Benzimidazole compounds, their preparation methods, pharmaceutical compositions and their applications

Abstract

This invention discloses benzimidazolone compounds, their preparation methods, pharmaceutical compositions, and their applications. Specifically, this invention discloses a pimozide derivative or a pharmaceutically acceptable salt thereof. The compounds of this invention possess favorable pharmacodynamic and pharmacokinetic properties and can be used to treat motor neuron degenerative diseases and improve neuromuscular function.

Description

Technical Field

[0001] This invention relates to benzimidazole ketone compounds, pharmaceutical compositions thereof, and their applications. Background Technology

[0002] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is the most common type of motor neuron disease. ALS ranks first among the "world's five most incurable diseases," with a survival period of 2 to 5 years after onset. There is no effective treatment, no biomarkers, and 10% of patients have gene mutations (including mutations in SOD1, TDP43, FUS, and C9orf72 genes). Other causes remain unclear, making its treatment a global challenge.

[0003] Advances in understanding the genetic etiology of ALS have spurred the development of animal models and sparked tremendous efforts, but unfortunately, these efforts have so far failed to find a cure for ALS. Treatments to slow the progression of ALS include riluzole and edaravone, but their effects are not significant. Therefore, the development and evaluation of new therapies for ALS is urgently needed, and this remains one of the greatest challenges in neurology. Neuromuscular junction (NMJ) delivery is a potential therapeutic target and biomarker.

[0004] NMJ dysfunction has been observed in animal models of ALS, leading to the general consensus that extensive neurogenesis and synaptic remodeling occur in NMJ well before the onset of symptoms. Early retraction or degeneration of presynaptic motor terminals has been observed in mice expressing mutant SOD1, prior to motor neuron death, and this has also been observed in TARDBP mice and tissues from ALS patients. Expression of mutant human TARDBP or FUS mRNA in zebrafish results in impaired transmission and reduced frequency of microendplate currents (mEPCs), as well as reduced quantization of NMJ transmission. Various trophic factors have been expressed in the muscle of SOD1 mice to test their ability to stabilize NMJ, but these therapies have shown limited success in mice and humans. Increased NMJ denervation has also been reported in rat and mouse models with TDP-43 gene mutations. Therefore, treatment targeting motor neuron function remains an interesting but unexplored approach for preventing or delaying disease onset and progression. Screening for small molecule drugs that can restore or improve motor function and NMJ conduction could lead to the discovery of new ALS treatments.

[0005] Other types of motor neurons include progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), and primary lateral sclerosis (PLS). Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a benzimidazole one compound, its preparation method, its pharmaceutical composition, and its application. The benzimidazole one compound of this invention has good pharmacodynamic and pharmacokinetic properties.

[0007] The inventors previously screened for the small molecule drug pimozide using an ALS gene mutation model, which can rescue the mutated phenotype. Not only in zebrafish and nematodes, but also in mouse Sod1 ALS models, pimozide enhances NMJ function. The inventors of this invention discovered that it works through T-type Ca... ++ Ion channels are involved, and a phase II clinical trial of the drug in ALS patients showed that it could improve neuromuscular function. However, because it was originally an antipsychotic drug, it has psychotropic side effects.

[0008] This invention modifies the structure of Pimozide to develop a new generation of drugs for treating ALS (Amyotrophic Lateral Sclerosis). The compound of this invention does not cross the blood-brain barrier, thus avoiding central nervous system side effects. The compound of this invention exhibits good efficacy and pharmacokinetic properties in animal model tests. This invention solves the above-mentioned technical problems through the following technical solutions.

[0009] This invention provides a compound or a pharmaceutically acceptable salt thereof as shown below:

[0010]

[0011]

[0012] In this invention, for A mixture.

[0013] In one aspect of the present invention, the "pharmaceutically acceptable salt" is a hydrochloride salt, and preferably, in the molecular structure of the hydrochloride salt, the molar ratio of the compound molecule to the hydrochloric acid molecule is 1:1.

[0014] In one aspect of this invention, the "pharmaceutically acceptable salt" is a salt as described below:

[0015]

[0016] The present invention also provides a pharmaceutical composition comprising the compound described in any of the above embodiments or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0017] The present invention also provides the use of any of the compounds described above or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described above, in the preparation of a medicament for the prevention and / or treatment of motor neurodegenerative diseases.

[0018] In one embodiment of the present invention, the motor neurodegenerative disease is amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, Kennedy's disease, or spinal muscular atrophy.

[0019] The present invention also provides a method for preventing and / or treating motor neurodegenerative diseases, comprising administering to an individual in need a therapeutically effective amount of the compound described in any of the above-described schemes or a pharmaceutically acceptable salt thereof.

[0020] In one embodiment of the present invention, the motor neurodegenerative disease is amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, Kennedy's disease, or spinal muscular atrophy.

[0021] The present invention also provides a method for preparing any of the above-mentioned compounds, which is one of the following methods: method 1, method 2, method 3, method 4 or method 5:

[0022] Method 1 comprises the following steps: under the action of an acid, a compound as shown in formula A-2 undergoes a deprotection reaction to prepare a compound as shown in formula D-3.

[0023]

[0024] Preferably, method 1 further comprises the following steps: in a solvent, under the action of a base, and in the presence of a phase transfer catalyst, pimozide undergoes a substitution reaction with a compound shown in formula A-1 to prepare a compound shown in formula A-2.

[0025] The compound represented by formula A-1 is prepared by the following method, comprising: in a solvent, under the action of PPh3 and a condensing agent, compound 1 A condensation reaction was carried out to prepare the compound shown in formula A-1;

[0026]

[0027] Method 2 comprises the following steps: in a solvent, under the action of a reducing agent, a compound as shown in Formula B-3 undergoes a reduction reaction to prepare a compound as shown in Formula 8-1.

[0028]

[0029] Preferably, method 2 further comprises the following step: in a solvent, under the action of a base, and in the presence of a phase transfer catalyst, pimozide undergoes a substitution reaction with a compound shown in formula B-2 to prepare a compound shown in formula B-3.

[0030] The compound shown in formula B-2 is prepared by the following method, comprising: a. 5-bromopent-1-ol and N3 in a solvent. - An azide reaction was performed to prepare the compound shown in Formula B-1. b. In the presence of triphenylphosphine and imidazole, the compound shown in formula B-1 was subjected to an iodination reaction with I2 to prepare the compound shown in formula B-2.

[0031]

[0032] Method 3 comprises the following steps: in the presence of a base, a compound of formula C1-2' undergoes a deprotection reaction to prepare a compound of formula M-10', wherein in the compound of formula C1-2', Pg is a hydroxyl protecting group.

[0033]

[0034] Preferably, method 3 further comprises the step of: in a solvent, in Cu 2+ In the presence of a nitrogen-containing tripentate ligand and a reducing agent, the compound shown in formula B-3 and the compound shown in formula C1-1' undergo an alkyne-azide click chemical reaction as shown below to prepare the compound shown in formula C1-2'.

[0035] Among them, the compound shown in formula B-3 is prepared by the method for preparing the compound shown in formula B-3 in method 2;

[0036] The compound shown in formula C1-1' is prepared by the following method, comprising: in a solvent, under the action of boron trifluoride diethyl ether, compound 3 The compound represented by formula C1-1' was prepared by condensation reaction with pento-4-yn-1-ol.

[0037]

[0038] Method 4, which includes the following steps: in a solvent, in Cu 2+In the presence of a nitrogen-containing tripentate ligand and a reducing agent, the compound shown in Formula B-3 and the compound shown in Formula B-4 undergo the alkyne-azide click chemistry reaction shown below to prepare compound 11'.

[0039]

[0040] Preferably, method 4 further comprises the following step: preparing the compound shown in formula B-3 by the method for preparing the compound shown in formula B-3 in method 2;

[0041] Method 5, which includes the following steps: in a solvent, in Cu 2+ In the presence of a nitrogen-containing tripentate ligand and a reducing agent, the compound shown in formula C3-1 undergoes an alkyne-azide click chemical reaction with the compound shown in formula B-3 as shown below to prepare compound 12'.

[0042]

[0043] Preferably, method 5 further comprises the following step: in a solvent, under the action of a base, and in the presence of DMAP and EDCI, propargyl-PEG2-amine undergoes an amide condensation reaction with biotin to prepare a compound as shown in formula C3-1.

[0044]

[0045] In method 1, the reaction conditions and operation of the substitution reaction can be the conventional reaction conditions and operation of such reactions in the art.

[0046] In method 1, the reaction conditions and operation of the condensation reaction can be the conventional reaction conditions and operation of such reactions in the art.

[0047] In method 1, the reaction conditions and operation of the deprotection reaction can be the conventional reaction conditions and operation of such reactions in the art.

[0048] In one aspect of the present invention, in method 1, the solvent in the substitution reaction is an amide solvent, such as DMF.

[0049] In one aspect of the present invention, in method 1, the base in the substitution reaction is an alkali metal carbonate, such as potassium carbonate.

[0050] In one aspect of the present invention, in method 1, the phase transfer catalyst is... Among them, R 1Independently, it is a C1-C6 alkyl group, such as butyl, and X is a halogen, such as Br; the phase transfer catalyst is, for example, TBAB.

[0051] In one aspect of the present invention, in method 1, the molar ratio of pimozide to the compound shown in formula A-1 in the substitution reaction is 1:(1.5 to 2.5), for example 1:2.

[0052] In one aspect of the present invention, in method 1, the molar ratio of the base to pimozide in the substitution reaction is 1:(1.5-2.5), for example 1:2.

[0053] In one aspect of the present invention, in method 1, the molar ratio of the phase transfer catalyst to the base in the substitution reaction is 1:(8-12), for example 1:10.

[0054] In one aspect of the present invention, in method 1, the reaction temperature of the substitution reaction is 80–100°C, for example, 90°C.

[0055] In one aspect of the present invention, in method 1, the solvent in the condensation reaction is an alkylbenzene solvent, such as toluene.

[0056] In one aspect of the present invention, in method 1, the condensation agent in the condensation reaction is DIAD or DEAD, for example, DIAD.

