4-amino-2,6-bis(phenylmethylene)cyclohexanone and its use

Abstract

The present invention relates to 4-amino-2,6-bis(phenylmethylene)cyclohexanones of general formula I and their pharmaceutically acceptable salts, addition salts, and solvates: 【Chemical 1】 JPEG2026525368000109.jpg38169 [wherein, R 1 , R 2 , R 3 and R 4 are each independently selected from the group consisting of a hydrogen atom, a hydroxy group, a C1-C3 alkoxy group, a trifluoromethoxy group, and a difluoromethoxy group; R 5 and R 6 are each independently selected from the group consisting of C1-C3 alkyl and a hydrogen atom; or, R 5 is a hydrogen atom, R 6 is an acyl group or a thioacyl group of general formula II, or a sulfonic acid group of general formula III: 【Chemical 2】 JPEG2026525368000110.jpg26169 where X is O or S; R 7 is selected from the group consisting of R 8 and NH-R 8 , R 8 is selected from the group consisting of C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 member heterocycloalkyl, (CH2CH2O) n -(C1-C3 alkyl), CH2O(CH2CH2O) n -(C1-C3 alkyl), C6-C12 aryl, 5-9 member heteroaryl, (C6-C12) aryl-(C1-C3) alkyl-, 5-7 member heteroaryl-(C1-C3) alkyl-, 5-7 member heteroaryl-O-(C1-C3) alkyl-, (C1-C3 alkyl)O-C(O)-(C1-C3) alkyl-; n is 1, 2, 3, 4 or 5, substituent R 8The following are optional: C1-C3 alkyl, C1-C3 alkoxy, OH, halogen, =O, NH2, NH2C(=O)-(C1-C3 alkoxy), NHR 9 It is substituted with at least one substituent selected from the group consisting of R 9 This includes C1-C3 alkyl and NR 10 Selected from 2, R 10 Each of these is independently selected from C1-C3 alkyl groups, or both R 10 These combine to form C2-C5 alkylenes. However, substituent R 1 and R 2 At least one of them is not a hydrogen atom, but a substituent R 3 and R 4 The present invention relates to a compound in which at least one of the atoms is not a hydrogen atom. The compound is suitable for the treatment of proteinopathy, viral diseases, and diseases directly associated with elevated levels of ferroptosis, such as stroke, rhabdomyolysis, non-alcoholic fatty liver disease, acute pancreatitis, and psoriasis.

Description

Detailed description of the invention

[0001] [Technical Field] This invention relates to novel compounds derived from 4-amino-2,6-bis(phenylmethylene)cyclohexanone, and to the use of these compounds to reduce proteotoxic stress in cells and tissues and to suppress ferroptosis.

[0002] [Background technology] Protein degradation within cells is a tightly regulated process essential for maintaining cellular homeostasis. Over 90% of cytoplasmic proteins are degraded by the so-called ubiquitin-proteasome system (UPS), which removes incompletely synthesized or misfolded proteins and regulates the amount of proteins directly involved in controlling their activity within cells. At the heart of the UPS system is the 26S proteasome, which degrades proteins covalently labeled with polyubiquitin chains (Lys48) into a mixture of peptides. The 26S proteasome consists of a 20S catalytic subunit with three catalytic sites exhibiting chymotrypsin-like, trypsin-like, or caspase-like activity, and one or two regulatory subunits designated as 19S. The aging process and the development of neurodegenerative diseases are closely linked to the accumulation of misfolded or damaged proteins, which can be toxic to cells or even directly induce apoptosis. A decrease in UPS activity is associated with both aging and the onset and progression of neurodegenerative diseases, which very often leads to the formation of intracellular protein aggregates. For these reasons, regulating or enhancing UPS activity is considered a very promising approach to delay the onset of or treat diseases associated with the accumulation of toxic protein forms, such as amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Kennedy disease, or Huntington's disease (Kleiger et al.: Trends in cell biology, 2014, 24, 6, 352-359; Ciechanover et al.: Experimental & molecular medicine, 2015, 47, 3, e147-e147; Calamini et al.: Nature chemical biology, 2012, 8.2, 185-196).

[0003] Increased UPS activity can be achieved, for example, by stimulating its catalytic activity with low molecular weight compounds. Unfortunately, however, compounds known to date exhibit this activity have shown only very limited effects at the cellular level (Trader et al.: Biochimica et Biophysica Acta (BBA)-General Subjects, 2017, 1861.4, 892-899; Leestemaker et al.: Cell chemical biology, 2017, 24.6, 725-736). The most promising way to enhance UPS capacity is to increase the synthesis of the proteasome itself, ideally by simultaneously activating the synthesis of heat shock proteins (HSPs). Both of these can be achieved by activating the stress transcription factors NRF1 (NFE2L1) and NRF2 (NFE2L2) from the so-called Cap-n-Collar (CNC) family of transcription factors (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, which induces the coordinated expression of all genes related to proteasome subunits in response to proteotoxic stress, is considered more appropriate. Important findings indicate that the activated NRF1 pathway prevents the formation of toxic protein aggregates compared to gene knockdown. Therefore, small molecular weight compounds that selectively activate this signaling pathway without interacting with UPS and without causing oxidative stress are considered the most promising future therapeutic approach for proteinopathy, including neurodegenerative diseases whose onset is associated with protein aggregate formation and proteotoxic stress (Njomen et al.: J Med Chem, 2019, 62.14, 6469-6481).

[0004] The transcription factor NRF1 preferentially induces the synthesis of all proteasome subunits when proteasome activity is inhibited (Kleiger et al.: Trends in cell biology, 2014, 24, 6, 352-359; Koizumi et al.: Proceedings of the Japan Academy. Series B, 2018, 325-336). NRF1 also increases the expression of the transcription factor HSF1, which is responsible for inducing the expression of heat shock proteins that ensure a cellular response to exposure to stress conditions. Heat shock proteins help misfolded or stress-damaged proteins regain their correct structure, and this function is now considered one of the essential defense mechanisms against the formation of aggregates or toxic protein morphologies, both at the cell culture level and in mouse models (Bose et al.: Ageing research reviews, 2017, 35, 155-175).

[0005] Recently, the NRF1 regulatory transcription pathway has been shown to protect cells and tissues from ferroptosis. Ferroptosis is a newly discovered type of regulated cell death that is morphologically, biochemically, and genetically distinct from other previously reported cell deaths. The main characteristic of ferroptosis is its iron dependence, characterized by the accumulation of lipid peroxides and reactive oxygen species induced by iron metabolism. In degenerative and ischemic diseases, ferroptosis is involved in their onset and pathogenesis, and suppressing ferroptosis is an interesting strategy to delay or directly prevent 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 protects against ferroptosis by inducing the expression of glutathione peroxidase 4 (GPX4), a key protein that prevents lethal lipid peroxidation, and by maintaining proteasome activity (Forcina et al.: Proceedings of the National Academy of Sciences, 119(11), e2118646119).

[0006] Currently, the most discussed compound capable of increasing proteasome activity while simultaneously increasing the expression of heat shock proteins regulated by the transcription factor HSF1 is the curcumin derivative ASC-JM17 ((1E,6E)-4-(cyclobutylmethyl)-1,7-bis(3,4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione). This has been shown to be a dual activator of both of the aforementioned 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 is approved by the European Medicines Agency (EMA) for the treatment of spinal muscular atrophy (known as Kennedy disease). However, low solubility under physiological conditions, and the resulting low bioavailability and relatively rapid degradation in tissues, are common characteristics and problems of curcumin and its derivatives.

[0007] Despite their immense therapeutic potential, only a few compounds capable of targeting the enhancement of cellular responses to proteotoxic stress have reached clinical use. Therefore, the development of novel drugs with higher efficacy, narrower targeted mechanisms of action, fewer side effects, and the lowest in vivo toxicity is essential.

[0008] [Disclosure of the Invention] The present invention aims to prevent and / or reduce the formation and deposition of metastable proteins in cells and tissues using novel compounds derived from 4-amino-2,6-bis(phenylmethylene)cyclohexanone, thereby addressing the undesirable effects of proteotoxic stress and ferroptosis in cells and tissues by controlling and mitigating them. Surprisingly, the novel compounds derived from 4-amino-2,6-bis(phenylmethylene)cyclohexanone described herein were found to specifically activate pathways controlled by the transcription factor NRF1 (encoded by the NFE2L1 gene). These pathways are therefore directly related to increased intracellular proteasome activity, increased synthesis of heat shock proteins directly involved in target protein degradation, and activation of autophagy. Increased NRF1 activity further inhibits ferroptosis. At the same time, these compounds exhibit extremely low cytotoxicity and endogenous toxicity at the relevant doses. It is an important and surprising finding that these compounds do not increase the generation of reactive oxygen species (ROS) and do not affect the cell cycle at the relevant concentrations, thereby reducing the risk of adverse side effects.

[0009] The object of the present invention relates to 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I and pharmaceutically acceptable salts, addition salts, and solvates thereof: [ka] [In the formula, R 1 , R 2 , R 3 and R 4 Each of these is independently selected from the group comprising a hydrogen atom, a hydroxyl group, a C1-C3 alkoxy group, a trifluoromethoxy group, and a difluoromethoxy group; R 5 and R 6 Each is independently selected from the group consisting of C1-C3 alkyl and hydrogen atoms; or R 5 R is a hydrogen atom, 6is an acyl or thioacyl group of general formula II, or a sulfonic acid group of general formula III: [Chemical formula] where X is O or S; R 7 is selected from the group consisting of R 8 and NH-R 8 R 8 is selected from C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 member heterocycloalkyl, (CH2CH2O) n -(C1-C3 alkyl), CH2O(CH2CH2O) n -(C1-C3 alkyl), CQ-C12 aryl, 5-9 member heteroaryl, (C6-C12) aryl-(C1-C3) alkyl-, 5-7 member heteroaryl-(C1-C3) alkyl-, 5-7 member heteroaryl-O-(C1-C3) alkyl-, (C1-C3 alkyl)O-C(O)-(C1-C3) alkyl-, n is 1, 2, 3, 4 or 5, substituent R 8 is 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 R 9 is selected from C1-CZ alkyl and NR 10 2, R- 10 is each independently selected from C1-C3 alkyl, or both R 10 together form C2-C5 alkylene, provided that at least one of substituents R 1 and R 2 is not a hydrogen atom, and at least one of substituents R 3 and R 4 is not a hydrogen atom).

[0010] Alkyls are saturated linear or branched hydrocarbons. Alkyls may include, but are not limited to, methyl, ethyl, propyl, and isopropyl hydrocarbons.

[0011] Cycloalkyls are saturated cyclic hydrocarbon residues. In some embodiments, all carbon atoms of the cycloalkyl are part of a ring. In some embodiments, some carbon atoms of the cycloalkyl are part of a ring, and some carbon atoms of the cycloalkyl form a linear or branched chain bonded to the ring. A cycloalkyl may contain one or more rings.

[0012] A heterocycloalkyl is a saturated cyclic hydrocarbon residue 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, some carbon atoms and heteroatoms of the heterocycle are part of the ring, and the carbon atoms and optionally heteroatoms that are part of the heterocycloalkyl form a linear or branched chain bonded to the ring. A heterocycloalkyl may contain one or more rings. Preferably, a heterocycloalkyl contains one to two heteroatoms. Examples of heterocycloalkyls include morpholinyl, piperazinyl, and morpholinyl ethyl.

[0013] Alkoxy groups are -O-alkyl groups. Examples of alkoxy groups include methoxy and ethoxy.

[0014] Aryls are aromatic cyclic hydrocarbons containing one or two rings. In particular, aryls are phenyl, naphthyl, and biphenyl, and preferably phenyl.

[0015] A heteroaryl 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 heteroatoms are preferably nitrogen atoms. Examples of heteroaryls include pyridyl, imidazolyl, and pyrazolopyrimidinyl.

[0016] The halogen is selected from the group consisting of fluorine, chlorine, bromine, and iodine.

[0017] In one embodiment, R 1 , R 2 , R 3 and R 4 Each of these is independently selected from the group comprising a hydroxyl group, a C1-C3 alkoxy group, a trifluoromethoxy group, and a difluoromethoxy group, preferably R 1 , R 2 , R 3 and R 4 Each of these is independently selected from the group containing a methoxy group and an ethoxy group.

[0018] In some embodiments, R 5 and R 6 Both are hydrogen atoms.

[0019] In some embodiments, R 5 R is a hydrogen atom, 6 R is an acyl group or thioacyl group of general formula II, or a sulfonic acid group of general formula III, 7 The group is selected from the following: [ka]

[0020] In some preferred embodiments, R 1 , R 2 , R 3 and R 4 R is a methoxy group or an ethoxy group, 5 R is a hydrogen atom, 6 R is an acyl group or thioacyl group of general formula II, or a sulfonic acid group of general formula III, 7 The alkyl group is C1-C6 alkyl, preferably methyl or ethyl.

[0021] In some embodiments, R 5 R is a hydrogen atom, 6 This is an acyl group or thioacyl group of general formula II.

[0022] In some embodiments, R 5 R is a hydrogen atom, 6 is a base of general formula II, and R 7 is R 8 and -NH-R 8 A selection is made from the group including R. 8 C1-C6 alkyl, C3-C8 cycloalkyl, piperazinyl, pyrrolidinyl, quinuclidinyl, phenyl, naphthyl, pyridyl, imidazolyl, thiazolyl, oxazolyl, pyrazolopyrimidinyl, (C6-C12)aryl-(C1-C3)alkyl-, (CH2CH2O) n -(C1~C3 alkyl), CH2O(CH2CH2O) n Selected from the group consisting of -(C1~C3 alkyl), 5~7 member heteroaryl-(C1~C3)alkyl-, and (C1~C3 alkyl)OC(O)-(C1~C3)alkyl-, n is 1, 2, 3, 4, or 5. Substituent R 8 The following are optional: C1-C3 alkyl, C1-C3 alkoxy, OH, halogen, =O, NH2, NHR 9 It is substituted with at least one substituent selected from the group consisting of R 9 This includes C1-C3 alkyl and NR 10 Selected from 2, R 10 Each of these is independently selected from C1-C3 alkyl groups, or both R 10 These combine to form C2-C5 alkylenes.

