4-amino-2, 6-bis (benzylidene) cyclohexanone and use thereof

Abstract

The present invention relates to 4-amino-2, 6-bis (benzylidene) cyclohexanone of general formula I wherein R1, R2, R3 and R4 are independently selected from the group comprising a hydrogen atom, a hydroxyl group, a C1-C3 alkoxy group, a trifluoromethoxy group and a difluoromethoxy group, and pharmaceutically acceptable salts, addition salts and solvates thereof; r < 5 > and R < 6 > are independently selected from the group consisting of C1-C3 alkyl groups and hydrogen atoms, or R < 5 > is a hydrogen atom and R < 6 > is an acyl or thioacyl group of general formula II, or a sulfonic acid group of general formula III, where X is O or S; r7 is selected from the group consisting of R8 and NH-R8, where R8 is selected from the group consisting of C1-C6 alkyl, C3-C8 cycloalkyl, ternary to eight membered heterocycloalkyl, (CH2CH2O) n-(C1-C3 alkyl), CH2O (CH2CH2O) n-(C1-C3 alkyl), C6-C12 aryl, five membered to nine membered heteroaryl, (C6-C12) aryl-(C1-C3) alkyl-, five membered to seven membered heteroaryl-(C1-C3) alkyl-, five membered to seven membered heteroaryl-O-(C1-C3) alkyl-, (C1-C3 alkyl) O-C (O)-(C1-C3) alkyl-, wherein n is 1, 2, 3, 4 or 5, where the substituent R8 is optionally substituted by at least one substituent selected from the group consisting of C1-C3 alkyl, C1-C3 alkoxy, OH, halogen, = O, NH2, NH2C (= O)-(C1-C3 alkoxy), NHR9, where R9 is selected from C1-C3 alkyl and NR102, where R10 is independently selected from C1-C3 alkyl, or both R10 are formed together by C2-C5 alkylene, with the proviso that at least one of substituents R1 and R2 is not a hydrogen atom, and at least one of substituents R1 and R2 is not a hydrogen atom. And at least one of substituents R3 and R4 is not a hydrogen atom. The compounds are suitable for use in the treatment of proteinopathies, viral diseases, and diseases directly associated with elevated levels of ferroptosis, such as stroke, rhabdomyolysis, non-alcoholic steatohepatitis, acute pancreatitis, and psoriasis.

Description

Technical Field

[0001] This invention relates to novel compounds derived from 4-amino-2,6-bis(benzylmethyl)cyclohexanone, and the use of these compounds in reducing protein toxicity stress in cells and tissues and inhibiting ferroptosis in cells and tissues. Background Technology

[0002] Intracellular protein degradation is a tightly regulated process crucial for maintaining cellular homeostasis. Over 90% of cytoplasmic proteins are degraded via the so-called ubiquitin-proteasome system (UPS), which removes synthetically defective or misfolded proteins and regulates intracellular protein levels, directly impacting protein activity. At the heart of the UPS system is the 26S proteasome, which 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 activity) and one or two regulatory subunits (called 19S). The aging process and the development of neurodegenerative diseases are closely linked to the accumulation of misfolded or damaged proteins, which can be cytotoxic or even directly induce apoptosis. Both aging and the development of neurodegenerative diseases are accompanied by a decline 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 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.). Cell Biology Trends , 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). The most promising way to improve UPS capacity is to increase the synthesis of the proteasome itself, ideally while simultaneously activating the synthesis of heat shock proteins (HSPs). Both 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). 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. An important 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 the future treatment of protein diseases, including neurodegenerative diseases, whose development is associated with the formation of protein aggregates and proteotoxic stress (Njomen et al.: 2016, 25.10, 1979-1989). J Med Chem , 2019, 62.14, 6469-6481).

[0004] When proteasome activity is inhibited, the transcription factor NRF1 preferentially induces the synthesis of all proteasome subunits (Kleiger et al.). Cell Biology Trends , 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, ensuring cellular responses to stress conditions. Heat shock proteins help misfolded or stress-damaged proteins achieve the correct conformation, which is currently considered one of the important defense mechanisms 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).

[0005] Recent findings suggest that NRF1-controlled transcriptional pathways protect cells and tissues from ferroptosis. Ferroptosis is a newly discovered type of regulated cell death, morphologically, biochemically, and genetically distinct from other currently described cell death types. Ferroptosis is characterized by iron dependence, marked by the accumulation of lipid peroxides and reactive oxygen species derived from iron metabolism. In degenerative and ischemic diseases, ferroptosis is involved in the development and pathogenesis of these diseases, and inhibiting ferroptosis represents an attractive strategy to slow or directly halt the progression of these chronic diseases (Liu et al.). Annals of Translational Medicine 2022 10 (6); Ryan et al.: Trends in Pharmacological Sciences , 2023; Pan et al.: Antioxidants , 11 (11), 2196). NRF1 prevents ferroptosis by inducing the expression of glutathione peroxidase 4 (GPX4), a key protein that prevents lethal lipid peroxidation, and NRF1 also prevents ferroptosis by maintaining proteasome activity (Forcina et al.: Proceedings of the National Academy of Sciences , 119 (11), e2118646119).

[0006] Currently, the most discussed compound that can increase proteasome activity and simultaneously increase the expression of heat shock proteins regulated by the transcription factor HSF1 is the curcumin derivative ASC-JM17 (1 E 6 E )-4-(cyclobutylmethyl)-1,7-bis(3,4-dimethoxyphenyl)hept-1,6-diene-3,5-dione, has been shown to be a dual activator of the above two signaling pathways (Koizumi et al.: Proceedings of the Japan Academy. Series B , 2018, 325-336; Bott et al.: Human Molecular Genetics (2016, 25.10, 1979-1989). This compound has been approved by the European Medicines Agency (EMA) for the treatment of spinal-bulbar muscular atrophy (known as Kennedy's disease). However, the low solubility and associated bioavailability of curcumin and its derivatives under physiological conditions, as well as their fairly rapid degradation in tissues (a common characteristic), are problematic.

[0007] 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

[0008] This invention aims to address the adverse effects of proteotoxic stress and ferroptosis in cells and tissues by employing novel compounds derived from 4-amino-2,6-bis(phenylmethylene)cyclohexanone to prevent and / or reduce the formation and deposition of metastable proteins in cells and tissues through controlled reduction of proteotoxic stress and ferroptosis. Surprisingly, the novel compounds derived from 4-amino-2,6-bis(phenylmethylene)cyclohexanone described herein specifically activate the transcription factor NRF1 (… NFE2L1 These pathways are controlled by genes. They are directly associated with enhanced intracellular proteasome activity, increased synthesis of heat shock proteins directly involved in protein degradation, and activation of autophagy. Enhanced NRF1 activity further inhibits ferroptosis. Simultaneously, these compounds exhibited extremely low cytotoxicity and in vivo toxicity at relevant doses. An important and surprising finding is that these compounds, at relevant concentrations, do not increase the production of reactive oxygen species (ROS) or affect the cell cycle, thus reducing the risk of adverse side effects.

[0009] The object of this invention is 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I and its pharmaceutically acceptable salts, addition salts and solvates.

[0010] in, R 1 R 2 R 3 and R 4 Independently selected from the group consisting of hydrogen atom, hydroxyl group, C1-C3 alkoxy group, trifluoromethoxy group and difluoromethoxy group; R 5 and R 6 Independently selected from the group consisting of C1-C3 alkyl groups and hydrogen atoms, or, R 5 It is a hydrogen atom, and R 6 It is an acyl or thioacyl group of general formula II, or a sulfonic acid group of general formula III.

[0011] in, X is either O or S; R 7 Choose freely R 8 and NH-R 8 The group consisting of R 8 Choose from the following groups: C1-C6 alkyl, C3-C8 cycloalkyl, ternary to octahedral heterocyclic alkyl, (CH2CH2O). n -(C1-C3 alkyl), CH2O(CH2CH2O) n-(C1-C3 alkyl), C6-C12 aryl, five- to nine-membered heteroaryl, (C6-C12)aryl-(C1-C3)alkyl-, five- to seven-membered heteroaryl-(C1-C3)alkyl-, five- to seven-membered heteroaryl-O-(C1-C3)alkyl-, (C1-C3 alkyl)OC(O)-(C1-C3)alkyl-, Where n is 1, 2, 3, 4 or 5, Among them, substituent R 8 Optionally substituted with at least one substituent selected from the group consisting of: C1-C3 alkyl, C1-C3 alkoxy, OH, halogen, =O, NH2, NH2C(=O)-(C1-C3 alkoxy), NHR 9 , where R 9 Selected from C1-C3 alkyl and NR 10 2, where R 10 Independently selected from C1-C3 alkyl groups, or two R groups 10 Together formed from C2-C5 alkylene groups, The condition is that the substituent R 1 and R 2 At least one of them is not a hydrogen atom, and the substituent R 3 and R 4 At least one of them is not a hydrogen atom.

[0012] Alkyl groups are saturated straight-chain or branched hydrocarbons. Alkyl groups can include, but are not limited to, methyl, ethyl, propyl, and isopropyl.

[0013] 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.

[0014] 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.

[0015] An alkoxy group is an -O-alkyl group. Examples of alkoxy groups are methoxy or ethoxy.

[0016] 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.

[0017] 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 pyridinyl, imidazolyl, and pyrazolopyrimidinyl.

[0018] Halogens are selected from the group consisting of fluorine, chlorine, bromine, and iodine.

[0019] In one implementation, R 1 R 2 R 3 and R 4 Independently selected from the group consisting of hydroxyl, C1-C3 alkoxy, trifluoromethoxy, and difluoromethoxy, preferably, R 1 R 2 R 3 and R 4 It is independently selected from the group including methoxy and ethoxy groups.

[0020] In some implementations, R 5 and R 6 Both are hydrogen atoms.

[0021] In some implementations, R 5 For hydrogen atoms, R 6 It is an acyl or thioacyl group of general formula II, or a sulfonic acid group of general formula III, and R 7 Choose from the following groups: .

[0022] In some preferred embodiments, R 1 R 2 R 3 and R 4 It is methoxy or ethoxy, R 5 For hydrogen atoms, R 6 It is an acyl or thioacyl group of general formula II, or a sulfonic acid group of general formula III, and R 7 It is a C1-C6 alkyl group, preferably methyl or ethyl.

[0023] In some implementations, R 5 It is a hydrogen atom, and R 6 It is an acyl or thioacyl group of general formula II.

[0024] In some implementations, R 5 For hydrogen atoms, R 6 It is a group of general formula II, and R 7 Selected from R 8 and -NH-R 8 The group, in which R 8Choose from the group consisting of: C1-C6 alkyl, C3-C8 cycloalkyl, piperazine, pyrrolyl, quinine cycloalkyl, phenyl, naphthyl, pyridinyl, imidazolyl, thiazolyl, oxazolyl, pyrazolopyrimidinyl, (C6-C12)aryl-(C1-C3)alkyl-, (CH2CH2O) n -(C1-C3 alkyl), CH2O(CH2CH2O) n -(C1-C3 alkyl), five- to seven-membered heteroaryl-(C1-C3)alkyl-, (C1-C3 alkyl)OC(O)-(C1-C3)alkyl-, Where n is 1, 2, 3, 4 or 5, Among them, substituent R 8 Optionally substituted with at least one substituent selected from the following groups: C1-C3 alkyl, C1-C3 alkoxy, OH, halogen, =O, NH2, NHR 9 , where R 9 Selected from C1-C3 alkyl and NR 10 2, where R 10 Independently selected from C1-C3 alkyl groups, or two R groups 10 They are formed together from C2-C5 alkylene groups.

[0025] Preferably, R 7 Selected from the group consisting of: methyl, ethyl, propyl, cyclopropyl, azirmonyl heptayl, morpholinyl, piperazine, phenyl, naphthyl, pyridinyl, imidazolyl, pyrrolyl, quininecycloyl, thiazolyl, oxazolyl, aminomethyl, aminoethyl, aminopropyl, N,N-dimethylaminopropyl, N,N-dimethylaminoethyl, N,N-dimethylaminomethyl, aminophenyl, diaminophenyl, N,N-dimethylaminophenyl, N,N-diethylaminomethyl, N-methylimidazolyl, (fluoro)pyrrolyl, (methoxymethyl)pyrrolyl, isopropylamino, N-methylpiperazine, aminocarbonylmethoxyphenyl, hydroxypyridinyl, methylpyridinyl, 6-hydroxy-4-methylpyridin-3-yl, (morpholin-4-yl)ethyl, difluoropyridinyl, (methoxyethoxy)ethoxymethyl, pyrazolo[1,5-] a Pyrimidinyl, piperidinylpyridine.

[0026] In a preferred embodiment, R 5 For hydrogen atoms, R 6 It is an acyl or thioacyl group of general formula II, and R 7 Choose from the following groups: .

[0027] In some implementations, R 5 It is a hydrogen atom, and R 6 A group of general formula II, wherein R7 It can be methyl, cyclopentyl, pyridine, or imidazole.

[0028] In some implementations, R 5 It is a hydrogen atom, and R 6 It is a sulfonic acid group of general formula III.

[0029] In some implementations, R 5 For hydrogen atoms, R 6 It is a sulfonic acid group of general formula III, and R 7 Selected from the group consisting of: methyl, ethyl, propyl, cyclopropyl, azirmonyl heptayl, morpholinyl, piperazine, phenyl, pyridinyl, imidazolyl, pyrrolyl, aminopropyl, aminoethyl, aminophenyl, diaminophenyl, N-methylimidazolyl, (fluoro)pyrrolyl, (methoxymethyl)pyrrolyl, isopropylamino, N-methylpiperazine, aminocarbonylmethoxyphenyl, hydroxypyridinyl, methylpyridinyl, 6-hydroxy-4-methylpyridin-3-yl, (morpholin-4-yl)ethyl.

[0030] In a preferred embodiment, R 5 For hydrogen atoms, R 6 It is a sulfonic acid group of general formula III, and R 7 Choose from the following groups: .

[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 relates to the use of compounds of general formula I as pharmaceuticals.

[0035] Compounds of formula I are particularly suitable for the treatment or prevention of protein disorders, especially neurodegenerative diseases such as polyglutamine disease, tau protein disease, synucleinosis, or amyotrophic lateral sclerosis (ALS); they are also suitable for the treatment or prevention of amyloidosis, cystic fibrosis, and diabetes. Compounds of formula I are also suitable for the treatment of viral diseases. It is assumed here that the ability of compounds of formula I to induce autophagy subsequently manifests as an increase in viral particle degradation, especially during viral entry. Furthermore, compounds of formula I are suitable for the treatment of diseases directly associated with elevated levels of: ferroptosis, particularly stroke; rhabdomyolysis—severe skeletal muscle damage; non-alcoholic steatohepatitis; acute pancreatitis; or psoriasis.

[0036] Proteins produced by polyglutamine diseases accumulate in the nuclei of neurons, forming intracellular inclusion bodies that lead to neuronal death. All polyglutamine diseases are progressive and fatal, typically beginning in adulthood and developing over 10 to 30 years. For a review of polyglutamine diseases, see the Journal of Molecular Cell Biology (2010), 2, 180-191. Polyglutamine diseases include Huntington's disease, spinobulbar muscular atrophy, spinocerebellar ataxia, and dentate nucleus-rubella-lewy body atrophy.

[0037] Tau proteinopathy and synucleinopathy are caused by the presence of characteristic plaques. Synucleinopathy is a group of distinct neurodegenerative diseases whose common pathological damage consists of aggregates of the insoluble protein α-synuclein in neurons and glial cells. This group of diseases includes Parkinson's disease and Lewy body-related dementia. In the absence of β-amyloid plaques, fibrous inclusions of tau protein and degeneration of brain tissue are typical features of neurodegenerative tau proteinopathy. Diseases belonging to this group include: Pick's disease, progressive supranuclear palsy (PSP), Parkinson's disease (Tau protein accumulation), Still-Richardson-Olshefsky disease, Guam disease (Guam Parkinson's disease-dementia syndrome), post-traumatic Parkinson's disease, and Alzheimer's disease.

[0038] Microscopically, PSP is characterized by the presence of neurofibrillary tangles (NFTs) with specific features. These are characteristically spherical (unlike NFTs in Alzheimer's disease) and composed of straight filaments approximately 15 nm long. NFTs are likely formed from abnormal protein formations called tau protein. Clinically, PSP is characterized by Parkinson's syndrome with predominantly axial muscle rigidity, postural instability with frequent falls, oculomotor disturbances, cognitive deficits, and subcortical dementia, with a characteristic onset and course, as well as many other typical symptoms.

[0039] Post-traumatic Parkinson's Disease: The deaths of boxers after World War II revealed numerous and fairly obvious signs of trauma in the athletes' brain tissue. Neurofibrillary changes in cortical neurons were very common. However, most importantly, pathological tau proteins were found in neurons, very similar to those found in PSP and other tau protein disorders, suggesting that the pathophysiology of this disease is more complex than a simple consequence of repeated microtrauma. Neurol 2000;20:179-185).

[0040] Alzheimer's disease: In Alzheimer's patients, there is hyperphosphorylation of tau protein and formation of neurofibrillary tangles in the brain tissue, as well as the aggregation of β-amyloid protein, which forms specific structures in the brain called "senile plaques." The neuropathological features of AD are "positive" lesions, such as amyloid plaques, cerebral amyloid angiopathy, and neurofibrillary tangles; and "negative" lesions, such as neuronal and synaptic loss. A form of AD with the Lewy body features of idiopathic Parkinson's disease present in various brain structures, including the cortex, is called a "Lewy body variant."

[0041] Amyloidosis includes systemic amyloidosis and organ-specific amyloidosis. These include familial amyloidosis without neuropathy, familial neuropathic amyloidosis, familial neurological amyloidosis, secondary systemic amyloidosis, and organ-specific amyloidosis.

[0042] The present invention also relates to the use of 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-leucocerebellar Lewy body atrophy, transthyretin familial amyloid polyneuropathy), as well as systemic or organ-specific amyloidosis, and / or cystic fibrosis or diabetes.

[0043] In another aspect, the present invention provides a compound 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), familial systemic amyloidosis or familial organ-specific amyloidosis, and / or cystic fibrosis).

[0044] In addition, compounds of general formula I are suitable for the treatment of viral diseases. Viral diseases include, in particular, those caused by the HBV virus, i.e., especially hepatitis B.

[0045] Furthermore, compounds of general formula I are suitable for treating diseases directly associated with elevated ferroptosis levels. In stroke, serine protease thrombin promotes arachidonic acid mobilization via esterification by acyl-CoA synthase (ACSL4), thereby participating in ferroptosis. Inhibition of this pathway reduces ischemic neuronal damage in stroke (Tuo et al.). Signal Transduct Target Ther 2022;7(1):59). Rhabdomyolysis is a relatively common and severe syndrome of skeletal muscle injury. Ferropreservation can exacerbate the development of rhabdomyolysis. Increased expression of GPX4, which binds to ACSL4, in muscle cells can reduce lipid peroxidation and inhibit the progression of the syndrome (Yuan et al.: 2016.03.016). In non-alcoholic steatohepatitis, RSL-3 significantly increases the levels of hepatitis-related inflammatory factors. Inhibiting ferropreservation is a novel approach to treating this disease (Qi et al.: 2022;7(1):59). Am J Pathol. 2020;190(1):68 - 81 Ferrocytosis is also associated with pancreatic disease: administration of the ferroptosis inhibitor liproxstatin-1 can alleviate acute pancreatitis (Li et al.). Chin Med J (Engl) .2022;135(17):2026-2034). Ferroprelation is also associated with psoriasis. Ferroprelation inhibitor ferrostatin-1 inhibited feroprelation-related changes in keratinocytes treated with erastin (a feroprelation inducer) and improved psoriatic-like dermatitis in an imiquimod-induced model (Shou et al.: 2022;135(17):2026-2034). Cell Death Dis. 2021; 12(11): 1009).

[0046] On the other hand, the object of the present invention is the use of compounds of general formula I for the treatment of diseases directly related to elevated levels of ferrodegeneration, said diseases preferably selected from the group comprising stroke, rhabdomyolysis, non-alcoholic steatohepatitis, acute pancreatitis and psoriasis.

[0047] In one embodiment, the compound of general formula I (wherein, R) 5 For hydrogen atoms, R 6 (The sulfonic acid group of general formula III as defined above) is used to treat diseases directly related to elevated ferroptosis levels, preferably selected from the group consisting of stroke, rhabdomyolysis, non-alcoholic steatohepatitis, acute pancreatitis, and psoriasis.