[0057] In one aspect of the present invention, in method 1, the molar ratio of PPh3 to compound 1 in the condensation reaction is (1-2):1, for example, 1.2:1.

[0058] In one aspect of the present invention, in method 1, the molar ratio of the condensing agent to compound 1 in the condensation reaction is (1-2):1, for example, 1.1:1.

[0059] In one aspect of the present invention, in method 1, the reaction temperature of the condensation reaction is such that the solvent is in a reflux state.

[0060] In one embodiment of the present invention, in method 1, the acid in the deprotection reaction is trifluoroacetic acid.

[0061] In one aspect of the present invention, in method 1, during the deprotection reaction, the molar ratio of the acid to the compound shown in formula A-2 is (0.05 to 0.15):1, for example, 0.08:1.

[0062] In one embodiment of the present invention, in method 1, the reaction system of the deprotection reaction further includes water, preferably, the volume ratio of water to acid is 1:(8-10), for example 1:9.

[0063] In method 2, the reaction conditions and operation of the substitution reaction can be the conventional reaction conditions and operation of such reactions in the art.

[0064] In method 2, the reaction conditions and operation of the azidation reaction can be the conventional reaction conditions and operation of such reactions in the art.

[0065] In method 2, the reaction conditions and operation of the iodination reaction can be the conventional reaction conditions and operation of such reactions in the art.

[0066] In one aspect of the present invention, in method 2, the solvent in the substitution reaction is an amide solvent, such as DMF.

[0067] In one aspect of the present invention, in method 2, the base in the substitution reaction is an alkali metal carbonate, such as potassium carbonate.

[0068] In one aspect of the present invention, in method 2, the phase transfer catalyst in the substitution reaction is... Among them, R 1 Independently, it is a C1-C6 alkyl group, such as butyl, and X is a halogen, such as Br; the phase transfer catalyst is, for example, TBAB.

[0069] In one aspect of the present invention, in method 2, the molar ratio of pimozide to the compound shown in formula B-2 in the substitution reaction is 1:(1.5 to 2.5), for example 1:2.

[0070] In one aspect of the present invention, in method 2, the molar ratio of the base to pimozide in the substitution reaction is (1.5-2.5):1, for example 2:1.

[0071] In one aspect of the present invention, in method 2, the molar ratio of the phase transfer catalyst to the base in the substitution reaction is 1:(8-12), for example 1:10.

[0072] In one aspect of the present invention, in method 2, the solvent in the azide reaction is an amide solvent, such as DMF.

[0073] In one aspect of the present invention, in method 2, during the azide reaction, the 5-bromopent-1-ol and N3 -The molar ratio is 1:(1.5~2.5), for example 1:2.

[0074] In one aspect of the present invention, in method 2, the reaction temperature of the azide reaction is 80-100°C, for example, 90°C.

[0075] In one aspect of the present invention, in method 2, during the azide reaction, the N3... - The N3 is prepared by the following method: reacting TMSN3 with a base in a solvent to prepare the N3. - The solvent may be an alcohol solvent, such as methanol; the base may be an alkali metal hydroxide, such as sodium hydroxide; and the molar ratio of TMSN3 to the base may be 1:(1 to 1.5), for example, 1:1.

[0076] In one aspect of the present invention, in method 2, during the iodination reaction, the molar ratio of the triphenylphosphine to the compound shown in formula B-1 is (1-2):1, for example, 1.5:1.

[0077] In one aspect of the present invention, in method 2, during the iodination reaction, the molar ratio of the imidazole to the compound shown in formula B-1 is (4-8):1, for example, 6:1.

[0078] In one aspect of the present invention, in method 2, during the iodination reaction, the molar ratio of I2 to the compound shown in formula B-1 is (1-2):1, for example, 1.5:1.

[0079] In one aspect of the present invention, in method 2, the reducing agent in the reduction reaction is H2. Preferably, the H2 is used in combination with Pd / C, and the Pd / C can be 10% Pd / C, where 10% represents the mass percentage of Pd in ​​Pd / C. The molar mass ratio of the compound shown in formula B-3 to Pd in ​​the Pd / C is 5 to 15 mmol / mg, for example, 8 mmol / mg.

[0080] In one aspect of the present invention, in method 2, the solvent in the reduction reaction is an alcohol solvent, such as methanol.

[0081] In method 3, the reaction conditions and operation of the alkyne-azide click chemistry reaction can be the conventional reaction conditions and operation of such reactions in the art.

[0082] In method 3, the reaction conditions and operation of the condensation reaction can be the conventional reaction conditions and operation of such reactions in the art.

[0083] In one aspect of the present invention, in method 3, the solvent in the alkyne-azide click chemistry reaction is an alcohol solvent, such as methanol.

[0084] In one aspect of the present invention, in method 3, during the alkyne-azide click chemistry reaction, the Cu... 2+ Provided by copper sulfate.

[0085] In one aspect of the present invention, in method 3, the nitrogen-containing tripentate ligand in the alkyne-azide click chemistry reaction is THPTA.

[0086] In one aspect of the present invention, in method 3, the reducing agent in the alkyne-azide click chemical reaction is sodium ascorbate.

[0087] In one aspect of the present invention, in method 3, the molar ratio of the compound shown in formula B-3 to the compound shown in formula C1-1' in the alkyne-azide click chemical reaction is 1:(1-2), for example 1:1.5.

[0088] In one aspect of the present invention, in method 3, during the alkyne-azide click chemistry reaction, the Cu... 2+ The molar ratio of the compound as shown in Formula B-3 is (0.01 to -0.1):1, for example, 0.05:1.

[0089] In one aspect of the present invention, in method 3, during the alkyne-azide click chemistry reaction, the nitrogen-containing tripentate ligand reacts with the Cu... 2+ The molar ratio is (1.5~2.5):1, for example 2:1.

[0090] In one aspect of the present invention, in method 3, the solvent in the condensation reaction is a haloalkane solvent, such as DCM.

[0091] In one aspect of the present invention, in method 3, the molar ratio of compound 3 to pent-4-yn-1-ol in the condensation reaction is 1:(3-5), for example 1:4.

[0092] In one aspect of the present invention, in method 3, the molar ratio of compound 3 to boron trifluoride diethyl ether compound in the condensation reaction is 1:(3-5), for example 1:4.

[0093] In one aspect of the present invention, in method 3, the base in the deprotection reaction is an alkali metal alkali alkali, such as sodium methoxide.

[0094] In one aspect of the present invention, in method 3, during the deprotection reaction, the molar ratio of the base to the compound represented by formula C1-2' is (1-1.5):1, for example, 1:1.

[0095] In one aspect of the present invention, in method 3, the deprotection reaction is carried out in an inert gas, such as N2.

[0096] In one aspect of the present invention, in method 3, in the deprotection reaction, in the compound represented by formula C1-2', Pg is independently (C1-C6 alkyl)C(=O)-, for example, acetyl.

[0097] In method 4, the reaction conditions and operation of the alkyne-azide click chemistry reaction can be the conventional reaction conditions and operation of such reactions in the art.

[0098] In one aspect of the present invention, in method 4, the solvent in the alkyne-azide click chemical reaction is an alcohol solvent and / or water, wherein the alcohol solvent is methanol; preferably, the solvent is an alcohol solvent and water, wherein the volume ratio of the alcohol solvent and water is (5-15):1, for example 9:1.

[0099] In one aspect of the present invention, in method 4, during the alkyne-azide click chemistry reaction, the Cu... 2+ Provided by copper sulfate.

[0100] In one aspect of the present invention, in method 4, the nitrogen-containing tripentate ligand in the alkyne-azide click chemistry reaction is THPTA (trihydroxypropyltriazolylmethylamine, CAS: 760952-88-3).

[0101] In one aspect of the present invention, in method 4, the reducing agent in the alkyne-azide click chemical reaction is sodium ascorbate.

[0102] In one aspect of the present invention, in method 4, the molar ratio of the compound shown in formula B-3 to the compound shown in formula B-4 in the alkyne-azide click chemical reaction is (1-2):1, for example 1.1:1 or 1.5:1.

[0103] In one aspect of the present invention, in method 4, during the alkyne-azide click chemistry reaction, the Cu... 2+ The molar ratio of the compound as shown in Formula B-3 is (0.01 to -0.1):1, for example, 0.05:1.

[0104] In one aspect of the present invention, in method 4, during the alkyne-azide click chemistry reaction, the nitrogen-containing tripentate ligand reacts with the Cu... 2+ The molar ratio is (1.5~2.5):1, for example 2:1.

[0105] In one aspect of the present invention, in method 4, during the alkyne-azide click chemistry reaction, the reducing agent reacts with the Cu... 2+ The molar ratio is (1.5~2.5):1, for example 2:1.

[0106] In one aspect of the present invention, in method 4, the alkyne-azide click chemistry reaction is carried out in an inert gas, such as N2.

[0107] In method 5, the reaction conditions and operation of the alkyne-azide click chemistry reaction can be the conventional reaction conditions and operation of such reactions in the art.

[0108] In method 5, the reaction conditions and operation of the amide condensation reaction can be the conventional reaction conditions and operation of such reactions in the art.

[0109] In one aspect of the present invention, in method 5, the solvent in the alkyne-azide click chemical reaction is an alcohol solvent and / or water, wherein the alcohol solvent is methanol; preferably, the solvent is an alcohol solvent and water, wherein the volume ratio of the alcohol solvent and water is (5-15):1, for example 9:1.

[0110] In one aspect of the present invention, in method 5, during the alkyne-azide click chemistry reaction, the Cu... 2+ Provided by copper sulfate.

[0111] In one embodiment of the present invention, in method 5, the nitrogen-containing tridentate ligand is THPTA (trihydroxypropyltriazolylmethylamine, CAS: 760952-88-3).

[0112] In one aspect of the present invention, in method 5, the reducing agent in the alkyne-azide click chemical reaction is sodium ascorbate.

