from [START_REF] Makhaeva | Kinetics and mechanism of inhibition of serine esterases by fluorinated aminophosphonates[END_REF].

Moreover, α-aminophosphonate derivatives containing a pyrazole moiety showed a significant inhibitory effect on AChE [START_REF] Ren | Synthesis and Biological Evaluation of α-Aminophosphonate Derivatives Containing a Pyrazole Moiety[END_REF]. Finally, another example of a potent inhibitor is the 2,3,4,5-tetrahydrobenzothiazepine-appended α-aminophosphonate derivatives which exhibited similar (BuChE) or better (AChE) inhibitory activity when compared to the standard drug that is galantamine [START_REF] Shaikh | characterization, in vitro cholinesterase and hRBCs hemolysis assay and computational evaluation of novel 2,3,4,5-tetrahydrobenzothiazepine appended α-aminophosphonates[END_REF].

Metallodrugs are also an efficient and important class of compounds. They are extensively used as active pharmaceutical ingredients (APIs) in the treatment of several pathologies including cancer [START_REF] Zaki | Current and future potential of metallo drugs: Revisiting DNA-binding of metal containing molecules and their diverse mechanism of action[END_REF]. The combination of metallic center and bioactive phosphonates could result in compounds with unprecedented structures, and they may potentially combine unique and synergistic properties. Few examples of AChE and BuChE metal-based inhibitors can be found in the literature. Interestingly, they can have better inhibition and pharmacokinetic profiles compared to their parent compounds [START_REF] Jawaria | Probing ferrocene-based thiosemicarbazones and their transition metal complexes as cholinesterase inhibitors[END_REF][START_REF] Trobec | Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug[END_REF]. Jawaria et al. reported ferrocene-based thiosemicarbazones and their transition metal complexes as cholinesterase inhibitors [START_REF] Jawaria | Probing ferrocene-based thiosemicarbazones and their transition metal complexes as cholinesterase inhibitors[END_REF]. Another example is the organoruthenium-nitrophen complex which was evaluated for its inhibitory activity against various cholinoesterases [START_REF] Trobec | Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug[END_REF]. Ristovski et al. found that the [(ⴄ 6 -p-cymene)Ru(pyrithionato)Cl] complex reversibly inhibited both AChE and BuChE [START_REF] Ristovski | Organoruthenium Prodrugs as a New Class of Cholinesterase and Glutathione-S-Transferase Inhibitors[END_REF].

In one of our works, we studied the metallocarbonyl phosphonate half-sandwich tungsten, iron, and molybdenum complexes obtained by phospha-Michael addition of diethyl-and diphenyl-phosphite to the maleimide ligand of complexes 1-4 (Figure 2). Iron complexes 5a-b were screened against AChE and BuChE. The experiments carried out suggested that the diphenyl phosphonate 5b reversibly inhibits BuChE according to a competitive mechanism and inactivates this enzyme in a time-dependent fashion by phosphonylation of its active site [START_REF] Rudolf | The phospha-Michael addition of dimethyl-and diphenylphosphites to the eta(1)-N-maleimidato ligand: Inhibition of serine hydrolases by half-sandwich metallocarbonyl azaphosphonates[END_REF]. To increase the potency of these inhibitors, we designed a hybrid structure based on a diethyl phosphonate motif linked through a spacer (methylenebenzylamine) with metallocarbonyl iron and ruthenium complexes. In this paper, we report an efficient threestep synthetic route to a series of (aminomethyl)benzylphosphonates starting from α,α'dibromo or dichloro-I-xylene (I = o, m, p). Subsequently, the (aminomethyl)benzylphosphonate 8a-c intermediates were reacted with the maleimide ligand of the CpM(CO)2(η 1 -N-maleimidato) (M = Fe, Ru) complex to give metallocarbonyl aminophosphonate derivatives 9a-c and 10a-c. The basic idea is the combination of phosphonate Scheme 1. Proposed mode of inhibition of a serine esterase (Enz-Ser-OH) by fluorinated aminophosphonates. Adopted from [START_REF] Makhaeva | Kinetics and mechanism of inhibition of serine esterases by fluorinated aminophosphonates[END_REF].

Moreover, α-aminophosphonate derivatives containing a pyrazole moiety showed a significant inhibitory effect on AChE [START_REF] Ren | Synthesis and Biological Evaluation of α-Aminophosphonate Derivatives Containing a Pyrazole Moiety[END_REF]. Finally, another example of a potent inhibitor is the 2,3,4,5-tetrahydrobenzothiazepine-appended α-aminophosphonate derivatives which exhibited similar (BuChE) or better (AChE) inhibitory activity when compared to the standard drug that is galantamine [START_REF] Shaikh | characterization, in vitro cholinesterase and hRBCs hemolysis assay and computational evaluation of novel 2,3,4,5-tetrahydrobenzothiazepine appended α-aminophosphonates[END_REF].

Metallodrugs are also an efficient and important class of compounds. They are extensively used as active pharmaceutical ingredients (APIs) in the treatment of several pathologies including cancer [START_REF] Zaki | Current and future potential of metallo drugs: Revisiting DNA-binding of metal containing molecules and their diverse mechanism of action[END_REF]. The combination of metallic center and bioactive phosphonates could result in compounds with unprecedented structures, and they may potentially combine unique and synergistic properties. Few examples of AChE and BuChE metal-based inhibitors can be found in the literature. Interestingly, they can have better inhibition and pharmacokinetic profiles compared to their parent compounds [START_REF] Jawaria | Probing ferrocene-based thiosemicarbazones and their transition metal complexes as cholinesterase inhibitors[END_REF][START_REF] Trobec | Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug[END_REF]. Jawaria et al. reported ferrocene-based thiosemicarbazones and their transition metal complexes as cholinesterase inhibitors [START_REF] Jawaria | Probing ferrocene-based thiosemicarbazones and their transition metal complexes as cholinesterase inhibitors[END_REF]. Another example is the organoruthenium-nitrophen complex which was evaluated for its inhibitory activity against various cholinoesterases [START_REF] Trobec | Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug[END_REF]. Ristovski et al. found that the [(η 6 -p-cymene)Ru(pyrithionato)Cl] complex reversibly inhibited both AChE and BuChE [START_REF] Ristovski | Organoruthenium Prodrugs as a New Class of Cholinesterase and Glutathione-S-Transferase Inhibitors[END_REF].

In one of our works, we studied the metallocarbonyl phosphonate half-sandwich tungsten, iron, and molybdenum complexes obtained by phospha-Michael addition of diethyl-and diphenyl-phosphite to the maleimide ligand of complexes 1-4 (Figure 2). Iron complexes 5a-b were screened against AChE and BuChE. The experiments carried out suggested that the diphenyl phosphonate 5b reversibly inhibits BuChE according to a competitive mechanism and inactivates this enzyme in a time-dependent fashion by phosphonylation of its active site [START_REF] Rudolf | The phospha-Michael addition of dimethyl-and diphenylphosphites to the eta(1)-N-maleimidato ligand: Inhibition of serine hydrolases by half-sandwich metallocarbonyl azaphosphonates[END_REF]. Moreover, α-aminophosphonate derivatives containing a pyrazole moiety showed a significant inhibitory effect on AChE [START_REF] Ren | Synthesis and Biological Evaluation of α-Aminophosphonate Derivatives Containing a Pyrazole Moiety[END_REF]. Finally, another example of a potent inhibitor is the 2,3,4,5-tetrahydrobenzothiazepine-appended α-aminophosphonate derivatives which exhibited similar (BuChE) or better (AChE) inhibitory activity when compared to the standard drug that is galantamine [START_REF] Shaikh | characterization, in vitro cholinesterase and hRBCs hemolysis assay and computational evaluation of novel 2,3,4,5-tetrahydrobenzothiazepine appended α-aminophosphonates[END_REF].

Metallodrugs are also an efficient and important class of compounds. They are extensively used as active pharmaceutical ingredients (APIs) in the treatment of several pathologies including cancer [START_REF] Zaki | Current and future potential of metallo drugs: Revisiting DNA-binding of metal containing molecules and their diverse mechanism of action[END_REF]. The combination of metallic center and bioactive phosphonates could result in compounds with unprecedented structures, and they may potentially combine unique and synergistic properties. Few examples of AChE and BuChE metal-based inhibitors can be found in the literature. Interestingly, they can have better inhibition and pharmacokinetic profiles compared to their parent compounds [START_REF] Jawaria | Probing ferrocene-based thiosemicarbazones and their transition metal complexes as cholinesterase inhibitors[END_REF][START_REF] Trobec | Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug[END_REF]. Jawaria et al. reported ferrocene-based thiosemicarbazones and their transition metal complexes as cholinesterase inhibitors [START_REF] Jawaria | Probing ferrocene-based thiosemicarbazones and their transition metal complexes as cholinesterase inhibitors[END_REF]. Another example is the organoruthenium-nitrophen complex which was evaluated for its inhibitory activity against various cholinoesterases [START_REF] Trobec | Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug[END_REF]. Ristovski et al. found that the [(ⴄ 6 -p-cymene)Ru(pyrithionato)Cl] complex reversibly inhibited both AChE and BuChE [START_REF] Ristovski | Organoruthenium Prodrugs as a New Class of Cholinesterase and Glutathione-S-Transferase Inhibitors[END_REF].

