EA048112B1 - COMPOUNDS FOR THE TREATMENT OF ALZHEIMER'S DISEASE - Google Patents

Abstract

The invention relates to the use of 8-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone (SUL-138) or a pharmaceutically acceptable salt thereof for improving memory function and/or reducing the number of plaques in a patient with Alzheimer's disease. The technical result achieved by the present invention consists in improving memory function and reducing the formation of beta plaques in patients suffering from Alzheimer's disease.

Description

I. Field of technology

The present invention relates to compounds for the treatment of Alzheimer's disease. The present invention also relates to chromanol compounds and derivatives thereof for improving memory function.

II. Description of the Prior Art

Alzheimer's disease is a progressive neurodegenerative disorder and a leading cause of dementia in older adults.

EP 2994160 B1 describes a method for the treatment of Alzheimer's disease in patients suffering from moderate Alzheimer's disease and/or in patients who are carriers of the ApoE4 allele by administering mixed immunoglobulin G.

EP 2892563 B1 describes methods for treating Alzheimer's disease as an adjunct to acetylcholinesterase therapy, comprising administering to a patient in need of such treatment 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 thereof, wherein the effective daily dose administered to the patient is from about 30 to about 60 mg.

EP 2937085 B1 describes that the combination of 6-[4-(1-cyclohexyl-1-tetrazol-5,5-yl)butoxy]-3,4-dihydrocarbostyril (cilostazol) or its salt and donepezil or its salt has a synergistic effect in the treatment of Alzheimer's disease.

WO 2002/043666 suggested that the use of antioxidants may prevent or reduce cognitive decline. Although antioxidants may indeed reduce oxidative stress in the mitochondria, no clear effect was found in the treatment of Alzheimer's disease.

Cai et al. in ACS Chemical Neuroscience (2017), 8:2496–2511 describe Trolox-based donepezil-based drugs proposed for use in the treatment of Alzheimer's disease. Several in vitro assays indicate some activity against some biomarkers of Alzheimer's disease.

Beta-amyloid (Λβ or Abeta) refers to peptides of 36-43 amino acids that are the main component of amyloid plaques found in the brains of people with Alzheimer's disease.

The peptides are formed from amyloid precursor protein (APP), which is cleaved by beta-secretase and gamma-secretase to form Aβ. Aβ molecules can aggregate to form flexible, soluble oligomers that can exist in several forms. It is now believed that some misfolded oligomers (known as seeds) can induce other Aβ molecules to also adopt the misfolded oligomeric form, causing a chain reaction that leads to plaque formation. Soluble oligomers are toxic to nerve cells, and soluble oligomers form plaques.

There remains a need for new compounds for the treatment of Alzheimer's disease and related diseases associated with deterioration of mitochondrial function and health, particularly compounds that have fewer side effects or preferably no side effects at all over the dosage range of such compound.

The object of the present invention is to provide compounds for the treatment of Alzheimer's disease.

Another object of the present invention is to provide compounds for improving memory function.

An additional object of the present invention is to provide compounds for reducing beta plaque formation in a patient suffering from Alzheimer's disease.

III. Essence of the invention

One or more of the above problems are solved by providing certain chromanol, quinone or hydroquinone compounds for one or more of the above methods of treatment.

The above mentioned problems are solved by the present invention by providing compounds of formula (I), (II), a hydroquinone analogue of formula (II) or a pharmaceutically acceptable salt thereof for use in the treatment of Alzheimer's disease or for improving memory function and/or for reducing the amount of plaques in a patient with Alzheimer's disease:

- 1 048112 si.

t.H,

wherein R1 is hydrogen or a prodrug moiety that can be removed in living tissue, and wherein either 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 containing from one to four N, O or S atoms, wherein R2 and R3 together contain from 3 to 12 carbon atoms;

or R2 is a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms, and R3 is an alkyl group optionally containing nitrogen or oxygen as a substituent, wherein said alkyl group contains from 3 to 12 carbon atoms, wherein the alkyl group in R3 comprises one or more non-aromatic cyclic structures and may contain linear and/or branched groups and one or more ethylene double bonds.

For the purposes of the present invention, the compound of formula (II) includes a hydrogenated quinone analogue (i.e., hydroquinone), although a quinone derivative is preferred from a stability standpoint.