[0113] In one aspect of the present invention, in method 5, the molar ratio of the compound represented by formula C3-1 to the compound represented by formula B-3 in the alkyne-azide click chemical reaction is (1-2):1, for example 1.1 or 1.5:1.

[0114] In one aspect of the present invention, in method 5, during the alkyne-azide click chemistry reaction, the Cu...2+ The molar ratio of the compound as shown in Formula B-3 is (0.01 to -0.1):1, for example, 0.05:1.

[0115] In one aspect of the present invention, in method 5, during the alkyne-azide click chemistry reaction, the nitrogen-containing tripentate ligand reacts with the Cu... 2+ The molar ratio is (1.5~2.5):1, for example 2:1.

[0116] In one aspect of the present invention, in method 5, during the alkyne-azide click chemistry reaction, the reducing agent reacts with the Cu... 2+ The molar ratio is (1.5~2.5):1, for example 2:1.

[0117] In one aspect of the present invention, in method 5, the alkyne-azide click chemistry reaction is carried out in an inert gas, such as N2.

[0118] In one aspect of the present invention, in method 5, the solvent in the amide condensation reaction is an amide solvent, such as DMF.

[0119] In one aspect of the present invention, in method 5, the base in the amide condensation reaction is an organic amine, for example... R 2 Independently, it is a C1-C6 alkyl group, such as ethyl, and the base is, for example, triethylamine.

[0120] In one embodiment of the present invention, in method 5, the molar ratio of propargyl-PEG2-amine to biotin in the amide condensation reaction is 1:(1-1.5), for example 1:1.2.

[0121] In one embodiment of the present invention, in method 5, the molar ratio of DMAP to propargyl-PEG2-amine in the amide condensation reaction is 1:(4-6), for example 1:5.

[0122] In one embodiment of the present invention, in method 5, the molar ratio of EDCI to propargyl-PEG2-amine in the amide condensation reaction is (1.5-3):1, for example 2:1.

[0123] The present invention also provides a compound as shown in Formula A-2, a compound as shown in Formula B-3, a compound as shown in Formula C1-2', a compound as shown in Formula B-4, a compound as shown in Formula C3-1, or a compound as shown in Formula C1-1':

[0124]

[0125]

[0126] In the compounds represented by formula C1-2', Pg is a hydroxyl protecting group, such as (C1-C6 alkyl)C(=O)-, or more specifically, an acetyl group.

[0127] When used in the specification and claims of this application, unless otherwise specifically indicated, the terms shall have the following meanings:

[0128] The term "pharmaceuticalally acceptable salt" refers to a salt prepared from the compounds of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or base. When the compounds of the present invention contain relatively acidic functional groups, a base addition salt can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, an acid addition salt can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts. See Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66:1-19 (1977), or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

[0129] The term "pharmaceuticalally acceptable excipient" refers to excipients or additives used in the manufacture of pharmaceutical products and the dispensing of prescriptions. It includes all substances contained in pharmaceutical preparations, excluding the active ingredient. See the Pharmacopoeia of the People's Republic of China (2020 Edition), Volume IV, or the Handbook of Pharmaceutical Excipients (Raymond C. Rowe, 2009 Sixth Edition).

[0130] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0131] The reagents and raw materials used in this invention are all commercially available.

[0132] The positive and progressive effects of this invention are as follows: This invention modifies the pimozide compound to make it less likely to cross the blood-brain barrier, thereby reducing the drug's side effects on the central nervous system and allowing for a higher drug dosage to improve peripheral neuromuscular function. The benzimidazole compounds of this invention can be used to treat amyotrophic lateral sclerosis (ALS) and other related motor neurodegenerative diseases; preferably, the compounds of this invention have good pharmacokinetic properties. Attached Figure Description

[0133] Figure 1 The results of the motility score test for C9orf72 orthologue alfa-1 nematodes after incubation with the compound.

[0134] Figure 2 The results of the motility score test for C9orf72-G4C2 nematodes after incubation with the compound.

[0135] Figure 3 The results of the motility score test for TDP-43(A315T) nematodes after incubation with the compound.

[0136] Figure 4 The results of the motility score test for SOD-1 (G93A) nematodes after incubation with the compound.

[0137] Figure 5 The results of the motility score test for FUS(S57Δ) nematodes after incubation with the compound.

[0138] Figure 6 Summary of the results of the test on the effectiveness of the compound on the movement of C. elegans Detailed Implementation

[0139] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0140] Example 1: Preparation of compound D-3 (anomeric mixture)

[0141]

[0142] (3aR,5S,6S,6aR)-2,2-dimethyl-5-((S)-epoxyethylene-2-yl)tetrahydrofurano[2,3-d][1,3]dioxacyclopentan-6-ol (A-1)

[0143] Under N2 atmosphere, PPh3 (7.15 g, 27.24 mmol) and DIAD (5.51 g, 24.24 mmol) were added to a stirred toluene solution of 1,2-O-isopropylidene-D-furanose (5.00 g, 22.70 mmol) cooled in an ice-water bath (50 mL). The reaction mixture was refluxed and stirred for 16 hours, then cooled and poured into ice water (100 mL). The resulting mixture was extracted with EtOAc (50 mL × 2). The combined organic layers were washed with brine (100 mL), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to give a residue, which was purified by column chromatography (silica gel, EtOAc / n-hexane = 1 / 2 (v / v)) to give a crude product. The crude product was recrystallized from i-Pr2O to give compound A-1, a white solid, 1.3 g (28%). 1 H NMR (500MHz, CDCl3) δ5.99(d,J=3.6Hz,1H),4.52(d,J=3.6Hz,1H),4.26(t,J=2.9Hz,1H),4.08(dd,J=4.3,2.6Hz,1H),3.44 (td,J=4.3,3.0Hz,1H),3.08(d,J=3.4Hz,1H),3.00(t,J=4.4Hz,1H),2.88(dd,J=4.6,2.9Hz,1H),1.48(s,3H),1.32(s,3H).

[0144] 1-(1-(4,4-bis(4-fluorophenyl)butyl)piperidin-4-yl)-3-((S)-2-hydroxy-2-((3aR,5R,6S,6aR)-6-hydroxy-2,2-dimethyltetrahydrofurano[2,3-d][1,3]dioxacyclopentan-5-yl)ethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (A-2)

[0145] Under a nitrogen atmosphere, K₂CO₃ (0.30 g, 2.16 mol) and TBAB (0.07 g, 0.21 mmol) were added to a stirred DMF solution of pimozide (0.50 g, 1.08 mmol) and compound A-1 (0.44 g, 2.16 mmol) in 5.0 mL. The reaction mixture was stirred at 90 °C for 5 hours, cooled, and poured into water (20 mL). The resulting mixture was extracted with EtOAc (10 mL × 2). The combined organic layers were washed with brine (10 mL × 4), dried over anhydrous MgSO₄, and concentrated on a rotary evaporator to obtain a residue, which was purified by column chromatography (silica gel, DCM / MeOH = 20 / 1 (v / v)) to give a white foam (crude A-2, 0.65 g), which could be used directly for the next step without further purification.

[0146] 1-(1-(4,4-bis(4-fluorophenyl)butyl)piperidin-4-yl)-3-((2R,3S,4S,5R)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-yl)methyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (D-3(anomeric mixture))

[0147] Under N2 atmosphere, a solution of compound A-2 (0.60 g, 0.90 mmol) in TFA (5.4 mL) and water (0.6 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated on a rotary evaporator to obtain a residue, which was purified by column chromatography (silica gel, DCM / MeOH = 10 / 1 (v / v)) to give a crude product. The crude product was ground with MTBE at room temperature to give compound D-3 (anomeric mixture). White solid, 110 mg (20%). mp 142.3℃-146.9℃. 1 H NMR(500MHz,DMSO)δ7.35(dd,J=8.6,5.6Hz,8H),7.28–7.14(m,4H),7.16–7.09(m,8H),7.02–7.01(m,4H),6.59(d,J=6.8Hz,1H),6.24 (d,J=4.7Hz,1H),5.19(d,J=5.4Hz,1H),5.15(d,J=5.5Hz,1H),4.96(d,J=4.8Hz,1H),4.88(d,J=4.7Hz,1H),4.81–4.78(t,J=4.5Hz,1H ),4.77(d,J=4.8Hz,1H),4.51(d,J=6.7Hz,1H),4.20–4.10(m,4H),4.01(t,J=7.5Hz,2H),3.92(m,1H),3.87–3.79(m,2H),3.47–3.41( m,2H),3.17–3.11(m,2H),3.06–2.98(m,2H),2.96–2.83(m,4H),2.42–2.24(m,6H),2.16–1.83(m,8H),1.63(s,4H),1.35–1.33(m,4H). 13CNMR (126MHz, DMSO) δ161.57, 159.65, 157.80 (q, J = 122.6Hz), 153.06, 153.03, 141.16, 129. 93,129.90,129.30,129.24,127.83,120.81,120.62,120.54,118.43,116.04,115.16,115. 00,109.08,108.84,108.59,96.76,92.18,76.14,74.60,73.90,72.72,72.58,72.13,69.50 ,57.11,52.57,50.77,48.61,42.95,32.68,28.67,26.80,25.07.ESI-HRMS:(m / z)calcd.for C 34 H 39 F2N3O6([M+H) + 624.2880, found: 624.2878. HPLC purity = 97.10% (composed of two peaks from two anomers).

