Compounds for the treatment of Alzheimer's disease

Abstract

The present invention relates to certain chromanol, quinone, or hydroquinone compounds and their derivatives for the treatment of Alzheimer's disease, improving memory function, and / or reducing plaque burden. Specifically, the present invention relates to chromanol compounds selected from (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-5yl)methanone, ((S)-6-hydroxy-2,5,7,8-tetramethyl-N-((R)-piperidin-3-yl)chroman-2-carboxamide hydrochloride, and S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone, and pharmaceutically acceptable salts thereof.

Description

Technical Field

[0001] The present invention relates to compounds for the treatment of Alzheimer's disease. The present invention further relates to chromanol compounds and their derivatives for improving memory function.

Background Art

[0002] Alzheimer's disease is a progressive neurodegenerative disease and the main cause of dementia in the elderly.

[0003] EP2994160B1 discloses a method for the treatment of Alzheimer's disease by administration of pooled immunoglobulin G in patients suffering from moderate Alzheimer's disease and / or carrying the ApoE4 allele.

[0004] EP2892563B1 describes a method for treating Alzheimer's disease as an adjuvant therapy to acetylcholinesterase therapy, which comprises administering an effective daily dose of N-(2-(6-fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine or a pharmaceutically acceptable salt to a patient in need of such treatment, wherein the effective daily dose administered to the patient is about 30 to about 60 mg.

[0005] EP2937085B1 describes a combination of 6-[4-(1-cyclohexyl-1-H-tetrazol-5-yl)butoxy]-3,4-dihydrocarbostyryl (cilostazol) or a salt thereof, and donepezil or a salt thereof, which shows a synergistic effect for treating Alzheimer's disease.

[0006] WO2002 / 043666 predictively suggests that the use of antioxidants can prevent or reduce mental deterioration. Although antioxidants may indeed reduce the oxidative burden on mitochondria, no clear effect in the treatment of Alzheimer's disease has been found.

[0007] Cai et al. described in ACS Chemical Neuroscience (2017) 8:2496-2511 a drug based on donepezil substituted with a trolox moiety proposed for use in the treatment of Alzheimer's disease. Some in vitro tests suggest the activity of some of the biomarkers of Alzheimer's disease.

[0008] Amyloid beta (Aβ or A-beta) refers to a 36-43 amino acid peptide that is the main component of amyloid plaques found in the brains of people with Alzheimer's disease.

[0009] The peptide is derived from the amyloid precursor protein (APP) and is cleaved by beta-secretase and gamma-secretase to produce Aβ. Aβ molecules can aggregate to form flexible soluble oligomers that can exist in several forms. Currently, certain misfolded oligomers (known as "seeds") are thought to induce other Aβ molecules to take on a misfolded oligomeric form, triggering a chain reaction that leads to plaque formation. Soluble oligomers are toxic to nerve cells, and plaques are formed from soluble oligomers.

[0010] There is still a need for new compounds for the treatment of Alzheimer's disease and related diseases associated with mitochondrial function and health deterioration, particularly compounds with few or preferably no side effects within the dosage range of such compounds.

[0011] An object of the present invention is to provide a compound for the treatment of Alzheimer's disease.

[0012] A further object of the present invention is to provide a compound for improving memory function.

[0013] A further object of the present invention is to provide a compound for reducing the occurrence of beta plaque load in patients experiencing Alzheimer's disease.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0015]

Non-Patent Document 1

Summary of the Invention

[0016] One or more of the above objects are satisfied by providing one or more of the aforementioned treatments with specific chromanol, quinone or hydroquinone compounds.

[0017] The above object is a compound according to formula (I), (II), a hydroquinone analogue of formula (II), or a pharmaceutically acceptable salt thereof for use in the treatment of Alzheimer's disease, for improving memory function and / or for reducing plaque load in Alzheimer's disease patients,

Chemical formula

Chemical formula

[0018] In the case of the present invention, the compounds according to formula (II) include hydroquinone (i.e., hydroquinone) analogs, but quinone derivatives are preferred from the viewpoint of stability.