[0023] Preferably, R 7The group is selected from the group including methyl, ethyl, propyl, cyclopropyl, azepanil, morpholinil, piperazinyl, phenyl, naphthyl, pyridyl, imidazolyl, pyrrolidinyl, quinuclidinyl, 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)pyrrolidinyl, (methoxymethyl)pyrrolidinyl, isopropylamino, N-methylpiperazinyl, aminocarbonylmethoxyphenyl, hydroxypyridyl, methylpyridyl, 6-hydroxy-4-methylpyridine-3-yl, (morpholine-4-yl)ethyl, difluoropyridyl, (methoxyethoxy)ethoxymethyl, pyrazolo[1,5-a]pyrimidinyl, and piperidinylpyridine.

[0024] In one preferred embodiment, R 5 R is a hydrogen atom, 6 R is an acyl group or thioacyl group of general formula II, 7 The group is selected from the following: [ka]

[0025] In some embodiments, R 5 R is a hydrogen atom, 6 is a base of general formula II, where R 7 These are methyl, cyclopentyl, pyridine, or imidazole.

[0026] In some embodiments, R 5 R is a hydrogen atom, 6 This is a sulfonic acid group of general formula III.

[0027] In some embodiments, R 5 R is a hydrogen atom, 6 R is a sulfonic acid group of general formula III, 7The group is selected from methyl, ethyl, propyl, cyclopropyl, azepanyl, morpholinyl, piperazinyl, phenyl, pyridyl, imidazolyl, pyrrolidinyl, aminopropyl, aminoethyl, aminophenyl, diaminophenyl, N-methylimidazolyl, (fluoro)pyrrolidinyl, (methoxymethyl)pyrrolidinyl, isopropylamino, N-methylpiperazinyl, aminocarbonylmethoxyphenyl, hydroxypyridyl, methylpyridyl, 6-hydroxy-4-methylpyridine-3-yl, and (morpholin-4-yl)ethyl.

[0028] In one preferred embodiment, R 5 R is a hydrogen atom, 6 R is a sulfonic acid group of general formula III, 7 The group is selected from the following: [ka]

[0029] If a compound of general formula I has a positive charge (is in cation form), the compound contains a counterion. This counterion is a pharmaceutically acceptable anion of an organic or inorganic acid, thereby forming a pharmaceutically acceptable salt. Such anions may be selected from the group including, for example, acetate, aspartate, benzenesulfonate, benzoate, besilate, bicarbonate, hydrogen tartrate, bromide, camusylate, carbonate, chloride, citrate, decanoate, edetate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolate, hexanoate, iodide, lactate, malate, maleate, mandelate, mesylate, methanesulfonate, naphusylate, nitrate, octanoate, oleate, palmoate, pantothenate, phosphate, polygalacturonate, propionate, salicylate, stearate, succinate, sulfate, tartrate, tosylate, and trifluoroacetate.

[0030] If the compound of formula I contains a chiral center, then formula I includes a mixture of enantiomers, including a pure enantiomer and a racemic mixture.

[0031] Formula I includes compounds of Formula I in their free form, salt form, addition salt form (with an acid or base), and / or solvates including hydrates or alcohol solvates.

[0032] Another object of the present invention relates to compounds of general formula I for use as pharmaceuticals.

[0033] Compounds of general formula I are suitable for use in the treatment or prevention of proteinopathy, particularly neurodegenerative diseases such as polyglutamine diseases, tauopathies, synucleinopathies, or amyotrophic lateral sclerosis, as well as in the treatment or prevention of amyloidosis, cystic fibrosis, and diabetes. Compounds of general formula I are also suitable for the treatment of viral diseases. Here, it is assumed that the ability of compounds of general formula I to induce autophagy manifests itself as an enhancement of viral particle degradation, particularly upon viral entry. Furthermore, compounds of general formula I are suitable for the treatment of diseases directly related to elevated levels of ferroptosis, particularly stroke, rhabdomyolysis (severe skeletal muscle injury), non-alcoholic fatty liver disease, acute pancreatitis, or psoriasis.

[0034] Polyglutamine diseases produce proteins that accumulate in the nuclei of neurons, forming intracellular inclusions and causing neuronal death. All polyglutamine diseases are progressive and fatal, usually beginning in adulthood and progressing over a period of 10 to 30 years. For a review of polyglutamine diseases, see Journal of Molecular Cell Biology (2010), 2, 180-191. Polyglutamine diseases include Huntington's disease, spinal muscular atrophy, spinocerebellar ataxia, and dentateblocal pallidus atrophy.

[0035] Tauopathies and synucleinopathy arise from the presence of characteristic plaques. Synucleinopathy is a diverse group of neurodegenerative diseases that share common pathological lesions consisting of aggregates of the insoluble protein α-synuclein in nerve cells and glial cells. This group includes Parkinson's disease and Lewy body dementia. Fibrous inclusions of tau protein and degeneration of brain tissue in the absence of β-amyloid plaques are typical features of neurodegenerative tauopathies. Diseases belonging to this group include Pick's disease, progressive supranuclear palsy (PSP), Parkinson's disease (tau accumulation), Steele-Richardson-Olshevsky disease, Guam disease (Guam parkinsonism-dementia complex), post-traumatic parkinsonism, and Alzheimer's disease.

[0036] PSP is microscopically characterized by the presence of neurofibrillary tangles (NFTs) with specific properties. These are spherical (different from NFTs in Alzheimer's disease) and consist of linear filaments approximately 15 nm long. The NFTs are likely formed from an abnormal protein structure called tau protein. Clinically, PSP is characterized by a Parkinsonian syndrome with axial rigidity as the dominant symptom, postural instability with frequent falls, oculomotor dysfunction, cognitive impairment, and subcortical dementia, following a characteristic onset and course. Several other typical symptoms are also observed.

[0037] Post-traumatic Parkinsonian syndrome: Following World War II, the deaths of boxers revealed numerous and remarkably prominent signs of trauma in their brain tissue. Neurofibrillary tangles in cortical neurons were very common. However, most importantly, the discovery of pathological tau proteins in the neurons that were very similar to those found in PSP and other tauopathies suggested a more complex pathophysiology than that resulting from simple repetitive microtrauma (Neurol 2000; 20: 179-185).

[0038] Alzheimer's disease: In patients with Alzheimer's disease, excessive phosphorylation of tau protein and formation of neurofibrillary tangles occur in brain tissue, along with aggregation of β-amyloid, forming specific structures called "senile plaques" in the brain. The neuropathological features of AD include "positive lesions" such as amyloid plaques, cerebral amyloid angiopathy, and neurofibrillary tangles, and "negative lesions" such as loss of nerve cells and synapses. The type of AD in which Lewy bodies, a characteristic of idiopathic Parkinson's disease, are found in various brain structures, including the cortex, is called the "Lewy body type."

[0039] Amyloidosis includes systemic and organ-specific amyloidosis. These include familial amyloidosis without neurological impairment, familial neuropathic amyloidosis, familial nervous system amyloidosis, secondary systemic amyloidosis, and organ-specific amyloidosis.

[0040] The object of the present invention is also to provide compounds of general formula I used for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), Kennedy disease (KD), Huntington's disease (HD), Creutzfeldt-Jakob disease (CJD), spinocerebellar degeneration (SCA), dentatorubral-pallidolar atrophy, transthyretin familial amyloid polyneuropathy, as well as systemic amyloidosis or organ-limited amyloidosis, and / or cystic fibrosis or diabetes.

[0041] In another aspect, the present invention provides compounds of general formula I for use in the prevention of genetically based forms of hereditary neurodegenerative diseases such as familial amyotrophic lateral sclerosis (ALS), familial Parkinson's disease (PD), familial Alzheimer's disease (AD), Kennedy disease (KD), Huntington's disease (HD), familial Creutzfeldt-Jakob disease (CJD), familial spinocerebellar degeneration (SCA), transthyretin familial amyloid polyneuropathy, familial dentatorubral-pallidoid atrophy, familial systemic amyloidosis or familial organ-specific amyloidosis, and / or cystic fibrosis.

[0042] Furthermore, compounds of general formula I are suitable for the treatment of viral diseases. Viral diseases include diseases caused by the HBV virus, particularly hepatitis B.

[0043] Furthermore, compounds of general formula I are suitable for treating diseases directly related to elevated levels of ferroptosis. In stroke, the serine protease thrombin is involved in ferroptosis by facilitating the recruitment of arachidonic acid through esterification of arachidonic acid by acyl-CoA synthase (ACSL4). Inhibition of this pathway therefore reduces ischemic neuronal damage in stroke (Tuo et al.: Signal Transduct Target Ther. 2022;7(1):59). Rhabdomyolysis is a relatively common syndrome of severe skeletal muscle injury. Ferroptosis may exacerbate the development of rhabdomyolysis. Increased expression of GPX4 in combination with ACSL4 in muscle cells can reduce lipid peroxidation and inhibit the progression of this syndrome (Yuan et al.: 2016.03.016). In non-alcoholic steatohepatitis, RSL-3 significantly increases levels of hepatitis-associated inflammatory factors. Inhibition of ferroptosis is a novel approach in the treatment of this disease (Qi et al.: Am J Pathol. 2020;190(1):68-81). Ferroptosis is also associated with pancreatic disease: administration of the ferroptosis inhibitor riproxstatin-1 reduces acute pancreatitis ((Li et al.: Chin Med J (Engl). 2022;135(17):2026-2034). Ferroptosis is also associated with psoriasis. The ferroptosis inhibitor ferrostatin-1 suppressed ferroptosis-related changes in keratinocytes treated with elastin (a ferroptosis inducer) and improved psoriasis-like dermatitis in an imiquimod-induced model (Shou et al.: Cell Death Dis. 2021;12(11):1009).

[0044] In another aspect, the object of the present invention is to provide compounds of general formula I for use in the treatment of diseases directly related to elevated levels of ferroptosis, preferably selected from the group consisting of stroke, rhabdomyolysis, non-alcoholic steatohepatitis, acute pancreatitis, and psoriasis.

[0045] In one embodiment, a compound of general formula I, where R 5 R is a hydrogen atom, 6 The sulfonic acid group of general formula III as defined above is used to treat diseases directly related to elevated levels of ferroptosis, preferably selected from the group consisting of stroke, rhabdomyolysis, non-alcoholic steatohepatitis, acute pancreatitis, and psoriasis.

[0046] Compounds of general formula I may be administered to animals or humans to activate cellular responses to proteotoxic stress caused by the presence or increase of metastable proteins within cells and by imbalances in protein homeostasis (e.g., due to aging).

[0047] Compounds of general formula I may be administered to animals or humans to activate cellular responses to elevated levels of ferroptosis in cells or tissues, for example, due to degenerative diseases, stroke, severe skeletal muscle injury, non-alcoholic fatty liver disease, acute pancreatitis, psoriasis, etc.

[0048] Compounds of general formula I can be formulated into pharmaceutical preparations with pharmaceutically acceptable excipients. These preparations may be in liquid, solid, or other forms such as aerosols. Liquid forms include, for example, solutions, suspensions, dispersions, emulsions, gels, and ointments formulated for injection or oral administration. Solid forms include, for example, capsules, tablets, coated tablets, powders, suppositories, and other forms well known to those skilled in the art.

[0049] Pharmaceutically acceptable excipients include solvents, solubility regulators, pH regulators, carriers, fillers, binders, lubricants, disintegrants, preservatives, adsorbents, viscosity regulators, and agents that affect the sensory properties of the product, such as taste, odor, and color.

[0050] In one embodiment, the present invention relates to a method for treating a disease directly related to an increase in ferroptosis levels, preferably a disease selected from the group including stroke, rhabdomyolysis, non-alcoholic steatohepatitis, acute pancreatitis, and psoriasis, and comprises the step of administering a pharmaceutical product comprising at least one compound of the general formula I as defined above to a subject in need of such treatment.

[0051] In one embodiment, the present invention relates to a method for treating or preventing neurodegenerative diseases such as proteinopathy, particularly polyglutamine diseases (Huntington's disease, spinocerbular muscular atrophy, spinocerebellar ataxia, dentatorubral-pallidal atrophy, etc.), tauopathy (Pick's disease, progressive supranuclear palsy (PSP), Parkinson's disease (tau body accumulation), Steele-Richardson-Olshevsky disease, Guam disease (Guam Parkinson's disease-dementia complex), post-traumatic parkinsonian syndrome, Alzheimer's disease), synucleinopathy (Parkinson's disease and Lewy body-associated dementia, etc.), or amyotrophic lateral sclerosis, comprising the step of administering a pharmaceutical product containing at least one compound of general formula I as defined above to a subject in need of such treatment.

[0052] In one embodiment, the present invention relates to a method for treating or preventing amyloidosis (such as familial amyloidosis without neurological impairment, familial neuropathic amyloidosis, familial nervous system amyloidosis, secondary systemic amyloidosis, organ-limited amyloidosis, etc.), cystic fibrosis, and diabetes mellitus, comprising the step of administering a pharmaceutical product comprising at least one compound of general formula I as defined above to a subject in need of such treatment.

[0053] In one embodiment, the present invention relates to a method for treating a viral disease (such as a disease caused by the HBV virus), and includes the step of administering a pharmaceutical product comprising at least one compound of general formula I as defined above to a subject in need of such treatment.

[0054] [Examples] [Table 1-1] [Table 1-2]

[0055] Example 1: Preparation of the compound All reactions were carried out in dry solvent under argon. Reverse-phase chromatography was performed using a Teledyne ISCO Combi Flash Rf+ system equipped with a RediSep Rf Gold C18 reverse-phase column. All starting materials were purchased directly from Sigma Aldrich, Combi-Blocks, and Fluorochem. The purity of the compounds and the composition of the reaction mixtures were tested using a Waters UPLC-MS Acquity with a QDa mass detector (flow rate 0.5 mL / min, gradient 0-100% MeCN / H2O (0.1% formic acid) over 7 minutes), using a 130 Å, 1.7 μm, 2.1 mm × 100 mm ACQUITY UPLC BEH C18 column with a 2.1 mm × 5 mm pre-column. High-resolution ESI mass spectra were recorded using a Thermo Scientific LTQ Orbitrap XL (Thermo Fisher Scientific) controlled with MassLynx software. The NMR spectra were recorded using a Bruker Avance III (trademark) HD ​​400MHz Prodigy NMR spectrometer.