[0148] (HPLC test conditions: Column: Waters XBridge C) 18 Column size: 50 mm × 4.6 mm, 3.5 μm; Mobile phase: 0.01 M HCO2NH4 in methanol solution (50 / 50 at 0 min, 70 / 30 at 10 min, 70 / 30 at 14 min, 50 / 50 at 20 min); Flow rate: 0.7 mL / min; UV detector: 210 nm; Column temperature: 25 °C

[0149] Example 2 Synthesis of Compound 8

[0150]

[0151] 5-Azide pentanol (B-1)

[0152] Under N2 atmosphere, in an ice-water bath, NaOH (2.39 g, 59.86 mmol) was added to a solution of TMSN3 (6.89 g, 59.86 mmol) in MeOH (50 mL). The reaction mixture was stirred at room temperature for 1 hour, then concentrated on a rotary evaporator to give the residue. The residue was dissolved in DMF (50 mL), and then 5-bromopentan-1-ol (5.0 g, 29.93 mmol) was added. The resulting mixture was stirred at 90 °C for 12 hours under N2 atmosphere. After cooling to room temperature, the mixture was poured into water (150 mL). The resulting mixture was extracted with EtOAc (50 mL × 3). The combined organic layers were washed with brine (50 mL × 4), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to give the residue, which was purified by column chromatography (silica gel, EtOAc / n-hexane = 1 / 3 (v / v)) to give compound B-1. Colorless oily substance, 2.12 g (55%). 1 H NMR (500MHz, CDCl3) δ3.67(t,J=6.5Hz,2H),3.29(t,J=6.9Hz,2H),1.70–1.56(m,4H),1.52–1.44(m,2H).

[0153] 1-Azide-5-iodopentane (B-2)

[0154] I2 (6.24 g, 24.62 mmol) and imidazole (6.70 g, 98.48 mmol) were added to a DCM (20 mL) stirred solution of PPh3 (6.45 g, 24.62 mmol) cooled in an ice-water bath, and the mixture was stirred under N2 atmosphere for 0.5 h. Compound B-1 (2.12 g, 16.41 mmol) was then added to the reaction mixture, and stirring was continued at room temperature under N2 atmosphere for 12 h. The reaction mixture was poured into water (50 mL), and the mixture was extracted with DCM (50 mL × 2). The combined extracts were washed with saturated sodium sulfite solution (50 mL × 1) and brine (50 mL × 1), dried over anhydrous MgSO4, concentrated on a rotary evaporator, and the residue was purified by column chromatography (silica gel, EtOAc / n-hexane = 1 / 15 (v / v)) to give compound B-2. Colorless oily substance, 2.10 g (54%). 1 H NMR (500MHz, CDCl3) δ3.29(t,J=6.8Hz,2H),3.20(t,J=6.9Hz,2H),1.93–1.79(m,2H),1.62(dd,J=14.8,7.1Hz,2H),1.53–1.44(m,2H).

[0155] 1-(5-Azidepentyl)-3-(1-(4,4-bis(4-fluorophenyl)butyl)piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (B-3)

[0156] Under a nitrogen atmosphere, K₂CO₃ (0.18 g, 1.30 mmol) and TBAB (0.04 g, 0.13 mmol) were added to a stirred DMF (3.0 mL) solution of pimozide (0.30 g, 0.65 mmol) and compound B-2 (0.31 g, 1.30 mmol). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 12 hours and then poured into water (10 mL). The resulting mixture was extracted with EtOAc (5 mL × 3). The combined extracts were washed with brine (10 mL × 4), dried over anhydrous MgSO₄, concentrated on a rotary evaporator, and the residue was purified by column chromatography (silica gel, EtOAc / n-hexane = 1 / 1 (v / v)) to give compound B-3. 0.24 g (65%) of a colorless oil. 1 H NMR(500MHz, CDCl3)δ7.27(d,J=7.7Hz,1H),7.19–7.15(m,4H),7.07–7.02(m,4H),6.99–6.95(m,5H),4.38–4.33(m,1H),3.90–3.86(m,3H),3 .26(t,J=6.9Hz,2H),2.99(d,J=11.2Hz,2H),2.44–2.37(m,4H),2.08– 2.00(m,4H),1.80–1.74(m,4H),1.69–1.61(m,2H),1.49–1.41(m,4H). 13 C NMR (126MHz, CDCl3) δ162.34,160.40,153.78,140.56,129.47(2C),129.1 4(2C),129.08(2C),128.24,120.80(d,J=5.7Hz,2C),115.38(2C),115.22 (2C),109.70,107.63,58.31,53.34,51.23(2C),51.11,49.77,40.88,33.86,29.72,29.27,28.54,27.94,25.65,24.03.ESI-HRMS:(m / z)calcd.for C 33 H 38 F2N6O([M+H)) + )573.3148,found:573.3152.

[0157] 5-(3-(1-(4,4-bis(4-fluorophenyl)butyl)piperidin-4-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)pent-1-ammonium chloride (compound 8)

[0158] A mixture of compound B-3 (0.24 g, 41.90 mmol) and 10% Pd / C (50 mg) in MeOH (3 mL) was subjected to standard hydrogenation overnight at room temperature and atmospheric pressure (balloon). TLC analysis indicated that the reaction was complete. The reaction mixture was filtered through a zeolite filter, and the filtrate was evaporated on a rotary evaporator to obtain a residue. The residue was purified by column chromatography (silica gel, DCM / MeOH = 10 / 1 (v / v)) to give a crude product. The crude product was dissolved in EtOAc (3 mL), and then a methanol solution of hydrogen chloride (0.5 mL, 0.5 M) was added. The resulting mixture was stirred at room temperature for 1 hour, and then evaporated on a rotary evaporator to give a residue. The residue was ground with EtOAc (5 mL) at room temperature to give compound 8. Yellow solid, 110 mg (46%). mp 76.0℃-79.8℃. 1 HNMR(500MHz,CD3OD)δ7.44(s,1H),7.32(dd,J=8.1,5.4Hz,4H),7.19–7.14 (m,3H),7.03(t,J=8.6Hz,4H),4.62–4.59(m,1H),4.04(t,J=7.1Hz,1H),3. 92(t,J=6.6Hz,2H),3.68–3.65(m,2H),3.22(s,4H),3.00–2.77(m,4H),2.1 5(d,J=4.6Hz,2H),2.04–2.00(m,2H),1.86–1.64(m,6H),1.46–1.43(m,2H). 13 C NMR(126MHz,MeOD)δ164.03,162.09,155.25,141.76,130.66(3C),130.59(3C),129.20,122.98(d,J=16.3Hz,2C),116.46(2C),116.29(2C),1 10.47,109.64,61.66,58.15,53.47,50.58,41.70,40.68,33.80,28.96,28.24,27.62,24.70,24.12,20.98,14.59.ESI-HRMS:(m / z)calcd.for C 33 H 40 F2N4O([M+H)) +)547.3243, found:547.3244. HPLC purity = 95.89%.

[0159] (HPLC test conditions: Column: Waters XBridge C) 18 Column size: 50mm × 4.6mm, 3.5μm; Mobile phase: 0.01M HCO2NH4 in methanol solution (60 / 40at 0min, 70 / 30at 8min, 70 / 30at 15min, 60 / 40at 22min, 60 / 40at 25min); Flow rate: 0.7mL / min; UV detector: 210nm; Column temperature: 25℃

[0160] Example 3 Synthesis of compound M-10'

[0161]

[0162] (2R,3R,4S,5S,6R)-2-(acetoxymethyl)-6-(pent-4-yn-1-yloxy)tetrahydro-2H-pyran-3,4,5-triacyltriacetate (C1-1)

[0163] To a stirred solution of 1,2,3,4,6-O-pentaacetyl-D-pyranomannose (5.00 g, 12.81 mmol) and pent-4-yn-1-ol (4.31 g, 51.23 mmol) in DCM (125 mL), boron trifluoride diethyl ether (7.27 g, 51.23 mmol) was added. The reaction mixture was stirred at room temperature for 12 hours under N2 atmosphere, then poured into water (100 mL). The resulting mixture was extracted with DCM (50 mL × 2). The combined organic layers were washed with saturated sodium bicarbonate solution (50 mL) and brine (50 mL), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to give the residue, which was purified by column chromatography (silica gel, EtOAc / n-hexane = 1 / 4 (v / v)) to give compound C1-1. 0.85 g (16%) of a yellow oil. 1H NMR (500MHz, CDCl3) δ5.35–5.24(m,3H),4.83(d,J=1.5Hz,1H),4.29(dd,J= 12.2,5.2Hz,1H),4.11(dd,J=12.2,2.4Hz,1H),4.03–4.00(m,1H),3.87–3.8 2(m,1H),3.56(dt,J=9.7,5.9Hz,1H),2.35–3.31(m,2H),2.16(s,3H),2.11( s,3H),2.05(s,3H),2.00(s,3H),1.97(t,J=2.7Hz,1H),1.87–1.79(m,2H)).

[0164] (2R,3R,4S,5S,6R)-2-(acetoxymethyl)-6-(3-(1-(5-(3-(4-bis(4-fluorophenyl)butyl)piperidin-4-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)pentyl)-1H-1,2,3-triazol-4-yl)propoxy)tetrahydro-2H-pyran-3,4,5-triacyltriacetate (C1-2)

[0165] THPTA (38 mg, 0.087 mmol) and CuSO4 (7 mg, 0.043 mmol) were dissolved in water (0.4 mL), and then added to a MeOH (8 mL) solution of B-3 (500 mg, 0.87 mmol) and compound C1-1 (540 mg, 1.31 mmol). Freshly prepared sodium ascorbate (18 mg, 0.087 mmol) was added to water (0.4 mL), and the mixture was stirred at room temperature under N2 atmosphere for 24 hours. The mixture was diluted with water (20 mL), and extracted with EtOAc (10 mL × 2). The combined extracts were washed with brine (50 mL), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to give the residue, which was purified by column chromatography (silica gel, DCM / MeOH = 10 / 1 (v / v)) to give compound C1-2. White foam, 0.75 g (87%). 1H NMR(500MHz, CDCl3)δ7.30(s,1H),7.26(s,2H),7.19–7.16(m,4H),7.08–7.02(m,2H),6.97(t,J=8.6Hz,4H), 5.34–5.22(m,3H),4.80(d,J=1.1Hz,1H),4.37–4.26(m,4H),4.10(dd,J=12.0,2.0Hz1H),4.02(m,1H),3.90–3 .84(m,3H),3.79–3.74(m,1H),3.58–3.42(m,1H),2.99(d,J=8.5Hz,2H),2.80(t,J=7.5Hz,2H),2.40(s,4H), 2.15(s,3H),2.09(s,3H),2.04(s,3H),1.99(s,3H),2.11–1.93(m,8H),1.80–1.75(m,6H),1.43–1.37(m,2H). 13 C NMR (126MHz, CDCl3) δ170.70,170.16,169.98,169.77,162.34,160.39,153.76,147.08,140.53,129.37, 129.13(2C),129.07(2C),128.19,120.84,120.80,115.38(2C),115.21(2C),109.71,107.63,97.59,69. 64,69.17,68.50,67.54,66.18,62.49,60.41,58.28(2C),53.31(2C),51.08,49.91,49.75,40.69,33.83 ,29.90,29.23,28.90,27.74,25.61,23.76,22.29,20.92,20.75,20.73(2C).ESI-HRMS:(m / z)calcd.for C 52 H 64 F2N6O 11 ([M+H)) + )987.4674,found:987.4687.

