WO2017021319A1 - Broad spectrum reactivators of opna-inhibition of human cholinesterases - Google Patents

Abstract

The present invention deals with novel compounds which are reactivators of human acetylcholinesterase, pharmaceutical compositions comprising said compounds, and their use in the treatment of a nervous and/or respiratory failure due to intoxication with at least one organophosphorous nerve agent such as warfare agents and pesticides. It also pertains to these compounds for use in the treatment of neurological diseases such as Alzheimer's disease.

Description

Broad spectrum reactivators of OPNA-inhibition of human cholinesterases Introduction The present invention deals with novel reactivators of human acetylcholinesterase (hAChE), pharmaceutical compositions comprising said compounds, and their use for reactivating human acetylcholinesterase inhibited by at least one organophosphorous nerve agent. Background of the invention

Organophosphorous nerve agents (OPNA) are extremely toxic compounds that comprise chemical warfare agents (CWA) including sarin, soman, cyclosarin, tabun, methylphosphonothioate (VX) and pesticides such as paraoxon, parathion and tetraethyl pyrophosphate (TEPP). Their acute toxicity results from the irreversible inhibition of acetylcholinesterase (AChE) through phosphylation of its catalytic serine, which results in the inability of the enzyme to hydro lyze acetylcholine (ACh). Accumulation of this neurotransmitter at cholinergic synapses occurs, leading to a permanent saturation of the muscarinic and nicotinic receptors which ultimately results in seizure and respiratory arrest. Depending on the class of OPNA and on the administrated dose, death can occur within a few minutes.

Due to the similarity between the chemical precursors of CWA and pesticides, and to the relatively simple chemistry involved in their synthesis, efforts to control the proliferation of these agents have proved of limited success. Illustrative examples include the terrorist attack in the Tokyo subway in 1995, the bombing of Kurd civilians during the Iraq-Iran war in 1988, and that of civilians in Syria, as reported in August 2013. Additionally, despite the international efforts aimed at regulating and lessening the use of these environmentally toxic compounds, ca. 100 different OPNA are still used intensively as pest control agents, with only anecdotal monitoring. This results in about 3,000,000 acute intoxications per year, 200,000 of which lead to death. Moreover, intoxications may also occur during the destruction of chemical weapons stockpiles. Therefore, the development of effective measures to counteract OPNA poisoning remains a challenging issue to protect and treat both civilian and military populations. The current treatment for OPNA poisoning consists of the administration of a combination of atropine (antimuscarinic agent) and diazepam (anticonvulsant drug), to limit convulsions, and of a standard pyridinium oxime (pralidoxime, trimedoxime, HI-6, obidoxime, or HLo-7) to reactivate AChE. Oximes exert their action on OPNA- inhibited AChE by attacking the phosphorous atom of the phosphylated serine, leading to the removal of the phosphonate and restoration of the enzyme's catalytic activity. Some of these known compounds have a pyridinium oxime-based structure coupled to a potential ligand for the peripheral site of the enzyme (PSL), the purpose of which is to increase the affinity of the reactivator for AChE (Mercey G. et al., Accounts of Chemical Research, 756-766, 2012, Vol. 45, No. 5).

The reactivation process can be illustrated by the following scheme:

reac vation

The efficiency of reactivators may be estimated by the second-order rate constant for reactivation k_r2, which is the ratio of the maximal reactivation rate constant (k_r) to the apparent dissociation constant of the reactivator - inhibited AChE complex (KD). As of today, none of the known oximes has proven equally effective against all types of OPNA-inhibited AChE, and most are ineffective against tabun-inhibited bAChE. This is because tabun-inhibited enzyme is resistant to reactivation due to weak electrophilicity and steric hindrance of the tabun-hAChE adduct.

Recently, the present inventors have synthetised bifunctional reactivators comprising a tetrahydroacridine (tacrine)-type peripheral site ligand bonded to an oxime moiety via a flexible linker (WO 2015/075082). These compounds have proven to be effective against hAChE inhibited by various OPNA and even more effective than trimedoxime against tabun-inhibited bAChE (Kliachyna M. et al., European Journal of Medicinal Chemistry, 78, 2014, 455-467). However, the reactivation efficiency of these compounds could still be improved.

Summary of the invention In this context, the Applicant found that bifunctional compounds comprising a specific peripheral site ligand (PSL) moiety of the amino-quinoline type had improved affinity for poisoned hAChE (thus, a lower KD) than the tacrine-based reactivators, which allowed them to be more potent reactivators of human AChE inhibited with any type of organophosphorous compounds. These novel compounds may thus be used at a lower concentration, which decreases the possible side effects and cost of the treatment.

This result was clearly unprecedented for at least three reasons. First, amino-quinoline is not known by itself as an inhibitor of bAChE. Secondly, the novel structure appears to be simplified compared to the known tacrine-based structure and essentially amounts to the removal of the tacrine moiety. By contrast, the literature suggests that the reactivation kinetics could be controlled by structural modification of the PSL moiety (Saint-Andre et al, Tetrahedron, 67, 2011, 6352-6361) which, according to drug design practice, rather amounts to grafting specific groups to a known structure. Thirdly, the reactivation mechanism of OPNA-inhibited bAChE is still not fully understood, such that it is not easy to design compounds having a proper KD ratio. The key to the improvement of the reactivation potency is finding the optimal balance between affinity of the functional reactivator for the enzyme and the positioning of the oxime function relative to the phosphorous atom of the OPNA-hAChE adduct. In the design of reactivators, one of the numerous issues to address is that some reactivation products, which are phosphyloximes, may themselves display high affinity for the enzyme. Therefore, the design of potent reactivators for the medical treatment of OPNA poisoning with a broad activity spectrum was not straightforward.

The compounds of this invention allow for effective and fast reactivation of hAChE without denaturing the same. They are thus useful for treating people poisoned by organophosphorous compounds and especially civilian and military people contaminated by warfare agents, farmers intoxicated by pest control agents and chemists who handle organophosphorous products. Moreover, these compounds may be produced under economically favorable conditions since they may be obtained using simple synthesis steps on a scale that may be easily adjusted.

Incidentally, these novel compounds have been shown to be able to inhibit native hAChE at a concentration range of micro- to nanomolar. They may thus be used in the treatment of various neurological diseases in which inhibitors of hAChE have proven to be useful, such as Alzheimer's disease.

A first object of the present invention is a compound of formula (I)

(I)

wherein the different groups are as defined in the detailed description below.

Another object of the present invention is a pharmaceutical composition comprising at least one compound of formula (I) and at least one pharmaceutically acceptable support.

Another object of the invention is a compound according to the invention, for use as a medicine.

A further object of the invention is a compound according to the invention for use in the treatment of a nervous and/or respiratory failure due to intoxication with at least one organophosphorous nerve agent. Still a further object of this invention is a compound of Formula (I) for use in the treatment of neurological diseases such as Alzheimer's disease.

Detailed description of the invention

A first object of the invention is a compound of formula (I):

(I)

wherein:

n is 0, 1, 2, 3 or 4

m is 0 or 1 ,

R¹ and R² are independently selected from the group consisting of a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, CF3, an aryl group and an heteroaryl group or R¹ and R² form together and with the carbon atoms linked thereto an aryl or heteroaryl group,

R³ and R⁴ are independently selected from the group consisting of a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, CF3 and an aryl group or R³ and R⁴ form together and with the carbon atoms linked thereto an aryl or heteroaryl group,

R⁵ is selected from the group consisting of a hydrogen atom, an alkyl group and an acyl group,

R⁶ is a hydrogen atom or an alkyl group, when present, and

R⁷ is selected from the group consisting of a hydrogen atom, a NH2 group and a CF3 group.

In the present invention, an "alkyl group" is a linear hydrocarbon group comprising from 1 to 6 carbon atoms, in particular from 1 to 3 carbon atoms, or a branched or cyclic hydrocarbon group comprising from 3 to 6 carbon atoms. Examples of alkyl groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, and cyclohexyl groups, and preferably methyl.

An "alkoxy group" is an -O-alkyl group wherein the alkyl moiety is as defined above. An "aryl group" refers to a monocyclic or polycyclic hydrocarbon aromatic group, with at least one of the rings having a system of conjugated pi electrons, which may be optionally substituted. Examples of aryl groups that can be used are optionally substituted phenyl, naphthalene and anthracene groups. Preferably, the aryl group is a phenyl group, which may be unsubstituted or substituted with one or more substituents independently selected from a halogen atom, an alkyl group, an alkoxy group and a CF3 group.

A "heteroaryl group" is an aryl group, wherein at least one carbon atom is replaced with a heteroatom such as N, O, P or S. Examples of heteroaryl groups include pyrrole, thiophene, furane, pyridine, pyrimidine, pyrazine, triazine, imidazole, thiazole, oxazole, and isoxazole groups.

An "acyl group" is an alkyl-CO- group wherein the alkyl moiety is as defined above. A "halogen atom" is an atom of F, CI, Br or I.

The compound of formula (I) may be labeled with one or more isotopes such as ¹⁵N, ²H (D) and ³H (T).

According to a preferred embodiment of this invention, m is 0. Neutral compounds are indeed preferred in this invention because they are able to cross the blood-brain barrier more easily in order to reactivate OPNA-inhibited AChE.

According to another embodiment of this invention, m = 1 and R⁶ = H. In this situation, preferred counter ions are CI^", Br, Γ, S0₄ ^2", AcO^", CF3CO2^", BzO^", CH3SO3^" and

Moreover, it is preferred that at least one and preferably all the following conditions are met:

• n is 2, 3 or 4, preferably n is 3,

· R¹ and R² form together and with the carbon atoms linked thereto an aryl group, preferably a substituted or non-substituted phenyl group, thereby forming a quinoline structure, • at least one, preferably at least two and more preferably each of R³, R⁵ and R⁷ is a hydrogen atom, preferably a hydrogen atom,

• R⁴ is a hydrogen atom or an alkyl group such as a methyl group,

• the pyridine ring in Formula (I) is substituted in position 6 by the quinoline- bearing group and/or in position 3 by the OH group.

Alternatively, the compound according to this invention may be such that at least one and preferably all the following conditions are met:

• n is 2, 3 or 4, preferably n is 2 or 3,

• R¹ and R² are independently selected from a hydrogen atom and an alkyl group, preferably each of R¹ and R² is a hydrogen atom,

at least one, preferably at least two and more preferably each of R , R and R is a hydrogen atom,

R⁴ is a hydrogen atom or an alkyl group such as a methyl group, preferably a hydrogen atom,

• the hydroxy-pyridine ring in Formula (I) is substituted in position 6 by the pyridine-bearing group and/or in position 3 by the OH group.