[0056] List of compounds tert-butyl(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate [ka] (4-oxocyclohexyl)carbamate tert-butyl (2 g, 9.38 mmol) was dissolved in EtOH (10 mL), and 20% NaOH aqueous solution (9.3 mL) was added dropwise. After stirring the solution for 5 minutes, 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, 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.

[0057] 1 H NMR (401 MHz, DMSO-d6) 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).

[0058] HRMS(ESI+): m / z calculation value (C 29 H 35 O7NNa) = 532.2306; Measured value = 523.2302 [M+Na] + .

[0059] 2,6-Bis(3,4-dimethoxybenzylidene)-4-(acetamide)cyclohexanone(1) [ka] 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. After stirring the solution for 5 minutes, 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 siRNA (2 × 10 mL). The organic layers were combined and washed with saturated brine and water. The solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (eluent: siRNA:MeOH = 10:1), followed by recrystallization from heated MeOH. The reaction yielded 0.320 g of the title compound in 14% yield.

[0060] 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).

[0061] HRMS(ESI+): m / z calculation value (C 26 H 30 O6N) = 452.2073; Measured value 452.2077 [M+H] + .

[0062] 3,5-Bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (2) [ka] (3,5-bis(E-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (1 g, 1.96 mmol) was dissolved in DCM (5 mL), cooled in an ice bath, and then TFA (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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.867 g of the title compound in 84% yield.

[0063] 1 H NMR (401 MHz, DMSO-d6) 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).

[0064] HRMS(ESI+): m / z calculated value (C 24 H 28 O5N) = 410.1962; Measured value = 410.1961 [M+H] + .

[0065] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)amino)-N,N-dimethyl-2-oxoethane-1-aminium trifluoroacetate (3) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) in DMF (3 mL) was added with HBTU (0.135 g, 0.35 mmol) and 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction gave 0.053 g of the title compound in 46% yield.

[0066] 1 1H 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 = 8.3 Hz, 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).

[0067] HRMS(ESI+): calculated for m / z (C 28 H 35 O6N2)=495.2490; found = 495.2487 "M+H" + .

[0068] 3-((3,5-Bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)quinuclidin-1-ium trifluoroacetate (4) [Chemical formula] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.11 mL, 0.79 mmol) in DMF (3 mL) was added with HBTU (0.109 g, 0.29 mmol) and 3-carboxyquinuclidin-1-ium hydrochloride (0.055 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction gave 0.071 g of the title compound in 56% yield.

[0069] 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).

[0070] HRMS(ESI+): m / z calculated for (C 32 H 39 O6N2)=547.2803; found = 547.2801 “M+H” + .

[0071] N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)-2-(2-(2-methoxyethoxy)ethoxy)acetamide (5) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and 2-(2-(2-methoxyethoxy)ethoxy)acetic acid (0.051 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 reverse-phase flash chromatography (eluent: H2O:ACN, gradient elution). The reaction yielded 0.080 g of the title compound in 74% yield.

[0072] 1 H NMR (401 MHz, DMSO-d6) 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).

[0073] HRMS(ESI+): m / z calculation value (C 31 H 39 O9NNa) = 592.2517; Measured value = 592.2513 [M+Na] + .

[0074] N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)cyclopentanecarboxamide(6) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) in DMF (3 mL) was to which HBTU (0.109 g, 0.29 mmol) and cyclopentanecarboxylic acid (0.033 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 reverse-phase flash chromatography (eluent: H2O:ACN, gradient elution). The reaction yielded 0.085 g of the title compound in 88% yield.

[0075] 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).

[0076] HRMS(ESI+): m / z calculation value (C 30 H 35 O6NNa) = 528.2357; Measured value = 528.2353 [M+Na] + .

[0077] 1-(2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)amino)-2-oxoethyl)-4-methylpiperazine-1,4-dium di(trifluoroacetate) salt (7) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and 2-(4-methylpiperazin-1-yl)acetic acid (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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.102 g of the title compound in 67% yield.

[0078] 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).

[0079] HRMS(ESI+): m / z calculation value (C 31 H 40 O6N3) = 550.2912; Measured value = 550.2910 [M+H] + .

[0080] 4-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)amino)-N,N-dimethyl-4-oxobutane-1-aminium trifluoroacetate (8) [ka] To a mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.11 mL, 0.79 mmol) in DMF (3 mL), HBTU (0.109 g, 0.29 mmol) and 3-carboxy-N,N-dimethylpropane-1-aminium hydrochloride (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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.065 g of the title compound in 53% yield.

[0081] 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).

[0082] HRMS(ESI+): m / z calculation value (C 30 H 39 O6N2) = 523.2803; Measured value = 523.2800 [M+H] + .

[0083] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)amino)-2-oxoethane-1-aminium trifluoroacetate (9) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and (tert-butoxycarbonyl)glycine (0.050 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The obtained solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and then 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.080 g of the title compound in 73% yield.

[0084] 1 H NMR (401 MHz, DMSO-d6) 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).

[0085] HRMS(ESI+): m / z calculation value (C 26 H 31 O6N2) = 467.2177; Measured value = 467.2175 [M+H] + .

[0086] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)-3-methylpyridine-1-iumtrifluoroacetate (10) [ka] To a mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) in DMF (3 mL), HBTU (0.109 g, 0.29 mmol) and 3-methyl picolinic acid (0.039 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.098 g of the title compound in 80% yield.

[0087] 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).

[0088] HRMS(ESI+): m / z calculation value (C 31 H 32 O6N2Na) = 551.2153; Measured value = 551.2151 [M+Na] + .

[0089] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (11) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and picolinic acid (0.035 g, 0.29 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.099 g of the title compound in 82% yield.

[0090] 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).

[0091] HRMS(ESI+): m / z calculation value (C 30 H 30 O6N2Na) = 537.1996; measured value = 537.1994 [M+Na] + .

[0092] 4-(2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)amino)-2-oxoethyl)-1H-imidazole-3-iumtrifluoroacetate (12) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.12 mL, 0.86 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and 2-(1H-imidazole-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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.079 g of the title compound in 66% yield.

[0093] 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).

[0094] HRMS(ESI+): m / z calculation value (C 29 H 32 O6N3) = 518.2286; Measured value = 518.2284 [M+H] + .

[0095] (S)-2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyrrolidine-1-iumtrifluoroacetate (13) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and (tert-butoxycarbonyl)-L-proline (0.062 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The obtained solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and then 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.068 g of the title compound in 58% yield.

[0096] 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).

[0097] HRMS(ESI+): m / z calculation value (C 29 H 32 O6N3) = 507.2490; Measured value = 507.2488 [M+H] + .

[0098] (S)-N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)-5-oxopyrrolidine-2-carboxamide(14) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and (S)-5-oxopyrrolidine-2-carboxylic acid (0.037 g, 0.29 mmol). The reaction mixture was stirred at ambient temperature for 2.5 hours. The solvent was evaporated under reduced pressure, and the residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.084 g of the title compound in 85% yield.

[0099] 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).

[0100] HRMS(ESI+): m / z calculation value (C 29 H 32 O7N2Na) = 543.2102; Measured value = 543.2100 [M+Na] + .

[0101] 1-(2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)amino)-2-oxoethyl)-1H-imidazole-1-iumtrifluoroacetate (15) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and 2-(1H-imidazole-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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.097 g of the title compound in 80% yield.

[0102] 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).

[0103] HRMS(ESI+): m / z calculation value (C 29 H 32 O6N3) = 518.2286; Measured value = 518.2284 [M+H] + .

[0104] 4-(2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)amino)-2-oxoethyl)morpholine-4-iumtrifluoroacetate (16) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.12 mL, 0.86 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and 2-morpholinoacetic acid (0.052 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.095 g of the title compound in 76% yield.

[0105] 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 = 8.4 Hz, 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).

[0106] HRMS(ESI+): m / z calculation value (C 30 H 37 O7N2) = 537.2595; Measured value = 537.2593 [M+H] + .

[0107] 4-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)amino)-4-oxobutane-1-aminium trifluoroacetate (17) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and 4-((tert-butoxycarbonyl)amino)butanoic acid (0.058 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The obtained solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and then 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.049 g of the title compound in 43% yield.

[0108] 1 H NMR (401 MHz, DMSO-d6) 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).

[0109] HRMS(ESI+): m / z calculation value (C 28 H 35 O6N2) = 495.2490; Measured value = 495.2487 [M+H] + .

[0110] 3-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)amino)-N,N-dimethyl-3-oxopropane-1-aminium trifluoroacetate (18) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.12 mL, 0.86 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and 2-carboxy-N,N-dimethylethane-1-aminium 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.093 g of the title compound in 78% yield.

[0111] 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).

[0112] HRMS(ESI+): m / z calculation value (C 29 H 37 O6N2) = 509.2646; Measured value = 509.2643 [M+H] + .

[0113] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)amino)-N,N-diethyl-2-oxoethane-1-aminium trifluoroacetate (19) [ka] To a mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.12 mL, 0.86 mmol) in DMF (3 mL), HBTU (0.109 g, 0.29 mmol) and N-(carboxymethyl)-N-ethylethananium hydrochloride (0.048 g, 0.29 mmol) were added. The reaction mixture was stirred at ambient temperature for 2 hours. The solvent was evaporated under reduced pressure, and the residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.096 g of the title compound in 79% yield.

[0114] 1H 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).

[0115] HRMS(ESI+): m / z calculation value (C 30 H 39 O6N2) = 523.2803; Measured value = 523.2799 [M+H] + .

[0116] 3-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)amino)-3-oxopropane-1-aminium trifluoroacetate (20) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.1 mL, 0.72 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and 3-((tert-butoxycarbonyl)amino)propanoic acid (0.058 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The obtained solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and then 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.059 g of the title compound in 52% yield.

[0117] 1 H NMR (401 MHz, DMSO-d6) 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).

[0118] HRMS(ESI+): m / z calculation value (C 27 H 33 O6N2) = 481.2333; Measured value = 481.2333 [M+H] + .

[0119] 1-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)-3-(pyridine-3-yl)thiourea(21) [Chemistry] To a mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexan-1-aminium trifluoroacetate (0.094 g, 0.18 mmol) and Et3N (0.04 mL, 0.27 mmol) in THF (3.5 mL), 3-isothiocyanatopyridine (0.027 g, 0.20 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 gave 0.070 g of the title compound in 72% yield.

[0120] 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).

[0121] HRMS(ESI+): m / z calculated for (C 30 H 31 O5N3SNa) = 568.1877; found = 568.1875 "M+Na" + [[ID=2"1]].

[0122] ((3,5-Bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)glycine ethyl (22) [Chemistry] To a mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.094 g, 0.18 mmol) and Et3N (0.04 mL, 0.27 mmol) in THF (3.5 mL), 2-isocyanatoethyl acetate (0.025 g, 0.20 mmol) was added, and the reaction mixture was stirred overnight. The solvent was evaporated, and the solid residue was recrystallized from heated MeOH. The reaction yielded 0.062 g of the title compound in 65% yield.

[0123] 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).

[0124] HRMS(ESI+): m / z calculation value (C 29 H 34 O8N2Na) = 561.2207; Measured value = 561.2207 [M+Na] + .

[0125] 3-(3-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)ureido)ethyl propanoate (23) [ka] To a mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.094 g, 0.18 mmol) and Et3N (0.04 mL, 0.27 mmol) in THF (3.5 mL), ethyl 3-isocyanatopropanoate (0.028 g, 0.20 mmol) was added, and the reaction mixture was stirred overnight. The solvent was evaporated, and the solid residue was recrystallized from heated MeOH. The reaction yielded 0.066 g of the title compound in 67% yield.

[0126] 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).

[0127] HRMS(ESI+): m / z calculation value (C 30 H 36 O8N2Na) = 575.2364; Measured value = 575.2361 [M+Na] + .

[0128] 1-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)-3-(3-methoxyphenyl)urea(24) [ka] To a mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.094 g, 0.18 mmol) and Et3N (0.04 mL, 0.27 mmol) in THF (3.5 mL), 1-isocyanato-3-methoxybenzene (0.029 g, 0.20 mmol) was added, and the reaction mixture was stirred overnight. The solvent was evaporated, and the solid residue was recrystallized from heated MeOH. The reaction yielded 0.079 g of the title compound in 79% yield.

[0129] 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).

[0130] HRMS(ESI+): m / z calculation value (C 32 H 34 O7N2Na) = 581.2258; Measured value = 581.2257 [M+Na] + .

[0131] 1-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)-3-(2-morpholinoethyl)thiourea(25) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.094 g, 0.18 mmol) and Et3N (0.04 mL, 0.27 mmol) in THF (3.5 mL) was added to 4-(2-isothiocyanatoethyl)morpholine (0.034 g, 0.20 mmol), and the reaction mixture was stirred overnight. The solvent was evaporated and the solid residue was recrystallized from hot MeOH. The reaction gave 0.080 g of the title compound in 77% yield.

[0132] 1 1H 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).

[0133] HRMS(ESI+): calculated for m / z (C 31 1H 39 6H3N3SNa)=604.2452; found = 604.2451 "M+Na" + .

[0134] 3-((3,5-Bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridin-1-ium trifluoroacetate (26)

Chemical Structure

[0135] 1 H NMR (401 MHz, DMSO-d6) 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).

[0136] HRMS(ESI+): m / z calculation value (C 30 H 30 O6N2Na) = 537.1996; measured value = 537.1992 [M+Na] + .

[0137] 3-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)-N,N-dimethylbenzeneaminium trifluoroacetate (27) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 a further 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 this mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred at ambient temperature for a further 2 hours. Evaporate the solvent and perform reverse-phase flash chromatography on the solid residue (eluent). : The compound was purified by H2O (0.1% TFA):ACN, gradient elution. The reaction yielded 0.231 g of the title compound in 88% yield.