In one of our works, we studied the metallocarbonyl phosphonate half-sandwich tungsten, iron, and molybdenum complexes obtained by phospha-Michael addition of diethyl-and diphenyl-phosphite to the maleimide ligand of complexes 1-4 (Figure 2). Iron complexes 5a-b were screened against AChE and BuChE. The experiments carried out suggested that the diphenyl phosphonate 5b reversibly inhibits BuChE according to a competitive mechanism and inactivates this enzyme in a time-dependent fashion by phosphonylation of its active site [START_REF] Rudolf | The phospha-Michael addition of dimethyl-and diphenylphosphites to the eta(1)-N-maleimidato ligand: Inhibition of serine hydrolases by half-sandwich metallocarbonyl azaphosphonates[END_REF]. To increase the potency of these inhibitors, we designed a hybrid structure based on a diethyl phosphonate motif linked through a spacer (methylenebenzylamine) with metallocarbonyl iron and ruthenium complexes. In this paper, we report an efficient threestep synthetic route to a series of (aminomethyl)benzylphosphonates starting from α,α'dibromo or dichloro-I-xylene (I = o, m, p). Subsequently, the (aminomethyl)benzylphosphonate 8a-c intermediates were reacted with the maleimide ligand of the CpM(CO)2(η 1 -N-maleimidato) (M = Fe, Ru) complex to give metallocarbonyl aminophosphonate derivatives 9a-c and 10a-c. The basic idea is the combination of phosphonate To increase the potency of these inhibitors, we designed a hybrid structure based on a diethyl phosphonate motif linked through a spacer (methylenebenzylamine) with metallocarbonyl iron and ruthenium complexes. In this paper, we report an efficient three-step synthetic route to a series of (aminomethyl)benzylphosphonates starting from α,α'-dibromo or dichloro-I-xylene (I = o, m, p). Subsequently, the (aminomethyl)benzylphosphonate 8a-c intermediates were reacted with the maleimide ligand of the CpM(CO) 2 (η 1 -N-maleimidato) (M = Fe, Ru) complex to give metallocarbonyl aminophosphonate derivatives 9a-c and 10a-c. The basic idea is the combination of phosphonate properties with the effectiveness of metallocarbonyl complexes as drugs. The overall approach to targeted metallocarbonyl aminophosphonates is depicted in Scheme 2. properties with the effectiveness of metallocarbonyl complexes as drugs. The overall approach to targeted metallocarbonyl aminophosphonates is depicted in Scheme 2.

Scheme 2. General approach to targeted metallocarbonyl aminophosphonates 9a-c and 10a-c.

The inhibitory activity against AChE and BuChE of the obtained (aminomethyl)benzylphosphonates 8a-c and metallocarbonyl analogs (9a-c, 10a-c) was evaluated. Interestingly, no data have been reported so far on the biochemical activity of metallocarbonyl complexes containing an aminophosphonate moiety, particularly on their ability to inhibit serine hydrolases. Cytotoxicity studies of compounds 8a-c, 9a-c, and 10a-c toward the V79 cell line were also carried out. Additionally, we have analyzed the interaction of selected phosphonate derivatives (8a-c) with the AChE protein by theoretical prediction and modeling.

Results and Discussion

Synthesis of Complexes 1-2

Half-sandwich complexes (η 5 -cyclopentadienyl)M(CO)2(η 1 -N-maleimidato) (1, M = Fe; 2, M = Ru), bearing the maleimide moiety, were obtained according to the previously reported photochemical reaction of (η 5 -cyclopentadienyl)M(CO)2I (M = Fe, Ru) with maleimide in the presence of diisopropylamine [START_REF] Rudolf | ETA(5)-cyclopentadienyl)Fe(CO)(2)-complex of maleimide anion-an organometallic carbonyl probe for biomolecules containing hs groups[END_REF][START_REF] Rudolf | Synthesis and characterization of new M(II) carbonyl complexes (M = Fe or Ru) including an eta(1)-N-maleimidato ligand. Reactivity studies with biological thiols[END_REF].

Synthesis of (Aminomethyl)benzylphosphonates 8a-c

Looking for new organometallic scaffolds bearing the aminophosphonate moiety, we designed (aminomethyl)benzylphosphonates 8a-c that are able to react with the maleimide double bond of metallocarbonyl complexes 1 and 2. The (aminomethyl)benzylphosphonates 8a-c were obtained in a three-step method depicted in Scheme 3. Scheme 3. Synthesis of (aminomethyl)benzylphosphonates 8a-c. The inhibitory activity against AChE and BuChE of the obtained (aminomethyl) benzylphosphonates 8a-c and metallocarbonyl analogs (9a-c, 10a-c) was evaluated. Interestingly, no data have been reported so far on the biochemical activity of metallocarbonyl complexes containing an aminophosphonate moiety, particularly on their ability to inhibit serine hydrolases. Cytotoxicity studies of compounds 8a-c, 9a-c, and 10a-c toward the V79 cell line were also carried out. Additionally, we have analyzed the interaction of selected phosphonate derivatives (8a-c) with the AChE protein by theoretical prediction and modeling.

Results and Discussion

Synthesis of Complexes 1-2

Half-sandwich complexes (η 5 -cyclopentadienyl)M(CO) 2 (η 1 -N-maleimidato) (1, M = Fe; 2, M = Ru), bearing the maleimide moiety, were obtained according to the previously reported photochemical reaction of (η 5 -cyclopentadienyl)M(CO) 2 I (M = Fe, Ru) with maleimide in the presence of diisopropylamine [START_REF] Rudolf | ETA(5)-cyclopentadienyl)Fe(CO)(2)-complex of maleimide anion-an organometallic carbonyl probe for biomolecules containing hs groups[END_REF][START_REF] Rudolf | Synthesis and characterization of new M(II) carbonyl complexes (M = Fe or Ru) including an eta(1)-N-maleimidato ligand. Reactivity studies with biological thiols[END_REF].

Synthesis of (Aminomethyl)benzylphosphonates 8a-c

Looking for new organometallic scaffolds bearing the aminophosphonate moiety, we designed (aminomethyl)benzylphosphonates 8a-c that are able to react with the maleimide double bond of metallocarbonyl complexes 1 and 2. The (aminomethyl)benzylphosphonates 8a-c were obtained in a three-step method depicted in Scheme 3. properties with the effectiveness of metallocarbonyl complexes as drugs. The overall approach to targeted metallocarbonyl aminophosphonates is depicted in Scheme 2.

Scheme 2. General approach to targeted metallocarbonyl aminophosphonates 9a-c and 10a-c.

The inhibitory activity against AChE and BuChE of the obtained (aminomethyl)benzylphosphonates 8a-c and metallocarbonyl analogs (9a-c, 10a-c) was evaluated. Interestingly, no data have been reported so far on the biochemical activity of metallocarbonyl complexes containing an aminophosphonate moiety, particularly on their ability to inhibit serine hydrolases. Cytotoxicity studies of compounds 8a-c, 9a-c, and 10a-c toward the V79 cell line were also carried out. Additionally, we have analyzed the interaction of selected phosphonate derivatives (8a-c) with the AChE protein by theoretical prediction and modeling.

Results and Discussion

Synthesis of Complexes 1-2

Half-sandwich complexes (η 5 -cyclopentadienyl)M(CO)2(η 1 -N-maleimidato) (1, M = Fe; 2, M = Ru), bearing the maleimide moiety, were obtained according to the previously reported photochemical reaction of (η 5 -cyclopentadienyl)M(CO)2I (M = Fe, Ru) with maleimide in the presence of diisopropylamine [START_REF] Rudolf | ETA(5)-cyclopentadienyl)Fe(CO)(2)-complex of maleimide anion-an organometallic carbonyl probe for biomolecules containing hs groups[END_REF][START_REF] Rudolf | Synthesis and characterization of new M(II) carbonyl complexes (M = Fe or Ru) including an eta(1)-N-maleimidato ligand. Reactivity studies with biological thiols[END_REF].