[0048] Compounds of general formula I can be administered to animals or humans to activate cellular responses to proteotoxic stress caused by the presence or elevated levels of metastable proteins within the cell and imbalances in protein homeostasis (protein homeostasis), such as due to aging.

[0049] Compounds of general formula I can be administered to animals or humans to activate cellular responses to elevated levels of ferroptosis in cells or tissues caused by conditions such as degenerative diseases, stroke, severe skeletal muscle injury, non-alcoholic steatohepatitis, acute pancreatitis, or psoriasis.

[0050] Compounds of general formula I can be formulated into pharmaceutical preparations with pharmaceutically acceptable excipients. These preparations can be in liquid, solid, or other forms, such as aerosols. Liquid forms include solutions, suspensions, dispersions, emulsions (e.g., formulated for injection or oral administration), gels, and ointments. Solid forms include, for example, capsules, tablets, coated tablets, powders, suppositories, and other forms known in the art.

[0051] Pharmaceutically acceptable excipients include solvents, solubility controls, pH controls, carriers, fillers, binders, flow aids, disintegrants, preservatives, adsorbents, viscosity controls, and agents that affect the sensory properties of a product (such as taste, odor, or color).

[0052] In one aspect, the present invention relates to a method for treating diseases directly associated with elevated ferrodeation levels (preferably selected from the group including stroke, rhabdomyolysis, non-alcoholic steatohepatitis, acute pancreatitis, and psoriasis), 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.

[0053] In one aspect, the present invention relates to a method for treating or preventing protein diseases (particularly neurodegenerative diseases such as polyglutamine diseases (e.g., Huntington's disease, spinobulbar muscular atrophy, spinocerebellar ataxia, and dentate nucleus-rubella-lewy body atrophy), tau protein diseases (e.g., Pick's disease, progressive supranuclear palsy (PSP), Parkinson's disease (Tau protein accumulation), Still-Richardson-Olshefsky disease, Guam disease (Guam Parkinson's disease-dementia syndrome), post-traumatic Parkinson's disease, Alzheimer's disease), synucleinic diseases (e.g., Parkinson's disease and Lewy body dementia), or amyotrophic lateral sclerosis), 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.

[0054] In one aspect, the present invention relates to a method for treating or preventing amyloidosis (e.g., familial amyloidosis without neuropathy, familial neuropathic amyloidosis, familial neurological amyloidosis, secondary systemic 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.

[0055] In one aspect, the present invention relates to a method for treating viral diseases (e.g., diseases caused by HBV virus), 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. Detailed Implementation

[0056] Examples

[0057] Abbreviations: ACN: Acetonitrile bs: Broad singlet (NMR) d: Bimodal (NMR) dd: Double doublet (NMR) ddd: Doublet of a doublet (NMR) dt: Double triplet (NMR) DCM: Dichloromethane DIPEA: N , N -Diisopropylethylamine DMF: Dimethylformamide DMSO: Dimethyl sulfoxide ESI: Electrospray Ionization Et3N: Triethylamine EtOAc: Ethyl acetate EtOH: Ethanol TLC: Thin-layer chromatography TMSOTf: Trimethylsilyl trifluoromethanesulfonate δ: Chemical shift (NMR) [δ] - ppm ALS: Amyotrophic Lateral Sclerosis AMC: 7-Amino-4-methylcoumarin BSA: Bovine serum albumin Dalton DMEM: Durbeco Modified Eagle Medium DMSO: Dimethyl sulfoxide DTT: Dithiothreitol EDTA: Ethylenediaminetetraacetic acid ESI: Electrospray Ionization GFP: Green fluorescent protein HRMS: High Resolution Mass Spectrometry HSF1: Heat shock factor 1 HSP: Heat Shock Protein NMR: Nuclear Magnetic Resonance fmoc: fluorenemethyloxycarbonyl Fmoc-Cl: fluorenemethyloxycarbonyl chloride HRMS: High Resolution Mass Spectrometry J: Coupling constant (NMR) m: Multiplet (NMR) m / z Mass-to-charge ratio (MS) MeOH: Methanol NMR: Nuclear Magnetic Resonance p: Quintet (NMR) q: Quartet (NMR) s: Single peak (NMR) SOCl2: thionyl chloride t: triplet (NMR) td: Triple doublet (NMR) TFA: Trifluoroacetic acid NRF1: Nuclear respiratory factor 1 NRF2: Nuclear Factor (Red Series 2) Related Factor 2 PBS: Phosphate-buffered saline PCR: Polymerase Chain Reaction PC 12: A pheochromocytoma cell line derived from the adrenal medulla of rats. PI: Propidium iodide ROS: Reactive oxygen species RT: Reverse transcription Ub: ubiquitin UPS: Ubiquitin-Proteasome System

[0058] Enzyme: Renilla luciferase: an enzyme derived from the sea kidney (Renilla reniformis). Firefly luciferase: an enzyme obtained from fireflies (Photinus pyralis). Example 1: Preparation of the compound 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.

[0059] Compound List

[0060] tert-butyl(3,5-bis(( E 3,4-Dimethoxybenzyl)-4-oxocyclohexyl)carbamate

[0061] tert-butyl(4-oxocyclohexyl)carbamate (2 g, 9.38 mmol) was dissolved in EtOH (10 mL), and 20% aqueous NaOH solution (9.3 mL) was added dropwise. The solution was stirred for 5 minutes, and 3,4-dimethoxybenzaldehyde (3.896 g, 46.89 mmol) was added. The reaction mixture was stirred overnight at ambient temperature. The resulting suspension was filtered off, and the filter cake was washed with water and cold EtOH and dried. The reaction yielded 3.449 g of the title compound in 72% yield.

[0062] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.89 – 7.83 (m, 2H), 7.12 – 7.05 (m, 2H), 7.00 (d, J = 2.0 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 4.14 – 4.01 (m, 1H), 3.92 (s, 6H), 3.91 (s, 6H), 3.23 – 3.13 (m, 2H), 3.08 – 3.04 (m, 2H), 1.35 (s, 9H).

[0063] HRMS (ESI+): m / z C 29 H 35 Calculated value of O7NNa = 532.2306; Measured value = 523.2302 [M+Na] + .

[0064] 2,6-Bis(3,4-dimethoxybenzylmethyl)-4-(acetamido)cyclohexanone (1)

[0065] Will N -(4-oxocyclohexyl)acetamide (0.775 g, 5 mmol) was dissolved in EtOH (10 mL), and KOH (0.11 g, 1.86 mmol) was added. The solution was stirred for 5 minutes, and 3,4-dimethoxybenzaldehyde (1.660 g, 10 mmol) was added. The reaction mixture was stirred overnight at ambient temperature. The solution was diluted with water (20 mL) and extracted with EtOAc (2 x 10 mL). The organic phases were combined and washed with brine and water. The solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent EtOAc:MeOH, 10:1), followed by recrystallization with hot MeOH. The reaction yielded 0.320 g of the title compound in 14% yield.

[0066] 1 H NMR (400 MHz, CDCl3) δ 7.91 (s, 2H), 7.12 (dd, J = 8.4, 1.9 Hz, 2H), 7.01 (d, J = 2.0 Hz, 2H), 6.93 (d, J= 8.4 Hz, 2H), 5.60 (d, J = 7.6 Hz,1H), 4.76 – 4.28 (m, 1H), 3.95 (s, 3H), 3.93 (s,31H), 3.17 (m, 4H), 1.91 (s,3H).

[0067] HRMS (ESI+): m / z C 26 H 30 Calculated value of O6N = 452.2073; Measured value = 452.2077 [M+H] + .

[0068] 3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (2)

[0069] tert-butyl(3,5-bis(( E 1 g (1.96 mmol) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate was dissolved in DCM (5 mL), cooled in an ice bath, and TFA (5 mL) was added dropwise. The solution was stirred at ambient temperature for 1.5 h. 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). The reaction yielded 0.867 g of the title compound in 84% yield.

[0070] 1 H NMR (401 MHz, DMSO- d 6) δ 8.11 (s, 3H), 7.74 (d, J = 2.3 Hz, 2H),7.18 – 7.11 (m, 4H), 7.08 (d, J = 8.3 Hz, 2H), 3.82 (s, 6H), 3.81 (s, 6H), 3.54 – 3.40 (m, 1H), 3.41 – 3.32 (m, 2H), 3.03 – 2.92 (m, 2H).

[0071] HRMS (ESI+): m / z C 24 H 28 Calculated value of O5N = 410.1962; Measured value = 410.1961 [M+H] + .

[0072] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)amino)- N,N -Dimethyl-2-oxoethane-1-ammonium trifluoroacetate (3)

[0073] 3,5-bis(( E Add HBTU (0.135 g, 0.35 mmol) to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol). N,N -Dimethylglycine (0.037 g, 0.36 mmol). The reaction mixture was stirred overnight at ambient temperature. 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). The reaction yielded 0.053 g of the title compound in 46% yield.

[0074] 1 H NMR (401 MHz, chloroform-) d ) δ 8.47 (d, J = 7.2 Hz, 1H), 7.82 (s, 2H), 7.05 (dd, J = 8.4, 2.0 Hz, 2H), 6.95 (d, J = 2.0 Hz, 2H), 6.88 (d, J = 8.3Hz, 2H), 4.21 – 4.13 (m, 1H), 3.90 (s, 6H), 3.88 (s, 6H), 3.71 (s, 2H), 3.23(dd, J = 15.9, 4.0 Hz, 2H), 3.00 – 2.85 (m, 2H), 2.82 (s, 6H).

[0075] HRMS (ESI+): m / z C 28 H 35 Calculated O6N2 value = 495.2490; Measured value = 495.2487 [M+H] + .

[0076] 3-((3,5-double(( E)-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)quinine ring-1-onium trifluoroacetate (4)

[0077] 3,5-bis(( E HBTU (0.109 g, 0.29 mmol) and 3-carboxyquinone-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) were added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.11 mL, 0.79 mmol). The reaction mixture was stirred overnight at ambient temperature. 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). The reaction yielded 0.071 g of the title compound in 56% yield.

[0078] 1 H NMR (401 MHz, chloroform-) d ) δ 11.87 (bs, 1H), 7.87 – 7.78 (m, 2H), 7.10– 7.02 (m, 2H), 6.98 – 6.92 (m, 2H), 6.89 (d, J = 8.5 Hz, 2H), 6.53 (d, J =7.2 Hz, 1H), 4.40 – 4.31 (m, 1H), 3.91 (s, 3H), 3.90 (s, 3H), 3.89 (s, 3H), 3.88 (s, 3H), 3.69 – 3.59 (m, 1H), 3.30 – 3.16 (m, 6H), 3.16 – 3.03 (m, 3H), 2.83 – 2.73 (m, 1H), 2.16 – 2.08 (m, 1H), 1.95 – 1.72 (m, 3H), 1.59 – 1.43(m, 1H).

[0079] HRMS (ESI+): m / z C 32 H 39 Calculated O6N2 value = 547.2803; Measured value = 547.2801 [M+H] + .

[0080] N -(3,5-double(( E)-3,4-dimethoxybenzyl)-4-oxocyclohexyl)-2-(2-(2-methoxyethoxy)ethoxy)acetamide (5)

[0081] 3,5-bis(( E To a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol), HBTU (0.109 g, 0.29 mmol) and 2-(2-(2-methoxyethoxy)ethoxy)acetic acid (0.051 g, 0.29 mmol) were added. The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (elution buffer H2O:ACN, gradient elution). The reaction yielded 0.080 g of the title compound in 74% yield.

[0082] 1 H NMR (401 MHz, DMSO- d 6) δ 7.79 (d, J = 7.5 Hz, 1H), 7.67 (s, 2H), 7.13 (d, J = 8.2 Hz, 4H), 7.05 (d, J = 8.2 Hz, 2H), 4.04 – 3.92 (m, 1H), 3.87(s, 2H), 3.81 (s, 6H), 3.80 (s, 6H), 3.59 – 3.49 (m, 6H), 3.46 – 3.39 (m, 2H), 3.21 (s, 3H), 3.19 – 3.10 (m, 2H), 3.03 – 2.91 (m, 2H).

[0083] HRMS (ESI+): m / z C 31 H 39 Calculated value of O9NNa = 592.2517; Measured value = 592.2513 [M+Na] + .

[0084] N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)cyclopentaneformamide (6)

[0085] 3,5-bis(( E HBTU (0.109 g, 0.29 mmol) and cyclopentanecarboxylic acid (0.033 g, 0.29 mmol) were added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol). The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (elution buffer H2O:ACN, gradient elution). The reaction yielded 0.085 g of the title compound in 88% yield.

[0086] 1 H NMR (401 MHz, chloroform-) d δ 7.88 (s, 2H), 7.09 (dd, J = 8.6, 1.8 Hz, 2H), 6.99 (d, J = 2.0 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 4.45 – 4.27 (m, 1H), 3.92 (s, 6H), 3.91 (s, 6H), 3.21 – 2.99 (m, 4H), 2.51 – 2.32 (m, 1H), 1.85 –1.56 (m, 6H), 1.56 – 1.45 (m, 2H).

[0087] HRMS (ESI+): m / z C 30 H 35 Calculated value of O6NNa = 528.2357; Measured value = 528.2353 [M+Na] + .

[0088] 1-(2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)amino)-2-oxoethyl)-4-methylpiperazine-1,4-dionium di(trifluoroacetate) (7)

[0089] 3,5-bis(( ETo a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol), HBTU (0.109 g, 0.29 mmol) and 2-(4-methylpiperazin-1-yl)acetic acid (0.045 g, 0.29 mmol) were added. The reaction mixture was stirred overnight at ambient temperature. 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). The reaction yielded 0.102 g of the title compound in 67% yield.

[0090] 1 H NMR (401 MHz, chloroform-) d ) δ 7.88 (s, 2H), 7.11 (dd, J = 8.5, 2.0 Hz,2H), 7.03 – 6.97 (m, 3H), 6.93 (d, J = 8.4 Hz, 2H), 4.55 – 4.46 (m, 1H), 3.93 (s, 6H), 3.92 (s, 6H), 3.34 – 3.24 (m, 2H), 3.16 – 3.07 (m, 2H), 3.07 (s, 2H), 2.82 (s, 3H), 2.79 (bs, 8H).

[0091] HRMS (ESI+): m / z C 31 H 40 Calculated O6N3 value = 550.2912; Measured value = 550.2910 [M+H] + .

[0092] 4-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)amino)- N,N -Dimethyl-4-oxobutane-1-ammonium trifluoroacetate (8)

[0093] 3,5-bis(( E HBTU (0.109 g, 0.29 mmol) and 3-carboxy-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) were added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.11 mL, 0.79 mmol). N,N-Dimethylpropane-1-ammonium hydrochloride (0.045 g, 0.29 mmol). The reaction mixture was stirred overnight at ambient temperature. 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). The reaction yielded 0.065 g of the title compound in 53% yield.

[0094] 1 H NMR (401 MHz, chloroform-) d ) δ 12.40 (s, 1H), 7.84 (s, 2H), 7.09 (dd, J =8.5, 2.0 Hz, 2H), 6.98 (d, J = 2.0 Hz, 2H), 6.94 – 6.87 (m, 3H), 4.30 – 4.18(m, 1H), 3.91 (s, 6H), 3.90 (s, 6H), 3.26 – 3.16 (m, 2H), 3.07 – 2.93 (m,4H), 2.78 – 2.73 (m, 6H), 2.38 – 2.30 (m, 2H), 2.01 (p, J = 6.9 Hz, 2H).

[0095] HRMS (ESI+: m / z C) 30 H 39 Calculated O6N2 value = 523.2803; Measured value = 523.2800 [M+H] + .

[0096] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)amino)-2-oxoethane-1-ammonium trifluoroacetate (9)

[0097] 3,5-bis(( EHBTU (0.109 g, 0.29 mmol) and (tert-butyloxycarbonyl)glycine (0.050 g, 0.29 mmol) were added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol). The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (eluent H2O (0.1% TFA): ACN, gradient elution). The resulting solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and TFA (0.5 mL) was added dropwise. The solution was stirred at ambient temperature for 1 hour. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (eluent H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.080 g of the title compound in 73% yield.

[0098] 1 H NMR (401 MHz, DMSO- d 6) δ 8.60 (d, J = 6.7 Hz, 1H), 7.95 (bs, 3H), 7.71 (s, 2H), 7.17 – 7.09 (m, 4H), 7.04 (d, J = 8.2 Hz, 2H), 4.05 – 3.93 (m,1H), 3.81 (s, 6H), 3.80 (s, 6H), 3.52 (s, 2H), 3.25 – 3.15 (m, 2H), 3.01 –2.86 (m, 2H).

[0099] HRMS (ESI+): m / z C 26 H 31 Calculated O6N2 value = 467.2177; Measured value = 467.2175 [M+H] + .

[0100] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)-3-methylpyridine-1-onium trifluoroacetate (10)

[0101] 3,5-bis(( EHBTU (0.109 g, 0.29 mmol) and 3-methylpyridinecarboxylic acid (0.039 g, 0.29 mmol) were added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol). The reaction mixture was stirred overnight at ambient temperature. 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). The reaction yielded 0.098 g of the title compound in 80% yield.

[0102] 1 H NMR (401 MHz, chloroform-) d ) δ 8.44 – 8.36 (m, 2H), 7.90 (s, 2H), 7.75 –7.68 (m, 1H), 7.42 (dd, J = 7.8, 4.9 Hz, 1H), 7.11 (dd, J = 8.4, 2.0 Hz, 2H), 7.01 (d, J = 2.0 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 4.54 – 4.41 (m, 1H), 3.91 (s, 6H), 3.90 (s, 6H), 3.40 – 3.30 (m, 2H), 3.17 – 3.06 (m, 2H), 2.63 (s, 3H).

[0103] HRMS (ESI+): m / z C 31 H 32 Calculated value of O6N2Na = 551.2153; Measured value = 551.2151 [M+Na] + .

[0104] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (11)

[0105] 3,5-bis(( EHBTU (0.109 g, 0.29 mmol) and pyridinecarboxylic acid (0.035 g, 0.29 mmol) were added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol). The reaction mixture was stirred at ambient temperature for 3 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). The reaction yielded 0.099 g of the title compound in 82% yield.

[0106] 1 H NMR (401 MHz, chloroform-) d ) δ 8.57 – 8.51 (m, 1H), 8.33 (d, J = 8.1 Hz, 1H), 8.14 (dt, J = 7.9, 1.1 Hz, 1H), 7.93 (s, 2H), 7.86 (td, J = 7.7, 1.7 Hz,1H), 7.50 – 7.42 (m, 1H), 7.23 (s, 1H), 7.12 (dd, J = 8.4, 1.8 Hz, 2H), 7.01(d, J = 2.0 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 4.62 – 4.49 (m, 1H), 3.91 (s,6H), 3.90 (s, 6H), 3.40 – 3.30 (m, 2H), 3.22 – 3.11 (m, 2H).

[0107] HRMS (ESI+): m / z C 30 H 30 Calculated value of O6N2Na = 537.1996; Measured value = 537.1994 [M+Na] + .

[0108] 4-(2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)amino)-2-oxoethyl)-1 H -Imidazole-3-onium trifluoroacetate (12)

[0109] 3,5-bis(( E HBTU (0.109 g, 0.29 mmol) and 2-(1-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) were added to a mixture of Et3N (0.12 mL, 0.86 mmol). H 4-Imidazol-4-yl)acetic acid (0.047 g, 0.29 mmol). The reaction mixture was stirred at ambient temperature for 1 hour. 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). The reaction yielded 0.079 g of the title compound in 66% yield.

[0110] 1 H NMR (401 MHz, chloroform-) d ) δ 8.29 – 8.25 (m, 1H), 7.79 (d, J = 7.1 Hz,1H), 7.75 (s, 2H), 7.06 – 7.01 (m, 1H), 6.99 (dd, J = 8.6, 1.9 Hz, 2H), 6.89(d, J = 1.9 Hz, 2H), 6.82 (d, J = 8.5 Hz, 2H), 4.16 (s, 1H), 3.84 (s, 6H), 3.82 (s, 6H), 3.57 (s, 2H), 3.21 – 3.12 (m, 2H), 3.05 – 2.95 (m, 2H).

[0111] HRMS (ESI+): m / z C 29 H 32 Calculated O6N3 value = 518.2286; Measured value = 518.2284 [M+H] + .