According to a preferred embodiment, the nitrogen may be an amine, quaternary amine, guanidine or imine, and the oxygen is hydroxyl, carbonyl or carboxylic acid and/or the oxygen and nitrogen together may form amide, urea or carbamate groups.

According to a preferred embodiment, R1 in formula (I) represents hydrogen or, together with oxygen in position 6, forms an ester group containing from 2 to 6 carbon atoms.

According to a preferred embodiment of any of the compounds of formula (I) or formula (II), R2 and R3 together with the N atom to which they are attached form a saturated ring including an additional N atom, wherein said ring is unsubstituted or contains as a substituent an alcohol or an alkanol group containing from 1 to 4 carbon atoms, such as ethylol.

According to another preferred embodiment, R2 represents a hydrogen atom and R3 comprises a saturated cyclic structure containing from 4 to 7 carbon atoms and containing one nitrogen atom, wherein said ring is unsubstituted or contains as a substituent an alcohol or an alkanol group containing from 1 to 4 carbon atoms, such as ethylol.

According to another preferred embodiment, the proposed compound is (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone (SUL-121), ((8)-6-hydroxy-2,5,7,8-tetramethyl-N-((B)-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).

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

According to a preferred embodiment of the invention, the molecular weight of the compound of formula (I) or formula (II) is less than 500 Da.

Trolox derivatives as such are described, for example, in WO 2014/098586, WO 2014/011047 and WO 2017/060432. However, memory function or plaque formation have not been investigated, nor has another type of in vivo or in vitro test been described that is directly relevant to the treatment of Alzheimer's disease.

WO 2019/101826 suggested that some compounds containing the trolox moiety may act as an inhibitor of MPGES (microsomal prostaglandin E synthase), which is believed to be useful in the treatment of inflammatory diseases. WO 2019/101826 suggested that Alzheimer's disease may act through MPGES, however, this study did not find any differences in expression in wild-type mice compared to APP/PS1 (presenilin-1) mice, indicating that MPGES is not relevant to Alzheimer's disease.

Memory function and plaque formation caused by amyloid-P polymerization are considered to be the main

-2048112 trolox derivatives may help improve memory function in Alzheimer's disease. Although some antioxidants may reduce the underlying oxidative pathways, no evidence has been provided that memory function can actually be improved. The present results suggest that certain trolox derivatives may represent an important new treatment option for Alzheimer's disease.

IV. Brief description of graphic materials

Fig. 1 shows how chronic treatment with SUL-138 enhances memory (freezing, %) in wild-type (WT) and APP mice.

Figure 2 shows how SUL-138 enhances LTP maintenance in both WT and APP mice.

Fig. 3 shows that treatment with SUL-138 reduces the number and size of plaques in APP/PS1 mice.

V. Detailed description of the invention

The present invention solves one or more of the above problems by providing compounds of formula (I) or (II) as shown above, or pharmaceutically acceptable salts thereof, for use in the treatment of Alzheimer's disease or for improving memory function and/or for reducing the amount of plaque in a patient suffering from Alzheimer's disease.

Memory function is preferably improved and at the same time plaque formation is reduced, thus allowing for further improvement in the treatment of Alzheimer's disease.

Since improving memory function is not considered a therapeutic treatment, the present invention also provides the use of said compounds as defined for improving memory function in a mammal. The mammal is preferably a human.

R1 may be a substituent that is easily removed in the human body, so that the proposed compound is a prodrug. R1 may be, for example, an amino acid derivative or an ester derivative and typically has a molecular weight of less than 100 Da.

According to a preferred embodiment, R1 in formula (I) is hydrogen or, together with oxygen in position 6, forms an ester group containing from 2 to 6 carbon atoms. The ester may contain one or more ether or alcohol groups. Suitable esters are acetate, butyrate, 3-hydroxybutyrate, etc.

According to a preferred embodiment of any of the compounds of formula (I) or formula (II), R2 and R3 together with the N atom to which they are attached form a saturated ring containing from 3 to 6 carbon atoms and including one additional N atom, which may contain as a substituent from 1 to 4 carbon atoms, which may contain oxygen, a carboxyl group or an amino group.

More preferably, R2 and R3 together with the N atom to which they are attached form a 5-7 membered ring containing one additional amino group, wherein said ring optionally contains methyl, ethyl or an alcohol containing methyl or ethyl as a substituent.