Synthesis of (Aminomethyl)benzylphosphonates 8a-c

Looking for new organometallic scaffolds bearing the aminophosphonate moiety, we designed (aminomethyl)benzylphosphonates 8a-c that are able to react with the maleimide double bond of metallocarbonyl complexes 1 and 2. The (aminomethyl)benzylphosphonates 8a-c were obtained in a three-step method depicted in Scheme 3. following the procedure developed by Bessieres et al. [START_REF] Bessières | synthesis and biological evaluation of 2-substituted-6-[(4-substituted-1-piperidyl)methyl]-1Hbenzimidazoles as inhibitors of ebola virus infection[END_REF]. Compounds 6a-c were obtained in high yields (71-84%) and were consecutively characterized by spectroscopic methods ( 1 H, 13 C NMR, IR) and mass spectrometry. For the second step, the diethyl(dioxoisoindolin-2-yl)methyl)benzylphosphonates 7a-c were obtained by reaction of 6a-c with the sodium salt of phthalimide in dry acetonitrile according to the procedure reported by Škopić et al. with modifications [START_REF] Klika Škopić | Exploration of a Au(i)-mediated threecomponent reaction for the synthesis of DNA-tagged highly substituted spiroheterocycles[END_REF]. The resulting products 7a-c were obtained in high yields ranging from 74% to 93%. The molecular composition of compounds 7a-c was established by 1 H, 13 C NMR, IR, and mass spectrometry, and in the case of compounds 7a and 7c, structures were determined by single-crystal X-ray diffraction (Figures 3 and4). In the first step, α,α'-dibromo or dichloro-I-xylene (I = o, m, p) undergoes the Arbuzov reaction with triethyl phosphite to give diethyl (halogenomethyl)benzylphosphonates 6ac following the procedure developed by Bessieres et al. [START_REF] Bessières | synthesis and biological evaluation of 2-substituted-6-[(4-substituted-1-piperidyl)methyl]-1Hbenzimidazoles as inhibitors of ebola virus infection[END_REF]. Compounds 6a-c were obtained in high yields (71-84%) and were consecutively characterized by spectroscopic methods ( 1 H, 13 C NMR, IR) and mass spectrometry. For the second step, the diethyl(dioxoisoindolin-2-yl)methyl)benzylphosphonates 7a-c were obtained by reaction of 6a-c with the sodium salt of phthalimide in dry acetonitrile according to the procedure reported by Škopić et al. with modifications [START_REF] Klika Škopić | Exploration of a Au(i)-mediated threecomponent reaction for the synthesis of DNA-tagged highly substituted spiroheterocycles[END_REF]. The resulting products 7a-c were obtained in high yields ranging from 74% to 93%. The molecular composition of compounds 7a-c was established by 1 H, 13 C NMR, IR, and mass spectrometry, and in the case of compounds 7a and 7c, structures were determined by single-crystal X-ray diffraction (Figures 3 and4). In the last step, the deprotection by hydrazine of 7a-c was carried out to afford the (aminomethyl)benzylphosphonates 8a-c (Scheme 3) [START_REF] Fernando | Breaking the Barrier to Slow Water Exchange Rates for Optimal Magnetic Resonance Detection of paraCEST Agents[END_REF]. Compounds 8b and 8c were already obtained in a similar way as reported in the patent by Charmant et al., where the α,α'-dibromo or dichloro-I-xylene (I = o, m, p) was reacted first with the sodium salt of phthalimide and then with triethylphosphite [START_REF] Charmot | Preparation of Isoquinolines and Related Compounds As Inhibitors of Nhe-Mediated Antiport Useful in the Treatment of Disorders Associated with Fluid Retention or Salt Overload and Gastrointestinal Tract Disorders[END_REF]. 1 H NMR and 31 P NMR data of compounds 8b and 8c agreed with those previously reported.

Crystal Structure Description of Compounds 7a and 7c

The molecular structures of both N-(phenylmethyl)phthalimide derivatives 7a and 7c are presented in Figure 3 and Figure 4, respectively. The asymmetric units in both crystal structures contain a single molecule of each compound. Three fragments can be distinguished in the molecules: a phthalimide part, a phenyl ring, and a diethyl phosphonate In the first step, α,α'-dibromo or dichloro-I-xylene (I = o, m, p) undergoes the Arbuzov reaction with triethyl phosphite to give diethyl (halogenomethyl)benzylphosphonates 6ac following the procedure developed by Bessieres et al. [START_REF] Bessières | synthesis and biological evaluation of 2-substituted-6-[(4-substituted-1-piperidyl)methyl]-1Hbenzimidazoles as inhibitors of ebola virus infection[END_REF]. Compounds 6a-c were obtained in high yields (71-84%) and were consecutively characterized by spectroscopic methods ( 1 H, 13 C NMR, IR) and mass spectrometry. For the second step, the diethyl(dioxoisoindolin-2-yl)methyl)benzylphosphonates 7a-c were obtained by reaction of 6a-c with the sodium salt of phthalimide in dry acetonitrile according to the procedure reported by Škopić et al. with modifications [START_REF] Klika Škopić | Exploration of a Au(i)-mediated threecomponent reaction for the synthesis of DNA-tagged highly substituted spiroheterocycles[END_REF]. The resulting products 7a-c were obtained in high yields ranging from 74% to 93%. The molecular composition of compounds 7a-c was established by 1 H, 13 C NMR, IR, and mass spectrometry, and in the case of compounds 7a and 7c, structures were determined by single-crystal X-ray diffraction (Figures 3 and4). In the last step, the deprotection by hydrazine of 7a-c was carried out to afford the (aminomethyl)benzylphosphonates 8a-c (Scheme 3) [START_REF] Fernando | Breaking the Barrier to Slow Water Exchange Rates for Optimal Magnetic Resonance Detection of paraCEST Agents[END_REF]. Compounds 8b and 8c were already obtained in a similar way as reported in the patent by Charmant et al., where the α,α'-dibromo or dichloro-I-xylene (I = o, m, p) was reacted first with the sodium salt of phthalimide and then with triethylphosphite [START_REF] Charmot | Preparation of Isoquinolines and Related Compounds As Inhibitors of Nhe-Mediated Antiport Useful in the Treatment of Disorders Associated with Fluid Retention or Salt Overload and Gastrointestinal Tract Disorders[END_REF]. 1 H NMR and 31 P NMR data of compounds 8b and 8c agreed with those previously reported.

Crystal Structure Description of Compounds 7a and 7c

The molecular structures of both N-(phenylmethyl)phthalimide derivatives 7a and 7c are presented in Figure 3 and Figure 4, respectively. The asymmetric units in both crystal structures contain a single molecule of each compound. Three fragments can be distinguished in the molecules: a phthalimide part, a phenyl ring, and a diethyl phosphonate In the last step, the deprotection by hydrazine of 7a-c was carried out to afford the (aminomethyl)benzylphosphonates 8a-c (Scheme 3) [START_REF] Fernando | Breaking the Barrier to Slow Water Exchange Rates for Optimal Magnetic Resonance Detection of paraCEST Agents[END_REF]. Compounds 8b and 8c were already obtained in a similar way as reported in the patent by Charmant et al., where the α,α'-dibromo or dichloro-I-xylene (I = o, m, p) was reacted first with the sodium salt of phthalimide and then with triethylphosphite [START_REF] Charmot | Preparation of Isoquinolines and Related Compounds As Inhibitors of Nhe-Mediated Antiport Useful in the Treatment of Disorders Associated with Fluid Retention or Salt Overload and Gastrointestinal Tract Disorders[END_REF]. 1 H NMR and 31 P NMR data of compounds 8b and 8c agreed with those previously reported.

Crystal Structure Description of Compounds 7a and 7c

The molecular structures of both N-(phenylmethyl)phthalimide derivatives 7a and 7c are presented in Figures 3 and4, respectively. The asymmetric units in both crystal structures contain a single molecule of each compound. Three fragments can be distinguished in the molecules: a phthalimide part, a phenyl ring, and a diethyl phosphonate group. They are connected to each other by methylene groups. In both crystal structures, the molecules interact with each other through weak C-H . . . O type hydrogen bonds (HBs), where most C-H donors are aliphatic.

The hydrogen bonds arrangement in the crystal structure of 7a, presented in Figure S57, is based on four C-H . . . O bonds. The first two form an intermolecular cyclic bifurcated acceptor type, where the C10-H10A and C17-H17B bonds of two methylene groups of one molecule play the role of the hydrogen donor, while one oxygen atom (O26) of a neighboring molecule phthalimide fragment acts as an acceptor for both. The second C-H bond of the mentioned methylene group creates the third HB C17-H17A . . . O27, and the oxygen atom O27 of a phthalimide fragment of another molecule acts as an acceptor. The fourth hydrogen bond is formed between the C20-H20B bond of the ethyl group and the O22 oxygen atom of the phosphonate group.

An intermolecular bifurcated acceptor type of HBs can also be observed in the crystal structure of 7c. The first intermolecular bifurcated acceptor HB connects three molecules, and it is formed by the C10-H10B bond of the methyl group of one molecule, the C4-H4 bond of the phenyl ring of the other, with the oxygen atom O27 of the third molecule acting as the acceptor for both donor bonds. The second intermolecular bifurcated acceptor HB is cyclic and consists of C17-H17B and C20-H20A bonds, with one of the methyl groups, and one of the ethyl groups of one molecule acting as the hydrogen donor and the oxygen atom O25 of the other molecule playing the role of an acceptor. Additionally, C12-H12 . . . O26 HB can be observed, where the C-H bond is aromatic and the oxygen atom O of a phthalimide fragment of another molecule acts as an acceptor. The extended network of hydrogen bonds in the crystal structure of 7c is shown in Figure S58.

Synthesis of Aminophosphonate Metal Complexes 9a-c and 10a-c

The reaction of metallocarbonyl complexes 1-2 with aminophosphonates 8a-c was carried out under mild conditions using a mixture of ethanol and distilled water (9:1) as a solvent at room temperature. Half-sandwich iron 9a-c and ruthenium 10a-c aminophosphonate complexes were obtained through the aza-Michael addition of (aminomethyl) benzylphosphonates 8a-c to the electron-deficient double bond of the maleimidato ligand of iron 1 and ruthenium 2 complexes using potassium carbonate as a base (Scheme 4). The aza-Michael reaction of primary amines to maleimide ligands was previously described by us [START_REF] Rudolf | Addition of imidazoles and aminoacids to the ethylenic bond in (eta(5)-C5H5)Fe(CO)(2)(eta(1)-Nmaleimidato)[END_REF] and found applications in the conjugation of the metallocarbonyl moiety to biomolecules and dendrimers [START_REF] Fischer-Durand | Site-specific conjugation of metal carbonyl dendrimer to antibody and its use as detection reagent in immunoassay[END_REF]. The hydrogen bonds arrangement in the crystal structure of 7a, presented in Figure S57, is based on four C-H … O bonds. The first two form an intermolecular cyclic bifurcated acceptor type, where the C10-H10A and C17-H17B bonds of two methylene groups of one molecule play the role of the hydrogen donor, while one oxygen atom (O26) of a neighboring molecule phthalimide fragment acts as an acceptor for both. The second C-H bond of the mentioned methylene group creates the third HB C17-H17A … O27, and the oxygen atom O27 of a phthalimide fragment of another molecule acts as an acceptor. The fourth hydrogen bond is formed between the C20-H20B bond of the ethyl group and the O22 oxygen atom of the phosphonate group.