[0112] ( S )-2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyrrolidine-1-onium trifluoroacetate (13)

[0113] 3,5-bis(( EHBTU (0.109 g, 0.29 mmol) and (tert-butyloxycarbonyl)-L-proline (0.062 g, 0.29 mmol) were added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol). The reaction mixture was stirred at ambient temperature for 1 hour. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (eluent H2O (0.1% TFA): ACN, gradient elution). The resulting solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and TFA (0.5 mL) was added dropwise. The solution was stirred at ambient temperature for 1.5 hours. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (eluent H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.068 g of the title compound in 58% yield.

[0114] 1 H NMR (401 MHz, chloroform-) d ) δ 11.65 (s, 1H), 8.22 (d, J = 6.7 Hz, 1H),7.84 – 7.77 (m, 2H), 7.08 – 7.00 (m, 2H), 6.94 (dd, J = 8.9, 1.9 Hz, 2H),6.91 – 6.84 (m, 2H), 4.51 (s, 1H), 4.19 – 4.07 (m, 1H), 3.91 (s, 3H), 3.89(s, 3H), 3.87 (s, 3H), 3.86 (s, 3H), 3.36 – 3.21 (m, 2H), 3.19 – 2.90 (m,4H), 2.35 – 2.28 (m, 1H), 1.97 – 1.75 (m, 3H).

[0115] HRMS (ESI+): m / z C 29 H 32 Calculated O6N3 value = 507.2490; Measured value = 507.2488 [M+H] + .

[0116] ( S )- N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)-5-oxopyrrolidine-2-carboxamide (14)

[0117] 3,5-bis(( E HBTU (0.109 g, 0.29 mmol) and ( )-3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) were added to a mixture of Et3N (0.1 mL, 0.72 mmol). S 5-O-pyrrolidine-2-carboxylic acid (0.037 g, 0.29 mmol). The reaction mixture was stirred at ambient temperature for 2.5 h. 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). The reaction yielded 0.084 g of the title compound in 85% yield.

[0118] 1 H NMR (401 MHz, chloroform-) d ) δ 7.85 – 7.76 (m, 2H), 7.10 – 7.00 (m, 2H), 7.00 – 6.88 (m, 4H), 6.90 – 6.81 (m, 2H), 4.47 – 4.38 (m, 1H), 4.14 – 4.06(m, 1H), 3.89 (s, 6H), 3.88 (s, 6H), 3.28 – 3.17 (m, 2H), 3.20 – 3.06 (m,2H), 2.47 – 2.32 (m, 1H), 2.21 (t, J = 8.0 Hz, 2H), 2.09 – 1.97 (m, 1H).

[0119] HRMS (ESI+): m / z C 29 H 32 Calculated value of O7N2Na = 543.2102; Measured value = 543.2100 [M+Na] + .

[0120] 1-(2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)amino)-2-oxoethyl)-1 H -Imidazole-1-onium trifluoroacetate (15)

[0121] 3,5-bis(( EHBTU (0.109 g, 0.29 mmol) and 2-(1-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) were added to a mixture. H -imidazol-1-yl)acetic acid (0.036 g, 0.29 mmol). 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). The reaction yielded 0.097 g of the title compound in 80% yield.

[0122] 1 H NMR (401 MHz, chloroform-) d ) δ 9.17 (s, 1H), 8.41 (d, J = 7.0 Hz, 1H),7.79 (s, 2H), 7.40 – 7.35 (m, 1H), 7.27 – 7.18 (m, 1H), 7.03 (dd, J = 8.5, 2.0 Hz, 2H), 6.94 (d, J = 2.0 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 4.97 (s, 2H), 4.29 – 4.20 (m, 1H), 3.90 (s, 6H), 3.87 (s, 6H), 3.26 – 3.17 (m, 2H), 3.11 – 3.01 (m, 2H).

[0123] HRMS (ESI+): m / z C 29 H 32 Calculated O6N3 value = 518.2286; Measured value = 518.2284 [M+H] + .

[0124] 4-(2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)amino)-2-oxoethyl)morpholine-4-onium trifluoroacetate (16)

[0125] 3,5-bis(( EHBTU (0.109 g, 0.29 mmol) and 2-morpholine acetic acid (0.052 g, 0.29 mmol) were added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.12 mL, 0.86 mmol). 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). The reaction yielded 0.095 g of the title compound in 76% yield.

[0126] 1 H NMR (401 MHz, chloroform-) d ) δ 8.30 (d, J = 7.2 Hz, 1H), 7.84 (s, 2H), 7.07 (dd, J = 8.4, 2.0 Hz, 2H), 6.97 (d, J = 2.1 Hz, 2H), 6.90 (d, J = 8.4Hz, 2H), 4.25 – 4.14 (m, 1H), 3.91 (s, 6H), 3.90 (s, 6H), 3.90 – 3.86 (m,4H), 3.57 (s, 2H), 3.22 (dd, J = 15.8, 3.9 Hz, 2H), 3.15 – 3.08 (m, 4H), 3.04 – 2.92 (m, 2H).

[0127] HRMS (ESI+): m / z C 30 H 37 Calculated O7N2 value = 537.2595; Measured value = 537.2593 [M+H] + .

[0128] 4-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)amino)-4-oxobutane-1-ammonium trifluoroacetate (17)

[0129] 3,5-bis(( EHBTU (0.109 g, 0.29 mmol) and 4-((tert-butoxycarbonyl)amino)butyric acid (0.058 g, 0.29 mmol) were added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol). The reaction mixture was stirred at ambient temperature for 1 hour. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (eluent H2O (0.1% TFA): ACN, gradient elution). The resulting solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and TFA (0.5 mL) was added dropwise. The solution was stirred at ambient temperature for 1.5 hours. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (eluent H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.049 g of the title compound in 43% yield.

[0130] 1 H NMR (401 MHz, DMSO- d 6) δ 8.17 (d, J = 6.6 Hz, 1H), 7.70 (bs, 3H), 7.67 (s, 2H), 7.17 – 7.08 (m, 4H), 7.04 (d, J = 8.2 Hz, 2H), 3.93 – 3.83 (m,1H), 3.81 (s, 6H), 3.80 (s, 6H), 3.15 (dd, J = 15.9, 3.9 Hz, 2H), 2.95 – 2.80 (m, 2H), 2.78 – 2.70 (m, 2H), 2.21 – 2.13 (m, 2H), 1.80 – 1.64 (m, 2H).

[0131] HRMS (ESI+): m / z C 28 H 35 Calculated O6N2 value = 495.2490; Measured value = 495.2487 [M+H] + .

[0132] 3-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)amino)- N,N -Dimethyl-3-oxopropane-1-ammonium trifluoroacetate (18)

[0133] 3,5-bis(( E HBTU (0.109 g, 0.29 mmol) and 2-carboxy-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) were added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.12 mL, 0.86 mmol). N,N -Dimethylethane-1-ammonium hydrochloride (0.044 g, 0.29 mmol). 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). The reaction yielded 0.093 g of the title compound in 78% yield.

[0134] 1 H NMR (401 MHz, chloroform-) d ) δ 12.55 (s, 1H), 7.84 (s, 2H), 7.14 (d, J =7.3 Hz, 1H), 7.07 (dd, J = 8.5, 2.0 Hz, 2H), 6.97 (d, J = 2.0 Hz, 2H), 6.89(d, J = 8.4 Hz, 2H), 4.28 – 4.17 (m, 1H), 3.91 (s, 6H), 3.90 (s, 6H), 3.30(t, J = 6.8 Hz, 2H), 3.26 – 3.16 (m, 2H), 3.06 – 2.95 (m, 2H), 2.72 (s, 6H), 2.71 (t, J = 6.8 Hz, 2H).

[0135] HRMS (ESI+): m / z C 29 H 37 Calculated O6N2 value = 509.2646; Measured value = 509.2643 [M+H] + .

[0136] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)amino)- N,N -Diethyl-2-oxoethane-1-ammonium trifluoroacetate (19)

[0137] 3,5-bis(( E HBTU (0.109 g, 0.29 mmol) was added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.12 mL, 0.86 mmol). N -(carboxymethyl) -N- Ethylethane ammonium hydrochloride (0.048 g, 0.29 mmol). 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). The reaction yielded 0.096 g of the title compound in 79% yield.

[0138] 1 H NMR (401 MHz, chloroform-) d ) δ 12.15 (s, 1H), 8.96 (d, J = 7.2 Hz, 1H), 7.86 (s, 2H), 7.09 (dd, J = 8.4, 1.9 Hz, 2H), 7.00 (d, J = 2.0 Hz, 2H), 6.92(d, J = 8.4 Hz, 2H), 4.25 – 4.12 (m, 1H), 3.93 (s, 6H), 3.92 (s, 6H), 3.73(s, 2H), 3.29 (dd, J = 15.5, 3.9 Hz, 2H), 3.22 – 3.19 (m, 4H), 3.00 – 2.89(m, 2H), 1.32 (t, J = 7.3 Hz, 6H).

[0139] HRMS (ESI+): m / z C 30 H 39 Calculated O6N2 value = 523.2803; Measured value = 523.2799 [M+H] + .

[0140] 3-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)amino)-3-oxopropane-1-ammonium trifluoroacetate (20)

[0141] 3,5-bis(( E HBTU (0.109 g, 0.29 mmol) and 3-((tert-butyloxycarbonyl)amino)propionic acid (0.058 g, 0.29 mmol) were added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol). 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 (eluent H2O (0.1% TFA): ACN, gradient elution). The resulting solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and TFA (0.5 mL) was added dropwise. The solution was stirred at ambient temperature for 1.5 hours. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (eluent H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.059 g of the title compound in 52% yield.

[0142] 1 H NMR (401 MHz, DMSO- d 6) δ 8.39 (d, J = 6.8 Hz, 1H), 7.70 (s, 3H), 7.68 (s, 2H), 7.16 – 7.09 (m, 4H), 7.05 (d, J = 8.2 Hz, 2H), 3.97 – 3.87 (m,1H), 3.81 (s, 6H), 3.80 (s, 6H), 3.17 (dd, J = 16.2, 4.0 Hz, 2H), 2.98 – 2.83(m, 4H), 2.44 (t, J = 6.9 Hz, 2H).

[0143] HRMS (ESI+): m / z C 27 H 33 Calculated value of O6N2 = 481.2333; Measured value = 481.2333 [M+H] + .

[0144] 1-(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)-3-(pyridin-3-yl)thiourea (21)

[0145] 3,5-bis(( E 3-Isothiocyanopyridine (0.027 g, 0.20 mmol) was added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.094 g, 0.18 mmol) and Et3N (0.04 mL, 0.27 mmol), and the reaction mixture was stirred overnight. The solvent was evaporated, and the solid residue was recrystallized from hot MeOH. The reaction yielded 0.070 g of the title compound in 72% yield.

[0146] 1 H NMR (401 MHz, chloroform-) d ) δ 8.90 (bs, 1H), 8.35 (d, J = 2.6 Hz, 1H),8.32 – 8.26 (m, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.72 (s, 2H), 7.45 (bs, 1H), 7.21 (dd, J = 8.3, 4.8 Hz, 1H), 6.98 (dd, J = 8.5, 2.0 Hz, 2H), 6.86 – 6.72(m, 4H), 5.22 – 5.11 (m, 1H), 3.89 (s, 6H), 3.80 (s, 6H), 3.54 (dd, J = 16.0,4.4 Hz, 2H), 3.23 – 3.10 (m, 2H).

[0147] HRMS (ESI+): m / z C 30 H 31 Calculated value of O5N3SNa = 568.1877; Measured value = 568.1875 [M+Na] + .

[0148] Ethyl((3,5-bis(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)glycine ester (22)

[0149] 3,5-bis(( EEthyl 2-isocyanate (0.025 g, 0.20 mmol) was added to a mixture of 0.094 g (0.18 mmol) of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate and Et3N (0.04 mL, 0.27 mmol), and the reaction mixture was stirred overnight. The solvent was evaporated, and the solid residue was recrystallized from hot MeOH. The reaction yielded 0.062 g of the title compound in 65% yield.

[0150] 1 H NMR (401 MHz, chloroform-) d δ 7.80 (s, 2H), 7.03 (dd, J = 8.6, 2.0 Hz, 2H), 6.93 (d, J = 1.9 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 5.28 (d, J = 7.7 Hz,1H), 5.23 – 5.17 (m, 1H), 4.41 – 4.35 (m, 1H), 4.10 (q, J = 7.2 Hz, 2H), 3.90(s, 8H), 3.86 (s, 6H), 3.24 – 3.05 (m, 4H), 1.19 (t, J = 7.1 Hz, 3H).

[0151] HRMS (ESI+): m / z C 29 H 34 Calculated value of O8N2Na = 561.2207; Measured value = 561.2207 [M+Na] + .

[0152] Ethyl 3-(3-(3,5-bis(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)ureo)propionate (23)

[0153] 3,5-bis(( EEthyl 3-isocyanopropionate (0.028 g, 0.20 mmol) was added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.094 g, 0.18 mmol) and Et3N (0.04 mL, 0.27 mmol), and the reaction mixture was stirred overnight. The solvent was evaporated, and the solid residue was recrystallized from hot MeOH. The reaction yielded 0.066 g of the title compound in 67% yield.

[0154] 1 H NMR (401 MHz, chloroform-) d δ 7.78 (s, 2H), 7.03 (dd, J = 8.3, 2.0 Hz, 2H), 6.93 (d, J = 2.0 Hz, 2H), 6.84 (d, J = 8.3 Hz, 2H), 5.11 (bs, 1H), 5.07– 5.00 (m, 1H), 4.35 – 4.29 (m, 1H), 3.99 (q, J = 7.1 Hz, 2H), 3.90 (s, 6H), 3.87 (s, 6H), 3.42 – 3.33 (m, 2H), 3.21 – 3.05 (m, 4H), 2.42 (t, J = 5.8 Hz, 2H), 1.15 (t, J = 7.1 Hz, 3H).

[0155] HRMS (ESI+): m / z C 30 H 36 Calculated value of O8N2Na = 575.2364; Measured value = 575.2361 [M+Na] + .

[0156] 1-(3,5-double(( E )-3,4-dimethoxyphenylmethylene)-4-oxocyclohexyl)-3-(3-methoxyphenyl)urea (24)

[0157] 3,5-bis(( ETo a mixture of 1-isocyano-3-methoxybenzene (0.029 g, 0.20 mmol) and Et3N (0.04 mL, 0.27 mmol), 0.094 g (0.094 g, 0.18 mmol) of 1-dimethoxybenzene-4-oxocyclohexane-1-ammonium trifluoroacetate (0.094 g, 0.18 mmol) was added, and the reaction mixture was stirred overnight. The solvent was evaporated, and the solid residue was recrystallized from hot MeOH. The reaction yielded 0.079 g of the title compound in 79% yield.

[0158] 1 H NMR (401 MHz, chloroform-) d ) δ 7.75 (s, 2H), 7.67 – 7.58 (m, 1H), 7.10 –7.01 (m, 2H), 6.98 – 6.91 (m, 2H), 6.84 – 6.78 (m, 2H), 6.77 – 6.70 (m, 3H),6.53 – 6.45 (m, 1H), 6.06 – 5.96 (m, 1H), 4.61 – 4.52 (m, 1H), 3.87 (s, 6H), 3.77 (s, 6H), 3.69 (s, 3H), 3.40 – 3.30 (m, 2H), 3.13 – 3.03 (m, 2H).

[0159] HRMS (ESI+): m / z C 32 H 34 Calculated value of O7N2Na = 581.2258; Measured value = 581.2257 [M+Na] + .

[0160] 1-(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)-3-(2-morpholinoethyl)thiourea (25)

[0161] 3,5-bis(( E 4-(2-isothiocyanoethyl)morpholine (0.034 g, 0.20 mmol) was added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.094 g, 0.18 mmol) and Et3N (0.04 mL, 0.27 mmol), and the reaction mixture was stirred overnight. The solvent was evaporated, and the solid residue was recrystallized from hot MeOH. The reaction yielded 0.080 g of the title compound in 77% yield.

[0162] 1 H NMR (401 MHz, chloroform-) d ) δ 7.72 (s, 2H), 7.05 – 6.98 (m, 2H), 6.86 –6.77 (m, 4H), 4.98 (bs, 1H), 3.91 (s, 6H), 3.84 (s, 6H), 3.61 – 3.51 (m, 2H),3.47 – 3.33 (m, 6H), 3.21 – 3.13 (m, 2H), 2.45 – 2.38 (m, 2H), 2.27 (bs, 4H).

[0163] HRMS (ESI+): m / z C 31 H 39 Calculated value of O6N3SNa = 604.2452; Measured value = 604.2451 [M+Na] + .

[0164] 3-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (26)

[0165] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1 hour. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 minutes. Nicotinyl chloride hydrochloride (0.140 g, 0.79 mmol) was added to the mixture, followed by DIPEA (0.21 mL, 1.18 mmol). The reaction mixture was stirred further at ambient temperature overnight. The reaction mixture was diluted with water (15 mL) and extracted with DCM (3 x 15 mL). The organic phases were combined, washed with brine, dried over Na2SO4, and filtered. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H₂O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.084 g of the title compound, in 34% yield.

[0166] 1 H NMR (401 MHz, DMSO-d 6) δ: 9.04 (dd, J = 2.2, 0.9 Hz, 1H), 8.91 (d, J = 6.8 Hz, 1H), 8.77 (dd, J = 5.0, 1.6 Hz, 1H), 8.31 (dt, J = 8.0, 1.9 Hz,1H), 7.69 (s, 2H), 7.63 (ddd, J = 8.0, 5.0, 0.8 Hz, 1H), 7.19 – 7.08 (m, 4H),7.04 (d, J = 8.3 Hz, 2H), 4.19 – 4.07 (m, 1H), 3.80 (s, 12H), 3.29 (dd, J =16.0, 4.1 Hz, 2H), 3.09 – 2.97 (m, 2H).

[0167] HRMS (ESI+): m / z C 30 H 30 Calculated value of O6N2Na = 537.1996; Measured value = 537.1992 [M+Na] + .

[0168] 3-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)- N,N -Dimethylphenylammonium trifluoroacetate (27)

[0169] tert-butyl(3,5-bis(( EA DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1 hour. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 3-(dimethylamino)benzoic acid (0.097 g, 0.59 mmol) was added to the mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred further at ambient temperature for 2 hours. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H₂O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.231 g of the title compound in 88% yield.

[0170] 1 H NMR (401 MHz, DMSO- d 6) δ: 8.52 (d, J = 7.0 Hz, 1H), 7.68 (s, 2H), 7.29 (t, J = 8.1 Hz, 1H), 7.20 (d, J = 6.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 4H), 7.04 (d, J = 8.2 Hz, 2H), 6.99 – 6.92 (m, 1H), 4.14 – 4.02 (m, 1H), 3.80 (s,12H), 3.26 (dd, J = 16.0, 4.1 Hz, 2H), 3.07 – 2.96 (m, 2H), 2.95 (s, 6H).

[0171] HRMS (ESI+): m / z C 33 H 36 Calculated value of O6N2Na = 579.2466; Measured value = 579.2462 [M+Na] + .

[0172] 5-((3,5-double(( E)-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)-2-(piperidin-1-onthium-1-yl)pyridine-1-onthium trifluoroacetate (28)

[0173] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1 hour. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 6-(piperidin-1-yl)nicotinic acid (0.121 g, 0.59 mmol) was added to the mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred further at ambient temperature for 1.5 hours. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H₂O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.168 g of the title compound in 52% yield.

[0174] 1 H NMR (401 MHz, DMSO- d 6) δ: 8.54 – 8.46 (m, 2H), 8.04 (dd, J = 9.3,2.5 Hz, 1H), 7.68 (s, 2H). 7.19 – 7.11 (m, 4H), 7.07 – 7.00 (m, 3H), 4.15 –4.02 (m, 1H), 3.80 (s, 12H), 3.64 (t, J = 5.4 Hz, 4H), 3.25 (dd, J = 16.0,4.1 Hz, 2H), 3.05 – 2.93 (m, 2H), 1.70 – 1.60 (m, 2H), 1.60 – 1.51 (m, 4H).

[0175] HRMS (ESI+): m / z C 35 H 40Calculated O6N3 value = 598.2912; Measured value = 598.2908 [M+H] + .