[0019] In a preferred embodiment, nitrogen can be an amine, quaternary amine, guanidine or imine, oxygen can be a hydroxyl, carbonyl or carboxylic acid, and / or oxygen and nitrogen together can form an amide, urea, or carbamate group.

[0020] In a preferred embodiment, R1 in formula (I) is hydrogen or forms an ester group having 2 to 6 carbon atoms together with 6-oxygen.

[0021] In a preferred embodiment of any of the compounds according to formula (I) or formula (II), R2 and R3, together with the N atom to which they are attached, form a saturated ring incorporating an additional N atom, which ring is unsubstituted or substituted with an alkanol group having 1 to 4 carbon atoms such as an alcohol or ethylol.

[0022] In another preferred embodiment, R2 is a hydrogen atom, R3 has 4 to 7 carbon atoms and contains a saturated cyclic structure having one nitrogen atom, and this ring is unsubstituted or substituted with an alkanol group having 1 to 4 carbon atoms such as alcohol or ethanol.

[0023] According to yet another preferred embodiment, the compound is either (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone (SUL-121), ((S)-6-hydroxy-2,5,7,8-tetramethyl-N-((R)-piperidin-3-yl)chromane-2-carboxamide hydrochloride (SUL-13), or (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone (SUL-109).

[0024] In the most preferred embodiment, the compound is the S-enantiomer of SUL-109, i.e., S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone (SUL-138).

[0025] In a preferred embodiment according to the present invention, any compound according to formula (I) or formula (II) has a molecular weight of less than 500 Da.

[0026] Therefore, for example, in WO2014 / 098586, WO2014 / 011047, and WO2017 / 060432, trolox derivatives are described. However, memory function and plaque formation have not been investigated, and no other types of in vivo or in vitro tests directly related to the treatment of Alzheimer's disease have been conducted.

[0027] WO2019 / 101826 suggests that some compounds containing a trolox moiety can act as MPGES inhibitors, which is suggested to be advantageous for the treatment of inflammatory diseases. WO2019 / 101826 suggests that Alzheimer's disease may act through MPGES, but in the studies of the present inventors, no difference in expression was found between wild-type mice and APP / PS1 mice, indicating that MPGES is not associated with Alzheimer's disease.

[0028] Memory function and plaque formation caused by the polymerization of amyloid-β are considered to be the main problems in Alzheimer's disease. Despite the fact that some antioxidants may reduce the underlying oxidative mechanism, no evidence has been provided that they can actually improve memory function. The current findings indicate that certain trolox derivatives may represent a useful new treatment option for treating Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0029]

Figure 1

Figure 2

Figure 3

[0030] One or more of the above objects are satisfied by the present invention by providing a compound of formula (I) or (II) as shown above, or a pharmaceutically acceptable salt thereof, for use in the treatment of Alzheimer's disease, or for improving memory function, and / or for reducing plaque burden in patients experiencing Alzheimer's disease.

[0031] Preferably, while the memory function is improved, plaque formation is also reduced, thereby enabling further improvement in the treatment of Alzheimer's disease.

[0032] Unless the improvement of the memory function is regarded as medical treatment, the present invention also provides the use of a compound defined for the improvement of the memory function in mammals. The mammal is preferably a human.

[0033] R1 can be a substituent that is easily removed in the human body, and thus the compound is a prodrug. R1 can be, for example, an amino acid derivative or an ester derivative, and generally has a molecular weight of less than 100 Daltons.

[0034] In a preferred embodiment, R1 of formula (I) is hydrogen or forms an ester group having 2 to 6 carbon atoms together with 6-oxygen. The ester can contain one or more ether or alcohol groups. Suitable esters are acetate, butyrate, 3-hydroxybutyrate, etc.

[0035] In a preferred embodiment of any compound according to formula (I) or formula (II), R2 and R3, together with the N atom to which they are attached, form a saturated ring having 3 to 6 carbon atoms and incorporating one additional N atom, which can be substituted with 1 to 4 carbon atoms that can contain an oxygen, carboxylic acid or amine group.