An intermolecular bifurcated acceptor type of HBs can also be observed in the crystal structure of 7c. The first intermolecular bifurcated acceptor HB connects three molecules, and it is formed by the C10-H10B bond of the methyl group of one molecule, the C4-H4 bond of the phenyl ring of the other, with the oxygen atom O27 of the third molecule acting as the acceptor for both donor bonds. The second intermolecular bifurcated acceptor HB is cyclic and consists of C17-H17B and C20-H20A bonds, with one of the methyl groups, and one of the ethyl groups of one molecule acting as the hydrogen donor and the oxygen atom O25 of the other molecule playing the role of an acceptor. Additionally, C12-H12…O26 HB can be observed, where the C-H bond is aromatic and the oxygen atom O of a phthalimide fragment of another molecule acts as an acceptor. The extended network of hydrogen bonds in the crystal structure of 7c is shown in Figure S58.

Synthesis of Aminophosphonate Metal Complexes 9a-c and 10a-c

The reaction of metallocarbonyl complexes 1-2 with aminophosphonates 8a-c was carried out under mild conditions using a mixture of ethanol and distilled water (9:1) as a solvent at room temperature. Half-sandwich iron 9a-c and ruthenium 10a-c aminophosphonate complexes were obtained through the aza-Michael addition of (aminomethyl)benzylphosphonates 8a-c to the electron-deficient double bond of the maleimidato ligand of iron 1 and ruthenium 2 complexes using potassium carbonate as a base (Scheme 4). The aza-Michael reaction of primary amines to maleimide ligands was previously described by us [START_REF] Rudolf | Addition of imidazoles and aminoacids to the ethylenic bond in (eta(5)-C5H5)Fe(CO)(2)(eta(1)-Nmaleimidato)[END_REF] and found applications in the conjugation of the metallocarbonyl moiety to biomolecules and dendrimers [START_REF] Fischer-Durand | Site-specific conjugation of metal carbonyl dendrimer to antibody and its use as detection reagent in immunoassay[END_REF]. The addition of aminophosphonate to the carbon-carbon double bond was confirmed by the disappearance of ethylenic protons at 6.7 ppm characteristic for the maleimide ligand of half-sandwich complexes 1-2 along with the appearance of complex and appropriate signals in the 1 H NMR spectra attributed to the 3 protons of the succinimide CH-CH2 motif (Figure S55). The splitting of the signal was compatible with an ABX spin system which was consistent with the formation of a stereogenic center. This observation is in line with our previously published works on aza-Michael additions [START_REF] Rudolf | Addition of imidazoles and aminoacids to the ethylenic bond in (eta(5)-C5H5)Fe(CO)(2)(eta(1)-Nmaleimidato)[END_REF][START_REF] Rudolf | Metallo-carbonyl complexes based on the CpFe(CO)(2)(eta(1)-N-imidato) system as protein labelling reagents: Reactivity and selectivity studies using bovine serum albumin as a model protein[END_REF].

The 31 P NMR spectra were also recorded for all the compounds. The (aminomethyl)benzylphosphonate compounds exhibited a singlet at 26.6 ppm (8a-ortho), 26.6 ppm The addition of aminophosphonate to the carbon-carbon double bond was confirmed by the disappearance of ethylenic protons at 6.7 ppm characteristic for the maleimide ligand of half-sandwich complexes 1-2 along with the appearance of complex and appropriate signals in the 1 H NMR spectra attributed to the 3 protons of the succinimide CH-CH 2 motif (Figure S55). The splitting of the signal was compatible with an ABX spin system which was consistent with the formation of a stereogenic center. This observation is in line with our previously published works on aza-Michael additions [START_REF] Rudolf | Addition of imidazoles and aminoacids to the ethylenic bond in (eta(5)-C5H5)Fe(CO)(2)(eta(1)-Nmaleimidato)[END_REF][START_REF] Rudolf | Metallo-carbonyl complexes based on the CpFe(CO)(2)(eta(1)-N-imidato) system as protein labelling reagents: Reactivity and selectivity studies using bovine serum albumin as a model protein[END_REF].

The 31 P NMR spectra were also recorded for all the compounds. The (aminomethyl) benzylphosphonate compounds exhibited a singlet at 26.6 ppm (8a-ortho), 26.6 ppm (8b-meta), and 26.5 ppm (8c-para), respectively, which was compatible with a phosphonate group connected to a C SP3 carbon atom. 31 P NMR signals for iron complexes 9a-c were unchanged and stayed at 27.0 ppm, 26.3 ppm, and 26.4 ppm, respectively, and for ruthenium complexes 10a-c at 26.8 ppm, 26.3 ppm, and 10c 26.3 ppm, respectively. In both types of metal complexes for ortho derivatives, the 31 P signal has been shifted downfield compared to the starting (aminomethyl)benzylphosphonate 8a. This was possibly due to the deshielding of phosphoryl group (P=O), occurring as a result of an intramolecular H-bond between NH and O=P, forming a stable 7-membered ring, whereas this type of H-bonding was missing in the other complexes.

The IR spectrum of these complexes displayed two intense ν(C≡O) bands at 2045 and 1994 cm -1 for the CpFe(CO) 2 moiety in iron complexes or at 2045 and 1990 cm -1 for the CpRu(CO) 2 moiety for ruthenium complexes and characteristic bands for phosphonate absorption around 1250, 1025, and 965 cm -1 (Figure S56) [START_REF] Rudolf | The phospha-Michael addition of dimethyl-and diphenylphosphites to the eta(1)-N-maleimidato ligand: Inhibition of serine hydrolases by half-sandwich metallocarbonyl azaphosphonates[END_REF]. The complexes were soluble in organic solvents and in water. Electrospray ionization mass spectra (ESI-MS) and elemental analysis confirmed the structures of iron (9a-c) and ruthenium (10a-c) complexes.

Biological Studies 2.4.1. Cholinesterase Inhibitory Activities

Obtained aminophosphonates were evaluated in vitro for their inhibitory activity for the electric eel AChE (eeAChE) using a modified Ellman assay [START_REF] Ellman | A new and rapid colorimetric determination of acetylcholinesterase activity[END_REF]. Furthermore, the activities of 8a-c to 10a-c towards BuChE (from equine serum) were also determined. Most of the compounds tested showed IC 50 greater than 1 mM. Compared to tacrine (positive control), the new compounds showed lower inhibitory activity for AChE and BuChE. With respect to BuChE inhibitory activities, the ruthenium complexes 10a and 10c were the most active inhibitors (respectively, IC 50 = 186 and 292 µM). Concerning iron complexes, only the ortho derivative 9a showed some activity (IC 50 = 331 µM) towards BuChE (Table 1). These results are consistent with previously published work in which the metallocarbonyl iron complex 5a showed a similar inhibitory activity toward BuChE (IC 50 = 220 µM).

The inhibitory tests against eeAChE showed that the ruthenium complex 10a was the only one of the metal complexes with some inhibitory potential (IC 50 = 164 µM).

Interestingly, the new ortho-substituted aminophosphonate 8a was found to be the most active of all tested compounds with an IC 50 value of 1.215 µM towards AChE. This result corroborates with our theoretical prediction, presented later, and is in good agreement with some recently reported activities of different aminophosphonates on the cholinergic system [START_REF] Shaikh | synthesis and evaluation of new chromone-derived aminophosphonates as potential acetylcholinesterase inhibitor[END_REF][START_REF] Shaikh | synthesis and biological evaluation of novel antipyrine based α-aminophosphonates as anti-Alzheimer and anti-inflammatory agent[END_REF][START_REF] Uparkar | synthesis and biological evaluation of furan based αaminophosphonate derivatives as anti-Alzheimer agent[END_REF]. For example, a chromone-derived aminophosphonate acts as an acetylcholinesterase inhibitor with IC 50 = 0.103 µM. The docking studies of N-substituted α-aminophosphonate bearing a chromone moiety reveal that it binds to the peripheral anionic site and catalytic anionic site of AChE. The protonated amino group present in this compound is responsible for a cation-π interaction with TYR334 and H-bonding interaction with ASP72 [START_REF] Shaikh | synthesis and evaluation of new chromone-derived aminophosphonates as potential acetylcholinesterase inhibitor[END_REF].

Further studies to get inside into the mechanism of binding of ortho-substituted aminophosphonate 8a to AChE should be performed.

Cell Culture and MTT Cytotoxicity Test

The benzylphosphonates derivatives 8a-c, the corresponding iron 9a-c, and ruthenium 10a-c complexes were tested for their potential cytotoxic effect on the V79 cell line, which has been widely used to study the toxicity, mutagenicity, and repair of a wide variety of DNA damaging agents. Due to the solubility issues and toxicity of DMSO (used to prepare the stock solutions of compounds), at a concentration higher than >0.2%, compounds were tested in the concentration range of 100-1000 µM. Cell viability below 70% indicates cytotoxicity. Compounds 8a-c did not exhibit cytotoxic activity in the tested range of concentrations (cell viability > 70%). Relative positions of phosphonomethyl and aminomethyl groups did not affect the cytotoxicity. Iron complex 9c was slightly cytotoxic at concentrations of 1000 µM and 750 µM, with viabilities of 58% and 69%, respectively (cell viability below 70%). Compounds 9a-b and 10a-c were slightly cytotoxic with IC 50 values of 690 µM, 909 µM, 725 µM, 787 µM, and 763 µM (Table 2, Figure 5). Compounds 9a and 10a had phosphonate groups in the ortho position, which turned out to induce the strongest toxicity. anionic site and catalytic anionic site of AChE. The protonated amino group present in this compound is responsible for a cation-π interaction with TYR334 and H-bonding interaction with ASP72 [START_REF] Shaikh | synthesis and evaluation of new chromone-derived aminophosphonates as potential acetylcholinesterase inhibitor[END_REF].

