Bis (benzylidene) cycloalkanones and heterocyclic analogs thereof, and their use as medicaments for treatment or prophylaxis of proteinopathies

Abstract

The present invention relates to the use of bis (benzylidene) cycloalkanones and heterocyclic analogs thereof in human and veterinary medicaments for the treatment of diseases caused by the presence or elevated level of intracellular metastable proteins, imbalanced protein homeostasis and proteotoxic stress, typically proteinopathies, such as human and veterinary diseases. In particular, they are useful in the treatment of neurodegenerative diseases (e.g. Amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), Kenyi's disease (KD), Huntington's disease (HD), Crash's disease (CJD), spinal cerebellar ataxia (SCA), dentonucleus red-nuclear and palliary-body atrophy, transthyretin familial amyloid polyneuropathy), systemic amyloidosis, neurodegenerative disease, neurodegenerative disease, neurodegenerative disease, neurodegenerative disease, neurodegenerative disease, neurodegenerative disease, neurodegenerative disease, neurodegenerative disease, neurodegenerative disease, neurodegenerative disease, neurodegenerative disease, neurodegenerative disease, neurodegenerative disease, neurodegenerative disease, and the like. The invention also relates to the use of the pharmaceutical composition for treating diabetes mellitus. In addition, the present invention provides novel bis (benzylidene) cycloalkanones.

Description

Technical Field

[0001] This invention relates to the use of bis(phenylmethylene)cycloalkanones and their heterocyclic analogues for the treatment of diseases caused by the presence or elevated levels of intracellular metastable proteins, imbalances in protein homeostasis (protein endocrine balance), and protein toxicity stress. These diseases are referred to as protein disorders. Background Technology

[0002] Intracellular protein degradation is a tightly regulated process essential for all vital functions and therefore necessary for maintaining cellular homeostasis. Over 90% of cytoplasmic proteins are degraded via the so-called ubiquitin-proteasome system (UPS). Defective or misfolded proteins are removed through this pathway, and the amount of protein within the cell is regulated in this way, which is directly related to the regulation of protein activity (Kleiger et al.). Trends in cell biology , 2014, 24, 6, 352-359; Ciechanover et al.: Experimental & molecular medicine (2015, 47, 3, e147-e147). The core of the UPS system is the 26S proteasome, a complex multi-protein catalytic complex (2500 kDa) that degrades covalently labeled polyubiquitin chains (Lys48) into a mixture of peptides. The 26S proteasome consists of a 20S catalytic subunit with three catalytic sites (possessing chymotrypsin-like, trypsin-like, or caspase-like activities) and one or two regulatory subunits (called 19S). It is well known that the aging process and the development of neurodegenerative diseases are closely related to the accumulation of misfolded or damaged proteins, which can be cytotoxic to cells or even directly induce apoptosis. The development of aging and neurodegenerative diseases is accompanied by a decrease in UPS activity, which is often accompanied by the formation of intracellular protein aggregates. For these reasons, modulating or enhancing UPS activity is considered a very promising approach to delaying the onset of diseases associated with the accumulation of toxic protein forms or to directly treat such diseases (e.g., amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Kennedy's disease, or Huntington's disease) (Kleiger et al.). Trends in cell biology , 2014, 24, 6, 352-359; Ciechanover et al.: Experimental & molecular medicine , 2015, 47, 3, e147-e147; Calamini et al.: Nature chemical biology (2012, 8.2, 185-196).

[0003] For example, the catalytic activity of UPS can be enhanced by using low molecular weight compounds to stimulate its catalytic activity. Unfortunately, compounds with this activity are currently known to have very limited effects at the intracellular level (Trader et al.). Biochimica et Biophysica Acta (BBA)-General Subjects , 2017, 1861.4, 892-899; Leestemakeret et al.: Cell chemical biology , 2017, 24.6, 725-736).

[0004] The most promising way to enhance UPS capacity is to increase the synthesis of the proteasome itself, accompanied by increased synthesis of heat shock proteins (HSPs). This can be achieved by activating the stress transcription factor NRF1 (Non-Stress Transcription 1), a transcription factor from the so-called Cap-n-Collar (CNC) family of transcription factors. NFE2L1 ) and NRF2 ( NFE2L2 ) to achieve (Huryn et al.: J Med Chem , 2019, 63.5, 1892-1907; Bott et al.: Human molecular genetics , 2016, 25.10, 1979-1989).

[0005] Among the compounds that activate the transcription factor NRF2, dimethyl fumarate is noteworthy; it is used clinically to treat multiple sclerosis (Yadav et al.). Journal of Molecular Medicine (2019, 97.4, 463-472). However, in addition to the proteasome subunit, NRF2 also increases the expression of many cell protection and antioxidant genes (Brandes et al.: ). ASN neuro (2020, 12, 1759091419899782), and NRF2 exhibits anti-inflammatory and immunomodulatory effects. Other transcription factors that activate NRF2 include sulforaphane (Chapple et al.: 2020, 12, 1759091419899782), and NRF2 also exhibits anti-inflammatory and immunomodulatory effects. Other transcription factors that activate NRF2 include sulforaphane (C The international Journal of Biochemistry & Cell Biology , 2012 44(8), 1315-1320).

[0006] Recently, activation of the transcription factor NRF1 appears to be more appropriate, as it is responsible for triggering the coordinated expression of all genes in the proteasome subunit in response to proteotoxic stress. A key finding is that, compared to gene knockdown, the activated NRF1 pathway prevents the formation of toxic protein aggregates. Therefore, low-molecular-weight compounds that selectively activate this signaling pathway without interacting with UPS and without inducing oxidative stress are considered the most promising approach for future treatment of neurodegenerative diseases (whose development is associated with protein aggregate formation and proteotoxic stress) (Njomen et al.). J Med Chem , 2019, 62.14, 6469-6481).

[0007] When proteasome activity is inhibited or absent, the transcription factor NRF1 preferentially induces the synthesis of all proteasome subunits; these subunits are functionally and structurally identical and cannot be substituted for one another (Kleiger et al.). Trends in cell biology , 2014, 24, 6, 352-359; Koizumi et al.: Proceedings of the Japan Academy. Series B (2018, 325-336). NRF1 also increases the expression of the transcription factor HSF1, which is responsible for inducing the expression of heat shock proteins, molecular chaperones that ensure cellular responses to stress conditions. Heat shock proteins help achieve the correct spatial conformation of misfolded or stress-damaged proteins, which is currently considered one of the important defense mechanisms in cells to prevent the formation of aggregates or toxic protein forms, both at the cell culture level and in mouse models (Bose et al., 2018, 325-336). Ageing research reviews , 2017, 35, 155-175).

[0008] Currently, the most discussed compound that can enhance both proteasome activity and the expression of heat shock proteins regulated by the transcription factor HSF1 is the curcumin derivative ASC-JM17 (1 E 6 E Curcumin (-4-(cyclobutylmethyl)-1,7-bis(3,4-dimethoxyphenyl)hept-1,6-diene-3,5-dione) has been shown to be a dual activator of both of the aforementioned signaling pathways. This compound has been approved by the European Medicines Agency for the treatment of spinal-bulbar muscular atrophy (so-called Kennedy's disease). However, curcumin and its derivatives are generally characterized by low bioavailability and relatively rapid degradation in tissues, which is problematic.

[0009] Although compounds that can target and enhance cellular responses to protein toxic stress have great therapeutic potential, very few such compounds have reached the clinical application stage. Therefore, it is crucial to develop new agents with higher efficacy, more targeted mechanisms of action, fewer side effects, and minimal in vivo toxicity. Summary of the Invention

[0010] This invention relates to bis(phenylmethylene)cycloalkanones and their heterocyclic analogs (whose activation is mediated by transcription factor NRF1). NFE2L1The invention relates to the use of compounds that control the synthesis of the proteasome (encoded by genes), VCP / p97 dissociative enzyme, and heat shock proteins for the treatment of diseases (typically protein disorders) caused by the presence or elevated levels of intracellular metastable proteins, protein homeostasis (protein endocrine imbalance), and protein toxicity stress. In particular, the compounds are associated with the treatment of neurodegenerative diseases, amyloidosis, cystic fibrosis, or diabetes. Furthermore, the invention provides novel bis(phenylmethylene)cycloalkanones.

[0011] The object of this invention is the use of compounds of general formula I as medicaments for the treatment or prevention of protein disorders (e.g., neurodegenerative diseases, amyloidosis, cystic fibrosis, and / or diabetes).

[0012] Among them, R 1 R 2 R 3 and R 4 Independently selected from the group consisting of hydrogen atom, fluorine atom, hydroxyl group, methoxy group, difluoromethoxy group, and trifluoromethoxy group. Optionally, R 1 and R 2 Together they form methylenedioxy and / or R 3 and R 4 Together they form methylenedioxy; A is a carbon atom or a nitrogen atom, and R 5 Selected from hydrogen atoms, deuterium, C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl and groups. The group, Among them, R 6 Selected from the group consisting of C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl, C1-C6 alkoxy, C3-C6 cycloalkoxy, C6-C10 aryloxy, C1-C6 alkylamino, C3-C6 cycloalkylamino, and C6-C10 arylamino. or A is selected from the group that includes oxygen atoms, sulfur atoms, and SO2 groups, and R 5 It does not exist; n is 1, 2, or 3.

[0013] In one aspect, the present invention relates to the use of compounds of general formula I as medicaments for the treatment or prevention of protein diseases, neurodegenerative diseases, amyloidosis, and / or cystic fibrosis or diabetes. Among them, R 1 R 2 R 3 and R 4Independently selected from the group consisting of hydrogen atom, fluorine atom, hydroxyl group, methoxy group, difluoromethoxy group, and trifluoromethoxy group. Optionally, R 1 and R 2 Together they form methylenedioxy and / or R 3 and R 4 Together they form methylenedioxy; A is a carbon atom or a nitrogen atom, and R 5 Selected from hydrogen atoms, deuterium, C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl and groups. The group, Among them, R 6 Selected from the group consisting of C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl, C1-C6 alkoxy, C3-C6 cycloalkoxy, C6-C10 aryloxy, C1-C6 alkylamino, C3-C6 cycloalkylamino, and C6-C10 arylamino. or A is selected from the group that includes oxygen atoms, sulfur atoms, and SO2 groups, and R 5 It does not exist; n is 1, 2, or 3; The condition is that when A is carbon and n is 1, then R 5 It is not a hydrogen atom or a deuterium atom; and / or the condition is that the substituent R 1 R 2 R 3 Or R 4 At least one substituent in it is not a hydroxyl group (four substituents R 1 To R 4 The maximum number of substituents in it can be -OH).

[0014] Observations revealed that when R 5 When hydrogen is not present, compounds of general formula I are more effective in activating the NRF1 transcriptional pathway. Therefore, in one embodiment, the present invention relates to the use of compounds of general formula I as medicaments for the treatment or prevention of protein disorders such as neurodegenerative diseases, amyloidosis, and / or cystic fibrosis or diabetes. Among them, R 1 R 2 R 3 and R 4 Independently selected from the group consisting of hydrogen atom, fluorine atom, hydroxyl group, methoxy group, difluoromethoxy group, and trifluoromethoxy group. Optionally, R 1 and R 2 Together they form methylenedioxy and / or R 3 and R 4 Together they form methylenedioxy; A is a carbon atom or a nitrogen atom, and R 5 Selected from C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl and group. The group, Among them, R 6 Selected from the group consisting of C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl, C1-C6 alkoxy, C3-C6 cycloalkoxy, C6-C10 aryloxy, C1-C6 alkylamino, C3-C6 cycloalkylamino, and C6-C10 arylamino. or A is selected from the group that includes oxygen atoms, sulfur atoms, and SO2 groups, and R 5 It does not exist; n is 1, 2, or 3. Preferably, the substituent R 1 R 2 R 3 Or R 4 At least one of the substituents is not a hydroxyl group.