For instance, the compound according to this invention may have the following formula (la):

in which n is 2 or 3 and R¹ and R² are each a hydrogen atom or R¹ and R² form together and with the carbon atoms linked thereto a phenyl group which may be substituted or non substituted.

The compounds of the invention are preferably selected from those listed in Table 1 below. Table 1

Exemplary reactivators

Table 1

Exemplary reactivators

specific embodiment, the compound of formula (I) is compound E or M Synthesis of the compounds of the invention

A compound of formula (I) according to the invention may be synthesized by any appropriate method known by anyone of ordinary skill in the art.

In particular, the process for the synthesis of compounds of formula (I) may comprise a step of Sonogashira coupling reaction between a halogenated pyridine derivative or a halogenated quinoline moiety, and an amino-compound comprising a terminal alkyne. Alternatively, an aromatic nucleophilic substitution (SNAr) reaction may be used with the same reagents, i.e. between a halopyridine scaffold or a halogenated quinoline moiety, and an amino-compound bearing a terminal alkyne. In the particular case of aminopyridine derivatives, the alkylation of the amino- group with an electrophilic alkyl-halide compound comprising a terminal alkyne can be used. The resulting alkyne may be reduced by reaction with hydrogen, for instance in presence of Pearlman's catalyst Pd(OH)₂/C.

For the compounds of formula (I) with R⁷ = H, the oxime may be formed from the corresponding aldehyde by reaction with NH₂OH, preferably as a hydrochloride, preferably in presence of a base, such as CFbCOONa.

For the compounds of formula (I) with R⁷ = NH₂, the amidoxime may be formed from the corresponding nitrile by reaction with NH₂OH, preferably as a hydrochloride, preferably in presence of a base, such as pyridine.

Pharmaceutical use of the compounds of the invention The compounds of this invention may be used in the treatment of a nervous and/or respiratory failure due to intoxication with at least one organophosphorous nerve agent which may preferably be selected from warfare agents such as O-ethyl S-[2- (diisopropylamino)ethyl] methylphosphonothioate (VX), tabun, sarin, cyclosarin and soman and pesticides such as paraoxon, parathion and tetraethyl pyrophosphate (TEPP). These compounds may alternatively be used in the treatment of diseases, which involve a reduced production of acetylcholine that may be overcome by the administration of acetylcholinesterase inhibitors. Examples of such diseases include in particular neurological diseases such as Alzheimer's disease.

The compound of this invention is usually included in a pharmaceutical composition comprising at least one compound according to the invention and a pharmaceutically acceptable support.

The amount of compound of formula (I) in the composition according to the invention may vary in a broad range depending upon the patient, the mode of administration and the expected effect.

The compound or composition according to the invention can be administered orally or non-orally, for instance via parenteral, intramuscular, intravenous, cutaneous, nasal or rectal route.

The pharmaceutical composition of the invention can present different forms including granules, powders, tablets, capsules, syrups, emulsions, suspensions, and forms used for non-oral administration, for instance injections, sprays, transdermal patches or suppositories. These pharmaceutical forms can be prepared via known conventional techniques.

The preparation of an orally administered solid pharmaceutical form can be for instance performed by the following process: an excipient (for example lactose, sucrose, starch or mannitol), a disintegrant (for example calcium carbonate, calcium carboxymethylcellulose, alginic acid, sodium carboxymethylcellulose, colloidal silicon dioxide, sodium croscarmellose, Crospovidone, guar gum, magnesium aluminium silicate, microcrystalline cellulose, cellulose powder, pregelatinised starch, sodium alginate or starch glycolate), a binder (for example alpha-starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, alginic acid, carbomer, dextrin, ethylcellulose, sodium alginate, maltodextrin, liquid glucose, magnesium aluminium silicate, hydroxyethylcellulose, methylcellulose or guar gum) and a lubricant (for example talc, magnesium stearate or polyethylene 6000) are added to the active principle and the mixture obtained is then tabletted. If necessary, the tablet can be coated via the known techniques, in order to mask the taste (for example with cocoa powder, mint, borneol or cinnamon powder) or to allow enteric dissolution or sustained release of the active principles. Coating products that can be used are, for example, ethylcellulose, hydroxymethylcellulose, polyoxyethylene glycol, cellulose acetophthalate, hydroxypropylmethylcellulose phthalate and Eudragit^® (methacrylic acid-acrylic acid copolymer), Opadry^® (hydroxypropylmethylcellulose + macrogol + titanium oxide + lactose monohydrate). Pharmaceutically acceptable colorants may be added (for example yellow iron oxide, red iron oxide or quinoline yellow lake).

Liquid pharmaceutical forms for oral administration include solutions, suspensions and emulsions. The aqueous solutions can be obtained by dissolving the active principle in water, followed by addition of flavourings, colorants, stabilisers and/or thickeners, if necessary. In order to improve the solubility, it is possible to add ethanol, propylene glycol or any other pharmaceutically acceptable non-aqueous solvent. The aqueous suspensions for oral use can be obtained by dispersing the finely divided active principle in water with a viscous product, such as a natural or synthetic gum or resin, methylcellulose or sodium carboxymethylcellulose.

The pharmaceutical forms for injection can be obtained, for example, by the following process. The active principle is dissolved, suspended or emulsified either in an aqueous medium (for example distilled water, physiological saline or Ringer's solution) or in an oily medium (for example olive oil, sesame seed oil, cottonseed oil, corn oil or propylene glycol), with a dispersant (for example Tween^® 80, HCO^® 60 (Nikko Chemicals), polyethylene glycol, carboxymethylcellulose or sodium alginate), a preserving agent (for example methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, benzyl alcohol, chlorobutanol or phenol), an isotonicity agent (for example sodium chloride, glycerol, sorbitol or glucose) and optionally other additives, such as, if desired, a solubilising agent (for example sodium salicylate or sodium acetate) or a stabiliser (for example human serum albumin).

Pharmaceutical forms for external use can be obtained from a solid, semi-solid or liquid composition containing the active principle. For example, to obtain a solid form, the active principle can be treated with excipients (for example lactose, mannitol, starch, microcrystalline cellulose or sucrose) and a thickener (for example natural gums, cellulose derivatives or acrylic polymers) so as to convert them into powder. The liquid pharmaceutical compositions are prepared in substantially the same way as the forms for injection, as indicated previously. The semi-solid pharmaceutical forms are preferably in the form of aqueous or oily gels or in the form of pomades. These compositions may optionally contain a pH regulator (for example carbonic acid, phosphoric acid, citric acid, hydrochloric acid or sodium hydroxide) and a preserving agent (for example a p-hydroxybenzoic acid ester, chlorobutanol or benzalkonium chloride).

A method for the treatment of a nervous and/or respiratory failure due to intoxication with at least one organophosphorous nerve agent, comprising administering at least one compound according to the invention is also described herein, as well as the use of a compound according to the invention for the preparation of a medicine for the treatment of a nervous and/or respiratory failure due to intoxication with at least one organophosphorous nerve agent.

Within the context of the invention, the term treatment denotes curative, symptomatic, and/or preventive treatments. In particular, it can refer to reducing the progression of the disease, reducing or suppressing at least one of its symptoms or complications, or improving in any way the state of health of patients.

The administration of the compounds or of the composition according to the invention may be performed before, during or after the exposition of the subject to the organophosphorous nerve agent.

In the present invention, the terms "subject" and "patient" are used indifferently and designate a human subject.

The amount of compound of formula (I) to be administered according to the invention may vary in a broad range depending upon the patient, the mode of administration and the expected effect. In particular, the amount of compound of formula (I) may be comprised between 200 mg and 4000 mg, with up to 3 daily intakes.

The compound or composition according to the invention may be co-administered with at least one other active agent, such as an antimuscarinic agent, in particular atropine, an anticonvulsant, in particular diazepam or one of its prodrugs, such as avizafone, and/or a bioscavenger able to capture and/or degrade OPNAs in blood, such as human butyrylcholinesterase. The term co-administered means that the administration of the compound or composition according to the invention and that of the other active agent can be simultaneous, sequential and /or separate. Other uses of the compounds of the invention

The compounds of this invention may further be used as screening tools for the identification of more potent hAChE reactivators. In this application, the compounds of formula (I) may include one or more isotopes, which will allow for their detection. The following examples are provided as illustrative, and not limitative, of the present invention.

Examples: Example 1 : Synthesis of Compound E according to the invention General procedure

Starting from the commercially available 4-bromoquinoline 1, a Buchwald-Hartwig amination coupling was carried out with l-amino-3-butyne to afford the terminal alkyne 2. After filtration on silica, the crude was subjected to Sonogashira coupling to afford the intermediate alkyne 3 in good yield after 2 steps (60%). Then the aldehyde 4 was isolated in excellent yield (69%) using a three-step sequence including: (a) "one-pot" alkyne reduction and O-debenzylation; (b) TBS protection of the phenolic function using TBSOTf and 2,6-lutidine; (c) reduction of the methyl ester into the corresponding aldehyde using DIBAL-H and deprotection of the silyloxy function. Finally, the desired oxime 5 was isolated by oximation using hydroxylamine hydrochloride and anhydrous sodium acetate in ethanol. It was recovered in 54% yield after purification by column chromatography and crystallization.

54%

Scheme 1 Synthesis of hybrid reactivator E.

Methyl 3-(benzyloxy)-6-(4-(quinolin-4-ylamino)but-l-yn-l-yl)picolinate (3).

A microwave tube containing a magnetic stirrer bar was charged with 4-bromoquinoline (143 mg, 0.690 mmol, 1.0 equiv), Pd₂(dba)₃ (25 mg, 0.028 mmol, 0.04 equiv), (±)- BINAP (37 mg, 0.055 mmol, 0.08 equiv) and Cs₂C0₃ (562 mg, 1.725 mmol, 2.5 equiv). The vessel was sealed with a microwave septum and purged with argon. Degassed 1 ,4- dioxane (2.3 mL) and l-amino-3-butyne (73 μΐ,, 0.897 mmol, 1.3 equiv) were introduced through the septum. The resulting mixture was heated to 100 °C for 1 h using a Biotage® Initiator Microwave Synthesizer Apparatus. After cooling, the reaction mixture was concentrated and filtrated through a small plug of silica. The crude material was directly used to the subsequent step.