[0166] 1-(1-(4,4-bis(4-fluorophenyl)butyl)piperidin-4-yl)-3-(5-(4-(3-(((2R,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)-1H-1,2,3-triazol-1-yl)pentyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (compound M-10')

[0167] Na (12 mg, 0.52 mmol) was dissolved in MeOH (5 mL) in an ice-water bath, and then compound C1-2 (0.50 g, 0.51 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. After completion, freshly activated cation exchange resin (H2O) was added. + Add 2.0 g of the mixture and continue stirring until the reaction mixture becomes neutral (pH = 7). Filter the reaction mixture, evaporate the filtrate on a rotary evaporator to give a crude product, which is purified by preparative HPLC to give compound M-10' as a white solid, 239 mg (58%). mp 79.8℃-84.0℃. 1 H NMR(500MHz, DMSO)δ7.79(s,1H),7.34(dd,J=8.5,5.7Hz,4H),7.24–7.22(m,1H),7.16–7.14(m,1H),7.11(t,J=8.8Hz,4H),7.06–6.98(m, 2H),4.71(dd,J=10.4,4.8Hz,2H),4.60(s,1H),4.56(d,J=5.8Hz,1H),4.45(t,J=5.9Hz,1H),4.25(t,J=7.0Hz,2H),4.18–4.07(m,1H),4.0 0(t,J=7.9Hz,1H),3.77(t,J=7.0Hz,2H),3.67–3.60(m,3H),3.48–3.42(m,2H),3.39–3.33(m,2H),3.33–3.30(m,1H),2.89(d,J=11.1Hz, 2H),2.66–2.58(m,2H),2.34–2.26(m,4H),2.02–1.94(m,4H),1.82–1 .79(m,4H),1.65–1.59(m,4H),1.36–1.30(m,2H),1.24–1.18(m,2H). 13C NMR(126MHz,DMSO)δ162.05,160.12,153.29,146.75,141.76,129.80(3C),129.74(3C), 129.44,128.42,122.12,121.08(d,J=13.8Hz,2C),115.64(3C),115.47(3C),109.29,10 8.42,100.26,74.51,71.52,70.86,67.50,65.98,61.79,57.81,53.18,51.29,49.44,49 .17,33.33,29.73,29.43,29.12,27.66,25.63,23.56,22.41.ESI-HRMS:(m / z)calcd.for C 44 H 56 F2N6O7([M+H) + 819.4251, found: 819.4252. HPLC purity = 99.67%.

[0168] (HPLC test conditions: Column: Waters XBridge C) 18 Column size: 50 mm × 4.6 mm, 3.5 μm; Mobile phase: 0.01 M HCO2NH4 in methanol solution (60 / 40 at 0 min, 70 / 30 at 8 min, 70 / 30 at 15 min, 60 / 40 at 20 min); Flow rate: 0.7 mL / min; UV detector: 210 nm; Column temperature: 25 °C

[0169] Example 4 Synthesis of Compound 11'

[0170]

[0171] 3-(2-((1-(5-(3-(1-(4,4-bis(4-fluorophenyl)butyl)piperidin-4-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)pentyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)propionic acid (compound 11')

[0172] THPTA (27 mg, 0.061 mmol) and CuSO4 (5 mg, 0.031 mmol) were dissolved in water (0.25 mL), and then added to a solution of B-3 (350 mg, 0.61 mmol) and propargyl-PEG2-acid (96 mg, 0.56 mmol) in MeOH (4.5 mL). A freshly prepared sodium ascorbate solution (12 mg, 0.061 mmol) in water (0.25 mL) was added, and the reaction mixture was stirred at room temperature under N2 atmosphere for 12 hours. The reaction mixture was diluted with water (10 mL), and the resulting mixture was extracted with EtOAc (10 mL × 2). The combined extracts were washed with brine (10 mL × 1), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to give the residue. The residue was purified by preparative HPLC to give compound 11' (n = 2). White solid, 113 mg (27%). mp 61.8℃-66.0℃. 1 H NMR(500MHz, DMSO)δ8.01(s,1H),7.34(dd,J=8.4,5.7Hz,4H),7.25–7.23(m,1H),7.16–7.12(m,1H),7.10(t,J=8 .8Hz,4H),7.04–7.01(m,2H),4.48(s,2H),4.30(t,J=7.0Hz,2H),4.16–4.11(m,1H),4.00(t,J=7.8Hz,1H),3.78( t,J=7.0Hz,2H),3.58(t,J=6.3Hz,2H),3.51(s,2H),3.50(s,2H),2.90(d,J=11.0Hz,2H),2.42(t,J=6.3Hz,2H),2 .36–7.26(m,4H),2.03–1.97(m,4H),1.86–1.80(m,2H),1.67–1.59(m,4H),1.35–1.32(m,2H),1.26–1.20(m,2H). 13C NMR(126MHz,DMSO)δ173.25,162.05,160.13,153.28,144.32,141.76,129.79(3C) ,129.73(3C),129.43,128.41,124.12,121.08(d,J=13.8Hz,2C),115.64(3C),115. 47(3C),109.29,108.42,70.00,69.31,66.82,64.02,57.74,53.12,51.21,49.57,4 9.16,35.39,33.31,29.73,29.04,27.65,25.55,23.54.ESI-HRMS:(m / z)calcd.for C 41 H 50 F2N6O5([M+H) + )745.3884, found:745.3882. HPLC purity = 96.35%.

[0173] (HPLC test conditions: Column: Waters XBridge C) 18 Column size: 50 mm × 4.6 mm, 3.5 μm; Mobile phase: 0.01 M HCO2NH4 in methanol solution = 60 / 40 at 0 min, 70 / 30 at 8 min, 70 / 30 at 15 min, 60 / 40 at 20 min; Flow rate: 0.7 mL / min; UV detector: 210 nm; Column temperature: 25 °C

[0174] Example 5 Synthesis of Compound 12'

[0175]

[0176] 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-N-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)pentanamide (C3-1)

[0177] To a stirred solution of propargyl-PEG2-amine (1.0 g, 6.98 mmol) and biotin (2.05 g, 8.39 mmol) in DMF (15 mL), Et3N (1.41 g, 13.96 mmol), DMAP (0.17 g, 1.39 mmol), and EDCI (2.68 g, 13.9 mmol) were added. The reaction mixture was stirred at room temperature for 12 hours under N2 atmosphere. The reaction mixture was concentrated on a rotary evaporator to obtain a residue, which was purified by column chromatography (silica gel, DCM / MeOH = 10 / 1 (v / v)) to give a white gel (crude C3-1, 1.20 g), which could be used directly for the next step without further purification.

[0178] N-(2-(2-((1-(5-(3-(1-(4,4-bis(4-fluorophenyl)butyl)piperidin-4-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)pentyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (compound 12'(n=2))

[0179] THPTA (31 mg, 0.070 mmol) and CuSO4 (6 mg, 0.035 mmol) were dissolved in water (0.30 mL), and then a MeOH solution (5.4 mL) of B-3 (400 mg, 0.69 mmol) and compound C3-1 (390 mg, 1.047 mmol) was added. A freshly prepared sodium ascorbate solution (14 mg, 0.070 mmol) in water (0.30 mL) was added, and the reaction mixture was stirred at room temperature under N2 atmosphere for 12 hours. The mixture was diluted with water (10 mL), and extracted with EtOAc (10 mL × 2). The combined organic layers were washed with brine (50 mL), dried over anhydrous MgSO4, and concentrated on a rotary evaporator to give the residue. The residue was purified by preparative HPLC to give compound 12', a white solid, 252 mg (38%). mp 72.1 °C–76.3 °C. 11H NMR (500 MHz, DMSO) δ 8.03 (s, 1H), 7.83 (t, J = 5.6 Hz, 1H), 7.36–7.33 (m, 4H), 7.24–7.23 (m, 1H), 7.16–7.14 (m, 1H), 7.12–7.08 (t, J = 9.00 Hz, 4H), 7.04–7.00 (m, 2H), 6.41 (s, 1H), 6.35 (s, 1H), 4.49 (s, 2H), 4.32–4.28 (m, 3H), 4.12–4.10 (m, 2H), 4.00 (t, J = 7.9 Hz, 1H), 3.78 (t, J = 7.1 Hz, 2H), 3.55–3.53 (m, 2H), 3.52–3.50 (m, 2H), 3.38 (t, J = 5.9 Hz, 2H), 3.18 (q, J = 5.8 Hz, 2H), 3.09–3.06 (m, 1H), 2.89 (d, J = 11.0 Hz, 2H), 2.80 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.34–2.26 (m, 4H), 2.07–2.04 (m, 2H), 2.02–1.95 (m, 4H), 1.86–1.80 (m, 2H), 1.67–1.59 (m, 5H), 1.52–1.41 (m, 3H), 1.36–1.27 (m, 6H). 13 13C NMR (126 MHz, DMSO) δ 172.59, 163.17, 162.05, 160.13, 153.29, 144.30, 141.77, 129.79 (3C), 129.73 (3C), 129.43, 128.44, 124.15, 121.09 (d, J = 14.7 Hz, 2C), 115.64 (3C), 115.47 (3C), 109.28, 108.42, 69.96, 69.67, 69.36, 64.02, 61.49, 59.66, 57.81, 55.89, 53.19, 51.30, 49.58, 49.18, 40.58, 38.91, 35.56, 33.33, 29.76, 29.13, 28.65, 28.50, 27.67, 25.72, 25.65, 23.56. ESI-HRMS: (m / z) calcd. for C 50 H 65 F2N9O5S ([M + H] + ) 492.4870, found: 492.4871. HPLC purity = 99.35%.