[0036] More preferably, R2 and R3, together with the N atom to which they are attached, form a 5- to 7-membered ring containing one additional amine group, and this ring is optionally substituted with methyl, ethyl, or alcohol-substituted methyl or ethyl.

[0037] In another preferred embodiment, R2 is a hydrogen atom and R3 contains a cyclic structure having 3 to 6 carbon atoms and one nitrogen atom.

[0038] More preferably, R2 is a hydrogen atom, R3 contains a 5- to 7-membered ring containing one additional amine group, this ring is bonded to the amide nitrogen and is optionally substituted with methyl, ethyl, or alcohol-substituted methyl or ethyl.

[0039] In any case, the ring (the cyclic structure formed by R2 and R3, or R3 only) can be unsubstituted or substituted with an alkanol group having 1 to 4 carbon atoms such as alkyl, alcohol, or ethynol having 1 to 4 carbon atoms.

[0040] In a preferred embodiment according to the present invention, any compound according to formula (I) or formula (II) has a molecular weight of less than 500 Da.

[0041] Certain chromanol compounds are described in WO2014 / 098586. The compounds described in detail have abbreviations referring to SUL-XXX (XXX is a two- or three-digit number). Many of these compounds are racemic mixtures, although some enantiomers have also been tested. Suitable methods for preparing the chromanol compounds according to the present invention are described in WO2014 / 098586 or WO2014 / 011047.

[0042] WO2017060432A1 discloses amide derivatives of 2-hydroxy-2-methyl-4-(3,5,6-trimethyl-1,4-benzoquinone-2-yl)-butanoic acid and methods for making such compounds.

[0043] Hydroquinone derivatives can be easily prepared by hydrogenation of the quinone structure.

[0044] According to yet another preferred embodiment, the compound is either (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone (SUL-121), ((S)-6-hydroxy-2,5,7,8-tetramethyl-N-((R)-piperidin-3-yl)chroman-2-carboxamide hydrochloride (SUL-13), or (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone (SUL-109).

[0045] In the most preferred embodiment, the compound is the S-enantiomer of SUL-109, namely S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone (SUL-138).

[0046] The counterion in the pharmaceutically acceptable salt can be a counterion known in the art. Preferably, the compound has at least one basic nitrogen, an amine, that can be protonated. The counterion is preferably a halogen such as chloride, sulfate, citrate, formate, most preferably chloride.

[0047] These compounds are effective as racemic mixtures or in substantially pure enantiomeric form. The compounds generally have one or two chiral centers, typically one or two.

[0048] Preferably, the compound is a substantially enantiomerically pure compound. Substantially enantiomerically pure means an enantiomeric excess of about 95% or more, more preferably about 98% enantiomeric excess, most preferably about 99% or more enantiomeric excess. Also, when the compound contains multiple chiral centers, these amounts apply.

[0049] The compound is preferably used in an effective amount to achieve improvement in memory function and / or to achieve treatment of Alzheimer's disease.

[0050] The term "treatment" encompasses reduction of the progression of a disease and / or improvement of the symptoms of a disease.

[0051] The effect is generally observed at an amount of about 1 μM in body fluids, but more amounts are preferably used. Preferred amounts are in vitro or in vivo concentrations of about 10 μM or more, more preferably about 20 μM or more. Generally, the concentration in humans is sufficient and safe at about 200 μM or less.

[0052] When used in humans, this means a dosage of about 10 mg or more, assuming a volume of distribution of 30 L, 100% efficacy, and a concentration of about 1 μM. Preferred amounts would result in a concentration of about 10 μM, for which a dosage of about 100 mg or more would be appropriate. Thus, preferably, dosage forms of about 20 mg or more, preferably 50 mg or more, preferably 100 mg or more are appropriate. Generally, solid, oral dosage forms contain a maximum of about 500 mg of the compound, preferably about 450 mg or less, to allow for excipients. More amounts can be administered by intravenous injection or other liquid dosage forms.