[0138] 1 H NMR (401 MHz, DMSO-d6) 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).

[0139] HRMS(ESI+): m / z calculation value (C 33 H 36 O6N2Na) = 579.2466; Measured value = 579.2462 [M+Na] + .

[0140] 5-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)-2-(piperidine-1-ium-1-yl)pyridine-1-iumtrifluoroacetate (28) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 a further 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 6-(piperidine-1-yl)nicotinic acid (0.121 g, 0.59 mmol) was added to this mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred at ambient temperature for a further 1.5 hours. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.168 g of the title compound in 52% yield.

[0141] 1H NMR (401 MHz, DMSO-d6) 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).

[0142] HRMS(ESI+): m / z calculation value (C 35 H 40 O6N3) = 598.2912; Measured value = 598.2908 [M+H] + .

[0143] 4-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (29) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 a further 30 minutes. Isonicotinol 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 at ambient temperature for a further 2 hours. The reaction mixture was diluted with water (15 mL) and extracted with DCM (3 × 15 mL). The organic layers were combined, washed with saturated brine, dried over Na₂SO₄, and filtered. Evaporate the solvent and perform reverse-phase flash chromatography on the solid residue (eluent).: The solution was purified by H2O (0.1% TFA):ACN, gradient elution. The reaction yielded 0.142 g of the title compound in 58% yield.

[0144] 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).

[0145] HRMS(ESI+): m / z calculation value (C 30 H 30 O6N2Na) = 537.1996; measured value = 537.1992 [M+Na] + .

[0146] 4-(2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)amino)-2-oxoethoxy)pyridine-1-iumtrifluoroacetate (30) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 a further 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 2-(pyridine-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 at ambient temperature for a further 2 hours. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.089 g of the title compound in 34% yield.

[0147] 1 H NMR (401 MHz, DMSO-d6) 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).

[0148] HRMS(ESI+): m / z calculation value (C 31 H 33 O7N2) = 545.2282; Measured value = 545.2277 [M+H] + .

[0149] 3-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)benzeneaminium trifluoroacetate (31) [ka] A solution of tert-butyl (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 a further 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 at ambient temperature for a further 2 hours. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The obtained solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and then 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.124 g of the title compound in 49% yield.

[0150] 1H NMR (401 MHz, DMSO-d6) 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).

[0151] HRMS(ESI+): m / z calculation value (C 31 H 32 O6N2Na) = 551.2153; Measured value = 551.2150 [M+Na] + .

[0152] 4-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)benzeneaminium trifluoroacetate (32) [ka] A solution of tert-butyl (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 a further 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 at ambient temperature for a further 2 hours. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The obtained solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and then 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.083 g of the title compound in 33% yield.

[0153] 1 H NMR (401 MHz, DMSO-d6) 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).

[0154] HRMS(ESI+): m / z calculation value (C 31 H 32 O6N2Na) = 551.2153; Measured value = 551.2149 [M+Na]+ .

[0155] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)benzeneaminium trifluoroacetate (33) [ka] A solution of tert-butyl (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 a further 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 at ambient temperature for a further 2 hours. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The obtained solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and then 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.093 g of the title compound in 37% yield.

[0156] 1H NMR (401 MHz, DMSO-d6) 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).

[0157] HRMS(ESI+): m / z calculation value (C 31 H 32 O6N2Na) = 551.2153; Measured value = 551.2149 [M+Na] + .

[0158] 2-((3,5-bis((E)-4-ethoxy-3-methoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (34) [ka] 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (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 then 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. The resulting filtered cake was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.067 g of the title compound in 34% yield.

[0159] 1H NMR (401 MHz, DMSO-d6) 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).

[0160] HRMS(ESI+): m / z calculation value (C 32 H 34 O6N2Na) = 565.2309; Measured value = 565.2305 [M+Na] + .

[0161] 2-((3,5-bis((E)-3-ethoxy-4-methoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (35) [ka] A mixture of 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (0.100 g, 0.30 mmol) and 3-ethoxy-4-methoxybenzaldehyde (0.108 g, 0.60 mmol) in glacial acetic acid (1 mL) was mixed with a 4 M hydrochloric acid dioxane solution (0.5 mL), and the reaction mixture was stirred overnight. The solvent was evaporated under reduced pressure, and the residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.080 g of the title compound in 41% yield.

[0162] 1H NMR (401 MHz, DMSO-d6) 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).

[0163] HRMS(ESI+): m / z calculation value (C 32 H 34 O6N2Na) = 565.2309; Measured value = 565.2306 [M+Na] + .

[0164] 2-((3,5-bis((E)-4-hydroxy-3-methoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (36) [ka] 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (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 then 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.076 g of the title compound in 42% yield.

[0165] 1H NMR (401 MHz, DMSO-d6) 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).

[0166] HRMS(ESI+): m / z calculation value (C 28 H 26 O6N2Na) = 509.1683; Measured value = 509.1680 [M+Na] + .

[0167] 2-((3,5-bis((E)-4-methoxy-3-(trifluoromethoxy)benzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (37) [ka] A mixture of 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (0.100 g, 0.30 mmol) and 4-methoxy-3-(trifluoromethoxy)benzaldehyde (0.166 g, 0.75 mmol) in glacial acetic acid (1 mL) was mixed with a 4M hydrochloric acid dioxane solution (0.15 mL), and the reaction mixture was stirred overnight. The solvent was evaporated under reduced pressure, and the residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.147 g of the title compound in 66% yield.

[0168] 1H NMR (401 MHz, DMSO-d6) 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).

[0169] HRMS(ESI+): m / z calculation value (C 30 H 24 O6N2F6Na) = 645.1431; Measured value = 645.1428 [M+Na] + .

[0170] 2-((3,5-bis((E)-4-hydroxy-3-(trifluoromethoxy)benzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (38) [ka] A mixture of 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (0.100 g, 0.30 mmol) and 4-hydroxy-3-(trifluoromethoxy)benzaldehyde (0.155 g, 0.75 mmol) in glacial acetic acid (1 mL) was mixed with a 4 M hydrochloric acid dioxane solution (1.5 mL), and the reaction mixture was stirred overnight. The solvent was evaporated under reduced pressure, and the residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.145 g of the title compound in 68% yield.

[0171] 1H NMR (401 MHz, DMSO-d6) 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).

[0172] HRMS(ESI+): m / z calculation value (C 28 H 20 O6N2F6Na) = 617.1118; Measured value = 617.1114 [M+Na] + .

[0173] 2-((3,5-bis((E)-4-(difluoromethoxy)-3-hydroxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (39) [ka] A mixture of 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (0.100 g, 0.30 mmol) and 4-(difluoromethoxy)-3-hydroxybenzaldehyde (0.142 g, 0.75 mmol) in glacial acetic acid (1 mL) was mixed with a 4 M hydrochloric acid dioxane solution (0.15 mL), and the reaction mixture was stirred overnight. The solvent was evaporated under reduced pressure, and the residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.136 g of the title compound in 67% yield.

[0174] 1H NMR (401 MHz, DMSO-d6) 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).

[0175] HRMS(ESI+): m / z calculation value (C 28 H 22 O6N2F4Na) = 581.1306; Measured value = 581.1309 [M+Na] + .

[0176] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)-3,5-difluoropyridine-1-iumtrifluoroacetate (40) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 hours. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for a further 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 3,5-difluoropicolinic 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 at ambient temperature for a further 1.5 hours. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.127 g of the title compound in 49% yield.

[0177] 1 H NMR (401 MHz, DMSO-d6) δ 8.81 (d, J = 7.3 Hz, 1H), 8.57 (d, J = 2.3 Hz, 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).

[0178] HRMS(ESI+): m / z calculation value (C 30 H 28 O6N2F2Na) = 573.1808; Measured value = 573.1805 [M+Na] + .

[0179] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)-6-hydroxypyridine-1-iumtrifluoroacetate (41) [ka] A mixture of 3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexane-1-aminium trifluoroacetate (0.100 g, 0.19 mmol) and Et3N (0.12 mL, 0.86 mmol) in DMF (3 mL) was mixed with HBTU (0.109 g, 0.29 mmol) and 6-hydroxypicolinic acid (0.040 g, 0.29 mmol). The reaction mixture was stirred at ambient temperature for 1.5 hours. The solvent was evaporated under reduced pressure, and the residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.051 g of the title compound in 41% yield.

[0180] 1 H NMR (401 MHz, DMSO-d6) 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).

[0181] HRMS(ESI+): m / z calculation value (C 30 H 30 O7N2Na) = 553.1945; Measured value = 553.1948 [M+Na] + .

[0182] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)-1H-imidazole-3-iumtrifluoroacetate (42) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 hours. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for a further 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 1H-imidazole-2-carboxylic acid (0.066 g, 0.59 mmol) was added to this mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred at ambient temperature for a further 1.5 hours. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.122 g of the title compound in 50% yield.

[0183] 1 H NMR (401 MHz, DMSO-d6) 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).

[0184] HRMS(ESI+): m / z calculation value (C 28 H 29 O6N3Na) = 526.1949; Measured value = 526.1945 [M+Na] + .

[0185] N-(3-((E)-3-methoxybenzylidene)-5-((E)-4-methoxybenzylidene)-4-oxocyclohexyl)picolinamide hydrochloride (43) [ka] A mixture of 2-((4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate (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) was mixed with 4M dioxane hydrochloride solution (0.45 mL), 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 (eluent: cHex: siRNA, 2:1). The reaction yielded 0.028 g of the title compound in 10% yield.

[0186] 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).

[0187] HRMS(ESI+): m / z calculated value (C 28 H 26O4N2Na) = 477.1785; Measured value = 477.1782 [M+Na] + .

[0188] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-ium sulfate (44) [ka] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate was dissolved in aqueous ammonia, and the resulting free base was extracted with DCM:iPrOH (1:10). The organic layers 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 and dried. The reaction yielded 0.143 g (0.23 mmol) of the title compound.

[0189] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-ium hydrochloride (45) [ka] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate was dissolved in aqueous ammonia, and the resulting free base was extracted with DCM:iPrOH (1:10). The organic layers 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 and dried. The reaction yielded 0.130 g (0.24 mmol) of the title compound.

[0190] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-ium hydrobromide (46) [ka] 2-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyridine-1-iumtrifluoroacetate was dissolved in aqueous ammonia, and the resulting free base was extracted with DCM:iPrOH (1:10). The organic layers 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 and dried. The reaction yielded 0.140 g (0.24 mmol) of the title compound.

[0191] 4-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)thiazole-3-iumtrifluoroacetate (47) [ka] A solution of tert-butyl (3,5-bis(E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 hours. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for a further 30 minutes. 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 this mixture, followed by DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred at ambient temperature for a further 1.5 hours. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.143 g of the title compound in 58% yield.

[0192] 1 H NMR (401 MHz, DMSO-d6) 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).

[0193] HRMS(ESI+): m / z calculated value (C 28 H 28 O6N2NaS) = 543.1560; Measured value = 543.1565 [M+Na] + .

[0194] N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)-2-methyloxazole-4-carboxamide(48) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 hours. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for a further 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). 2-methyloxazole-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 at ambient temperature for a further 2 hours. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.092 g of the title compound in 45% yield.

[0195] 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).

[0196] HRMS(ESI+): m / z calculation value (C 29 H 30 O7N2Na) = 541.1945; Measured value = 541.1946 [M+Na] + .

[0197] 3-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamoyl)pyrrolidine-1-iumtrifluoroacetate (49) [ka] A solution of tert-butyl (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 a further 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 this mixture, followed by the addition of DIPEA (0.21 mL, 1.18 mmol) and HBTU (0.223 g, 0.59 mmol). The reaction mixture was stirred at ambient temperature for a further 1.5 hours. The solvent was evaporated under reduced pressure, and the solid residue was purified by flash chromatography (eluent: cHex: siRNA, gradient elution 40-85%). The obtained solid was dissolved in DCM (0.5 mL), cooled in an ice bath, and then 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 reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.123 g of the title compound in 65% yield.

[0198] 1H 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).

[0199] HRMS(ESI+): m / z calculation value (C 29 H 35 O6N2) = 507.2490; Measured value = 507.2488 [M+H] + .

[0200] N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide(50) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 hours. DIPEA (0.14 mL, 0.79 mmol) was added dropwise, and stirring was continued at ambient temperature for a further 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in DMF (5 mL). Pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (0.096 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 at ambient temperature for a further 2 hours. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.108 g of the title compound in 50% yield.

[0201] 1 H NMR (401 MHz, DMSO-d6) 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).

[0202] HRMS(ESI+): m / z calculation value (C 31 H 30 O6N4Na) = 577.2058; Measured value = 577.2062 [M+Na] + .

[0203] N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)cyclopropanesulfonamide(51) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 hours. DIPEA (0.21 mL, 1.18 mmol) was added dropwise, and stirring was continued at ambient temperature for a further 30 minutes. The reaction mixture was cooled again in an ice bath, cyclopropanesulfonyl chloride (0.055 g, 0.39 mmol) was added, followed by Et3N (0.06 mL, 0.39 mmol). The solution was stirred at ambient temperature overnight. The reaction was quenched with water and extracted with DCM. The organic layers were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.110 g of the title compound in 55% yield.

[0204] 1 H NMR (401 MHz, DMSO-d6) 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).

[0205] HRMS(ESI+): m / z calculation value (C 27 H31 O7NNaS) = 536.1713; Measured value = 536.1711 [M+Na] + .

[0206] N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)ethanesulfonamide(52) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 hours. DIPEA (0.21 mL, 1.18 mmol) was added dropwise, and stirring was continued at ambient temperature for a further 30 minutes. 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 at ambient temperature overnight. The reaction was quenched with water and extracted with DCM. The organic layers were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.063 g of the title compound in 32% yield.

[0207] 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).

[0208] HRMS(ESI+): m / z calculation value (C 26 H 31 O7NNaS) = 524.1713; Measured value = 524.1711 [M+Na] + .

