Treating Dementia

Abstract

Provided herein are methods for treating, delaying the onset of, or ameliorating at least one symptom of dementia associated with β-amyloid (Aβ) accumulation, and methods for improving memory in a subject suffering from dementia associated with Aβ accumulation, comprising administering to a subject in need thereof an effective amount of an inhibitor of LIMK1, wherein the inhibitor comprises a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt thereof. Also provided are methods for reducing Aβ toxicity in neurons, comprising exposing neurons to an effective amount of an inhibitor of LIMK1, wherein the inhibitor comprises a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt thereof.

Description

[Technical field]

[0001] The present disclosure relates generally to methods of treating dementias associated with β-amyloid accumulation, in particular Alzheimer's disease, using inhibitors of LIM domain kinase 1 (LIMK1) as defined herein. [Background technology]

[0002] Alzheimer's disease is the most common form of senile and presenile dementia worldwide, accounting for more than 50% of all cases of dementia in people aged 65 years or older. Clinically, Alzheimer's disease is characterized by a gradual progressive decline in cognitive function, typically manifesting as loss of memory, intellectual abilities, and increased language impairment (dysphasia). On average, death occurs about 9 years after diagnosis. The incidence of Alzheimer's disease increases dramatically with age, and it is estimated that more than 50% of people aged 85 years or older worldwide suffer from Alzheimer's disease.

[0003] Alzheimer's disease is pathologically characterized by the deposition of β-amyloid peptide (Aβ) in extracellular plaques and the accumulation of the microtubule-associated protein tau in intraneuronal neurofibrillary tangles. At the cellular level, neuronal connections are progressively impaired, leading to synaptic dysfunction and loss prior to cell loss. These neuropathological changes occur in parallel with the progressive loss of memory.

[0004] Aβ is a peptide of 38-43 amino acids generated by cleavage from the neuronal transmembrane protein amyloid precursor protein (APP) by β-secretase and γ-secretase. Aβ is released into the extracellular space where it aggregates to form plaques. Aβ is harmful to neurons, causing pore formation that leads to disruption of intracellular calcium balance and loss of membrane potential, promoting apoptosis, causing synaptic loss, and disrupting the cytoskeleton. Increasing evidence indicates that soluble oligomers of Aβ cause the majority of Aβ toxicity and are responsible for cognitive dysfunction and decline. Summary of the Invention [Problem to be solved by the invention]

[0005] The prognosis for patients with Alzheimer's disease is poor and treatments are limited. There is a clear need to develop new methods for treating this debilitating disease and related forms of dementia and cognitive decline. [Means for solving the problem]

[0006] This disclosure is based on the inventors' discovery that genetic depletion and pharmacological inhibition of LIM domain kinase 1 (LIMK1) ameliorates memory deficits and neuronal network abnormalities in mouse models of Alzheimer's disease and protects mice from β-amyloid (Aβ) toxicity and excitotoxicity.

[0007] According to a first aspect of the present disclosure, there is provided a method for treating, delaying the onset of, or ameliorating at least one symptom of dementia associated with β-amyloid (Aβ) accumulation, comprising administering to a subject in need thereof an effective amount of an inhibitor of LIMK1, the inhibitor having formula (I): [ka] (In the formula, Z is selected from the group consisting of optionally substituted cycloalkylene, optionally substituted arylene, and optionally substituted aniline; R 1 , R 2 and R 3 is independently selected from the group consisting of H, halogen, nitro, cyano, hydroxyl, optionally substituted alkoxy, optionally substituted amine, optionally substituted alkyl, optionally substituted heteroalkyl, and optionally substituted alkenyl; Y is selected from the group consisting of O, S, NCN, NCS, and NSO2Me; Ar is selected from the group consisting of optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl), or a pharma- ceutically acceptable salt thereof.

[0008] In some embodiments, Z has the structure: [ka] (In the formula, R 4 , R 5 , R 6 and R 7 is the above R 1 , R 2 and R 3 has the same definition as X is CH or N; R 8 is H or optionally substituted alkyl.

[0009] In certain embodiments, the compound of formula (I) has the formula (Ia): [ka] where the variable moiety R 1 ~R 7 , X, Y and Ar are as defined above), or a pharma- ceutically acceptable salt thereof.