[0015] Alkyl groups are saturated linear or branched hydrocarbons. Alkyl groups can include, but are not limited to, methyl, ethyl, propyl, and isopropyl.

[0016] A cycloalkyl group is a saturated cyclic hydrocarbon residue. In some embodiments, all carbon atoms of the cycloalkyl group are part of a ring. In some embodiments, some carbon atoms of the cycloalkyl group are part of a ring, while some carbon atoms of the cycloalkyl group form a straight chain or branched chain attached to the ring. A cycloalkyl group may contain one or more rings.

[0017] Heterocyclic alkyl groups are saturated cyclic hydrocarbon residues containing at least one heteroatom selected from O, S, and N. In some embodiments, all carbon atoms and heteroatoms of the heterocycle are part of the ring. In some embodiments, certain carbon atoms and heteroatoms of the heterocycle are part of the ring, while certain carbon atoms of the heterocyclic alkyl group, and optionally certain heteroatoms, form a straight or branched chain attached to the ring. Heterocyclic alkyl groups may contain one or more rings. Preferably, the heterocyclic alkyl group contains one to two heteroatoms. Examples of heterocyclic alkyl groups include morpholino, piperazine, and morpholinoethyl.

[0018] An alkoxy group is an -O-alkyl group. An example of an alkoxy group is the methoxy group.

[0019] An aryl group is an aromatic cyclic hydrocarbon containing one or two rings. In particular, the aryl group can be phenyl, naphthyl, or biphenyl, with phenyl being preferred.

[0020] A heteroaryl group is an aromatic cyclic or bicyclic hydrocarbon containing one or more heteroatoms selected from O, S, and N, preferably one, two, or three heteroatoms. The heteroatom is preferably a nitrogen atom. Examples of heteroaryl groups are pyridyl and imidazolyl.

[0021] Halogens are selected from the group consisting of fluorine, chlorine, bromine, and iodine.

[0022] In one implementation, the substituent R 1 Unlike hydroxyl.

[0023] In one implementation, A is a carbon atom or a nitrogen atom, and R 5 Selected from C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl and group. The group, in which R 6 It is selected from the group consisting of C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl, C1-C6 alkoxy, C3-C6 cycloalkoxy, C6-C10 aryloxy, C1-C6 alkylamino, C3-C6 cycloalkylamino and C6-C10 arylamino.

[0024] In one implementation, A is a carbon atom or a nitrogen atom, and R... 5 It is a group And R 6 It is selected from the group consisting of methyl, ethyl, propyl, cyclopropyl, trifluoromethyl, benzyl, dimethoxybenzyl, aminoethyl, aminopropyl, N,N-dimethylaminoethyl, and N,N-dimethylaminopropyl.

[0025] In one implementation, A is a carbon atom or a nitrogen atom, and R 5 Selected from the group consisting of H, CH3, CF3 and benzyl, more preferably, R 5 Selected from the group including CH3, CF3 and benzyl.

[0026] In one embodiment, A is selected from the group consisting of oxygen atoms, sulfur atoms, and SO2 groups, and R 5 It does not exist.

[0027] In one implementation, R 1 Selected from the group consisting of hydrogen atom, fluorine atom, methoxy group, difluoromethoxy group, and trifluoromethoxy group, or R 1 and R 2 Together they form a methylenedioxy group, where A is a carbon or nitrogen atom, and R... 5 Selected from C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl and group. The group, in which R 6 It is selected from the group consisting of C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl, C1-C6 alkoxy, C3-C6 cycloalkoxy, C6-C10 aryloxy, C1-C6 alkylamino, C3-C6 cycloalkylamino and C6-C10 arylamino.

[0028] In one embodiment, A is selected from the group consisting of oxygen atoms, sulfur atoms, and SO2 groups, and R 5 It does not exist, and R 1 Selected from the group consisting of hydrogen atom, fluorine atom, methoxy group, difluoromethoxy group, and trifluoromethoxy group, or R 1 and R 2 Together they form methylenedioxy.

[0029] Preferably, R 1 R 2 R 3 and R 4 It is independently selected from the group consisting of fluorine atoms, methoxy groups, ethoxy groups, difluoromethoxy groups, and trifluoromethoxy groups.

[0030] In a preferred embodiment, n is 1.

[0031] When a compound of general formula I is positively charged (in cationic form), the compound contains a counterion, which can be an anion of a pharmaceutically acceptable organic or inorganic acid, thereby forming a pharmaceutically acceptable salt. For example, such anion can be selected from the group including: acetate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate, tartrate, bromide, camphorsulfonate, carbonate, chloride, citrate, decanoate, ethylenediaminetetraacetic acid, ethanesulfonate, fumarate, gluconate, gluconate, glutamate, glycolate, hexanoate, iodide, lactate, malate, maleate, mandelic acid, methanesulfonate, methylsulfate, naphthalenesulfonate, nitrate, caprylate, oleate, palmitate, pantothenate, phosphate, polygalacturonic acid, propionate, salicylate, stearate, succinate, sulfate, tartrate, toluenesulfonate, trifluoroacetate.

[0032] If a compound of Formula I contains a chiral center, then Formula I includes pure enantiomers and mixtures of enantiomers (including racemates).

[0033] Formula I includes compounds of Formula I in their free form, as well as compounds of Formula I in the form of salts, addition salts (with acids or bases), and / or solvates (including hydrates or alcohol solvates).

[0034] Another object of the present invention is the use of compounds of general formula I for the treatment of diseases caused by the presence or elevated levels of intracellular metastable proteins, imbalance of protein homeostasis (protein endothermic state), and protein toxic stress. These diseases are known as protein diseases.

[0035] Furthermore, the object of this invention is the use of compounds of general formula I for the treatment of neurodegenerative diseases, amyloidosis, and / or cystic fibrosis or diabetes.

[0036] The purpose of this invention is to use the compounds of formula I for the treatment of neurodegenerative diseases (e.g., amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), Kennedy's disease (KD), Huntington's disease (HD), Creutzfeldt-Jakob disease (CJD), spinocerebellar ataxia (SCA), dentate nucleus-rubella-lewy body atrophy, transthyretin familial amyloid polyneuropathy), as well as systemic or organ-specific amyloidosis, and / or cystic fibrosis or diabetes.

[0037] On the other hand, the present invention relates to the use of compounds of general formula I for the prevention of genetically based neurodegenerative diseases (e.g., familial amyotrophic lateral sclerosis (ALS), familial Parkinson's disease (PD), familial Alzheimer's disease (AD), Kennedy's disease (KD), Huntington's disease (HD), familial Creutzfeldt-Jakob disease (CJD), familial spinocerebellar ataxia (SCA), transthyretin familial amyloid polyneuropathy, familial dentate nucleus-rubella-lewy body atrophy), as well as familial systemic amyloidosis or familial organ-specific amyloidosis, and / or cystic fibrosis.

[0038] Another object of the present invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of formula I and at least one pharmaceutically acceptable carrier, filler and / or diluent and / or excipient.

[0039] Another object of the present invention is compounds selected from general formula I, which include the group consisting of: 2,6-Bis(3-fluoro-4-(trifluoromethoxy)benzylmethyl)-4-methylcyclohexanone, 1-Acetyl-3,5-bis(3,4-methylenedioxybenzylmethyl)piperidin-4-one 3,5-Bis(3,4-dimethoxybenzylmethyl)-1-cyclopropylcarbonylpiperidin-4-one, 3,5-Bis(3,4-dimethoxybenzylmethyl)-1-trifluoroacetylpiperidin-4-one, 3,5-Bis(3,4-dimethoxybenzyl)-1-(3,4-dimethoxybenzoyl)piperidin-4-one 1-Cyclopropanecarbonyl-3,5-bis(4-hydroxy-3-methoxybenzylmethyl)piperidin-4-one 2,4-Bis(3,4-dimethoxybenzyl)-8-methyl-8-azabicyclo[3.2.1]oct-3-one, 2,6-Bis(3,4-dimethoxybenzyl)-4-trifluoromethylcyclohexanone, 2,6-Bis(4-methoxy-3-(trifluoromethoxy)benzylmethyl)-4-methylcyclohexanone, 3,5-Bis(4-methoxy-3-(trifluoromethoxy)benzylmethyl)-1-acetylpiperidin-4-one 3,5-Bis(4-methoxy-3-(trifluoromethoxy)benzylmethyl)-1-(3,4-dimethoxybenzoyl)piperidin-4-one, 3,5-Bis(3,4-Di(difluoromethoxy)benzylmethyl)-1-acetylpiperidin-4-one 2,6-Bis(3,4-Di(difluoromethoxy)benzyl)-4-methylcyclohexanone, 2,6-Bis(4-hydroxy-3-(trifluoromethoxy)benzyl)-4-methylcyclohexanone, 3,5-bis(3,4-bis(difluoromethoxy)benzylmethyl)tetrahydrothiaran-4-one.

[0040] In one aspect, the present invention relates to a method for treating a disease directly related to a protein disease (e.g., neurodegenerative disease, amyloidosis, cystic fibrosis, or diabetes), the method comprising administering to a subject requiring such treatment a medicament comprising at least one compound of general formula I as defined above.

[0041] In one aspect, the present invention relates to a method for treating protein diseases, particularly neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), Kennedy's disease (KD), Huntington's disease (HD), Creutzfeldt-Jakob disease (CJD), spinocerebellar ataxia (SCA), dentate nucleus-rubella-lewy body atrophy, transthyretin familial amyloid polyneuropathy, and systemic or organ-specific amyloidosis, and / or cystic fibrosis or diabetes), the method comprising administering to a subject requiring such treatment a medicament comprising at least one compound of general formula I as defined above.

[0042] In one aspect, the present invention relates to a method for treating amyloidosis (e.g., familial amyloidosis without neuropathy, familial neuropathic amyloidosis, familial neurological amyloidosis, secondary generalized amyloidosis, and organ-specific amyloidosis), cystic fibrosis, and diabetes, the method comprising the step of administering to a subject requiring such treatment a medicament comprising at least one compound of general formula I as defined above.

[0043] In another aspect, the present invention relates to a method for preventing genetically based neurodegenerative diseases (e.g., familial amyotrophic lateral sclerosis (ALS), familial Parkinson's disease (PD), familial Alzheimer's disease (AD), Kennedy's disease (KD), Huntington's disease (HD), familial Creutzfeldt-Jakob disease (CJD), familial spinocerebellar ataxia (SCA), transthyretin familial amyloid polyneuropathy, familial dentate nucleus-rubella-lewy body atrophy), as well as familial systemic amyloidosis or familial organ-specific amyloidosis, and / or cystic fibrosis), the method comprising the step of administering to a subject requiring such prevention a medicament comprising at least one compound of general formula I as defined above.