[0053] Examples of dosages that can be used are dosages of 0.2 mg / kg or more of the compound of the present invention, for example, preferably in the range of about 1 mg / kg to about 100 mg / kg, or in the range of about 2 mg / kg to about 40 mg / kg body weight, or in the range of about 3 mg / kg to about 30 mg / kg body weight, or in the range of about 4 mg / kg to about 15 mg / kg body weight. The compounds of the present invention can be administered in a once-daily dose, or the total daily dosage can be administered in divided doses of 2, 3, or 4 times a day.

[0054] The compounds described herein can be prepared as pharmaceutical compositions by incorporating pharmaceutically or physiologically acceptable excipient carriers, and additives such as vehicles.

[0055] Suitable pharmaceutical or physiologically acceptable excipients, carriers and vehicles include, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-P-cyclodextrin, polyvinylpyrrolidone, low melting wax, etc., as well as processing agents, drug delivery regulators and enhancers such as any combination of two or more of them. Other suitable pharmaceutically acceptable excipients are described in "Remington’s Pharmaceutical Sciences", Mack Pub.Co., New Jersey (1991).

[0056] The pharmaceutical composition preferably includes unit dosage formulations, and the unit dosage is a dosage sufficient to have a therapeutic effect. The unit dosage can be a dosage that is regularly administered during the process of treating or suppressing the disorder.

[0057] Furthermore, the unit dosage can be a dosage that is regularly administered during the process of treatment to improve the original cognitive function related to memory.

[0058] The compounds of the present invention can be administered enterally, orally, parenterally, sublingually, by inhalation (e.g., as a mist or spray), rectally, or topically, in dosage unit formulations containing conventional non-toxic pharmaceutically or physiologically acceptable carriers, adjuvants, and vehicles, as required. As used herein, the term parenterally includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. The compounds are mixed with pharmaceutically acceptable carriers, adjuvants, and vehicles suitable for the desired route of administration.

[0059] Oral administration is the preferred route of administration, and formulations suitable for oral administration are the preferred formulations.

[0060] The compounds described for use herein can be administered in solid form, liquid form, aerosol form, or in the form of tablets, pills, powder mixtures, capsules, granules, injection solutions, creams, liquids, suppositories, enemas, colonics, emulsions, dispersions, food premixes, and other suitable forms. The compounds can also be administered in liposomal formulations.

[0061] Injectable formulations, for example, sterile aqueous or oleaginous suspensions, can be prepared according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable formulations can also be sterile injectable solutions or suspensions in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in propylene glycol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any bland fixed oil containing synthetic monoglycerides or diglycerides can be used. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0062] Suppositories for rectal administration of a drug can be prepared by mixing the drug with suitable non-irritating excipients such as cocoa butter and polyethylene glycol, which are solid at room temperature but liquid at rectal temperature and therefore melt in the rectum to release the drug.

[0063] Solid dosage forms for oral administration can include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound can be mixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms can also include additional substances other than the inert diluent, for example, lubricants such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms can also include buffering agents. Tablets and pills can be further prepared with enteric coatings.

[0064] For oral administration, liquid dosage forms may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art such as water. Such compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, cyclodextrins, sweetening agents, flavoring agents, and perfumes.

[0065] The amount of active ingredient that can be combined with a carrier material to produce a single-dose form will vary depending on the host to which the active ingredient is administered and the particular mode of administration. The selected unit dose is usually manufactured and administered to provide a defined final concentration of the drug in the blood, tissue, organ, or other target area of the body. The effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician or artisan.

[0066] The present invention will be further illustrated using the following examples. In the examples, reference is made to the figures.

Example

[0067] Example 1 The effectiveness of the compounds according to the present invention for the treatment of Alzheimer's disease was tested by two independent tests, one reflecting memory and the other showing synaptic connections.