[0010] In an embodiment, the compound of formula (I) is a compound of formula (Ia) or a pharma- ceutically acceptable salt thereof: R 1 , R 3 , R 5 , R 6 and R 7 is H, R 2 is methyl, R 3 is (S)-methyl, X is N, Y is NCN, Ar is 3-bromophenyl.

[0011] The compound may be a selective inhibitor of LIMK1.The compound may be a specific inhibitor of LIMK1.

[0012] In an embodiment, the compound of formula (Ia) has the following structure: [ka] has.

[0013] In certain embodiments, the dementia is Alzheimer's disease.

[0014] The at least one symptom of dementia may include a clinical or pathological symptom. In certain embodiments, the at least one symptom may include memory deficits and / or abnormalities or disruptions in neuronal networks. The abnormalities or disruptions in neuronal networks may be associated with excitotoxicity or Aβ toxicity.

[0015] According to a second aspect of the present disclosure, there is provided a method of improving memory in a subject suffering from dementia associated with β-amyloid (Aβ) accumulation, comprising administering to a subject in need thereof an effective amount of an inhibitor of LIMK1, wherein the inhibitor comprises a compound of formula (I) as defined above or a pharma- ceutically acceptable salt thereof.

[0016] According to a third aspect of the present disclosure, there is provided a method of reducing Aβ toxicity in a neuron, comprising exposing the neuron to an effective amount of an inhibitor of LIMK1, the inhibitor comprising a compound of formula (I) as defined above or a pharma- ceutically acceptable salt thereof.

[0017] A fourth aspect of the present disclosure provides the use of an inhibitor of LIMK1 in the manufacture of a medicament for treating, delaying the onset of, or ameliorating at least one symptom of dementia associated with β-amyloid (Aβ) accumulation, wherein the inhibitor comprises a compound of formula (I) as defined above or a pharma- ceutically acceptable salt thereof.

[0018] A fifth aspect of the present disclosure provides the use of an inhibitor of LIMK1 in the manufacture of a medicament for improving memory in a subject suffering from dementia associated with β-amyloid (Aβ) accumulation, wherein the inhibitor comprises a compound of formula (I) as defined above or a pharma- ceutically acceptable salt thereof.

[0019] A sixth aspect of the present disclosure provides the use of an inhibitor of LIMK1 in the manufacture of a medicament for reducing Aβ toxicity in neurons, wherein the inhibitor comprises a compound of formula (I) as defined above or a pharma- ceutically acceptable salt thereof.

[0020] Aspects and embodiments of the present disclosure are herein described, by way of non-limiting example only, and with reference to the accompanying drawings. [Brief description of the drawings]