[0209] 4-(N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)sulfamoyl)-1-methylpiperazine-1-trifluoroacetate (53) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 at ambient temperature for a further 30 minutes. 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 at ambient temperature overnight. The reaction was quenched with water and extracted with DCM. The organic layers were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.175 g of the title compound in 65% yield.

[0210] 1H NMR (401 MHz, DMSO-d6) 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).

[0211] HRMS(ESI+): m / z calculated value (C 29 H 38 O7N3S) = 572.2425; Measured value = 572.2422 [M+H] + .

[0212] N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)morpholine-4-sulfonamide(54) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 at ambient temperature for a further 30 minutes. 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 at ambient temperature overnight. The reaction was quenched with water and extracted with DCM. The organic layers were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.076 g of the title compound in 35% yield.

[0213] 1 H NMR (401 MHz, DMSO-d6) 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).

[0214] HRMS(ESI+): m / z calculated value (C 28 H 34 O8N2NaS) = 581.1928; Measured value = 581.1927 [M+Na] + .

[0215] N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)-1-methyl-1H-imidazole-4-sulfonamide(55) [ka] A solution of tert-butyl (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 at ambient temperature for a further 30 minutes. The reaction mixture was cooled again in an ice bath, and 1-methyl-1H-imidazole-4-sulfonyl chloride (0.250 g, 1.38 mmol) was added. The solution was stirred at ambient temperature for a further 1.5 hours. The reaction was quenched with water and extracted with DCM. The organic layers were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.140 g of the title compound in 64% yield.

[0216] 1 H NMR (401 MHz, DMSO-d6) 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).

[0217] HRMS(ESI+): m / z calculated value (C 28 H 31 O7N3NaS) = 576.1775; Measured value = 576.1771 [M+Na] + .

[0218] N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)-2-(methoxymethyl)pyrrolidine-1-sulfonamide(56) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 at ambient temperature for a further 30 minutes. The reaction mixture was cooled again in an ice bath, and 2-(methoxymethyl)pyrrolidine-1-sulfonyl chloride (0.250 g, 1.17 mmol) was added. The solution was stirred at ambient temperature overnight. The reaction was quenched with water and extracted with DCM. The organic layers were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.169 g of the title compound in 73% yield.

[0219] 1 H NMR (401 MHz, DMSO-d6) 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).

[0220] HRMS(ESI+): m / z calculation value (C 30 H 38 O8N2NaS) = 609.2241; Measured value = 609.2239 [M+Na] + .

[0221] (R)-N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)-3-fluoropyrrolidine-1-sulfonamide(57) [ka] (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) was cooled in a DCM (3.5 mL) solution 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 at ambient temperature for a further 30 minutes. The reaction mixture was cooled again in an ice bath, and (R)-3-fluoropyrrolidine-1-sulfonyl chloride (0.250 g, 1.33 mmol) was added. The solution was stirred at ambient temperature overnight. The reaction was quenched with water and extracted with DCM. The organic layers were combined, dried over MgSO4, and filtered. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.145 g of the title compound in 66% yield.

[0222] 1 H NMR (401 MHz, DMSO-d6) 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).

[0223] HRMS(ESI+): m / z calculated value (C 28 H 33O7N2FNaS) = 583.1885; Measured value = 583.1882 [M+Na] + .

[0224] N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)azepan-1-sulfonamide(58) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 at ambient temperature for a further 30 minutes. The reaction mixture was cooled again in an ice bath, and azepan-1-sulfonyl chloride (0.100 g, 0.51 mmol) was added. The solution was stirred at ambient temperature for a further 12 days. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution), and then recrystallized from MeOH. The reaction yielded 0.044 g of the title compound in 20% yield.

[0225] 1 H NMR (401 MHz, DMSO-d6) 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 = 5.8 Hz, 4H), 2.96 - 2.84 (m, 2H), 1.52 - 1.44 (m, 4H), 1.47 - 1.32 (m, 4H).

[0226] HRMS(ESI+): m / z calculation value (C 30 H 38 O7N2NaS) = 593.2292; Measured value = 593.2291 [M+Na] + .

[0227] 3-(N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)sulfamoyl)pyridine-1-iumtrifluoroacetate (59) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 (0.59 mL, 2.75 mmol) was added dropwise, and stirring was continued at ambient temperature for a further 30 minutes. The reaction mixture was cooled again in an ice bath, and pyridine-3-sulfonyl chloride hydrochloride (0.250 g, 1.17 mmol) was added. The solution was stirred overnight at ambient temperature. The reaction was quenched with water and extracted with DCM. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.090 g of the title compound in a 35% yield.

[0228] 1H NMR (401 MHz, DMSO-d6) 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).

[0229] HRMS(ESI+): m / z calculated value (C 29 H 30 O7N2NaS) = 573.1666; Measured value = 573.1664 [M+Na] + .

[0230] 2-(4-(N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)sulfamoyl)phenoxy)acetamide (60) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 at ambient temperature for a further 30 minutes. The reaction mixture was cooled again in an ice bath, and 4-(2-amino-2-oxoethoxy)benzenesulfonyl chloride (0.294 g, 1.18 mmol) was added. The solution was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.126 g of the title compound in 52% yield.

[0231] 1 H NMR (401 MHz, DMSO-d6) 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).

[0232] HRMS(ESI+): m / z calculation value (C 32 H 34 O9N2NaS) = 645.1877; Measured value = 645.1876 [M+Na] + .

[0233] 3-((3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)-isopropylsulfamide(61) [ka] A solution of (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate tert-butyl (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 at ambient temperature for a further 30 minutes. The reaction mixture was cooled again in an ice bath, and isopropylsulfamoyl chloride (0.250 g, 1.59 mmol) was added. The solution was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.154 g of the title compound in 74% yield.

[0234] 1 H NMR (401 MHz, DMSO-d6) 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 = 6.5Hz, 6H).

[0235] HRMS(ESI+): m / z calculation value (C 27 H 34 O7N2NaS) = 553.1979; measured value = 553.1976 [M+Na] + .

[0236] N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)-6-hydroxy-4-methylpyridine-3-sulfonamide(62) [ka] A solution of tert-butyl (3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)carbamate (0.200 g, 0.39 mmol) in DCM (3.5 mL) 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 at ambient temperature for a further 30 minutes. The reaction mixture was cooled again in an ice bath, and 6-hydroxy-4-methylpyridine-3-sulfonyl chloride (0.250 g, 1.59 mmol) was added. The solution was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.036 g of the title compound in 16% yield.

[0237] 1 H NMR (401 MHz, DMSO-d6) 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).

[0238] HRMS(ESI+): m / z calculation value (C 30 H 32 O8N2NaS) = 603.1772; Measured value = 603.1770 [M+Na] + .

[0239] 4-(2-(N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)sulfamoyl)ethyl)morpholine-4-ium trifluoroacetate (63) [ka] A solution of 2-morpholinoethane-1-sulfonic acid (0.191 g, 0.98 mmol) and DMF (0.008 mL, 0.10 mmol) in SOCl2 (1.8 mL) was sealed in a test tube and stirred at 80°C for 40 minutes. Conversion was confirmed by TLC. During the reaction, the generated gas had to be vented through the septum. The reaction mixture was cooled and transferred to a round-bottom flask, where it was evaporated three times with toluene. The residue was suspended in DCM (3 mL), and 4-amino-2,6-bis((E)-3,4-dimethoxybenzylidene)cyclohexane-1-one (free base) (0.100 g, 0.24 mmol) and DIPEA (0.17 mL, 0.98 mmol) were added. The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA):ACN, gradient elution). The reaction yielded 0.060 g of the title compound in 35% yield.

[0240] 1 H NMR (401 MHz, DMSO-d6) 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).

[0241] HRMS(ESI+): m / z calculated value (C 30 H 39O8N2S) = 587.2422; Measured value = 587.2420 [M+H] + .

[0242] 3-(N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)sulfamoyl)propane-1-aminium trifluoroacetate (64) [ka] To a 1:1 (8 mL) mixed solvent of 3-aminopropane-1-sulfonic acid (0.222 g, 1.60 mmol) and NaHCO3 (0.282 g, 3.36 mmol) in H2O:1,4-dioxane, fmoc-Cl (0.455 g, 1.76 mmol) was added in small amounts. The reaction mixture was stirred at ambient temperature over the weekend. Amberlite IR 120 H was added to the solution, and the mixture was stirred for a further 10 minutes. The amberlite was filtered off, and the filtrate was diluted with water. The aqueous layer was extracted with ELISA (3 times). The organic layers were combined and extracted with water (1 time). The aqueous layers of all extracts were combined and freeze-dried. The reaction yielded sodium 3-(fmoc-amino)propane-1-sulfonate in good yield.

[0243] 1 H NMR (401 MHz, DMSO-d6) 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).

[0244] A solution of 3-(fmoc-amino)propane-1-sulfonate sodium (0.345 g, 0.90 mmol) and DMF (0.007 mL, 0.09 mmol) in SOCl2 (2 mL) was sealed in a test tube and stirred at 80°C for 1.5 hours. Conversion was confirmed by TLC. During the reaction, the generated gas had to be vented through the septum. The reaction mixture was cooled, transferred to a round-bottom flask, and evaporated three times with toluene. The residue was suspended in DCM (4.5 mL), and 4-amino-2,6-bis((E)-3,4-dimethoxybenzylidene)cyclohexane-1-one (free base) (0.123 g, 0.30 mmol) and DIPEA (0.16 mL, 0.90 mmol) were added. The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The obtained 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 hours. The solvent was evaporated under reduced pressure, and the residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.040 g of the title compound in 21% yield.

[0245] 1 H NMR (401 MHz, DMSO-d6) 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).

[0246] HRMS(ESI+): m / z calculation value (C 27 H 35O7N2S) = 531.2160; Measured value = 531.2158 [M+H] + .

[0247] 2-(N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)sulfamoyl)ethane-1-aminium trifluoroacetate (65) [ka] To a 1:1 (8 mL) solution of a mixed solvent H2O:1,4-dioxane containing 2-aminoethane-1-sulfonic acid (0.200 g, 1.60 mmol) and NaHCO3 (0.282 g, 3.36 mmol), fmoc-Cl (0.455 g, 1.76 mmol) was added in small amounts. The reaction mixture was stirred at ambient temperature over the weekend. Amberlite IR 120H was added to the solution, and the mixture was stirred for a further 10 minutes. The amberlite was filtered off, and the filtrate was diluted with water. The aqueous layer was extracted three times with ELISA. The organic layers were combined and extracted once with water. The aqueous layers of all extractions were combined and freeze-dried. The reaction yielded sodium 2-(fmoc-amino)ethane-1-sulfonate in good yield.

[0248] 1 H NMR (401 MHz, DMSO-d6) 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).

[0249] A solution of 2-(fmoc-amino)ethane-1-sulfonate sodium (0.333 g, 0.90 mmol) and DMF (0.007 mL, 0.09 mmol) in SOCl2 (2 mL) was sealed in a test tube and stirred at 80°C for 1.5 hours. Conversion was confirmed by TLC. During the reaction, the generated gas had to be vented through the septum. The reaction mixture was cooled, transferred to a round-bottom flask, and evaporated three times with toluene. The residue was suspended in DCM (3.5 mL), and 4-amino-2,6-bis((E)-3,4-dimethoxybenzylidene)cyclohexane-1-one (free base) (0.123 g, 0.30 mmol) and DIPEA (0.16 mL, 0.90 mmol) were added. The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The obtained solid was dissolved in DCM (1 mL), and piperidine (0.16 mL, 1.62 mmol) was added. The reaction mixture was stirred at ambient temperature for 30 minutes. The solvent was evaporated under reduced pressure, and the residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.101 g of the title compound in 53% yield.

[0250] 1 H NMR (401 MHz, DMSO-d6) 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).

[0251] HRMS(ESI+): m / z calculation value (C 26 H 33 O7N2S) = 517.2003; Measured value = 517.2000 [M+H] + .

[0252] 2-(N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)sulfamoyl)benzeneaminium trifluoroacetate (66) [ka] To a 1:1 (8 mL) mixed solvent solution of 2-aminobenzenesulfonic acid (0.277 g, 1.60 mmol) and NaHCO3 (0.671 g, 8.00 mmol) in H2O:1,4-dioxane, fmoc-Cl (0.455 g, 1.76 mmol) was added in small amounts. The reaction mixture was stirred at ambient temperature over the weekend. Amberlite IR 120H was added to the solution, and the mixture was stirred for a further 10 minutes. The amberlite was filtered off, and the filtrate was freeze-dried. The resulting solid was purified by flash chromatography (eluent DCM:MeOH, 9:1). The reaction yielded 0.529 g of sodium 2-(fmoc-amino)benzenesulfonate in 84% yield.

[0253] 1 H NMR (401 MHz, DMSO-d6) 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, 1.6 Hz, 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).

[0254] A solution of 2-(fmoc-amino)benzenesulfonate sodium (0.376 g, 0.90 mmol) and DMF (0.007 mL, 0.09 mmol) in SOCl2 (2 mL) was sealed in a test tube and stirred at 80°C for 1.5 hours. Conversion was confirmed by TLC. During the reaction, the generated gas had to be vented through the septum. The reaction mixture was cooled, transferred to a round-bottom flask, and evaporated three times with toluene. The residue was suspended in DCM (3.5 mL), and 4-amino-2,6-bis((E)-3,4-dimethoxybenzylidene)cyclohexane-1-one (free base) (0.123 g, 0.30 mmol) and DIPEA (0.16 mL, 0.90 mmol) were added. The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The obtained 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 hour. The solvent was evaporated under reduced pressure, and the residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.029 g of the title compound in 14% yield.

[0255] 1 H NMR (401 MHz, DMSO-d6) 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).

[0256] HRMS(ESI+): m / z calculation value (C 30 H 33 O7N2S) = 565.2003; Measured value = 565.2000 [M+H] + .

