Novel compounds capable of modulating the synapsin-3 / alpha-synuclein interaction and the therapeutic use thereof as disease-modifying agents for synucleinopathies

Abstract

Described herein are novel compounds capable of binding to the protein synapsin III and reducing the fibrillar aggregates of said protein with alpha-synuclein. The novel compounds of the invention are therefore suitable for use in the therapeutic treatment of neurodegenerative diseases such as, in particular, Parkinson's disease, Lewy body dementia, multiple system atrophy, and the Lewy body variant of Alzheimer's disease. A pharmaceutical composition comprising the compounds according to the invention in a pharmaceutically acceptable carrier, as well as in combination with optional adjuvants, stabilizers and / or preservatives, is also described.

Description

[0001] Novel compounds capable of modulating the synapsin-3 / alpha-synuclein interaction and the therapeutic use thereof as disease-modifying agents for synucleinopathies

[0002] The present invention refers to the field of heterocyclic organic compounds for use as disease-modifying therapeutic agents in neurodegenerative diseases, particularly Parkinson's disease, Lewy body dementia, multiple system atrophy, and the Lewy body variant of Alzheimer’s disease.

[0003] Synucleinopathies affect more than ten million people worldwide. These diseases are characterized by complex pathological processes, including the formation of aggregates of alpha-synuclein, a protein widely expressed in the central nervous system, particularly in presynaptic endings, into neurotoxic fibrils (Lewy bodies and cytoplasmic glial aggregates). This protein is involved in modulating the cycle of synaptic vesicles, affecting the synthesis, release and recycling of neurotransmitters, particularly dopamine.

[0004] Lewy body formation contributes to synapse dysfunction and progressive neuronal loss in Parkinson's disease and Lewy body dementia (Spillantini MG et al., Nature 1997 Aug 28;388(6645):839-40. doi: 10.1038 / 42166.; Spillantini MG and Goedert M., Cell Tissue Res. 2018 Jul;373(1):137-148. doi: 10.1007 / s00441-017-2706-9; Bellucci A. et al., Neuropathol Appl Neurobiol. 2016 Feb;42(1):77-94. doi: 10.1111 / nan.12297).

[0005] Recent studies have shown that the formation of these fibrillary aggregates is related to a pathological interaction between alpha-synuclein and synapsin III (also commonly referred to as synapsin 3), another protein that participates in the recycling of synaptic vesicles. These two proteins, under physiological conditions, cooperate in regulating the dynamics of synaptic vesicles and the release of neurotransmitters. In particular, under normal conditions, alpha-synuclein is a soluble protein, lacking a fixed three-dimensional structure, which gives it high conformational flexibility and allows it to bind to synaptic vesicle membranes, assuming an alpha-helical conformation that facilitates interaction with other synaptic proteins, including synapsin III. This interaction is dynamic and reversible and is essential for proper synaptic function (Zaltieri et al., J. Cell. Sci., 2015, 128(13):2231-43).Otherwise, under pathological conditions, interaction between alpha-synuclein and synapsin III may become dysfunctional. In fact, synapsin III was found to be essential to allow alpha-synuclein aggregation: in its presence, toxic protein aggregates form, which impair the normal cycle of synaptic vesicles and the release of neurotransmitters, contributing to synaptic dysfunction and neurodegeneration (Faustini G. et al., Acta Neuropathol. 2018 Oct;136(4):621-639. doi: 10.1007 / s00401-018-1892-1). Conversely, in the absence of synapsin III, the alpha-synuclein protein cannot form fibrillary aggregates even if overexpressed, and consequently neuronal degeneration is prevented (Faustini et al., 2018).

[0006] Furthermore, it has been shown that the reduction in synapsin III levels obtained through gene silencing can destabilize and reduce alpha-synuclein aggregates even when these are already abundantly deposited and can impair synaptic function (Faustini G. et al., Mol Ther.

[0007] 2022 Apr. 6;30(4):1465-1483. doi: 10.1016 / j.ymthe.2022.01.021). As a result of the reduction in alpha-synuclein aggregates induced by synapsin III gene silencing, neurodegeneration is blocked (Faustini et al., 2022).

[0008] Methylphenidate (threo-MPH), a monoamine reuptake inhibitor used to treat attention deficit and hyperactivity disorder (ADHD), was recently found to be able to bind to synapsin III, resulting capable of stimulating alpha-synuclein-mediated dopamine neurotransmission. In particular, it was found that the compound threo-MPH, regardless of its inhibitory action on the dopamine transporter, is able to interact with synapsin III and thus stimulate interaction between synapsin III and alpha-synuclein in its alpha-helical conformation, which serves to mediate the mobilization of synaptic vesicles (Faustini et al., Neurobiology of disease, 2020, 138, 104789; Casiraghi et al., Chem. Med. Chem. 2020, 15, 1330).

[0009] However, therapeutic application of threo-MPH is limited due to the weak affinity and selectivity for synapsin III observed for that compound. In fact, threo-MPH tends to interact more with monoamine transporters, such as dopamine and noradrenaline transporters, which are the classic targets for this molecule. This interaction may lead to the onset of off-target effects, therefore non-specific and undesirable effects, including possible disruptions of monoaminergic neurotransmission and potential cardiovascular side effects, limiting the applicability of threo-MPH as a specific therapeutic agent for the treatment ofsynucleinopathies (Devos et al., CNS Drugs (2013), 27:1-14; Liang et al., Int. J. Environ. Res. Public Health (2018), 15(8)).

[0010] Patent application WO2022029151 describes structural threo-analogues of threo-MPH, which are capable of binding and sequestering the synapsin III protein from alpha-synuclein aggregates and in parallel of stimulating interaction between synapsin III and soluble alpha-synuclein in the alpha-helical conformation, but with a potency significantly higher than threo-MPH. Through this mechanism, the structural threo-analogues of threo-MPH described in patent application WO2022029151 are also significantly more potent than threo-MPH in reducing alpha-synuclein aggregates. In addition, these compounds lack the ability to interact with monoamine transporters, thus significantly decreasing the possibility of causing undesirable off-target effects.

[0011] In this context, there is a need to improve the prior art compounds by increasing their potency while maintaining their high selectivity of action, thereby increasing their effectiveness and safety profile.

[0012] This and other needs are now met by a compound of Formula (I) as defined in the appended claim 1.

[0013] Further objects of the invention are a pharmaceutical composition comprising a compound of Formula (I) and the therapeutic use thereof, as defined in the attached independent claims.

[0014] Additional features and advantages of the invention are defined in the dependent claims, which form an integral part of the specification.

[0015] As illustrated in the experimental section below, the present inventors, using a multidisciplinary approach combining computational modelling, chemical synthesis, and biological analysis, devised and synthesized novel molecules derived from VII-threo (WO2022029151), verifying their therapeutic potential. In particular, as shown in the graph in Figure 1, the inventors initially verified, through the use of FRET methodology on neuronal-like cells, that said novel molecules were able to significantly improve the abilityto stimulate physiological interaction between synapsin III and soluble alpha-synuclein compared to the original molecule.

[0016] In vitro studies subsequently carried out by the inventors have also demonstrated the ability of the molecules object of the invention to advantageously bind to synapsin III and significantly reduce the size of alpha-synuclein fibrillary aggregates in human neuroblastoma cells. Surprisingly, this activity exhibited by the molecules of the invention is significantly higher than the structural t / zreo-MPH analogues described in the state of the art.

[0017] Therefore, one object of the present invention is a compound of Formula (I),

[0018]

[0019] (I)

[0020] wherein

[0021] R₁ is C₁-C₆ alkyl;

[0022] R₂ is selected from a hydrogen atom (H), C₁-C₆ alkyl and O-C₁-C₆ alkoxy;

[0023] R₃ is selected from the group consisting of:

[0024]

[0025] wherein

[0026] R₄ is selected from a hydrogen atom (H), C₁-C₆ alkyl,

[0027] R₅ is a hydrogen atom (H) or C₁-C₆ alkyl,

[0028] wherein said alkyl and / or alkoxy groups are each independently a saturated or unsaturated, linear or branched C₁-C₆ chain and / or O-C₁-C₆ chain optionally independently substituted with one or more substituents,including isomers and racemic mixtures thereof,

[0029] including pharmaceutically acceptable salts thereof.

[0030] According to the invention, the compound of Formula (I) includes isomers thereof, such as for example threo and erythro racemates, individual threo (R, R) and (S, S) stereoisomers as well as racemic mixtures thereof.

[0031] The term “isomer”, as defined above, includes the threo and erythro diastereomers, mixtures of the threo and erythro configurations, enantiomers of the threo and erythro forms, and equimolar mixtures of the (R, R) and (S, S) enantiomers for the threo configuration, as well as that of the (S, R) and (R, S) erythro enantiomers.

[0032] In addition, the compound of Formula (I) according to the invention includes pharmaceutically acceptable salts thereof.

[0033] According to the invention, the compound of Formula (I) also includes the salts of any isomer, such as for example the salts of threo and erythro racemates thereof and the enantiomers of the threo (R, R) or (S, S) configuration, as well as the salts of racemic mixtures thereof.

[0034] The term "alkyl", as used herein, refers to a linear or branched, saturated or unsaturated hydrocarbon radical comprising one to six carbon atoms (C₁-C₆), wherein the alkyl radical can optionally be independently substituted with one or more substituents as indicated above.