Further studies to get inside into the mechanism of binding of ortho-substituted aminophosphonate 8a to AChE should be performed.

Cell Culture and MTT Cytotoxicity Test

The benzylphosphonates derivatives 8a-c, the corresponding iron 9a-c, and ruthenium 10a-c complexes were tested for their potential cytotoxic effect on the V79 cell line, which has been widely used to study the toxicity, mutagenicity, and repair of a wide variety of DNA damaging agents. Due to the solubility issues and toxicity of DMSO (used to prepare the stock solutions of compounds), at a concentration higher than >0.2%, compounds were tested in the concentration range of 100-1000 µM. Cell viability below 70% indicates cytotoxicity. Compounds 8a-c did not exhibit cytotoxic activity in the tested range of concentrations (cell viability > 70%). Relative positions of phosphonomethyl and aminomethyl groups did not affect the cytotoxicity. Iron complex 9c was slightly cytotoxic at concentrations of 1000 µM and 750 µM, with viabilities of 58% and 69%, respectively (cell viability below 70%). Compounds 9a-b and 10a-c were slightly cytotoxic with IC50 values of 690 µM, 909 µM, 725 µM, 787 µM, and 763 µM (Table 2, Figure 5). Compounds 9a and 10a had phosphonate groups in the ortho position, which turned out to induce the strongest toxicity.

Theoretical Prediction Analysis of Ligands 8a-c with AChE

To confirm the experimental results obtained from the cholinesterase assay, we analyzed the results of the theoretical prediction of ligand-protein interactions. Although the most interesting would be to see the effect of these new synthetic compounds on the human AChE (hAChE), the most relevant here is to compare it to the same enzyme, electric eel AChE (eeAChE). The crystal structure of the electric eel acetylcholinesterase (eeAChE)

Theoretical Prediction Analysis of Ligands 8a-c with AChE

To confirm the experimental results obtained from the cholinesterase assay, we analyzed the results of the theoretical prediction of ligand-protein interactions. Although the most interesting would be to see the effect of these new synthetic compounds on the human AChE (hAChE), the most relevant here is to compare it to the same enzyme, electric eel AChE (eeAChE). The crystal structure of the electric eel acetylcholinesterase (eeAChE) (PDB ID: 1C2O) was completed using modeling and aligned with the structure of human hAChE (PDB ID: 1B41) in order to compare the structural similarity (Figure S59) and the observed RMSD is less than 1.0A. Furthermore, after optimization using Gaussian-163, the structure of eeAChE was used for molecular docking by AutoDock 4 (ADT) with further refinement using the AutoDock tool. During the simulation, the ligands were used flexibly, and root-mean-square deviation (RMSD) values less than 1.0A were considered optimal for clustering. In the results, the ligand with the highest binding energy (most negative) was observed to have the highest binding affinity (Table 3). The overall structure of the protein with all three ligands is presented in Figure 6a; it is a relatively compact protein with two extended α-helixes (Figure S62). Each of the 8a-c compounds was positioned and fitted nicely in the active center (Figure 6b and Figure S60), which is a small pocket led by the tight canal (Figure 6c). All three compounds are in the active center; however, the conformation of each is different (Figure 6c,d andFigures S64-S66). When comparing the docking energy of the eeAChE-interacted ligand complexes, the 8a-interacted eeAChE complex has the highest docking energy of -7.80 kcal/mol. The maximum and minimum occupancy of hydrogen bonds was found to be 5 and 2, respectively. The 8a-eeAChE complex has the highest number of hydrogen bond formations of 5 compared to the other ligands as shown in Figure S63. These results corroborate well with the experimental data, which states that ligand 8a has a strong interaction with the active site residues of eeAChE.

(PDB ID: 1C2O) was completed using modeling and aligned with the structure of human hAChE (PDB ID: 1B41) in order to compare the structural similarity (Figure S59) and the observed RMSD is less than 1.0A. Furthermore, after optimization using Gaussian-163, the structure of eeAChE was used for molecular docking by AutoDock 4 (ADT) with further refinement using the AutoDock tool. During the simulation, the ligands were used flexibly, and root-mean-square deviation (RMSD) values less than 1.0A were considered optimal for clustering. In the results, the ligand with the highest binding energy (most negative) was observed to have the highest binding affinity (Table 3). The overall structure of the protein with all three ligands is presented in Figure 6a; it is a relatively compact protein with two extended α-helixes (Figure S62). Each of the 8a-c compounds was positioned and fitted nicely in the active center (Figures 6b andS60), which is a small pocket led by the tight canal (Figure 6c). All three compounds are in the active center; however, the conformation of each is different (Figures 6c,d andS64-S66). When comparing the docking energy of the eeAChE-interacted ligand complexes, the 8ainteracted eeAChE complex has the highest docking energy of -7.80 kcal/mol. The maximum and minimum occupancy of hydrogen bonds was found to be 5 and 2, respectively. The 8a-eeAChE complex has the highest number of hydrogen bond formations of 5 compared to the other ligands as shown in Figure S63. These results corroborate well with the experimental data, which states that ligand 8a has a strong interaction with the active site residues of eeAChE.

Materials and Methods

Materials

All the reactions were carried out under a nitrogen or argon atmosphere using standard Schlenk techniques. All solvents were distilled over standard drying agents directly before use. The starting materials were purchased from commercial suppliers (Sigma-Aldrich (Darmstadt, Germany), Merck (Darmstadt, Germany), Alfa Aesar (Kandel, Germany), Trichemicals (Newark, U.S)). All reagents were used as received unless otherwise stated. Acetonitrile was freshly distilled and stored over molecular sieves. Chromatographic purifications were performed on Silica gel Merck 60 (230-400 mesh). Complexes (η 5 -C 5 H 5 )M(CO) 2 (η 1 -N-maleimidato) (M = Fe, Ru) 1-2 were synthesized as previously described [START_REF] Nachon | Crystal structures of human cholinesterases in complex with huprine W and tacrine: Elements of specificity for anti-Alzheimer's drugs targeting acetyl-and butyrylcholinesterase[END_REF][START_REF] Radić | Three distinct domains in the cholinesterase molecule confer selectivity for acetyl-and butyrylcholinesterase inhibitors[END_REF].

General Methods

IR spectra were recorded using a NICOLET iS50 instrument equipped with a liquid N 2 -cooled mercury-cadmium-telluride (MCT-A) detector. A flat crystal plate Zn-Se ATR unit (crystal thickness 4 mm) was used. The incidence angle was 45 • . The ATR accessory was obtained from Pike Technologies.

1 H NMR, 13 C NMR, and 31 P{H}NMR spectra were recorded on BRUKER Ultra Shield 400 Plus (400 MHz), BRUKER Avance III (600 MHz), and Varian Gemini (200 MHz) spectrometers, unless otherwise noted. Measurements were made in CDCl 3 or DMSO-d 6 solutions. Coupling constants were expressed in Hertz (Hz). 31 P NMR spectra were obtained with the use of broadband 1 H decoupling. Copies of IR and NMR spectra are presented at Supplementary Materials Figures S1-S54. Electrospray ionization mass spectra were performed on a Varian 500-MS LC ion trap (University of Lodz). Elemental analysis was performed on a Micro Vario Cube elemental analyzer (Elementar).

Inhibition of AChE and BuChE

The method of Ellman et al. [START_REF] Ellman | A new and rapid colorimetric determination of acetylcholinesterase activity[END_REF] with modifications was followed. Nine different concentrations of each compound were used in order to measure inhibition of AChE or BuChE activity. The assay solution consisted of a 0.1 M phosphate buffer pH 8.0, with the addition of 0.4 mg/mL 5,5 -dithio-bis(2-nitrobenzoic acid), 2 unit/mL of EeAChE or 4 unit/mL of EqBuChE (Sigma Aldrich, Darmstadt, Germany), and 1 mM or 2 mM AChE or BuChE substrate (acetylthiocholine iodide or butyrylthiocholine iodide), respectively. Assays were performed with a blank containing all components except AChE or BuChE. The reaction rates were compared and the percentage of inhibition due to the presence of the test compound was calculated. Each concentration was analyzed in triplicate and repeated three times, and IC 50 values were determined graphically from log concentrationinhibition curves.

Cell Culture and MTT Cytotoxicity Test

V79 cells (Cricetulus griseus lung fibroblast cell line) (European Collection of Cell Culture) were grown in MEM (PAN-Biotech, Aidenbach, Germany) containing 2 mM glutamine (Sigma Aldrich, Darmstadt, Germany)), 1% non-essential amino acids (Biological Industries, Cromwell, U.S), 10% FBS (Biowest, Nuaillé, France), and 100 units/mL penicillin and 100 mg/mL streptomycin (Biological Industries, Cromwell, U.S). Cells were plated and grown in an incubator at 37 • C with 5% CO 2 to 80% confluence before the initiation of the assay.

V79 cells were seeded at a density of 1 × 10 4 cells per well and were incubated for 24 h. After 24 h, the medium was removed from the wells, and cells were exposed to the compound solutions over a range of concentrations or nothing but culture medium (blank control). Cells were further incubated for an additional 24 h. At the end, the medium was removed and MTT solution (0.75 mg/mL) was added. Next, plates were incubated in the dark for 2 h at 37 • C. Then, the MTT solution was removed and DMSO was added to each well. Plates were incubated for 10 min at room temperature. The absorbance at 570 nm was measured on a microplate reader. Cell viability was expressed as a percentage of the control values (blank).