To a degassed solution of methyl 3-benzyloxy-6-bromopicolinate (244 mg, 0.759 mmol, 1.1 equiv) in THF/EtsN (7 mL/ 5 mL), Pd[PPh₃]4 (79 mg, 0.069 mmol, 0.1 equiv) and Cul (26 mg, 0.138 mmol, 0.2 equiv) were added. After degasing the reaction mixture for 5 min at room temperature, a degassed solution of the previous N-(but-3-yn-l- yl)quinolin-4-amine 2 (0.690 mmol, 1 equiv) in THF (3 mL) was added dropwise and the reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography (CIHhCk/MeOH 9: 1) to afford the desired methyl 3- (benzyloxy)-6-(4-(quinolin-4-ylamino)but-l-yn-l-yl)picolinate 3 as white foam (181 mg, 0.414 mmol, 60%).

TLC R_f (CftCk/MeOH 8:2) 0.30.

IR Vmax neat (cm^"1) 3234 (m, b), 3034 (b), 2950 (m), 2881 (m), 2231 (w), 1733 (s), 1583 (s), 1451 (s), 1383 (m), 1341 (m), 1273 (s), 1211 (s), 1099 (s).

¾ NMR (400 MHz, CDC ) δ (ppm) 8.38 (d, J = 6.1 Hz, 1H, 6), 8.33 (d, J = 8.3 Hz, 1H, 8), 8.07 (d, J = 8.6 Hz, 1H, 2), 7.66 (br s, 1H, 10), 7.64 (t, J= 7.7 Hz, 1H, 9), 7.49- 7.26 (m, 8H, 1+16+17+24+25+26+27+28), 6.54 (d, J = 6.4 Hz, 1H, 5), 5.18 (s, 2H, 22), 3.94 (s, 3H, 21), 3.72 (q, J= 6.0 Hz, 2H, 11), 2.89 (t, J= 6.9 Hz, 2H, 12).

1³C NMR (101 MHz, CDCb) δ (ppm) 164.98 (20), 153.46 (6), 153.12 (18), 145.67 (7), 142.91 (4), 140.26 (19), 135.50 (15), 134.66 (23), 131.52 (9), 130.27 (16), 128.89 (2C, 25+27), 128.45 (26), 127.06 (2C, 24+28), 126.14 (8), 124.93 (1), 121.99 (17), 121.95 (2), 118.13 (3), 98.26 (5), 86.31 (16), 81.50 (14), 71.01 (22), 52.90 (21), 41.93 (11), 19.76 (12).

LRMS (ESI⁺) m/z 438.0 ([M+H]⁺).

HRMS (ESI⁺) m/z calcd for C27H₂4N₃03⁺ 438.1812 found 438.1804 Da. -Hydroxy-6-(4-(quinolin-4-ylamino)butyl)picolinaldehyde (4).

To a degassed solution of methyl 3-(benzyloxy)-6-(4-(quinolin-4-ylamino)but-l-yn-l- yl)picolinate 3 (358 mg, 0.820 mmol, 1.0 equiv) in dry MeOH (60 mL), Pearlman's catalyst Pd(OH)₂/C (20% wt. with 50% moisture, 230 mg, 0.164 mmol, 0.2 equiv) was added. After evaporating and flushing with H2 five times, the reaction mixture was stirred for 7 h at room temperature under H2 (1 arm.). Upon completion, the catalyst was removed by filtration through a short column of celite, the solvent was evaporated and the residue was directly subjected to the following step without purification. The presence of the desired reduced product was supported by TLC and LRMS (ESI⁺) m/z 351.9 ([M+H]⁺).

To a solution of methyl 3-hydroxy-6-(4-(quinolin-4-ylamino)butyl)picolinate (288 mg, 0.820 mmol, 1.0 equiv) in dry CH2CI2 (8.2 mL), 2,6-lutidine (286

2.459 mmol, 3.0 equiv), and fert-butyldimethylsilyltrifluoromethanesulfonate (564 μί, 2.459 mmol, 3.0 equiv) were successively added and the reaction mixture was stirred at room temperature under argon atmosphere for 1 h 30. After completion, the reaction mixture was quenched with a saturated aqueous CuS0₄ solution (10 mL). After separation, the aqueous phase was extracted with CH2CI2 and the combined organic was then dried over Na2S0₄ and concentrated under reduced pressure. The presence of the desired silylether was supported by TLC and LRMS (ESI⁺) m/z 466.1 ([M+H]⁺). After drying in vacuo, the residue was directly subjected to the following step.

To the solution of methyl 3-((tert-butyldimethylsilyl)oxy)-6-(4-(quinolin-4- ylamino)butyl)picolinate (0.820 mmol, 1.0 equiv) in dry CH2CI2 (8.2 mL) at -78 °C, DIBAL-H (2.46 mL of a 1.0 M solution in CH2CI2, 2.459 mmol, 3.0 equiv) was added dropwise. The reaction mixture was stirred at -78 °C for 12 min, then the reaction was quenched with MeOH (3 mL), and the cooling bath was removed. When the mixture warmed to room temperature the organic phase was washed with an aqueous solution of NaOH (1.0 M). The aqueous phase was then extracted with CH2CI2 and the organics were dried over Na2S0₄ and concentrated under reduced pressure. After drying in vacuo, the residue was purified by column chromatography (CffcCk/MeOH 8:2) to afford the desired 3-hydroxy-6-(4-(quinolin-4-ylamino)butyl)picolinaldehyde 4 as bright yellow wax (182 mg, 0.566 mmol, 69% over 3 steps).

TLC Rf (CH₂Cl₂/MeOH 8:2) 0.14.

IR Vmax neat (cm^"1) 3275 (b), 2924 (m), 2853 (m), 1668 (m), 1584 (s), 1457 (s), 1340 (m), 1223 (m), 1139 (m).

¾ NMR (400 MHz, CDCI3) δ (ppm) 10.66 (br s, 1H, 21), 10.04 (s, 1H, 20), 8.56 (d, J = 4.9 Hz, 1H, 6), 7.98 (d, J = 8.6 Hz, 1H, 8), 7.74 (d, J = 8.1 Hz, 1H, 2), 7.63 (t, J = 7.6 Hz, 1H, 9), 7.42 (t, J = 7.5 Hz, 1H, 1), 7.34-7.29 (m, 2H, 16+17), 6.43 (d, J = 5.1 Hz, 1H, 5), 5.07 (br s, 1H, 10), 3.39 (q, J= 6.6 Hz, 2H, 11), 2.90 (t, J= 7.5 Hz, 2H, 14), 2.05-1.79 (m, 4H, 12+13).

¹³C NMR (101 MHz, CDC ) δ (ppm) 198.75 (20), 154.27 (18), 151.27 (6), 149.69 (4), 130.31 (9), 129.95 (8), 129.14 (1), 126.76 (16), 124.76 (2), 119.28 (17), 98.98 (5), 43.31 (11), 36.88 (14), 28.40 (12), 27.28 (13). Short run was made due to the stability of aldehyde, thus some quaternary carbons were not visible (3, 7, 15 and 19)

LRMS (ESI⁺) m/z 322.0 ([M+H]⁺).

HRMS (ESI⁺) m/z calcd for Ci9H₂oN30₂ ⁺ 322.1550 found 322.1544 Da.

3-Hydroxy-6-(4-(quinolin-4-ylamino)butyl)picolinaldehyde oxime acetate (E).

.mol^"1

A solution of 3-hydroxy-6-(4-(quinolin-4-ylamino)butyl)picolinaldehyde 4 (102 mg, 0.319 mmol, 1.0 equiv), hydroxylamine hydrochloride (33 mg, 0.479 mmol, 1.5 equiv), and CHsCC Na (39 mg, 0.479 mmol, 1.5 equiv) in dry EtOH (10 mL) was stirred at reflux for 15 h. After concentration under reduced pressure, the crude product was purified by the column chromatography (CH₂Cl₂/MeOH 9: 1) and precipitated in methanol/hexane (2:3) to afford the desired 3-hydroxy-6-(4-(quinolin-4- ylamino)butyl)picolinaldehyde oxime as white solid (58 mg, 0.037 mmol, 54%).

TLC Rf (CH₂Cl₂/MeOH 8:2) 0.13.

MP 197 °C (methanol/hexane 2:3)

IR Vmax neat (cm ¹) 3420 (w), 2928 (w), 2860 (w), 2570 (w, b), 1586 (s), 1538 (s), 1460 (m), 1347 (m), 1262 (s), 1037 (s).

¾ NMR (500 MHz, CD3OD) δ (ppm) 8.33 (d, J = 6.5 Hz, 1H, 6), 8.25 (dt, J = 8.5, 1.0 Hz, 1H, 8), 8.23 (s, 1H, 20), 7.83-7.79 (m, 2H, 2+9), 7.63-7.56 (m, 1H, 1), 7.25 (d, J = 8.5 Hz, 1H, 17), 7.16 (d, J= 8.5 Hz, 1H, 16), 6.69 (d, J = 6.6 Hz, 1H, 5), 3.54 (t, J = 6.8 Hz, 2H, 11), 2.81 (t, J = 7.2 Hz, 2H, 14), 1.92 (s, 3H, 23), 1.90-1.77 (m, 4H, 12+13).

¹³C NMR (125.8 MHz, CD₃OD) δ (ppm) 155.80 (4), 154.21 (15), 153.85 (18), 152.71 (20), 146.03 (6), 142.88 (7), 136.38 (19), 133.21 (9), 127.18 (1), 126.05 (17), 125.45 (16), 123.98 (2), 123.14 (8), 119.09 (3), 99.17 (5), 44.18 (11), 37.27 (14), 28.47 (12), 28.41 (13), 23.38 (23).

LRMS (ESI⁺) m/z 337.1 ([M+H]⁺).