[0035] Examples of C₁-C₆ alkyl groups include, but are not limited to, methyl, ethyl, propyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl.

[0036] In one embodiment, R₁ is a linear or branched C₁-C₄ alkyl chain.

[0037] Preferably, R₁ is selected from a methyl group, an ethyl group, a propyl group, and an isopropyl group, more preferably R₁ is a methyl group.According to another embodiment of the invention, R₂ is hydrogen (H) or C₁-C₆ alkyl, preferably hydrogen (H) or methyl, even more preferably hydrogen (H).

[0038] In a further embodiment, R₅ is hydrogen (H) or C₁-C₆ alkyl, more preferably hydrogen (H) or methyl, even more preferably hydrogen (H).

[0039] In a still further embodiment, R₄ is hydrogen (H).

[0040] The preferred embodiments described above can be combined with each other as required, and the implementation of these combinations falls within the skills of the person skilled in the art.

[0041] Particularly preferred compounds within the scope of the invention are:

[0042]

[0043] (III); and

[0044]

[0045] The inventors found that the compounds of Formula (II), (III), and (IV) show remarkable efficiency in removing synapsin III from the fibrillary aggregates of this protein with alpha-synuclein, promoting their disruption.

[0046] In particular, the compound of Formula (II) has a tetrahydroquinoline structure.

[0047] Instead, the compounds of Formula (III) and (IV) have a tetrahydroisoquinoline structure binding the Formula (I) in position 1 or 3, respectively.

[0048] These compounds of Formula (II), (III), (IV) exhibit different steric hindrances, which affect their interactions with biological targets.

[0049] In fact, the inventors have observed that the presence of tetrahydroquinoline and tetrahydroisoquinoline cores in the compound of Formula (I) increases the ability to reduce the aggregation of alpha-synuclein by stimulating functional interaction of synapsin III with alpha-synuclein.

[0050] In particular, due to the nature of the R3 substituent, the compound of Formula (I) according to the invention exhibits greater steric hindrance located near the nitrogen atom, a characteristic that could affect its binding to proteins.

[0051] In a preferred embodiment of the invention, the compounds of Formula (II), (III) and (IV) are threo diastereomers.Advantageously, the threo form of the compounds of Formula (II), (III) and (IV) of the invention is suitable for action on synapsin III, thus enhancing the anti -aggregation potency on alpha-synuclein and consequently the disease-modifying effect.

[0052] The spatial configuration of the threo form is particularly suitable for effectively removing synapsin III from fibrillary aggregates, improving the overall pharmacological activity of the compound of the invention.

[0053] In a preferred embodiment, the compound of Formula (I) according to the invention is in the form of a pharmacologically acceptable salt, preferably in the hydrochloride salt (HC1) form.

[0054] The compound of Formula (I) according to the invention in the hydrochloride salt (HC1) form is particularly preferred for its properties of chemical stability, water solubility and compatibility with conventional pharmaceutical formulations, which make it suitable for therapeutic use.

[0055] As illustrated above, the modifications introduced by the present inventors to the phenyl moiety and the piperidine core of / A / w-MPH advantageously promote the binding of the compound of Formula (I) with synapsin III, thereby sequestering this protein from alpha-synuclein aggregates while facilitating functional physiological interaction between nonaggregated soluble alpha-synuclein and synapsin III. In other words, the compound of Formula (I) according to the invention advantageously operates by disrupting a stage of the pathogenic mechanism underlying the neurodegeneration process, thus exhibiting a diseasemodifying activity.

[0056] In this context, the compound of the invention also surprisingly showed higher efficacy compared to the structural treo-MPH analogues described in WO2022029151.

[0057] A further object of the present invention is a compound having the general Formula (I),

[0058]

[0059] (I)

[0060] wherein

[0061] R₁ is C₁-C₆ alkyl;

[0062] R2 is selected from a hydrogen atom (H), Ci-Ce alkyl, and O-Ci-Ce alkoxy;

[0063] R₃ is selected from the group consisting of:

[0064]

[0065] R₅

[0066] wherein

[0067] R₄ is selected from a hydrogen atom (H), C₁-C₆ alkyl, and O-Ci-Ce alkoxy;

[0068] R₅ is a hydrogen atom (H) or C₁-C₆ alkyl,

[0069] wherein said alkyl and / or alkoxy groups are each independently a saturated or unsaturated, linear or branched C₁-C₆ chain and / or O-C₁-C₆ chain optionally independently substituted with one or more substituents,

[0070] including isomers and racemic mixtures thereof,

[0071] including pharmaceutically acceptable salts thereof,

[0072] for use as a medicament.

[0073] In the context of the present invention, the term "disease-modifying" is intended to mean a treatment that can directly intervene on the pathogenic mechanisms underlying a neurodegenerative disease, blocking its progression, and restoring normal physiological function.

[0074] In particular, in the case of synucleinopathies such as, for example, Parkinson’s disease or Lewy body dementia, this effect manifests itself through the reduction of pathological alpha-synuclein aggregates, the restoration of the functional physiological interaction of alpha-synuclein with other proteins, and the prevention of neuronal degeneration. The compounds of the present invention, which are suitable for achieving this effect, clearly differ from symptomatic treatments that act only by mitigating the symptoms without affecting a salient point of the pathogenic mechanism of the disease.

[0075] In addition, in the context of the present invention, the term "aggregates of alpha-synuclein and synapsin III" is intended to refer to abnormal accumulations of these proteins within neurons, known as Lewy bodies and Lewy neurites. Their morphology ranges from spherical inclusions (Lewy bodies) to filamentous structures (Lewy neurites).

[0076] The term “alpha-synuclein particles,” as used herein, refers to microaggregates of this protein that can be deposited in neuronal cells and can act as seeds for the formation of Lewy bodies and Lewy neurites. Therefore, these particles, by acting as precursors, can trigger the formation of larger structures and are considered early indicators of the development of neurodegenerative diseases. The presence of such particles is associated with, for example, progression of Parkinson's disease and Lewy body dementia, as their growth progressively impairs neuronal viability.

[0077] Due to their beneficial activities on reducing alpha-synuclein aggregation and stimulating functional interaction between this protein and synapsin III, the compounds of Formula (I) as defined above have significant therapeutic potential for use as a medicament.

[0078] In particular, the compound of Formula (I) for use according to the invention is particularly suitable for use in the therapeutic treatment of a neurodegenerative disease, in particular synucleinopathies such as Parkinson’s disease, Lewy body dementia, multiple system atrophy, and the Lewy body variant of Alzheimer’s disease.

[0079] Preferably, in the compound of Formula (I) for use as a medicament R₁ is methyl; R₂ and R₄ are independently H or C₁-C₆ alkyl; R₅ is H or methyl.

[0080] More preferably, in the compound of Formula (I) for use as a medicament R₁ is methyl; R₂, R₄ and R₅ are each a hydrogen atom (H).Even more preferably, the compound of Formula (I) for use as a medicament is selected from:

[0081]

[0082] (II) (III) (IV)

[0083] A pharmaceutical composition comprising a compound of Formula (I), as defined above with reference to its use as a medicament, and at least one pharmaceutically and / or nutraceutically acceptable carrier, excipient and / or diluent, is also within the scope of the invention.

[0084] The pharmaceutical composition according to the invention is suitable for use in the above therapeutic medical applications relating to the compound according to the invention.

[0085] The pharmaceutical composition of the invention may include, for example, com starch, dibasic calcium phosphate, lactose monohydrate, magnesium stearate, and / or colloidal silica.

[0086] The presence of corn starch, dibasic calcium phosphate, lactose monohydrate, magnesium stearate and / or colloidal silica improves the administration, stability and bioavailability characteristics of the compound according to the invention.

[0087] In addition, the pharmaceutically acceptable carriers, excipients and / or diluents of the pharmaceutical composition according to the invention may modulate the release of the active ingredient, promoting a more gradual and controlled administration, or contribute to reducing local or systemic side effects, improving the tolerability of the treatment.

[0088] The pharmaceutical composition of the present invention can be formulated into any dosageform suitable for oral administration.

[0089] The inclusion of selected carriers, excipients, or diluents, known to those skilled in the art, allows for customization of the formulation, ensuring optimal therapeutic effectiveness and greater patient compliance.

[0090] The following are practical examples illustrating, in a detailed but non-limiting manner, the chemical synthesis of some embodiments of the compound of the invention. Furthermore, experimental results are reported which illustrate the biological activity of some preferred embodiments of the invention, as well as demonstrate the potential effectiveness of the disclosed compounds as medicaments.