[0044] Compared to the known compound ASC-JM17, the compound of the present invention specifically and significantly activates the transcription factor NRF1 (which is produced by...). NFE2L1 The pathways controlled by the gene encoding the protein are as shown in Example 2. Furthermore, based on the analysis disclosed in Example 3, it was surprisingly found that the compounds of the present invention did not have the negative impact on protein homeostasis as observed in ASC-JM17. For the treatment of diseases caused by protein toxic stress (particularly neurodegenerative diseases) using these compounds, affecting protein homeostasis by prolonging protein degradation time is an undesirable effect. Compared to the DMSO control, all tested compounds showed values ​​ranging from a decrease of -0.15-fold to an increase of +0.35-fold, which are very favorable values ​​for potentially affecting cellular protein homeostasis. In contrast, ASC-JM17 showed a 4.24-fold increase in observed fluorescence (i.e., prolonged degradation time) compared to the control group.

[0045] Furthermore, it was demonstrated that the compounds according to the invention significantly increased the monitored mRNA levels of proteasome subunits compared to controls, and were significantly better than compound ASC-JM17 (see Example 4). It was also shown that the compounds resulted in a surprisingly strong increase in the levels of mRNA encoding heat shock proteins.

[0046] The protective effect of the compounds according to the invention against protein toxicity stress was illustrated in Example 7. Cell lines that excessively produced synuclein were treated with rotenone, which induces protein toxicity stress, manifested as a significant decrease in the viability of the test cell lines. This toxic effect was inhibited when cells were treated with all the test compounds.

[0047] A crucial aspect of treatment for protein disorders is that the compounds used do not induce the formation of unwanted reactive oxygen species (ROS). As shown in Example 8, the compounds were tested at a concentration of 5 μmol·L⁻¹. -1Or even at concentrations 5 times higher (25 μmol·L⁻¹) -1 Neither of these methods induced the formation of reactive oxygen species (ROS), which is in stark contrast to ASC-JM17, which significantly increased ROS compared to the DMSO control.

[0048] Another important aspect of the ultimate treatment of protein disorders is testing the minimal effect of compounds on the cell cycle. None of the tested compounds showed any effect on the cell cycle. In contrast, the control compound ASC-JM17, compared to the control, caused a significant proportion of cells to enter the G2 / M phase, indicating cell cycle arrest at the G2 checkpoint, and demonstrating significant toxicity of this comparative compound (see Example 9).

[0049] Synthetic schemes for bis(benzyl)cycloalkanones and their heterocyclic analogs. Starting cycloalkanones or their heterocyclic analogs are converted to bis(benzyl) derivatives by aldol condensation with the corresponding benzaldehyde. For 3,5-bis(benzyl)piperidin-4-one derivatives, the nitrogen atom is further acylated with a suitable reagent.

[0050] Detailed Implementation

[0051] Examples

[0052] List of abbreviations

[0053] ALS: Amyotrophic Lateral Sclerosis

[0054] AMC: 7-Amino-4-methylcoumarin

[0055] BSA: Bovine serum albumin

[0056] Dalton

[0057] DMEM: Durbeco Modified Eagle Medium

[0058] DMSO: Dimethyl sulfoxide

[0059] DTT: Dithiothreitol

[0060] EDTA: Ethylenediaminetetraacetic acid

[0061] ESI: Electrospray Ionization

[0062] GFP: Green fluorescent protein

[0063] HRMS: High Resolution Mass Spectrometry

[0064] HSF1: Heat shock transcription factor 1

[0065] HSP: Heat Shock Protein

[0066] NMR: Nuclear Magnetic Resonance

[0067] NRF1: Nuclear Factor Erythroid 2-related Factor 1

[0068] NRF2: Nuclear Factor Erythroid 2-related Factor 2

[0069] PBS: Phosphate-buffered saline

[0070] PCR: Polymerase Chain Reaction

[0071] PC 12: A pheochromocytoma cell line derived from the adrenal medulla of rats.

[0072] EtOAc: Ethyl acetate

[0073] HBTU: Benzotriazole tetramethylurea hexafluorophosphate

[0074] HCl: Hydrochloric acid

[0075] HR-ESI-MS: High-resolution mass spectrometry with electrospray ionization

[0076] J Coupling constant (NMR)

[0077] m: Multiplet (NMR)

[0078] MeOH: Methanol

[0079] MeOD- d4 Deuterated methanol

[0080] PI: Propidium iodide

[0081] ROS: Reactive oxygen species

[0082] RT: Reverse transcription

[0083] Ub: ubiquitin

[0084] UPS: Ubiquitin-Proteasome System

[0085] Renilla Luciferase: an enzyme derived from the sea kidney (Renilla reniformis)

[0086] Firefly luciferase: an enzyme derived from the firefly (Photinus pyralis).

[0087] ACN: Acetonitrile

[0088] bs: Broad singlet (NMR)

[0089] CDCl3: Deuterated chloroform

[0090] d: Bimodal (NMR)

[0091] dd: Double doublet (NMR)

[0092] ddd: Doublet of a doublet (NMR)

[0093] dt: Double triplet (NMR)

[0094] dtd: Doublet of a double triplet (NMR)

[0095] dd: Double doublet (NMR)

[0096] DMAP: 4-Dimethylaminopyridine

[0097] DMF: Dimethylformamide

[0098] DMSO- d6 Deuterated dimethyl sulfoxide

[0099] MCPBA: m-chloroperoxybenzoic acid

[0100] NMR: Nuclear Magnetic Resonance

[0101] q: Quartet (NMR)

[0102] s: Single peak (NMR)

[0103] t: triplet (NMR)

[0104] td: Triple doublet (NMR)

[0105] TFA: Trifluoroacetic acid

[0106] δ: Chemical shift (NMR) [δ] - ppm

[0107] Example 1: Preparation of the compound

[0108] All reactions were carried out under argon atmosphere in a dry solvent. Reversed-phase chromatography was performed using a Teledyne ISCO Combi Flash Rf+ rapid chromatography system equipped with a RediSep Rf Gold C18 reversed-phase column. All starting materials were purchased from Sigma Aldrich, Combi-Blocks, and Fluorochem and used as is. The purity of the compounds and the composition of the reaction mixtures were determined on a Waters UPLC-MS Acquity equipped with a QDa mass spectrometer (over 7 minutes, flow rate 0.5 mL / min, gradient 0–100% MeCN / H₂O (0.1% formic acid)) using an ACQUITY UPLC BEH C18 column, 130 Å, 1.7 μm, 2.1 mm × 100 mm, with a 2.1 mm × 5 mm pre-column. ESI high-resolution mass spectra were recorded using a Thermo Scientific LTQ Orbitrap XL (Thermo Fisher Scientific) controlled by MassLynx software. Using a Bruker Avance III NMR spectrometer TM HD 400 MHz Prodigy record NMR spectrum.

[0109] List of compounds

[0110] General Procedure A: A mixture of cycloalkanone (5.0 mmol), benzaldehyde (10.0 mmol), and potassium hydroxide (0.11 g, 1.86 mmol) in ethanol (10 mL) was stirred overnight and then quenched with water (20 mL). The product was extracted with ethyl acetate (2 × 10 mL), and the combined organic layers were washed with water and brine. The solvent was evaporated, and the residue was recrystallized from methanol or purified by column chromatography and then recrystallized from alcohol.

[0111] 2,6-Bis(benzo[1,3]dioxacyclopenten-5-ylmethylene)cyclohexyl-1-one (1)

[0112] The title compound was prepared by aldol condensation of cyclohexanone and piperonal according to general procedure A. The residue after evaporation was purified by recrystallization from methanol to give 0.520 g of yellow solid (yield 29%). 1 H NMR (400 MHz, CDCl3) δ 7.73 (s, 2H), 7.02 (m, 4H), 6.88 (dd, J= 7.7, 0.7 Hz, 2H), 6.03 (s,4H), 2.94 – 2.83 (m, 4H), 1.83 (m, 2H). HR-ESI-MS:C 22 H 19 O5 (M + H) + Calculated value: 363.1232; measured value: 363.1235.

[0113] 2,6-Bis(3,4-dimethoxybenzylmethyl)cyclohexyl-1-one (2)

[0114] The title compound was prepared by aldol condensation of cyclohexanone and 3,4-dimethoxybenzaldehyde according to general procedure A. The residue after evaporation was purified by recrystallization from ethanol to give 1.02 g of a yellow solid (yield 51%). 1 H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 2.0 Hz, 2H), 7.14 (ddd, J = 8.4, 2.0, 0.7 Hz,2H), 7.05 (d, J = 1.9 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H), 3.95 (s, 6H), 3.94(s, 6H), 3.11 – 2.75 (m, 4H), 1.86 (m, 2H). HR-ESI-MS:C 22 H 27 O5 (M + H) + Calculated value: 395.1853, measured value: 395.1853.

[0115] 2,6-Bis(3,4-dimethoxybenzyl)-4-methylcyclohexane-1-one (3)

[0116] The title compound was prepared by aldol condensation of 4-methylcyclohexanone and 3,4-dimethoxybenzaldehyde according to general procedure A. The residue after evaporation was purified by recrystallization from ethanol to give 1.66 g of a yellow solid (yield 81%). 1 HNMR (400 MHz, CDCl3) δ 7.78 (d, J = 2.5 Hz, 2H), 7.14 (dd, J = 8.7, 1.9 Hz, 2H), 7.04 (d, J = 1.9 Hz, 2H), 6.94 (d, J= 8.4 Hz, 2H), 3.95 (s, 3H), 3.93(s, 3H), 3.29 – 2.94 (m, 2H), 2.54 (m, 2H), 1.93 (m, 1H), 1.13 (d, J = 6.6Hz, 3H). HR-ESI-MS: C 25 H 29 O5 (M + H) + Calculated value: 409.2009; Measured value: 409.2009.

[0117] 2-(3,5-bis(3,4-dimethoxybenzylmethyl)-4-oxopiperidin-1-carbonyl)pyridine-1-onium trifluoroacetate (4)

[0118] The title compound was prepared by aldol condensation of 1-pyridinecarboxylpiperidin-4-one and 3,4-dimethoxybenzaldehyde according to general procedure A. The residue after evaporation was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution) to give 0.11 g of yellow solid (yield 35%). 1 H NMR (401 MHz, CDCl3) δ 8.45 –8.39 (m, 1H), 7.86 (s, 1H), 7.77 (s, 1H), 7.69 (td, J = 7.8, 1.7 Hz, 1H), 7.51 (dt, J = 7.7, 1.1 Hz, 1H), 7.33 (ddd, J = 7.7, 5.0, 1.2 Hz, 1H), 7.17(d, J = 8.4 Hz, 1H), 7.09 (s, 1H), 7.00 – 6.94 (m, 1H), 6.81 – 6.74 (m, 3H), 5.11 (s, 2H), 4.85 (s, 2H), 3.95 (s, 6H), 3.90 (s, 4H), 3.84 (s, 3H). HR-ESI-MS:C 29 H 28 O6N2Na (M + Na) + Calculated value: 523.1840; Measured value: 523.1845.

[0119] 3,5-Bis(3,4-dimethoxybenzylmethyl)tetrahydro-4 H -thiaran-4-one (5)

[0120] Following standard procedure A, via tetrahydro-4 H The title compound was prepared by aldol condensation of 1,4-thiaran-4-one and 3,4-dimethoxybenzaldehyde. The residue after evaporation was purified by recrystallization from methanol to give 0.73 g of a yellow solid (35% yield). 1 H NMR (400 MHz, CDCl3) δ 7.76 (s, 2H), 7.13 – 7.01 (m, 2H), 7.00 – 6.88 (m, 4H), 3.98 (m, 4H), 3.95 (s, 3H), 3.93 (s, 3H). HR-ESI-MS:C 23 H 25 O5S (M + H) + Calculated value: 413.1417; Measured value: 413.1413.