[0068] Details of the method and experiment The APP / PS1 mouse model is a model of Aβ pathology (one of the two major neuropathological features of Alzheimer's disease (AD)), which is widely used in Alzheimer's disease (AD). These mice contain human transgenes of APP (Swedish mutation) and PSEN1 (L166P mutation), which lead to pathological amyloid deposition in the brain starting at about 3 months of age (3 moa) and impairment of hippocampal-dependent memory and long-term potentiation (LTP).

[0069] The effectiveness of SUL-138 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone) in alleviating / preventing common pathological conditions was tested in the APP / PS1 model. The effect on memory was tested in a hippocampus-dependent contextual test (fear conditioning (FC)), and synaptic connections were tested by electrophysiological LTP (long-term potentiation) measurements. In this mouse model, both are impaired under basal conditions. Furthermore, the Phenotyper (Sylics) was used to exclude that SUL-138 induces stereotyped behavior after chronic oral treatment.

[0070] Wild-type (WT) and APP / PS1 mice were each divided into two groups and given either vehicle or SUL-138 via their diet. The group size was 12. Based on the mouse body weight of approximately 30 g, feed intake of approximately 5 g / day, and desired oral intake of 30 mg / day / kg, the feed pellets were sprayed with SUL-138 dissolved in water containing 0.0145% ethanol at 1 g of SUL-138 per 5 kg of feed. The vehicle diet was prepared by spraying the same amount of water containing 0.0145% ethanol.

[0071] Prior to the test, the mice were chronically treated at 2.5 moa (pre-pathology / memory impairment) to 6 moa (the age at which obvious neuropathology and memory impairment occur).

[0072] FC: After exposing the mice to the context for 2 minutes, a 0.7 mA foot shock was given. Thirty seconds after the foot shock, the mice were returned to their home cages. Twenty-four hours later, the mice were placed in the same context and the freezing level was measured for 2 minutes.

[0073] LTP: Acute coronal hippocampal slices were maintained in artificial CSF and LTP was measured after 3 × 100 Hz stimulation.

[0074] Phenotyper (provided by Sylics, Amsterdam, Netherlands): Mice were housed in the Phenotyper for 3 days, during which spontaneous behaviors: activity, dark / light, habituation, kinematics, light / dark transition pattern, and avoidance were measured.

[0075] Results Overall well-being was monitored and showed no difference between vehicle-treated animals and SUL-138-treated animals, with similar weight gain in all groups.

[0076] Memory was evaluated at 6 moa by measuring freezing after context acquisition.

[0077] Figure 1 shows how chronic SUL-138 treatment increased memory (freezing %) in WT and APP mice. SUL-138 treatment increased freezing levels (memory) in both WT and APP mice. Student's t-test, *: p < 0.05, **: p < 0.01.

[0078] When treated with a control diet, APP / PS1 mice showed a predicted decrease in freezing compared to WT mice. Memory after chronic SUL-138 treatment in APP / PS1 mice recovered to WT levels. This indicates that SUL-138 is effective in preventing or improving Alzheimer's disease and / or its symptoms.

[0079] Interestingly, WT mice administered SUL-138 also showed excellent performance in the memory task. This indicates that SUL-138 is also effective in improving the memory function of healthy mammals.

[0080] Figure 2 shows how SUL-138 increases LTP maintenance in both WT and APP mice. LTP was induced by 1 s of 3 × 100 Hz stimulation (tetanus) at 20 s intervals in 8 - 14 hippocampal slices per group (2A: WT control, WT SUL-138, 2B: APP control, APP SUL-138). The slope was measured for 60 minutes. LTP was expressed as a percentage of the baseline. All LTP data analysis was performed blindly. Maintenance of LTP (30 - 60 minutes) was significantly (p < 0.05) higher in SUL-138 animals (both WT and APP). Student's t - test, *P < 0.05; 2C.

[0081] Chronic SUL-138 treatment did not induce differences in spontaneous behavior, and activity, dark / light, habituation, kinematics, light / dark transition pattern, and withdrawal were measured.