[0091] In the experimental section below, reference is made to the accompanying drawings, wherein:

[0092] - Figure 1 shows a graph summarizing the FRET efficiency under basal conditions and after treatment with a 10 pM concentration of the compounds threo MPH, threo PK7 (also referred to as threo XVII), threo XII, threo XVII, of the prior art and the threo compound of Formula (II) of the present invention (key to the statistical values obtained: *P < 0.05; ****p < 0.0001; #P < 0.05; ##P < 0.01; ###P < 0.001, ####P < 0.0001, one-way ANOVA + Tukey’s test);

[0093] - Figure 2 shows micrographs acquired with a Zeiss LSM900 confocal microscope (Carl Zeiss) representative of alpha-synuclein immunostaining in SK-N-SH cells overexpressing human alpha-synuclein, under basal (untreated) conditions and after a 24-hour treatment with the prior art compound threo PK7 (also referred to as threo XVII) or with the threo compound of Formula (II) of the invention, each tested at increasing concentrations of 25, 50 and 100 nM;

[0094] - Figure 3 shows the in vitro effects of the compound of the invention on alpha-synuclein fibrillary aggregates, (a) Histogram representative of the total alpha-synuclein (a-Syn)-positive area of aggregates in SK-N-SH cells overexpressing human alpha-synuclein, these cells having been treated with the prior art compound threo PK7 (also referred to as threo XVII) and with the threo compound of Formula (II) of the invention at different concentrations, (b) Histogram representative of the mean size of a-Syn-positive particles ofSK-N-SH cells overexpressing human alpha-synuclein, these cells having been treated with the prior art compound threo PK7 and with the threo compound of Formula (II) of the invention at different concentrations (key to the statistical values obtained in Figures 3a and 3b: *P<0.05; **P<0.001; ****p < 0.0001, one-way ANOVA + Tukey’s test).

[0095] - Figure 4 illustrates the results of the MTT cytotoxicity assay on primary cortical neuronal cultures treated for 24 hours with increasing concentrations (0.01–100 μM) of the threo compound of Formula (II). Data are expressed as absorbance values at 570 nm (*** P<0.001 vs CTRL, one-way ANOVA + Dunnett' s multiple comparisons test).

[0096] CHEMICAL SYNTHESIS

[0097] MATERIALS AND METHODS

[0098] For all examples and experimental tests below, experimental data were obtained as follows: - melting points were measured by thermal analysis (TA) in particular with an Instruments Q20 DSC differential scanning calorimeter;

[0099] - nuclear magnetic resonance spectra were recorded with the Varian Mercury 300 spectrophotometer, and were acquired for1H-NMR at 300 MHz and for13C-NMR at 75 MHz;

[0100] - thin layer chromatography (TLC) was performed on standard analytical silica gel layers (thickness 0.20 mm; Macherey-Nagel ALUGRAM SIL G / UV254);

[0101] - chromatographic purifications were performed, in the normal phase, using Sepachrom Puriflash XS 420 on different flash chromatography cartridges filled with Merck Silica Gel 60 (0.040-0.063 pm).

[0102] The purity of the final compounds was assessed by high performance liquid chromatography (HPLC) using the Elite LaChrom HPLC system with diode array detector (190-400 nm) and a Waters XBridge TM C-18 column (5 pm, 4.6x150 mm).

[0103] The specific method is reported herein and was found to be effective in separating the threo and erythro isomers.Time % Solvents

[0104] Flow (ml / min) (minutes) | Water + l%o TFA | ACN + l%o TFA D < 0. |'90% f l"o"% f'l. A.. P

[0105] 25 fl0%. f'90% |"1.

[0106] -30''"'''''''''''''^^ \Z1-

[0107]

[0108] The HPLC method was set up using a Waters XBridgeTM C-18 column conditioned with freshly prepared 90% Water / 10% ACN + l%o TFA until the column pressure was stable. All tested samples were prepared by dissolving the purified products in the selected mobile phase, at concentrations of approximately 1 mg / ml, filtered through a 0.45 pm filter and analysed. The injection volume was 20 pl. Due to the presence of the substituted phenyl ring in each compound, purity was assessed on chromatograms recorded at 217 nm and expressed as Area%.

[0109] Abbreviations used herein: ACN: acetonitrile; BnBr: Benzyl bromide; DCM: di chloromethane; DMF: dimethylformamide; IPA: isopropyl alcohol; m-CPBA: meta-chloroperbenzoic acid; m.p.: melting point; MW: molecular weight; n-BuLi: normal-butyl lithium; NaBH3CN: sodium cyano borohydride; RT: room temperature; TEA: tri ethylamine; TFA: trifluoroacetic acid; THF: tetrahydrofuran.

[0110] Table 1 shows, as an example, the structures of the compounds of Formula (II), (III) and (IV), for which appropriate synthetic routes have been developed.

[0111] Table 1

[0112] Cpd# Structure Cpd# Structure

[0113] Formula no? Formula

[0114] (II) 1 Z

[0115] H (II)

[0116] (threo) (erythro)

[0117] HCI

[0118]

[0119] Formula Formula

[0120] (HI) k (HI

[0121] 1X: Ao-Me)

[0122] 1 - {threo) H {erythro) H \

[0123] HCI [1 J HCI || J

[0124] Formula Formula

[0125] (IV) (IV) ^N'"^<XOCH3

[0126] {threo) {erythro) 1 =

[0127]

[0128] H^0^

[0129] The procedures developed to obtain the compounds according to the invention are described below. p j

[0130] Some exemplary schemes of the synthetic reactions performed for each of the compounds of Formulas (II), (III), and (IV) as a racemic mixture of two isomers, threo and erythro, are also shown.

[0131] The subsequent process steps performed on the two obtained isomers are the same for both isomers; however, for brevity, they are described for an individual isomer.

[0132] SYNTHESIS OF THE COMPOUND OF FORMULA (II)

[0133] The synthesis scheme 1 below schematically shows the synthesis process for the compound of Formula (II) according to the invention by carrying out the process steps (a), (b), (c), (d), (e), (f) and (g) performed consecutively.

[0134] Said synthesis process uses quinoline as the starting reagent which is transformed by process step (a) into a reaction intermediate (1) (i.e., the corresponding N-oxide) by a method known per se (Ochiai, The Journal of Organic Chemistry 1953 18 (5), 534-551).

[0135] Subsequently, the reaction intermediate (1) is transformed into methyl quinolin-2-yl acetate(reaction intermediate (2)) by the reaction process step (b), similar to what is reported in the state of the art for isoquinoline derivatives (Funakoshi, Chemical and Pharmaceutical Bulletin, 1984, 32, 12, 4731-4739).

[0136] Then, by means of process step (c), an alkylation reaction of the reaction intermediate (2) is performed with / / -butyl lithium and benzyl bromide in THF.

[0137] This reaction leads to the reaction intermediate (3) which by partial reduction (process step (d)) provides the compound of Formula (II) as a mixture of the threo and erythro isomers.

[0138] The inventors also noted that these isomers can subsequently be separated by flash chromatography; however, in order to obtain higher separation yields, it is preferable to derivatize them as N-Bocs (i.e., the corresponding tert-butylcarbamates) according to process step (e) to optimize the separation itself (process step (f)) and then remove the protective group using process step (g).

[0139] Lastly, the treatment of the individual isomers, obtained by chromatography, with methanolic HC1 allows the crystallization of the corresponding hydrochlorides.

[0140] Below is a detailed description of the steps in the synthesis process.

[0141] SCHEME 1: process for the synthesis of the compound of Formula (II) and for the separation of its isomers

[0142]

[0143] N-Boc of Formula II Formula II threo threo

[0144]

[0145] N-Boc of the Compound of Formula (II)

[0146]

[0147] N-Boc of Formula II Formula II erythro erythro

[0148] Reagents and conditions: (a) m-CPBA, DCM, 0°C; (b) Methyl acetoacetate, Acetic anhydride, DMF, RT; 10% HC1; (c) n-BuLi, THF, BnBr, 0°C; (d) NaBH3CN, Acetic acid, RT; (e) di-tert-butyl dicarbonate, DCM, TEA, RT; (f) chromatographic separation (g) TFA, DCM.

[0149] As mentioned above, process steps (a) and (b) are known per se.

[0150] The scheme below represents an extract of the synthesis scheme 1 corresponding to process step (c).

[0151]

[0152] In reaction step (c), there is essentially an alkylation reaction leading from reaction intermediate (2) to reaction intermediate (3).

[0153] This process step (c) was carried out by slowly adding a 2.5 M w-butyl lithium solution in hexane (8.80 ml) to a solution of methyl quinol-2-yl acetate (4.02 g; 20.00 mmol) in THF (40 ml), said solution being cooled to 0°C. Subsequently, still maintaining a temperature of 0°C, a solution of benzyl bromide (3.56 g, 21.00 mmol, 2.50 ml) in THF (15 ml) was added. The reaction was then stirred at the same temperature until complete conversion of the starting reaction intermediate. The reaction was then treated, again at 0°C, with 10% HC1 (50 ml).

[0154] After phase separation, the organic phase was washed with 10% HC1 (15 ml). The pooled acidic phases were added with AcOEt (50 ml) and 30% NaOH up to pH 10. The aqueous phase was extracted two more times with AcOEt (20 ml), and the organic phases were pooled. After washing with water (3x20 ml), the organic phase was dried over NaSO4and evaporated under reduced pressure to obtain 5.3 g of reaction intermediate (3).

[0155] Subsequently, to obtain the compound of Formula (II), process step (d) was performed, an extract of which from synthesis scheme 1 is shown below.

[0156] NaBH3CN

[0157] Acetic acid

[0158] RT

[0159] 2 hours

[0160]

[0161] In process step (d), 0.216 g of NaBH3CN was added to a solution of the compound (3) (0.50 g, 1.72 mmol) in acetic acid (20 ml). The resulting mixture was reacted at room temperature for 2 hours.

[0162] Once the reaction was completed, the acidity was buffered with a saturated sodium bicarbonate solution, and the product was extracted with DCM (3x10 ml).

[0163] The organic phase was washed with brine, dried over NaSO4, filtered, and evaporated under reduced pressure, yielding 0.420 g of methyl 3-phenyl-2-(l,2,3,4-tetrahydroquinol-2-yl)propanoate, i.e., the compound of Formula (II) as a mixture of threo and erythro isomers.