[0021] [Figure 1]Figure 1: Limk1 depletion prevents memory deficits and neuronal network abnormalities in a mouse model of Alzheimer's disease. (A) Left graph: Temporal improvement of escape latency was delayed in Limk1- / - mice and was significantly impaired in APP transgenic APP23 (APP23 / Limk1+ / +) mice compared to non-mutated littermates (Limk1+ / +). APP23 / Limk1- / - mice performed similarly to Limk1- / - mice, indicating improved learning. Right graph: Significant memory consolidation impairment during the probe test in Limk1- / -, APP23 / Limk1+ / + and APP23 / Limk1- / - mice compared to Limk1+ / + controls. n=8-10, *p<0.05, **p<0.01 (Student's t-test). From left to right: Limk1+ / +; Limk1- / -; APP23 / Limk1+ / +; APP23 / Limk1- / -. (B) APP23 / Limk1+ / + mice fail to learn the location of the escape platform 24 h after acquisition in a water T maze, whereas APP23 / Limk1- / - mice perform similarly to Limk1+ / + and Limk1+ / + with significantly fewer failures after 24 h. n=7 or 8, *p<0.05, **p<0.01 (ANOVA). The left column is acquisition, the middle column is 2 h after acquisition, and the right column is 24 h after acquisition for Limk1+ / +, Limk1- / -, APP23 / Limk1+ / +, and APP23 / Limk1- / -, respectively. (C) Left graph: Significant increase in spikes per minute in APP transgenic APP23 mice (APP23 / Limk1+ / +) compared to non-transgenic (Limk1+ / +) and Limk1 knockout (Limk1- / -) littermates during telemetry hippocampal electroencephalography (EEG) recordings in freely moving mice. Spike frequency was significantly lower in EEG recordings from APP23 / Limk1- / - mice compared to APP23 / Limk1+ / + littermates and was not significantly different from Limk1+ / + and Limk1- / - controls (ns). n=11, *p<0.05, ***p<0.001 (Student's t-test).Right graph: Representation of spike frequencies of EEG recordings in individual Limk1+ / +, Limk1- / -, APP23 / Limk1+ / + and APP23 / Limk1- / - mice statistically analyzed in the left graph. (D) Significant reduction in modulation index of cross frequency coupling in APP23 / Limk1+ / + mice compared to APP23 / Limk1- / - littermates and Limk1+ / + and Limk1- / - controls. n=8, ***p<0.001 (Student's t-test). For (C) and (D), from left to right: Limk1+ / +; Limk1- / -; APP23 / Limk1+ / +; APP23 / Limk1- / -. (E) Significant disruption of amplitude and phase in EEG recordings from APP23 / Limk1+ / + mice compared to APP23 / Limk1- / - littermates and Limk1+ / + and Limk1- / - controls, n=8, *p<0.05, **p<0.01 (ANOVA). [Diagram 2] Figure 1: Elevated Limk1 activity in human Alzheimer's disease and mouse models of Alzheimer's disease. (A) Representative immunohistochemical staining of human Alzheimer's disease (left) and human mutant APP transgenic APP23 mice (right) brains. Cells with accumulation of phosphorylated LIMK1 substrate ADF / cofilin (p-ADF / cofilin; red; arrows) around amyloid-β (Aβ) plaques (green) stained with Aβ antibody 6E10. Nuclei were stained with DAPI. Scale bar, 50 μm. (B) Quantification of the marked reduction in the number of ADF / cofilin aggregates in the hippocampus of aged APP23 / Limk1- / - mice compared to APP23 littermates. [Diagram 3] 1 shows that LIMK1 inhibitor (LIMKi) efficiently inhibits LIMK1 activity. In vitro LIMK1 activity assay demonstrates kinase inhibition by LIMKi at all concentrations tested (100 μM, 10 μM, 1 μM, 0.1 μM). DMSO (solvent) did not inhibit LIMK1 activity. [Figure 4]Figure 1: LIMK1 inhibition reduced susceptibility to seizure induction and alleviated neuronal network hypersynchrony and memory deficits in a mouse model of Alzheimer's disease. (A) Excitotoxic seizures were induced by acute intraperitoneal injection of 50 mg / kg body weight pentylenetetrazole (PTZ), followed by timing and scoring of seizure onset (0=no seizures to 7=end-stage status epilepticus). Mice were treated intraperitoneally with vehicle or 10 mg / kg body weight LIMK1 inhibitor (LIMKi) 30 min prior to seizure induction. Left graph: Delayed latency to onset of mild seizures (i.e., score <5) in LIMKi-treated mice (thick line) compared to vehicle controls. Only vehicle-treated mice develop severe seizure phase over time (i.e., score = 5). Right graph: Significant reduction in seizure severity in LIMKi-treated mice (right column) compared to vehicle controls. n=9, **p<0.001 (Student's t-test). (B) Composite hippocampal electroencephalography (EEG) recordings over 4 hours in non-transgenic (wt) and APP transgenic (APP23) mice, including 2 hours before (Pre) and 2 hours after (Post) LIMKi or vehicle administration. Time points of intraperitoneal LIMKi and vehicle administration are indicated by arrows. Left: Absence of obvious spiking activity before and after vehicle or LIMKi injection in wt mice. Frequent spikes before and after vehicle administration in APP23 mice. In contrast, LIMKi injection alleviates spiking activity in APP23 mice after injection. Right graph: Absence of significant change in spike frequency in vehicle-treated APP23 compared to pre- and post-spike counts and significant reduction in spike frequency in LIMKi-treated APP23 mice. n=8, *p<0.05 (Student's t-test). (C) A significant reduction in the modulation index of inter-frequency coupling in APP23 mice (vehicle) was corrected 1 h after LIMKi administration and was no longer different from wt controls (vehicle / LIMKi-treated). n=8, **p<0.01 (Student's t-test). (D) A significant disruption of amplitude and phase in EEG recordings in APP23 mice (vehicle) was corrected 1 h after LIMKi delivery and was no longer different from wt controls (vehicle / LIMKi-treated). n=8, *p<0.05 (ANOVA).(E) Chronic Limk1 inhibition reverses memory deficits in AD mice. APP23 mice develop memory deficits in the Morris Water Maze (MWM) paradigm at 3 months of age. ip treatment with 10 mg / kg LIMKi (3 or 4 times per week) was initiated at 3 months of age and continued for 4 months before MWM testing at 7 months of age. Left graph: vehicle-treated APP23 mice showed delayed learning compared to vehicle-treated controls. LIMKi-treated controls showed moderate delayed learning, but LIMKi treatment improved learning in APP23 mice to vehicle control levels. Right graph: vehicle-treated APP23 spent significantly less time in the target quadrant in the MWM probe test, indicating deficits in memory formation. LIMKi-treated APP23 showed no significant difference from controls. n=8-10, *p<0.05 (ANOVA). For the bar graphs in (C) and (E), from left to right: non-Tg+vehicle; non-Tg+LIMKi; APP23+vehicle; APP23+LIMKi. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, patent applications, published applications and publications, databases, websites, and other published materials referenced throughout this disclosure are incorporated herein by reference in their entirety unless otherwise specified. In the event of a plurality of definitions of a term, the definitions in this section prevail. When referring to a URL or other such identifier or address, it is understood that such identifiers may change and specific information on the Internet may shift, but equivalent information may be found by an Internet search. Reference to an identifier evidences the availability and public dissemination of such information.