[0082] Conclusion The examples show that SUL-138 increases memory and LTP in both WT and APP / PS1 mice and effectively restores memory and LTP to control levels in APP / PS1 mice.

[0083] The increase in both of these parameters reflects a general increase in plasticity / LTP facilitation processes stimulated by SUL-138. This finding means that SUL-138 can be used to alleviate the symptoms of neurological diseases that show reduced synaptic strength or plasticity.

[0084] The effect of SUL-138 seems to be specific to memory improvement since no stereotyped behavior was introduced into the mice by the treatment after 3 months of chronic treatment. Furthermore, no differences in body weight were measured during 3 months of chronic oral treatment that could indicate aversion or toxic behavior towards the SUL-138 - treated diet, or changes in major physiological functions.

[0085] Finally, no issues of animal welfare and no differences between groups occurred throughout the experiment.

[0086] Example 2 Reduction of plaque burden in APP / PS1 mice after intervention with SUL-138 APP / PS1 (n = 10) and WT mice (WT, n = 10) were treated with either vehicle or SUL-138. Mice were treated with either SUL-138 or vehicle diet pellets starting at 3 months of age for 3 months. Mice were sacrificed at 6 months of age, an age at which hippocampus-dependent memory impairment and overt plaque burden are expected.

[0087] 4% PFA-perfused brains stored in sucrose were sectioned at 35 μM using a cryostat (-20 °C, Leica). Hippocampal sections (n = 2 / animal, 5 animals / group) were washed with 1× PBS 3× for 10 minutes and then blocked with blocking solution (10 mL of 1× PBS + 500 μL of normal goat serum + 0.250 g of bovine serum albumin + 20 μL of Triton-100) for 1 hour. Sections were incubated overnight with anti-amyloid beta (6E10) (ITK Diagnostics, 1:400), washed with 1× PBS 3× for 10 minutes, and then incubated with secondary goat anti-mouse Alexa Fluor 488 antibody (Sigma-Aldrich, 1:250) for 2 hours. Next, sections were washed with 1× PBS 3× for 10 minutes and mounted on slides.

[0088] Sections were imaged using a Zeiss Cell Discover 7 high-content microscope equipped with an LSM900 confocal head. Using Fiji, both hippocampi were separately selected for 5 animals per group (yellow lines in Figure 3A), and the number and size of plaques were measured (Figure 3B, C). Mean plaque number and plaque size per animal were used for statistical analysis with GraphPad 8 using Student's one-sided t-test.

[0089] Oral SUL-138 for 3 months reduced both the plaque number (Figure 3B, p = 0.0138) and plaque size (Figure 3C, p = 0.0021) in APP / PS1 mice compared to vehicle-treated mice. SUL-138 and vehicle-treated WT animals did not show plaques.

[0090] These data indicate that SUL-138, together with rescuing memory and increasing synaptic transmission (long-term potentiation) in APP / PS1 mice observed in Example 1, is a promising therapeutic option for Alzheimer's disease.

[0091] The bioavailability of SUL-138 in the brain appears to be high, thereby overcoming the problems of other mitochondrial target compounds and becoming a more suitable therapeutic option for future clinical applications.

[0092] Example 3 In vitro assays demonstrating the activity of the compounds according to the invention.

[0093] Excitotoxicity is a process in which neurons are damaged or die when, otherwise necessary and safe, neurotransmitter levels become pathologically high, resulting in overstimulation of receptors. Excitotoxicity may be involved in neurodegenerative diseases of the central nervous system such as Alzheimer's disease.

[0094] In vitro assays for examining excitotoxicity utilize inducers with well-defined characteristics of neuronal death (e.g., glutamate, dopamine, or NDMA) and quantification of the cell viability of stimulated neuron-like cells. The SH-SY5Y cell line derived from human neuroblastoma can be differentiated in vitro to morphologically and biochemically resemble mature neurons. Furthermore, differentiated SH-SY5Y neuron-like cells are sensitive to excitotoxicity induced particularly by glutamate and dopamine.