[0164] The two diastereomers can be separated by chromatography to isolate the pure isomers. In particular, the inventors noted that chromatographic separation yields could be increased by preferably operating as described below.

[0165] As is known to those skilled in the art, chromatographic separation can be optimized by transforming the two isomers into the corresponding tert-butylcarbamates. The separation of the two diastereomers is performed by silica gel chromatography, eluting with a 99 / 1 toluene / AcOEt mixture. The stereochemistry of the threo form (first eluted peak) and the erythro form (second eluted peak) was determined by NMR analysis.

[0166] The N-Boc derivatives of the compound of Formula (II) were obtained by performing the process step (e) shown below:

[0167]

[0168] Compound of Formula (II) N-Boc of the Compound of Formula (

[0169]

[0170] II)

[0171] In process step (e), the compound of Formula (II) (1.00 g, 3.40 mmol) was added to a solution of di -tert-butyl dicarbonate (1.13 g, 5.18 mmol) in THF (2 mL), and the resulting mixture was allowed to react to reflux until disappearance of the starting product. Once the reaction was completed, the mixture was taken up with AcOEt, and the resulting solution was washed with brine, dried over NaSO4, filtered, and evaporated under reduced pressure, yielding 1.207 g of crude product as a light yellow oil.

[0172] In the next step (f) of the process, a chromatographic separation was performed, which allowed the isolation of the pure isomers.

[0173] Process (g) (scheme below), starting from the individual threo or erythro isomer, makes it possible to obtain the corresponding compounds of Formula II (threo and erythro) by proceeding as follows for the threo isomer:

[0174]

[0175] N-Boc of the compound

[0176] Compound of Formula (II) threo

[0177] of Formula (II) threoTFA (2.0 ml) was added to a solution of the threo N-Boc-compound of Formula (II) (0.573 g, 1.45 mmol) in DCM (6 ml) at 0°C. The resulting solution was maintained at RT for 1 h. After removal of the solvent, the residue was transformed into the corresponding hydrochloride by treatment with MeOH (1.5 ml) and methanolic HC1 (1 ml), yielding 0.330 g of the hydrochloride of the threo compound Formula (II).

[0178] Similarly, starting from the erythro N-Boc-compound of Formula (II) (0.420 g, 1.06 mmol), 0.280 g of the hydrochloride of the erythro compound Formula (II) is obtained.

[0179] Synthesis scheme 1 makes it possible to obtain other compounds of the invention, starting from the quinoline as is or appropriately substituted at positions 5, 6, 7, or 8 and / or using benzyl bromide as is or bearing the appropriate substituent on the aromatic ring.

[0180] Synthesis scheme 1 was monitored and then verified by NMR analysis of the various reaction intermediates and the final product; in some cases, the melting point and / or molecular weight values were also determined.

[0181] Data of the Reaction Intermediates:

[0182] Reaction intermediate (1) Quinoline N-oxide".

[0183] 1H NMR (300 MHz, CDCl3): δ 8.76 (s, 1H), 8.13 (dd, J = 7.1, 1.8 Hz, 1H), 7.82-7.76 (m, 1H), 7.76-7.69 (m, 1H), 7.66 (d, J= 7.3 Hz, 1H), 7.63-7.54 (m, 2H).

[0184] Melting point: 61°C.

[0185] Reaction intermediate (2) Methyl quinol-2-yl acetate.

[0186] 1H NMR (300 MHz, CDCl3): δ 8.10 (d, J= 8.2 Hz, 1H), 8.04 (d, J= 8.2 Hz, 1 H), 7.80-7.63 (m, 2H), 7.53-7.46 (m, 1H), 7.40 (d, J= 8.2 Hz, 1H), 4.04 (s, 2H), 3.71 (s, 3 H).

[0187] MW: 201.22.

[0188] Reaction intermediate (3) Methyl 2-(quinol-2-yl)-3-phenylpropanoate'.

[0189] 1H NMR (300 MHz, CDCl3): δ 8.12 (m, 2H), 7.78 (m, 1H), 7.71 (m, 1H), 7.54 (m, 1H), 7.39 (m, 1H), 7.19 (m, 5H), 4.36 (t, J= 7.7 Hz, 1H), 3.64 (s, 3H), 3.56 (dd, J= 13.9, 7.7 Hz, 1H),3.38 (dd, J= 13.9, 7.7 Hz, 1H).

[0190] MW: 291.34.

[0191] Data of the Product Compound of Formula (II):

[0192] Compound of Formula (II) - as a mixture of threo and erythro isomers-:

[0193] 1H NMR (300 MHz, CD3OD): δ 7.39 – 7.09 (m, 5H), 6.86 (m, 2H), 6.53 (m, 2H), 3.54-3.51 (m, 4H), 3.12 - 2.74 (m, 5H), 2.06 – 1.73 (m, 2H).

[0194] MW: 295.38.

[0195] N-Boc of the compound of Formula (Il)-threo

[0196] White solid with m.p. of 85°C after crystallization with IPA.

[0197] 1H NMR (300 MHz, CD3OD): δ 7.36 – 7.02 (m, 5H), 6.96 (dd, J = 7.8, 1.4 Hz, 2H), 4.78 – 4.65 (m, 1H), 3.43 (s, 3H), 2.97 – 2.53 (m, 6H), 2.35 – 2.10 (m, 2H), 1.78 (dt, J = 13.3, 6.3 Hz, 1H), 1.49 (s, 9H).

[0198] N-Boc of the compound of Formula (IV)-erythro

[0199] 1H NMR (300 MHz, CD3OD): δ 7.43 – 6.94 (m, 7H), 4.95 – 4.71 (m, 1H), 3.47 (s, 3H), 3.00 – 2.54 (m, 6H), 2.37 – 2.10 (m, 2H), 2.03 – 1.83 (m, 1H), 1.48 (s, 9H).

[0200] Once the separation of the two diastereomers was completed, the stereochemistry of the threo and erythro forms was determined by NMR analysis. These NMR data are shown below:

[0201] Compound of Formula (IV)-threo Free Base:

[0202] 1H NMR (300 MHz, CD3OD): δ 7.30-7.09 (m, 5H), 7.05-6.93 (m, 2H), 6.77-6.65 (m, 2H), 3.64-3.43 (m, 1H), 3.54 (s, 3H), 3.17-3.06 (m, 1H), 3.00-2.86 (m, 2H), 2.86-2.66 (m, 2H), 2.09-1.93 (m, 1H), 1.85-1.68 (m, 1H).

[0203] Compound of Formula (IV)-erythro Free Base:

[0204] 1H NMR (300 MHz, CD3OD): δ 7.35-7.08 (m, 5H), 6.95-6.82 (m, 2H), 6.62-6.46 (m, 2H), 3.65-3.45 (m, 1H), 3.53 (s, 3H), 3.10-2.81 (m, 3H), 2.81-2.65 (m, 2H), 2.08-1.77 (m, 2H).PREPARATION OF THE HYDROCHLORIDE SALTS OF THE THREO AND ERYTHRO FORMS OF THE COMPOUND OF FORMULA (II)

[0205] For the preparation of the hydrochlorides, the individual free bases were dissolved in MeOH (5 ml) and added with 3M HC1 in MeOH (1 ml), and then concentrated to a solid dry residue.

[0206] This salification step can be carried out in a similar manner, as can be easily inferred by a person skilled in the art, for racemic mixtures of other compounds according to the invention.

[0207] The characterization of the hydrochloride salts of the two diastereomers of the compound of Formula (II) was performed by NMR analysis, the data of which are given below, and by measuring the melting points and / or molecular weights:

[0208] Compound of Formula (ll)-threo Hydrochloride:

[0209] m.p. 144°C.

[0210] 1H NMR (300 MHz, CD3OD): δ 7.40 – 7.07 (m, 9H), 3.70 (m, 1H), 3.61 (s, 3H), 3.25 (m, 1H), 3.11 (m, 2H), 2.98 (m, 2H), 2.30 (m, 1H), 1.96 (m, 1H).

[0211] MW: 331.84.

[0212] Compound of Formula (ll)-erythro Hydrochloride:

[0213] m.p. 124°C.

[0214] 1H NMR (300 MHz, CD3OD): δ 7.60 – 6.69 (m, 9H), 3.85 (m, 1H), 3.58 (s, 3H), 3.21 – 2.86 (m, 5H), 2.37 (m, 1H), 1.98 (m, 1H).

[0215] MW: 331.84.

[0216] SYNTHESIS OF THE COMPOUND OF FORMULA (III)

[0217] The synthesis scheme 2 below schematically shows the synthesis process for the compound of Formula (III) according to the invention by carrying out the process steps (a’), (b’), (c’), (d’), and (e’) performed consecutively, i.e., one after the other.The first reagent in the synthesis is the commercially available methyl isoquinolin-3-yl acetate, which undergoes an alkylation reaction by performing process step (a’) using n-butyl lithium and benzyl bromide in THF. This process step (a’) yields the reaction intermediate (4).

[0218] By performing process step (b'), a subsequent partial reduction occurs, yielding the tetrahydroisoquinoline compound of Formula (III) as a mixture of threo and erythro isomers.

[0219] These isomers are subsequently separated by performing the chromatographic process step, preferably derivatizing them as N-Bocs (step (c’)) to optimize the separation itself (step d’) and then remove the protective group by means of step (e’).