[0023] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0024] In the context of this specification, the term "about" is understood to refer to a range of numbers that one of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[0025] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" or "comprising" are understood to mean the inclusion of a stated integer or step, or group of integers or steps, but not to the exclusion of any other integer or step, or group of integers or steps.

[0026] The term "optionally" is used herein to mean that a subsequently described feature may or may not be present, or that a subsequently described event or circumstance may or may not occur. Thus, the specification is understood to include and encompass embodiments in which a feature is present and embodiments in which a feature is not present, as well as embodiments in which an event or circumstance occurs and embodiments in which the event or circumstance does not occur.

[0027] The term "inhibitor", as used herein, refers to an agent that directly or indirectly reduces or inhibits at least one function or biological activity of a target molecule, such as LIMK1. The term "selective" and grammatical variations thereof are used herein to refer to an agent that inhibits a target molecule without substantially inhibiting the function of another molecule.

[0028] In the context of the present disclosure, the term "inhibit" and grammatical equivalents do not necessarily mean complete inhibition of a specified event, activity or function. Rather, inhibition may be to an extent and / or duration sufficient to produce a desired effect. Inhibition may be prevention, delay, reduction or otherwise impediment of an event, activity or function. Such inhibition may be of a magnitude and / or temporal nature. In certain contexts, the terms "inhibit" and "prevent" and variations thereof may be used interchangeably. Similarly, the terms "inhibit", "reduce" and "reduce" may be used interchangeably with respect to a level or value of a substance, phenomenon, function or activity in a second sample or at a second time point that is lower than the level or value of the substance, phenomenon, function or activity in a first sample or at a first time point. Reduction may be determined or measured subjectively or objectively, or according to statistical analysis methods recognized in the art.

[0029] The use of the term "associated with" herein describes a temporal, physical or spatial relationship between events, symptoms or conditions. Thus, for example, in the context of the present disclosure, dementia associated with Aβ accumulation means that the dementia is at least partially characterized or caused by, directly or indirectly, the accumulation, usually extracellular accumulation, of Aβ. The dementia may occur or begin upon accumulation of Aβ. Alternatively, Aβ accumulation and dementia may be separated in time such that the onset of dementia is minutes, hours, days, weeks, months or years after the onset of Aβ accumulation.

[0030] As used herein, the terms "treat", "treatment" and grammatical equivalents refer to any and all applications of improving the described neurodegenerative disease, preventing, retarding or slowing the establishment of the disease, or otherwise preventing, hindering, retarding or reversing the progression of the disease. Thus, terms such as "treat" should be considered in their broadest context. For example, treatment does not necessarily mean that the patient is treated until complete recovery. In cases where a disease exhibits or is characterized by multiple symptoms, treatment or prevention does not necessarily have to improve, prevent, hinder, retard or reverse all of the symptoms, but may prevent, hinder, retard or reverse one or more of the symptoms.