HRMS (ESI⁺) m/z calcd for Ci9H₂iN40₂ ⁺ 337.1659 found 337.1657 Da

Synthesis of the bis acetate of Compound E

Compound E was dissolved in glacial acetic acid and stirred for 10 min at room temperature. Lyophilisation of the homogenous solution led to the acetate salt of E. Alternatively, the solution can be concentrated using a rotavapor and the residue can be dried in vacuum to afford the desired compound. ¾ NMR (500 MHz, CD3OD) δ (ppm) 8.33 (d, J = 6.6 Hz, 1H, 6), 8.29 (m, 1H,

8), 8.21 (s, 1H, 20), 7.89-7.85 (m, 1H, 9), 7.83-7.81 (m, 1H, 2), 7.64 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H, 1), 7.25 (d, J = 8.3 Hz, 1H, 17), 7.16 (d, J = 8.3 Hz, 1H, 16), 6.74 (d, J = 6.7 Hz, 1H, 5), 3.57 (t, J = 6.8 Hz, 2H, 11), 2.81 (t, J = 7.2 Hz, 2H, 14), 1.93 (s, 6H, 23), 1.90-1.81 (m, 4H, 12+13).

¹³C MR (125.8 MHz, CD3OD) δ (ppm) 156.56 (4), 154.15 (15), 153.84 (18),

152.68 (20), 144.77 (6), 136.39 (19), 133.88 (9), 127.58 (1), 126.05 (17), 125.46 (16), 123.37 (2), 122.80 (8), 118.80 (3), 99.17 (5), 44.30 (11), 37.23 (14), 28.39 (12), 28.34 (13), 23.02 (23). Example 2: Synthesis of Compound M according to the invention

84 % for 3 steps

Scheme 2: Synthesis of hybrid reactivator M

Synthesis of N-(4-Pyridyl)Benzyl Carbamate (7): bz

To a solution of 4-aminopyridine 1 (3.2 g, 34.0 mmol) in CH2CI2 (80 mL) at 0° C. was added triethylamine (9.50 mL, 68.0 mmol) and benzyl chloroformate (7.3 mL, 51.0 mmol). The ice bath was removed and the resulting mixture was allowed to warm to roomtemperature overnight before being poured into saturated aqueous NaHCCte. The product was extracted with CH2CI2, dried (Na₂S0₄) and concentrated. The crude product was recrystallized from EtOAc to give 6.4 g (83% yield) of pure benzyl carbamate 7. ¾ NMR (400 MHz, CDCb) δ (ppm) 5.22 (s, 2H), 7.32-7.42 (m, 7H), 8.45 (d, J= 5.9 Hz, 2H).

Ref: From U.S. Pat. Appl. Publ, 20110130429, 02 Jun 2011

Synthesis of benzyl prop-2-yn-l-yl(pyridin-4-yl)carbamate (8):

To a stirred solution of NaH (632 mg, 26.30 mmol) in DMF (15 mL) at 0° C. was added benzyl carbamate 7 (2 g, 8.77 mmol) in DMF (15 mL) and stirred it for 30 min at 0° C. Then propargyl bromide (1.15 mL, 13.15 mmol) was added slowly at 0° C. Warmed the reaction mixture to room temperature and stirred it for 4 h. after completion of the reaction, the reaction mixture was quenched with saturated NH4CI solution and extracted with EtOAc. The combined organic layers are dried over MgS0₄. The solids were filtered off and the solvent is evaporated. The crude product was purified by silica gel column chromatography (EtOAc/cyclo hexane 1 :4) to give the propargyl carbamate 8 (1.64 g, 70 %) as a white solid.

Rf (pure EtOAc) 0.60; IR (neat) v_max 3165, 1717, 1589, 1389, 1212, 1040, 751, 695, 548, 506 cm^"1; ¾ NMR (400 MHz, CDCb) δ (ppm) 8.54 (d, J= 5.5 Hz, 2H, Ar), 7.42- 7.29 (m, 7H, Ar), 5.25 (s, 2H, -CH₂Ph), 4.49 (d, J= 2.4 Hz, 2H, Hs), 2.34 (t, J= 2.4 Hz, 1H, H10). ¹³C NMR (100 MHz, CDCb) δ (ppm) 153.57, 150. 36, 148.56, 135.39, 128.49, 128.29, 127.95, 118.17 (Ar), 78.58 (C9), 73.03 (CIO), 68.31 (CH2PI1), 38.69 (C8); HRMS (ESI⁺) m/z calcd for Ci6Hi₅N₂0₂ ⁺ 267.1103 found 267.1128. Synthesis of methyl 3-(benzyloxy)-6-(3-(((benzyloxy)carbonyl)(pyridin-4- yl)amino)prop-l-yn-l-yl)picolinate (10):

To a degassed solution of methyl 3-benzyloxy-6-bromopicolinate 9 (1.21 mg, 3.76 mmol, 1 equiv) in THF/EtsN (20 mL/ 20 mL), Pd[PPh₃]4 (435 mg, 0.376 mmol, 0.1 equiv) and Cul (143 mg, 0.752 mmol, 0.2 equiv) were added. After degassing the reaction mixture for 5 min at room temperature, a degassed solution of the previous carbamate 8 (1 g, 3.76 mmol, 1 equiv) in THF (10 mL) was added dropwise and the reaction mixture was stirred at the room temperature for 16 h. After completion, the reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography (CffcCk/MeOH 9: 1) to afford the desired picolinate 10 as white foam (1.71 g, 90%).

Rf (pure EtOAc) 0.36; IR (neat) v_max 1722, 1588, 1387, 1209, 1097, 825, 734, 695 cm^{" l}; Ή NMR (400 MHz, CDCb) δ (ppm) 8.57 (d, J = 5.3 Hz, 2H, Ar), 7.50-7.23 (m, 15H, Ar), 5.32 (s, 2H, -CH₂Ph), 5.23 (s, 2H, -COCH₂Ph), 4.74 (s, 2H, H₉), 3.99 (s, 3H, His); ¹³C NMR (100 MHz, CDCb) δ (ppm) 164.45 (C18), 153.43 (-COCftPh), 153.25, 150.09, 148.17, 140.23, 135.35, 135.11, 133.60, 130.25, 128.56, 128.41, 128.17, 128.11, 127.92, 126.70, 121.39 (Ar), 83.41 (C7), 82.86 (C8), 70.61 (-CH2PI1), 68.22 (- COCftPh), 52.55 (CI 8), 39.24 (C9); HRMS (ESI⁺) m/z calcd for C₃oH₂6N₃0₅ ⁺ 508.1844 found 508.1867.

Synthesis of methyl 3-hydroxy-6-(3-(pyridin-4-ylamino)propyl)picolinate (11):

To a degassed solution of picolinate 10 (1.7 g, 0.820 mmol, 1 equiv) in dry MeOH (150 mL), Pearlman's catalyst Pd(OH)₂/C (20% with 50% moisture, 188 mg, 1.342 mmol, 0.4 equiv) was added. After evaporating and flushing with H₂ three times, the reaction mixture was stirred at room temperature under H2 (1 atm.) for 16 h. Upon completion, the catalyst was removed by filtration through a short column of celite, the solvent was evaporated, and the residue was directly subjected for the following step without purification.

Rf (pure MeOH) 0.20; IR (neat) v_max 2922, 2853, 1725, 1651, 1543, 1464, 1194, 1095, 792, 721, 673, 541, 507 cm^"1; ¾ NMR (400 MHz, CDCb) δ (ppm) 8.90 (br t, J = 5.0 Hz, 1H, H10), 7.90 (d, J= 6.3 Hz, 2H, H13, His), 7.40 (d, J = 8.6 Hz, 1H, H₅), 7.30 (d, J = 8.6 Hz, 1H, H₄), 6.88, (br d, J = 5.0 Hz, 2H, H12, Hie), 4.03 (s, 3H, His), 3.33 (q, J = 6.5 Hz, 2H, H₉), 2.95 (t, J = 7.1 Hz, 2H, Hv), 2.15 (p, J = 6.5 Hz, 7.0 Hz, 2H, Hs); ¹³C NMR (100 MHz, CDCb) δ (ppm) 169.61 (C17), 158.00, 157.32, 152.31, 139.09, 129.80, 127.11 (Ar), 53.17 (C18), 42.71 (C9), 34.30 (C7), 27.40 (C8); HRMS (ESI⁺) m/z calcd for Ci₅Hi₈N303⁺ 288.1330 found 288.1343.

Synthesis of (Z)-3-hydroxy-6-(3-(pyridin-4-ylamino)propyl)picolinaldehyde oxime (M):

To a solution of deprotected picolinate (240 mg, 0.836 mmol, 1 equiv) in dry CH2CI2 (5 mL), 2,6-lutidine (290 μί, 2.508 mmol, 3 equiv), and tert- butyldimethylsilyltrifluoromethanesul-fonate (293 μΐ,, 1.672 mmol, 2 equiv) were successively added and the reaction mixture was stirred at the room temperature under argon atmosphere during 5 h. After completion, the reaction mixture was directly concentrated under reduced pressure to give silylated compound. LRMS (ESI⁺) m/z 466.1 [M+H]⁺. After drying in vacuo, the residue was directly subjected to the following step.

To the solution of silylated compound (160 mg 0.399 mmol, 1 equiv) in dry CH2CI2 (5 mL) at -78 °C, DIBAL-H (1M solution in CH2CI2, 2.99 mL, 2.394 mmol, 6 equiv) was added dropwise. The reaction mixture was stirred at -78 °C for 1 h, then the reaction was quenched with MeOH (3 mL), and the cooling bath was removed. When the mixture was warmed to room temperature the sovents in reaction mixture was evoporated under reduced pressure to give aldehyde compound as crude along with Aluminium salts, the salts were filtered by washing with CH2CI2 (50 mL). The filtrate was evaporated, and the residue was directly subjected for the next step without purification.

A solution of picolinaldehyde (0.399 mmol, 1 equiv), hydroxylamine hydrochloride (56 mg, 0.798 mmol, 2 equiv), and CftCChNa (65 mg, 0.798 mmol, 2 equiv) in dry ethanol (10 mL) was stirred at reflux during 16 h. After concentration under reduced pressure, the crude product was washed with CH2CI2 (5 * 10 mL) to remove all the impurities carrying from the last two reactions. The existing compound in the round bottom flask was picolinaldehyde oxime, which was dried in high vacuo (95 mg, 84%) and confirmed by 1H NMR.