[0121] 1-Benzyl-3,5-bis(3,4-dimethoxybenzylmethyl)piperidin-4-one (6)

[0122] The title compound was prepared by aldol condensation of 1-benzyl-4-piperidinone and 3,4-dimethoxybenzaldehyde according to general procedure A. The residue after evaporation was purified by recrystallization from methanol to give 0.55 g of a yellow solid (yield 22%). 1 H NMR (400 MHz, CDCl3) δ 7.78 (s, 2H), 7.27 (m, 5H), 7.07 – 6.94 (m, 2H), 6.94 – 6.81 (m, 4H), 3.94 (s, 6H), 3.88 (m, 10H), 3.77 (s, 2H). HR-ESI-MS:C 30 H 32 O5N (M + H) + Calculated value: 486.2275; Measured value: 486.2274.

[0123] General Procedure B: 4-Piperidinone hydrochloride monohydrate (3.25 mmol) and benzaldehyde (6.50 mmol) were dissolved in glacial acetic acid (7 mL). A dioxane solution of 4 M HCl (2 mL) was added dropwise, and the reaction mixture was stirred overnight at ambient temperature. The resulting suspension was filtered, and the solid was purified by recrystallization from hot methanol to give the product as hydrochloride.

[0124] 3,5-Bis(3,4-dimethoxybenzylmethyl)piperidin-4-one (7)

[0125] The title compound was prepared by aldol condensation of 4-piperidinone hydrochloride monohydrate and 3,4-dimethoxybenzaldehyde according to general procedure B. The residue after evaporation was purified by recrystallization from methanol to give 0.80 g of yellow solid (yield 57%). 1 H NMR (400 MHz, MeOD- d 4) δ 8.02 (s, 2H), 7.11 (m, 4H), 7.07 (d, J =1.4 Hz, 2H), 4.67 (d, J = 1.9 Hz, 4H), 3.92 (s, 6H), 3.91 (s, 6H). HR-ESI-MS:C 23 H 26 O5N (M + H) + Calculated value: 396.1805; measured value: 396.1801.

[0126] 3,5-Bis(3,4-methylenedioxybenzyl)piperidin-4-one (8)

[0127] The title compound was prepared by aldol condensation of 4-piperidinone hydrochloride monohydrate and piperonal according to general procedure B. The residue after evaporation was purified by recrystallization from methanol to give 0.70 g of yellow solid (yield 53%). 1 HNMR (400 MHz, DMSO- d 6) δ 9.82 (s, 2H), 7.79 (s, 2H), 7.16 (d, J = 1.1 Hz, 2H), 7.10 (d, J = 1.0 Hz, 4H), 6.14 (s, 4H), 4.76 – 4.35 (m, 4H). HR-ESI-MS:C 21 H 18 O5N (M + H) + The calculated value is 364.1179, and the measured value is 364.1177.

[0128] 3,5-Bis(3,4-dihydroxybenzyl)piperidin-4-one (9)

[0129] The title compound was prepared by aldol condensation of 4-piperidinone hydrochloride monohydrate and 3,4-dihydroxybenzaldehyde according to general procedure B. The residue after evaporation was purified by recrystallization from methanol to give 0.55 g of a yellow solid (yield 45%). 1 H NMR (400 MHz, DMSO- d6) δ 9.85 (bs, 2H), 9.74 (bs, 2H), 9.44 (bs, 2H), 7.70 (s, 2H), 7.10 – 6.65 (m, 6H), 4.43 (bs, 4H). HR-ESI-MS:C 19 H 18 O5N (M + H) + Calculated value: 340.1180; measured value: 340.1182.

[0130] 3,5-Bis(4-hydroxy-3-methoxybenzyl)piperidin-4-one (10)

[0131] The title compound was prepared by aldol condensation of 4-piperidinone hydrochloride monohydrate and vanillin, following standard procedure B. The residue after evaporation was purified by recrystallization from methanol to give 0.63 g of a yellow solid (48% yield). 1 HNMR (400 MHz, DMSO- d 6) δ 9.91 (bs, 2H), 9.67 (bs, 2H), 7.82 (d, J = 1.8 Hz, 2H), 7.14 (d, J = 1.9 Hz, 2H), 7.07 – 6.74 (m, 4H), 4.50 (bs, 4H), 3.84 (s, 6H). HR-ESI-MS:C 21 H 22 O5N (M + H) + The calculated value is 368.1492, and the measured value is 368.1491.

[0132] 2,6-Bis(3,4-dihydroxybenzyl)-4-methylcyclohexanone (11)

[0133] The title compound was prepared by aldol condensation of 4-methylcyclohexanone and 3,4-dihydroxybenzaldehyde according to general procedure B. The residue after evaporation was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution) to give 0.86 g of brownish-green solid (yield 55%). 1 H NMR (400 MHz, DMSO- d 6) δ 9.45 (bs, 2H),9.16 (bs, 2H), 7.46 (d, J = 2.3 Hz, 2H), 6.99 (d, J= 2.0 Hz, 2H), 6.93 –6.86 (m, 2H), 6.86 – 6.69 (m, 2H), 2.97 (dd, J = 16.1, 3.7 Hz, 2H), 1.80(dtd, J = 10.7, 6.8, 3.6 Hz, 1H), 1.07 (d, J = 6.5 Hz, 3H). HR-ESI-MS: C 21 H 21 O5(M + H) + Calculated value: 353.1383; measured value: 353.1385.

[0134] 2,6-Bis(3-fluoro-4-(trifluoromethoxy)benzyl)-4-methylcyclohexanone (12)

[0135] The title compound was prepared by aldol condensation of 4-methylcyclohexanone and 3-fluoro-4-(trifluoromethoxy)benzaldehyde according to general procedure B. The residue after evaporation was purified by recrystallization from methanol to give 0.13 g of a yellow solid (yield 15%). 1 H NMR (400 MHz, CDCl3) δ 7.71 (s, 2H), 7.43 – 7.09 (m, 6H), 3.24 – 2.96 (m, 2H), 2.52 (ddd, J = 16.1, 11.1, 2.8 Hz, 2H), 1.94 (m, 1H), 1.14 (d, J =6.6 Hz, 3H). HR-ESI-MS: C 23 H 17 O3F8 (M + H) + Calculated value: 493.1050; Measured value: 493.1038.

[0136] General procedure C

[0137] A mixture of bis(benzyl)piperidin-4-one hydrochloride (1 equivalent), acylation agent (1.5 equivalent), triethylamine (3.0 equivalent), and DMAP (0.05 equivalent) in DMF was stirred overnight and then quenched with water. The product was extracted with ethyl acetate, and the combined organic layers were washed with water and brine. The solvent was evaporated, the residue was purified by column chromatography, and then recrystallized from alcohol.

[0138] 1-Acetyl-3,5-bis(4-hydroxy-3-methoxybenzylmethyl)piperidin-4-one (13)

[0139] The title compound was prepared by reacting 3,5-bis(4-hydroxy-3-methoxybenzyl)piperidin-4-one (0.28 g, 0.69 mmol) with acetic anhydride (0.07 g, 0.69 mmol) according to general procedure C. The residue after evaporation was purified by column chromatography (eluent EtOAc:MeOH / 20:1) and then recrystallized from ethanol to give 0.12 g of a yellow solid (yield 42%). 1 H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.78 (s, 1H), 7.21 – 6.72 (m,6H), 5.96 (s, 1H), 4.94 (s, 2H), 4.74 (s, 2H), 3.97 (s, 6H), 2.00 (s, 3H). HR-ESI-MS:C 23 H 24 O6N (M + H) + Calculated value: 410.1604; measured value: 410.1606.

[0140] 1-Acetyl-3,5-bis(3,4-methylenedioxybenzyl)piperidin-4-one (14)

[0141] The title compound was prepared by reacting 3,5-bis(3,4-methylenedioxybenzyl)piperidin-4-one (0.28 g, 0.69 mmol) with acetic anhydride (0.11 g, 1.04 mmol) according to general procedure C. The residue after evaporation was purified by column chromatography (elution buffer EtOAc) and then recrystallized from ethanol to give 0.15 g of yellow solid (yield 53%). 1 H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 1.8 Hz, 1H), 7.75 (s, 1H), 7.12 – 6.79 (m,6H), 6.07 (s, 2H), 6.05 (s, 2H), 4.91 (d, J = 2.1 Hz, 2H), 4.70 (s, 2H), 2.00 (s, 3H). HR-ESI-MS:C 23 H 20 O6N (M + H) + Calculated value: 406.1285; measured value: 406.1282.

[0142] 1-Acetyl-3,5-bis(3,4-dimethoxybenzylmethyl)piperidin-4-one (15)

[0143] The title compound was prepared by reacting 3,5-bis(3,4-dimethoxybenzyl)piperidin-4-one (0.30 g, 0.69 mmol) with acetic anhydride (0.11 g, 1.04 mmol) according to general procedure C. The residue after evaporation was purified by column chromatography (elution buffer EtOAc:MeOH / 10:1) and then recrystallized from ethanol to give 0.25 g of yellow solid (yield 82%). 1 H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.79 (d, J = 2.1 Hz, 1H), 7.14(dd, J = 8.4, 2.0 Hz, 1H), 7.10 – 7.04 (m, 1H), 7.02 – 6.86 (m, 4H), 4.96 (s,2H), 4.75 (s, 2H), 3.97 (s, 3H), 3.96 (s, 3H), 3.95 (s, 6H), 2.00 (s, 3H). HR-ESI-MS:C 25 H 28 O6N (M + H) + Calculated value: 438.1911; measured value: 438.1910.

[0144] 3,5-Bis(3,4-dimethoxybenzylmethyl)-1-cyclopropylcarbonylpiperidin-4-one (16)

[0145] The title compound was prepared by reacting 3,5-bis(3,4-dimethoxybenzyl)piperidin-4-one (0.50 g, 1.16 mmol) with cyclopropane carbonyl chloride (0.16 g, 1.50 mmol) according to general procedure C. The residue after evaporation was purified by column chromatography (elution buffer EtOAc) and then recrystallized from ethanol to give 0.43 g of yellow solid (yield 80%). 1 HNMR (400 MHz, CDCl3) δ 7.82 (s, 2H), 7.15 – 6.84 (m, 6H), 4.97 (d, J = 1.8Hz, 4H), 3.96 (s, 6H), 3.95 (s, 6H), 1.53 (ddd, J = 8.0, 4.7, 3.3 Hz, 1H), 1.12 – 0.83 (m, 2H), 0.77 – 0.53 (m, 2H). HR-ESI-MS:C 27 H 30 O6N (M + H)+ Calculated value: 464.2073; Measured value: 464.2076.

[0146] 3,5-Bis(3,4-dimethoxybenzylmethyl)-1-trifluoroacetylpiperidin-4-one (17)

[0147] The title compound was prepared by reacting 3,5-bis(3,4-dimethoxybenzyl)piperidin-4-one (0.30 g, 0.69 mmol) with trifluoroacetic anhydride (0.22 g, 1.04 mmol) according to general procedure C. The residue after evaporation was purified by column chromatography (elution buffer EtOAc:cyclohexane / 1:1) and then recrystallized from ethanol to give 0.21 g of yellow solid (61% yield). 1 H NMR (400 MHz, CDCl3) δ 7.90 (s, 1H), 7.88 (s, 1H), 7.19 – 7.08 (m,1H), 7.04 (d, J = 2.0 Hz, 1H), 7.02 – 6.91 (m, 1H), 5.06 – 4.97 (m, 2H), 4.94(s, 2H), 3.98 (s, 3H), 3.97 (s, 3H), 3.96 (s, 3H), 3.94 (s, 3H). HR-ESI-MS:C 25 H 25 O6NF3 (M + H) + Calculated value: 492.1634; measured value: 492.1636.