According to another preferred embodiment, R2 represents a hydrogen atom and R3 comprises a cyclic structure containing from 3 to 6 carbon atoms and containing one nitrogen atom.

More preferably, R2 is a hydrogen atom and R3 comprises a 5-7 membered ring containing one additional amino group, said ring being attached to the amide nitrogen, and said ring optionally containing methyl, ethyl or an alcohol containing methyl or ethyl as a substituent.

In any case, said ring (the cyclic structure formed by R2 and R3 or R3 alone) may be unsubstituted or may contain as a substituent an alkyl group containing from 1 to 4 carbon atoms, an alcohol or an alkanol group containing from 1 to 4 carbon atoms, such as ethylol.

According to a preferred embodiment of the invention, the molecular weight of the compound of formula (I) or formula (II) is less than 500 Da.

Some chromanol compounds have been described in WO 2014/098586. These compounds are abbreviated to SUL-XXX (where XXX is a two- or three-digit number). Many of these compounds are racemic mixtures, although some enantiomers have also been investigated. Suitable methods for preparing the chromanol compounds of the present invention are described in WO 2014/098586 or WO 2014/011047.

WO 2017060432 A1 describes amide derivatives of 2-hydroxy-2-methyl-4-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)butanoic acid and methods for preparing such compounds.

Hydrogenated quinone derivatives can be easily prepared by hydrogenating the quinone structure.

According to another preferred embodiment, the proposed compound is (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone (SUL-121), hydro

- 3 048112 chloride (^)-6-hydroxy-2,5,7,8-tetramethyl-N-(^)-piperidin-3-yl)chroman-2-carboxamide (SUL-13) or (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone (SUL-109).

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

The counterion in the pharmaceutically acceptable salt may be a counterion known in the art. The present compounds preferably contain at least one basic nitrogen, an amine, which may be protonated. The counterion is preferably a halogen such as chloride, sulfate, citrate, formate, etc., most preferably chloride.

The compounds disclosed are effective as a racemic mixture or in substantially pure enantiomeric form. The compounds disclosed have one or more chiral centers, typically one or two.

The proposed compound is preferably a substantially enantiomerically pure compound. Substantially enantiomerically pure corresponds to an enantiomeric excess of about 95% or more, more preferably an enantiomeric excess of about 98%, and most preferably an enantiomeric excess of about 99% or more. In addition, in the case where the proposed compound contains more than one chiral center, the indicated amounts are used.

The proposed compounds are preferably used in effective amounts to provide an improvement in memory function and/or to provide treatment for Alzheimer's disease.

The term treatment includes slowing the progression of the disease and/or relieving the symptoms of the disease.

Generally, effects are observed at amounts of about 1 μM in body fluids, but higher amounts are preferably used. Preferred amounts are in vivo or in vitro concentrations of about 10 μM or higher, more preferably about 20 μM or higher. Generally, a concentration in humans of about 200 μM or lower should be sufficient and safe.

For human use, this would mean - assuming a distribution volume of 30 l, an availability of 100% and a concentration of about 1 μM - a dosage of about 10 mg or more. Preferred amounts provide a concentration of about 10 μM, for which a dose of about 100 mg or more would be suitable. Therefore, dosage forms of about 20 mg or more, preferably 50 mg or more, preferably 100 mg or more are preferably suitable. Solid oral dosage forms typically contain a maximum of about 500 mg of compound, preferably about 450 mg or less, taking into account excipients. Larger amounts can be administered when other liquid forms are administered intravenously.

Examples of dosages that can be used are an effective amount of the compounds of the present invention in a dose of 0.2 mg/kg or higher, for example, preferably in the range of about 1 to about 100 mg/kg, or in the range of about 2 to about 40 mg/kg body weight, or in the range of about 3 to about 30 mg/kg body weight, or in the range of about 4 to about 15 mg/kg body weight. The compounds of the present invention can be administered in a single daily dose, or the total daily dose can be administered in a divided dose of two, three or four times a day.

The compounds described herein can be prepared as pharmaceutical compositions by formulating a mixture with additives such as pharmaceutically or physiologically acceptable excipients, carriers and vehicles.