Rf (pure MeOH) 0.20; IR (neat) v_max 3282, 2944, 1650, 1544, 1257, 1228, 1170, 1023, 629, 515 cm^"1; ^XH NMR (400 MHz, CDCb) δ (ppm) 8.21 (s, 1H, H17), 7.97, 7.79 (2 br d, J= 6.6 Hz, 2H, His, His), 7.35, 7.27 (2 d, J= 8.6 Hz, 2H, H₄, H₅), 6.68, 6.57 (2 br d, J = 6.1 Hz, 2H, H12, Hie), 3.36 (t, J = 6.5 Hz, 2H, H₉), 2.86 (t, J = 6.9 Hz, 2H, Hv), 2.08 (p, J = 6.5 Hz, 6.9 Hz, 2H, Hs); ¹³C NMR (100 MHz, CDCb) δ (ppm) 159.94 (- COCH3), 152.74 (C17), 153.93, 153.41, 140.47, 136.63, 126.18, 125.63, 123.41, 120.25 (Ar), 43.35 (C9), 34.93 (C7), 29.48 (C8), 22.94 (-COCH3); HRMS (ESI⁺) m/z calcd for Ci₄HivN₄0₂ ⁺ 273.1345 found 273.1346.

Example 3 : Synthesis of Compound K according to the invention

Scheme 2: Synthesis of hybrid reactivator K

Synthesis of 8-methoxyquinolin-4-ol (14):

To a solution of o-anisidine (2.29 mL, 20.3 mmol, 1 equiv) in ethanol (20 mL) were added Meldrum's acid (3.57 g, 24.8 mmol, 1.2 equiv) and triethyl orthoformate (8.0 mL, 48.0 mmol, 2.4 equiv). The mixture was heated to reflux for 2.5 h. The reaction was cooled to 0 °C and the solid was filtered and washed with cold ethanol. The dried solid was added portiowise over 5 mins to boiling diphenyl ether (100 mL). Reflux was maintained for an additional 3 mins. The reaction was stirred at room temperature for 30 mins before petroleum ether (25 mL) was added and the solid was filtered and dried. The crude product was purified by column chromatography on silica gel (EtOAc/MeOH 95 :5), to afford 8-methoxyquinolin-4-ol (2.70 g, 15.4 mmol, 76%).

The analyses were consistent with the data reported in the literature.

Ref: WO2010/123995 (2010)

TLC R_f 0.06 (EtOAc/MeOH 95 :5)

^!H MR (400MHZ, DMSO-de) δ (ppm) 1 1.35 (s, 1H, OH), 7.58-7.70 (m, 1H,

NCH), 7.38-7.43 (m, 1H, ArH), 7.23-7.27 (m, 2H, OHCCCH, OMeCCH), 6.95-7.08 (m, 1H, OHCCH), 3.99 (s, 3H, OMe) ppm

¹³CNMR (101 MHz, DMSO-de) δ (ppm) 177.1 1 (7), 149.0 (4), 139.3 (10),

130.5 (2), 123.2 (5), 1 19.1 (3), 1 16.7 (1), 1 1 1.4 (6), 109.6 (8), 56.6 (13) ppm

IR (neat) _v max 2921 (br), 2851 (m), 1272 (s), 1041 (s) cm^"1

LCMS (ESI⁺) m/z 176.1 [M+H]⁺

(ESI⁺) m/z 198.1 [M+Na]⁺

11 RMS (ESI⁺) m/z calcd. 176.0706 m/z meas. 176.0707 [M+H]⁺

Mpt 168 °C Synthesis of 4-bromo-8-methoxyquinoline (15):

To a solution of 8-methoxyquinolin-4-ol (2.67 g, 15.2 mmol, 1 equiv) in N,N- dimethylformamide (20 mL) at 60 °C was added phosphorus tribromide (1.65 mL, 17.5 mmol, 1.2 equiv). The mixture was cooled to 45 °C for 45 mins. After cooling to room temperature, the reaction was diluted with water (25 mL) and the pH was adjusted to ~ 10 with saturated aqueous NaHCCb. The resulting solid was washed in water, filtered and taken up in EtOAc and concentrated in vacuo, to afford 8-methoxy-4- bromoquinoline (2.44 g, 10.3 mmol, 67%).

The analyses were consistent with the data reported in the literature.

Ref: WO2010/152603 (2008)

TLC R_f 0.51 (EtOAc/ MeOH 75:25)

!H MR (400MHz, CDCb) δ (ppm) 8.69 (d, J=4.6 Hz, 1H, NCH), 7.78 (d,

J=8.1 Hz, 1H, BrCCCH), 7.75 (d, J=4.8 Hz, 1H, BrCCH), 7.58 (t, J=8.2 Hz, 1H, ArH), 7.14 (d, J=8.0 Hz, 1H, ArH), 4.12 (s, 3H, OMe) ppm

¹³CNMR (101 MHz, CDCb) δ (ppm) 155.5 (4), 148.5 (10), 134.1 (2), 129.0

(7), 128.0 (1), 125.8 (5,8), 118.5 (3), 108.5 (6), 56.3 (13) ppm

IR (neat) v max 1252 (s), 1085 (s) cm^"1

LCMS (ESI⁺) m/z 238.0 [M⁷⁹Br+H]⁺, 340.0 [M⁸¹Br+H]⁺

(ESI⁺) m/z 260.0 [M⁷⁹Br+Na]⁺, 262.0 [M⁸¹Br+Na]⁺

11 RMS (ESI⁺) m/z calcd. 237.9862 meas. 237.9865 [M+H]⁺

Mpt 99 - 101 °C

Synthesis of N-(but-3-yn-l-yl)-8-methoxyquinolin-4-amine (16):

3-Butyn-l-amine (0.86 mL, 10.5 mmol, 5 eq.) was mixed with 4-bromo-8- methoxyquinoline (500 mg, 2.1 mmol, 1 eq.) to form a paste. The paste was heated to 80 °C for 1 hour without stirring. The temperature was then raised to 140 °C and the reaction was stirred overnight. The reaction was cooled to room temperature and NaOH (1 M, 2 mL) was added. The organic product was basified with 10%> aqueous NaOH and was extracted (CH2CI2). The organic layers were combined, washed, dried (MgS0₄), filtered and concentrated in vacuo. The crude product was purified by column chromatography (AI2O3, 10% MeOH in EtOAc), affording N-(but-3-yn-l-yl)-8- methoxyquinolin-4-amine (250 mg, 2.17 mmol, 53%).

Ref: Austin et al, J ACS, 1489-1497 (1958)

TLC R_f 0.73 (MeOH/ EtOAc 1 :9, AI2O3)

!H MR (400MHz, DMSO-de) δ (ppm) 8.36 (d, J=5.4 Hz, 1H, NCH), 7.73

(d, J=7.8 Hz, 1H, MeOCCHCHCH), 7.35 (t, J=8.1 Hz, 1H, MeOCCHCH), 7.10 (d, J=7.3 Hz, 1H, OMeCCH), 6.55 (d, J=5.4 Hz, 1H, NCHCH), 3.91 (s, 3H, OMe), 3.39-3.53 (m, 2H, CH2CH2CCH), 2.89 (t, J=2.6 Hz, 1H, CH2CH2CCH), 2.56 (td, J=7.1, 2.7 Hz, 2H, CH2CH2CCH) ppm

CNMR (101 MHz, DMSO-de) δ (ppm) 155.2 (3), 149.9 (9), 148.8 (8), 139.6

(1), 124.3 (4), 119.6 (6), 113.3 (2), 108.5 (5), 99.1 (10), 82.5 (16), 72.6 (17), 55.8 (12), 41.5 (14), 18.0 (15) ppm

IR (neat) v max 3279 (m), 2938 (m), 753 (s) cm^"1

LCMS (ESI⁺) m/z 227.1 [M+H]⁺

11 RMS (ESI⁺) m/z calcd 227.1179 meas. 227.1183 [M+H]⁺

Mpt 154 - 155 °C

Synthesis of methyl 3-(benzyloxy)-6-(4-((8-methoxyquinolin-4-yl)amino)but-l-yn- 1-yl) picolinate (17):

To a degassed solution of methyl 3-(benzyloxy)-6-bromopyridine-2-carboxylate (391 mg, 1.22 mmol, 1.1 eq.) in THF/Et₃N (7 mL/3 mL), tetrakis(triphenylphosphine) palladium (127 mg, 0.11 mmol, 10 mol%>) and copper iodide (48 mg, 0.22 mmol, 20 mol%) were added. After bubbling the reaction mixture with argon for 5 minutes at room temperature, a degassed solution of N-(but-3-yn-l-yl)-8-methoxyquinolin-4- amine (250 mg, 1.10 mmol, 1 eq.) in THF (3 mL) was added dropwise and the reaction mixture was stirred for 16 hours at room temperature. Upon completion, the reaction mixture was concentrated in vacuo and purified by column chromatography (AI2O3, 10% MeOH in EtOAc), affording methyl 3-(benzyloxy)-6-(4-((8-methoxyquinolin-4- yl)amino)but-l-yn-l-yl) picolinate (405 mg, 0.86 mmol, 78%).

Ref: Mien de Sousa Thesis, 2.49, 196 (2015)

TLC R_f 0.80 (MeOH/ EtOAc 1 :9, AI2O3)

!H MR (400MHz, DMSO-de) δ (ppm) 8.36 (br. d, J=5.1 Hz, 1H, NCH),

7.67-7.81 (m, 2H, NCCH), 7.57 (d, J=8.8, 1H, NCCHCH), 7.28- 7.47 (m, 7H, ArH), 7.08 (br. d, J=7.6 Hz, 1H, MeOCCH), 6.60 (br. d, J=5.1 Hz, 2H, NCHCH) 5.26 (s, 2H, PhCH₂), 3.90 (s, 3H, C0₂CH₃), 3.84 (s, 3H, OMe), 3.60-3.50 (m, 2H, NHCH₂CH₂), 2.82 (br. t, J=7.1 Hz, 2H, NHCH2CH2) ppm

¹³CNMR (101 MHz, DMSO-de) δ (ppm) 165.2 (19), 152.6 (1), 141.4 (18),

136.6 (6), 134.8 (8), 132.3 (17), 132.0 (22), 131.9 (14), 130.1 (9), 129.1 (4), 129.0 (24), 128.9 (5), 128.4 (15), 127.7 (16), 126.0 (23), 123.4 (3), 111.6 (2), 101.2 (7), 88.0 (12), 81.4 (13), 71.2 (21), 57.0 (25), 52.5 (20), 42.3 (10), 19.3 (11) ppm