[0148] 3,5-Bis(3,4-dimethoxybenzylmethyl)-1-ethoxycarbonylpiperidin-4-one (18)

[0149] The title compound was prepared by reacting 3,5-bis(3,4-dimethoxybenzyl)piperidin-4-one (0.43 g, 1.00 mmol) with ethyl chloroformate (0.16 g, 1.50 mmol) according to general procedure C. The residue after evaporation was purified by column chromatography (elution buffer EtOAc:cyclohexane / 1:1) and then recrystallized from ethanol to give 0.40 g of yellow solid (yield 85%). 1 H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 1.9 Hz, 2H), 7.18 – 7.04 (m,2H), 7.04 – 6.86 (m, 4H), 4.83 (s, 4H), 4.12 (q, J= 7.1 Hz, 2H), 3.96 (s,6H), 3.95 (s, 6H), 1.18 (t, J = 7.1 Hz, 3H). HR-ESI-MS: C 26 H 30 O7N (M + H) + Calculated value: 468.2022; Measured value: 468.2024.

[0150] 1-(3,4-dimethoxybenzoyl)-3,5-bis(3,4-dimethoxybenzyl)piperidin-4-one (19)

[0151] The title compound was prepared by reacting 3,5-bis(3,4-dimethoxybenzyl)piperidin-4-one (0.30 g, 0.69 mmol) with 3,4-dimethoxybenzoyl chloride (0.21 g, 1.10 mmol) according to general procedure C. The residue after evaporation was purified by column chromatography (elution buffer EtOAc) and then recrystallized from ethanol to give 0.34 g of yellow solid (yield 88%). 1 H NMR (400 MHz, CDCl3) δ 7.86 (s, 2H), 7.13 – 6.84 (m, 7H), 6.79 (dd, J = 8.2, 1.9 Hz, 1H), 6.45 (d, J = 8.3 Hz, 1H), 4.92 (bs, 4H), 3.94 (s, 6H), 3.91 (s, 6H), 3.80 (s, 3H), 3.76 (s, 3H). HR-ESI-MS:C 32 H 34 O8N (M + H) + Calculated value: 560.2278; Measured value: 560.2280.

[0152] 1-Cyclopropanecarbonyl-3,5-bis(4-hydroxy-3-methoxybenzylmethyl)piperidin-4-one (20)

[0153] The title compound was prepared by reacting 3,5-bis(4-hydroxy-3-methoxybenzyl)piperidin-4-one (0.30 g, 0.74 mmol) with cyclopropane carbonyl chloride (0.077 g, 0.74 mmol) according to general procedure C. The residue after evaporation was purified by column chromatography (elution buffer EtOAc) and then recrystallized from ethanol to give 0.15 g of yellow solid (yield 46%). 1H NMR (400 MHz, CDCl3) δ 9.70 (s, 2H), 7.64 (s, 2H), 7.15 (s, 2H), 7.10– 6.97 (m, 2H), 6.90 (d, J = 8.2 Hz, 2H), 5.05 (bs, 2H), 4.85 (bs, 2H), 3.84(s, 6H), 1.78 (ddd, J = 9.5, 6.6, 3.9 Hz, 1H), 0.62 (ddt, J = 14.2, 5.4, 3.0Hz, 4H). HR-ESI-MS:C 25 H 26 O6N (M + H) + Calculated value: 436.1454; Measured value: 436.1455.

[0154] 2,4-bis(3,4-dimethoxybenzyl)-8-methyl-8-azabicyclo[3.2.1]oct-3-one (21)

[0155] The title compound was prepared by aldol condensation of tropinone and 3,4-dimethoxybenzaldehyde according to general procedure A. The residue after evaporation was purified by column chromatography (elution buffer EtOAc) and then recrystallized from ethanol to give 0.25 g of yellow solid (yield 28%). 1 H NMR (400 MHz, CDCl3) δ 7.83 (s, 2H), 7.04 (d, J = 1.9 Hz, 2H), 7.02 – 6.86 (m, 4H), 4.66 – 4.41 (m, 2H), 3.96 (s, 6H), 3.94 (s, 6H), 2.65 (dd, J = 9.0, 4.7 Hz, 2H), 2.37 (s, 3H), 2.07 (m, 2H). HR-ESI-MS:C 26 H 30 O5N(M + H) + Calculated value: 436.2118; measured value: 436.2117.

[0156] 2,6-Bis(3,4-dimethoxybenzyl)-4-trifluoromethylcyclohexanone (22)

[0157] The title compound was prepared by aldol condensation of 4-trifluoromethylcyclohexanone and 3,4-dimethoxybenzaldehyde according to general procedure A. The residue after evaporation was purified by recrystallization from methanol to give 0.61 g of a yellow solid (yield 26%). 1 H NMR (400 MHz, CDCl3) δ 7.89 (m, 2H), 7.12 (dt, J = 8.4, 1.2 Hz, 2H), 7.01 (d, J = 1.9 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H), 3.96 (s, 6H), 3.94 (s,6H), 3.39 (dd, J = 15.6, 3.6 Hz, 2H), 2.87 (ddd, J = 15.7, 12.5, 2.9 Hz, 2H),2.50 (ddt, J = 16.3, 8.2, 4.0 Hz, 1H). HR-ESI-MS:C 25 H 26 O5F3 (M + H) + Calculated value: 463.1727; measured value: 463.1721.

[0158] 3,5-Bis(3,4-dimethoxybenzylmethyl)-1,1-di(oxide)tetra(hydro)-4 H -Thiaran-4-one (23)

[0159] In DCM, 3,5-bis(3,4-dimethoxybenzyl)tetrahydro-4-di ... H The title compound was prepared by oxidation of 4-thiaran-4-one (0.30 g, 0.73 mmol). The residue was purified by column chromatography (eluent EtOAc:cyclohexane / 1:1) and then recrystallized from ethanol to give 0.20 g of yellow solid (yield 21%). 1 H NMR (400 MHz, CDCl3) δ 7.95 (s, 2H), 7.12 – 7.02 (m, 4H), 6.96 (d, J =8.3 Hz, 2H), 4.50 (s, 2H), 3.96 (s, 6H), 3.95 (s, 6H). HR-ESI-MS:C 23 H 27 O7SNa (M+ Na)+ Calculated value: 467.1135; measured value: 467.1136.

[0160] 2,7-Bis(3,4-dimethoxybenzyl)cycloheptanone (24)

[0161] The title compound was prepared by aldol condensation of cycloheptanone and 3,4-dimethoxybenzaldehyde according to general procedure A. The reaction mixture was refluxed overnight. The residue after evaporation was purified by column chromatography (eluent EtOAc:cyclohexane / 1:2) and then recrystallized from ethanol to give 0.50 g of yellow solid (60% yield). 1 H NMR (400 MHz, CDCl3) δ 7.37(s, 2H), 7.18 – 7.06 (m, 2H), 7.04 (d, J = 2.1 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 3.94 (s, 6H), 3.93 (s, 6H), 2.88 – 2.52 (m, 4H), 2.80 – 1.86 (m, 4H). HR-ESI-MS:C 25 H 29 O5 (M + H) + Calculated value: 409.2009; Measured value: 409.2010.

[0162] 2,6-Bis(4-methoxy-3-(trifluoromethoxy)benzyl)-4-methylcyclohexane-1-one (25)

[0163] The title compound was prepared by aldol condensation of 4-methylcyclohexane-1-one and 4-methoxy-3-(trifluoromethoxy)benzaldehyde according to general procedure A. The reaction mixture was refluxed overnight. The residue after evaporation was purified by column chromatography (eluent EtOAc:cyclohexane / 1:3) and then recrystallized from methanol to give 0.43 g of a yellow solid (yield 36%). 1 H NMR (400 MHz, CDCl3) δ 7.77 – 7.65 (m, 2H), 7.43 (m, 1H), 7.41 (m, 3H), 7.06 (d, J = 9.1 Hz, 2H), 3.95 (s, 6H), 3.15 – 2.91 (m, 2H), 2.52 (ddd, J = 16.0,11.2, 2.7 Hz, 2H), 2.19 – 1.83 (m, 1H), 1.13 (d, J= 6.6 Hz, 3H). HR-ESI-MS: C 25 H 23 O5F6 (M + H) + Calculated value: 517.1444; Measured value: 517.1444.

[0164] 1-Acetyl-3,5-bis(4-methoxy-3-(trifluoromethoxy)benzylmethyl)piperidin-4-one (26)

[0165] The title compound was prepared by reacting 3,5-bis(4-methoxy-3-(trifluoromethoxy)benzylmethyl)piperidin-4-one (0.21 g, 0.39 mmol) with acetic anhydride (0.06 g, 0.58 mmol) according to general procedure C. The residue after evaporation was purified by column chromatography (elution buffer EtOAc:cyclohexane / 1:1) and then recrystallized from methanol to give 0.12 g of yellow solid (yield 56%). 1 H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.74 (s, 1H), 7.51 –7.32 (m, 3H), 7.30 (s, 1H), 7.09 (dd, J = 13.2, 8.6 Hz, 2H), 5.21 – 4.80 (m,2H), 4.72 (s, 2H), 3.98 (s, 3H), 3.96 (s, 3H), 1.99 (s, 3H). HR-ESI-MS:C 25 H 22 O6NF6(M + H) + Calculated value: 546.1345; measured value: 546.1341.

[0166] 1-(3,4-dimethoxybenzoyl)-3,5-bis(4-methoxy-3-(trifluoromethoxy)benzylmethyl)piperidin-4-one (27)

[0167] The title compound was prepared by reacting 3,5-bis(4-methoxy-3-(trifluoromethoxy)benzylmethyl)piperidin-4-one (0.21 g, 0.39 mmol) with 3,4-dimethoxybenzoyl chloride (0.12 g, 0.58 mmol) according to general procedure C. The residue after evaporation was purified by column chromatography (elution buffer EtOAc) and then recrystallized from methanol to give 0.15 g of yellow solid (yield 57%). 1 H NMR (400 MHz, CDCl3) δ 7.80 (d, J= 1.9 Hz, 2H), 7.42 -7.20 (m, 4H), 7.03 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 2.0 Hz, 2H), 6.77 (dd, J = 8.2, 2.0 Hz, 1H), 6.47 (d, J = 8.3 Hz, 1H), 4.88 (bs, 4H), 3.95 (s, 6H), 3.79 (s, 3H), 3.76 (s, 3H). HR-ESI-MS:C 32 H 28 O8NF6(M + H) + The calculated value is 668.1713, and the measured value is 668.1711.

[0168] 1-Acetyl-3,5-bis(3,4-bis(difluoromethoxy)benzylmethyl)piperidin-4-one (28)

[0169] The title compound was prepared by reacting 3,5-bis(3,4-bis(difluoromethoxy)benzylmethyl)piperidin-4-one (0.25 g, 0.44 mmol) with acetic anhydride (0.07 g, 0.65 mmol) according to general procedure C. The residue after evaporation was purified by column chromatography (eluent EtOAc:cyclohexane / 1:1) and then recrystallized from methanol to give 0.18 g of a yellow solid (71% yield). 1 H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.75 (s, 1H), 7.55 – 7.21 (m,6H), 6.62 (t, J = 73.1 Hz, 4H), 4.93 – 4.76 (m, 2H), 4.78 – 4.63 (m, 2H), 2.00 (s, 3H). HR-ESI-MS:C 25 H 20 O6NF8(M + H) + Calculated value: 582.1155; Actual measured value: 582.1155.