[0176] 4-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (29)

[0177] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1 hour. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 minutes. Isonicotinyl chloride hydrochloride (0.140 g, 0.79 mmol) was added to the mixture, followed by DIPEA (0.21 mL, 1.18 mmol). The reaction mixture was stirred further at ambient temperature for 2 hours. The reaction mixture was diluted with water (15 mL) and extracted with DCM (3 x 15 mL). The organic phases were combined, washed with brine, dried over Na2SO4, and filtered. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H₂O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.142 g of the title compound in 58% yield.

[0178] 1 H NMR (401 MHz, chloroform-) d ) δ: 8.79 – 8.73 (m, 2H), 7.99 – 7.93 (m, 2H), 7.83 (s, 2H), 7.30 (d, J = 7.3 Hz, 1H), 7.04 (dd, J = 8.6, 2.0 Hz, 2H), 6.93(d, J = 2.0 Hz, 2H), 6.87 (d, J = 8.5 Hz, 2H), 4.68 – 4.59 (m, 1H), 3.90 (s, 6H), 3.87 (s, 6H), 3.36 – 3.21 (m, 4H).

[0179] HRMS (ESI+):m / z C 30 H 30 Calculated value of O6N2Na = 537.1996; Measured value = 537.1992 [M+Na] + .

[0180] 4-(2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)amino)-2-oxoethoxy)pyridine-1-onium trifluoroacetate (30)

[0181] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1 hour. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 2-(pyridin-4-yloxy)acetic acid (0.090 g, 0.59 mmol) was added to the mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred further at ambient temperature for 2 hours. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H₂O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.089 g of the title compound in 34% yield.

[0182] 1 H NMR (401 MHz, DMSO- d 6) δ: 8.86 (d, J = 6.7 Hz, 1H), 8.47 – 8.40 (m,1H), 8.37 (dt, J = 5.9, 1.3 Hz, 1H), 8.03 – 7.93 (m, 1H), 7.97 – 7.85 (m,1H), 7.71 (s, 2H), 7.19 – 7.07 (m, 4H), 7.04 (d, J= 8.2 Hz, 2H), 5.30 (s, 2H), 4.05 – 3.94 (m, 1H), 3.81 (s, 6H), 3.79 (s, 6H), 3.26 – 3.17 (m, 2H), 3.04 – 2.92 (m, 2H).

[0183] HRMS (ESI+): m / z C 31 H 33 Calculated O7N2 value = 545.2282; Measured value = 545.2277 [M+H] + .

[0184] 3-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)phenylammonium trifluoroacetate (31)

[0185] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1 hour. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 3-((tert-Butoxycarbonyl)amino)benzoic acid (0.140 g, 0.59 mmol) was added to the mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred further at ambient temperature for 2 hours. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (eluent H₂O (0.1% TFA): ACN, gradient elution). The resulting solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and TFA (0.5 mL) was added dropwise. The solution was stirred at ambient temperature for 3 hours. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (eluent H₂O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.124 g of the title compound, in 49% yield.

[0186] 1 H NMR (401 MHz, DMSO- d 6) δ 8.52 (d,J = 6.9 Hz, 1H), 7.68 (s, 2H), 7.30 – 7.20 (m, 3H), 7.19 – 7.08 (m, 4H), 7.04 (d, J = 8.2 Hz, 2H), 6.98 –6.89 (m, 1H), 4.13 – 4.01 (m, 1H), 3.80 (s, 12H), 3.25 (dd, J = 15.8, 4.0 Hz, 2H), 3.06 – 2.94 (m, 2H).

[0187] HRMS (ESI+): m / z C 31 H 32 Calculated value of O6N2Na = 551.2153; Measured value = 551.2150 [M+Na] + .

[0188] 4-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)phenylammonium trifluoroacetate (32)

[0189] tert-butyl(3,5-bis(( EA DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1 hour. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 4-((tert-Butoxycarbonyl)amino)benzoic acid (0.140 g, 0.59 mmol) was added to the mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred further at ambient temperature for 2 hours. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (eluent H₂O (0.1% TFA): ACN, gradient elution). The resulting solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and TFA (0.5 mL) was added dropwise. The solution was stirred at ambient temperature for 3 hours. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (eluent H₂O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.083 g of the title compound, in 33% yield.

[0190] 1 H NMR (401 MHz, DMSO- d 6) δ 8.14 (d, J = 6.9 Hz, 1H), 7.67 (s, 2H), 7.64 – 7.56 (m, 2H), 7.19 – 7.08 (m, 4H), 7.04 (d, J = 8.2 Hz, 2H), 6.63 –6.55 (m, 2H), 4.09 – 3.99 (m, 1H), 3.80 (s, 12H), 3.23 (dd, J = 16.1, 4.1 Hz, 2H), 3.03 – 2.91 (m, 2H).

[0191] HRMS (ESI+): m / z C 31 H 32 Calculated value of O6N2Na = 551.2153; Measured value = 551.2149 [M+Na] + .

[0192] 2-((3,5-double(( E)-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)phenylammonium trifluoroacetate (33)

[0193] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1 hour. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 2-((tert-Butoxycarbonyl)amino)benzoic acid (0.140 g, 0.59 mmol) was added to the mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred further at ambient temperature for 2 hours. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (eluent H₂O (0.1% TFA): ACN, gradient elution). The resulting solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and TFA (0.5 mL) was added dropwise. The solution was stirred at ambient temperature for 3 hours. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (eluent H₂O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.093 g of the title compound, in 37% yield.

[0194] 1 H NMR (401 MHz, DMSO- d 6) δ 8.42 (d, J = 6.8 Hz, 1H), 7.68 (s, 2H), 7.56 (dd, J = 8.0, 1.5 Hz, 1H), 7.24 – 7.12 (m, 5H), 7.08 – 7.01 (m, 2H), 6.76 (dd, J = 8.2, 1.2 Hz. 1H), 6.69 – 6.60 (m, 1H), 4.12 – 3.99 (m, 1H), 3.80 (s, 12H), 3.26 (dd, J = 15.9, 4.0 Hz, 2H), 3.00 (ddd, J= 15.9, 11.0, 2.7 Hz, 2H).

[0195] HRMS (ESI+): m / z C 31 H 32 Calculated value of O6N2Na = 551.2153; Measured value = 551.2149 [M+Na] + .

[0196] 2-((3,5-double(( E )-4-ethoxy-3-methoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (34)

[0197] 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (0.115 g, 0.35 mmol) was dissolved in EtOH (4 mL), and 20% NaOH aqueous solution (0.25 mL) was added dropwise. The mixture was stirred for 5 minutes, and 4-ethoxy-3-methoxybenzaldehyde (0.156 g, 0.87 mmol) was added. The reaction mixture was stirred overnight at ambient temperature. Water was added to the reaction mixture, and the resulting suspension was filtered off. The resulting filter cake was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.067 g of the title compound in 34% yield.

[0198] 1 H NMR (401 MHz, DMSO- d 6) δ 8.85 (d, J = 7.7 Hz, 1H), 8.65 (dt, J =4.8, 1.4 Hz, 1H), 8.03 – 7.92 (m, 2H), 7.70 (s, 2H), 7.65 – 7.56 (m, 1H),7.18 – 7.08 (m, 4H), 7.02 (d, J = 8.3 Hz, 2H), 4.23 – 4.12 (m, 1H), 4.05 (q, J = 7.0 Hz, 4H), 3.79 (s, 6H), 3.28 – 3.10 (m, 4H), 1.33 (t, J = 7.0 Hz, 6H).

[0199] HRMS (ESI+):m / z C 32 H 34 Calculated value of O6N2Na = 565.2309; Measured value = 565.2305 [M+Na] + .

[0200] 2-((3,5-double(( E )-3-ethoxy-4-methoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (35)

[0201] To a mixture of 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (0.100 g, 0.30 mmol) and 3-ethoxy-4-methoxybenzaldehyde (0.108 g, 0.60 mmol) in glacial acetic acid (1 mL), 0.5 mL of a 4 M solution of dioxane in hydrogen chloride was added, and the reaction mixture was stirred overnight. 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). The reaction yielded 0.080 g of the title compound in 41% yield.

[0202] 1 H NMR (401 MHz, DMSO- d 6) δ 8.84 (d, J = 7.8 Hz, 1H), 8.66 (dt, J =4.8, 1.4 Hz, 1H), 8.07 – 7.94 (m, 2H), 7.69 (s, 2H), 7.65 – 7.57 (m, 1H),7.18 – 7.11 (m, 4H), 7.03 (d, J = 8.3 Hz, 2H), 4.23 – 4.11 (m, 1H), 4.05 (q, J = 7.0 Hz, 4H), 3.80 (s, 6H), 3.27 – 3.09 (m, 4H), 1.32 (t, J = 6.9 Hz, 6H).

[0203] HRMS (ESI+): m / z C 32 H 34 Calculated value of O6N2Na = 565.2309; Measured value = 565.2306 [M+Na] + .

[0204] 2-((3,5-double(( E )-4-hydroxy-3-methoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (36)

[0205] 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (0.100 g, 0.30 mmol) was dissolved in EtOH (1.3 mL), and 20% NaOH aqueous solution (0.17 mL) was added dropwise. The mixture was stirred for 5 minutes, and 4-hydroxy-3-methoxybenzaldehyde (0.114 g, 0.75 mmol) was added. The reaction mixture was stirred overnight at ambient temperature. 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). The reaction yielded 0.076 g of the title compound in 42% yield.

[0206] 1 H NMR (401 MHz, DMSO- d 6) δ 9.58 (s, 2H), 8.83 (d, J = 7.7 Hz, 1H), 8.65 (dt, J = 4.8, 1.4 Hz, 1H), 8.03 – 7.94 (m, 2H), 7.68 (s, 2H), 7.65 –7.57 (m, 1H), 7.14 (d, J = 2.0 Hz, 2H), 7.05 (dd, J = 8.6, 2.0 Hz, 2H), 6.85(d, J = 8.2 Hz, 2H), 4.27 – 4.09 (m, 1H), 3.80 (s, 6H), 3.27 – 3.07 (m, 4H).

[0207] HRMS (ESI+): m / z C 28 H 26 Calculated value of O6N2Na = 509.1683; Measured value = 509.1680 [M+Na] + .

[0208] 2-((3,5-double(( E )-4-methoxy-3-(trifluoromethoxy)benzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (37)

[0209] To a mixture of 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (0.100 g, 0.30 mmol) and 4-methoxy-3-(trifluoromethoxy)benzaldehyde (0.166 g, 0.75 mmol) in glacial acetic acid (1 mL), 0.5 mL of a 4 M solution of dioxane in hydrogen chloride was added, and the reaction mixture was stirred overnight. 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). The reaction yielded 0.147 g of the title compound in 66% yield.

[0210] 1 H NMR (401 MHz, DMSO- d 6) δ 8.88 (d, J = 7.6 Hz, 1H), 8.66 (dt, J =4.8, 1.4 Hz, 1H), 8.04 – 7.95 (m, 2H), 7.68 (s, 2H), 7.66 – 7.51 (m, 5H),7.32 (d, J = 8.6 Hz, 2H), 4.24 – 4.11 (m, 1H), 3.90 (s, 6H), 3.21 – 3.10 (m, 4H).

[0211] HRMS (ESI+): m / z C 30 H 24 Calculated value of O6N2F6Na = 645.1431; Measured value = 645.1428 [M+Na] + .

[0212] 2-((3,5-double(( E )-4-hydroxy-3-(trifluoromethoxy)benzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (38)

[0213] To a mixture of 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (0.100 g, 0.30 mmol) and 4-hydroxy-3-(trifluoromethoxy)benzaldehyde (0.155 g, 0.75 mmol) in glacial acetic acid (1 mL), a 4 M solution of dioxane in hydrogen chloride (1.5 mL) was added, and the reaction mixture was stirred overnight. 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). The reaction yielded 0.145 g of the title compound in 68% yield.

[0214] 1 H NMR (401 MHz, DMSO- d 6) δ 10.77 (s, 2H), 8.87 (d, J = 7.6 Hz, 1H), 8.66 (dt, J = 4.8, 1.4 Hz, 1H), 8.04 – 7.94 (m, 2H), 7.67 – 7.57 (m, 3H), 7.52 – 7.42 (m, 4H), 7.10 (d, J = 8.5 Hz, 2H), 4.21 – 4.11 (m, 1H), 3.22 –3.07 (m, 4H).

[0215] HRMS (ESI+): m / z C 28 H 20 Calculated value of O6N2F6Na = 617.1118; Measured value = 617.1114 [M+Na] + .

[0216] 2-((3,5-double(( E )-4-(difluoromethoxy)-3-hydroxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (39)

[0217] To a mixture of 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (0.100 g, 0.30 mmol) and 4-(difluoromethoxy)-3-hydroxybenzaldehyde (0.142 g, 0.75 mmol) in glacial acetic acid (1 mL), 0.15 mL of a 4 M solution of dioxane in hydrogen chloride was added, and the reaction mixture was stirred overnight. 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). The reaction yielded 0.136 g of the title compound in 67% yield.

[0218] 1 H NMR (401 MHz, DMSO- d 6) δ 10.13 (s, 2H), 8.88 (d, J = 7.9 Hz, 1H), 8.66 (dt, J = 4.7, 1.4 Hz, 1H), 8.04 – 7.94 (m, 2H), 7.67 – 7.57 (m, 3H), 7.20 – 7.12 (m, 4H), 7.10 (t, J = 74.7 Hz, 2H) 7.05 – 6.98 (m, 2H), 4.22 –4.12 (m, 1H), 3.24 – 3.08 (m, 4H).

[0219] HRMS (ESI+): m / z C 28 H 22 Calculated value of O6N2F4Na = 581.1306; Measured value = 581.1309 [M+Na] + .

[0220] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)-3,5-difluoropyridine-1-onium trifluoroacetate (40)

[0221] tert-butyl(3,5-bis(( EA DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1.5 h. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 3,5-Difluoropyridinecarboxylic acid (0.094 g, 0.59 mmol) was added to the mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred further at ambient temperature for 1.5 h. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H₂O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.127 g of the title compound in 49% yield.

[0222] 1 H NMR (401 MHz, DMSO- d 6) δ 8.81 (d, J = 7.3 Hz, 1H), 8.57 (d, J = 2.3Hz, 1H), 8.12 – 8.02 (m, 1H), 7.70 (s, 2H), 7.18 – 7.10 (m, 4H), 7.05 (d, J =8.2 Hz, 2H), 4.18 – 4.06 (m, 1H), 3.80 (s, 6H), 3.80 (s, 6H), 3.24 (dd, J =15.9, 4.1 Hz, 2H), 3.14 – 3.03 (m, 2H).

[0223] HRMS (ESI+): m / z C 30 H 28 Calculated value of O6N2F2Na = 573.1808; Measured value = 573.1805 [M+Na] + .

[0224] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)-6-hydroxypyridine-1-onium trifluoroacetate (41)

[0225] 3,5-bis(( E HBTU (0.109 g, 0.29 mmol) and 6-hydroxypyridinecarboxylic acid (0.040 g, 0.29 mmol) were added to a mixture of 3,4-dimethoxybenzyl)-4-oxocyclohexane-1-ammonium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.12 mL, 0.86 mmol). The reaction mixture was stirred at ambient temperature for 1.5 h. 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). The reaction yielded 0.051 g of the title compound in 41% yield.

[0226] 1 H NMR (401 MHz, DMSO- d 6) δ 8.47 (d, J = 6.3 Hz, 1H), 7.74 – 7.64 (m,3H), 7.25 – 7.21 (m, 1H), 7.18 – 7.10 (m, 4H), 7.08 – 6.99 (m, 2H), 6.73 (d, J = 8.6 Hz, 1H), 4.19 – 4.08 (m, 1H), 3.80 (s, 6H), 3.80 (s, 6H), 3.29 – 3.20 (m, 2H), 3.14 – 3.03 (m, 2H).

[0227] HRMS (ESI+): m / z C 30 H 30 Calculated value of O7N2Na = 553.1945; Measured value = 553.1948 [M+Na] + .

[0228] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)-1 H -Imidazole-3-onium trifluoroacetate (42)

[0229] tert-butyl(3,5-bis(( EA DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1.5 h. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued for an additional 30 min at ambient temperature. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 1 H -Imidazole-2-carboxylic acid (0.066 g, 0.59 mmol) was added, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred further at ambient temperature for 1.5 h. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.122 g of the title compound in 50% yield.

[0230] 1 H NMR (401 MHz, DMSO- d 6) δ 8.60 (d, J = 7.5 Hz, 1H), 7.70 (s, 2H),7.25 (s, 2H), 7.18 – 7.12 (m, 4H), 7.07 – 7.00 (m, 2H), 4.17 – 4.05 (m, 1H),3.80 (s, 11H), 3.23 (dd, J = 16.0, 4.2 Hz, 2H), 3.16 – 3.05 (m, 2H).

[0231] HRMS (ESI+): m / z C 28 H 29 Calculated value of O6N3Na = 526.1949; Measured value = 526.1945 [M+Na] + .

[0232] N -(3-(( E )-3-methoxybenzyl)-5-(( E )-4-methoxybenzyl)-4-oxocyclohexyl)pyridinecarboxamide hydrochloride (43)

[0233] To a mixture of 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate (0.200 g, 0.60 mmol), 3-methoxybenzaldehyde (0.088 g, 0.72 mmol), and 4-methoxybenzaldehyde (0.088 g, 0.72 mmol) in glacial acetic acid (1.2 mL), 0.45 mL of a 4 M solution of dioxane in hydrogen chloride was added, and the reaction mixture was stirred for 6 hours. The solvent was evaporated under reduced pressure, and the residue was immediately purified by column chromatography (elution buffer cHex:EtOAc, 2:1). The reaction yielded 0.028 g of the title compound in 10% yield.

[0234] 1 H NMR (400 MHz, chloroform-) d ) δ 8.56 – 8.49 (m, 1H), 8.19 (d, J = 8.3 Hz, 1H), 8.12 (dt, J = 7.8, 1.1 Hz, 1H), 7.91 (s, 2H), 7.81 (td, J = 7.7, 1.7 Hz,1H), 7.50 – 7.41 (m, 2H), 7.45 – 7.37 (m, 1H), 7.35 – 7.26 (m, 1H), 7.08 –7.01 (m, 1H), 7.00 – 6.94 (m, 1H), 6.98 – 6.89 (m, 2H), 6.93 – 6.84 (m, 1H), 4.57 – 4.44 (m, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 3.43 – 3.27 (m, 2H), 3.16 –3.04 (m, 2H).

[0235] HRMS (ESI+): m / z C 28 H 26 Calculated value of O4N2Na = 477.1785; Measured value = 477.1782 [M+Na] + .

[0236] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium hydrogen sulfate (44)

[0237] 2-((3,5-double(( E)-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate was dissolved in ammonia water, and the resulting free base was treated with DCM: i Extracted with PrOH (1:10). The organic phases were combined, dried over Na₂SO₄, and filtered. The solvent was evaporated to obtain the free base of the title compound. The free base (0.123 g, 0.24 mmol) was dissolved in Et₂O (2 mL), and H₂SO₄ (0.12 mL, 2 M aqueous solution) was added dropwise. The resulting suspension was filtered off and dried. The reaction yielded 0.143 g (0.23 mmol) of the title compound.

[0238] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium hydrochloride (45)

[0239] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate was dissolved in ammonia water, and the resulting free base was treated with DCM: i Extracted with PrOH (1:10). The organic phases were combined, dried over Na2SO4, and filtered. The solvent was evaporated to obtain the free base of the title compound. The free base (0.123 g, 0.24 mmol) was dissolved in Et2O (2 mL), and HCl (0.06 mL, 4 M dioxane solution) was added dropwise. The resulting suspension was filtered off and dried. The reaction yielded 0.130 g (0.24 mmol) of the title compound.

[0240] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium hydrobromide (46)

[0241] 2-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyridine-1-onium trifluoroacetate was dissolved in ammonia water, and the resulting free base was treated with DCM: iExtracted with PrOH (1:10). The organic phases were combined, dried over Na₂SO₄, and filtered. The solvent was evaporated to obtain the free base of the title compound. The free base (0.123 g, 0.24 mmol) was dissolved in Et₂O (2 mL), and HBr (3.8 M aqueous solution) was added dropwise. The resulting suspension was filtered off and dried. The reaction yielded 0.140 g (0.24 mmol) of the title compound.

[0242] 4-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)thiazole-3-onium trifluoroacetate (47)

[0243] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1.5 h. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). Thiazole-4-carboxylic acid (0.076 g, 0.59 mmol) was added to the mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred further at ambient temperature for 1.5 h. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H₂O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.143 g of the title compound in 58% yield.