[0220] Below is a detailed description of the steps in the synthesis process.

[0221] SCHEME 2: process for the synthesis of the compound of Formula (III) and for the separation of its isomers

[0222]

[0223] Compound of Formula (!!!) (three and erythro)

[0224] Formula (11!) (three)

[0225] M< Boc of Formula (HI) {three and erythro) e’

[0226]

[0227] -Boc of Formula (ill) Formula (IH) (erythro) (erythro)

[0228] Reagents and conditions: (a’) w-BuLi, THF, BnBr, 0°C; (b’) NaBH3CN, acetic acid, RT; (c’) di -tert-butyl dicarbonate, DCM, TEA, RT; (d’) chromatographic separation; (e’) TFA, DCM.

[0229] Below is an extract of the synthesis scheme 2, corresponding to process step (a’).

[0230]

[0231] C12H11NO2C19H17NO2In process step (a1), a 2.5 M / / -Butyl lithium solution in hexane (1.30 ml) was slowly added to a solution of methyl isoquinol -3 -yl acetate (0.60 g; 2.98 mmol) in THF (4 ml), cooled to 0°C.

[0232] Subsequently, while maintaining a temperature of 0°C, a solution of benzyl bromide (0.51 g, 2.98 mmol, 0.35 ml) in THF (2 ml) was added. The reaction was then stirred at the same temperature until complete conversion of the starting reagent. The reaction was then treated, again at 0°C, with 10% HC1 (20 ml). After phase separation, the organic phase was washed with 10% HC1 (10 ml). The pooled acidic phases were added with AcOEt (20 ml) and 30% NaOH up to pH 10. The aqueous phase was extracted two more times with AcOEt (15 ml), and the organic phases were pooled. After washing with water (3x10 ml), the organic phase was dried over NaSO4 and evaporated under reduced pressure to obtain 0.79 g of reaction intermediate (4) consisting of methyl 3-phenyl-2-(isoquinolin-3-yl)propanoate.

[0233] Below is an extract of the synthesis scheme 2, corresponding to process step (b’), performed after step (a’).

[0234] b’

[0235] NaBH3CN

[0236] Acetic acid

[0237] RT

[0238] compound of Formula (III)

[0239]

[0240] C19H21NO2

[0241] In this step (b’), 0.17 g of NaBHsCN was added to a solution of the reaction intermediate (4) (0.39 g, 1.34 mmol) in acetic acid (4 ml). The resulting mixture was reacted at room temperature for 18 hours.

[0242] Once the reaction was completed, the acidity was buffered with a saturated sodium bicarbonate solution, and the product was extracted with DCM (3x10 ml). The organic phasewas washed with brine, dried over NaSCh, filtered, and evaporated under reduced pressure.

[0243] The resulting crude product consisting of the compound of Formula (III) (i.e., methyl 3-phenyl-2-(l,2,3,4-tetrahydroisoquinol-3-yl)propanoate) was obtained as a mixture of threo and erythro isomers.

[0244] In this case, too, as is known to those skilled in the art, in order to optimize the chromatographic separation, it is possible to transform the isomers into the corresponding tert-butylcarbamates, and after the separation, restore the corresponding free bases, that is, the threo compound of Formula (III) and the erythro compound of Formula (III), by treatment with trifluoroacetic acid.

[0245] The resulting compound of Formula (III) was then transformed into the N-Boc derivative by process (c’) as described below: the compound of Formula (III) (0.90 g, 3.04 mmol) and TEA (0.69 g, 4.61 mmol) were added to a solution of di -tert-butyl dicarbonate (0.664 g, 3.04 mmol) in DCM (10 ml), maintained at 0°C, and the resulting mixture was allowed to react under reflux for 4 h. Once the reaction was completed, the mixture was taken up with DCM, and the resulting solution was washed with a 10% NaHCOs solution, dried over NaSO4, filtered, and evaporated under reduced pressure, yielding 1.09 g of crude product.

[0246] In the next step (d’) of the process, chromatographic separation on silica gel was carried out by eluting with a 95 / 5 cyclohexane / AcOEt mixture. Under these conditions, the first isomer to be eluted is the threo N-Boc derivative of Formula (III). The stereochemistry of the N-Boc-Formula (III) isomers was determined by NMR analysis.

[0247] Subsequently, process step (e’) (scheme below), starting from the N-Boc derivatives of the individual threo or erythro isomers, makes it possible to obtain the threo and erythro compounds of Formula (III) and their hydrochlorides by proceeding as follows for the threo isomer.

[0248]

[0249] N-Boc of Formula (III) Formula (III) (threo)

[0250] (threo)

[0251] In particular, TFA (1.0 ml) was added to a solution of the threo N-Boc-compound of Formula (III) (0.500 g, 1.26 mmol) in DCM (5 ml) at 0°C. The resulting solution was maintained at RT for 1 h. After removal of the solvent, the residue was transformed into the corresponding hydrochloride by treatment with MeOH (1.5 ml) and methanolic HC1 (1 ml), yielding 0.43 g of the hydrochloride of the threo compound Formula (III).

[0252] Similarly, starting from the erythro N-Boc-compound of Formula (III) (0.20 g, 0.51 mmol), 0.16 g of the hydrochloride of the erythro compound Formula (III) is obtained.

[0253] The molecular weight (MW) of the compound of Formula (III) was found to be 295.38.

[0254] Synthesis scheme 2 was monitored and then verified by NMR analysis of the reaction intermediates and final product; in some cases, the melting point and / or molecular weight values were determined.

[0255] It is possible to synthesize other compounds of Formula (I) (including for use as a medicament) according to the invention by following synthesis scheme 2, starting from the isoquinoline as is or appropriately substituted at positions 5, 6, 7, or 8 and / or using benzyl bromide as is or bearing the appropriate substituent on the aromatic ring.

[0256] Data o f the Reaction Intermediates:

[0257] Reaction intermediate (4) Methyl 3-phenyl-2-isoquinolin-3-yl acetate:

[0258] XHNMR (300 MHz, CDCh): 69.23 (s, 1H), 7.97 (dd, J= 8.2, 1.1 Hz, 1H), 7.80 (d, J= 8.2Hz, 1H), 7.72-7.67 (m, 1H), 7.65 (s, 1H), 7.61-7.56 (m, 1H), 7.30-7.09 (m, 5H), 4.61 (m, 1H), 3.70 (m, 1H), 3.63 (s, 3H), 3.40 (m, 1H).

[0259] MW: 291.34.

[0260] N-Boc of the Compound of Formula (IU)-threo

[0261] 1H-NMR (300 MHz, CDCl3): δ 7.54-6.78 (m, 9H), 5.10- 4.59 (m, 2H), 4.17 (d, J= 17.4 Hz, 1H), 3.45 (s, 3H), 3.10 (dd, J= 16.4, 5.8 Hz, 1H), 2.95 (m, 1H), 2.77 (m, 2H), 2.61 (d, J= 16.2 Hz, 1H), 1.55 (s, 9H).

[0262] N-Boc of the Compound of Formula (IIT)-erythro

[0263] 1H-NMR (300 MHz, CDCl3): δ 7.42-6.83 (m, 9H), 5.04-4.62 (m, 2H), 4.35 (t, 1H, J= 18.9 Hz, 1H), 3.47 (s, 3H), 3.19-2.65 (m, 5H), 1.49 (s, 9H).

[0264] Data of the Reaction Product Compound of Formula (III):

[0265] As mentioned above, chromatography allows the separation of the two isomers of the compound of Formula (III) as free bases, the threo and erythro stereochemistry of which were determined by NMR analysis. These NMR data are shown below:

[0266] Compound of Formula (III)-threo Free Base:

[0267] 1H NMR (300 MHz, CD3OD): δ 7.30-7.09 (m, 5H), 7.05-6.93 (m, 2H), 6.77-6.65 (m, 2H), 3.98 (s, 2H), 3.54 (s, 3H), 3.06-2.54 (m, 6H).

[0268] Compound of Formula (III)-erythro Free Base:

[0269] 1H NMR (300 MHz, CD3OD): δ 7.35-7.08 (m, 5H), 6.95-6.82 (m, 2H), 6.62-6.46 (m, 2H), 3.95 (s, 2H), 3.53 (s, 3H), 3.10-2.44 (m, 6H).

[0270] PREPARATION OF THE HYDROCHLORIDE SALTS OF THE THREO AND ERYTHRO FORMS OF THE COMPOUND OF FORMULA (III)

[0271] For the preparation of the hydrochlorides, the individual free bases were dissolved in MeOH (5 ml) and added with 3M HC1 in MeOH (1 ml), and then concentrated to a solid dry residue.This salification step can be carried out in a similar manner as described for the hydrochloride salts of the isomers of the compound of Formula (II).

[0272] The hydrochlorides of the threo and erythro forms of the compound of Formula (III) were also verified by melting point analysis and NMR analysis, the data of which are given below:

[0273] Compound of Formula (IIT)-threo Hydrochloride:

[0274] m.p. 179°C.

[0275] 1H NMR (300 MHz, CD3OD): δ 7.38–7.17 (m, 9H), 4.52–4.36 (m, 2H), 3.76 (dt, 1H), 3.67 (s, 3H), 3.39-3.32 (m, 1H), 3.26–2.96 (m, 4H).