[0170] 2,6-Bis(3,4-bis(difluoromethoxy)benzyl)-4-methylcyclohexane-1-one (29)

[0171] The title compound was prepared by aldol condensation of 4-methylcyclohexane-1-one and 3,4-bis(difluoromethoxy)benzaldehyde according to general procedure A. The residue after evaporation was purified by column chromatography (eluent EtOAc:cyclohexane / 1:3) and then recrystallized from methanol to give 0.31 g of yellow solid (yield 26%). 1 H NMR (400 MHz, CDCl3) δ 7.84 –7.66 (m, 2H), 7.43 – 7.37 (m, 2H), 7.37 – 7.31 (m, 4H), 6.59 (td, J = 73.3,4.5 Hz, 4H), 3.15 – 2.90 (m, 2H), 2.52 (m, 2H), 1.94 (m, 1H), 1.12 (d, J =6.6 Hz, 3H). HR-ESI-MS: C 25 H 21 O5F8 (M + H) + Calculated value: 553.1255; Measured value: 553.1253.

[0172] 2,6-Bis(4-hydroxy-3-(trifluoromethoxy)benzyl)-4-methylcyclohexane-1-one (30)

[0173] The title compound was prepared by aldol condensation of 4-methylcyclohexanone and 4-hydroxy-3-(trifluoromethoxy)benzaldehyde according to general procedure B. The residue after evaporation was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution) to give 0.42 g of yellow solid (yield 71%). 1 H NMR (400 MHz, CDCl3) δ 7.77 –7.63 (m, 2H), 7.47 – 7.30 (m, 4H), 7.11 (d, J = 8.4 Hz, 2H), 5.67 (bs, 2H), 3.16 – 2.95 (m, 2H), 2.51 (ddd, J = 16.0, 11.2, 2.7 Hz, 2H), 1.93 (dtd, J =11.0, 7.0, 3.8 Hz, 1H), 1.13 (d, J = 6.5 Hz, 3H). HR-ESI-MS: C 23 H 19 O5F6 (M + H) +Calculated value: 489.1131; Measured value: 489.1123.

[0174] 3,5-bis(3,4-bis(difluoromethoxy)benzylmethyl)tetrahydro-4 H -Thiaran-4-one (31)

[0175] Following standard procedure A, via tetrahydro-4 H The title compound was prepared by aldol condensation of 3,4-thiaran-4-one and 3,4-bis(difluoromethoxy)benzaldehyde. The residue after evaporation was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution) to give 0.36 g of yellow solid (yield 62%). 1 H NMR (400 MHz, CDCl3) δ 7.80 –7.59 (m, 2H), 7.43 – 7.27 (m, 6H), 6.59 (td, J = 73.1, 4.2 Hz, 4H), 3.90 (d, J = 1.3 Hz, 4H). HR-ESI-MS: C 23 H 16 O5F8NaS (M + Na) + The calculated value is 579.0483, and the measured value is 579.0482.

[0176] 3,5-Bis(3,4-dihydroxybenzyl)tetrahydro-4 H -Thiaran-4-one (32)

[0177] Following standard procedure B, via tetrahydro-4 H The title compound was prepared by aldol condensation of 1,4-thiaran-4-one and 3,4-dihydroxybenzaldehyde. The residue after evaporation was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution) to give 0.32 g of yellow solid (yield 27%). 1 H NMR (400 MHz, CDCl3) δ 9.51 (s, 2H), 9.19 (s, 2H), 7.45 (s, 2H), 6.95 (d, J = 2.0 Hz, 2H), 6.87 (dd, J = 8.4, 2.0Hz, 2H), 6.82 (d, J = 8.2 Hz, 2H), 3.96 (d, J = 1.3 Hz, 4H). HR-ESI-MS: C 19 H 17O5S(M + H) + Calculated value: 357.0791, measured value: 357.0787.

[0178] 2-(3,5-bis(3,4-dimethoxybenzyl)-4-oxopiperidin-1-carbonyl)-3-methylpyridine-1-onium trifluoroacetate (33)

[0179] To a mixture of 3,5-bis(3,4-dimethoxybenzyl)-4-oxopiperidin-1-onium hydrochloride (0.090 g, 0.21 mmol) and triethylamine (0.10 mL, 0.75 mmol) in DMF (3 mL), HBTU (0.119 g, 0.31 mmol) and 3-methylpyridinecarboxylic acid (0.043 g, 0.31 mmol) were added. The reaction mixture was stirred at ambient temperature for 2 hours. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (elution buffer H₂O (0.1% TFA): ACN, gradient elution) to give 0.105 g of yellow solid (80% yield). 1 H NMR (401 MHz, CDCl3) δ 8.40 – 8.34 (m,1H), 7.86 (t, J = 2.2 Hz, 1H), 7.73 – 7.68 (m, 1H), 7.54 – 7.46 (m, 1H), 7.30– 7.22 (m, 1H), 7.17 (dd, J = 8.5, 2.0 Hz, 1H), 7.06 (d, J = 2.0 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.74 (d, J = 8.4 Hz, 1H), 6.68 (dd, J = 8.4, 2.0Hz, 1H), 6.62 (d, J = 2.0 Hz, 1H), 5.14 (d, J = 2.2 Hz, 2H), 4.48 (s, 2H), 3.95 (s, 6H), 3.88 (s, 3H), 3.79 (s, 3H), 2.15 (s, 3H). HR-ESI-MS:C 30 H 31 O6N2 (M+ H) + Calculated value: 515.2177; Measured value: 515.2174.

[0180] 3-(3,5-bis(3,4-dimethoxybenzylmethyl)-4-oxopiperidin-1-yl)- N , N -Dimethyl-3-oxopropane-1-trifluoroacetate ammonium (34)

[0181] To a mixture of 3,5-bis(3,4-dimethoxybenzyl)-4-oxopiperidin-1-onium hydrochloride (0.090 g, 0.21 mmol) and triethylamine (0.13 mL, 0.94 mmol) in DMF (3 mL), HBTU (0.119 g, 0.31 mmol) and 3-(dimethylamino)propionate (0.048 g, 0.31 mmol) were added. The reaction mixture was stirred at ambient temperature for 2 hours. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (elution buffer H₂O (0.1% TFA): ACN, gradient elution) to give 0.102 g of yellow solid (yield 85%). 1 H NMR (401 MHz, CDCl3) δ 7.81 –7.74 (m, 2H), 7.11 – 7.01 (m, 2H), 7.01 – 6.95 (m, 2H), 6.92 (dd, J = 5.2,3.2 Hz, 2H), 4.90 (s, 2H), 4.79 (s, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.91 (s,6H), 3.32 (t, J = 6.5 Hz, 2H), 2.88 (t, J = 6.6 Hz, 2H), 2.73 (s, 6H). HR-ESI-MS:C 28 H 35 O6N2 (M + H) + Calculated value: 495.2490; Measured value: 495.2485.

[0182] Example 2: Activation of NRF1 by bis(phenylmethylene)cycloalkanones and their heterocyclic analogs monitored by cell-based reporter gene assays. NFE2L1 Functions of transcription pathways

[0183] In a solution containing 10% FBS, 2 mmol·L -1 Stable cell lines derived from HEK293 cells were cultured in DMEM (Durbeco Modified Eagle Medium) containing L-glutamine, 50 μg / ml penicillin, and 50 μg / ml streptomycin. These cells contained human-derived... PSMA4The promoter of the gene contains the ARE (antioxidant response element) response element 3xPSMA4-ARE / minP / luc2P / reporter gene, which can monitor the activation of the NRF1 pathway. Cells were cultured in a CO2 incubator at 37°C under a 5% CO2 atmosphere. When cell coverage reached approximately 70%, the reporter gene line was transfected with the standardized Renilla reporter gene pRL-TK (product number E2241; Promega, Hercules, CA). For transfection alone, polyethyleneimine (PEI) was dissolved in OptiMEM medium at a 3:1 PEI / DNA ratio. Cells were then injected at 10 x 10⁻⁶ cells per well in a volume of 25 μL. 3 The concentration was seeded in 384-well plates, and after equilibration for 16 hours, the test compound (concentration of 10 µmol·L⁻¹) was dissolved in dimethyl sulfoxide (DMSO). -1 Cells were treated. The experiment was performed in four technical replicates and two biological replicates. After incubation for 16 hours, the culture medium was removed, and the cells were incubated in 5 μL of 1X lysis buffer (25 mmol / L). -1 Tris-phosphate pH 7.8; 2 mmol·L -1 Dithiothreitol (DTT); 2 mmol·L -1 Cells were lysed in 2,2',2'',2'"-(ethane-1,2-diyldionitrile)tetraacetic acid; 10% (all values ​​are by volume percentage) glycerol and 1% Triton X-100. After incubation on a shaker for 10 minutes, firefly / photinus luciferase substrate (200 mM Tris-HCl; 15 mmol·L⁻¹) was added to each well in a volume of 20 μL. -1 MgSO4; 0.1 mmol·L -1 EDTA; 25 mmol·L -1 DTT; 1 mmol·L -1 ATP; 0.2 mmol·L -1 Coenzyme A; and 200 mmol·L -1 D-luciferin (pH 8.0) was used to measure luminescence. The activity of firefly luciferase was determined, and then buffer (25 mmol·L⁻¹) was added to the same volume. -1 Na₄P₂O₇; 10 mmol·L⁻¹ -1 AcONa; 15 mmol·L -1 EDTA; 500 mmol·L -1 Na₂SO₄; 500 mmol·L⁻¹ -1 NaCl; 25 μmol·L -1Phenylenol-benzothiazole; 4 μmol.l -1 The renin and 0.04% BSA (pH 5.0) were mixed and then the renin activity was measured. The results (Table 1) are the ratios of renin activity to firefly luciferase activity, representing the average of two biological replicates and four technical replicates, with standard deviations given.

[0184] The activation levels of the NRF1-controlled pathway in the tested compounds ranged from 1 to 19, representing an increase of up to 1900% compared to the control; this was observed in compound 19. A total of 13 tested compounds showed higher NRF1 pathway activation values ​​than the control compound ASC-JM17. EC 50 The value is in the submicromolar range.

[0185] Table 1: NRF1 (NonRF1) monitored by cell-based reporter gene assay NFE2L1 The efficacy of transcription pathway activation

[0186] N represents the "bimodal effect, EC" 50 Unable to determine Concentration of 7 μmol·L -1 E at time 最大 value

[0187] Example 3: Effects of bis(phenylmethylene)cycloalkanones and their heterocyclic analogues on cellular protein homeostasis (protein homeostasis)

[0188] In a CO2 incubator at 37°C, under an atmosphere containing 5% CO2, and in an atmosphere containing 10% FBS and 2 mmol·L⁻¹, -1 U2OS cells stably expressing the Ub(G76V)-GFP reporter gene were cultured in DMEM medium containing L-glutamine, 50 μg / ml penicillin, and 50 μg / ml streptomycin. The cells were first cultured at 10 x 10⁻⁶ cells / ml. 3Cells / well were seeded in 384-well plates in DMEM medium without phenol red. The test compound was added the following day. The experiment was performed at least three times. Eight hours later, the GFP signal (excitation λ = 400 nm, emission λ = 510 nm) was measured, and the cytotoxicity of the compound was then measured using the Resazurin / Almar blue assay according to the manufacturer's protocol (Thermo Fisher Scientific, product number DAL1025). GFP intensity was normalized to cell viability according to the formula: (compound GFP intensity) / (baseline GFP intensity). Baseline GFP intensity corresponds to cells exposed only to the DMSO control. The results are then expressed as a percentage of the DMSO control.