[0244] 1 H NMR (401 MHz, DMSO- d 6) δ: 9.19 (d, J = 2.0 Hz, 1H), 8.54 (d, J =7.6 Hz, 1H), 8.30 (d, J= 2.0 Hz, 1H), 7.72 – 7.67 (m, 2H), 7.18 – 7.12 (m,4H), 7.07 – 7.00 (m, 2H), 4.18 – 4.09 (m, 1H), 3.80 (s, 6H), 3.79 (s, 6H),3.27 – 3.06 (m, 4H).

[0245] HRMS (ESI+): m / z C 28 H 28 Calculated value of O6N2NaS = 543.1560; Measured value = 543.1565 [M+Na] + .

[0246] N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)-2-methyloxazol-4-carboxamide (48)

[0247] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1.5 h. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 2-Methyloxazol-4-carboxylic acid (0.075 g, 0.59 mmol) was added to the mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred further at ambient temperature for 2 h. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H₂O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.092 g of the title compound in 45% yield.

[0248] 1 H NMR (401 MHz, chloroform-) d δ: 8.43 (s, 1H), 8.30 (d, J= 7.6 Hz, 1H),7.71 – 7.65 (m, 2H), 7.18 – 7.07 (m, 4H), 7.07 – 6.99 (m, 2H), 4.11 – 4.03(m, 1H), 3.80 (s, 6H), 3.80 (s, 6H), 3.23 – 3.13 (m, 2H), 3.13 – 3.02 (m, 2H), 2.46 (s, 3H).

[0249] HRMS (ESI+): m / z C 29 H 30 Calculated value of O7N2Na = 541.1945; Measured value = 541.1946 [M+Na] + .

[0250] 3-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamoyl)pyrrolidine-1-onium trifluoroacetate (49)

[0251] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1 hour. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 1-(tert-Butoxycarbonyl)pyrrolidine-3-carboxylic acid (0.066 g, 0.31 mmol) was added to the mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred further at ambient temperature for 1.5 hours. The solvent was evaporated under reduced pressure, and the solid residue was purified by rapid chromatography (elution buffer: cHex: EtOAc, gradient elution 40%-85%). The resulting solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and TFA (0.5 mL) was added dropwise. The solution 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). The reaction yielded 0.123 g of the title compound, in 65% yield.

[0252] 1 H NMR (401 MHz, chloroform-) d δ: 8.81 (bs, 2H), 8.43 (d, J = 6.6 Hz, 1H),7.70 – 7.65 (m, 2H), 7.16 – 7.07 (m, 4H), 7.07 – 7.00 (m, 2H), 3.93 (s, 1H), 3.81 (s, 6H), 3.80 (s, 3H), 3.80 (s, 3H), 3.31 – 2.85 (m, 9H), 2.15 – 2.02(m, 1H), 1.91 – 1.77 (m, 1H).

[0253] HRMS (ESI+): S C 29 H 35 Calculated O6N2 value = 507.2490; Measured value = 507.2488 [M+H] + .

[0254] N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)pyrazolo[1,5- a ]Pyrimidine-3-carboxamide (50)

[0255] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1.5 h. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued for an additional 30 min at ambient temperature. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). Pyrazolo[1,5-]pyrazolium chloride was added to the mixture. aPyrimidine-3-carboxylic acid (0.096 g, 0.59 mmol) was added, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred further at ambient temperature for 2 hours. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.108 g of the title compound in 50% yield.

[0256] 1 H NMR (401 MHz, DMSO- d 6) δ: 9.28 (dd, J = 7.0, 1.7 Hz, 1H), 8.75 (dd, J = 4.2, 1.7 Hz, 1H), 8.50 (s, 1H), 8.08 (d, J = 7.0 Hz, 1H), 7.79 – 7.73 (m,2H), 7.24 (dd, J = 7.0, 4.2 Hz, 1H), 7.21 – 7.12 (m, 4H), 7.12 – 7.01 (m,2H), 4.38 – 4.28 (m, 1H), 3.79 (s, 12H), 3.32 – 3.15 (m, 4H).

[0257] HRMS (ESI+): m / z C 31 H 30 Calculated value of O6N4Na = 577.2058; Measured value = 577.2062 [M+Na] + .

[0258] N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)cyclopropanesulfonamide (51)

[0259] tert-butyl(3,5-bis(( EA DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1.5 h. DIPEA (0.21 mL, 1.18 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The reaction mixture was cooled in an ice bath again, and cyclopropanesulfonyl chloride (0.055 g, 0.39 mmol) was added, followed by Et3N (0.06 mL, 0.39 mmol). The solution was stirred further at ambient temperature overnight. The reaction was quenched with water and extracted with DCM. The organic phases were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.110 g of the title compound in 55% yield.

[0260] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.93 – 7.88 (m, 2H), 7.12 – 7.04 (m, 2H), 6.98 (d, J = 2.0 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 4.52 (bs, 1H), 4.01 –3.94 (m, 1H), 3.92 (s, 6H), 3.91 (s, 6H), 3.32 – 3.23 (m, 2H), 3.19 – 3.11(m, 2H), 2.17 (tt, J = 7.9, 4.8 Hz, 1H), 1.07 – 0.98 (m, 2H), 0.79 – 0.66 (m, 2H).

[0261] HRMS (ESI+): m / z C 27 H 31 Calculated value of O7NNaS = 536.1713; Measured value = 536.1711 [M+Na] + .

[0262] N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)ethanesulfonamide (52)

[0263] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1.5 h. DIPEA (0.21 mL, 1.18 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The reaction mixture was cooled again in an ice bath, and ethanesulfonyl chloride (0.050 g, 0.39 mmol) was added. The solution was stirred further at ambient temperature overnight. The reaction was quenched with water and extracted with DCM. The organic phases were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.063 g of the title compound in 32% yield.

[0264] 1 H NMR (401 MHz, chloroform-) d ) δ: 7.93 – 7.88 (m, 2H), 7.11 – 7.04 (m, 2H), 6.98 (d, J = 2.0 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 4.40 (d, J = 6.6 Hz, 1H),4.04 – 3.95 (m, 1H), 3.93 (s, 6H), 3.91 (s, 6H), 3.28 – 3.09 (m, 4H), 2.83(q, J = 7.4 Hz, 2H), 1.18 (t, J = 7.4 Hz, 3H).

[0265] HRMS (ESI+): m / z C 26 H 31 Calculated value of O7NNaS = 524.1713; Measured value = 524.1711 [M+Na] + .

[0266] 4-( N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)sulfonamide)-1-methylpiperazine-1-trifluoroacetate (53)

[0267] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 3 h. DIPEA (0.27 mL, 1.57 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The reaction mixture was cooled again in an ice bath, and 4-methylpiperazine-1-sulfonyl chloride (0.234 g, 1.18 mmol) was added. The solution was stirred further at ambient temperature overnight. The reaction was quenched with water and extracted with DCM. The organic phases were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.175 g of the title compound in 65% yield.

[0268] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.90 – 7.84 (m, 2H), 7.12 – 7.04 (m, 2H), 6.98 (d, J = 2.0 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 5.03 (d, J = 6.2 Hz, 1H), 3.95 – 3.91 (m, 7H), 3.90 (s, 6H), 3.60 (bs, 2H), 3.33 (bs, 2H), 3.26 – 3.11(m, 6H), 2.70 – 2.60 (m, 5H).

[0269] HRMS (ESI+): m / z C 29 H 38 Calculated value of O7N3S = 572.2425; Measured value = 572.2422 [M+H] + .

[0270] N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)morpholine-4-sulfonamide (54)

[0271] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 3 h. DIPEA (0.27 mL, 1.57 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The reaction mixture was cooled again in an ice bath, and morpholine-4-sulfonyl chloride (0.219 g, 1.18 mmol) was added. The solution was stirred further at ambient temperature overnight. The reaction was quenched with water and extracted with DCM. The organic phases were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.076 g of the title compound in 35% yield.

[0272] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.94 – 7.88 (m, 2H), 7.13 – 7.06 (m, 2H), 6.99 (d, J = 2.0 Hz, 2H), 6.92 (d, J = 8.4 Hz, 2H), 4.33 (bs, 1H), 4.00 –3.93 (m, 1H), 3.93 (s, 6H), 3.92 (s, 6H), 3.50 – 3.43 (m, 4H), 3.19 (dd, J =5.1, 1.8 Hz, 4H), 3.02 – 2.95 (m, 4H).

[0273] HRMS (ESI+): m / z C 28 H 34 Calculated value of O8N2NaS = 581.1928; Measured value = 581.1927 [M+Na] + .

[0274] N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)-1-methyl-1 H -Imidazole-4-sulfonamide (55)

[0275] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 3 hours. DIPEA (0.27 mL, 1.57 mmol) was added dropwise, and stirring was continued for an additional 30 minutes at ambient temperature. The reaction mixture was cooled in an ice bath again, and 1-methyl-1 H -Imidazole-4-sulfonyl chloride (0.250 g, 1.38 mmol). The solution was stirred further at ambient temperature for 1.5 h. The reaction was quenched with water and extracted with DCM. The organic phases were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.140 g of the title compound in 64% yield.

[0276] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.89 (d, J = 6.2 Hz, 1H), 7.63 (d, J =1.4 Hz, 1H), 7.62 (d, J = 1.4 Hz, 1H), 7.61 – 7.59 (m, 2H), 7.12 – 7.08 (m,2H), 7.07 – 7.04 (m, 4H), 3.83 (s, 6H), 3.80 (s, 6H), 3.50 (s, 3H), 3.35 –3.31 (m, 1H), 3.15 – 3.06 (m, 2H), 2.87 – 2.75 (m, 2H).

[0277] HRMS (ESI+): m / z C 28 H 31 Calculated value of O7N3NaS = 576.1775; Measured value = 576.1771 [M+Na] + .

[0278] N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)-2-(methoxymethyl)pyrrolidine-1-sulfonamide (56)

[0279] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 3 h. DIPEA (0.27 mL, 1.57 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The reaction mixture was cooled in an ice bath again, and 2-(methoxymethyl)pyrrolidine-1-sulfonyl chloride (0.250 g, 1.17 mmol) was added. The solution was stirred further at ambient temperature overnight. The reaction was quenched with water and extracted with DCM. The organic phases were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.169 g of the title compound in 73% yield.

[0280] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.93 – 7.83 (m, 2H), 7.13 – 7.04 (m, 2H), 7.00 (dd, J = 4.5, 2.0 Hz, 2H), 6.90 (dd, J = 8.4, 2.2 Hz, 2H), 3.92 (s, 6H), 3.91 (s, 6H), 3.90 – 3.75 (m, 2H), 3.19 (s, 3H), 3.40 – 2.99 (m, 8H), 1.87 –1.73 (m, 1H), 1.73 – 1.59 (m, 3H).

[0281] HRMS (ESI+): m / z C 30 H 38 Calculated value of O8N2NaS = 609.2241; Measured value = 609.2239 [M+Na] + .

[0282] ( R )- N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)-3-fluoropyrrolidine-1-sulfonamide (57)

[0283] tert-butyl(3,5-bis(( E A solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) in DCM was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 3 hours. DIPEA (0.27 mL, 1.57 mmol) was added dropwise, and stirring was continued for an additional 30 minutes at ambient temperature. The reaction mixture was cooled in an ice bath again, and ( R 1,3-Fluoropyrrolidine-1-sulfonyl chloride (0.250 g, 1.33 mmol). The solution was stirred overnight at ambient temperature. The reaction was quenched with water and extracted with DCM. The organic phases were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.145 g of the title compound in 66% yield.

[0284] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.91 – 7.86 (m, 2H), 7.08 (dd, J = 8.5, 2.0 Hz, 2H), 6.99 (d, J = 2.0 Hz, 2H), 6.94 – 6.87 (m, 2H), 5.05 (dt, J =53.0, 3.9 Hz, 1H), 4.44 (bs, 1H), 3.92 (s, 6H), 3.92 – 3.90 (m, 7H), 3.51 –3.13 (m, 8H), 2.10 – 1.94 (m, 1H), 1.92 – 1.69 (m, 1H).

[0285] HRMS (ESI+): m / z C 28 H 33 Calculated value of O7N2FNaS = 583.1885; Measured value = 583.1882 [M+Na] + .

[0286] N -(3,5-double(( E)-3,4-dimethoxybenzyl)-4-oxocyclohexyl)-3-azacycloheptane-1-sulfonamide (58)

[0287] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 3 h. DIPEA (0.27 mL, 1.57 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The reaction mixture was cooled in an ice bath again, and azircycloheptan-1-sulfonyl chloride (0.100 g, 0.51 mmol) was added. The solution was stirred further at ambient temperature for 12 days. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution), followed by recrystallization with MeOH. The reaction yielded 0.044 g of the title compound in 20% yield.

[0288] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.67 – 7.62 (m, 2H), 7.36 (d, J = 5.7 Hz,1H), 7.17 – 7.09 (m, 4H), 7.06 (d, J = 8.2 Hz, 2H), 3.81 (s, 6H), 3.81 (s, 6H), 3.38 – 3.28 (m, 1H), 3.24 (dd, J = 16.1, 3.9 Hz, 2H), 3.07 (t, J = 5.8Hz, 4H), 2.96 – 2.84 (m, 2H), 1.52 – 1.44 (m, 4H), 1.47 – 1.32 (m, 4H).

[0289] HRMS (ESI+): m / z C 30 H 38 Calculated value of O7N2NaS = 593.2292; Measured value = 593.2291 [M+Na] + .

[0290] 3-( N-(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)sulfonamide)pyridine-1-onium trifluoroacetate (59)

[0291] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 3 h. DIPEA (0.59 mL, 2.75 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The reaction mixture was cooled in an ice bath again, and pyridine-3-sulfonyl chloride hydrochloride (0.250 g, 1.17 mmol) was added. The solution was stirred further at ambient temperature overnight. The reaction was quenched with water and extracted with DCM. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.090 g of the title compound in 35% yield.

[0292] 1 H NMR (401 MHz, DMSO- d 6) δ: 9.00 (dd, J = 2.3, 0.8 Hz, 1H), 8.52 (dd, J = 5.0, 1.6 Hz, 1H), 8.03 (ddd, J = 8.1, 2.3, 1.6 Hz, 1H), 7.84 – 7.78 (m,2H), 7.19 (ddd, J = 8.1, 5.0, 0.8 Hz, 1H), 6.92 (dd, J = 8.5, 2.0 Hz, 2H), 6.89 – 6.82 (m, 4H), 5.37 (d, J = 7.3 Hz, 1H), 4.05 – 3.97 (m, 1H), 3.94 (s, 6H), 3.88 (s, 6H), 3.10 – 2.95 (m, 4H).

[0293] HRMS (ESI+): m / z C 29 H 30Calculated value of O7N2NaS = 573.1666; Measured value = 573.1664 [M+Na] + .

[0294] 2-(4-( N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)sulfonamide)phenoxy)acetamide (60)

[0295] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 3 h. DIPEA (0.27 mL, 1.57 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The reaction mixture was cooled in an ice bath again, and 4-(2-amino-2-oxoethoxy)benzenesulfonyl chloride (0.294 g, 1.18 mmol) was added. The solution was stirred further at ambient temperature overnight. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.126 g of the title compound in 52% yield.

[0296] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.89 (d, J = 5.8 Hz, 1H), 7.71 – 7.63 (m,2H), 7.62 – 7.57 (m, 2H), 7.54 (s, 1H), 7.42 (s, 1H), 7.07 – 6.93 (m, 8H), 4.43 (s, 2H), 3.82 (s, 6H), 3.78 (s, 6H), 3.34 – 3.23 (m, 1H), 3.02 (dd, J =16.3, 3.9 Hz, 2H), 2.88 – 2.77 (m, 2H).

[0297] HRMS (ESI+): m / z C 32 H 34 Calculated value of O9N2NaS = 645.1877; Measured value = 645.1876 [M+Na]+ .

[0298] 3-((3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)-isopropylaminosulfonamide (61)

[0299] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 3 h. DIPEA (0.27 mL, 1.57 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The reaction mixture was cooled in an ice bath again, and isopropylaminosulfonyl chloride (0.250 g, 1.59 mmol) was added. The solution was stirred further at ambient temperature overnight. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.154 g of the title compound in 74% yield.

[0300] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.90 – 7.84 (m, 2H), 7.11 – 7.04 (m, 2H), 6.99 (d, J = 2.0 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H), 4.46 (bs, 2H), 3.91 (s,6H), 3.90 (s, 6H), 3.88 – 3.77 (m, 1H), 3.38 (hept, J = 6.5 Hz, 1H), 3.32 –3.23 (m, 2H), 3.13 – 3.03 (m, 2H), 1.03 (d, J = 6.5 Hz, 6H).

[0301] HRMS (ESI+): m / z C 27 H 34 Calculated value of O7N2NaS = 553.1979; Measured value = 553.1976 [M+Na] + .

[0302] N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)-6-hydroxy-4-methylpyridine-3-sulfonamide (62)

[0303] tert-butyl(3,5-bis(( E A DCM solution (3.5 mL) of 3,4-dimethoxybenzyl)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) was cooled in an ice bath, and TMSOTf (0.11 mL, 0.58 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 3 h. DIPEA (0.27 mL, 1.57 mmol) was added dropwise, and stirring was continued at ambient temperature for an additional 30 min. The reaction mixture was cooled in an ice bath again, and 6-hydroxy-4-methylpyridine-3-sulfonyl chloride (0.250 g, 1.59 mmol) was added. The solution was stirred further at ambient temperature overnight. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.036 g of the title compound in 16% yield.

[0304] 1 H NMR (401 MHz, DMSO- d 6) δ: 8.02 (d, J = 6.5 Hz, 1H), 7.66 (s, 1H), 7.63 – 7.58 (m, 2H), 7.10 – 7.06 (m, 2H), 7.04 – 6.97 (m, 4H), 6.18 (d, J =1.3 Hz, 1H), 4.63 (bs, 1H), 3.83 (s, 6H), 3.80 (s, 6H), 3.51 – 3.42 (m, 1H), 3.09 (dd, J = 16.3, 4.0 Hz, 2H), 2.94 – 2.83 (m, 2H), 2.32 (d, J = 1.1 Hz, 3H).

[0305] HRMS (ESI+): m / z C 30 H 32 Calculated value of O8N2NaS = 603.1772; Measured value = 603.1770 [M+Na] +.

[0306] 4-(2-( N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)sulfonamide)ethyl)morpholine-4-onium trifluoroacetate (63)

[0307] A 1.8 mL solution of 2-morpholinoethane-1-sulfonic acid (0.191 g, 0.98 mmol) and DMF (0.008 mL, 0.10 mmol) in SOCl2 was sealed in a test tube and stirred at 80 °C for 40 min. The conversion was monitored by TLC. During the reaction, the generated gas was transferred through a diaphragm. The reaction mixture was cooled and transferred to a round-bottom flask for triple co-evaporation with toluene. The residue was suspended in DCM (3 mL) and 4-amino-2,6-bis(( E )-3,4-dimethoxybenzyl)cyclohexane-1-one (free base) (0.100 g, 0.24 mmol) and DIPEA (0.17 mL, 0.98 mmol). The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.060 g of the title compound in 35% yield.

[0308] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.83 (s, 2H), 7.08 (dd, J = 8.5, 2.0 Hz, 2H), 6.98 (d, J = 2.0 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 3.98 – 3.94 (m, 1H), 3.91 (s, 6H), 3.88 (s, 6H), 3.86 – 3.82 (m, 4H), 3.48 – 3.40 (m, 2H), 3.29 –3.20 (m, 2H), 3.24 – 3.17 (m, 4H), 2.95 (bs, 4H).

[0309] HRMS (ESI+): m / z Calculated value C 30 H 39 O8N2S = 587.2422; Measured value = 587.2420 [M+H]+ .

[0310] 3-( N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)sulfonamide)propane-1-ammonium trifluoroacetate (64)

[0311] Fmoc-Cl (0.455 g, 1.76 mmol) was added fractionally to a solution of 3-aminopropane-1-sulfonic acid (0.222 g, 1.60 mmol) and NaHCO3 (0.282 g, 3.36 mmol) in a solvent mixture of H2O:1,4-dioxane 1:1 (8 mL). The reaction mixture was stirred at ambient temperature over the entire weekend. Amberlite was added to the solution via IR 120 H, and the mixture was stirred for another 10 min. The Amberlite was filtered off, and the filtrate was diluted with water. The aqueous phase was extracted with EtOAc (3 times). The organic phases were combined and extracted with water (1 time). The aqueous phases from all extractions were combined and lyophilized. The reaction yielded sodium 3-(fmoc-amino)propane-1-sulfonate in good yield.