[0276] Compound of Formula (IIT)-erythro Hydrochloride:

[0277] m.p. 244°C.

[0278] 1H NMR (300 MHz, CD3OD): δ 77.37–7.18 (m, 9H), 4.55–4.35 (m, 2H), 3.82 (dt, 1H), 3.62 (s, 3H), 3.28–3.03 (m, 5H).

[0279] SYNTHESIS OF THE COMPOUND OF FORMULA (IV)

[0280] The synthesis scheme 3 below schematically shows the synthesis process for the compound of Formula (IV) according to the invention by carrying out the process steps (a”), (b”), (c”), (d”), (e”), (f”) and (g”) performed consecutively.

[0281] The synthesis process uses isoquinoline as the starting reagent which is transformed into the corresponding N-oxide (reaction intermediate (5)) by performing process step (a”) as reported in the state of the art (Ochiai, The Journal of Organic Chemistry 1953 18 (5), 534-551).

[0282] Subsequently, by performing process step (b”), the reaction intermediate (5) is transformed into methyl isoquinol- 1-yl acetate (reaction intermediate (6)) as described in the state of the art (Funakoshi, Chemical and Pharmaceutical Bulletin, 1984, 32, 12, 4731-4739).Process step (c”) essentially consists of an alkylation reaction, performed with / / -Butyl lithium and benzyl bromide in THF, and leads to the reaction intermediate (7).

[0283] The latter reaction intermediate (7), by performing process step (d”), through partial hydrogenation, provides the compound of Formula (IV) as a mixture of threo and erythro isomers.

[0284] These isomers can be separated by flash chromatography; however, it is preferable to derivatize them as N-Bocs, process (e”), to optimize the separation itself (f”), and then remove the protective group by means of process (g”).

[0285] Below is a detailed description of the steps in the synthesis process.

[0286] SCHEME 3: process for the synthesis of the compound of Formula (IV) and for the separation of its isomers

[0287]

[0288] e” N-Boc Formula (IV) Formula (IV) 'C.r‘ (threo) (threo)

[0289] Compound of Formula (IV) N-Boc Formula (IV) (threo and erythro) (threo and erythro)

[0290]

[0291] N-Boc Formula (IV) Formula (IV) (erythro) (erythro)Reagents and conditions: (a“) m-CPBA, DCM, 0°C; (b“) I) Methyl acetoacetate, Acetic anhydride, DMF, 40°C, II) 10%HCl; (c“) w-BuLi, THF, BnBr, 0°C; (d“)NaBH3CN, Acetic acid, RT; (e“) di-tert-butyl dicarbonate, DCM, TEA, RT; (f”) chromatographic separation (g”) TFA, DCM.

[0292] Isoquinoline-N-oxide (reaction intermediate (5)) was prepared by performing process step (a”), as described in the literature (Ochiai, The Journal of Organic Chemistry, 1953, 18 (5), 534-551).

[0293] Methyl isoquinol- 1-yl acetate (reaction intermediate (6)) was prepared by performing process step (b”), as described in the literature (Funakoshi, Chemical and Pharmaceutical Bulletin, 1984, 32, 12, 4731-4739).

[0294] Below is an extract of the synthesis scheme 3, corresponding to process step (c”).

[0295]

[0296] In process step (c”), to obtain methyl 3-phenyl-2-(isoquinol-l-yl)propanoate (reaction intermediate (7)), starting from reaction intermediate (6), a 2.5 M n-Butyl lithium solution in hexane (4.06 ml) was slowly added to a solution of methyl isoquinol-2-yl acetate (2.04 g; 10.13 mmol) in THF (20 ml), cooled to 0°C.

[0297] Subsequently, again at 0°C, a solution of benzyl bromide (1.73 g, 10.13 mmol, 1.20 ml) in THF (10 ml) was added. The reaction was then stirred at the same temperature until complete conversion of the starting product. The reaction was then treated, again at 0°C, with 10% HC1 (20 ml). After phase separation, the organic phase was washed with 10% HC1 (10 ml).The pooled acidic phases were added with AcOEt (20 ml) and 30% NaOH up to pH 10. The aqueous phase was extracted two more times with AcOEt (15 ml), and the organic phases were pooled. After washing with water (3x10 ml), the organic phase was dried over NaSO4 and evaporated under reduced pressure to obtain 2.29 g of crude product consisting of reaction intermediate (7), i.e., methyl 3-phenyl-2-(isoquinolin-l-yl)propanoate.

[0298] Below is an extract of the synthesis scheme 3, corresponding to process step (d”), performed after step (c”).

[0299] d”

[0300] - NaBH3CN

[0301] Acetic acid

[0302] RT

[0303]

[0304] compound of Formula (IV)

[0305]

[0306] C19H21NO2

[0307] In process step (d”), 0.98 g of NaBHsCN was added to a solution of the reaction intermediate (7) (2.29 g, 7.86 mmol) in acetic acid (20 ml). The resulting mixture was reacted at room temperature for 18 hours.

[0308] Once the reaction was completed, the acidity was buffered with a saturated sodium bicarbonate solution, and the product was extracted with DCM (3x10 ml). The organic phase was washed with brine, dried over NaSO4, filtered, and evaporated under reduced pressure, thereby yielding the compound of Formula (IV) as a mixture of threo and erythro isomers.

[0309] The subsequent transformation into the corresponding N-Boc derivatives of Formula (IV), a threo and erythro mixture, and the subsequent chromatographic separation were performed using processes (e”) and (f”). An exemplary scheme of step (e”) is shown below:

[0310]

[0311] Compound of Formula (IV) N-Boc Formula (IV)

[0312] (threo and erythro) (threo and erythro

[0313] In process step (e”), the compound of Formula (IV) (1.38 g, 4.67 mmol) and TEA (1.05 g, 7.01 mmol) were added to a solution of di-tert-butyl dicarbonate (1.02 g, 4.67 mmol) in DCM (15 ml), maintained at 0°C. The resulting mixture was allowed to react at RT for 4 h. Once the reaction was completed, the mixture was taken up with DCNM, and the resulting solution was washed with brine, dried over NaSO4, filtered, and evaporated under reduced pressure, yielding 1.89 g of crude product as a light-yellow oil.

[0314] In the next step (f”) of the process, the separation of the threo and erythro isomers was performed by silica gel chromatography, eluting with 95 / 5 Cyclohexane / AcOEt. The threo isomer is the first eluted compound and the stereochemistry of the isolated isomers was determined by NMR analysis.

[0315] Process (g”), as shown in an exemplary scheme below, starting from the N-Boc derivatives of the individual threo or erythro isomers, makes it possible to obtain the corresponding compounds of Formula (IV) (threo and erythro) by proceeding as follows for the threo isomer:

[0316]

[0317] N-Boc Formula (IV) Formula (IV)

[0318] (threo) (threo)

[0319] TFA (1.0 ml) was added to a solution of the threo N-Boc-compound of Formula (IV) (0.419 g, 1.06 mmol) in DCM (5 ml) at 0°C. The resulting solution was maintained at RT for 1 h. After removal of the solvent, the residue was transformed into the corresponding hydrochloride by treatment with MeOH (1.5 ml) and methanolic HC1 (1 ml), yielding 0.330 g of the hydrochloride of the threo compound Formula (IV).

[0320] Similarly, starting from the erythro N-Boc-compound of Formula (IV) (0.420 g, 1.06 mmol), 0.280 g of the hydrochloride of the erythro compound Formula (IV) is obtained.

[0321] The molecular weight (MW) of the compound of Formula (IV) was found to be 295.38.

[0322] Synthesis scheme 3 was monitored and then verified by NMR analysis of the reaction intermediates and final product, and the melting point and / or molecular weight values were determined for some compounds.

[0323] Data of the Reaction Intermediates:

[0324] Reaction intermediate (5), Isoquinoline-N-oxide:

[0325] 1H NMR (300 MHz, CDCl3): δ 8.70 (d, J= 8.8 Hz, 1H), 8.49 (dd, J= 6.1, 0.5 Hz, 1H), 7.82 (d, J= 8.3 Hz, 1H), 7.72 (m, 2H), 7.60 (dt, J = 7.0, 1.1 Hz, 1H), 7.25 (dd, J= 8.4, 6.1 Hz, 1H).

[0326] Reaction intermediate (6), Methyl isoquinol-l-yl acetate'.1H NMR (300 MHz, CDCl3): δ 8.47 (d, J= 5.7 Hz, 1H), 8.06 (t, J = 8.6 Hz, 1H), 7.83 (d, J= 8.4 Hz, 1H), 7.71-7.58 (m, 3H), 4.35 (s, 2H), 3.71 (s, 3H).

[0327] MW: 201.22.

[0328] Reaction intermediate (7), Methyl 3-phenyl-2-(isoquinol-l-yl)propanoate:

[0329] 1H NMR (300 MHz, CDCl3): δ 8.54 (d, J = 5.7 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 8.2 Hz, 1H), 7.64 (m, 1H), 7.57 (d, J = 5.7 Hz, 1H), 7.29-7.12 (m, 6H), 4.92 (dd, J = 8.1, 6.7 Hz, 1H), 3.68 (dd, J = 13.9, 8.1 Hz, 1H), 3.63 (s, 3H), 3.38 (dd, J = 13.9, 6.7 Hz, 1H). MW: 291.34.