General Procedures for the Synthesis of 6a-c

Triethylphosphite (1 equiv) was dropwise added to α,α'-dibromo or dichloro-I-xylene (I = o, m, p) to a two-necked flask fitted with a Dean-Stark separator to remove bromoethane or chloroethane. The reaction mixture was heated to 110 • C for 6 h and then cooled to rt. The product was purified by column chromatography on silica gel (ethyl acetate to ethyl acetate/MeOH 80:20) to give diethyl (bromo or chloromethyl)benzylphosphonates (6a-c) in yields ranging from 71% to 84%. Details-Supplementary Data (Table S1).

General Procedures for the Synthesis of 7a-c

A mixture of bromomethyl or chloromethylbenzylphosphonate 6a-c (1 equiv), phthalimide (1.2 equiv), and NaH (60% in mineral oil, 1.2 equiv) in dry acetonitrile was heated to 110 • C for 8 h. When the solution was cooled to rt, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography on silica gel (ethyl acetate to ethyl acetate/MeOH 40:60). The diethyl (dioxoisoidolin-2yl)methyl)benzyl-phosphonates were obtained (7a-c) in yields ranging from 74% to 93%. Details-Supplementary Data (Table S2).

General Procedures for the Synthesis of Diethyl(aminomethyl)benzylphosphonates 8a-c

A mixture of diethyl(1,3-dioxoisoidolin-2-yl)methylbenzylphosphonate (7a-c) and hydrazine monohydrate in EtOH was stirred at reflux for 3 h. After the mixture was cooled to 25 • C, the resulting precipitate was filtered and then washed 3 times with toluene. The filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (ethyl acetate to ethyl acetate/MeOH 90:10) to give pure diethyl (aminomethyl) benzylphosphonates (8a-c) in yields ranging from 69% to 93%. Details-Supplementary Data (Table S3).

Reactions of Complexes 1-2 with (Aminomethyl)benzylphosphonates 8a-c

To an argon-saturated solution of metallocarbonyl complex CpM(CO) 2 (η 1 -N-maleimidato) (M = Fe 1, Ru 2) (1 equiv) and diethyl(aminomethyl)benzylphosphonate 8a-c (1.5 equiv) in a mixture of EtOH and distilled water (9:1), K 2 CO 3 (5 equiv) was added. The reaction mixture was stirred for 24 h at rt. The mixture was extracted with CHCl 3 . The CHCl 3 extracts were concentrated in vacuo. The residue was purified by column chromatography on silica gel (CHCl 3 to CHCl 3 /MeOH 90:10) to give 9a-c and 10a-c metallocarbonyl complexes in moderate to good yields (40-77%).

Crystallographic Data

Single crystals of 7a and 7c were obtained after slow evaporation of dichloromethane solution at 5 • C. Suitable crystals were sequentially mounted on a fiber loop and used for X-ray diffraction measurement. X-ray data were collected on an Oxford Diffraction SuperNova DualSource diffractometer with the use of the monochromated CuKα X-ray source (λ = 1.54184). The crystals were kept at 100 K during data collection. Data reduction and analytical absorption correction were performed with CrysAlis PRO [46]. Using Olex2 [START_REF] Dolomanov | OLEX2: A complete structure solution, refinement and analysis program[END_REF], the structures were solved with the ShelXS [START_REF] Sheldrick | A short history of SHELX[END_REF] structure solution program using direct methods and refined with the ShelXL [START_REF] Sheldrick | A short history of SHELX[END_REF] refinement package using least squares minimization.

The non-hydrogen atoms were refined anisotropically. Hydrogen atoms were introduced in calculated positions with idealized geometry and constrained using a rigid body model with isotropic displacement parameters equal to 1.2 of the equivalent displacement parameter of the parent atoms. A summary of relevant crystallographic data is given in Table S4 in Supplementary Data. The geometry of hydrogen bonds was calculated by Mercury 4.10 [49] and gathered in Table S5 in Supplementary Data along with the symmetry codes of the described intermolecular non-covalent interactions.

Molecular Docking

The crystal structure of the electric eel acetylcholinesterase (eeAChE) was downloaded from the RCSB protein data bank (PDB ID: 1C2O) [START_REF] Bourne | Conformational flexibility of the acetylcholinesterase tetramer suggested by x-ray crystallography[END_REF]. To complete the whole protein structure, the sequence was supplemented and modeled using the blastp suite [START_REF] Boratyn | Domain enhanced lookup time accelerated BLAST[END_REF]. The geometry of the complete modeled eeAChE protein was compared to the X-ray structure of eeAChE (PDB ID: 1C2O) in order to confirm the methods utilized in this study, and the observed RMSD were less than 1A. Furthermore, optimization was carried out using Gaussian-16 [START_REF] Frisch | Revision C.01[END_REF] software with the WB97XD [START_REF] Sahoo | Charge transport and prototypical optical absorptions in functionalized zinc phthalocyanine compounds: A density functional study[END_REF] level of DFT (density functional theory)/LANL2DZ [START_REF] Kafoumba | Theoretical Determination of Influence of the Metallic State of Oxidation toward Cytotoxic Activity: Case of Ruthenium Complexes[END_REF] level of theory to minimize steric clashes in ligand structures. To find the stability of the structure, harmonic vibrational frequency analyses were carried out at the same computational level of theory. The chemical shift calculations for NMR were performed using the gauge-independent atomic orbital (GIAO) [START_REF] Kupka | Theory and computation of nuclear shielding[END_REF] method at the WB97XD LANL2DZ. All of the stable structures were found to be at energy minima with the lowest possible positive frequencies. Initially, the presence of solvent molecules and ligands was cleared by AutoDock 4 (ADT), which is mainly used to assign charge to the protein and ligand complexes prior to analysis or docking. The molecular docking of the protein molecule was further refined using the AutoDock tool. The ligands were considered to be flexible throughout the docking process, while the protein was considered to be rigid. Positional root-mean-square deviation (RMSD) values less than 1.0A were considered optimal and clustered together to identify the best binding. The ligand with the highest binding energy (most negative) was observed to have the highest binding affinity. The data of the top 5 complexes are listed in Supplementary Table S6. The docking predicted binding conformations for the ligands are shown in Figure 6a-d and their corresponding results are presented in Table 3. The computer-generated 3D structure of selected ligands 8a-c are presented at Figure S61.

The complete data set for the crystal structures under study has been deposited at the Cambridge Crystallographic Data Centre (CCDC 2154277 and CCDC 2154278).

Conclusions

In conclusion, we report the design, synthesis, and characterization of (aminomethyl) benzylphosphonates 8a-c and their metallocarbonyl complexes 9a-c (Fe) and 10a-c (Ru) obtained from phosphonates 6a-c and 7a-c. All obtained compounds were characterized by spectroscopic methods ( 1 H-, 13 C-, 31 P{ 1 H}-NMR, IR) and mass spectrometry. The structures of compounds 7a and 7c were also confirmed by single-crystal X-ray diffraction analysis.

The (aminomethyl)benzylphosphonates 8a-c and their metallocarbonyl complexes 9a-c (Fe) and 10a-c (Ru) were tested for inhibitory potentials against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) by determination of IC 50. Inhibitory studies showed that the best inhibitor of the studied compounds was the ortho-substituted (aminomethyl)benzylphosphonate 8a with IC 50 = 1.215 µM against AChE. The docking studies performed for complexes 8a-c reveal that the 8a-eeAChE complex has the highest number of hydrogen bond formations, which is well correlated with experimental results.

The IC 50 values of metallocarbonyl complexes 9a-c (Fe) and 10a-c (Ru) are much higher compared to the reference inhibitor (tacrine); however, complexes 9a, 10a, and 10c show inhibitory activities towards BuChE similar to that of the previously reported iron metallocarbonyl complex 5b [START_REF] Rudolf | The phospha-Michael addition of dimethyl-and diphenylphosphites to the eta(1)-N-maleimidato ligand: Inhibition of serine hydrolases by half-sandwich metallocarbonyl azaphosphonates[END_REF].

Cytotoxicity studies towards the V79 cell line show no cytotoxicity of the phosphonates 8a-c, and some cytotoxicity for the metallocarbonyl complexes 9a-c and 10a-c. The most cytotoxic were the ortho-derivatives 9a and 10a.

This study has presented a novel approach to accessing some (aminomethyl) benzylphosphonates and their metallocarbonyl derivatives. Our findings are in good agreement with the results of other research teams on aminophosphonates [START_REF] Shaikh | synthesis and evaluation of new chromone-derived aminophosphonates as potential acetylcholinesterase inhibitor[END_REF][START_REF] Shaikh | synthesis and biological evaluation of novel antipyrine based α-aminophosphonates as anti-Alzheimer and anti-inflammatory agent[END_REF][START_REF] Uparkar | synthesis and biological evaluation of furan based αaminophosphonate derivatives as anti-Alzheimer agent[END_REF]. They clearly indicate that the aminophosphonates provide a good basis for the development of an effective substance that inhibits the cholinergic system. The (aminomethyl)benzylphosphonate 8a should be further carefully examined in terms of the inhibition mechanism of AChE. In our previous work, we found that the half-sandwich metallocarbonylphosphonic acids showed an inhibiting activity against certain serine hydrolases [START_REF] Rudolf | The phospha-Michael addition of dimethyl-and diphenylphosphites to the eta(1)-N-maleimidato ligand: Inhibition of serine hydrolases by half-sandwich metallocarbonyl azaphosphonates[END_REF]. Therefore, we plan to synthesize the corresponding phosphonic acid of compounds 8a-c, 9a-c, and 10a-c and test their inhibitory activity towards AChE and BuChE.