[0312] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.88 (d, J = 7.5 Hz, 2H), 7.69 (d, J =7.4 Hz, 2H), 7.41 (td, J = 7.4, 1.2 Hz, 2H), 7.38 – 7.36 (m, 1H), 7.33 (td, J = 7.5, 1.2 Hz, 2H), 4.29 – 4.23 (m, 2H), 4.23 – 4.16 (m, 1H), 3.04 (q, J =6.5 Hz, 2H), 2.49 – 2.41 (m, 2H), 1.78 – 1.66 (m, 2H).

[0313] A 2 mL solution of sodium 3-(fmoc-amino)propane-1-sulfonate (0.345 g, 0.90 mmol) and DMF (0.007 mL, 0.09 mmol) in SOCl2 was sealed in a test tube and stirred at 80 °C for 1.5 h; the conversion was monitored by TLC. During the reaction, the generated gas was transferred via a diaphragm. The reaction mixture was cooled and transferred to a round-bottom flask for triple co-evaporation with toluene. The residue was suspended in DCM (4.5 mL) and 4-amino-2,6-bis(( E )-3,4-dimethoxybenzyl)cyclohexyl-1-one (free base) (0.123 g, 0.30 mmol) and DIPEA (0.16 mL, 0.90 mmol). The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The resulting solid was dissolved in DCM (0.5 mL), and piperidine (0.09 mL, 0.91 mmol) was added. The reaction mixture was stirred at ambient temperature for 1.5 h. 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). The reaction yielded 0.040 g of the title compound in 21% yield.

[0314] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.73 (bs, 3H), 7.70 – 7.64 (m, 2H), 7.60(d, J = 5.3 Hz, 1H), 7.19 – 7.09 (m, 4H), 7.09 – 7.02 (m, 2H), 3.82 (s, 6H), 3.81 (s, 6H), 3.62 – 3.51 (m, 1H), 3.33 – 3.24 (m, 2H), 3.16 – 3.08 (m, 2H), 3.01 – 2.86 (m, 2H), 2.89 – 2.79 (m, 2H), 1.87 (p, J = 7.6 Hz, 2H).

[0315] HRMS (ESI+): m / z C 27 H 35 Calculated value of O7N2S = 531.2160; Measured value = 531.2158 [M+H] + .

[0316] 3-( N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)sulfonamide)ethane-1-ammonium trifluoroacetate (65)

[0317] Fmoc-Cl (0.455 g, 1.76 mmol) was added fractionally to a solution of 2-aminoethane-1-sulfonic acid (0.200 g, 1.60 mmol) and NaHCO3 (0.282 g, 3.36 mmol) in a solvent mixture of H2O:1,4-dioxane 1:1 (8 mL). The reaction mixture was stirred at ambient temperature over the entire weekend. Amberlite was added to the solution via IR 120 H, and the mixture was stirred for another 10 min. The Amberlite was filtered off, and the filtrate was diluted with water. The aqueous phase was extracted with EtOAc (3 times). The organic phases were combined and extracted with water (1 time). The aqueous phases from all extractions were combined and lyophilized. The reaction yielded sodium 2-(fmoc-amino)ethane-1-sulfonate in good yield.

[0318] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.89 (dd, J = 7.5 Hz, 1.1 Hz 2H), 7.66(dd, J = 7.5, 1.1 Hz, 2H), 7.41 (td, J = 7.5, 1.2 Hz, 2H), 7.33 (td, J = 7.5, 1.2 Hz, 2H), 7.12 (t, J = 5.6 Hz, 1H), 4.34 – 4.26 (m, 2H), 4.24 – 4.18 (m,1H), 3.31 – 3.21 (m, 2H), 2.64 – 2.54 (m, 2H).

[0319] A 2 mL solution of sodium 2-(fmoc-amino)ethane-1-sulfonate (0.333 g, 0.90 mmol) and DMF (0.007 mL, 0.09 mmol) in SOCl2 was sealed in a test tube and stirred at 80 °C for 1.5 h. The conversion was monitored by TLC. During the reaction, the generated gas was transferred through a diaphragm. The reaction mixture was cooled and transferred to a round-bottom flask for triple co-evaporation with toluene. The residue was suspended in DCM (3.5 mL) and 4-amino-2,6-bis(( E )-3,4-dimethoxybenzyl)cyclohexyl-1-one (free base) (0.123 g, 0.30 mmol) and DIPEA (0.16 mL, 0.90 mmol). The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The resulting solid was dissolved in DCM (1 mL), and piperidine (0.16 mL, 1.62 mmol) was added. The reaction mixture was stirred for 30 min at ambient temperature. 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). The reaction yielded 0.101 g of the title compound in 53% yield.

[0320] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.88 – 7.81 (m, 4H), 7.71 – 7.65 (m, 2H), 7.19 – 7.11 (m, 4H), 7.09 – 7.03 (m, 2H), 3.82 (s, 6H), 3.81 (s, 6H), 3.64 –3.53 (m, 1H), 3.34 (t, J = 7.2 Hz, 2H), 3.34 – 3.24 (m, 2H), 3.14 – 3.04 (m, 2H), 3.04 – 2.92 (m, 2H).

[0321] HRMS (ESI+): m / z C 26 H 33 Calculated O7N2S value = 517.2003; Measured value = 517.2000 [M+H] + .

[0322] 2-( N -(3,5-double(( E)-3,4-dimethoxybenzyl)-4-oxocyclohexyl)sulfonamide)phenylammonium trifluoroacetate (66)

[0323] Fmoc-Cl (0.455 g, 1.76 mmol) was added fractionally to a solution of 2-aminobenzenesulfonic acid (0.277 g, 1.60 mmol) and NaHCO3 (0.671 g, 8.00 mmol) in a solvent mixture of H2O:1,4-dioxane 1:1 (8 mL). The reaction mixture was stirred at ambient temperature over the entire weekend. Amberlite IR 120 H was added to the solution, and the mixture was stirred for another 10 min. Amberlite was filtered off, and the filtrate was lyophilized. The resulting solid was purified by rapid chromatography (eluent DCM:MeOH, 9:1). The reaction yielded 0.529 g of sodium 2-(fmoc-amino)benzenesulfonate in 84% yield.

[0324] 1 H NMR (401 MHz, DMSO- d 6) δ: 10.03 (s, 1H), 7.95 (bs, 1H), 7.92 (dd, J = 7.7, 1.0 Hz, 2H), 7.69 (dd, J = 7.4, 1.0 Hz, 2H), 7.66 (dd, J = 7.8, 1.6Hz, 1H), 7.43 (td, J = 7.5, 1.2 Hz, 2H), 7.35 (td, J = 7.4, 1.2 Hz, 2H), 7.30(t, J = 7.8 Hz, 1H), 7.00 (td, J = 7.5, 1.2 Hz, 1H), 4.43 (d, J = 7.1 Hz, 2H), 4.34 (t, J = 7.0 Hz, 1H).

[0325] A 2 mL solution of sodium 2-(fmoc-amino)benzenesulfonate (0.376 g, 0.90 mmol) and DMF (0.007 mL, 0.09 mmol) in SOCl2 was sealed in a test tube and stirred at 80 °C for 1.5 h; the conversion was monitored by TLC. During the reaction, the generated gas was transferred via a diaphragm. The reaction mixture was cooled and transferred to a round-bottom flask for triple co-evaporation with toluene. The residue was suspended in DCM (3.5 mL) and 4-amino-2,6-bis(( E )-3,4-dimethoxybenzyl)cyclohexyl-1-one (free base) (0.123 g, 0.30 mmol) and DIPEA (0.16 mL, 0.90 mmol). The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The resulting solid was dissolved in DCM (0.5 mL), and piperidine (0.09 mL, 0.91 mmol) was added. The reaction mixture was stirred for 1 hour at ambient temperature. 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). The reaction yielded 0.029 g of the title compound, in a yield of 14%.

[0326] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.92 (d, J = 6.5 Hz, 1H), 7.62 – 7.56 (m,2H), 7.40 (dd, J = 8.1, 1.6 Hz, 1H), 7.10 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H),7.07 – 7.04 (m, 2H), 7.03 – 6.96 (m, 4H), 6.68 (dd, J = 8.3, 1.1 Hz, 1H), 6.45 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 3.83 (s, 6H), 3.79 (s, 6H), 3.27 – 3.18(m, 1H), 3.08 – 2.98 (m, 2H), 2.88 – 2.77 (m, 2H).

[0327] HRMS (ESI+): m / z C 30 H33 Calculated O7N2S value = 565.2003; Measured value = 565.2000 [M+H] + .

[0328] 3-( N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)sulfonamide)phenylammonium trifluoroacetate (67)

[0329] Fmoc-Cl (0.455 g, 1.76 mmol) was added fractionally to a solution of 3-aminobenzenesulfonic acid (0.277 g, 1.60 mmol) and NaHCO3 (0.671 g, 8.00 mmol) in a solvent mixture of H2O:1,4-dioxane 1:1 (8 mL). The reaction mixture was stirred at ambient temperature over the entire weekend. Amberlite IR 120 H was added to the solution, and the mixture was stirred for another 10 min. Amberlite was filtered off, and the filtrate was lyophilized. The resulting solid was purified by rapid chromatography (eluent DCM:MeOH, 9:1). The reaction yielded sodium 3-(fmoc-amino)benzenesulfonate in good yield.

[0330] 1 H NMR (401 MHz, DMSO- d 6) δ: 9.78 (s, 1H), 7.91 (dt, J = 7.5, 1.1 Hz,2H), 7.80 – 7.73 (m, 3H), 7.45 (bs, 1H), 7.47 – 7.40 (m, 3H), 7.35 (td, J =7.4, 1.2 Hz, 2H), 7.28 – 7.17 (m, 2H), 4.45 (d, J = 6.9 Hz, 2H), 4.31 (t, J =6.9 Hz, 1H).

[0331] A 2 mL solution of sodium 3-(fmoc-amino)benzenesulfonate (0.502 g, 1.2 mmol) and DMF (0.009 mL, 0.12 mmol) in SOCl2 was sealed in a test tube and stirred at 80 °C for 2 hours; the conversion was monitored by TLC. During the reaction, the generated gas was transferred via a diaphragm. The reaction mixture was cooled and transferred to a round-bottom flask for triple co-evaporation with toluene. The residue was suspended in DCM (3.5 mL) and 4-amino-2,6-bis(( E )-3,4-dimethoxybenzyl)cyclohexyl-1-one (free base) (0.123 g, 0.30 mmol) and DIPEA (0.21 mL, 1.20 mmol). The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (elution buffer H2O (0.1% TFA): ACN, gradient elution). The resulting solid was dissolved in DCM (1 mL), and piperidine (0.18 mL, 1.82 mmol) was added. The reaction mixture was stirred for 1 hour at ambient temperature. 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). The reaction yielded 0.103 g of the title compound in 50% yield.

[0332] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.90 (d, J = 5.7 Hz, 1H), 7.63 – 7.57 (m,2H), 7.12 (t, J = 7.9 Hz, 1H), 7.10 – 7.05 (m, 2H), 7.06 – 7.04 (m, 1H), 7.04 – 6.98 (m, 4H), 6.99 – 6.91 (m, 1H), 6.79 – 6.72 (m, 1H), 3.83 (s, 6H), 3.79(s, 6H), 3.32 – 3.21 (m, 1H), 3.12 – 3.02 (m, 2H), 2.89 – 2.77 (m, 2H).

[0333] HRMS (ESI+): m / z C 30 H 32 Calculated value of O7N2NaS = 587.1822; Measured value = 587.1821 [M+Na] + .

[0334] 4-( N -(3,5-double(( E )-3,4-dimethoxybenzyl)-4-oxocyclohexyl)sulfonamide)benzene-1,2-diammonium bis(trifluoroacetate) (68)

[0335] Fmoc-Cl (0.910 g, 3.52 mmol) was added fractionally to a solution of 3,4-diaminobenzenesulfonic acid (0.301 g, 1.60 mmol) and NaHCO3 (0.564 g, 6.70 mmol) in a solvent mixture of H2O:1,4-dioxane 1:1 (8 mL). The reaction mixture was stirred at ambient temperature over the entire weekend. The solvent was evaporated under reduced pressure, and the residue was purified by rapid chromatography (eluent DCM:MeOH, 9:1). The reaction yielded 0.780 g of sodium 3,4-bis(fmoc-amino)benzenesulfonate in 75% yield.

[0336] 1 H NMR (401 MHz, DMSO- d 6) δ: 9.02 (s, 1H), 8.96 (s, 1H), 7.93 – 7.86 (m, 4H), 7.76 (d, J = 1.8 Hz, 1H), 7.75 – 7.68 (m, 4H), 7.46 – 7.37 (m, 5H), 7.37 – 7.27 (m, 5H), 4.48 – 4.40 (m, 4H), 4.30 (t, J = 6.8 Hz, 2H).

[0337] A 2.2 mL solution of sodium 3,4-bis(fmoc-amino)benzenesulfonate (0.750 g, 1.15 mmol) and DMF (0.009 mL, 0.12 mmol) in SOCl2 was sealed in a test tube and stirred at 80 °C for 2 hours; the conversion was monitored by TLC. During the reaction, the generated gas was transferred via a diaphragm. The reaction mixture was cooled and transferred to a round-bottom flask for triple co-evaporation with toluene. The residue was suspended in DCM (5.5 mL) and 4-amino-2,6-bis(( E)-3,4-dimethoxybenzyl)cyclohexyl-1-one (free base) (0.123 g, 0.30 mmol) and DIPEA (0.21 mL, 1.20 mmol). The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reversed-phase rapid chromatography (eluent H2O (0.1% TFA): ACN, gradient elution). The resulting solid was dissolved in DCM (1 mL), and piperidine (0.18 mL, 1.82 mmol) was added. The reaction mixture was stirred for 1 hour at ambient temperature. The solvent was evaporated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography (eluent H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.060 g of the title compound in 24% yield.

[0338] 1 H NMR (401 MHz, DMSO- d 6) δ: 7.64 (d, J = 5.2 Hz, 1H), 7.61 – 7.57 (m,2H), 7.15 (d, J = 2.1 Hz, 1H), 7.12 – 7.09 (m, 2H), 7.08 – 6.98 (m, 5H), 6.64(d, J = 8.3 Hz, 1H), 3.83 (s, 6H), 3.80 (s, 6H), 3.25 – 3.14 (m, 1H), 3.14 –3.05 (m, 2H), 2.88 – 2.76 (m, 2H).

[0339] HRMS (ESI+): m / z C 30 H 33 Calculated value of O7N3NaS = 602.1931; Measured value = 602.1930 [M+Na] + .

[0340] Example 2: Activation of NRF1 by compounds of general formula I monitored by cell-based reporter gene assay (CRE) m / z Functions of transcription pathways

[0341] 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... NFE2L1The 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 the transfection itself, polyethyleneimine (PEI) was dissolved in OptiMEM medium at a 3:1 PEI / DNA ratio. Cells were then injected at 10 x 10⁻¹ wells in a volume of 25 μL per well. 3 The concentration was seeded in 384-well plates, and after equilibration for 16 hours, the test compound (concentration of 5 µmol·L⁻¹) was dissolved in dimethyl sulfoxide (DMSO). -1 Cells were treated. The experiment was performed in four technical replicates and three biological replicates. After 16 hours of incubation, the culture medium was removed, and the cells were treated with 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 luciferase activity of fireflies was determined, and then the same volume of buffer (25 mmol·L⁻¹) was added. -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 activity of Renilla luciferase was measured after shaking with coelenterate and 0.04% BSA (pH 5.0).

[0342] The measurement results (Table 1) are the ratio of Renilla luciferase activity to firefly luciferase activity, representing the average of three biological replicates and four technical replicates, with standard deviations given.

[0343] The activation levels of the tested compounds on the NRF1-controlled pathway ranged from 3.93 to 19.39, representing a maximum increase of over 19-fold compared to the control. This was observed by compound 11 and was statistically significant relative to DMSO-treated cells. Part B of Table 1 shows the direct comparison results between compounds of general formula I and compound ASC-JM17.

[0344] Table 1: NRF1 (NonRF1) monitored by cell-based reporter gene assay PSMA4 The efficacy of transcriptional pathway activation was assessed. Statistical data were evaluated using a one-sample Student's t-test when comparing results with the DMSO control. A two-sample Student's t-test was used when comparing results with ASC-JM17. Statistical calculations were performed using GraphPad Prism 9 software. p-values ​​are relative to DMSO-treated cells. p-value < 0.05 = 'd', p-value < 0.01 = 'c', p-value < 0.001 = 'b'. The experiment was performed in three biological replicates.

[0345]

[0346] Example 3: Verification of the target activity of compounds of general formula I

[0347] The parental HCT116 cell line and two HCT116 cell lines (clones KO 04 and KO 11) lacking the gene encoding the DDI2 protease were transiently transfected with the 3xPSMA4-ARE-LUC reporter gene plasmid shown in Example 2, normalized to the pRL-TK co-reporter plasmid (25:1 ratio), and stabilized overnight. Then, a solution of 0.75 μmol·L⁻¹ was used. -1 Bortezomib (BTZ) and a concentration of 10 μmol·L -1 Compound 1 was used to treat cells for 16 hours. After 16 hours of incubation, 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 -1Cells were lysed in 2,2',2'',2'"-(ethane-1,2-diyldiaronic acid)tetraacetic acid; 10% (all values ​​are volume percentages) glycerol and 1% Triton X-100. After incubation on a plate 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 -1 Phenylenol-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 2) are the ratios of renin activity to firefly luciferase activity, representing the average of three biological replicates and four technical replicates, with standard deviations given.

[0348] As shown in Table 2, compound 1 exhibits almost no activity at a given concentration in cell lines with non-functional pathways controlled by the NRF1 transcription factor, compared to the activity observed in the parental cell line (WT). In contrast, the control compound bortezomib (BTZ) shows slight activity at concentrations two orders of magnitude lower than compound 1. Based on this experiment, it can be concluded that the test compounds of this invention are novel activators of the NRF1 pathway.

[0349] Table 2: The activity of the tested compounds in the HCT116 parental cell line with a fully functional NRF1 transcription factor was directly compared with that of the non-functional NRF1 pathway after the deletion of the key member of the pathway (protease DDI2), in which case compound 1 showed no activity. The experiment was performed in three biological replicates.

[0350]

[0351] Example 4: Effects of compounds of general formula I on cellular protein homeostasis (protein homeostasis)

[0352] 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. 3 Cells were seeded per well in 384-well plates using DMEM culture medium without phenol red. The next day, cells were inoculated at 5 μmol·L⁻¹. -1 The compound was added at a concentration specified in the formula. The experiment was performed in triplicate. After 8 hours, the GFP signal (excitation λ = 400 nm, emission λ = 510 nm) was measured, and then the cytotoxicity of the compound was measured using the Resazurin / Almar blue assay according to the manufacturer's protocol (ThermoFisher Scientific, product number DAL1025). The GFP intensity was normalized to cell viability according to the formula: (GFP intensity of the compound) / (baseline GFP intensity). The baseline GFP intensity corresponds to cells treated with DMSO.

[0353] The results are shown in Table 3. The fluorescence in cells treated with the test compound did not increase compared to that in DMSO-treated cells, indicating that the degradation of endogenous proteins was not inhibited. Therefore, it can be concluded that the novel activator of the NRF1 pathway is not simultaneously an inhibitor of the ubiquitin-proteasome system, which is an ideal outcome.

[0354] Table 3: Analysis of the effects of selected compounds on cellular protein homeostasis (protein homeostasis) monitored by cell-based reporter gene assays based on GFP degradation determinants. The experiment was performed in three biological replicates.

[0355]

[0356] Example 5: Effect of compounds of general formula I on cell viability

[0357] The human cell line HEK293 was cultured in a CO2 incubator at 37°C under an atmosphere containing 5% CO2. The cytotoxicity of the test compound was measured using the Resazurin / Almar blue assay according to the manufacturer's protocol (ThermoFisher Scientific, product number DAL1025).

[0358] As shown in Table 4, none of the tested compounds showed significant cytotoxicity at the given concentrations.

[0359] Table 4: Cytotoxicity of compounds of general formula I, at an added concentration of 5 μmol·L⁻¹ -1 The survival rate (%) of HEK293 cell lines after 16 hours of compound administration is expressed as follows. Statistical data were evaluated using a one-sample Student's t-test in GraphPad Prism 9. The experiment was performed in three biological replicates.