Suitable pharmaceutically or physiologically acceptable excipients, carriers and vehicles include emulsifiers and drug delivery modifiers and enhancers such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low melting waxes and the like, as well as combinations of any two or more of these substances. Other suitable pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991).

The pharmaceutical composition preferably comprises a dosage form in the form of a single dose, wherein said single dose is a dose sufficient to provide a therapeutic effect. The single dose may be a dose administered periodically during the treatment or suppression of the disorder.

In addition, a single dose may be a dose administered periodically during treatment to improve innate cognitive functions related to memory.

The compounds of the present invention may be administered enterally, orally, parenterally, sublingually, by inhalation (e.g., as aerosols or sprays), rectally, or topically in unit dosage forms containing conventional non-toxic pharmaceutically or physiologically acceptable carriers, adjuvants, and excipients, as desired. As used herein, the term

- 4 048112 parenteral includes subcutaneous injections, intravenous, intramuscular, intranasal injections or infusion methods. The proposed compounds are mixed with pharmaceutically acceptable carriers, adjuvants and excipients suitable for the required route of administration.

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

The compounds described for use in the present invention can be administered in solid form, in liquid form, in aerosol form, or in the form of tablets, pills, powder mixtures, capsules, granules, injections, creams, solutions, suppositories, enemas, intestinal irrigations, emulsions, dispersions, food mixtures and other suitable forms. The proposed compounds can also be administered in liposomal formulations.

Injectable preparations, for example sterile aqueous or oleaginous injection suspensions, can be prepared according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injection solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in propylene glycol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are usually used as a solvent or suspending medium. Any bland fixed oil can be used for this purpose, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectable dosage forms.

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

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

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water. These compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, cyclodextrins and sweetening, flavoring and aromatizing additives.

The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will vary depending on the host to which the active ingredient is administered and the particular route of administration. Typically, a selected single dosage is prepared and administered to provide a specific final concentration of the drug in the blood, tissues, organs, or other target areas of the body. An effective amount for a particular situation can be readily determined by routine experimentation and is within the skill and judgment of an ordinary clinician or skilled in the art.

The present invention will be further illustrated by means of the following examples. In these examples, reference is made to the graphic materials.

VI. Examples

Example 1.

The efficacy of the compounds of the present invention for the treatment of Alzheimer's disease was investigated using two independent tests: a reflex memory test and a test showing the configuration of synaptic connections.

Methods and details of the experiment.

The APP/PS1 mouse model is a widely used A-beta-based pathology model for Alzheimer's disease (AD) (1 of the 2 major neuropathological hallmarks of AD). These mice contain human transgenes for APP (Swedish mutation) and PSEN1 (L166P mutation), resulting in abnormal amyloid deposition in the brain and impairments in hippocampus-dependent memory and long-term potentiation (LTP) beginning at ~3 months of age (3 moa).

The efficacy of SUL-138 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone) in alleviating/preventing global pathology in the APP/PS1 model was investigated. The effects on memory were examined using a hippocampus-dependent contextual test (fear conditioning (FC)), and synaptic connectivity was examined by electrophysiological measurements of LTP (long-term potential).

- 5 048112 ciations). Both tests showed abnormalities in the indicated mouse model under standard conditions. In addition, Phenotyper cells (Sylics) were used to exclude the possibility that SUL-138 causes atypical behavior after long-term oral administration.

All wild type (WT) and APP/PS1 mice were divided into two groups receiving either vehicle or SUL-138 via their food. Group size was 12 animals. Based on a mouse weight of ~30 g, food intake of ~5 g/day, and a required oral intake of 30 mg/day/kg, food pellets were sprayed with a solution of SUL-138 in water containing 0.0145% ethanol, at a SUL-138 content of 1 g per 5 kg of food. Food as vehicle was prepared by spraying the same volume of water containing 0.0145% ethanol.

Prior to the study, mice were subjected to long-term treatment ranging from 2.5 months (pre-pathology/memory impairment) to 6 months (age at which overt neuropathology and memory impairment occur).

FC: mice were exposed to the context for 2 min, after which they received a 0.7 mA foot shock. 30 s after the foot shock, mice were returned to their home cage. After 24 h, mice were placed in the same context and freezing was measured for 2 min.

LTP: Acute coronal hippocampal slices were stored in artificial cerebrospinal fluid (CSF) and LTP was measured after 3x100 Hz stimulation.