[0257] 3-(N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)sulfamoyl)benzeneaminium trifluoroacetate (67) [ka] To a 1:1 (8 mL) mixed solvent solution of 3-aminobenzenesulfonic acid (0.277 g, 1.60 mmol) and NaHCO3 (0.671 g, 8.00 mmol) in H2O:1,4-dioxane, fmoc-Cl (0.455 g, 1.76 mmol) was added in small amounts. The reaction mixture was stirred at ambient temperature over the weekend. Amberlite IR 120H was added to the solution, and the mixture was stirred for a further 10 minutes. The amberlite was filtered off, and the filtrate was freeze-dried. The resulting solid was purified by flash chromatography (eluent: DCM:MeOH, 9:1). The reaction yielded sodium 3-(fmoc-amino)benzenesulfonate in good yield.

[0258] 1 H NMR (401 MHz, DMSO-d6) 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).

[0259] A solution of sodium 3-(fmoc-amino)benzenesulfonate (0.502 g, 1.2 mmol) and DMF (0.009 mL, 0.12 mmol) in SOCl2 (2 mL) was sealed in a test tube and stirred at 80°C for 2 hours. Conversion was confirmed by TLC. During the reaction, the gas generated had to be vented through the septum. The reaction mixture was cooled and transferred to a round-bottom flask, where it was evaporated three times with toluene. The residue was suspended in DCM (3.5 mL), and 4-amino-2,6-bis((E)-3,4-dimethoxybenzylidene)cyclohexane-1-one (free base) (0.123 g, 0.30 mmol) and DIPEA (0.21 mL, 1.20 mmol) were added. The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The obtained solid was dissolved in DCM (1 mL), and piperidine (0.18 mL, 1.82 mmol) was added. The reaction mixture was stirred at ambient temperature for 1 hour. The solvent was evaporated under reduced pressure, and the residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.103 g of the title compound in 50% yield.

[0260] 1 H NMR (401 MHz, DMSO-d6) 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).

[0261] HRMS(ESI+): m / z calculation value (C 30 H 32O7N2NaS) = 587.1822; Measured value = 587.1821 [M+Na] + .

[0262] 4-(N-(3,5-bis((E)-3,4-dimethoxybenzylidene)-4-oxocyclohexyl)sulfamoyl)benzene-1,2-diaminium bis(trifluoroacetic acid) salt (68) [ka] To a 1:1 (8 mL) mixed solvent solution of 3,4-diaminobenzenesulfonic acid (0.301 g, 1.60 mmol) and NaHCO3 (0.564 g, 6.70 mmol) in H2O:1.4-dioxane, Fmoc-Cl (0.910 g, 3.52 mmol) was added in small amounts. The reaction mixture was stirred at ambient temperature over the weekend. The solvent was evaporated under reduced pressure, and the residue was purified by flash chromatography (eluent DCM:MeOH, 9:1). The reaction yielded 0.780 g of sodium 3,4-bis(Fmoc-amino)benzenesulfonate in 75% yield.

[0263] 1 H NMR (401 MHz, DMSO-d6) 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).

[0264] A solution of 3,4-bis(fmoc-amino)benzenesulfonate sodium (0.750 g, 1.15 mmol) and DMF (0.009 mL, 0.12 mmol) in SOCl2 (2.2 mL) was sealed in a test tube and stirred at 80°C for 2 hours. Conversion was confirmed by TLC. During the reaction, the generated gas had to be vented through the septum. The reaction mixture was cooled, transferred to a round-bottom flask, and evaporated three times with toluene. The residue was suspended in DCM (5.5 mL), and 4-amino-2,6-bis((E)-3,4-dimethoxybenzylidene)cyclohexane-1-one (free base) (0.123 g, 0.30 mmol) and DIPEA (0.21 mL, 1.20 mmol) were added. The reaction mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the solid residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The obtained solid was dissolved in DCM (1 mL), and piperidine (0.18 mL, 1.82 mmol) was added. The reaction mixture was stirred at ambient temperature for 1 hour. The solvent was evaporated under reduced pressure, and the residue was purified by reverse-phase flash chromatography (eluent: H2O (0.1% TFA): ACN, gradient elution). The reaction yielded 0.060 g of the title compound in 24% yield.

[0265] 1 H NMR (401 MHz, DMSO-d6) 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).

[0266] HRMS(ESI+): m / z calculated value (C 30 H 33 O7N3NaS) = 602.1931; Measured value = 602.1930 [M+Na] + .

[0267] Example 2: Efficacy of compound with general formula I in activation of the NRF1 (NFE2L1) transcription pathway observed by cell-based reporter assay. A stable cell line derived from HEK293 cells containing a 3xPSMA4-ARE / minP / luc2P / reporter based on an ARE (antioxidant response element) response element derived from the human PSMA4 gene promoter, enabling observation of NRF1 pathway activation, was cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS, 2 mmol / l L-glutamine, 50 μg / mL penicillin, and 50 μg / mL streptomycin. The cells were cultured in a 37°C CO2 incubator under an atmosphere containing 5% CO2. When this reporter strain reached approximately 70% confluence, it was transfected with the normalized reporter Renira reporter pRL-TK (catalog number E2241; Promega, Hercules, CA). Polyethyleneimine (PEI) was used for transfection in OptiMEM medium at a PEI / DNA ratio of 3:1. Next, the cells are placed in a 384-well plate in a 10x 3Seeds were seeded at 25 μL / well at the specified concentration, and after 16 hours of equilibration, the cells were treated with the test compound dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 μmol / l. The experiment was repeated in a technical 4-stage and a biological 3-stage series. After 16 hours of incubation, the medium was removed and the cells were dissolved in 5 μL 1× lysis buffer (25 mmol / l, Tris-phosphate pH 7.8; 2 mmol / l dithiotrate (DTT); 2 mmol / l 2,2′,2′′,2′′′-(ethane-1,2-diyldinitrile)tetraacetic acid; 10% (hereinafter always expressed as volume percentage) glycerol and 1% Triton X-100). After incubation on a shaker for 10 minutes, the firefly / photinusluciferase substrate (200 mM Tris-HCl; 15 mmol / l MgSO4; 0.1 mmol / l EDTA; 25 mmol / l DTT; 1 mmol / l ATP; 0.2 mmol / l coenzyme A; and 200 mmol / l D-luciferin, pH 8.0) was added in 20 μL per well, and luminescence was measured. After measuring the firefly luciferase activity, the same amount of buffer (25 mmol / l Na4P2O7; 10 mmol / l AcONa; 15 mmol / l EDTA; 500 mmol / l Na2SO4; 500 mmol / l NaCl; 25 μmol / l phenylbenzothiazole; 4 μmol / l corenterazine; and 0.04% BSA, pH 5.0) was added, and after shaking, the reniralciferase activity was measured.

[0268] The measurement results (Table 1) represent the ratio of reniral luciferase to firefly luciferase activity, showing the mean and standard deviation from the biological 3-step series using a technical 4-step series.

[0269] In the test compounds, activation of the pathway regulated by the NRF1 transcription factor ranged from 3.93 to 19.39. This represents an increase of more than 19 times compared to the control, demonstrating that compound 11 was statistically significant in DMSO-treated cells. Part B of Table 1 shows the results for compounds of general formula I, directly compared with compound ASC-JM17.

[0270] [Table 2]

[0271] Example 3: Verification of the target activity of compounds of general formula I The parental HCT116 cell line and HCT116 cell lines (two clones, KO04 and KO11) lacking a gene encoding the DDI2 protease were transiently transfected with the 3xPSMA4-ARE-LUC reporter plasmid shown in Example 2, normalized with a pRL-TK reporter (25:1 ratio), and stabilized overnight. The cells were then treated with bortezomib (BTZ) at a concentration of 0.75 μmol / l and compound 1 at a concentration of 10 μmol / l for 16 hours. After 16 hours of incubation, the culture medium was removed and the cells were lysed in 5 μL of 1× lysis buffer (25 mmol / l Tris-phosphate pH 7.8; 2 mmol / l dithiotrate (DTT); 2 mmol / l 2,2′,2′′,2′′′-(ethane-1,2-diyldinitrile)tetraacetic acid; 10% (hereinafter always expressed as volume percentage) glycerol; and 1% Triton X-100). After incubation on a shaking plate for 10 minutes, the firefly / photinusluciferase substrate (200 mM Tris-HCl; 15 mmol / l MgSO4; 0.1 mmol / l EDTA; 25 mmol / l DTT, 1 mmol / l ATP, 0.2 mmol / l coenzyme A, and 200 mmol / l D-luciferin, pH 8.0) was added in 20 μL per well, and luminescence was measured. After measuring firefly luciferase activity, the same amount of buffer (25 mmol / l Na4P2O7; 10 mmol / l AcONa; 15 mmol / l EDTA; 500 mmol / l Na2SO4; 500 mmol / l NaCl; 25 μmol / l phenylbenzothiazole, 4 μmol / l corenterazine, and 0.04% BSA, pH 5.0) was added, and after shaking, reniral luciferase activity was measured. The measurement results (Table 2) show the ratio of reniral luciferase activity to firefly luciferase activity, and the mean and standard deviation from the biological 3-series based on a technical 4-series is shown.

[0272] As is clear from the results shown in Table 2, compound 1 at a predetermined concentration showed almost no activity in cell lines with a non-functional pathway controlled by the NRF1 transcription factor, compared to the activity observed in the parental cell line (WT). In contrast, the control compound bortezomib (BTZ) already showed slight activation at a concentration two orders of magnitude lower than that of compound 1. From these experimental results, we can conclude that our test compound is a novel activator of the NRF1 pathway.

[0273] [Table 3]

[0274] Example 4: Effects of compounds of general formula I on cellular protein homeostasis (proteostasis) U2OS cells stably expressing the Ub(G76V)-GFP reporter were cultured in DMEM medium containing 10% FBS, 2 mmol / l L-glutamine, 50 μg / mL penicillin, and 50 μg / mL streptomycin in a CO2 incubator at 37°C under a 5% CO2 atmosphere. The cells were first cultured in 384-well plates in DMEM medium without phenol red, in 10 × 10⁶ wells. 3 Cells were seeded in wells. On day 2, the compound was added at a concentration of 5 μmol / l. The experiment was performed in three measurements. After 8 hours, the GFP signal was measured (excitation wavelength λ=400 nm, emission wavelength λ=510 nm), and the cytotoxic effect of the compound was measured using the Lezazlin / Almar Blue assay according to the manufacturer's protocol (ThermoFisher Scientific, catalog number DAL1025). The GFP intensity was normalized by cell viability based on the following formula: (GFP intensity of the compound) / (basal GFP intensity). The basal GFP intensity corresponds to cells treated with DMSO.

[0275] The results are shown in Table 3. Fluorescence in cells treated with the test compound did not increase compared to DMSO-treated cells, and therefore endogenous protein degradation was not inhibited. Thus, it can be concluded that the novel activator of the NRF1 pathway is not simultaneously an inhibitor of the ubiquitin-proteasome system, which is a desirable result. [Table 4]

[0276] Example 5: Effect of compound of general formula I on cell viability Human cell line HEK293 was cultured in a CO2 incubator at 37°C under an atmosphere containing 5% CO2. The cytotoxic effects of the test compound were measured using the Lezazlin / Almar Blue assay according to the manufacturer's protocol (ThermoFisher Scientific, catalog number DAL1025). As shown in Table 4, none of the tested compounds showed significant cytotoxicity at the specified concentrations.

[0277] [Table 5]

[0278] Example 6: Effect of compound of general formula I on the expression of genes regulated by the transcription factor NRF1 (NFE2L1) Human neuroblastoma cell line SH-SY5Y was cultured in DMEM medium containing 10% FBS and 2 mmol / l L-glutamine in a CO2 incubator at 37°C under a 5% CO2 atmosphere. Compound 1 was administered in 4 × 10⁶ doses in both the technical 2-stage and biological 3-stage series. 5The reagent was added to individual cells. After 16 hours, the cells were lysed, and mRNA was isolated using the NucleoSpin RNA Kit (Macherey-Nagel, catalog number 740955.250) according to the manufacturer's protocol. The mRNA was then transcribed into cDNA using the TATTA grandScript cDNA Supermix Kit (TATAAbiocenter) according to the manufacturer's recommendations. Quantitative RT-PCR (RT-qPCR) was performed using a LightCycler 480 (Roche Life Science). The primers for each gene used in the RT-qPCR reaction are shown in Table 5. mRNA encoding GAPDH was used for data normalization.

[0279] The analysis results are shown in Table 6. After treatment of the human neuroblastoma cell line SH-SY5Y with compound 1, mRNA levels of all observed genes encoding proteasome subunits (PSMB7, PSMD12, or PSMC4) increased compared to the control (DMSO). When mRNA levels encoding heat shock protein (HSP1A1) and ubiquinone (NQO1) were observed, a significant increase in mRNA levels was observed after cell treatment with compound 1 compared to the control.

[0280] [Table 6] [Table 7]

[0281] Example 7: Effect of compound of general formula I on the expression of proteins regulated by the transcription factor NRF1 (NFE2L1) Next, the expression levels of HA-NRF1 in HEK-293 cells overexpressing HA-tagged NRF1 protein (HA-NRF1), and the expression levels of HSPA1A, NQO1, and PSMB7 proteins in SH-SY5Y cells overexpressing α-synuclein were observed by Western blotting. Cells were treated with dimethyl sulfoxide (DMSO) as a negative control, or with compound 1 at a concentration of 5 μmol / l for 16 hours.

[0282] After cell treatment with compound 1, the expression levels of all tested proteins increased compared to the control (Table 7). [Table 8]

[0283] Example 8: Effect of compound of general formula I on proteasome activity in cells The proteolytic activity of the 20S proteasome was measured using a fluorescent substrate (Suc-LLVY-AMC, Bachem I-1395). Human HEK293 cell lines were incubated with the test compound at a concentration of 7.5 μmol / l for 16 hours. The cells were lysed in lysis buffer (50 mmol / l HEPES, pH 7.5; 5 mmol / l EDTA; 150 mmol / l NaCl; 2 mmol / l ATP; 1% Triton), and the lysates were incubated in 100 μL of buffer containing 200 μmol / l Suc-LLVY-AMC, a fluorescent substrate for measuring chymotrypsin activity (50 mmol / l Tris, pH 8.0; 10 mmol / l MgCl2; 1 mmol / l ATP; 1 mmol / l DTT) in three technical sets for 30 minutes at 37°C in the dark. The fluorescence of the resulting AMC was measured using a fluorometer at excitation wavelength λ=360 nm and emission wavelength λ=460 nm. Proteasome activity was measured relative to a control group affected only by DMSO.