IR (neat) v max 2945 (m), 1722 (s), 695 (s) cm^"1

LCMS (ESI⁺) m/z 468.2 [M+H]⁺

11 RMS (ESI⁺) m/z calcd 468.1931 meas. 468.1930 [M+H]⁺

Mpt 181 - 183 °C

Synthesis of methyl 6-(4-((8-methoxyquinolin-4-yl)amino)butyl)-3- hydroxypicolinate (18):

To a degassed solution of methyl 3-(benzyloxy)-6-(4-((8-methoxyquinolin-4- yl)amino)but-l-yn-l-yl) picolinate (200 mg, 0.43 mmol, 1 eq.) in dry MeOH (30 mL), was added Pearlman's catalyst (12 mg, 0.09 mmol, 20 mol%). After evacuating and flushing with hydrogen five times, the reaction mixture was stirred at room temperature, under a hydrogen atmosphere for 7 hours. Upon completion, the catalyst was removed by filtration through Celite and the solvent was removed in vacuo, affording methyl 6- (4-((8-methoxyquinolin-4-yl)amino)butyl)-3-hydroxypicolinate (163 mg, 0.42 mmol, 100%). Ref: Julien de Sousa Thesis, 2.49, 196 (2015)

TLC R_f 0.00 (MeOH/ EtOAc 1 :9, AI2O3)

^!H MR (400MHz, CDCb) δ (ppm) 8.50 (br. d, J=4.6 Hz, 1H, NCH), 7.23-

7.41 (m, 4H, ArH), 7.00 (d, J=7.8, 1H, MeOCCH), 6.42 (br. d, J=5.9 Hz, 1H, NCHCH), 3.98 (s, 3H, C0₂CH₃), 3.97 (s, 3H, OMe),

3.42 (br. s, 2H, NHCH2CH2CH2), 2.88 (t, J=7.2 Hz, 2H, NHCH2CH2CH2), 1.79-1.92 (m, 4H, NHCH2CH2CH2) ppm

¹³CNMR (101 MHz, CDCb) δ (ppm) 169.9 (19), 157.2 (14), 153.5 (18), 153.2

(1), 151.6 (6), 146.7 (8), 136.5 (9), 129.3 (4), 128.7 (17), 126.9 (15), 125.1 (16), 1 19.0 (3), 1 12.6 (5), 108.6 (2), 98.6 (7), 55.9 (25), 53.0 (20), 43.3 (10), 36.7 (13), 27.6 (12), 27.5 (11) ppm

IR (neat) v max 2927 (m), 2359 (m), 1675 (s), 725 (s) cm^"1

LCMS (ESI⁺) m/z 382.4 [M+H]⁺

11 RMS (ESI⁺) m/z calcd 382.1775 meas. 382.1771 [M+H]⁺

Mpt 156 - 159 °C

Synthesis of (E)-3-hydroxy-6-(4-((8-methoxyquinolin-4-yl)amino)butyl) picolinaldehyde oxime (K):

To a degassed solution of methyl 6-(4-((8-methoxyquinolin-4-yl)amino)butyl)-3- hydroxypicolinate (50 mg, 0.13 mmol, 1 eq.) in dry CH2CI2 (5 mL), was added 2,6- lutidine (42 mg, 0.05 mL, 0.39 mmol, 3 eq.) and TBSOTf (69 mg, 0.06 mL, 0.26 mmol, 2 eq.) successively and the reaction was stirred at room temperature for 5 hours under argon. The reaction mixture was concentrated in vacuo. To a solution of the residue in dry CH2CI2 (5 mL) at -78 °C was added DIBAL-H (1 M in CH2CI2) (56 mg, 0.40 mL, 0.40 mmol, 3 eq.) dropwise. The reaction was stirred at -78 °C for 1 hour and the reaction was quenched at -78 °C with MeOH (3 mL). The mixture was warmed to room temperature and the solvent was removed in vacuo. The aluminium salts were removed by filtration in CH2CI2. The filtrate was concentrated in vacuo and the residue was dissolved in dry ethanol (7 mL). Hydroxylamine hydrochloride (18 mg, 0.26 mmol, 2 eq.) and sodium acetate (22 mg, 0.26 mmol, 2 eq.) were added and the solution was stirred at reflux for 16 hours. After concentration in vacuo, the crude product was washed with CH2CI2. The crude product was purified by column chromatography on silica gel (10% MeOH in CH2CI2) to afford (E)-3-hydroxy-6-(4-((8-methoxyquinolin-4- yl)amino)butyl) picolinaldehyde oxime (40 mg, 0.11 mmol, 83%).

Ref: Mien de Sousa Thesis, 2.49, 196 (2015)

TLC R_f 0.19 (MeOH/ CH2CI2 1 :9, S1O2)

!H MR (400MHz, MeOD-d₄) δ (ppm) 8.16 (d, J=7.1 Hz, IH, NCH), 8.10 (s, 2H,

CH2NOH), 7.78 (d, J=8.1 Hz, IH, CHCOMe), 7.54 (t, J=8.3 Hz, IH, CHCHCOMe), 7.38 (d, J=7.6 Hz, IH, CHCHCHCOMe), 7.07-7.21 (m, 2H, NCCHCH), 6.76 (d, J=7.3 Hz, IH, NCHCH), 4.07 (s, 3H, OMe), 3.54 (t, J=7.1 Hz, 2H, NHCH2CH2), 2.74 (t, J=7.0 Hz, 2H, NHCH2CH2CH2CH2), 1.73-1.88 (m, 4H, NHCH2CH2CH2CH2) ppm

CNMR (101 MHz, MeOD-d₄) δ (ppm) 157.4 (1), 154.2 (19), 153.9 (6), 152.8

(14), 151.1 (18), 142.1 (8), 136.4 (17), 128.5 (9), 126.1 (15), 125.7 (16), 123.5 (3), 120.3 (2), 114.6 (5), 113.3 (4), 99.7 (7), 57.4 (20), 44.5 (10), 37.3 (13), 28.3 (12), 28.5 (11) ppm

IR (neat) v max 3451 (br), 1621 (s), 1043 (s) cm^"1

LCMS (ESI⁺) m/z 355.3 [M+H]⁺

11 RMS (ESl⁺) m/z calcd 367.1765 meas. 367.1765 [M+H]⁺

Mpt 157 - 159 °C

Example 4: Synthesis of Compound H according to the invention

NH,

20 21 22

Scheme 3 : Synthesis of Compound H

Synthesis of 6-fluoroquinolin-4-ol (21):

To a solution of p-fluoroaniline (3.03 mL, 31.5 mmol, 1 equiv) in ethanol (30 mL) were added Meldrum's acid (5.54 g, 38.4 mmol, 1.2 equiv) and triethyl orthoformate (12.4 mL, 74.5 mmol, 2.4 equiv). The mixture was heated to reflux for 2.5 h. The reaction was cooled to 0 °C and the solid was filtered and washed with cold ethanol. The dried solid was added portiowise over 5 mins to boiling diphenyl ether (100 mL). Reflux was maintained for an additional 3 mins. The reaction was stirred at room temperature for 30 mins before petroleum ether (25 mL) was added and the solid was filtered and dried. The crude product was purified by column chromatography on silica gel (EtOAc/MeOH 95:5), to afford 6-fluoroquinolin-4-ol (2.36 g, 14.5 mmol, 46%).

The analyses were consistent with the data reported in the literature.

Ref: WO2010/123995 (2010)

TLC R_f 0.41 (EtOAc/MeOH 95:5)

¹HNMR (400MHz, DMSO-de) δ (ppm) 11.90 (s, 1H, OH), 7.94 (d, J=7.6, 1H,

NCH), 7.73 (m, 1H, NCCH), 7.53-7.67 (m, 2H, FCCH, FCCH), 6.04 (d, J=7.3, 1H, OHCCH) ppm

CNMR (101 MHz, DMSO-de) δ (ppm) 159.9 (7), 157.7 (5), 140.0 (2), 137.2

(10), 127.3 (4), 121.1 (6), 120.8 (1), 109.5 (3), 108.3 (8) ppm IR (neat) v max 2768 (br), 1594 (m) cm^"1

LCMS (ESI⁺) m/z 164.1 [M+H]⁺, 186.1 [M+Na]⁺

11 RMS (ESI⁺) m/z calcd. 164.0506 m/z meas. 164.0509 [M+H]

Mpt 227 - 228 °C

Synthesis of AMI0016 - 4-bromo-6-fluoroquinoline (22):

To a solution of AMI0015 (2.30 g, 14.1 mmol, 1 equiv) in N,N-dimethylformamide (20 mL) at 60 °C was added phosphorus tribromide (1.52 mL, 16.2 mmol, 1.2 equiv). The mixture was cooled to 45 °C for 45 mins. After cooling to room temperature, the reaction was diluted with water (25 mL) and the pH was adjusted to ~ 10 with saturated aqueous NaHCCte. The resulting solid was washed in water, filtered and taken up in EtOAc and concentrated in vacuo, to afford 4-bromo-6-fluoroquinoline (3.12 g, 13.8 mmol, 98%).

The analyses were consistent with the data reported in the literature.

Ref: WO2010/152603 (2008)

TLC R_f 0.64 (EtOAc/MeOH 8:2)

^!H MR (400MHZ, DMSO-de) δ (ppm) 8.73 (d, J=4.6 Hz, 1H, NCH), 8.18 (m, 1H,

NCCH), 8.00 (d, J=4.6 Hz, 1H, BrCCH), 7.77-7.88 (m, 2H, FCCH, FCCH) ppm

¹³CNMR (101 MHz, DMSO-de) δ (ppm) 162.5 (5), 150.5 (10), 146.0 (2), 133.5 (6),

128.6 (7), 128.5 (4), 126.6 (1), 121.2 (8), 1 10.5 (3) ppm

IR (neat) _v max 1585 (m) cm^"1

LCMS (ESI⁺) m/z 227.0 [M⁷⁹Br+H]⁺, 229.0 [M⁸¹Br+H]⁺

11 RMS (ESI⁺) m/z calcd. 225.9662 m/z meas. 225.9962 [M+H]⁺ Synthesis of N-(but-3-yn-l-yl)-6-fluoroquinolin-4-amine (23):

3-Butyn-l -amine (764 mg, 0.91 mL, 1 1.1 mmol, 5 eq.) was mixed with 4-bromo-6- fluoroquinoline (500 mg, 2.21 mmol, 1 eq.) to form a paste, which was heated to 80 °C for 1 hour without stirring, the temperature was raised to 140 °C and the reaction was left overnight with stirring. After cooling to room temperature, NaOH (1M, 2 mL) was added. The organic product was basified (10% NaOH) and extracted with CH2CI2. The organic extracts were combined, dried (MgS04), filtered and concentrated in vacuo. The crude product was purified by column chromatography (AI2O3, 10%> MeOH in EtOAc), affording N-(but-3-yn-l-yl)-6-fluoroquinolin-4-amine (465 mg, 2.17 mmol, 98%>).