[0180] (HPLC test conditions: Column: Waters XBridge C) 18 50mm × 4.6mL, 3.5μm; Mobile phase: 0.01M HCO2NH4 in methanol solution = 60 / 40at 0min, 70 / 30at 8min, 70 / 30at 15min, 60 / 40at 20min; Flow rate: 0.7mL / min; UV detector: 210nm; Column temperature: 25℃

[0181] Example 1: Effectiveness test of the compound on the movement of C. elegans

[0182] 1.1 Handling of C.elegans

[0183] C. elegans was preserved on Escherichia coli OP50 bacterial growth on NGM agar plates at 20°C. Experiments using the invertebrate C. elegans do not require ethical approval.

[0184] 1.2 C. elegans phenotype

[0185] To determine the relevant kinetic defect phenotypes, the kinetic behavior of mutant strains and N2 worms was first compared in M9 liquid screening using the wMicrotracker screening system.

[0186] 1.3 Worm Preparation

[0187] To obtain an age-synchronized worm population, approximately 20 hermaphroditic adults were transferred to *E. coli* OP50 bacterial colonies on NGM plates (16 x 92 mm petri dishes, Sarstedt Inc., Montreal) and stored at 20°C for 3–4 days. Pregnant adults were collected using M9 buffer (1 M KH₂PO₄, 1 M Na₂HPO₄, 1 M NaCl, 1 M MgSO₄) and centrifuged at 4000 rpm (A-4-81 rotor, Eppendorf 580R) for 2 minutes at 4°C. After centrifugation, the precipitate was resuspended for 5 minutes in a solution containing 1 M NaOH and bleach (1:1), followed by vigorous vortexing to degrade the worms, leaving a precipitate containing only eggs. The precipitate was washed four times with 50 mL of M9 buffer and then centrifuged at 4000 rpm for 1 minute at 4°C. The eggs were cultured on bacteria-free NGM plates and stored overnight at 20°C. Wash four times with 50 mL M9 buffer, then centrifuge at 4000 rpm for 1 minute at 4°C. Culture the eggs on bacteria-free NGM plates and incubate overnight at 20°C. The next day, collect the hatched L1 worms in M9 buffer, transfer to plates coated with OP50 E. coli, and incubate at 20°C until they reach the L4 stage. Optimize the worm / well ratio by counting the number of worms in 10 μL, then adjust the volume to obtain 5 worms / 10 μL. Distribute approximately 50 age-synchronized L4-juvenile adults into 100 μL of M9 buffer + 2% DMSO (v:v) (untreated) or the desired concentration of the compound (treated), and distribute in 96-well plates (BioLite 96-well multi-well Thermo Scientific Canada). The final concentration of dimethyl sulfoxide (DMSO) is 2% v / v.

[0188] 1.4 Drug Screening

[0189] Microplates were evaluated for up to 10 hours at 20°C using a 96-channel infrared tracking device (WMicrotracker ONE, PhylumTech, Argentina) (Ref. Simonetta SH, Golombek DA. An automated tracking system for Caenorhabditis elegans locomotor behavior and circadian studies application. J NeurosciMethods. 2007 Apr 15; 161(2):273-80.). This machine was able to automatically track the dynamic activity of Caenorhabditis elegans in 96-well microplates. Measurements were repeated three times. Two infrared beams passed through each well from top to bottom, and the detector determined the frequency at which the beams were interrupted by the worms moving in the wells. This signal was used to calculate the movement score, i.e., the amount of movement of the animal within a fixed time period. The mean movement score was compared with the mean movement score of the untreated control group and the entire plate. Drugs that showed significant improvement in the mutant phenotype were considered positive candidates.

[0190] Results were analyzed using GraphPad Prism6 (La Jolla, CA 92037, USA). Each point corresponds to the activity count / 30-minute mean of the replicated sample. One-way ANOVA multiple comparisons were performed using the Dunnett test to assess significance.

[0191] 1.5C. elegans strain

[0192] The nematode strains used in these embodiments were purchased from the Caenorhabditis Genetics Center (CGC) in the United States or as cited in the relevant references.

[0193] KRA315 (C9orf72-G4C2, or C9orf72+)(See: Sonobe Y, Aburas J, Krishnan G, Fleming AC, Ghadge G, Islam P, Warren EC, Gu Y, Kankel MW, Brown AEX, Kiskinis E, Gendron TF, Gao FB, Roos RP, Kratsios P. A C. elegans model of C9orf72-associated ALS / FTD uncovers a conserved role for eIF2D in RAN translation. Nat Commun. 2021 Oct 15;12(1):6025.)

[0194] C9orf72 orthologue alfa-1 (C9orf72-KO)(See: Therrien M, Rouleau GA, Dion PA, Parker JA. Deletion of C9ORF72 results in motor neuron degeneration and stress sensitivity in C. elegans. PLoS One. 2013 Dec 12;8(12):e83450.)

[0195] SOD-1(G93A)(See: Baskoylu SN, Yersak J, O'Hern P, Grosser S, Simon J, Kim S, Schuch K, Dimitriadi M, Yanagi KS, Lins J, Hart AC. Single copy / knock-in models of ALS SOD1 in C. elegans suggest loss and gain of function have different contributions to cholinergic and glutamatergic neurodegeneration. PLoS Genet. 2018 Oct 8;14(10):e1007682.)

[0196] TDP-43(A315T)(Please refer to: Vaccaro A, Tauffenberger A, Aggad D, Rouleau G, Drapeau P, Parker JA.Mutant TDP-43and FUS cause age-dependent paralysis and neurodegeneration in C.elegans.PLoS One.2012;7(2):e31321.)

[0197] FUS(S57Δ)(See: Labarre A, Tossing G, Maios C, Doyle JJ, Parker JA. Asingle copy transgenic mutant FUS strain reproduces age-dependent ALSphenotypes in C.elegans.MicroPubl Biol.2021Sep22; 2021:10.17912 / micropub.biology.000473.)

[0198] N2 wild type: C. elegans var. Bristol. (See: https: / / cgc.umn.edu / strain / N2)

[0199] 1.6 ALS models of various C. elegans species and results of motility score tests after compound incubation

[0200] The ALS models of each C. elegans and the results of the motility score tests after compound incubation are shown in the figure. Figures 1-5 And Tables 1-1 to 1-5.

[0201] Figures 1-5 And in Tables 1-1-1-5: comp 1 is compound D-3; comp 2 is compound 8; comp 3 is compound M-10'; comp 4 is compound 11'; comp 5 is compound 12'; comp 6 is glucose; comp 7 is pimozide; pimozide represents pimozide; edaravone represents edaravone.

[0202] Figure 1 The results of the motility score test for C9orf72 orthologue alfa-1 nematodes after incubation with the compound.

[0203] The experimental results of each experimental group and the control group were compared using Dunnett's multiple comparisons method. Figure 1 In the example: C9orf72 vs. comp 2 (10uM), * indicates a p-value of 0.0196; C9orf72 vs. comp 4 (10uM), ** indicates a p-value of 0.0027; C9orf72 vs. comp 6 (10uM), * indicates a p-value of 0.0438; C9orf72 vs. comp 4 (1uM), * indicates a p-value of 0.0327; C9orf72 vs. comp 7 (1uM), * indicates a p-value of 0.0213; C9orf72 vs. N2, **** indicates a p-value < 0.0001.

[0204] Table 1-1 Motility scores of C9orf72 orthologue alfa-1 nematodes after incubation with the compound.

[0205] Group Nematode model Exercise score comp1 10uM C9orf72 orthologue alfa-1 365.40 comp2 10uM C9orf72 orthologue alfa-1 392.0 comp3 10uM C9orf72 orthologue alfa-1 376.4 comp4 10uM C9orf72 orthologue alfa-1 408.5 comp5 10uM C9orf72 orthologue alfa-1 373.1 comp6 10uM C9orf72 orthologue alfa-1 384.2 comp7 10uM C9orf72 orthologue alfa-1 374.9 DMSO 2% C9orf72 orthologue alfa-1 302.9 comp 1 1uM C9orf72 orthologue alfa-1 351.3 comp 2 1uM C9orf72 orthologue alfa-1 379.8 comp 3 1uM C9orf72 orthologue alfa-1 377.7 comp 4 1uM C9orf72 orthologue alfa-1 387.1 comp 5 1uM C9orf72 orthologue alfa-1 379.0 comp 6 1uM C9orf72 orthologue alfa-1 363.9 comp 7 1uM C9orf72 orthologue alfa-1 391.2 / N2 459.2

[0206] Figure 2 The results of the motility score test for C9orf72-G4C2 nematodes after incubation with the compound.

[0207] The experimental results of each experimental group and the control group were compared using Dunnett's multiple comparisons method. Figure 2 In the example: C9 G4C2 vs. comp4 (10uM), ** indicates a p-value of 0.0017; C9 G4C2 vs. comp6 (10uM), ** indicates a p-value of 0.0033; C9 G4C2 vs. comp7 (10uM), *** indicates a p-value of 0.0002; C9G4C2 vs. comp2 (1uM), * indicates a p-value of 0.0221; C9 G4C2 vs. comp3 (1uM), * indicates a p-value of 0.0266; C9G4C2 vs. N2, **** indicates a p-value < 0.0001.

[0208] Table 1-2 Motility scores of C9orf72-G4C2 nematodes after incubation with the compound

[0209]

[0210]

[0211] Figure 3 The results of the motility score test for TDP-43(A315T) nematodes after incubation with the compound.