[0031] As used herein, the term "effective amount" includes within its meaning a non-toxic but sufficient amount or dose of an agent or compound to produce the desired effect. The exact amount or dose required will vary from subject to subject, depending on factors such as the species being treated, the age, size, weight and general condition of the subject, the severity of the disease or condition being treated, the specific agent being administered, and the method of administration. For this reason, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0032] The term "subject" as used herein refers to a mammal, including humans, primates, livestock animals (e.g., sheep, pigs, cows, horses, donkeys), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs), show and show animals (e.g., horses, livestock, dogs, cats), companion animals (e.g., dogs, cats), and captive wild animals. Preferably, the mammal is a human or a laboratory test animal. Even more preferably, the mammal is a human.

[0033] The LIM domain family of protein kinases includes LIM domain kinase 1 (LIMK1) and LIM domain kinase 2 (LIMK2). LIM kinases are serine / threonine protein kinases that affect the organization of the actin cytoskeleton by binding to actin and regulating the activity of cofilin proteins through phosphorylation. Neuronal LIMK1, the most highly expressed in the brain, is an established regulator of synapse morphology. Prior to the present invention, little was known about the functional role of LIMK1 in neurodegenerative diseases. Human LIMK1 is a 647 amino acid protein (UniProt protein database accession number P53667) produced from the LIMK1 gene encoded on chromosome 7. Alternative splicing generates four isoforms of LIMK1.

[0034] As exemplified herein, the inventors have demonstrated the effects of Limk1 depletion and LIMK1 inhibition on memory, neuronal network activity, and neuropathological changes of Aβ formation in transgenic mice. The data described herein establish a novel role for LIMK1 in Alzheimer's disease and provide a novel potential therapeutic approach to ameliorate functional impairment in Alzheimer's disease and related forms of dementia by inhibiting LIMK1.

[0035] In one aspect, the disclosure provides a method of treating, delaying the onset of, or ameliorating at least one symptom of dementia associated with β-amyloid (Aβ) accumulation, comprising administering to a subject in need thereof an effective amount of an inhibitor of LIMK1, the inhibitor having formula (I): [ka] (In the formula, Z is selected from the group consisting of optionally substituted cycloalkylene, optionally substituted arylene, and optionally substituted aniline; R 1 , R 2 and R 3is independently selected from the group consisting of H, halogen, nitro, cyano, hydroxyl, optionally substituted alkoxy, optionally substituted amine, optionally substituted alkyl, optionally substituted heteroalkyl, and optionally substituted alkenyl; Y is selected from the group consisting of O, S, NCN, NCS, and NSO2Me; Ar is selected from the group consisting of optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl, or a pharma- ceutically acceptable salt thereof.

[0036] In some embodiments, Z has the structure: [ka] (In the formula, R 4 , R 5 , R 6 and R 7 is the above R 1 , R 2 and R 3 has the same definition as X is CH or N; R 8 is H or optionally substituted alkyl.

[0037] "Alkyl" refers to monovalent alkyl groups which may be linear or branched and preferably have 1 to 10 carbon atoms, or more preferably 1 to 6 carbon atoms. Examples of such groups include methyl, ethyl, n-isopropyl, isopropyl, n-butyl, isobutyl, n-hexyl, and the like.

[0038] "Alkenyl" refers to a monovalent aliphatic carbocyclic group having at least one carbon-carbon double bond, which may be linear or branched, and preferably having 2 to 10 carbon atoms. Examples of such groups include vinyl or ethenyl groups (-CH=CH2), n-propenyl (-CH2CH=CH2), isopropenyl (-C(CH3)=CH2), but-2-enyl (-CH2CH=CHCH3), and the like.

[0039] "Alkynyl" refers to a monovalent aliphatic carbocyclic group having at least one carbon-carbon triple bond, which may be linear or branched, and preferably having 2 to 10 carbon atoms. Examples of such groups include acetylene or ethynyl groups (-C≡CH), propargyl (-CHC≡CH), and the like.

[0040] "Aryl" refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl, anthracenyl), preferably having 6 to 14 carbon atoms. Examples of aryl groups include phenyl, naphthyl, anthracenyl, and the like.