Ref: Austin et al, J ACS, 1489-1497 (1958)

TLC R_f 0.75 (MeOH/ EtOAc 1 :9, AI2O3)

^!H MR (400MHZ, DMSO-de) δ (ppm) 8.41 (d, J=5.1 Hz, 1H, NCH), 7.89-8.08

(m, 1H, FCCHCH), 7.85 (dd, J=9.3, 5.9 Hz, 1H, FCCHCH), 7.53 (td, J=8.7, 2.8 Hz, 1H, FCCHC), 6.54 (d, J=5.4 Hz, 1H, NCHCH), 3.42-3.51 (m, 2H, CH2CH2CCH), 2.90 (t, J=2.6 Hz, 1H, CH2CH2CCH), 2.56 (td, J=7.1 , 2.7 Hz, 2H, CH2CH2CCH) ppm

¹³CNMR (101 MHz, DMSO-de) δ (ppm) 150.5 (4), 150.2 (7), 149.1 (9), 140.0 (1),

124.6 (2), 1 19.9 (3), 1 13.6 (6), 108.8 (5), 99.4 (8), 72.9 (16), 56.0 (13), 41.8 (11), 18.3 (12) ppm

IR (neat) v max 3387 (m), 2830 (m), 663 (s) cm^"1

LCMS (ESI⁺) m/z 215.1 [M+H]⁺

11 RMS (ESI⁺) m/z calcd. 215.0979 m/z meas. 215.0982 [M+H]⁺

Mpt 148 - 150 °C

Synthesis of methyl 3-(benzyloxy)-6-(4-((6-fluoroquinolin-4-yl)amino)but-l-yn-l- yl) picolinate (24):

To a degassed solution of methyl 3-(benzyloxy)-6-bromopyridine-2-carboxylate (496 mg, 1.54 mmol, 1.1 eq.) in THF/Et₃N (7 mL/3 mL), tetrakis(triphenylphosphine) palladium (161 mg, 1.54 mmol, 10 mol%>) and copper iodide (53 mg, 0.28 mmol, 20 mol%) were added. After bubbling the reaction mixture with argon for 5 minutes at room temperature, a degassed solution of N-(but-3-yn-l-yl)-6-fluoroquinolin-4-amine (330 mg, 1.4 mmol, 1 eq.) in THF (3 mL) was added dropwise and the reaction mixture was stirred for 16 hours at room temperature. Upon completion, the reaction mixture was concentrated in vacuo and purified by column chromatography (S1O2, 10%> MeOH in EtOAc), affording methyl 3-(benzyloxy)-6-(4-((6-fluoroquinolin-4-yl)amino)but-l- yn-l-yl) (5 15 mg, 1 .13 mmol, 81 %).

Ref: Mien de Sousa Thesis, 2.49, 196 (2015)

TLC R_f 0.20 (MeOH/EtOAc 1 :9, S1O2)

^!H MR (400MHZ, DMSO-de) δ (ppm) 8.00-8.14 (m, 1H, NCH), 7.69 (d,

J=8.8 Hz, 1H, NCCHCHCF), 7.57 (d, J=8.8, 1H, NCCH), 7.29-7.52 (m, 8H, ArH), 6.61 (br. s, 1H, NCHCH), 5.26 (s, 2H, PhCH₂), 3.84 (s, 3H, C0₂CH₃), 3.53-3.61 (m, 2H, NHCH₂CH₂), 2.83 (t, J=7.0 Hz, 2H, NHCH₂CH₂) ppm

¹³CNMR (101 MHz, DMSO-de) δ (ppm) 165.2 (19), 160.3 (18), 157.9 (3), 152.5

(6) , 150.7 (9), 149.7 (8), 140.6 (17), 136.5 (14), 134.3 (22), 130.4 (15), 129.0 (5), 127.7 (24), 123.0 (16), 1 19.0 (2), 1 18.7 (23), 106.4 (4), 106. 1

(7) , 88.0 (12), 80.8 (13), 70.4 (21), 52.8 (20), 41 .7 (10), 19.0 (11) ppm IR (neat) v max 3255 (m), 295 1 (m), 1725 (s), 721 (s) cm^"1

LCMS (ESI⁺) m/z 456.2 [M+H]⁺

11 RMS (ESI⁺) m/z calcd 456.1718 m/z meas. 456. 1720 [M+H]⁺

Mpt 156 - 157 °C

Synthesis of methyl 6-(4-((6-fluoroquinolin-4-yl)amino)butyl)-3-hydroxypicolinate (25):

To a degassed solution of methyl 3-(benzyloxy)-6-(4-((6-fluoroquinolin-4- yl)amino)but-l-yn-l-yl) picolinate (200 mg, 0.44 mmol, 1 eq.) in dry MeOH (40 mL), was added Pearlman's catalyst (12.3 mg, 0.087 mmol, 20 mol%). After evacuating and flushing with hydrogen five times, the reaction mixture was stirred at room temperature, under a hydrogen atmosphere for 7 hours. Upon completion, the catalyst was removed by filtration through Celite and the solvent was removed in vacuo, affording methyl 6- (4-((6-f uoroquinolin-4-yl)amino)butyl)-3-hydroxypicolinate (160 mg, 0.433 mmol, 99%).

Ref: Mien de Sousa Thesis, 2.49, 196 (2015) TLC R_f 0.00 (EtOAc, AI2O3)

!H MR (400MHz, DMSO-de) δ (ppm) 8.35 (d, J=5.1 Hz, 1H, NCH), 8.05

(dd, J=l l . l, 2.8 Hz, 1H, NCCH), 7.81 (dd, J=9.3, 5.9 Hz, 2H, FCCHCH), 7.49 (td, J=8.7, 2.7 Hz, 1H, FCCH), 7.28-7.40 (m, 2H, NCCHCHCOH), 6.44 (d, J=5.4 Hz, 1H, NCHCH), 3.85 (s, 3H, C02CH3), 3.27-3.30 (m, 2H, HNCH2CH2CH2CH2), 2.67-2.78 (m, 2H, NHCH2CH2CH2CH2), 1.62-1.83 (m, 4H,

CNMR (101 MHz, DMSO-de) δ (ppm) 169.2 (19), 160.6 (3), 158.2 (14),

157.2 (18), 153.2 (6), 150.9 (8), 144.5 (9), 130.0 (17), 129.0 (15), 127.1 (1), 124.7 (16), 118.6 (2), 105.2 (5), 105.0 (4), 98.0 (7), 51.8 (20), 42.3 (10), 35.9 (13), 29.3 (11), 27.3 (12) ppm

IR (neat) r max 3240 (m), 2951 (m), 2860 (m), 2361 (s), 2342 (s), 1674 (s),

719 (s) cm^"1

LCMS (ESI⁺) m/z 370.2 [M+H]⁺

11 RMS (ESI⁺) m/z calcd. 370.1561 m/z meas. 370.1568 [M+H]⁺

Mpt 155 - 157 °C

Synthesis of (E)-6-(4-((6-fluoroquinolin-4-yl)amino)butyl)-3- hydroxypicolinaldehyde oxime (H):

To a degassed solution of methyl 6-(4-((6-fluoroquinolin-4-yl)amino)butyl)-3- hydroxypicolinate (100 mg, 0.27 mmol, 1 eq.) in dry CH2CI2 (5 mL), was added 2,6- lutidine (87 mg, 0.09 mL, 0.81 mmol, 3 eq.) and TBSOTf (143 mg, 0.12 mL, 0.54 mmol, 2 eq.) successively and the reaction was stirred at room temperature for 5 hours under argon. The reaction mixture was concentrated in vacuo. To a solution of the residue in dry CH2CI2 (5 mL) at -78 °C was added DIBAL-H (1 M in CH2CI2) (115 mg, 0.81 mL, 0.81 mmol, 3 eq.) dropwise. The reaction was stirred at -78 °C for 1 hour and the reaction was quenched at -78 °C with MeOH (3 mL). The mixture was warmed to room temperature and the solvent was removed in vacuo. The aluminium salts were removed by filtration in CH2CI2. The filtrate was concentrated in vacuo and the residue was dissolved in dry ethanol (7 mL). Hydroxylamine hydrochloride (37.6 mg, 0.54 mmol, 2 eq.) and sodium acetate (44.4 mg, 0.54 mmol, 2 eq.) were added and the solution was stirred at reflux for 16 hours. After concentration in vacuo, the crude product was washed with CH2CI2. The crude product was purified by column chromatography (S1O2, 10% MeOH in CH2CI2) to afford (E)-6-(4-((6-fiuoroquinolin-4- yl)amino)butyl)-3-hydroxypicolinaldehyde oxime (80 mg, 0.27 mmol, 90%).

Ref: Mien de Sousa Thesis, 2.49, 196 (2015)

TLC R_f 0.19 (MeOH/CftCk 1 :9, S1O2)

^!H MR (400MHZ, MeOD-d₄) δ (ppm) 8.33 (d, J=6.6 Hz, 1H, NCH), 8.21 (s, 2H,

CH2NOH), 8.05 (dd, J=10.3, 2.7 Hz, 1H, NCCH), 7.88 (dd, J=9.2, 5.0 Hz, 1H, FCCHC), 7.67 (m, 1H, FCCHCH), 7.09-7.30 (m, 2H, NCCHCH), 6.71 (d, J=6.6 Hz, 1H, NCHCH), 3.47-3.56 (m, 2H, NHCH2CH2), 2.80 (t, J=7.1 Hz, 2H, NHCH2CH2CH2CH2), 1.75-1.96 (m, 4H, NHCH2CH2CH2CH2) ppm

¹³CNMR (101 MHz, MeOD-d₄) δ (ppm) 163.2 (19), 160.6 (3), 156.4 (15), 154.5

(8), 154.1 (10), 153.0 (20), 144.9 (9), 136.6 (17), 126.8 (1), 125.9 (18), 123.6 (16), 120.5 (2), 1 17.3 (5), 108.3 (4), 99.6 (7), 44.7 (11), 37.5 (14), 28.6 (13), 28.5 (12) ppm

IR (neat) _v max 3673 (br), 1602 (s), 1033 (s) cm^"1

LCMS (ESI⁺) m/z 355.3 [M+H]⁺

11 RMS (ESI⁺) m/z calcd. 355.1561 m/z meas. 355.1561 [M+H]⁺

Mpt 157 - 159 °C Example 5 : Reactivation of hAChE inhibited by OPNAs

Materials and methods

IC50 measurements. Recombinant hAChE was produced and purified as previously described [Carletti et al 2008 JAmChemSoc 130(47): 1601 1-20). Oximes were dissolved in MeOH to make 5 mM or 10 mM stock solution and further diluted in phosphate buffer (sodium phosphate 0.1 M, pH 7.4). Recombinant hAChE activity was measured spectrophotometrically (absorbance at 412 nm) in the presence of various concentrations of oximes in 1 mL Ellman's buffer (sodium phosphate 0.1 M, pH 7.4, 0.1% BSA, 0.5 mM DTNB, 25 °C). Measurements were performed at least in duplicate for each concentration tested. The concentration of oxime producing 50% of enzyme inhibition was determined by non-linear fitting using ProFit (Quantumsoft) using the standard ICso equation: %Activity=100*IC5o/(IC5o+[Ox]).