Phenotypers cages (provided by Sylics, Amsterdam, The Netherlands): Mice were housed in Phenotyper cages for 3 days, during which spontaneous behavior was assessed: activity, effects of light/dark cycles on behavior, adaptation, kinematics, light/dark phase transition profile, and defensive behavior.

Results.

General well-being was monitored and showed no differences between vehicle-treated and SUL-138-treated animals, with all groups showing similar weight gain.

Memory was assessed at 6 months of age by measuring freezing after learning a context.

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

As expected, when fed the control diet, APP/PS1 mice exhibited less freezing behavior than wild-type mice. After long-term treatment with SUL-138, memory in APP/PS1 mice was restored to wild-type levels. This suggests that SUL-138 is effective in preventing or alleviating Alzheimer's disease and/or its symptoms.

Interestingly, WT mice treated with SUL-138 also performed better on the memory task, indicating that SUL-138 is also effective in improving memory function in healthy mammals.

Figure 2 shows how SUL-138 enhances LTP maintenance in both WT and APP mice. Eight to 14 hippocampal slices per group (2A: WT Ctrl, WT SUL-138, 2B: APP Ctrl, APP SUL-138) were subjected to LTP evoked by 3x100 Hz (tetanus) stimulation for 1 s at 20 s intervals. Slopes were measured over 60 min. LTP was expressed as a percentage of baseline. All LTP data were analyzed in a blinded manner. LTP maintenance (30 to 60 min) was significantly (p<0.05) higher in SUL-138-treated animals (both WT and APP mice); Student's t-test*, P<0.05; Figure 2C.

Chronic treatment with SUL-138 did not cause differences in spontaneous behavior: activity, light/dark cycle effects on behavior, adaptation, kinematics, light/dark phase transition profile, and defensive behavior were assessed.

Conclusions.

These examples showed that SUL-138 enhances memory and LTP in both WT and APP/PS1 mice, and effectively restores memory and LTP in APP/PS1 mice to control levels.

The increase in both of these parameters reflects the general process of increased plasticity/LTP facilitation that is promoted by SUL-138. This finding suggests that SUL-138 may be useful in alleviating symptoms of neurological diseases that exhibit decreased synaptic strength or plasticity.

The effects of SUL-138 appear to be specific to memory enhancement, as the treatment did not induce atypical behavior in mice after 3 months of chronic treatment. In addition, measurements showed no differences in weight over 3 months of chronic oral treatment, which may indicate aversion or habituation to SUL-138-treated food or changes in basic physiological functions.

Finally, there were no problems with the animals' well-being throughout the experiment.

- 6 048112 or differences between groups.

Example 2.

Reduction in plaque burden in APP/PS1 mice following SUL-138 intervention.

APP/PS1 mice (n=10) and wild-type (WT, n=10) mice were treated with either vehicle or SUL-138. Mice were treated for 3 months beginning at 3 months of age with either SUL-138 or vehicle-treated food pellets. Mice were sacrificed at 6 months of age, the age at which (among other things) hippocampus-dependent memory impairment and marked plaque accumulation are expected.

Brains perfused with 4% PFA (paraformaldehyde), stored on sucrose, were cut into thin slices at 35 pM using a cryostat (-20°C; Leica). Hippocampal slices (n=2/animal; 5 animals/group) were washed 3 times for 10 min with 1x PBS (phosphate-buffered saline) and then incubated for 1 h in blocking solution (10 ml 1x PBS + 500 μl normal goat serum + 0.250 g bovine serum albumin + 20 μl Triton-100). Sections were incubated overnight with anti-beta-amyloid antibody (6E10) (ITK Diagnostics, 1:400), washed 3 x 10 min with 1x PBS, and then incubated with Alexa 488-labeled goat anti-mouse secondary fluorescent antibody (Sigma-Aldrich, 1:250) for 2 h. Sections were then washed 3 x 10 min with 1x PBS and mounted on glass slides.

Sections were imaged using a Zeiss Cell Discover 7 high-performance microscope with an LSM900 confocal head. Using Fiji, both hippocampi were selected separately for 5 animals per group (yellow line in Fig. 3A) and plaque number and size were measured (Fig. 3B, C). The mean plaque number and plaque size per animal were used for statistical analysis in GraphPad 8 using a one-tailed Student's t-test.