[0284] The results are shown in Table 8. It is clear that all test compounds increased proteasome chymotrypsin activity to an extent equivalent to or greater than that of the comparative compound ASC-JM17.

[0285] [Table 9]

[0286] Example 9: Protective effect of compounds of general formula I against proteotoxic stress in SH-SY5Y cells in which aggregation of overproduced α-synuclein is induced by rotenone. The protective effect of the test compounds against proteotoxic stress was further investigated in the SH SY5Y-SNCA (synuclein overproduction) cell line compared to the parental SH-SY5Y cell line. Both cell lines were treated with DMSO, rotenone [1.125 μmol / l] (which specifically induces proteotoxic stress), the comparative compound ASC-JM17, and compound 1 [concentration series 2.5, 1.25, 0.625, and 0.3125 μmol / l]. Measured viability is relative to the DMSO control and is expressed as mean ± standard deviation [%].

[0287] All test compounds showed protective effects against α-synuclein-induced proteotoxic stress (Table 9).

[0288] [Table 10]

[0289] Example 10: Effect of compounds of general formula I on the generation of reactive oxygen species in cells The generation of reactive oxygen species (ROS) and associated oxidative stress in cells were measured using a 2',7'-dichlorofluorescein diacetate (H2DCFDA) probe (catalog number D6883-50MG, Sigma-Aldrich) to detect intracellular ROS generation. For plate preparation, 10,000 SH-SY5Y cells were seeded per well in a 96-well plate, and the cells were allowed to adhere overnight. The following day, the test compounds were added at two concentrations, 5 and 25 μmol / l; rotenone (Merck, catalog number R8875-1G) and TBHP organic peroxide were used as controls for ROS induction in cells. After 2 hours, the cells were washed with 1×PBS (137 mmol / l NaCl; 2.7 mmol / l KCl; 1.5 mmol / l KH2PO4; 10 mmol / l Na2HPO4), and then 100 μL of H2DCFDA probe dissolved in 1×PBS to a final concentration of 100 μmol / l was added per well for 30 minutes. Free probe was then removed by washing. In the final step, 50 μL of 1×PBS was added per well, and the fluorescence signal was measured using a Tecan infinite M1000 reader at an excitation wavelength of 480 nm and an emission wavelength of 535 nm. The experiments were performed in a biological triad consisting of four technical triads; data are shown as a percentage relative to the DMSO control.

[0290] The results are shown in Table 10. Compound 1 does not induce the formation of reactive oxygen species at a concentration of 5 μmol / l or five times that concentration (25 μmol / l). This is in contrast to the comparative compound ASC-JM17, which significantly increases ROS concentration compared to the DMSO control.

[0291] [Table 11]

[0292] Example 11: Effects of compounds of general formula I on the cell cycle After treating SH-SY5Y cells with compound 1, cell cycle analysis was performed using flow cytometry, comparing them to negative control DMSO and comparison compound ASC-JM17. Different stages of the cell cycle were determined by staining with propidium iodide (P4170-250MG, Sigma-Aldrich). This propidium iodide was inserted into double-stranded DNA as a fluorescent dye, allowing for semi-quantitative measurement of total DNA amount and the distribution of cell populations in different stages of the cell cycle: G0 / G1 phase (amount of unreplicated DNA), S phase (DNA synthesis), and G2 / M phase. (2.5 × 10⁶ cells in a 6-well plate) 5 Cells at a density of cells / mL were exposed to a 5 μmol / L concentration of the test compound in a CO2 incubator for 16 hours. Subsequently, the cells were collected in a flow cytometry tube, centrifuged at 500 g for 5 minutes, washed with non-sterile PBS, and fixed in 70% ethanol at 4°C. The prepared sample was stored at -20°C. Next, 500 μL of propidium iodide solution was added, mixed and incubated in a 37°C water bath in the dark for 15 minutes, then 200 μL of ribonuclease A solution (0.7 mol / L, 060M7000V, Sigma-Aldrich, USA) was added, and the sample was incubated again under the same conditions for 15 minutes. The incubated sample was kept at 4°C for at least 1 hour. Analysis was performed using a BD LSR Fortessa flow cytometer with an argon laser (excitation wavelength 488 nm).

[0293] The results are shown in Table 11. Compound 1 did not affect cell cycle progression. In contrast, the comparative compound ASC-JM17 caused a significant proportion of cells to remain in the G2 / M phase compared to the control. This suggests cell cycle arrest at the G2 checkpoint and also indicates significant toxicity of this comparative compound.

[0294] [Table 12]

[0295] Example 12: Effect of compound of general formula I on the expression level of proteins involved in autophagy The efficacy of the novel NRF1 pathway activator was observed by analyzing the expression levels of p62 and LC3 proteins involved in autophagy using Western blotting. Human HEK239 cell lines were treated with compound 1 in the same manner as in Example 7.

[0296] As shown in Table 12 below, compound 1 at a concentration of 10 μmol / l significantly increases the level of endogenous p62. At the same concentration, it significantly decreases the level of LC3-I and increases the level of LC3-II. Therefore, this indicates activation that is largely involved in autophagy, i.e., the degradation of abnormally folded proteins within cells.

[0297] [Table 13]

[0298] Example 13: Effect of compound of general formula I on the formation of Lewy body-like aggregates in SH-SY5Y cells overexpressing SNCA-GFP fusion protein. The efficacy of a novel NRF1 pathway activator was further observed by analyzing Lewy body-like aggregates in SH-SY5Y cells overexpressing SNCA-GFP fusion protein. Cells were treated with DMSO (negative control) or test compound 1 (C=5 μmol / l) for 72 hours. One hour after the addition of the above compound, recombinant α-synuclein was added to a final concentration of 50 μmol / l to induce the formation of SNCA-GFP aggregates.

[0299] The results are shown in Table 13, representing the percentage (%) of cells containing Lewy body-like aggregates relative to the total number of cells observed in the analyzed microscopic images. Test compound 1 significantly reduced the percentage of cells containing Lewy body-like aggregates compared to the DMSO control.

[0300] [Table 14]

[0301] Example 14: Effect of compound of general formula I on the formation of poly-Q aggregates in cells To visualize and quantify the amount of expansive poly-Q aggregates, U2OS cells were grown on slides divided into four independent compartments pre-treated with poly-D-lysine solution. 40,000 cells in 450 μL of phenol red-free DMEM culture medium were seeded into individual chambers on these slides. Subsequently, the cells were transfected with the Htt EGFP-Q74 plasmid using Lipofectamine 2000 (Thermo Fisher Scientific, USA) to generate elongation repeats. 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. The treated cells were then cultured for 24 hours. Subsequently, the culture medium was aspirated, the cells were washed with PBS, 400 μL of 4% paraformaldehyde fixative was added to each chamber, and the slides were incubated in a hood for 20 minutes. The experiment was performed in a biological triplicate for each compound, including the control. Next, the slides were washed twice more with PBS, and the fixed cells were permeabilized for 30 minutes with a 0.3% Triton X100 and 0.1% FBS solution in PBS. Subsequently, the cell nuclei were stained with Hoechst 33255 solution (ThermoFisher Scientific, catalog number H3569) at a concentration of 1 μg / mL for 10 minutes. Finally, the solution was aspirated and replaced with fresh PBS. Fluorescence images were acquired using a Zeiss LSM 780 confocal microscope. Selected fluorescent dyes were captured as follows: EGFP excitation wavelength λ=488 nm; emission wavelength λ=495~555 nm (at 25% laser power), and for Hoechst 33255, excitation wavelength λ=405 nm; fluorescence wavelength λ=410~435 nm (at 10% laser power). Images were taken of more than 60 randomly selected cells for each test compound and the control sample (DMSO). Subsequently, image analysis was performed on the acquired images using ZEN (Zeiss, Germany) and ImageJ (National Institutes of Health, Bethesda USA) software. This quantified the EGFP signal intensity in positive cells and determined the number and size of expansive Htt-Q74 aggregates within GFP-positive cells.

[0302] The results are shown in Table 14. Analysis of the number of aggregates in cells treated with the test compound revealed that the fluorescence intensity of a single cell decreased by up to 42% when cells were treated with compound 1. This is a significant decrease compared to the comparative compound ASC-JM17. Furthermore, the number of individual cells containing aggregates also decreased significantly by up to 46%.

[0303] [Table 15]

[0304] Example 15: Effect of compound of general formula I on the activity of transcription factor SKN-1A (NRF1) in vivo The in vivo activity of compound 1 was observed using the C. elegans reporter strain GR2183. In this strain, activation of the transcription factor SKN-1A (NRF1 in C. elegans) is observed via the expression of the induced rpt-3P::GFP reporter. This strain was obtained from the Caenorhabditis Genetic Centre (CGC, University of Minnesota). Nematode worms were maintained at 20°C under standard culture conditions. The nematodes were castrated, and the instar-synchronized offspring were grown at 20°C until adulthood, thereby avoiding reproduction and eliminating the need for fluorodeoxyuridine treatment. L4 larvae / young adults were cultured in E. coli OP50 suspension in S-Complete medium, then treated with compound 1 (concentrations of 10 μmol / l and 50 μmol / l), and pipetted into 96-well plates (approximately 15 worms per well) in liquid culture state. The plates were sealed to prevent evaporation of the culture medium and kept at 20°C for 24 hours. Fluorescence intensity was evaluated using a microscope equipped with an epifluorescence spectrometer (Zeiss Axio). Image analysis was performed using ImageJ software (NIH, https: / / imagej.nih.gov / ij / ).

[0305] The results shown in Table 15 indicate that compound 1 significantly induced the SKN1A(NRF1) regulatory pathway at both concentrations tested.

[0306] [Table 16]

[0307] Example 16: Effect of compound of general formula I on the formation of expanded protein aggregates in vivo C. elegans AM140 strains producing Htt-Q35::YFP were cultured for 6 days in DMSO (negative control) or with final concentrations of test compound 1 at 10, 20, or 50 μmol / l, as in Example 14. Aggregate size was evaluated by analyzing microscopic data using the ImageJ program (NIH, https: / / imagej.nih.gov / ij / ).

[0308] The results of the in vivo experiment are shown in Table 16. Compound 1 significantly reduced the size of expanded protein aggregates compared to the DMSO control.

[0309] [Table 17]

[0310] Example 17: In vivo toxicity of compounds of general formula I The in vivo toxicity of compound 1 was observed in the wild-type strain N2 of C. elegans. This strain was obtained from the Caenorhabditis Genetic Centre (CGC, University of Minnesota). The nematodes were maintained at 20°C under standard culture conditions. Compound 1 was tested for baseline toxicity and worm reproductive capacity. Reproductive capacity was measured by a chitinase test, and the results were expressed as a percentage of vitality. Visual evaluation of the nematodes was performed under a microscope. Briefly, L1 larvae (instar-synchronized) were diluted to a concentration of 200-300 individuals per 1 mL, fed with a 3 mg / mL bacterial suspension, and transferred to a 96-well plate. The larvae were treated with the test compound in DMSO, or with the same amount of DMSO alone. The population was grown at 20°C for 4 days. At this point, healthy worms had become adults and begun laying eggs. Healthy embryos produce chitinase at hatching. A fluorescent substrate (4-methylumberferyl-β-DN,N',N''-triacetylchitotrioside, 20 μM) was added to the wells, and the plate was incubated for 1 hour (37°C). Chitinase activity was then evaluated. The reaction was then stopped by adding alkaline buffer (1 M glycine / 1 M NaOH, pH 10.6), and fluorescence intensity was measured at 360 / 460λm. Results are shown as a percentage relative to DMSO.

[0311] The results in Table 17 show that compound 1 did not exhibit any measurable toxicity at any of the concentrations tested.

[0312] [Table 18]

[0313] Example 18: Effect of compound of general formula I on intracellular viral protein loss in viral infection Human hepatocellular carcinoma-derived cell lines (HepG2-NTCP) constitutively expressing the NTCP cotransporter were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 6-well plates (5.5 x 10⁶ cells per well in 2 mL of culture medium). 5Cells were cultured at 37°C under 5% CO2 conditions. The cells were infected with HBV virus, and on day 7 post-infection, they were treated with compound 1 (or DMSO) for 16 hours. Subsequently, the cells were harvested, the supernatant was saved, and extracellular capsid proteins were measured by ELISA. The harvested cells were lysed, and the lysate was subjected to Western blot analysis. The experiment was performed using three independent measurements.

[0314] The results in Table 18 show that in established HBV infections, infected cells treated with compound 1 showed a significant loss of intracellular viral capsid protein compared to control (DMSO) treated cells. Table 19 shows that in established HBV infections, extracellular viral capsid protein was significantly lost compared to the control.

[0315] [Table 19]

[0316] [Table 20]

[0317] Example 19: Effect of compound of general formula I on intracellular viral protein loss in initial viral infection Human hepatocellular carcinoma-derived cell lines (HepG2-NTCP) constitutively expressing the NTCP cotransporter were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 6-well plates (5.5 × 10⁶ cells per well in 2 mL of culture medium). 5 Cells were cultured at 37°C under 5% CO2 conditions. The cells were treated with compound 1 (or DMSO) for 8 hours, then infected with HBV virus, and retreated with compound 1 (or DMSO) for 16 hours on day 3 post-infection. The cells were harvested on day 7, and the supernatant was saved for extracellular capsid protein analysis by ELISA. The harvested cells were lysed, and the lysate was subjected to Western blot analysis. The experiment was performed once.

[0318] Table 20 shows that, compared to the control (DMSO), there is a clear loss of intracellular viral capsid protein in the early stages of HBV infection after treating infected cells with compound 1. Similarly, Table 21 shows a clear loss of extracellular viral capsid protein in the early stages of HBV infection compared to the control.

[0319] [Table 21]

[0320] [Table 22]

[0321] Example 20: Solubility of compounds of general formula I in the physiological environment The maximum solubility of compounds in DMEM (Dulbecc's modified Eagle medium) cell culture medium was determined by UV spectroscopy after centrifugation. The degree of precipitation was estimated by comparing the spectrum of the precipitate dissolved in DMSO with the spectrum of the compound in DMSO at known concentrations.