[0095] In this current study, the effectiveness of SUL-11, SUL-127, SUL-13, SUL-138 (and its primary metabolite SUL-138M2), SUL-150 and SUL-151 in inhibiting glutamate and dopamine-induced excitotoxicity in human SH-SY5Y neuronal-like cells was investigated. SUL-11 is trolox and SUL-127 is the methyl ester of trolox. These two compounds were used as references.

[0096] The compounds used in this study are shown in Table 1 below.

Table 1

Table 2

[0097] Human SH-SY5Y neuroblastoma cells (ATCC#CRL-2266) were maintained in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (#P4333, Sigma-Aldrich, St. Louis, MO) and passaged until the cultures reached 70% confluence. Prior to the experiment, SH-SY5Y cells were differentiated by serum reduction (to 1%) and stimulation with 10 μM retinoic acid (#R7882, Sigma-Aldrich, St. Louis, MO) for 72 h. Differentiated SH-SY5Y cells were seeded at 0.6×10 5 cells / cm 2 in all experiments.

[0098] Differentiated SH-SY5Y cells were treated with SUL compounds (dose range 8×10 -4 ~1×10 -8Pre-incubated with (M) for 30 minutes under standard culture conditions and then further stimulated with either 1-glutamic acid (60 mM, #12843-0, Sigma-Aldrich, St. Louis, MO) or dopamine (100 μM, #H8502, Sigma, St. Louis, MO) for 24 hours. Neutral Red assay solution (#N2889, Sigma-Aldrich, St. Louis, MO) was added to the cultures at a concentration of 10% (v / v) during the last 4 hours of the culture. Cells were washed with warm PBS and Neutral Red solubilized in acidic ethanol (1% acetic acid in 50% EtOH). Absorbance was recorded at 540 nm using a CLARIOStar Plus plate reader (BMG Labtech, Germany). Cell viability was normalized to the absorbance measurements of untreated cultures (100% viability) and absorbance measurements of cell-free samples (0% viability).

[0099] All experiments were performed in triplicate for each condition and averaged. Data obtained from two separate experiments were used for evaluation with GraphPad Prism 8.0 (GraphPad Software Inc, Ca). Four-parameter non-linear regression was used to determine the efficacy and potency of SUL compounds that reduce excitotoxicity induced by either 1-glutamic acid or dopamine. The efficacy of SUL compounds that inhibit excitotoxicity was calculated as follows: E max = 100 * V (処置済み) - V (ビヒクル) ) / 100% - V (ビヒクル) ) where V is the observed viability and E max is the maximum effect caused by the SUL compound treatment.

[0100] When SUL-compounds in the molar ranges shown in the table below were used, cytotoxicity was not observed as a decrease in viability.

[0101] SH-SY5Y neuroblastoma cells were differentiated into neuron-like cells according to an established protocol and stimulated with 60 mM glutamate to induce excitotoxicity. Glutamate decreased the viability of SH-SY5Y cells from 100 ± 1.63% of vehicle-treated control cells to 55.4 ± 1.7% of SH-SY5Y cells exposed to glutamate for 24 h (p < 0.0001). Differentiated SH-SY5Y cells were pre-incubated with SUL compounds (10 -3 ~10 -8 M), and the cell viability of SH-SY5Y cells challenged with glutamate increased dose-dependently, although at different levels. As shown in Table 2 below, trolox and the methyl ester of trolox were clearly less effective than the other SUL compounds.

[0102] Differentiated SH-SY5Y neuroblastoma cells were stimulated with 150 μM dopamine to induce excitotoxicity. Dopamine decreased the viability of SH-SY5Y cells from 100 ± 0.8% in vehicle-treated control cells to 50.5 ± 1.0% in SH-SY5Y cells exposed to dopamine for 24 h (p < 0.0001). Differentiated SH-SY5Y cells were pre-incubated with SUL compounds (10 -3 ~10 -8 M), and as shown in Table 2 below, the effectiveness varied, but the cell viability of dopamine-challenged SH-SY5Y cells increased dose-dependently. In this model, all compounds decreased cell viability at a dose level of 10 -3 M.