[0330] N-Boc of the compound of Formula (IV)-threo

[0331] 1H NMR (300 MHz, CD3OD): δ 7.41–6.88 (m, 9H), 5.34 (d, J = 9.2 Hz, 1H), 3.64 (ddd, J = 26.2, 20.1, 17.0 Hz, 2H), 3.47 (s, 3H), 3.20–2.82 (m, 5H), 2.69 (t, J = 12.3 Hz, 1H), 1.48 (s, 9H).

[0332] N-Boc of the compound of Formula (IV)-erythro

[0333] XH NMR (300 MHz, CD3OD): 7.42-6.85 (m, 9H), 5.42 (dd, J = 32.1, 9.0 Hz, 1H), 4.04-3.75 (m, 1H), 3.55-3.40 (m, 1H), 3.37 (d, J = 8.6 Hz, 3H), 3.17-2.72 (m, 6H), 1.46 (s, 9H).

[0334] Data of the Reaction Product Compound of Formula (IV):

[0335] Compound of Formula (IV)-threo Free Base:

[0336] 1H NMR (300 MHz, CD3OD): δ 7.24–7.06 (m, 9H), 4.24 (d, J= 5.7 Hz, 1H), 3.38 (s, 3H), 3.40–3.23 (m, 2H), 3.07–2.67 (m, 5H).

[0337] Compound of Formula (IV)-erythro Free Base:

[0338] 1H NMR (300 MHz, CD3OD): δ 7.24–7.06 (m, 9H), 4.39 (d, J= 5.5 Hz, 1H), 3.52 (s, 3H), 3.41–3.27 (m, 2H), 3.05–2.62 (m, 5H).

[0339] PREPARATION OF THE HYDROCHLORIDE SALTS OF THE THREO AND ERYTHRO FORMS OF THE COMPOUND OF FORMULA (IV)For the preparation of the hydrochlorides, the individual free bases were dissolved in MeOH (5 ml) and added with 3M HC1 in MeOH (1 ml), and then concentrated to a solid dry residue.

[0340] This salification step can be carried out in a similar manner as described for the hydrochloride salts of the isomers of the compound of Formula (II). Their characterization is as follows:

[0341] Hydrochloride of the compound of Formula (IV)-threo:

[0342] m.p. 182°C.

[0343]

[0344] 1H NMR (300 MHz, CD3OD): δ 7.47–7.18 (m, 9H), 4.94 (d, J = 4.3 Hz, 1H), 3.79–3.65 (m, 2H), 3.27 (s, 3H), 3.39–3.02 (m, 5H).

[0345] Hydrochloride of the compound of Formula (IV)-erythro:

[0346] m.p. 165°C.

[0347] 1H NMR (300 MHz, CD3OD): δ 7.45–7.02 (m, 9H), 5.02 (d, J= 4.4 Hz, 1H), 3.53 (s, 3H), 3.79–2.98 (m, 6H), 2.73 (dd, J= 13.3, 3.6 Hz, 1H).

[0348] BIOLOGICAL ACTIVITY

[0349] MATERIALS AND METHODS OF THE BIOLOGICAL ACTIVITY EXPERIMENTS:

[0350] Cell cultures

[0351] Human neuroblastoma SK-N-SH cells overexpressing human alpha-synuclein were grown in complete medium comprising Dulbecco's modified Eagle's medium with 1000 mg / 1 glucose (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Thermo-Fisher), 100 pg / ml penicillin and 100 pg / ml streptomycin (Sigma- Aldrich). The cells were maintained at 37°C in a humidified atmosphere with 5% CO2and 95% O2.

[0352] Acceptor photobleaching FRET (Fluorescent Resonance Energy Transfer) studies

[0353] For FRET studies, SK-N-SH cells were seeded on 13 mm poly-D-lysine-coated coverslipsin 24-well plates (15,000 cells per coverslip) and maintained in differentiation medium for ten days, adding 10 pM retinoic acid to the medium daily. On Day 7, the cells were transiently transfected with human pGFP-synapsin III (synapsin III labelled with human green fluorescent protein (GFP)) and pCMV6-RFP-alpha-synuclein (alpha-synuclein labelled with red fluorescent protein (RFP)) constructs, using Lipofectamine 3000 (Thermo Fisher), according to the manufacturer's instructions. Cells transfected with pCMV6-RFP-alpha-synuclein were used as negative controls during FRET experiments. Three days after transfection, the cells were treated for 15 minutes with the vehicle (0.9 % normal saline -control), 10 μM MPH (d-threo), 10 pM PK7 -threo, 10 μM XII-threo compound, 10 μM XVII-threo compound, 10 pM threo compound of Formula (II) of the present invention; then immediately fixed with Immunofix (Diapath) for 15 minutes and subsequently mounted on slides.

[0354] The fixed cells were analysed using a Zeiss LSM 880 confocal laser microscope (Carl Zeiss) with the laser set at λ=488–543. After identifying double-positive cells, three to six regions of interest (ROI) were analysed for 4 series. Two baseline (pre-bleaching) images were acquired prior to bleaching the RFP acceptor fluorophore with the 543 laser set at 65% power. Two more images were subsequently acquired after bleaching (post-bleaching).

[0355] FRET efficiency (intended as GFP recovery after RFP photobleaching) was measured using Zen black software (Carl Zeiss). The average background intensity (outside the cell) was subtracted from the average ROI intensity, and all FRET values resulting from the different ROIs were used for statistical analysis.

[0356] Immunocytochemistry

[0357] Stable clones of alpha-synuclein-containing SK-N-SH human neuroblastoma were seeded on poly-D-lysine-coated glass coverslips in 24-well plates and grown in complete medium comprising Dulbecco's modified Eagle's medium with 1000 mg / 1 glucose (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Thermo Fisher), 100 pg / ml penicillin and 100 pg / ml streptomycin (Sigma-Aldrich). The cells were maintained at 37°C in a humidified atmosphere with 5% CO2and 95% O2. This medium was then removedand replaced with either complete medium or complete medium plus 25, 50, or 100 nM PK7-threo or 25, 50, or 100 nM of the compound of Formula (II) (threo). After 24 hours of treatment, the cells were fixed by incubation for 15 minutes in 4% paraformaldehyde with 4% sucrose in 1 M PBS, pH 7.4 (Sigma-Aldrich). The fixed cells were then stored in PBS containing 0.05% sodium azide (Sigma-Aldrich). The slides were incubated for 1 hour at room temperature in a blocking solution [2% w / v bovine serum albumin (BSA, Sigma-Aldrich) plus 3% v / v normal goat serum (Vector Laboratories) in PBS], then overnight at 4°C with the primary antibody (Syn211 antibody against alpha-synuclein, Thermo-Fisher Cat. No. 32-8100) at the optimal working dilution.

[0358] The following day, the cells were incubated for 1 hour at room temperature with Cy3-conjugated secondary antibody (Vector Laboratories) diluted in 0.1% Triton X-100 PBS plus 1 mg / ml BSA.

[0359] Finally, the cell nuclei were counterstained with Hoechst 33258 stain (Sigma-Aldrich) and the coverslips were mounted on slides using Vectashield.

[0360] RESULTS

[0361] Assessment of the ability of the compounds of the invention to stimulate functional interaction between alpha-synuclein and synapsin III in neuronal-like cells exhibiting alpha-synuclein / synapsin III co-aggregates by acceptor photobleaching florescence resonance energy transfer (FRET)

[0362] The ability of the compounds of the invention to stimulate physiological alpha-synuclein / synapsin III functional interaction was assessed by acceptor photobleaching FRET in neuronal-like cells. In particular, neuronally differentiated SK-N-SH neuroblastoma cells were used and transiently transfected with wild-type human alpha-synuclein labelled with red fluorescent protein (RFP) and with the 63-kDa human synapsin III isoform labelled with green fluorescent protein (GFP).

[0363] Acceptor photobleaching FRET microscopy allows accurate assessment of protein-proteininteractions by measuring FRET efficiency. This parameter is indicative of the increase in donor fluorescence after complete photobleaching of acceptor fluorescence, within a FRET pair of fluorophores (where the emission wavelength of a donor falls within the excitation wavelength of an acceptor). Since FRET efficiency is only detectable when the two fluorophores are close enough to ensure that their bound proteins interact, the molecular proximity of the latter is strictly dependent on this parameter (Ishikawa-Ankerhold et al., 2012; Bajar BT, et al. Sensors (Basel). 2016 Sep 14; 16(9): 1488. doi: 10.3390 / sl6091488). Therefore, the fluorescence of the donor (GFP-Synapsin III) after complete photobleaching of the acceptor (RFP-alpha-synuclein) was measured in cells that were treated for 15 minutes with the compounds of the present invention by calculating the FRET efficiency (%) according to the method described by Bajar BT, et al. Sensors (Basel). 2016 Sep 14;16(9):1488. doi: 10.3390 / sl6091488.

[0364] Figure 1 shows as an example a comparison between the FRET efficiency (%) of the compound of Formula (II) (threo) of the present invention and compounds described in the state of the art, such as MPH threo, and the compounds described in patent application WO2022029151 referred to as PK7 threo, XII threo, XVII threo, respectively.

[0365] In the experiments reported herein, all the following compounds were used in the hydrochloride salt form.

[0366] The structural formulas of the tested and compared compounds are shown below.