[0189] Table 2 also shows the results for the comparative compound ASC-JM17, which exhibited a strong inhibitory effect on protein degradation, up to eight times that of the DMSO control, thus negatively impacting cellular protein homeostasis. Conversely, bis(phenylenemethyl)cycloalkanones and their heterocyclic analogs did not inhibit intracellular protein degradation, and protein homeostasis remained unchanged.

[0190] Table 2: Effects of selected bis(phenylmethylene)cycloalkanones and their heterocyclic analogs on cellular protein homeostasis (protein homeostasis) using reporter gene detection based on GFP degradation determinants.

[0191] N represents the "bimodal effect, EC" 50 Unable to determine

[0192] Example 4: The effect of bis(phenylmethylene)cycloalkanones and their heterocyclic analogs on transcription factor NRF1 ( NFE2L1 The influence of gene expression controlled by )

[0193] In a CO2 incubator at 37°C, under an atmosphere containing 5% CO2, and in an atmosphere containing 10% FBS and 2 mmol·L⁻¹, -1 The human neuroblastoma cell line SH-SY5Y was cultured in DMEM medium containing L-glutamine. Each time, 4 x 10⁻⁶ cells were added. 5Compounds were added to cells, and two technical replicates and three biological replicates were performed. After 16 hours, cells were lysed, and mRNA was isolated using the NucleoSpin RNA Kit (Macherey-Nagel, product number 740955.250) according to the manufacturer's protocol. Then, the mRNA was transcribed into cDNA using the TATTA GrandScript cDNA Supermix Kit (TATAAbiocenter) according to the manufacturer's recommended specifications. Quantitative RT-PCR (RT-qPCR) was performed using a LightCycler 480 (Roche Life Science). Table 3 shows the primers used for each gene in the RT-qPCR reaction. Encoding... GAPDH Data standardization was performed on the mRNA.

[0194] The analytical results are shown in Table 4. Compared with the control, all tested compounds, after treatment, encoded the monitored proteasome subunits. PSMB7 , PSMD12 or PSMC4 The levels of mRNA were elevated. Furthermore, compared to controls, these compounds increased the levels of mRNA encoding heat shock proteins (…). HSP1A1 , DNAJA1 and HSPB1 The effects were significant at the level of compounds 8, 15, and 17. These effects were most pronounced and significantly outweighed those of the comparative compound ASC-JM17.

[0195] Table 3: RT-qPCR primer sequences

[0196] Table 4: Effects of the bis(phenylenemethyl)cycloalkanones and their heterocyclic analogues on transcription factor NRF1 ( NFE2L1 The effects of the compounds on the controlled gene expression were monitored by quantitative RT-PCR. The efficiency of the selected compounds in activating the target gene was expressed as the expression level of the target gene in cells treated with the test compound, compared to the expression level of the target gene in cells treated with dimethyl sulfoxide (DMSO) as a negative control or in cells treated with ASC-JM17. Statistical data were assessed using Welch's corrected one-tailed unpaired t-test in GraphPadPrism 9. The p-value represents the increase in a given mRNA level and is relative to DMSO-treated cells or ASC-JM17; p-values ​​< 0.0001 are indicated by the letter "a", p < 0.001 = "b", p < 0.01 = "c", and p < 0.05 = "d". The experiment was performed in six biological replicates.

[0197]

[0198] Example 5: The effects of the bis(phenylmethylene)cycloalkanone and its heterocyclic analogues on transcription factor NRF1 ( NFE2L1 Effects of controlled protein expression

[0199] The expression levels of the studied proteins HA-NRF1, HSPA1A, NQO1, and PSMB7 in HEK-293 cells (which overproduced labeled HA-NRF1) and SH-SY5Y cells (which overproduced α-synuclein) were analyzed using Western blotting. The concentrations were 5 μmol·L⁻¹. -1 The concentration was determined by treating cells for 16 hours with dimethyl sulfoxide (DMSO), ASC-JM17, or the test compound as a negative control.

[0200] Of the five compounds studied, four showed a significant increase in NRF1 protein levels. Similar results were obtained when monitoring HSPA1A protein expression. Treatment of cells with compounds 13 and 17 resulted in a significant increase in ubiquinone (NQO1) expression. Compound 8 showed an increasing trend in the level of the proteasome subunit PSMB7.

[0201] Table 5: The effects of the bis(phenylmethylene)cycloalkanones and their heterocyclic analogues on NRF1 ( NFE2L1 The effects of transcription factor-controlled protein expression were investigated (monitored by Western blotting). Expression levels of the studied proteins were calculated from the integrated fluorescence of the corresponding bands and normalized to β-actin. Statistical analysis was performed using a one-sample Student's t-test in GraphPad Prism 9 software; p-values ​​are relative to DMSO-treated cells. p-values ​​< 0.0001 were denoted by the letter "a", p < 0.001 = "b", p < 0.01 = "c", and p < 0.05 = "d". Experiments were performed in four biological replicates (six biological replicates for HA-NRF1).

[0202]

[0203] Example 6: Effects of the bis(phenylmethylene)cycloalkanones and their heterocyclic analogues described herein on intracellular proteasome activity.

[0204] The proteolytic activity of the 20S proteasome was determined using a fluorescent substrate (Suc-LLVY-AMC, Bachem I-1395). PC12, HEK293, MCF, and SH-SY5Y-SNCA cell lines were subjected to a 5 µmol·L⁻¹ solution. -1The test compounds were incubated together for 16 hours. In lysis buffer (50 mmol / L) -1 HEPES, pH 7.5; 5 mmol·L -1 EDTA; 150 mmol·L -1 NaCl; 2 mmol·L -1 Cells were lysed in ATP (1% Triton) and the lysates were dissolved in 100 μL buffer (50 mmol·L⁻¹) at 37°C in the dark, with each technical replicate performed in triplicate. -1 Tris, pH 8.0; 10 mmol·L -1 MgCl2; 1 mmol·L -1 ATP; 1 mmol·L -1 In DTT), with 200 μmol·l -1 The sample was incubated for 30 minutes with the Suc-LLVY-AMC fluorescent substrate used to measure chymotrypsin activity. The fluorescence of the formed AMCs was measured using a fluorometer at excitation wavelength λ = 360 nm and emission wavelength λ = 460 nm. Proteasome activity is relative to a control group affected only by DMSO.

[0205] The results are shown in Table 6. Compared with the control, compounds 13, 15, and 17 showed a significant increase in proteasome activity in each cell line. For the other compounds, an increasing trend was observed.

[0206] Table 6. Relative chymotrypsin activity of the proteasome in HEK 293, MCF, PC-12, and SH-SY5Y cell lines expressing SNCA 16 hours after treatment with the compound. Statistical data were evaluated using the one-sample Student's test in GraphPad Prism 9 software. p-values ​​are relative to DMSO-treated cells; p-values ​​< 0.0001 are indicated by the letter "a", p < 0.001 = "b", p < 0.01 = "c", and p < 0.05 = "d". Experiments were performed in three or four biological replicates.

[0207]

[0208] Example 7: The protective effect of the bis(phenylmethylene)cycloalkanone and its heterocyclic analogues against protein toxicity stress in SH-SY5Y cells, in which the accumulation of excessive α-synuclein is induced by rotenone.

[0209] The protective effect of the compound against protein toxicity stress was further investigated in the SH-SY5Y-SNCA (synuclein overproduction) cell line compared with the parental SH-SY5Y cell line. Both cell lines were treated with DMSO (as a negative control) and rotenone [1.125 μmol·L⁻¹]. -1 [It induces protein toxicity stress] and test compounds [concentration series 7.5 μmol·L] -1 5 μmol·l -1 3.75 μmol·l -1 2.5 μmol·l -1 1.25 μmol·l -1 and 0.625 μmol·l -1 The activity values ​​were processed. The measured activity values ​​are relative to the DMSO control and are expressed as mean ± standard deviation [%].

[0210] As shown in Table 7, all tested compounds exhibited protective effects against protein toxicity stress induced by α-synuclein.

[0211] Table 7: Protective effects of the tested compounds against protein toxicity stress. SH-SY5Y cells and SH-SY5Y SNCA cells were tested using DMSO (as a negative control) and rotenone [1.125 μmol·L⁻¹]. -1 ] and test compounds [concentration series 7.5 μmol·L -1 5 μmol·l -1 3.75 μmol·l -1 2.5 μmol·l -1 1.25 μmol·l -1 and 0.625 μmol·l -1 Cells were treated with rotenone alone. Viability values ​​were measured relative to the DMSO control and expressed as mean ± standard deviation [%]. Statistical data were assessed using Welch's ANOVA in GraphPad Prism 9, followed by multiple comparisons using Dunnett's T3 test. p-values ​​are relative to cells treated with rotenone alone; p < 0.0001 is indicated by the letter "a", p < 0.001 = "b", p < 0.01 = "c", p < 0.05 = "d". Experiments were performed in triplicate.

[0212]

[0213] Example 8: Determination of the potential effects of the bis(phenylmethylene)cycloalkanones and their heterocyclic analogues described herein on the formation of reactive oxygen species in cells.

[0214] Intracellular reactive oxygen species (ROS) generation was detected using a 2',7'-dichlorofluorescein diacetate (H2DCFDA) probe (product number D6883-50MG, Sigma-Aldrich) to measure cellular ROS production and associated oxidative stress. For culture plate preparation, 10,000 SH-SY5Y cells were seeded into each well of a 96-well plate. Cells adhered overnight, and the next day, they were inoculated at two concentrations (5 μmol·L⁻¹). -1 and 25 μmol·l -1 The test compounds were added; rotenone (Merck, product number R8875-1G) and TBHP organic peroxide were used as controls for ROS-induced reactions in cells. Two hours later, the cells were treated with 1xPBS (137 mmol·L⁻¹). -1 NaCl; 2.7 mmol·L -1 KCl; 1.5 mmol·L -1 KH2PO4; 10 mmol·L -1 Cells were washed with Na2HPO4, and then 100 μL of H2DCFDA probe dissolved in 1xPBS (final concentration 100 μmol·L⁻¹) was added to each well. -1 30 minutes. The free probe was then rinsed off. In the final step, 50 μL of 1xPBS was added to each well, and fluorescence signals were measured using a Tecan Infinite M1000 reader at excitation and emission wavelengths of 480 nm and 535 nm. Experiments were performed in triplicate (biological replicates) and each experiment consisted of four technical replicates; data are presented as a percentage of DMSO control.

[0215] The results are shown in Table 6. The tested compounds were tested at a concentration of 5 μmol·L⁻¹. -1 Or at a concentration 5 times higher (25 μmol·L -1 None of them led to the formation of reactive oxygen species, which is in stark contrast to ASC-JM17, which significantly increased ROS compared to the DMSO control.

[0216] Table 6: Effects of selected compounds on intracellular reactive oxygen species (ROS) formation, as determined by the DCF-DA assay. SH-SY5Y cells were treated with either DMSO (as a negative control) or ASC-JM17 (as a control compound). Statistical data were assessed using a one-tailed Student's t-test (for DMSO) and a one-tailed unpaired t-test with Welch's correction (ASC-JM17). p-values ​​represent significant changes in ROS levels and are relative to cells treated with DMSO or ASC-JM17; p-values ​​< 0.0001 are indicated by the letter "a", p < 0.001 = "b", p < 0.01 = "c", and p < 0.05 = "d". The experiments were performed in triplicate.