Figure 1 .

 1 Figure 1. Structures of some organophosphorus-based serine esterase inhibitors.

Figure 1 .

 1 Figure 1. Structures of some organophosphorus-based serine esterase inhibitors.

Figure 2 .

 2 Figure 2. Structure of the metallocarbonyl complexes CpM(CO)n(η 1 -N-maleimidato) (M = Fe, n = 2 (1), M = Ru, n = 2 (2), M = Mo, n = 3 (3), M = W, n = 3 (4)) and half-sandwich iron phosphonate complexes (5a-b).

16 Scheme 1 .

 161 Scheme 1. Proposed mode of inhibition of a serine esterase (Enz-Ser-OH) by fluorinated aminophosphonates. Adopted from[START_REF] Makhaeva | Kinetics and mechanism of inhibition of serine esterases by fluorinated aminophosphonates[END_REF].

Figure 2 .

 2 Figure 2. Structure of the metallocarbonyl complexes CpM(CO)n(η 1 -N-maleimidato) (M = Fe, n = 2 (1), M = Ru, n = 2 (2), M = Mo, n = 3 (3), M = W, n = 3 (4)) and half-sandwich iron phosphonate complexes (5a-b).

Figure 2 .

 2 Figure 2. Structure of the metallocarbonyl complexes CpM(CO) n (η 1 -N-maleimidato) (M = Fe, n = 2 (1), M = Ru, n = 2 (2), M = Mo, n = 3 (3), M = W, n = 3 (4)) and half-sandwich iron phosphonate complexes (5a-b).

  Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 16

Scheme 2 .

 2 Scheme 2. General approach to targeted metallocarbonyl aminophosphonates 9a-c and 10a-c.

  Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 16

Scheme 3 .

 3 Scheme 3. Synthesis of (aminomethyl)benzylphosphonates 8a-c. Scheme 3. Synthesis of (aminomethyl)benzylphosphonates 8a-c. In the first step, α,α'-dibromo or dichloro-I-xylene (I = o, m, p) undergoes the Arbuzov reaction with triethyl phosphite to give diethyl (halogenomethyl)benzylphosphonates 6a-c

  Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 16

Figure 3 .

 3 Figure 3. Molecular structure of 7a with atom-labeling scheme.

Figure 4 .

 4 Figure 4. Molecular structure of 7c with atom-labeling scheme.

Figure 3 .

 3 Figure 3. Molecular structure of 7a with atom-labeling scheme.

Figure 3 .

 3 Figure 3. Molecular structure of 7a with atom-labeling scheme.

Figure 4 .

 4 Figure 4. Molecular structure of 7c with atom-labeling scheme.

Figure 4 .

 4 Figure 4. Molecular structure of 7c with atom-labeling scheme.

Scheme 4 .

 4 Scheme 4. Synthesis of new organometallic phosphonates 9a-c and 10a-c.

Scheme 4 .

 4 Scheme 4. Synthesis of new organometallic phosphonates 9a-c and 10a-c.

Figure 5 .

 5 Figure 5. Percentage of cell viability after 24 h incubation with compounds as function of the concentration with phosphonates 8a-c, 9a-c, 10a-c.

Figure 5 .

 5 Figure 5. Percentage of cell viability after 24 h incubation with compounds as function of the concentration with phosphonates 8a-c, 9a-c, 10a-c.

Figure 6 .

 6 Figure 6. (a-d) The structure of electric eel AChE with the three ligands 8a-c. (a) The overall structure of the protein with secondary structure projection and protein surface; (b) the protein projection with all three compounds, 8a-c; (c,d) the active side of the eeAChE with three ligands.

  Int. J.Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 16 group. They are connected to each other by methylene groups. In both crystal structures, the molecules interact with each other through weak C-H … O type hydrogen bonds (HBs), where most C-H donors are aliphatic.

Table 1 .

 1 Inhibitory effects of compounds 8a-c, 9a-c, 10a-c against AChE and BuChE.

	Metal	Compound	IC 50 AChE ± SD (µM) a	IC 50 BuChE ± SD (µM) b
	-	8a	1.215 ± 0.006	>1000
	-	8b	>1000	>1000
	-	8c	>1000	363 ± 29
	Fe	9a	>1000	331 ± 41
	Fe	9b	>1000	>1000
	Fe	9c	>1000	>1000
	Ru	10a	>1000	186 ± 21
	Ru	10b	>1000	389 ± 26
	Ru	10c	164 ± 21	292 ± 31
	-	Tacrine	0.056 ± 0.005	0.018 ± 0.002

a IC 50 : 50% inhibitory concentration (means ± SD of three independent experiments) AChE. b IC 50 : 50% inhibitory concentration (means ± SD of three independent experiments) BuChE.

Table 2 .

 2 Viability of the V79 cell line after treatment with compounds.

	Compound	9a	9b	9c	10a	10b	10c	8a 8b 8c
	Cytotoxicity	IC 50 = 690 ± 42 µM	IC 50 = 909 ± 53 µM	Toxic at concentrations of 750 and 1000 µM-viability of 69% and 58%, respectively	IC 50 = 725 ± 62 µM	IC 50 = 787 ± 113 µM	IC 50 = 763 ± 77 µM	Non-toxic

Table 2 .

 2 Viability of the V79 cell line after treatment with compounds.

Table 3 .

 3 Molecular docking analysis of AChE and ligand complexes.

	Ligand	Binding Energy Kcal/mol	Intermolecular Energy Kcal/mol	Total Internal Energy Kcal/mol	Torsional Free Energy Kcal/mol	Unbound Systems Energy Kcal/mol
	8a	-7.80	-10.18	-1.90	2.39	-1.90
	8b	-6.36	-8.74	-0.64	2.39	-0.64
	8c	-6.74	-9.13	0	2.39	0

Table 3 .

 3 Molecular docking analysis of AChE and ligand complexes.

	Ligand	Binding Energy Kcal/mol	Intermolecular Energy Kcal/mol	Total Internal Energy Kcal/mol	Torsional Free Energy Kcal/mol	Unbound Systems Energy Kcal/mol
	8a	-7.80	-10.18	-1.90	2.39	-1.90
	8b	-6.36	-8.74	-0.64	2.39	-0.64
	8c	-6.74	-9.13	0	2.39	0

Supplementary Materials:The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms23158091/s1.

Acknowledgments: ERASMUS + programme of the European Union for funding internship at ENSCM, Montpellier (A.K.).

Data Availability Statement: Not applicable.

Conflicts of Interest:

The authors declare no conflict of interest.


Dr Rémy Ferlet

Dr Mathieu Pucheault

Laurent Chabaud
email: laurent.chabaud@u-bordeaux.fr

Jean-François Paquin
email: jean-francois.paquin@chm.ulaval.ca

Jean-François Paquin

Prof ] R Ferlet

PROTEO J.-F Paquin

Metal-Free Defluoroborylation of 3,3-Difluoropropenes

Keywords: C-F activation, borylation, defluoroborylation, metal-free reaction, monofluoroalkene

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The widespread applications of fluorine chemistry in various fields can be attributed to the unique effects achieved by introducing one or more fluorine atoms into materials or bioactive molecules. [1] The popularity of fluorine's new applications has led chemists to diversify their synthetic methods of organofluorine compounds. [START_REF] Campbell | Selected reviews on the synthesis of fluorinated compounds: a)[END_REF] One approach involves breaking down a polyfluorinated precursor to obtain a partially defluorinated target compound of interest. [START_REF] Kiplinger | For a selection of reviews on C-F bond activation[END_REF] This "top-down" strategy is a formidable challenge, particularly due to the necessity to overcome the strength of the carbon-fluorine bond (C-F). In terms of synthetic strategy, it offers an alternative to conventional C-F bond formation protocols by using selective C-F bond functionalization of a molecule to reach new fluorinated motifs. [START_REF] Campbell | Selected reviews on the synthesis of fluorinated compounds: a)[END_REF] Of the many existing fluorinated motifs, monofluoroalkenes are of particular interest. This is particularly true in medicinal chemistry as they can potentially act as an amide bond isostere or as a mimic of enols (Scheme 1A). [START_REF] Van Steenis | For reviews on the use and synthesis of mono-fluoroalkenes[END_REF] While many approaches to monofluoroalkenes have been reported, our group has been particularly interested in their synthesis from 3,3-difluoropropenes [ 5 ] using either transition-metal-catalyzed [ 6 ] or metal-free reactions [7] (Scheme 1B). [START_REF] Hamel | For a similar approach using gem-chlorofluoropropenes[END_REF] In terms of nucleophiles, these methods allowed the introduction of aliphatic or aromatic amines, malonates and derivatives, alkyl, vinyl, alkynyl and aryl groups. Notably missing from this list are boron-based nucleophiles as organoboron compounds are not only valuable synthetic handles, [9] but also key substituents in some bioactive compounds. [10] A number of studies have already been reported on the metal-catalyzedc SN2' defluoroborylation of fluoroallyl groups using B2pin2 (Scheme 1C). [ 11 ] Recently, an electrochemical method has also been described. [12] Herein, we disclose the first metal-free defluorinative borylation of 3,3difluoropropenes using a combination of B2pin2 and fluoride source to provide β-boryl-substituted monofluoroalkenes (Scheme 1D).