[0322] The results shown in Table 22 indicate that the solubility of the new compound is increased compared to the comparative compound ASC-JM17.

[0323] [Table 23]

[0324] Example 21: Efficacy of compound with general formula I in activation of the NRF1 (NFE2L1) transcription pathway observed by cell-based reporter assay. A stable cell line derived from HEK293 cells containing a 3xPSMA4-ARE / minP / luc2P / reporter based on the human PSMA4 gene promoter's ARE (antioxidant response element), enabling observation of NRF1 pathway activation, was cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum, 2 mmol / l L-glutamine, 50 μg / mL penicillin, and 50 μg / mL streptomycin. Cells were cultured in a CO2 incubator at 37°C under a 5% CO2 atmosphere. When the reporter cell line reached approximately 70% confluence, it was transfected with the normalized reporter, Renira reporter pRL-TK (catalog number E2241; Promega, Hercules, CA). Polyethyleneimine (PEI) was used in optiMEM medium at a PEI / DNA ratio of 3:1. Cells were then transferred to 384-well plates in 10 × 10⁶ wells. 3Seeds were seeded at 25 μL / well at the specified concentration, and 16 hours after stabilization, the cells were treated with a 5 μmol / l concentration of the test compound dissolved in dimethyl sulfoxide (DMSO). The experiment was repeated in a technical 4-sequence and a biological 3-sequence. After 16 hours of incubation, the medium was removed and the cells were dissolved in 1 × lysis buffer (25 mmol / l, Tris-phosphate pH 7.8; 2 mmol / l dithiotrate (DTT); 2 mmol / l 2,2′,2′′,2′′′-(ethane-1,2-diyldinitrile)tetraacetic acid; 10% (hereinafter always expressed as volume percentage) glycerol, and 1% Triton X-100). After incubation on a shaker for 10 minutes, the firefly / photinus luciferase substrate was added in 20 μL per well (200 mM Tris-HCl; 15 mmol / l MgSO4; 0.1 mmol / l EDTA; 25 mmol / l DTT; 1 mmol / l ATP; 0.2 mmol / l coenzyme A; and 200 mmol / l D-luciferin, pH 8.0), and luminescence was measured. After measuring the firefly luciferase activity, the same amount of buffer (25 mmol / l Na4P2O7; 10 mmol / l AcONa; 15 mmol / l EDTA; 500 mmol / l Na2SO4; 500 mmol / l NaCl; 25 μmol / l phenylbenzothiazole; 4 μmol / l corenterazine and 0.04% BSA, pH 5.0) was added, and after shaking, the renilurus luciferase activity was measured. The measurement results (Table 23) represent the ratio of reniral luciferase activity to firefly luciferase activity, showing the mean and standard deviation from a biological triple with a technical quadruple.

[0325] In the test compounds, activation of the pathway regulated by the transcription factor NRF1 ranged from 3.98 to 13.33, representing an increase of up to 13 times compared to the control, indicating that most of the test compounds were statistically significant in DMSO-treated cells.

[0326] [Table 24]

[0327] Example 22: Effect of compound of general formula I on cell viability Human HEK293 cell lines were cultured in a CO2 incubator at 37°C under a 5% CO2 atmosphere. The cytotoxic effects of the tested compounds were measured using the Lezazlin / Almar Blue assay according to the manufacturer's protocol (ThermoFisher Scientific, catalog number DAL1025). As shown in Table 24, none of the tested compounds showed significant cytotoxicity at the specified concentrations.

[0328] [Table 25]

[0329] Example 23: Effects of compounds of general formula I on cellular protein homeostasis (proteostasis) U2OS cells stably expressing the Ub(G76V)-GFP reporter were cultured in DMEM medium containing 10% FBS, 2 mmol / l L-glutamine, 50 μg / mL penicillin, and 50 μg / mL streptomycin in a CO2 incubator at 37°C under a 5% CO2 atmosphere. Initially, the cells were cultured in DMEM culture medium without phenol red in a 384-well plate at a rate of 10 × 10⁴ 3 Cells were seeded. On day 2, the test compound was added at a concentration of 10 μmol / l. After 6 hours, the GFP signal (excitation wavelength λ=400 nm, emission wavelength λ=510 nm) was measured. After subtracting the autofluorescence of the test compound, the resulting signal was correlated with the basal GFP intensity of DMSO-treated cells and expressed as a percentage.

[0330] Table 25 shows the test results. Fluorescence in cells treated with the test compound did not increase compared to fluorescence in DMSO-treated cells, and therefore endogenous protein degradation was not inhibited. It can be concluded that novel activators of the NRF1 pathway are not inhibitors of the ubiquitin-proteasome system.

[0331] [Table 26]

[0332] Example 24: Protective effect of compounds of general formula I against elastin-induced ferroptosis cell death in SH-SY5Y cells Elastin is a small molecule that can initiate ferroptotic cell death by activating voltage-dependent anion channels (VDACs) and functionally inhibiting the cystine-glutamate antiporter system Xc-. Cells treated with elastin become depleted of cysteine ​​and the synthesis of the antioxidant glutathione is inhibited. Intracellular glutathione depletion leads to the accumulation of free reactive oxygen species (ROS), followed by lipid peroxidation. This ultimately leads to iron-dependent ferroptotic cell death.

[0333] The protective effect of compounds of general formula I against elastin-induced ferroptosis was measured in the neuroblastoma cell line SH-SY5Y. These cells were placed in a 384-well plate in approximately 3 × 10⁶ wells. 3 Cells were seeded at a cell / well density. After stabilization, cells were treated with the test compound at a concentration of 3 μmol / l for 24 hours, followed by the addition of elastin at a concentration of 20 μmol / l for another 24 hours. Cell viability was measured by the rezazlin / Almar Blue assay according to the manufacturer's protocol (ThermoFisher Scientific, catalog number DAL1025). 2 μl of Almar Blue / rezazlin solution at a concentration of 0.15 mg / mL was added to each well. After incubation at 37°C and 5% CO2 for 1 hour, resorphine fluorescence (excitation / emission: 560 / 590 nm) was measured using a Tecan Infinite M1000 instrument. The protective effect shown by cell viability was expressed as the percentage of cells exposed to elastin, or to elastin and the test compound, compared to control cells exposed to DMSO (Table 26).

[0334] [Table 27] Example 25: Effect of compound of general formula I on ROS levels in SH-SY5Y cell line Reactive oxygen species (ROS) induction in SH-SY5Y neuroblastoma cell lines was detected using a fluorescence assay based on 2',7'-dichlorofluorescein diacetate (DCFH-DA). Cells were cultured in 96-well plates and washed with preheated HBSS buffer. DCFH-DA solution was added to the cells at a final concentration of 100 μmol / l, followed by incubation for 30 minutes. The cells were then treated for a further 2 hours with compound 1 or ASC-JM17 lysed in HBSS buffer at final concentrations of 5 μmol / l or 25 μmol / l, respectively. Control cells were given a DMSO vehicle as a negative control, or two stress inducers serving as positive controls: rotenone at a concentration of 250 μmol / l, or tert-butyl hydroperoxide (tBHP) at concentrations of 25 μmol / l or 50 μmol / l. Fluorescence was measured at 485 / 530 nm.

[0335] Table 27 shows that treatment with tBHP-positive controls induced 6-fold and 10-fold changes in fluorescence intensity compared to DMSO-treated cells. The comparative compound ASC-JM17 increased fluorescence intensity, particularly at high concentrations. Compound 1, on the other hand, showed no effect at either of the tested concentrations. Based on these results, it can be concluded that compound 1 does not induce ROS.

[0336] [Table 28]

[0337] Example 26: Effect of compound of general formula I on jugron-induced stress in vivo The protective effect of compound 1 against oxidative stress was tested in wild-type N2 strain of Caenorhabditis elegans exposed to juglon, a solid stress factor and pro-oxidant. This strain was obtained from the Center for Nematode Genetics (CGC) at the University of Minnesota. Isolated nematode eggs were cultured in liquid S-basal medium using E. coli OP50 strain as a food source. Growing L4 larvae (young adults) were treated with compound 1 at a final concentration of 50 μmol / l, or the same amount of DMSO (solvent, negative control), and incubated at 20°C for 24 hours. Subsequently, approximately 120 individuals per group were transferred to plates containing juglon at a final concentration of 200 μmol / l to induce lethal oxidative stress. Mortality was counted hourly for 15 hours. The obtained data were analyzed using Kaplan-Meier survival analysis. Survival curves were compared using the Logrank (Mantel-Cox) test in GraphPad Prism software (v.10.2.2).

[0338] The results in Table 28 show that treatment with compound 1 has a positive effect on the survival of individuals subjected to jugron stress.

[0339] [Table 29]

[0340] Example 27: Effect of compound of general formula I on the motility of an animal model of Huntington's disease Synchronized individuals of the C. elegans strain AM140, in which polyglutamic acid repeats are fused with a yellow fluorescent protein (Q35::YFP), were cultured for 7 days at 19°C in S-Complete medium supplemented with compound 1 or DMSO vehicle (negative control) at a final concentration of 50 μmol / l. Subsequently, gently washed individuals were transferred to clear 384-well plates in droplets of M9 buffer at two different time points, allowed to adapt for 30 seconds, and then the number of body flexions / trashes was counted over 60 seconds. Body flexion was defined as a change in the direction of flexion in the midsection of the individual's body. In total, three independent experiments were performed.

[0341] The results of the in vivo experiment are shown in Table 29. Compound 1 significantly increased the motility of the organisms compared to the DMSO control.

[0342] [Table 30]

Claims

1. 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I and pharmaceutically acceptable salts, addition salts, and solvates thereof: 【Chemistry 1】 [In the formula, R １ , R ２ , R ３ and R ４ Each of these is independently selected from the group comprising a hydrogen atom, a hydroxyl group, a C1-C3 alkoxy group, a trifluoromethoxy group, and a difluoromethoxy group; R ５ and R ６ Each is independently selected from the group consisting of C1-C3 alkyl and hydrogen atoms; or R ５ is a hydrogen atom, R ６ is an acyl group or thioacyl group of general formula II, or a sulfonic acid group of general formula III: 【Chemistry 2】 Here X is either O or S; R ７ is selected from the group consisting of R ８ and NH-R ８ where R ８ is selected from the group consisting of C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 member heterocycloalkyl, (CH ２ CH ２ O) ｎ -(C1-C3 alkyl), CH ２ O(CH ２ CH ２ O) ｎ -(C1-C3 alkyl), C6-C12 aryl, 5-9 member heteroaryl, (C6-C12) aryl-(C1-C3) alkyl-, 5-7 member heteroaryl-(C1-C3) alkyl-, 5-7 member heteroaryl-O-(C1-C3) alkyl-, (C1-C3 alkyl)O-C(O)-(C1-C3) alkyl- n is 1, 2, 3, 4, or 5. Substituent R ８ The following are optional: C1-C3 alkyl, C1-C3 alkoxy, OH, halogen, =O, NH ２ NH ２ C(=O)-(C1-C3 alkoxy), NHR ９ It is substituted with at least one substituent selected from the group consisting of R ９ C1-C3 alkyl and NR １０ ２ Selected from, R １０ Each of these is independently selected from C1-C3 alkyl groups, or both R １０ These combine to form C2-C5 alkylenes. However, substituent R １ and R ２ At least one of them is not a hydrogen atom, but a substituent R ３ and R ４ [The condition is that at least one of them is not a hydrogen atom.]

2. R １ , R ２ , R ３ and R ４ Each of these is independently selected from the group comprising a methoxy group and an ethoxy group, comprising 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I according to claim 1.

3. R ５ is a hydrogen atom, R ６ is a base of general formula II, and R ７ Methyl, ethyl, propyl, cyclopropyl, azepanyl, morpholinyl, piperazinyl, phenyl, naphthyl, pyridyl, imidazolyl, pyrrolidinyl, quinuclidinyl, 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)pyrrolidine 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I according to claim 1 or 2, selected from the group comprising nyl, (methoxymethyl)pyrrolidinyl, isopropylamino, N-methylpiperazinyl, aminocarbonylmethoxyphenyl, hydroxypyridyl, methylpyridyl, 6-hydroxy-4-methylpyridine-3-yl, (morpholine-4-yl)ethyl, difluoropyridyl, (methoxyethoxy)ethoxymethyl, pyrazolo[1,5-a]pyrimidinyl, and piperidinylpyridine.

4. R ５ is a hydrogen atom, R ６ R is a sulfonic acid group of general formula III, ７ 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I as described in claim 1 or 2, selected from the group comprising methyl, ethyl, propyl, cyclopropyl, azepanyl, morpholinyl, piperazinyl, phenyl, pyridyl, imidazolyl, pyrrolidinyl, aminopropyl, aminoethyl, aminophenyl, diaminophenyl, N-methylimidazolyl, (fluoro)pyrrolidinyl, (methoxymethyl)pyrrolidinyl, isopropylamino, N-methylpiperazinyl, aminocarbonylmethoxyphenyl, hydroxypyridyl, methylpyridyl, 6-hydroxy-4-methylpyridine-3-yl, and (morpholin-4-yl)ethyl.

5. 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I as described in any one of claims 1 to 4 for use as a pharmaceutical.

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

7. A 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I according to any one of claims 1 to 4, for use in the treatment of a neurodegenerative disease selected from polyglutamine diseases, tauopathies, synucleinopathy, amyotrophic lateral sclerosis, amyloidosis, and cystic fibrosis.

8. 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I according to any one of claims 1 to 4, for use in the prevention of familial forms of neurodegenerative diseases selected from polyglutamine diseases, tauopathies, synucleinopathy, amyotrophic lateral sclerosis, familial amyloidosis, and cystic fibrosis.

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

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

11. A pharmaceutical preparation characterized by containing at least one 4-amino-2,6-bis(phenylmethylene)cyclohexanone of general formula I as described in any one of claims 1 to 4, and at least one pharmaceutically acceptable excipient.

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