Table 3

[0103] The results in the table indicate that the SUL compounds according to the present invention exhibit either improved EC50 (i.e., active at lower concentrations) and / or improved Emax (i.e., a higher level of recovery from toxicity). Thereby, this example shows that, next to SUL-138, other claimed SUL compounds are also likely to show the advantage of improving memory function and / or reducing plaque formation, i.e., generally being advantageous for the treatment of Alzheimer's disease.

[0104] Reference experiment A The hippocampal tissues of WT and APP / PS1 mice were examined for the protein expression of prostaglandin synthase and thromboxane synthase A. Peptides similar to prostaglandin synthases PTGS1, PTGES2, PTGES3, and PTGFS were found in the hippocampal tissues of both WT and APP / PS1 mice (Table 4). Protein fragments of PTGS2, PTGDS, PTGES1, PTGIS, and TXA were not found. Treatment with SUL-138 in either WT or APP / PS1 mice did not change the protein expression of prostaglandin synthase.

Claims

1. A composition for treating Alzheimer's disease, improving memory function, and / or reducing plaque burden in Alzheimer's disease patients, comprising a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof, wherein: 【Chemical 1】 【Chemical 2】 wherein R1 is hydrogen or forms an ester group having 2 to 6 carbon atoms together with 6-oxygen; R2 and R3 together with the N atom to which they are attached form a saturated or unsaturated, non-aromatic, optionally substituted 5- to 8-membered ring having 1 to 4 N, O, or S atoms, and R2 and R3 together contain 3 to 12 carbon atoms, or or R2 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R3 is an alkyl group optionally substituted with nitrogen or oxygen, said alkyl group contains 3 to 12 carbon atoms, and the alkyl group in R3 contains one or more non-aromatic cyclic structures and may contain linear and / or branched groups and one or more ethylenic unsaturations.

2. In any compound of formula (I) or formula (II), R2 and R3 together with the N atom to which they are attached form a saturated ring incorporating an additional N atom, and this ring is unsubstituted or substituted with an alcohol or an alkanol group having 1 to 4 carbon atoms. The composition according to claim 1.

3. The composition according to claim 2, wherein the compound is a compound of formula I.

4. R2 and R3 together with the N atom to which they are attached form a 5- to 7-membered ring containing one additional amine group, and this ring is optionally substituted with methyl, ethyl, or alcohol-substituted methyl or ethyl. The composition according to claim 3.

5. The composition according to claim 1, wherein R2 is a hydrogen atom, R3 has 4 to 7 carbon atoms and contains a saturated cyclic structure having 1 nitrogen atom, and this ring can be substituted with a group having 1 to 4 carbon atoms which may contain an alkyl group, an alcohol group, or an oxygen, carboxylic acid or amine group.

6. The composition according to claim 5, wherein the compound is a compound of formula II, wherein R2 is a hydrogen atom, R3 has 4 to 6 carbon atoms and contains a cyclic structure having 1 nitrogen atom, and this ring is optionally substituted with methyl, ethyl, or alcohol-substituted methyl or ethyl.

7. The composition according to claim 1, wherein the compound is (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone (SUL-121), ((S)-6-hydroxy-2,5,7,8-tetramethyl-N-((R)-piperidin-3-yl)chroman-2-carboxamide hydrochloride (SUL-13) or (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone (SUL-109).

8. The composition according to claim 7, wherein the compound is the S-enantiomer of SUL-109: S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone (SUL-138).

9. The composition according to any one of claims 1 to 6, wherein the compound according to formula (I) or formula (II) has a molecular weight of less than 500 Da.

10. The composition according to any one of claims 1 to 9 for treating Alzheimer's disease.

11. The composition according to any one of claims 1 to 10 for improving memory function.

12. The composition according to any one of claims 1 to 11 for reducing plaque load in Alzheimer's disease patients.

13. The composition according to claim 11 for improving memory function in mammals.

14. The composition according to claim 13, wherein the mammal is a human.

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