Results and Discussion

To begin our investigation on the metal-free defluorinative borylation reaction, we choose 3,3-difluoropropene 1a as a model substrate and bis(pinacolato)diboron (B2pin2) as a readily available nucleophilic source of boron. The influence of the Lewis base, neutral or charged, was assessed in a systematic manner to form in situ a sp 2 -sp 3 adduct of B2pin2, [13] already reported as a boron source in metal-free borylation of alkenes including allylic alcohols. [14] After a survey of different Lewis bases (Table 1, see Supporting Information for more details), such as alkoxides (MeOK, MeONa), amines (DBU, DMAP, etc.), and fluoride sources, the organic-based fluorides such as TBAF (1 M solution in THF) and TBAT provided the best results as judged by NMR with the exclusive formation of the (2-fluoroallyl)boronate 2a product. Because the 1 M solution of TBAF in THF allowed for more reproducible results, the optimization was further pursued with this Lewis base. Lewis base [a] NMR Yield [b] Alkoxide : (MeOK, MeONa Since the purification of (2-fluoroallyl)boronate 2a can be tedious due to the absorption of the product on silica gel, for the rest of the optimization, the defluorinative borylation reaction was followed by an oxidation step in order to generate and isolate the corresponding alcohol 3a. Selected results are shown in Table 2.The fluorinated allyl alcohol 3a could be obtained, after 24 h of reaction at 60 °C, in 42% yield over two steps with two equivalents of TBAF and B2Pin2 (Table 1, entry 1). It has been demonstrated that C-F bond-cleavage can be assisted by a silicon-based reagent. [15] We thus investigated hexamethyldisiloxane (TMS2O) and hexadimethylsilazane (HMDS) as additives, and found that the product 3a was obtained in 50% and 79% yield, respectively (Table 1, entries 2-3). After modification of the TBAF/HMDS ratio (Table 1, entries 4-6), we obtained a good 75 %yield of 3a with 1.5 equiv. of TBAF and 1.4 equiv. of HMDS (Table 1, entry 6). Pleasingly, decreasing the amount of B2Pin2 to 1.5 equivalents afforded 3a in 81% (Table 1, entry 7). However, attempt to reduce to 1.1 equivalents of B2Pin2 erodes the yield of 3a to 58% (Table 1, entry 8). To ensure the effect of the Lewis base and the additive onto the defluoroborylation process, we performed the control reactions without TBAF or HMDS (Table 1, entries 9-10). In the absence of fluoride, the desired product 2a was not observed. Whereas in the absence of HMDS, a modest yield of 56% was obtained that ascertain the effect of the silicon-based additive. Therefore, the conditions of entry 7 were used as the optimal ones. -56

[a] The reactions were performed on a 0.3 mmol scale. [b] Isolated yields of 3a.

The substrate scope was next documented with various 3,3difluoropropenes and the results are shown in Scheme 2. A bromo-substituted tetralone derivative gave a good yield of 70 % (3b) whereas with a methoxy group, a moderate yield of 49 % (3c). We next surveyed substrates derived from 3a and bearing an heteroelement in the non-aromatic cyclic core. Compound 3d with a sulfur atom led to complete degradation of the (fluoroallyl)boronates intermediate after 24 h. Decreasing the reaction time to 6 h, allowed us to isolate the desired compound 3d in a low 15% yield, although full conversion was observed. In contrast, compound 3e containing an oxygen atom gave a better isolated yield (38%) in 6 h of reaction. Interestingly, from a trisubstituted alkene, the reaction afforded the desired product 3f in moderate yield (38%) from a Z/E (40:60) mixture, while a slightly better yield of 48 % was obtained when starting from the Z olefin (Z/E > 97:3). We also examined compound 1g derived from indanone. A modest yield of 40 % was obtained in 6 h of reaction, that could be attributed to partial degradation of the allylboronate intermediate, and the low stability of the corresponding alcohol 3g even when stored under argon at -20 °C. Finally, acyclic gem-difluoropropenes are well tolerated to the reaction conditions as demonstrated with compound 3h-k isolated in moderate to good yields (46 to 74%) always as a mixture of isomers. However, disubstituted alkenes furnished the corresponding alcohols 3l and 3m in lower yields due to the formation of unidentifiable side products during the defluoroborylation step. Gem-difluoropropene substituted by an alkyl group bearing an alcohol (i.e., 1n) or perfluoroalkyl group (3o) were not reactive under the optimized conditions. Scheme 2. Scope for the defluorinative borylation of 3,3-difluoropropenes. All reactions were conducted on a 0.3 mmol scale. Yield of the isolated products are provided. [a] Reaction time was 6 h. [b] From 1f (Z/E = 40:60). [c] From 1f (Z/E > 97:3). [d] Ratio of isomers. The E and Z configuration could not be unambiguously assigned. [e] Only one isomer isolated (stereochemistry not determined).

The (2-fluoroallyl)boronates products can undergo a wide range of transformations through organoboron chemistry allowing a direct access to various valuable mono-fluoroalkene products.

To further demonstrate the scalability of the protocol, the synthesis at a larger scale was explored. First, the reaction conditions were successfully applied on 1.5 mmol scale on the allylic difluoride 1a, affording the (2-fluoroallyl)boronate 2a with a satisfactory 65% isolated yield (Scheme 3). The compound 2a was oxidized into 3a, obtained in 81% yield. Functional group exchange on the boron atom can be performed by treatment of 2a with a saturated aqueous solution of KHF2 yielding the potassium trifluoroborate salt 4a in 73% yield. [ 16 ] Finally, homologation of allylboronate 2a by a Matteson reaction led to αchloro-borane 5a, [17] that could be used for further transformation thanks to boron chemistry. A plausible mechanism is depicted in Scheme 4. Reaction of the fluoride on the bis(pinacolato)diboron generates a polarized sp 3 -sp 2 diborane adduct, which develops a nucleophilic character on the sp 2 boron atom. [ 18 ] Such adduct can then perform a diborylation reaction of the alkene 1 to produce 6. [ 19 ] This intermediate 6 could not be detected during the reaction, and probably undergoes a fast B-F elimination promoted by a fluoride anion. [20] Alternatively, a SN2' reaction pathway, as observed for organolithium compounds and lithium amides, [7] can occur leading directly to the allylboronate. Interestingly, the formation of the pinBF2 -is invariably observed at the end of the reaction, indicating that the released Lewis acid FBpin can either activate the C-F bond [ 21 ] of the allyldifluoride or trap the expulsed fluoride. [START_REF]An equilibrium between [pinB-B(F)pin]and FBpin leading to B2pin2 and pinBF2is also possible[END_REF] The role of the silylated additive is not clear yet. hypothesis is that it also act as an activating agent of the C-F bond, [15] or a trapping reagent of the water contained in the TBAF solution. Further studies are undergoing to insights into the mechanism.

Conclusion

We have developed a metal-free defluorinative borylation of 3,3difluoropropenes to synthesize -boryl-substituted monofluoroalkenes using a B2pin2/TBAF system in combination with HMDS as a promoter. A direct oxidation of the intermediate boronates allowed the isolation of the corresponding alcohols in low to good yields (981%), depending on the structure of the starting 3,3-difluoropropenes. Useful transformations of a (2fluoroallyl)boronate were efficiently carried out, demonstrating the versatility of a single common intermediate for the synthesis of a variety of monofluoroalkenes.

Experimental Section

General procedure for the defluoroborylation/oxidation reactions: In a 2-5 mL dried microwave reaction vials, the gemdifluoropropene (1 equiv., 0.30 mmol), bis(pinacolato)diboron (1.5 equiv., 114.3 mg, 0.45 mmol), and a PTFE-coated stir bars are added, and the vial was sealed and purged three times with argon. A cold 1 M TBAF solution in THF was added in one portion (1.5 equiv., 0.45 mL), followed by hexamethyldisilazane (1.4 equiv., 0.88 L). The reaction tube was immersed in an oil bath preheated at 60 °C. After 24 h of reaction, the reaction medium was brought to room temperature under argon and then diluted with 1 mL of anhydrous THF. The reaction mixture was then added dropwise to a solution of NaOH/H2O2 (30% in water) (10 equiv.) at 0 °C and brought to room temperature under stirring upon completion determined by TLC. Then, the mixture was quenched with a saturated aq. solution of NaHCO3 and extracted twice with EtOAc. The combined organic layers were dried over Na2SO4, filtered and volatile were evaporated in vacuo. The crude product was purified on flash silica gel chromatography.

Scheme 1 .

 1 Scheme 1. Context, previous work and metal-free borylation of 3,3difluoropropenes.

  : tetrabutylammonium.[b] Estimated by1 H NMR with mesitylene as the internal standard.

Scheme 3 .

 3 Scheme 3. Transformations on (2-fluoroallyl)boronate 2a.

Scheme 4 .

 4 Scheme 4. Proposed mechanism.

Table 1 .

 1 Preliminary screening of Lewis bases.

Table 2 .

 2 Optimization of the reaction conditions.

Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), Université Laval, Université de Bordeaux and CNRS (International Emerging Action). RF thanks IDEX International Doctorate program (Université de Bordeaux) for financial support.

Entry for the Table of Contents

A metal-free defluoroborylation of 3,3-difluoropropenes has been developed. The reaction involves the convenient borylation agent B2pin2 and a combination of both TBAF and HMDS as an activating system. The fluorinated allylboronates were used as a platform to prepare functionalized monofluoroalkenes.

Institute and/or researcher Twitter usernames: @jfluorine

Supporting Information

The authors have cited additional references within the Supporting Information. [START_REF] Harris | [END_REF]24,25]