[0217]

[0218] Example 9: The effect of the bis(phenylmethylene)cycloalkanone and its heterocyclic analogues described herein on the cell cycle

[0219] Cell cycle analysis was performed on SH-SY5Y cells treated with the test compound, compared to the negative control DMSO and the control compound ASC-JM17, using flow cytometry. The cell cycle phases were determined by propidium iodide (P4170-250MG, Sigma-Aldrich) staining. Propidium iodide, as a fluorescent dye, is inserted into double-stranded DNA, allowing for semi-quantitative determination of total DNA content and cell population distribution at different phases of the cycle: G0 / G1 phase (unreplicated DNA), S phase (DNA synthesis), and G2 / M phase. Cells were incubated in a CO2 incubator at 2.5 × 10⁻⁶. 5 A density of cells / ml was used to expose cells in a 6-well plate to a concentration of 5 μmol·L⁻¹. -1 The compound was applied for 16 hours. Subsequently, cells were collected into cell counting tubes, centrifuged at 500 g for 5 minutes, washed with non-sterile PBS, and fixed with 70% ethanol at 4°C. The prepared sample was stored at -20°C. Next, 500 μL of propidium iodide solution was added, and the mixture was incubated in a 37°C water bath in the dark for 15 minutes, followed by the addition of 200 μL of ribonuclease A solution (0.7 mol·L⁻¹). -1 Samples were incubated at 060M7000V (Sigma-Aldrich, USA) and then incubated again under the same conditions for 15 minutes. Samples incubated in this manner were stored at 4°C for at least 1 hour. Analysis was performed using an argon laser (excitation wavelength 488 nm) on a BD LSR Fortessa flow cytometer.

[0220] The results are shown in Table 7. None of the tested compounds had any effect on cell cycle progression. In contrast, the control compound ASC-JM17 caused a significant proportion of cells to enter the G2 / M phase, indicating cell cycle arrest at the G2 checkpoint, thus demonstrating the significant toxicity of this comparison compound.

[0221] Table 7: Cell cycle analysis of SH-SY5Y cells after treatment with the test compound at a concentration of 5 μM for 16 hours. SH-SY5Y cells were treated with DMSO as a negative control or with the comparative compound ASC-JM17. Values ​​are expressed as mean ± standard deviation (%).

[0222]

[0223] Example 10: The effect of the bis(phenylmethylene)cycloalkanone and its heterocyclic analogues described herein on the formation of intracellular polyQ aggregates.

[0224] To visualize and quantify the number of amplified polyQ aggregates, U2OS cells were grown on slides pre-coated with poly-D-lysine solution, the slides being divided into four individual compartments. 40,000 cells were seeded into each compartment of these slides in 450 μL of phenol red-free DMEM medium. Cells were then allowed to adhere to the walls until the next day. Subsequently, the cells were transfected with the Lipofectamine 2000 (Thermo Fisher Scientific, USA) Htt EGFP-Q74 plasmid (Addgene, product number 40262, gifted by David Rubinsztein) to generate amplified repetitive sequences. Four hours after transfection, the medium was replaced with clean medium, and the test compound was added to a final concentration of 5 μmol·L⁻¹. -1Cells treated in this manner were then cultured for 24 hours. The culture medium was then aspirated, cells were washed with PBS, and 400 μL of 4% paraformaldehyde fixation solution was added to each chamber. The slides were incubated in a fume hood for 20 minutes. Three biological replicates were performed for each compound (including controls). The slides were then washed twice more with PBS, and the fixed cells were permeabilized with a PBS solution of 0.3% Triton X100 and 0.1% FBS for half an hour. The nuclei were then stained with 1 μg / ml Hoechst 33255 solution (ThermoFisher Scientific, product number H3569) for 10 minutes. Finally, the solution was aspirated and replaced with fresh PBS. Fluorescence images were captured using a Zeiss LSM 780 confocal microscope. Selected fluorophores were captured: EGFP excitation λ = 488 nm; emission λ = 495–555 nm at 25% laser power; Hoechst 33255 excitation λ = 405 nm; emission λ = 410–435 nm at 10% laser power. For each compound and control sample (DMSO), images of ≥60 randomly selected cells were captured, and the resulting images were then analyzed using ZEN (Zeiss, Germany) and ImageJ (National Institutes of Health, Bethesda, USA) software to quantify the EGFP signal intensity in positive cells and to determine the number and size of Htt-Q74 aggregates amplified within GFP-positive cells.

[0225] The results are shown in Table 8. Analysis of the number of amplified aggregates in cells treated with the test compounds clearly showed that compound 8 reduced the relative fluorescence intensity of one cell by 66%, while the other compounds reduced it by 57% to 37%. In contrast, the comparative compound ASC-JM17 reduced the number of aggregates to only 67%. The total area occupied by intracellular protein aggregates remained unchanged. However, surprisingly, a significant decrease was observed with compound 17 compared to the control. All test compounds significantly reduced the number of GFP-positive cells containing protein aggregates, and compound 17 again showed a significant decrease compared to the control.

[0226] Table 8: Comparison with DMSO and ASC-JM17 control compounds, using a concentration of 5 μmol·L⁻¹ -1 Analysis of amplified Htt-Q74 aggregates in U2OS cells 24 hours after treatment with the test compound. Fluorescence intensity of the test cells is relative to the DMSO control. The total area of ​​intracellular aggregates is expressed in µm. 2The number of GFP-positive cells containing aggregates is expressed as a percentage of the total number of cells examined. The relative fluorescence intensity and total aggregate area were statistically analyzed using the Kolomorov-Smirnov test; in GraphPad Prism 9, the proportion of GFP-positive cells containing aggregates was analyzed using a one-tailed unpaired t-test with Welch's correction. p-values ​​< 0.0001 are denoted by the letter "a", p < 0.001 = "b", p < 0.01 = "c", and p < 0.05 = "d".

[0227]

[0228] Industrial applicability

[0229] This invention relates to the use of bis(phenylmethylene)cycloalkanones and their heterocyclic analogues in human and veterinary pharmaceuticals for the treatment and / or prevention of diseases caused by the presence of misfolded proteins in cells, protein homeostasis imbalance, and protein toxicity stress, typically protein diseases, especially neurodegenerative diseases (e.g., amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), Kennedy's disease (KD), Huntington's disease (HD), Creutzfeldt-Jakob disease (CJD), desminopathy, spinocerebellar ataxia (SCA), transthyretin familial amyloid polyneuropathy, dentate nucleus-rubella-lewy body atrophy), as well as systemic amyloidosis, organ-specific amyloidosis, cystic fibrosis, or diabetes.

Claims

1. Compounds of general formula I Used as a medicine for the treatment or prevention of protein disorders, such as neurodegenerative diseases, amyloidosis, cystic fibrosis, or diabetes. in, R 1 R 2 R 3 and R 4 Independently selected from the group consisting of hydrogen atom, fluorine atom, hydroxyl group, methoxy group, difluoromethoxy group, and trifluoromethoxy group, optionally, R 1 and R 2 Together they form methylenedioxy and / or R 3 and R 4 Together they form methylenedioxy; A is a carbon atom or a nitrogen atom, and R 5 Selected from hydrogen atoms, deuterium, C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl and groups. The group, Among them, R 6 Selected from the group consisting of C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl, C1-C6 alkoxy, C3-C6 cycloalkoxy, C6-C10 aryloxy, C1-C6 alkylamino, C3-C6 cycloalkylamino, and C6-C10 arylamino. or A is selected from the group that includes oxygen atoms, sulfur atoms, and SO2 groups, and R 5 It does not exist; n is 1, 2, or 3.

2. Use of the compound of general formula I according to claim 1, wherein, When A is a carbon atom or a nitrogen atom, then R 5 Selected from C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl and group. The group, Among them, R 6 It is selected from the group consisting of C1-C6 alkyl, C3-C6 cycloalkyl, C6-C10 aryl, trifluoromethyl, C1-C6 alkoxy, C3-C6 cycloalkoxy, C6-C10 aryloxy, C1-C6 alkylamino, C3-C6 cycloalkylamino and C6-C10 arylamino.

3. Use of the compound of general formula I according to claim 1, wherein, Substituent R 1 R 2 R 3 Or R 4 At least one of the substituents is not a hydroxyl group.

4. Use of the compound of formula I according to claim 1, wherein the compound is used as a medicament for treating neurodegenerative diseases, systemic amyloidosis, organ-specific amyloidosis, cystic fibrosis, and diabetes, wherein the neurodegenerative diseases are selected from the group comprising: amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), Kennedy's disease (KD), Huntington's disease (HD), Creutzfeldt-Jakob disease (CJD), spinocerebellar ataxia (SCA), dentate nucleus-rubella-lewy body atrophy, and transthyretin familial amyloid polyneuropathy.

5. Use of the compound of formula I according to claim 1, wherein the compound is used as a medicament for the prevention of genetically based neurodegenerative diseases, familial systemic amyloidosis, familial organ-specific amyloidosis, and cystic fibrosis, wherein the genetically based neurodegenerative diseases are selected from the group comprising: familial amyotrophic lateral sclerosis (ALS), familial Parkinson's disease (PD), familial Alzheimer's disease (AD), Kennedy's disease (KD), Huntington's disease (HD), familial Creutzfeldt-Jakob disease (CJD), familial spinocerebellar ataxia (SCA), transthyretin familial amyloid polyneuropathy, and familial dentate nucleus-rubella-lewy body atrophy.

6. Compounds of general formula I, selected from the group consisting of: 2,6-Bis(3-fluoro-4-(trifluoromethoxy)benzylmethyl)-4-methylcyclohexanone, 1-Acetyl-3,5-bis(3,4-methylenedioxybenzylmethyl)piperidin-4-one 3,5-Bis(3,4-dimethoxybenzylmethyl)-1-cyclopropylcarbonylpiperidin-4-one, 3,5-Bis(3,4-dimethoxybenzylmethyl)-1-trifluoroacetylpiperidin-4-one, 3,5-Bis(3,4-dimethoxybenzyl)-1-(3,4-dimethoxybenzoyl)piperidin-4-one 1-Cyclopropanecarbonyl-3,5-bis(4-hydroxy-3-methoxybenzylmethyl)piperidin-4-one 2,4-Bis(3,4-dimethoxybenzyl)-8-methyl-8-azabicyclo[3.2.1]oct-3-one, 2,6-Bis(3,4-dimethoxybenzyl)-4-trifluoromethylcyclohexanone, 2,6-Bis(4-methoxy-3-(trifluoromethoxy)benzylmethyl)-4-methylcyclohexanone, 3,5-Bis(4-methoxy-3-(trifluoromethoxy)benzylmethyl)-1-acetylpiperidin-4-one 3,5-Bis(4-methoxy-3-(trifluoromethoxy)benzylmethyl)-1-(3,4-dimethoxybenzoyl)piperidin-4-one, 3,5-Bis(3,4-Di(difluoromethoxy)benzylmethyl)-1-acetylpiperidin-4-one 2,6-Bis(3,4-Di(difluoromethoxy)benzyl)-4-methylcyclohexanone, 2,6-Bis(4-hydroxy-3-(trifluoromethoxy)benzyl)-4-methylcyclohexanone, 3,5-bis(3,4-bis(difluoromethoxy)benzylmethyl)tetrahydrothiaran-4-one.

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