Three months of oral SUL-138 treatment resulted in a reduction in both plaque number (Fig. 3B; p=0.0138) and plaque size (Fig. 3C; p=0.0021) in APP/PS1 mice compared to vehicle-treated mice. No plaques were detected in wild-type animals treated with SUL-138 and vehicle.

These data, together with the memory enhancement with SUL-138 and the enhancement of synaptic transmission (long-term potentiation) in APP/PS1 mice observed in Example 1, indicate that SUL-138 is a potential therapeutic agent for Alzheimer's disease.

The bioavailability of SUL-138 in the brain appears to be high, thereby overcoming the problems of other mitochondria-targeting compounds and making it a more suitable treatment for future clinical use.

Example 3.

In vitro assays demonstrating that the compounds of the present invention are active.

Excitotoxicity is a process in which nerve cells are damaged or killed when levels of seemingly necessary and harmless neurotransmitters become abnormally high, leading to overstimulation of their receptors. Excitotoxicity may be associated with neurodegenerative diseases of the central nervous system, such as Alzheimer's disease.

In vitro assays for studying excitotoxicity employ well-characterized inducers of neuronal cell death (e.g., glutamate, dopamine, or NDMA (N-nitrosodimethylamine)) and quantification of the viability of stimulated neuronal-like cells. The SH-SY5Y cell line, derived from human neuroblastoma, can be differentiated in vitro to resemble morphologically and biochemically mature neurons. In addition, differentiated SH-SY5Y neuronal-like cells are sensitive to excitotoxicity induced by, among others, glutamate and dopamine.

In this current study, the efficacy of SUL-11, SUL-127, SUL-13, SUL-138 (and its major 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, while SUL-127 is trolox methyl ester. These two compounds were used as a reference.

The compounds used in this study are shown in Table 1.

- 7 048112

Table 1

Connection Chemical name Formula Structure MW Reference connections SlJL-11 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid CuHinOj p o X 250.3 SUL-I27 methyl 6-hydroxy-2,5,7,8-tetramethyl l-3,4-dihydro-2 [ I- 1-benzopyran-2-carboxylate WITH HO, 0 XQv 264.3 Compounds according to the present invention • • • SIJL-13 (S )-<y-hydroxy-2,5,7,8- tetramethyl-]Ch-((K}- piperidin-3-yl)chroman-2-carboxamide C.JTiNiO, I Ί 1 and ^{1 0} Μ Η 332.4 SUL-I38 (5)-(6-hydroxy-2,5,7,8- tetramethylchroman-2-yl X4(2-hydroxyethyl)piperazin1-yl)methanone CzuHauNiO* VYn 362,5 SUL- 13 8M2 4-(2,5-dihydroxy-3,4,6-trimethylphenyl)-2-hydroxy144-(2-hydroxyethyl)piperazin-1 ylX2-methylbutan-1-one _C3BH3 , _N , _OS 5 O 380,5 SUL-I5O ( R)-( 6-hydroxy-2,5,7,8- tetramethylchroman-2- yl Chpiperazin-1-yl }methanone C _ls JbNzOj 4 ⁰ ¼ 318.4 SUL-I5I (3)-(6-hydroxy-2,5,7,8- tetramethylchroman-2- silt Hpiperazine -1 -yl }methanone C _ls ]l _M N _z Oj 318.4

SH-SY5Y human neuroblastoma cells (ATCC #CRL-2266) were maintained in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin (#P4333, Sigma-Aldrich, St. Louis, MO) and passaged when the cultures reached 70% confluency. Before experiments, SH-SY5Y cells were differentiated by serum reduction (to 1%) and stimulation with 10 pM retinoic acid (#R7882, Sigma-Aldrich, St. Louis, MO) for 72 h. Differentiated SH-SY5Y cells were seeded at a density of 0.6 x 10 ⁵ cells/cm ² for all experiments.

Differentiated SH-SY5Y cells were preincubated with SUL compounds (dose range from 8 x 10’ ⁴ to 1 x 10“ ⁸ M) under standard culture conditions for 30 min and then stimulated with either l-glutamate (60 mM; #12843-0, Sigma-Aldrich, St. Louis, MO) or dopamine (100 μM; #H8502, Sigma, St. Louis, MO) for an additional 24 h. 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 h of culture. Cells were washed with warm PBS and solubilized with neutral red in acidic ethanol (1% acetic acid in 50% E1OH). Optical densities were recorded at 540 nm using a CLARIOStar Plus plate reader (BMG Labtech, Germany). Cell viability was normalized to optical densities measurements.