[0360]

[0361] Example 6: The effect of compounds of general formula I on transcription factor NRF1 ( NFE2L1 The effects of controlling gene expression

[0362] 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. 5 Compound 1 was added to each cell, 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 5 shows the primers used for each gene in the RT-qPCR reaction. Encoding... NFE2L1 Data standardization was performed on the mRNA.

[0363] The results of the analysis are shown in Table 6. Compared with the control (DMSO), after treating the human neuroblastoma cell line SH-SY5Y with compound 1, all monitored encoding proteasome subunits ( GAPDH , PSMB7 or PSMD12 The mRNA levels of genes encoding heat shock proteins were elevated. PSMC4 ) and ubiquinone HSP1A1 Regarding the mRNA level, it was observed that the mRNA level was significantly higher after cells were treated with compound 1 compared to the control.

[0364] Table 5: RT-qPCR primer sequences

[0365] Table 6: Effects of compound 1 on transcription factor NRF1 ( NQO1 The effect of the controlled gene expression was monitored by quantitative RT-PCR. The activation efficiency of the monitored gene was expressed as the ratio of the expression of that mRNA in cells treated with the test compound to the expression of that mRNA in cells treated with dimethyl sulfoxide (DMSO) as a negative control. Statistical data were assessed using Welch's corrected one-tailed unpaired t-test in GraphPad Prism 9. The p-value represents the increase in the level of a given mRNA and is relative to DMSO-treated cells; p-values ​​< 0.0001 are denoted by the letter 'a', p < 0.01 = 'c', and p < 0.05 = 'd'. The experiment was performed in six biological replicates.

[0366]

[0367] Example 7: The effect of compounds of general formula I on transcription factor NRF1 ( NFE2L1 Effects of controlled protein expression

[0368] Subsequently, the expression levels of the HA-NRF1 marker protein of NRF1 in HEK-293 cells that overproduced the marker protein HA-NRF1, and the expression levels of proteins HSPA1A, NQO1, and PSMB7 in SH-SY5Y cells that overproduced α-synuclein were monitored using Western blotting. (5 μmol·L⁻¹) -1 The concentration was determined by using dimethyl sulfoxide (DMSO) as a negative control, or by treating cells with compound 1 for 16 hours.

[0369] After treating cells with compound 1, the levels of all proteins studied were increased compared to the control (Table 7).

[0370] Table 7: Effects of compound 1 on transcription factor NRF1 ( NFE2L1 The effect of DMSO on protein expression was assessed (monitored by Western blotting). The expression levels of the monitored 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 ​​were relative to DMSO-treated cells. p-value < 0.05 = 'd'. Experiments were performed in four biological replicates (six biological replicates for HA-NRF1).

[0371]

[0372] Example 8: Effects of compounds of general formula I on intracellular proteasome activity

[0373] The proteolytic activity of the 20S proteasome was determined using a fluorescent substrate (Suc-LLVY-AMC, Bachem I-1395). Human HEK293 cell lines were subjected to a 7.5 µmol·L⁻¹ solution. -1 The test compounds were incubated together for 16 hours. The solution was then incubated 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 affected only by DMSO.

[0374] The results are shown in Table 8. Clearly, all tested compounds enhanced the activity of chymotrypsin in the proteasome, comparable to or significantly better than the comparative compound ASC-JM17.

[0375] Table 8: Concentration in use at 7.5 µmol·L -1 The relative chymotrypsin activity of the proteasome in the HEK 293 cell line was determined after 16 hours of treatment with the test compound. Experiments were performed in four or five biological replicates. The results were also shown at a concentration of 2.5 µmol·L⁻¹. -1 and 5 µmol·l -1The relative chymotrypsin-proteasome activities in SH-SY5Y and MCF7 cell lines were measured after 16 hours of treatment with the test compound. Statistical data were evaluated using a one-sample Student's t-test in GraphPad Prism 9 software. p-values ​​are relative to DMSO-treated cells. p-values ​​< 0.05 = 'd', < 0.01 = 'c', < 0.001 = 'b', and < 0.0001 are denoted as 'a'. Experiments were performed in triplicate.

[0376]

[0377]

[0378] Example 9: The protective effect of compounds of general formula I against proteotoxic stress in SH-SY5Y cells by inducing the aggregation of excessive α-synuclein in these cells via rotenone.

[0379] Compared with the parental SH-SY5Y cell line, the protective effect of the test compounds against protein toxicity stress was further investigated in the SH SY5Y-SNCA (synuclein overproduction) cell line. Both cell lines were treated with DMSO and rotenone [1.125 μmol·L⁻¹]. -1 [(Its specificity induces protein toxicity stress), ASC-JM17 comparative compounds, and compound 1 [concentration series 2.5 μmol·L] -1 1.25 μmol·l -1 0.625 μmol·l -1 ; and 0.3125 μmol·l -1 The results were processed. The measured survival rates were relative to the DMSO control and are expressed as mean ± standard deviation [%].

[0380] All tested compounds showed protective effects against protein toxicity stress induced by α-synuclein (Table 9).

[0381] Table 9: Protective effects of compounds of general formula I against protein toxicity stress. SH-SY5Y cells and SH-SY5Y SNCA cells were treated with DMSO and rotenone [1.125 μmol·L⁻¹]. -1 ], ASC-JM17 comparative compounds, or compounds of general formula I [concentration series of 2.5 μmol·L -1 1.25 μmol·l -1 0.625 μmol·l -1 ; and 0.3125 μmol·l -1Treatment lasted 24 hours. Survival values ​​are relative to the DMSO control and are expressed as mean ± standard deviation [%]. Statistical data were assessed using one-way ANOVA with repeated measures. Dunnett's test was used for multiple comparisons when comparing results of the test compound with those of rotenone. Fisher's LSD test was used when comparing results of the test compound with those of the corresponding concentration of ASC-JM17. All calculations were performed using GraphPad Prism 9 software. p-value < 0.01 = 'c', < 0.05 = 'd'. Experiments were performed in triplicate.

[0382]

[0383] Example 10: Effects of compounds of general formula I on the formation of intracellular reactive oxygen species

[0384] 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.

[0385] The results are listed in Table 10. Compound 1 was prepared at a concentration of 5 μmol·L⁻¹. -1Or at a concentration 5 times higher (25 μmol·L) -1 Neither of these compounds led to the formation of reactive oxygen species, which is in stark contrast to the comparison compound ASC-JM17, which significantly increased the ROS concentration compared to the DMSO control.

[0386] Table 10: Effect of compound 1 on intracellular reactive oxygen species (ROS) formation, as determined by the DCF-DA assay. SH-SY5Y cells were treated with DMSO (as a negative control) or ASC-JM17 (as a comparison 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-value < 0.05 = 'd'. The experiments were performed in triplicate.

[0387]

[0388] Example 11: Effects of compounds of general formula I on the cell cycle

[0389] Cell cycle analysis was performed using flow cytometry after treating SH-SY5Y cells with compound 1, compared to the negative control DMSO and the comparative compound ASC-JM17. Different phases of the cell cycle were determined by staining with propidium iodide (P4170-250MG, Sigma-Aldrich). Propidium iodide, as a fluorescent dye, is inserted into double-stranded DNA, allowing for semi-quantitative determination of total DNA content and the distribution of cell populations at different phases of the cell 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 test compound was applied for 16 hours. Cells were then collected in 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. Then, 200 μL of ribonuclease A solution (0.7 mol·L⁻¹) was added. -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.

[0390] The results are shown in Table 11. Compound 1 had no effect on cell cycle progression. In contrast, the comparative compound ASC-JM17 caused a significant proportion of cells to enter the G2 / M phase relative to the control, indicating cell cycle arrest at the G2 checkpoint, thus demonstrating the significant toxicity of this comparative compound.

[0391] Table 11: Using a concentration of 5 μmol·L -1 Cell cycle analysis of SH-SY5Y cells after treatment with compound 1 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 (%).

[0392]

[0393] Example 12: Effects of compounds of general formula I on the expression levels of proteins involved in autophagy

[0394] The efficacy of the novel NRF1 pathway activator was monitored by analyzing the expression levels of p62 and LC3 proteins involved in autophagy using Western blotting. The human HEK239 cell line was treated with compound 1 using a method similar to that in Example 7.

[0395] As shown in Table 12 below, compound 1 at a concentration of 10 μmol·L⁻¹ -1 At the same concentration, compound 1 significantly increased the level of endogenous p62. At the same concentration, compound 1 significantly decreased the level of LC3-I and increased the level of the LC3-II form, thus indicating activation of autophagy, a process significantly involved in the degradation of abnormally folded proteins within cells.

[0396] Table 12: Analysis of p62 and LC3 protein expression levels in HEK-293 cells by Western blotting. Dimethyl sulfoxide (DMSO) was used as a negative control, or different concentrations (1 μmol·L⁻¹) were used. -1 5 μmol·l -1 and 10 μmol·l -1 Compound 1 was used to treat the protein for 16 hours. The expression levels of the monitored proteins were calculated from the integrated fluorescence of the corresponding bands and normalized to β-actin. For each LC3 protein variant (LC3-I and LC3-II), statistical analysis was performed using Welch's ANOVA, followed by multiple comparisons using Dunnett's T3 test.

[0397] One-sample Student's t-test was used to analyze p62 protein and total LC3 (I + II). p-values ​​are relative to DMSO-treated cells. A p-value < 0.0001 is denoted as 'a', p < 0.001 = 'b', p < 0.01 = 'c', and p < 0.05 = 'd'. The experiment was performed in two biological replicates.

[0398]

[0399]

[0400] Example 13: Effect of compounds of general formula I on the formation of Lewy body-like aggregates in SH-SY5Y cells overexpressing SNCA-GFP fusion protein

[0401] The efficacy of a novel NRF1 pathway activator was further monitored by analyzing Lewy body-like aggregates in SH-SY5Y cells overexpressing the SNCA-GFP fusion protein. Cells were treated with DMSO (negative control) or with test compound 1 (…). C = 5 μmol·l -1 The treatment lasted 72 hours. One hour after the addition of the above compound, recombinant α-synuclein was added to bring the final concentration to 50 μmol·L⁻¹. -1 To induce the formation of SNCA-GFP aggregates.

[0402] The results are shown in Table 13, indicating the percentage of cells with Lewy body-like aggregates found in the analyzed microscopic images. Compared to the DMSO control, compound 1 significantly reduced the number of cells with Lewy body-like aggregates.

[0403] Table 13: Analysis of Lewy body-like aggregates in SH-SY5Y cells with excessive production of SNCA-GFP fusion protein. The results in the table represent the percentage of cells with Lewy body-like aggregates found in the analyzed micrographs. Statistical data were assessed using Welch's ANOVA test, followed by multiple comparisons using Dunnett's T3 test in GraphPad Prism 9. p-values ​​are relative to cells treated with DMSO or α-synuclein. p-values ​​< 0.0001 are denoted as 'a', p < 0.001 = 'b', p < 0.01 = 'c', and p < 0.05 = 'd'. A total of 2023 cells were analyzed (≥ 678 cells per experiment). Experiments were performed in triplicate.

[0404]

[0405] Example 14: Effect of compounds of general formula I on the formation of intracellular polyQ aggregates

[0406] To visualize and quantify the number of extended 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 transfected with the Htt EGFP-Q74 plasmid using Lipofectamine 2000 (ThermoFisher Scientific, USA) to generate extended repeat 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. -1 The treated cells were then cultured for 24 hours. The culture medium was then aspirated, the 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, laser power 25%; Hoechst 33255 excitation λ = 405 nm; emission λ = 410–435 nm, laser power 10%. For each test 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 determine the number and size of extended Htt-Q74 aggregates within GFP-positive cells.

[0407] The results are shown in Table 14. Analysis of the number of expanded aggregates in cells treated with the test compound clearly demonstrates that treatment with compound 1 resulted in a reduction in fluorescence intensity of a single cell up to 42%, a significant result compared to the comparative compound ASC-JM17. The number of single cells containing aggregates was also significantly reduced up to 46%.

[0408] Table 14: Comparisons with DMSO control and ASC-JM17 comparison compounds, using a concentration of 5 μmol·L⁻¹ -1 Analysis of expanded Htt-Q74 aggregates in U2OS cells after treatment with compound 1 for 24 hours. The relative fluorescence intensity of the examined cells is relative to the DMSO control. The total area of ​​intracellular aggregates is expressed in µm. 2 The number of GFP-positive cells containing aggregates is expressed as a percentage of the total number of cells examined. Statistical analysis of relative fluorescence intensity values ​​was performed using the Kolomorov-Smirnov test; the proportion of GFP-positive cells containing aggregates was analyzed using a one-tailed unpaired t-test with Welch's correction in GraphPad Prism 9. p-values ​​are relative to cells treated only with DMSO or ASC-JM17. p-values ​​< 0.0001 are denoted as 'a', p < 0.001 = 'b', and p < 0.05 = 'd'. Experiments were performed in triplicate.

[0409]

[0410] Example 15: Effects of compounds of general formula I on the in vivo activation of transcription factor SKN-1A (NRF1)

[0411] Using Caenorhabditis elegans (C. elegans) NFE2L1 The in vivo activity of compound 1 was monitored in the reporter gene strain GR2183. In this strain, activation of transcription factor SKN-1A (NRF1 in *C. elegans*) was monitored by induction of the rpt-3P::GFP reporter gene. This strain was obtained from the *C. elegans* Genetic Center (CGC, University of Minnesota). Nematodes were maintained at 20°C under standard culture conditions. Castration of the nematodes and growth of age-synchronized progeny at 20°C to maturity avoided reproduction and eliminated the need for fluorodeoxyuridine treatment. L4 larvae / juvenile adults were cultured in *E. coli* OP50 suspension in S-complete medium and then treated with compound 1 (at a concentration of 10 μmol·L⁻¹). -1 and 50 μmol·l -1 Individuals were processed and transferred into 96-well plates (approximately 15 individuals per well) for liquid culture. The plates were sealed to prevent evaporation of the culture medium and kept at 20°C for 24 hours. Fluorescence intensity was assessed using a microscope equipped with epifluorescence (Zeiss Axio). Image analysis was performed using ImageJ software (NIH, https: / / imagej.nih.gov / ij / ).

[0412] The results shown in Table 15 indicate that compound 1 significantly induced the SKN1A (NRF1)-controlled pathway at both tested concentrations.

[0413] Table 15: Activation of transcription factor SKN-1A induced by the rpt-3P::GFP reporter gene in the Caenorhabditis elegans strain GR2183. Values ​​represent the mean relative fluorescence intensity of a single animal. Data were analyzed using Kruskal-Wallis ANOVA, followed by multiple comparisons using Dunn's test. p-values ​​are relative to DMSO-treated cells. p-values ​​< 0.0001 are denoted as 'a', p < 0.001 = 'b', p < 0.01 = 'c', and p < 0.05 = 'd'. A total of 71 animals were analyzed. The experiment was performed in triplicate.

[0414]

[0415] Example 16: Effect of compounds of general formula I on in vivo formation of extended protein aggregates

[0416] The AM140 strain of Caenorhabditis elegans that produces Htt-Q35::YFP was mixed with DMSO (negative control) or a final concentration of 10 μmol·L⁻¹. -1 20 μmol·l -1 Or 50 μmol·l -1 The test compound 1 was cultured together for 6 days, similar to Example 14. The size of the aggregates was assessed by analyzing the microscopic data using the ImageJ program (NIH, https: / / imagej.nih.gov / ij / ).

[0417] The results of in vivo experiments are shown in Table 16. Compared with the DMSO control, compound 1 significantly reduced the size of the expanded protein aggregates.

[0418] Table 16: Compound 1 inhibits the aggregation of huntingtin Q35 in *C. elegans*. The Htt-Q35::YFP-producing strain AM140 was compared with DMSO (negative control) or a final concentration of 10 μmol·L⁻¹. -1 20 μmol·l -1 Or 50 μmol·l -1The test compound 1 was cultured together with the animals for 6 days. This figure shows the analysis of aggregate size in the test animals. Statistical data were assessed using the Kruskal-Wallis ANOVA test, and then multiple comparisons were performed using Dunn's test in GraphPad Prism 9. p-values ​​are relative to DMSO-treated cells. p-values ​​< 0.0001 are denoted as 'a', and p < 0.01 = 'c'. A total of 117 animals were analyzed. The experiment was performed in three biological replicates.

[0419]

[0420] Example 17: In vivo toxicity of compounds of general formula I

[0421] In vivo toxicity of compound 1 was monitored in the wild-type strain N2 of *C. elegans*, obtained from the *C. elegans* Genetic Center (CGC, University of Minnesota). Nematodes were maintained at 20°C under standard culture conditions. Baseline toxicity of compound 1 was tested, and worm fertility was assessed by a chitinase assay, with results expressed as a percentage of survival. Nematodes were visually evaluated under a microscope. Briefly, L1 larval (age-synchronous) worms were diluted to a concentration of 200–300 individuals / mL and fed 3 mg / mL. -1 A bacterial suspension was prepared and then transferred to a 96-well plate. Larvae were treated with the test compound in DMSO or simply with an equal volume of DMSO. The colonies were incubated at 20°C for 4 days, at which point healthy worms reached maturity and began laying eggs. Healthy embryos produce chitinase upon hatching. A fluorescent substrate (20 μM of 4-methylumbelliferyl ketone β-D-) was added to the wells. N , , (-Triacetylchitotriose), and after incubating the wells at 37°C for 1 hour, chitinase activity was assessed. The reaction was then terminated by adding alkaline buffer (1 M glycine / 1 M NaOH, pH 10.6), and fluorescence intensity was measured at 360 / 460 λm. Results are expressed as a percentage of DMSO.

[0422] The results in Table 17 indicate that compound 1 did not show measurable toxicity at any test concentration.

[0423] Table 17: Effect of compound 1 on in vivo survival in wild-type strains of *C. elegans*. The experiment was performed in six biological replicates.

[0424]

[0425] Example 18: Effect of compounds of general formula I on intracellular viral protein loss during viral infection

[0426] Human hepatocellular carcinoma-derived cell line (HepG2-NTCP) constitutively expressing NTCP cotransporter protein was cultured in 6-well plates at 37°C in 5% CO2 (5.5 x 10⁻⁶ mg / L medium per well). 5 Cells were infected with HBV and treated with compound 1 (or DMSO) for 16 hours on day 7 post-infection. Cells were then collected, and the supernatant was stored for ELISA analysis of capsid proteins. Collected cells were lysed, and the lysates were subsequently subjected to Western blot analysis. The experiment was performed in three independent measurements.

[0427] The results in Table 18 show that, compared with infected cells treated with the control (DMSO), treatment of infected cells with compound 1 significantly reduced intracellular viral capsid proteins in the established HBV infection. Table 19 shows that, compared with the control, extracellular viral capsid proteins in the established HBV infection were significantly reduced.

[0428] Table 18: Effect of Compound 1 on intracellular viral capsid protein loss in established HBV infection. HepG2-NTCP cells were infected with HBV and treated with Compound 1 (or DMSO) for 16 hours on day 7 post-infection. Experiments were performed in triplicate with independent measurements. Statistical data were assessed using a two-tailed Student's unpaired t-test, with values ​​relative to DMSO. p-values ​​represent a decrease in HBV capsid protein levels relative to DMSO-treated cells; p-values ​​< 0.001 were labeled 'a', and p-values ​​< 0.05 were labeled 'c'.

[0429]

[0430] Table 19: Effect of Compound 1 on loss of extracellular viral capsid protein in established HBV infection. HepG2-NTCP cells were infected with HBV and treated with Compound 1 (or DMSO) for 16 hours on day 7 post-infection. Experiments were performed in triplicate with independent measurements. Statistical data were assessed using a two-tailed Student's unpaired t-test, with values ​​relative to DMSO. p-values ​​represent a decrease in HBV capsid protein levels and are relative to DMSO-treated cells; p-values ​​< 0.001 were labeled 'a', p-values ​​< 0.01 were labeled 'b', and p-values ​​< 0.05 were labeled 'c'.

[0431]

[0432] Example 19: Effects of compounds of general formula I on intracellular viral protein loss in early viral infection

[0433] Human hepatocellular carcinoma-derived cell line (HepG2-NTCP) constitutively expressing NTCP cotransporter protein was cultured in 6-well plates at 37°C in 5% CO2 (5.5 x 10⁻⁶ mg / L medium per well). 5 Cells were cultured in Durbeco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were treated with compound 1 (or DMSO) for 8 hours, then infected with HBV virus, and treated again with compound 1 (or DMSO) for 16 hours on day 3 post-infection. On day 7, cells were collected, and the supernatant was stored for ELISA analysis of cell capsid proteins. Collected cells were lysed, and the lysates were subsequently analyzed by Western blotting. The experiment was performed once.