[0212] The experimental results of each experimental group and the control group were compared using Dunnett's multiple comparisons method. Figure 3 In the example, TDP43 vs. comp2 (10uM), ** indicates a p-value of 0.0095; TDP43 vs. comp3 (10uM), *** indicates a p-value of 0.0004; TDP43 vs. comp4 (10uM), ** indicates a p-value of 0.0091; TDP43 vs. comp5 (10uM), *** indicates a p-value of 0.0001; TDP43 vs. comp6 (10uM), * indicates a p-value of 0.0122; TDP43 vs. comp7 (10uM), ** indicates a p-value of 0.0069; TDP43 vs. pimozide (10uM), **** indicates a p-value < 0.0001; TDP43 vs. edaravone (10uM), *** indicates a p-value of 0.0005; TDP43 vs. N2, **** indicates a p-value < 0.0001; TDP43 `vs.comp1(1uM)`, *** indicates a p-value of 0.0007; `TDP43 vs.comp2(1uM)`, ** indicates a p-value of 0.0022; `TDP43 vs.comp3(1uM)`, **** indicates a p-value < 0.0001; `TDP43 vs.comp5(1uM)`, **** indicates a p-value < 0.0001; `TDP43 vs.comp7(1uM)`, * indicates a p-value of 0.0124; `TDP43 vs.pimozide(1uM)`, *** indicates a p-value of 0.0009.

[0213] Table 1-3 Motility scores of TDP-43 (A315T) nematodes after incubation with the compound

[0214]

[0215]

[0216] Figure 4 The results of the motility score test for SOD-1 (G93A) nematodes after incubation with the compound.

[0217] The experimental results of each experimental group and the control group were compared using Dunnett's multiple comparisons method. Figure 4In the table, SOD-1(G93A) vs. comp2(10uM), * indicates a p-value of 0.0219; SOD-1(G93A) vs. comp3(10uM), **** indicates a p-value < 0.0001; SOD-1(G93A) vs. comp4(10uM), *** indicates a p-value of 0.0008; SOD-1(G93A) vs. comp5(10uM), *** indicates a p-value of 0.0005; SOD-1(G93A) vs. comp7(10uM), **** indicates a p-value < 0.0001; SOD-1(G93A) vs. comp 1 (1uM), ** indicates a p-value of 0.0072; SOD-1(G93A) vs. comp4 (1uM), ** indicates a p-value of 0.0021; SOD-1(G93A) vs. comp6 (1uM), * indicates a p-value of 0.0307; SOD-1(G93A) vs. comp7 (1uM), *** indicates a p-value of 0.0006; SOD-1(G93A) vs. N2, **** indicates a p-value < 0.0001.

[0218] Table 1-4 Motility scores of SOD-1 (G93A) nematodes after incubation with the compound

[0219]

[0220]

[0221] Figure 5 The results of the motility score test for FUS(S57Δ) nematodes after incubation with the compound.

[0222] The experimental results of each experimental group and the control group were compared using Dunnett's multiple comparisons method. Figure 5In the code, XQ472FUS(s57Δ)vs.comp3(10uM), * indicates a p-value of 0.0160; XQ472FUS(s57Δ)vs.comp1(1uM), ** indicates a p-value of 0.0026; XQ472FUS(s57Δ)vs.comp2(1uM), * indicates a p-value of 0.0361; XQ472FUS(s57Δ)vs.comp3(1uM), * indicates a p-value of 0.0173; XQ472FUS(s57Δ)vs.comp4(1uM), *** indicates a p-value of 0.0002; XQ472FUS (s57Δ)vs.comp5(1uM), ** indicates a p-value of 0.0024; XQ472FUS(s57Δ)vs.comp6(1uM), * indicates a p-value of 0.0388; XQ472FUS(s57Δ)vs.comp7(1uM), **** indicates a p-value < 0.0001; XQ472FUS(s57Δ)vs.N2, **** indicates a p-value < 0.0001.

[0223] Table 1-5 Motility scores of FUS(S57Δ) nematodes after incubation with compounds

[0224] Group Nematode model Exercise score comp1 10uM FUS(S57Δ) 364.0 comp2 10uM FUS(S57Δ) 377.5 comp3 10uM FUS(S57Δ) 387.0 comp4 10uM FUS(S57Δ) 370.4 comp5 10uM FUS(S57Δ) 352.9 comp6 10uM FUS(S57Δ) 365.8 comp7 10uM FUS(S57Δ) 376.2 DMSO 2% FUS(S57Δ) 353.0 comp 1 1uM FUS(S57Δ) 392.7 comp 2 1uM FUS(S57Δ) 384.2 comp 3 1uM FUS(S57Δ) 386.8 comp 4 1uM FUS(S57Δ) 399.8 comp 5 1uM FUS(S57Δ) 392.9 comp 6 1uM FUS(S57Δ) 383.9 comp 7 1uM FUS(S57Δ) 401.2 / N2 535.7 .

[0225] 1.7 Summary of the results of the test on the effectiveness of the compound on the locomotion of C. elegans

[0226] Figure 6 This is a summary of the experimental results in Section 1.6. The p-values ​​above are comparisons between each group and the model control group without medication. NS indicates "no significance".

[0227] The results showed that the five newly synthesized compounds were effective in experimental treatment in at least three of the five nematode ALS models, with compounds 8 and 11' showing the best efficacy, being effective in all five tested models.

[0228] Example 2: Pharmacokinetic study of ICR mice after administration of compounds (compounds M-10', 11' and 8)

[0229] 2.1 Test Plan

[0230]

[0231] Experimental Design:

[0232]

[0233]

[0234] Note: Fasting for 12 hours is required before the experiment, but free access to water is allowed. Food should be consumed 4 hours after drug administration.

[0235] Intravenous injection / gastric administration of solvent: 5% DMSO + 50% propylene glycol aqueous solution

[0236] Note: The solvent can be adjusted according to the actual situation.

[0237]

[0238] 2.2 Test Results

[0239] 2.2.1 Pharmacokinetic parameters of compound M-10' administered to ICR mice

[0240] Table 1

[0241]

[0242] Table 2

[0243]

[0244] 2.2.2 Pharmacokinetic parameters of compound 11' administered to ICR mice

[0245] Table 3

[0246]

[0247] Table 4

[0248]

[0249] 2.2.3 Pharmacokinetic parameters of compound 8 administered to ICR mice

[0250] Table 5

[0251]

[0252] Table 6

[0253]

[0254]

[0255] 2.2.4 Pharmacokinetic parameters of compound D-3 administered to ICR mice

[0256] Table 7

[0257]

[0258] Table 8

[0259]

[0260] 2.2.5 Summary of the identification of metabolites of compounds 8, M-10', 11' and D-3 in ICR mouse plasma

[0261] (1) Sample pretreatment:

[0262] After plasma samples are extracted and thawed at room temperature, plasma samples from different individuals at different sampling time points are combined according to the table below:

[0263]

[0264] Take the combined plasma sample, take 60 μL of the sample, add 180 μL of acetonitrile, vortex at 2300 rpm for 3 min, centrifuge at 14000 rpm for 5 min, transfer 180 μL of supernatant for LC-HRMS analysis.

[0265] (2) LC-HRMS analysis:

[0266] Instruments and equipment:

[0267] UPLC-UV / Orbitrap MS liquid chromatography system (including degasser, binary pump, autosampler and column oven, model Vanquish Flex, Thermo Fisher Scientific);

[0268] HRMS system (Q-Exactive Plus Orbitrap MS, equipped with an electrospray ionization source (ESI source), ThermoFisher Scientific, Bremen, Germany);

[0269] Data acquisition and processing: Xcalibur;

[0270] Data Analysis: Freestyle and Compound Discover 3.3

[0271] Chromatographic conditions The chromatographic column is ACQUITY. TM HSS T3 C18 column (100×2.1mm ID, 1.8μm particle size), Waters Corporation, USA; column temperature 40℃; flow rate 0.35mL / min; UV detection wavelength 254nm; mobile phase and elution gradient are shown in the table below.

[0272] Time (min) A (5mM ammonium acetate contains 0.1% formic acid in water, %) B (acetonitrile, %) 0 70 30 2 70 30 6 0 100 7 0 100 9 70 30

[0273] Mass spectrometry conditions: The ion source was an electrospray ionization (ESI) source, employing both positive and negative ion modes. Ion source parameters are shown in the table below:

[0274] parameter Positive ion mode Ionization voltage (V) 3500 Capillary temperature (°C) 320 Sheath Gas 45 Aux Gas 8 Probe Heater Temp (°C) 350

[0275] Full Mass-dd MS2 scanning The mode is a full scan with a scanning range of 100-1500 m / z, a resolution of 70000, and an automatic gain of 3e. 6 The maximum injection time is 200ms; the target scan separation window for dd MS2 is ±2Da, the resolution is 17500, and the automatic gain is set to 2e. 5 The maximum injection time is 50ms.

[0276] (3) Data collection and analysis

[0277] Data acquisition was performed using Xcalibur software from Thermo Scientific, and data analysis was performed using Freestyle and Compound Discoverer 3.3 software from Thermo Fisher Scientific. The LC-MS peak area represents the quasi-molecular ion peak [M+H]. + The peak area of ​​the extracted ion chromatography.

[0278] (4) Test Results

[0279] ①Relevant information on the metabolism of compound 8 in mouse plasma

[0280] Table 9. UPLC-HRMS information on metabolites of compound 8 in mouse plasma.

[0281]

[0282]

[0283] The metabolic pathway of compound 8 is as follows:

[0284]

[0285] ②Relevant information on the metabolism of compound M-10' in mouse plasma

[0286] Table 10. UPLC-HRMS information on metabolites of compound M-10' in mouse plasma.

[0287]

[0288] The metabolic pathway of compound M-10' is as follows:

[0289]

[0290] ③Relevant information on the metabolism of compound 11' in mouse plasma

[0291] Table 11 UPLC-HRMS information of compound 11' metabolites in mouse plasma

[0292]

[0293]

[0294] The metabolic pathway of compound 11' is as follows:

[0295]

[0296] The analysis of the metabolites above shows that the N-alkyl chains of compounds M-10' and 11' are unstable and will detach from the N-alkyl chains to form the pimozide drug within 1-24 hours after intravenous injection. However, the N-side chain of compound 8 is more stable in the blood, and the oxidized and sulfated structures with the side chain are the main forms within 1-24 hours after intravenous injection.