Claims (2)

the optical density of untreated cultures (100% viability) and the optical density measurements of cell-free samples (0% viability). All experiments were performed in triplicate per condition and averaged. Data from two separate experiments were used for evaluation in GraphPad Prism 8.0 (GraphPad Software Inc, California). Four-parameter nonlinear regression was used to determine the potency and ability of SUL compounds to reduce excitotoxicity induced by either l-glutamate or dopamine. The potency of SUL compounds to inhibit excitotoxicity was calculated as E _max = 100 x V( _treated ) - V( _vehicle )) / 100% - V( _vehicle )), where V is the observed viability and E _max is the maximal effect induced by the SEIL compound. No cellular toxicity in the form of decreased viability was observed when SUL compounds were used in the molar range shown in Table 1 below. 2. SH-SY5Y neuroblastoma cells were differentiated into neuron-like cells according to standard protocols and stimulated with 60 mM glutamate to induce excitotoxicity. Glutamate reduced the viability of SH-SY5Y cells from 100 ± 1.63% in vehicle-treated control cells to 55.4 ± 1.7% in SH-SY5Y cells exposed to glutamate for 24 h (p < 0.0001). Preincubation of differentiated SH-SY5Y cells with SEIL compounds (10 ^-3 to 10 ^-8 M) resulted in a dose-dependent increase in the viability of glutamate-exposed SH-SY5Y cells, although at different levels. Trolox and trolox methyl ester were clearly less effective than the other SEL compounds, as shown in Table 2 below. Differentiated SH-SY5Y neuroblastoma cells were stimulated with 150 pM dopamine to induce excitotoxicity. Dopamine reduced 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). Preincubation of differentiated SH-SY5Y cells with SEL compounds (10 ^-3 to 10 ^-8 M) resulted in a dose-dependent increase in the viability of dopamine-exposed SH-SY5Y cells, although with different potencies, as shown in Table 2. In this model, all compounds reduced cell viability at the 10 ^-3 M dose level. Table 2 Glutamate excitotoxicity Dopamine excitotoxicity Compound ECso (M) Emax (%) ECso (M) Emax (%) SEL-11 4.62 Yu' ⁶ 76.7 8.35 Yu' ⁶ 79.5 SUL-127 2.01 10' ⁵ 90.4 3.53 10' ⁶ 69.5 SUL-13 3.82 10' ⁶ 100.0 3.24 10' ⁷ 91.9 SUL-138 1.42 10' ⁶ 100.0 6.60 10' ⁷ 100.0 SUL-138M2 4.43 10' ⁶ 100.0 1.69 10' ⁶ 94.7 SUL-150 1.22 10' ⁷ 100.0 5.55 10' ⁸ 100.0 SUL-151 9.61 10' ⁸ 100.0 6.92 10' ⁸ 100.0 The results in the table above show that the SUL compounds of the present invention exhibit either an improved EC ₅₀ (i.e., are active at a lower concentration) and/or an improved E _max (i.e., toxicity recovery occurs at a higher level). Thus, this example shows that, in addition to SUL-138, the other claimed SUL compounds are also likely to have benefits related to improving memory function and/or reducing plaque formation; i.e., are generally effective in the treatment of Alzheimer's disease. A.'s reference experiment. Hippocampal tissue from wild-type and APP/PS1 mice was examined for prostaglandin synthase and thromboxane synthase A protein expression. Prostaglandin synthase-like peptides PTGS1, PTGES2, PTGES3, and PTGFS were detected in hippocampal tissue from both wild-type and APP/PS1 mice. Protein fragments of PTGS2, PTGDS, PTGES1, PTGIS, and TXA were not detected. SUL-138 treatment of wild-type or APP/PS1 mice did not alter protein expression of prostaglandin synthesizing enzymes. CLAUSE OF INVENTION Use of S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1 yl)methanone (SUL-138) or a pharmaceutically acceptable salt thereof for improving memory function and/or reducing plaque burden in a patient with Alzheimer's disease. -