[0041] "Alkoxy" and "aryloxy" refer respectively to the "-O-alkyl" and "-O-aryl" groups in which the alkyl and aryl groups are as defined above.

[0042] "Halogen" refers to fluoro, chloro, bromo and iodo groups.

[0043] "Heteroaryl" refers to a monovalent aromatic carbocyclic group preferably having from 6 to 14 carbon atoms and from 1 to 4 heteroatoms within the ring and independently selected from oxygen, nitrogen, and sulfur. Such heteroaryl groups can have a single ring (e.g., pyridyl, pyrrolyl, or furyl) or multiple condensed rings (e.g., indolyl and benzofuryl).

[0044] "Heterocyclyl" refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, preferably having 4 to 10 carbon atoms and 1 to 4 heteroatoms independently selected from nitrogen, sulfur, oxygen, selenium, and phosphorus.

[0045] Examples of heterocyclyl and heteroaryl groups include, but are not limited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0046] As used herein, the term "optionally substituted" in connection with a particular group is taken to mean that the group may be unsubstituted or further substituted with one or more groups selected from hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, alkylaryl, aryl, aryloxy, carboxyl, acylamino, cyano, halogen, nitro, sulfate, phosphate, phosphine, heteroaryl, heterocyclyl, oxyacyl, oxyacylamino, aminoacyloxy, trihalomethyl, and the like.

[0047] Examples of particularly suitable optional substituents include F, Cl, Br, I, CH3, CH2CH3, OH, OCH3, CF3, OCF3, NO2, NH2, COCH3, and CN.

[0048] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound, including pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of the compounds of formula (I) can be prepared from inorganic or organic acids. Examples of inorganic acids include hydrochloric acid, sulfuric acid, and phosphoric acid. Examples of organic acids include aliphatic, alicyclic, aromatic, heterocyclic carboxylic and sulfonic organic acids, such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, guluronic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, maleic acid, alkylsulfonic acid, and arylsulfonic acid. When the compounds of formula (I) are solids, the compounds and their salts may exist in one or more different crystalline or polymorphic forms, all of which are intended to be included within the scope of formula (I).

[0049] In certain embodiments, the compound of formula (I) has the formula (Ia): [ka] where the variable moiety R 1 ~R 7 , X, Y and Ar are as defined above), or a pharma- ceutically acceptable salt thereof.

[0050] In an embodiment, the compound of formula (I) is a compound of formula (Ia) or a pharma- ceutically acceptable salt thereof: R 1 , R 3 , R 5 , R 6 and R 7 is H, R 2 is methyl, R 3 is (S)-methyl, X is N, Y is NCN, Ar is 3-bromophenyl.

[0051] In an exemplary embodiment of the disclosure, the compound of formula (Ia) has the following structure (also referred to herein as LIMKi): [ka]

[0052] The inhibitor may be a specific inhibitor of LIMK1 or may be selective for LIMK1. Therefore, the inhibitor may also exhibit inhibitory activity against LIMK2. The inhibitor may exhibit inhibitory activity against one or more isoforms of LIMK1.

[0053] The embodiments of the present disclosure are applicable to the treatment of dementia associated with β-amyloid (Aβ) accumulation. Treatment can result in the improvement of one or more clinical symptoms of dementia, for example, improvement of memory, including reduction or prevention of memory deficits or memory impairment associated with dementia. Treatment can result in the improvement of one or more pathological features of dementia, for example, reduction of Aβ toxicity, or abnormality or disruption of neuronal network.

[0054] Typically, the dementia is Alzheimer's disease. Alternatively, the dementia may include or be associated with amyloidopathy (Alzheimer's dementia) or cerebral amyloid angiopathy. The subject may have been diagnosed with dementia or may have one or more symptoms of dementia. The subject with dementia may be asymptomatic, and the treatment of the present disclosure delays the onset of the disease or its symptoms. Symptoms of dementia include memory deficits and neuronal abnormalities or destruction characteristic of or associated with the disease. The subject may be susceptible to dementia or at risk of developing dementia.