[0367]

[0368] The "baseline" control sample in Figure 1 corresponds to untreated neuronally differentiated SK-N-SH cells that still express the two proteins GFP-Synapsin III and RFP-alpha-synuclein; the subsequent samples, in Figure 1 named MPH threo, PK7 threo, XII threo, XVII threo, and Formula (II) threo, were treated for 15 minutes with the prior art compounds MPH threo, PK7 three. XII three, and XVII threo and the compound of Formula (II) threo of the invention, respectively.

[0369] As can be seen from the graph shown in Figure 1, the compound of Formula (II) of the invention retains the ability to significantly promote the functional physiological interaction between alpha-synuclein and synapsin III, exhibiting an improved effect compared to both the “baseline” sample of untreated cells alone and the MPH threo sample. Importantly, the results show a 5.8% variation compared to MPH threo treatment, with a statistical significance level P < 0.001, calculated by one-way ANOVA variance analysis and Tukey's post-hoc multiple comparison test, confirming that the observed variation is ascribable to the effect of the compound of the invention and not to random variability or other factors.

[0370] These data indicate a significant and reproducible response, confirming the efficacy of the inventive compound under the experimental conditions tested.

[0371] Assessment of the ability of the compounds of the present invention to reduce alpha-synuclein aggregation in dopaminergic differentiated SK-N-SH cells

[0372] To assess the ability of the compounds of the present invention to reduce the deposition of alpha-synuclein inclusions, differentiated SK-N-SH cells overexpressing alpha-synuclein and synapsin III were used. The process details for obtaining the cells used in these experiments are illustrated in the previous section “MATERIALS AND METHODS OF THE BIOLOGICAL ACTIVITY EXPERIMENTS”.

[0373] In particular, micrographs representative of alpha-synuclein immunolabeling in SK-N-SH cells overexpressing alpha-synuclein and human synapsin III were first recorded under basal (untreated) conditions, and then after treatment for 24 hours with PK7 threo or the compound of Formula (II) threo, both at increasing concentrations of 25, 50, and 100 nM.The total positive area for alpha-synuclein and the average particle size (to be understood as the size of the aggregates) of alpha-synuclein were then measured from the micrographs using Fiji software (ImageJ).

[0374] As can be seen even just by observing the micrographs illustrated in Figure 2, surprisingly the compound of Formula (II) threo of the present invention, already at a concentration of 25 nM, is able to reduce the deposits of alpha-synuclein fibrillary aggregates, thus showing a markedly greater ability compared to PK7 threo to disrupt the pathogenic protein aggregates of alpha-synuclein.

[0375] On the other hand, PK7 threo shows aggregate disruption ability only at significantly higher concentrations.

[0376] Furthermore, as can be seen from the graph shown in Figure 3a, the compound of Formula (II) (threo of the present invention, already at a concentration of 25 nM, was also shown to be able to reduce the area of alpha-synuclein aggregates more significantly than the prior art compound PK7 threo.

[0377] In particular, treatment of cells overexpressing alpha-synuclein and synapsin III with the compound of Formula (II) of the invention at a concentration of 25 nM resulted in a reduction in the alpha-synuclein-positive area of –324.9 μm2with a statistical significance of P < 0.05 (one-way ANOVA + Tukey's post-hoc multiple comparison test).

[0378] It is also possible to see from Figure 3b that the compound of Formula (II) threo of the invention exhibits an improved effect compared to the prior art compound PK7 threo also in reducing the average particle size of alpha-synuclein. Indeed, as shown in the graph in Figure 3b, treatment with the compound of Formula (II) threo of the invention, already at a concentration of 25 nM, induced a decrease in the average alpha-synuclein particle size of -0.17 μm2with a statistical significance of P < 0.05 (one-way ANOVA + Tukey's post-hoc multiple comparison test).Therefore, while PK7 threo showed a dose-dependent effect, surprisingly the compound of Formula (II) (threo) of the present invention shows the highest efficiency in reducing fibrillary aggregates already at a concentration of 25 nM, indicating that this compound of the invention exhibits a higher pharmacological potency than the prior art compound PK7 threo.

[0379] The reported experimental data show that the compound of Formula (I) of the invention has a striking disease-modifying effect, as previously defined. This effect is distinct from that of the well-known compounds used for the treatment of Parkinson's disease and Lewy body dementia, such as for example dopaminergic drugs, which only improve motor symptoms but do not slow down or stop nerve cell death. In fact, while dopaminergic drugs compensate for the lack of dopamine and only benefit the symptoms of the disease, the compounds of the invention promote the physiological functional interaction between alpha-synuclein and synapsin III and are therefore able to limit the progression of neurodegeneration, as demonstrated by the reduction of aggregates and the preservation of dopaminergic fibres in the striatum.

[0380] Assessment of the cytotoxicity of the compounds of the present invention by MTT assay

[0381] The present inventors carried out experimental studies to ascertain the absence of cytotoxic effects of the present compounds. Primary cortical neuronal cultures from C57BL / 6J embryos at day 19 of gestation were prepared for this purpose. After tissue dissociation and counting of the cells resuspended in Neurobasal medium supplemented with penicillin / streptomycin, glutamine and B27, the cortical cells were seeded on poly-D-lysine pre-treated slides in 24-well plates (100,000 cells / well) and maintained at 37°C, 5% CO2and 95% O2for 10 days in vitro (DIV 10).

[0382] At DIV 10, the cultures were treated for 24 hours with increasing concentrations (0.01–100 μM) of the compound of Formula (II) threo. After treatment, the MTT assay was performed: the culture medium was removed and 300 μl of 0.5 mg / ml MTT diluted in culture medium was added for 90 minutes at 37°C; after that, the medium added with MTT was removed and the formazan salts were solubilized with 250 μl of DMSO per well.The absorbance was finally measured in a 96-well plate with a microplate reader at 570 nm. These absorbance values for the tested concentrations of the compound of Formula (II) threo are shown in the graph in Figure 4. The results showed that the compound of Formula (II) threo of the present invention does not show toxic effects at working concentrations that reduce alpha-synuclein aggregation or stimulate interaction between synapsin III and alpha-synuclein. The compound of Formula (II) threo was found to be toxic only at a concentration of 100 μM (Figure 4, *** P<0.001 vs CTRL, one-way ANOVA + Dunnett's multiple comparisons test).

Claims

CLAIMS1. A compound having the general formula (I),"r r -J _RRX'-'(I)whereinR₁ is C₁-C₆ alkyl;R₂ is selected from a hydrogen atom (H), C₁-C₆ alkyl and O-C₁-C₆ alkoxy;R₃ is selected from the group consisting of:whereinR4 is selected from a hydrogen atom (H) and Ci-Ce alkyl;R₅ is a hydrogen atom (H) or C₁-C₆ alkyl,wherein said alkyl and / or alkoxy groups are each, independently, a saturated or unsaturated, linear or branched C₁-C₆ chain and / or O-C₁-C₆ chain optionally independently substituted with one or more substituents,including isomers and racemic mixtures thereof,including pharmaceutically acceptable salts thereof.

2. The compound according to claim 1, whereinRi is a methyl group;R2 and R4 are each, independently, H or Ci-Ce alkyl;R5 is H or a methyl group.

3. The compound according to claim 1 or 2, whereinRi is a methyl group;R2, R4, and R5 are each a hydrogen atom (H).

4. The compound according to any one of claims 1 to 3, having a formula selected from Formula (II), Formula (III), and Formula (IV)5. The compound according to any one of claims 1 to 4, wherein the pharmaceutically acceptable salt is a hydrochloride salt (HC1).

6. The compound according to any of claims 1 to 5, for use as a medicament.

7. A compound having the general formula (I),"r r -J _RRX'-'(I)whereinR₁ is C₁-C₆ alkyl;R₂ is selected from a hydrogen atom (H), C₁-C₆ alkyl and O-C₁-C₆ alkoxy;R₃ is selected from the group consisting of:whereinR₄ is selected from a hydrogen atom (H), C₁-C₆ alkyl, and O-Ci-Ce alkoxy;R₅ is a hydrogen atom (H) or C₁-C₆ alkyl,wherein said alkyl and / or alkoxy groups are each, independently, a saturated or unsaturated, linear or branched C₁-C₆ chain and / or O-C₁-C₆ chain optionally independently substituted with one or more substituents,including isomers and racemic mixtures thereof,including pharmaceutically acceptable salts thereof,for use as a medicament.

8. The compound according to claim 6 or 7, for use in the therapeutic treatment of a neurodegenerative disease.

9. The compound for use according to claim 8, wherein the neurodegenerative disease is selected from the group consisting of synucleinopathies, Parkinson's disease, Lewybody dementia, multiple system atrophy, and the Lewy body variant of Alzheimer’s disease.

10. A pharmaceutical composition comprising a compound according to any of claims 1 to 5, and at least one pharmaceutically acceptable carrier, excipient and / or diluent.

11. A pharmaceutical composition comprising a compound according to claim 7 and at least one pharmaceutically acceptable carrier, excipient and / or diluent.

12. The pharmaceutical composition according to claim 10 or 11, which is in a form suitable for oral administration.

13. The pharmaceutical composition according to any one of claims 10 to 12, for use as a medicament.

14. The pharmaceutical composition according to claim 13, for use in the therapeutic treatment of a neurodeg enerative disease.

15. The pharmaceutical composition for use according to claim 14, wherein the neurodegenerative disease is selected from the group consisting of synucleinopathies, Parkinson's disease, Lewy body dementia, multiple system atrophy, and the Lewy body variant of Alzheimer’s disease.

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