[0297] ④ Relevant information on the metabolism of compound D-3 in mouse plasma

[0298] Analysis of the metabolites of compound D-3 in mouse plasma showed that compound D-3 is relatively stable in blood, and no N-dealkylation chain products were detected.

[0299] Example 3: Tissue distribution of compounds 8 and D-3 in rats

[0300] 3.1 Test Plan

[0301]

[0302] Experimental Design:

[0303]

[0304] Note: Fasting for 12 hours is required before the experiment, but free access to water is allowed. Food should be consumed 4 hours after drug administration.

[0305] Intravenous solvent: 4% DMSO + 4% HS15 + 92% normal saline

[0306] Note: The solvent can be adjusted according to the actual situation.

[0307]

[0308] 3.2 Test Results

[0309] 3.2.1 Tissue distribution of rats after intravenous injection of compound 8

[0310] (1) Average drug concentration in various tissues of rats after intravenous injection of 5 mg / kg of compound 8

[0311] Table 12. Average drug concentrations (ng / g or ng / mL) in various tissues of rats after intravenous injection of compound 8 at a dose of 5 mg / kg.

[0312]

[0313] (2) Average drug concentration of Pimozide in various tissues of rats after intravenous injection of 5 mg / kg of compound 8

[0314] Table 13. Average Pimozide concentrations (ng / g or ng / mL) in various tissues of rats after intravenous injection of compound 8 at a dose of 5 mg / kg.

[0315]

[0316] (3) Comparison of parent drug exposure and plasma exposure (AUC) in different tissues after intravenous injection of 5 mg / kg compound 8 in rats

[0317] Table 14 Comparison of parent drug exposure and plasma exposure (AUC) in various tissues after intravenous injection of 5 mg / kg compound 8 in rats

[0318]

[0319] (4) Comparison of Pimozide exposure and plasma exposure (AUC) in different tissues after intravenous injection of 5 mg / kg compound 8 in rats

[0320] Table 15 Comparison of Pimozide exposure and plasma exposure (AUC) in various tissues of rats after intravenous injection of 5 mg / kg compound 8

[0321]

[0322]

[0323] 3.2.2 Tissue distribution of rats after intravenous injection of compound D-3

[0324] (1) Average drug concentration in various tissues of rats after intravenous injection of 8 mg / kg D-3

[0325] Table 16. Mean drug concentrations (ng / g or ng / mL) in various tissues of rats after intravenous injection of 8 mg / kg D-3.

[0326]

[0327] (2) Average Pimozide concentration in various tissues of rats after intravenous injection of 8 mg / kg compound D-3

[0328] The results showed that no Pimozide was detected in any of the organizations. things.

[0329] (3) Comparison of drug exposure in various tissues and plasma exposure (AUC) in rats after intravenous injection of 8 mg / kg D-3

[0330] Table 17 Comparison of drug exposure and plasma exposure (AUC) in various tissues of rats after intravenous injection of 8 mg / kg D-3

[0331]

[0332]

[0333] The results in 3.2.1 and 3.2.2 above show that the tissue distribution of compounds 8 and D-3 in brain tissue is very low, ranging from one-tenth to one-hundredth of that in other tissues.

[0334] 3.2.3 Summary of Detection Results of Compound 8 Metabolites in Rat Tissue Samples

[0335] (1) Sample pretreatment:

[0336] Take the tissue distribution test samples of compound 8 (plasma, liver tissue, and skeletal muscle homogenates, 24h samples), thaw them at room temperature, take 100 μL of the sample, add 300 μL of acetonitrile, vortex at 2300 rpm for 3 min, centrifuge at 14000 rpm for 5 min, transfer 200 μL of the supernatant for LC-HRMS analysis.

[0337] (2) LC-HRMS analysis:

[0338] Instruments and equipment:

[0339] UPLC-UV / Orbitrap MS liquid chromatography system (including degasser, binary pump, autosampler and column oven, model Vanquish Flex, Thermo Fisher Scientific);

[0340] HRMS system (Q-Exactive Plus Orbitrap MS, equipped with an electrospray ionization source (ESI source), ThermoFisher Scientific, Bremen, Germany);

[0341] Data acquisition and processing: Xcalibur;

[0342] Data Analysis: Freestyle and Compound Discover 3.3

[0343] Chromatographic conditions The chromatographic column is ACQUITY. TM HSS T3 C18 column (100×2.1mm ID, 1.8μm particle size), Waters Corporation, USA; column temperature 40℃; flow rate 0.35mL / min; UV detection wavelength 254nm; mobile phase and elution gradient are shown in the table below.

[0344]

[0345] Mass spectrometry conditions The ion source is an electrospray ionization (ESI) source, employing both positive and negative ion modes. Ion source parameters are shown in the table below:

[0346] parameter Positive ion mode Ionization voltage (V) 3500 Capillary temperature (°C) 320 Sheath Gas 45 Aux Gas 8 Probe Heater Temp (°C) 350

[0347] Full Mass-dd MS2 scanning The mode is set to full scan with a scanning range of 80-1000 m / z, a resolution of 70000, and an automatic gain of 3e. 6 The maximum injection time is 100ms; the target scan separation window for dd MS2 is ±1Da, the resolution is 17500, and the automatic gain is set to 2e. 5 The maximum injection time is 50ms.

[0348] (3) Data collection and analysis

[0349] Data acquisition was performed using Xcalibur software from Thermo Scientific, and data analysis was performed using Freestyle and Compound Discoverer 3.3 software from Thermo Fisher Scientific.

[0350] The LC-MS peak area corresponds to the quasi-molecular ion peak [M+H]. + The peak area of ​​the extracted ion chromatography was determined. Three parallel samples were tested for plasma, liver, and skeletal muscle, and the statistical result was the average peak area of ​​the three samples.

[0351] (4) Experimental results:

[0352] Table 18 Results of UPLC-HRMS detection of metabolites of compound 8 in rat tissue samples

[0353]

[0354] The metabolic pathway of compound 8 is as follows (P: plasma; L: liver; S: skeletal muscle):

[0355]

[0356] The tissue distribution experiment of the drug in rats showed that the main forms of compound 8 in rat liver and skeletal muscle were the original drug and its oxidized form, while the N-dealkylated form was less common; under experimental conditions, the double oxide deamination structure (M8-2A, M8-2B or / and M8-2C) was present in plasma and liver.

[0357] Compounds M8-2A, M8-2B, and M8-2C have the same effect as compound 8.

[0358] Conclusion: To develop next-generation drugs for treating motor neurodegenerative diseases (such as ALS), this invention designed and modified the Pimozide molecule to prevent the new drug from crossing the blood-brain barrier, thereby reducing Pimozide's side effects on the central nervous system. We designed and synthesized five novel compound molecules (NCEs) and verified their efficacy using multiple nematode ALS models. Pharmacokinetic analysis confirmed that compounds 8 and D-3 are structurally stable in vivo and have good half-lives. Tissue distribution experiments confirmed low levels in brain tissue, meeting the requirements of molecular design. This invention also identified new drug metabolite molecules. Because these drugs do not easily cross the blood-brain barrier, limiting psychiatric side effects, higher drug doses can be used clinically to improve neuromuscular function for the treatment of ALS and related neurodegenerative diseases.

Claims

1. A compound as shown below, or a pharmaceutically acceptable salt thereof: 、 、 、 、 、 、 、 , or 。 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The pharmaceutically acceptable salts mentioned are hydrochloride salts.

3. The compound of claim 2 or a pharmaceutically acceptable salt thereof, characterized in that, In the aforementioned hydrochloride molecule structure, the molar ratio of the compound molecule to the hydrochloric acid molecule is 1:

1.

4. A compound as shown below: 。 5. A pharmaceutical composition comprising the compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

6. The use of a compound as described in any one of claims 1-4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 5, in the preparation of a medicament for the prevention and / or treatment of motor neurodegenerative diseases.

7. The application as described in claim 6, characterized in that, The aforementioned motor neurodegenerative diseases are amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, Kennedy's disease, or spinal muscular atrophy.

8. A method for preparing a compound as shown in Formula D-3, characterized in that, It includes the following steps: under the action of acid, the compound shown in formula A-2 undergoes a deprotection reaction to prepare the compound shown in formula D-3. 。 9. A method for preparing a compound as shown in Formula 8-1, characterized in that, It includes the following steps: in a solvent, under the action of a reducing agent, the compound shown in Formula B-3 undergoes a reduction reaction to prepare the compound shown in Formula 8-1. 。 10. A compound as shown in formula M-10', characterized in that, It comprises the following steps: in the presence of a base, a compound as shown in formula C1-2' undergoes a deprotection reaction to prepare a compound as shown in formula M-10', wherein in the compound as shown in formula C1-2', Pg is a hydroxyl protecting group. 。 11. A method for preparing compound 11', characterized in that, It includes the following steps: in a solvent, in Cu 2+ In the presence of a nitrogen-containing tripentate ligand and a reducing agent, the compound shown in Formula B-3 and the compound shown in Formula B-4 undergo the alkyne-azide click chemistry reaction shown below to prepare compound 11'. 。 12. A method for preparing compound 12', characterized in that, It includes the following steps: in a solvent, in Cu 2+ In the presence of a nitrogen-containing tripentate ligand and a reducing agent, the compound shown in formula C3-1 undergoes an alkyne-azide click chemical reaction with the compound shown in formula B-3 as shown below to prepare compound 12'. 。 13. Any one of the following compounds: ; in, In the compounds shown in formula C1-2', Pg is a hydroxyl protecting group.

14. The compound according to claim 13, characterized in that, In the compounds shown in formula C1-2', Pg is (C1-C6 alkyl)C(=O)-.

15. The compound according to claim 13, characterized in that, In the compounds shown in formula C1-2', Pg is an acetyl group.

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