Inhibition of hAChE by OPNAs. Recombinant hAChE was produced and purified as previously described (see reference: http://www.ncbi.nlm.nih.gov/pubmed/18975951) VX and tabun were from DGA maitrise NRBC (Vert le Petit, France). Paraoxon-ethyl was purchased from Sigma-Aldrich. HI-6 was from Pharmacie Centrale des Armees (Orleans, France). All other chemicals including paraoxon were from Sigma. Stock solution of VX and tabun were 5 mM in isopropanol. The inhibition of 120 μΜ hAChE was carried out with a 5 -fold excess of OPNAs and was performed in tris buffer (20 mM, pH 7.4, 0.1% BSA) at 25 °C. After a 20-minute incubation, inhibited hAChE was desalted on PD-10 column (GE Healthcare).

Reactivation of hAC hE inhibited by OPNAs.

OPNA-inhibited hAChE was incubated at 37 °C with at least 4 or 5 concentrations of oxime in phosphate buffer (0.1 M, pH 7.4, 0.1% BSA). The final concentration of MeOH in the incubation mix was below 2% and had no influence on the enzyme stability. At time intervals ranging from 1 to 10 minutes depending on the reactivation rate, 10 aliquots of each solution containing the different concentrations of oxime were transferred to cuvettes containing 1 mM acetylthiocholine in 1 mL Ellman's buffer (phosphate 0.1 M, pH 7.4, 0.1% BSA, 0.5 mM DTNB, 25 °C) for measurement of hAChE activity.

The enzyme activity in the control remained constant during the experiment. The percentage of reactivated enzyme (%E_react) was calculated as the ratio of the recovered enzyme activity and activity in the control. The apparent reactivation rate kobs for each oxime concentration, the dissociation constant ΚΌ of inhibited enzyme -oxime complex (E-POx) and the maximal reactivation rate constant ki, were calculated by non-linear fit with ProFit (Quantumsoft) using the standard oxime concentration-dependent reactivation equation derived from the following κ D

E-P + Ox E-POx POx

k_r[Ox]

%E_react = 100■ (l-e^k°^bs- ' ) and k_obs

K_D

Results

The ability of the following oximes to reactivate in vitro hAChE poisoned with various OPNAs was studied by spectrophotometry using Ellman's reagent and compared to known reactivators such as pralidoxime (also called 2-PAM), obidoxime (also called LuH-6), HLo-7, trimedoxime (also called TMB4) and HI-6:

KM 297 JDS 207

E

Compound E is as described in Example 1.

Compound KM 297 is described in Eur. J. Med. Chem. 2014, 6, 455-467 as compound 12.

JDS 207 is described in WO 2015/075082 as compound 15.

The results are provided in Table 1 below. Table 1

In vitro reactivation of AChE inhibited by various OPNAs

Significantly, the quinoline-based reactivator according to this invention outperformed most reactivators for VX- and tabun-inhibited hAChE. These data demonstrate in particular that it outclassed HI-6, which is known to be the most effective reactivator for VX poisoning to date, as well as trimedoxime, which is known to be the only reactivator moderately effective against tabun poisoning. Compound E further exhibited much higher in vitro reactivation potency of WFA-inhibited AChE compared to tacrine -based oximes (KM297 and JDS 207) due to a significantly improved affinity toward the inhibited enzyme (KD). Moreover, this compound also effectively reactivated paraoxon- and sarin-inhibited AChE, which allows this compound to be applied against a broad spectrum of neurotoxic agents.

An additional experiment was performed to determine the first order reactivation rate constant, kobs, of compound M according to this invention, compared with HI-6, at a concentration of 100 μΜ. The results obtained are summarized in Table 2 below.

Table 2

In vitro reactivation of AChE inhibited by various OPNAs

As shown in this table, Compound M displays higher kobs than the commercial reactivator (HI-6) and this compound has thus the potential to be a good reactivator of OP-inhibited AchE.

Example 6: Inhibition of hAChE with compounds of the invention

As shown in Table 3, compound E was found to inhibit native hAChE.

Table 3

Compound ICso (μΜ)

Tacrine 0.205±18

(human erythrocyte

AChE)^a

E 1.4 ± 0.1 mM

(hAChE)

Claims

1. Compound of formula (I)

n is 0, 1, 2, 3 or 4

m is 0 or 1 ,

R¹ and R² are independently selected from the group consisting of a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, CF3, an aryl group and an heteroaryl group or R¹ and R² form together and with the carbon atoms linked thereto an aryl or heteroaryl group,

R³ and R⁴ are independently selected from the group consisting of a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, CF3 and an aryl group or R³ and R⁴ form together and with the carbon atoms linked thereto an aryl or heteroaryl group, R⁵ is selected from the group consisting of a hydrogen atom, an alkyl group and an acyl group,

R⁶ is a hydrogen atom or an alkyl group, when present, and

R⁷ is selected from the group consisting of a hydrogen atom, a NH2 group and a CF3 group.

2. Compound according to claim 1, wherein m is 0.

3. Compound according to claim 1, wherein m is 1 and R⁶ = H.

4. Compound according to any one of claims 1 to 3, wherein least one and preferably all the following conditions are met:

• n is 2, 3 or 4, preferably n is 3, R¹ and R² form together and with the carbon atoms linked thereto an aryl group, preferably a substituted or non-substituted phenyl group, thereby forming a quinoline structure,

at least one, preferably at least two and more preferably each of R³, R⁵ and R⁷ is a hydrogen atom,

R⁴ is a hydrogen atom or an alkyl group such as a methyl group, preferably a hydrogen atom,

• the pyridine ring in Formula (I) is substituted in position 6 by the quinoline- bearing group and/or in position 3 by the OH group.

5. Compound according to any one of claims 1 to 3, wherein at least one and preferably all the following conditions are met:

• n is 2, 3 or 4, preferably n is 2 or 3,

• R¹ and R² are independently selected from a hydrogen atom and an alkyl group, preferably each of R¹ and R² is a hydrogen atom,

• at least one, preferably at least two and more preferably each of R³, R⁵ and R⁷ is a hydrogen atom,

• R⁴ is a hydrogen atom or an alkyl group such as a methyl group, preferably a hydrogen atom,

• the hydroxy-pyridine ring in Formula (I) is substituted in position 6 by the pyridine-bearing group and/or in position 3 by the OH group.

6. Compound according to claim 1, which has the following formula (la):

in which n is 2 or 3 and R¹ and R² are each a hydrogen atom or R¹ and R² form together and with the carbon atoms linked thereto a phenyl group which may be substituted or non substituted.

7. Compound according to any one of claims 1 to 6, which is selected from the group consisting of:

• (E)-6-(4-((7-chloroquinolin-4-yl) amino)butyl)-3-hydroxypicolinaldehyde oxime · (E)-6-(4-((7-fluoroquinolin-4-yl) amino)butyl)-3-hydroxypicolinaldehyde oxime

• (E)-6-(4-((7-(trifluoromethyl)quinolin-4-yl)amino)butyl)-3-hydroxy

picolinaldehyde oxime

• (E)-6-(4-((6-methoxyquinolin-4-yl) amino)butyl)-3 -hydroxypicolinaldehyde oxime

· (E)-6-(4-(quinolin-4-yl)amino)butyl)-3 -hydroxy picolinaldehyde oxime

• (E)-6-(4-((2,6-dimethylpyridin-4-yl) amino)butyl)-3-hydroxypicolinaldehyde oxime

• (E)-6-(4-((6-chloro-8-methylquinolin-4-yl)amino)butyl)-3-hydroxy

picolinaldehyde oxime

· (E)-6-(4-((6-fluoroquinolin-4-yl)amino)butyl)-3-hydroxy picolinaldehyde oxime

• (E)-6-(4-((8-fluoroquinolin-4-yl)amino)butyl)-3-hydroxy picolinaldehyde oxime

• (E)-6-(4-((6-(trifluoromethyl)quinolin-4-yl)amino)butyl)-3-hydroxy

picolinaldehyde oxime

• (E)-6-(4-((8-methoxyquinolin-4-yl) amino)butyl)-3-hydroxypicolinaldehyde oxime

• (E)-6-(4-(pyridin-4-ylamino)butyl)-3 -hydroxypicolinaldehyde oxime

• (E)-6-(4-(pyridin-4-ylamino)propyl)-3 -hydroxypicolinaldehyde oxime.

8. Pharmaceutical composition comprising at least one compound of formula (I) or (la) as defined in any one of claims 1 to 7 and at least one pharmaceutically acceptable support.

9. Compound according to any one of claims 1 to 7 for use as a medicine.

10. Compound according to any one of claims 1 to 7 for use in the treatment of a nervous and/or respiratory failure due to intoxication with at least one organophosphorous nerve agent.

11. Compound according to any one of claims 1 to 7 for use according to claim 10, wherein said organophosphorous nerve agent is selected from warfare agents such as O- ethyl 5'-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX), tabun, sarin, cyclosarin and soman and pesticides such as paraoxon, parathion and tetraethyl pyrophosphate (TEPP).

12. Compound according to any one of claims 1 to 7, for use in the treatment of neurological diseases such as Alzheimer's disease.