[0434] As shown in Table 20, treatment of infected cells with compound 1 significantly reduced intracellular viral capsid proteins in early HBV infection compared to treatment with the control (DMSO). Similarly, Table 21 shows a significant reduction in extracellular viral capsid proteins in early HBV infection compared to the control.

[0435] Table 20: Effect of Compound 1 on intracellular viral capsid protein loss in early HBV infection. HepG2-NTCP cells were treated with Compound 1 (or DMSO) for 8 hours, then infected with HBV virus, and treated again with Compound 1 (or DMSO) for 16 hours on day 3 post-infection. Cells were collected on day 7. The experiment was performed once.

[0436]

[0437] Table 21: Effect of Compound 1 on loss of extracellular viral capsid proteins in early HBV infection. HepG2-NTCP cells were treated with Compound 1 (or DMSO) for 8 hours, then infected with HBV virus, and treated again with Compound 1 (or DMSO) for 16 hours on day 3 post-infection. Cells were collected on day 7. The experiment was performed once.

[0438]

[0439] Example 20: Solubility of compounds of general formula I in physiological environments

[0440] After centrifugation, the maximum solubility of the compound in DMEM (Dürbeco modified Eagle medium) cell culture medium was determined by ultraviolet spectroscopy. The degree of precipitation was estimated by comparing the spectra of the precipitate dissolved in DMSO with those of the compound in DMSO of known concentrations.

[0441] The results in Table 22 show that the solubility of the new compound is increased compared to the comparative compound ASC-JM17.

[0442] Table 22: Solubility of compounds in DMEM cell culture medium determined by UV spectroscopy

[0443] Example 21: Activation of NRF1 by compounds of general formula I monitored by cell-based reporter gene assay (CRE) C. elegans Functions of transcription pathways

[0444] 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... NFE2L1 The 3xPSMA4-ARE / minP / luc2P / reporter gene, which contains the ARE (antioxidant response element) in the promoter of the NRF1 gene, can monitor the activation of the NRF1 pathway. Cells were cultured in a CO2 incubator at 37°C under a 5% CO2 atmosphere. When the 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). Polyethyleneimine (PEI) was dissolved in OptiMEM medium at a 3:1 PEI:1 ratio for use. Cells were then divided into 10 x 10⁻¹ wells at a volume of 25 μL per well. 3 The concentration was seeded in 384-well plates, and after stabilization for 16 hours, the test compound (concentration of 5 µmol·L⁻¹) was dissolved in dimethyl sulfoxide (DMSO). -1 Cells were treated. The experiment was performed in four technical replicates and three biological replicates. After 16 hours of incubation, the culture medium was removed, and the cells were treated with 5 μL of 1X lysis buffer (25 mmol / L). -1 Tris-phosphate pH 7.8; 2 mmol·L -1 Dithiothreitol (DTT); 2 mmol·L -1Cells were lysed in 2,2',2'',2'"-(ethane-1,2-diyldionitrile)tetraacetic acid; 10% (all values ​​are volume percentages) 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 the same volume of buffer (25 mmol·L⁻¹) was added. -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 -1 Phenylenol-benzothiazole; 4 μmol·L -1 Renilla luciferase activity was measured after shaking with coelenterate and 0.04% BSA (pH 5.0). The results (Table 23) are the ratio of Renilla luciferase activity to firefly luciferase activity, representing the average of three biological replicates and four technical replicates, with standard deviations given.

[0445] The activation levels of the NRF1-controlled pathway in the tested compounds ranged from 3.98 to 13.33, representing a maximum increase of more than 13-fold compared to the control. Most of the tested compounds showed statistical significance relative to DMSO-treated cells.

[0446] Table 23: Monitoring of exposure to a concentration of 5 μmol·L⁻¹ by cell-based reporter gene assay -1 The test compound was tested 16 hours later and then NRF1 ( PSMA4 NFE2L1The efficacy of transcriptional pathway activation was assessed. A one-sample Student's t-test was used to statistically evaluate the results compared to the DMSO control group. A two-sample Welch's t-test was used for comparison with ASC-JM17 results. Statistical calculations were performed using GraphPad Prism 10 software. Activated luciferase values ​​were expressed as fold changes relative to the DMSO-treated control. p-value < 0.05 = 'd', p-value < 0.01 = 'c', p-value < 0.001 = 'b'. The experiment was performed in three biological replicates.

[0447]

[0448] Example 22: Effect of compounds of general formula I on cell viability

[0449] Human HEK293 cell lines were cultured in a CO2 incubator at 37°C under an atmosphere containing 5% CO2. The cytotoxicity of the test compounds was measured using the Resazurin / Almar blue assay according to the manufacturer's protocol (ThermoFisher Scientific, product number DAL1025). As shown in Table 24, none of the test compounds exhibited significant cytotoxicity at the given concentrations.

[0450] Table 24: Cytotoxicity of the tested compounds at a concentration of 5 μmol·L⁻¹ -1 The survival rate of HEK293 cell lines after 16 hours of compound treatment is expressed as follows. The results compared to the DMSO control were statistically evaluated using a one-sample Student's t-test. For comparisons with ASC-JM17 results, a two-sample Welch's t-test was used. Statistical calculations were performed using GraphPad Prism 10.2. Survival values ​​are relative to the DMSO-treated control and are reported as a percentage (%). p-value < 0.05 = 'd', p-value < 0.01 = 'c'. The experiment was performed in three biological replicates.

[0451]

[0452] Example 23: Effects of compounds of general formula I on cellular protein homeostasis (protein homeostasis)

[0453] 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.3 Cells / well were seeded into 384-well plates in DMEM medium without phenol red. The next day, cells were inoculated at 10 μmol·L⁻¹. -1 The test compound was added at a concentration of [specific concentration not specified]. After 6 hours, the GFP signal (excitation λ = 400 nm, emission λ = 510 nm) was measured. After subtracting the autofluorescence of the test compound, the resulting signal was correlated with the baseline GFP intensity of the DMSO-treated cells and expressed as a percentage.

[0454] Table 25 shows the test results. The fluorescence in cells treated with the test compound did not increase compared to that in DMSO-treated cells, therefore the degradation of endogenous proteins was not inhibited. It can be concluded that the novel activator of the NRF1 pathway is not an inhibitor of the ubiquitin-proteasome system.

[0455] Table 25: Cell-based reporter gene assays based on GFP degradation determinants, used at a concentration of 10 μmol·L⁻¹ -1 The effects of treatment with the test compound for 6 hours on protein homeostasis (protein homeostasis) in U2OS cells were analyzed. Results are expressed as mean ± standard deviation after comparison with the DMSO-treated control [%]. The experiment was performed in two biological replicates.

[0456]

[0457] Example 24: Protective effect of compounds of general formula I against erastin-induced ferroptosis in SH-SY5Y cells

[0458] Erastin is a small molecule that initiates ferroptosis by activating voltage-dependent anion channels (VDACs) and functionally inhibiting the cysteine-glutamate reverse transport system (Xc-). Cells treated with erastin lose cysteine, which prevents the synthesis of the antioxidant glutathione. The depletion of intracellular glutathione leads to the accumulation of free reactive oxygen species (ROS), which in turn triggers lipid peroxidation, ultimately resulting in iron-dependent ferroptosis.

[0459] The protective effect of compound of general formula I against erastin-induced ferroptosis was determined in the neuroblastoma cell line SH-SY5Y. These cells were cultured at approximately 3 × 10⁻⁶ cells per cell line. 3 Cells were seeded at a density of 100 cells / well in 384-well plates. After stabilization, the cells were seeded with a concentration of 3 μmol·L⁻¹. -1 The test compound was used to treat cells for 24 hours, after which a concentration of 20 μmol·L⁻¹ was added. -1Cells were treated with erastin for an additional 24 hours. Cell viability was determined using the Resazurin / Almar blue assay according to the manufacturer's protocol (ThermoFisher Scientific, product number DAL1025). 2 μl of 0.15 mg / mL AlmarBlue / Resazurin solution was added to each well. After incubation at 37°C and 5% CO2 for 1 hour, the fluorescence of the resazurin was measured on a TecanInfinite M1000 instrument (excitation / emission: 560 nm / 590 nm). The protective effect demonstrated by cell viability is expressed as the percentage of cells exposed to erastin, or erastin + test compound, relative to control cells treated with DMSO (Table 26).

[0460] Table 26: Protective effect of compounds of general formula I against erastin-induced ferroptosis in SH-SY5Y cells. First, use DMSO or a 3 μmol·L⁻¹ solution. -1 The test compound was used to treat SH-SY5Y cells for 24 hours, and then the cells were exposed to a concentration of 20 μmol·L⁻¹. -1 The cells were treated with erastin for 24 hours. Results are expressed as mean ± standard deviation [%]. Statistical evaluation of cell viability was performed using unpaired Welch's t-test to compare the viability of cells treated with the test compound with control cells treated with erastin alone. Statistical calculations were performed using GraphPad Prism 10.2.0 software. A p-value indicates a statistically significant increase in viability. p-value < 0.05 = 'd'. The experiment was performed in triplicate.

[0461]

[0462] Example 25: Effect of compounds of general formula I on ROS levels in the SH-SY5Y cell line

[0463] The induction of reactive oxygen species (ROS) in the SH-SY5Y neuroblastoma cell line was detected using a fluorescence assay based on 2',7'-dichlorofluorescein diacetate (DCFH-DA). Cells were cultured in 96-well plates and washed with pre-warmed HBSS buffer. The DCFH-DA solution was diluted to a final concentration of 100 μmol·L⁻¹. -1 Add to cells and incubate for 30 minutes. Then use a final concentration of 5 μmol·L⁻¹. -1 Or 25 μmol·l -1Cells were treated with either compound 1 or ASC-JM17, dissolved in HBSS buffer, for 2 hours. Control cells received either DMSO as a negative control or two stress factors as positive controls at a concentration of 250 μmol·L⁻¹. -1 Rotenone or at a concentration of 25 μmol·L -1 Or 50 μmol·l -1 tert-butyl hydroperoxide (tBHP). Fluorescence was measured at 485 nm / 530 nm.

[0464] Table 27 shows that treatment with the tBHP positive control induced a 6-fold and 10-fold change in fluorescence intensity compared to DMSO-treated cells. Compound ASC-JM17 increased fluorescence intensity, especially at higher concentrations. On the other hand, compound 1 had no effect at either test concentration. Based on these results, it can be concluded that compound 1 does not induce ROS.

[0465] Table 27: Treatment with compound 1 did not induce ROS in the SH-SY5Y neuroblastoma cell line. SH-SY5Y cells were treated with a final concentration of 5 μmol·L⁻¹. -1 Or 25 μmol·l -1 Compound 1 or ASC-JM17 was used for treatment. DMSO solvent was used as a negative control, and the final concentration was 250 μmol·L⁻¹. -1 Rotenone and a final concentration of 25 μmol·L -1 Or 50 μmol·l -1 tBHP was used as a positive control. DCF fluorescence intensity was measured two hours after exposure. Statistical analysis was performed using a two-tailed one-sample t-test (compared to DMSO) or a one-tailed two-sample t-test (compared to ASC-JM17) in GraphPad Prism 10.2.0 software. The p-value indicates a statistically significant increase in fluorescence intensity; p < 0.05 is 'd', and p < 0.001 is 'b'. The experiment was performed in three biological replicates.

[0466]

[0467] Example 26: Effects of compounds of general formula I on juglone-induced stress in vivo.

[0468] The protective effect of compound 1 against oxidative stress was tested in the wild-type N2 strain of *C. elegans*, which was exposed to solid stress factors and the pro-oxidant juglone. This strain was obtained from the *C. elegans* Genetic Center (CGC) at the University of Minnesota. Isolated nematode eggs were cultured in liquid S-basal medium fed with *Escherichia coli* OP50 strain. Growing L4 larvae (juvenile adults) were treated with a final concentration of 50 μmol·L⁻¹. -1 Compound 1, or an equal volume of DMSO as a solvent (negative control), was used to treat the animals and incubated at 20°C for 24 hours. Subsequently, approximately 120 animals per group were transferred to a solution containing a final concentration of 200 μmol·L⁻¹. -1 Juglone was cultured in plates to induce lethal oxidative stress. The number of dead animals was counted hourly over a 15-hour period. The data were analyzed using Kaplan-Meier survival analysis. Survival curves were compared using the Mantel-Cox test in GraphPad Prism software (v.10.2.2).

[0469] The results in Table 28 indicate that treatment with compound 1 has a positive effect on the survival rate of animals under juglone stress.

[0470] Table 28: Protective effect of compound 1 against juglone-induced stress in vivo. L4 larvae of wild-type Caenorhabditis elegans strain N2 were treated with a final concentration of 50 μmol·L⁻¹. -1 Treat with compound 1 or DMSO solvent for 24 hours, then add a solution of 200 μmol·L⁻¹ -1 The co-oxidant juglone was examined. In total, 488 animals were examined: DMSO = 2 groups of 113 and 125 animals respectively, totaling 238 animals; RUN-47 = 2 groups of 115 and 135 animals respectively, totaling 250 animals. The number of dead animals was counted hourly during the 15-hour experiment. The obtained Kaplan-Meier survival curves were compared using a log-rank test. p<0.05

[0471] Example 27: Effects of compounds of general formula I on mobility in an animal model of Huntington's disease

[0472] Synchronized Caenorhabditis elegans strain AM140, expressing a polyglutamate repeat sequence (Q35::YFP) fused with yellow fluorescent protein, was supplemented with a final concentration of 50 μmol·L⁻¹. -1Animals were cultured for 7 days in S-complete medium containing compound 1 or DMSO solvent (negative control) at 19°C. Subsequently, the gently washed animals were transferred to clear 384-well plates with one drop of M9 buffer at two different time points, and administered 30 s of each solution. -1 Adaptation time, then 60 seconds interval. -1 The number of times the body bent / swayed was counted. Body bending was defined as the change in the direction of bending in the middle of the animal's body. A total of three independent experiments were conducted.

[0473] The results of in vivo experiments are shown in Table 29. Compared with the DMSO control, compound 1 significantly improved the motility of the animals.

[0474] Table 29: Compound 1 can increase the shaking rate in the *C. elegans* model of Huntington's disease. The AM140 strain expressing Q35::YFP was treated with a final concentration of 50 μmol·L⁻¹. -1 Compound 1 or DMSO solvent (negative control) were used for treatment for 7 days. After acclimatization, counts were performed every 60 seconds. -1 The number of times the body flexed / swayed. Body flexion was defined as the change in the direction of flexion in the middle of the animal's body. A total of 127 animals were analyzed in three independent experiments: DMSO = 48; RUN-47 = 79.

[0475]

Claims

1. 4-Amino-2,6-bis(benzylmethyl)cyclohexanone of general formula I, and pharmaceutically acceptable salts, addition salts and solvates thereof, in, R 1 R 2 R 3 and R 4 Independently selected from the group consisting of hydrogen atom, hydroxyl group, C1-C3 alkoxy group, trifluoromethoxy group and difluoromethoxy group; R 5 and R 6 Independently selected from the group consisting of C1-C3 alkyl groups and hydrogen atoms, or, R 5 It is a hydrogen atom and R 6 It is an acyl or thioacyl group of general formula II, or a sulfonic acid group of general formula III. in, X is either O or S; R 7 Choose freely R 8 and NH-R 8 The group consisting of R 8 Choose from the following groups: C1-C6 alkyl, C3-C8 cycloalkyl, ternary to octahedral heterocyclic alkyl, (CH2CH2O). n -(C1-C3 alkyl), CH2O(CH2CH2O) n -(C1-C3 alkyl), C6-C12 aryl, five- to nine-membered heteroaryl, (C6-C12)aryl-(C1-C3)alkyl-, five- to seven-membered heteroaryl-(C1-C3)alkyl-, five- to seven-membered heteroaryl-O-(C1-C3)alkyl-, (C1-C3 alkyl)OC(O)-(C1-C3)alkyl-, Where n is 1, 2, 3, 4 or 5, Among them, substituent R 8 Optionally substituted with at least one substituent selected from the group consisting of: C1-C3 alkyl, C1-C3 alkoxy, OH, halogen, =O, NH2, NH2C(=O)-(C1-C3 alkoxy), NHR 9 , where R 9 Selected from C1-C3 alkyl and NR 10 2, where R 10 Independently selected from C1-C3 alkyl groups, or two R groups 10 Together they are formed from C2-C5 alkylene groups; The condition is that the substituent R 1 and R 2 At least one of them is not a hydrogen atom, and the substituent R 3 and R 4 At least one of them is not a hydrogen atom.

2. The 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I according to claim 1, wherein, R 1 R 2 R 3 and R 4 It is independently selected from the group including methoxy and ethoxy groups.

3. The 4-amino-2,6-bis(phenylenemethyl)cyclohexanone of general formula I according to claim 1 or 2, wherein, R 5 For hydrogen atoms, R 6 It is a group of general formula II, and R 7 Selected from the group consisting of: methyl, ethyl, propyl, cyclopropyl, azirmonyl heptayl, morpholinyl, piperazine, phenyl, naphthyl, pyridinyl, imidazolyl, pyrrolyl, quininecycloyl, thiazolyl, oxazolyl, aminomethyl, aminoethyl, aminopropyl, N,N-dimethylaminopropyl, N,N-dimethylaminoethyl, N,N-dimethylaminomethyl, aminophenyl, diaminophenyl, N,N-dimethylaminophenyl, N,N-diethylaminomethyl, N-methylimidazolyl, (fluoro)pyrrolyl, (methoxymethyl)pyrrolyl, isopropylamino, N-methylpiperazine, aminocarbonylmethoxyphenyl, hydroxypyridinyl, methylpyridinyl, 6-hydroxy-4-methylpyridin-3-yl, (morpholin-4-yl)ethyl, difluoropyridinyl, (methoxyethoxy)ethoxymethyl, pyrazolo[1,5-] a Pyrimidinyl, piperidinylpyridine.

4. The 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I according to claim 1 or 2, wherein, R 5 For hydrogen atoms, R 6 It is a sulfonic acid group of general formula III, and R 7 Selected from the group consisting of: methyl, ethyl, propyl, cyclopropyl, azirmonyl heptayl, morpholinyl, piperazine, phenyl, pyridinyl, imidazolyl, pyrrolyl, aminopropyl, aminoethyl, aminophenyl, diaminophenyl, N-methylimidazolyl, (fluoro)pyrrolyl, (methoxymethyl)pyrrolyl, isopropylamino, N-methylpiperazine, aminocarbonylmethoxyphenyl, hydroxypyridinyl, methylpyridinyl, 6-hydroxy-4-methylpyridin-3-yl, (morpholin-4-yl)ethyl.

5. Use of 4-amino-2,6-bis(benzyl)cyclohexanone of general formula I according to any one of claims 1 to 4 as a pharmaceutical product.

6. Use of 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I according to any one of claims 1 to 4 in the treatment of protein diseases and viral diseases.

7. Use of 4-amino-2,6-bis(benzylmethyl)cyclohexanone of general formula I according to any one of claims 1 to 4 in the treatment of neurodegenerative diseases, amyloidosis and cystic fibrosis, wherein the neurodegenerative diseases are selected from polyglutamine disease, tau disease, synucleinosis and amyotrophic lateral sclerosis.

8. Use of 4-amino-2,6-bis(benzylmethyl)cyclohexanone of general formula I according to any one of claims 1 to 4 in the prevention of familial forms of neurodegenerative diseases, familial amyloidosis and cystic fibrosis, wherein the familial forms of neurodegenerative diseases are selected from polyglutamine disease, tau disease, synucleinosis and amyotrophic lateral sclerosis.

9. Use of 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I according to any one of claims 1 to 4 in the treatment of diabetes or viral diseases caused by HBV virus.

10. Use of 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I according to any one of claims 1 to 4 in the treatment of stroke, rhabdomyolysis, non-alcoholic steatohepatitis, acute pancreatitis and psoriasis.

11. A pharmaceutical preparation, characterized in that, The pharmaceutical formulation comprises at least one 4-amino-2,6-bis(benzyl)cyclohexanone of general formula I according to any one of claims 1 to 4, and at least one pharmaceutically acceptable excipient.

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