[0055] The embodiments of the present disclosure contemplate delivery of the LIMK1 inhibitor to a subject in need of treatment by any suitable means, typically in the form of a pharmaceutical composition, which may include one or more pharma- ceutically acceptable carriers, additives, or diluents. Such compositions may be administered by any convenient or suitable route, for example parenterally (e.g., intraperitoneally, subcutaneously, intraarterially, intravenously, intramuscularly, intrathecally, intracerebrally, intraocularly), orally (including sublingually), nasally, mucosally, or topically. In situations where it is necessary to deliver an appropriate concentration of the molecule directly to the site in the body to be treated, administration may be local rather than systemic. Local administration provides the ability to deliver a very high local concentration of the molecule to the required site, thus avoiding exposure of other organs of the body to the vector and the molecule, and thus is suitable for achieving the desired therapeutic or prophylactic effect while potentially reducing side effects.

[0056] As will be understood by those skilled in the art, the selection of pharma- ceutically acceptable carrier or diluent depends on the route of administration and the nature of the pathology and subject to be treated.Specific carrier or diluent and route of administration can be easily determined by those skilled in the art.Carrier or diluent and route of administration should be carefully selected so that the activity of the compound is not depleted during preparation of the formulation and the compound can reach the site of action intact.

[0057] Examples of pharma- ceutically acceptable carriers or diluents are demineralized or distilled water; saline; peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, arachis oil, oils of vegetable origin such as coconut oil or coconut oil; silicone oils including polysiloxanes such as methylpolysiloxane, phenylpolysiloxane and methylphenylpolysiloxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methylcellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols such as ethanol or isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; gum tragacanth or gum arabic, and petrolatum. Typically, the carrier(s) will comprise from 10% to 99.9% by weight of the composition.

[0058] Those of skill in the art can readily determine the appropriate formulation of the compound to be administered using conventional approaches. Formulation and administration techniques can be found, for example, in Remington (1980) Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition and Niazi (2009) Handbook of Pharmaceutical Manufacturing Formulations, Informa Healthcare, New York, second edition, the contents of which are incorporated herein by reference in their entireties.

[0059] The identification of preferred pH ranges (if appropriate) and suitable excipients is routine in the art, for example as described in Katdare and Chaubel (2006) Excipient Development for Pharmaceutical, Biotechnology and Drug Delivery Systems (CRC Press).

[0060] In some embodiments, the compound of formula I is formulated for oral administration in a dosage form such as a tablet, pill, capsule, liquid, gel, syrup, slurry, suspension, lozenge, etc. for oral ingestion by a subject. In certain embodiments, the compound of formula I is formulated for oral administration in a solid dosage form such as a tablet, pill, lozenge, or capsule. In such embodiments, the pharmaceutically acceptable carrier may include a number of additives including, but not limited to, diluents, disintegrants, binders, lubricants, etc.

[0061] Suitable diluents (also referred to as "fillers") include, but are not limited to, lactose (including lactose monohydrate, spray dried monohydrate, anhydrous, etc.), mannitol, xylitol, dextrose, sucrose, sorbitol, compressible sugar, isomalt, microcrystalline cellulose, powdered cellulose, starch, pregelatinized starch, dextrates, dextran, dextrin, dextrose, maltodextrin, calcium carbonate, dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate, magnesium carbonate, magnesium oxide, poloxamer, polyethylene oxide, hydroxypropyl methylcellulose, silicates (e.g., silicon dioxide), polyvinyl alcohol, talc, and combinations thereof.

[0062] Suitable disintegrants include, but are not limited to, sodium carboxymethylcellulose, pregelatinized starch, calcium carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, sodium starch glycolate, microcrystalline cellulose, lower alkyl-substituted hydroxypropylcellulose, starch, sodium alginate, and combinations thereof.Suitable binders include, but are not limited to, microcrystalline cellulose, gelatin, sugar, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropylcellulose, hydroxypropylmethylcellulose, and combinations thereof.Suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, polyethylene glycol, and combinations thereof.

[0063] Pharmaceutical preparations for parenteral administration include aqueous solutions of the compound of formula I in water-soluble form. Furthermore, suspensions of the compound of formula I can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may contain suitable stabilizers or agents that increase the solubility of the compound, allowing for the preparation of highly concentrated solutions.

[0064] Sterile solutions can be prepared by combining the required amount of the compound in an appropriate solvent with other additives as required, as described above, followed by sterilization, such as filtration. In general, dispersions are prepared by incorporating the various sterilized active compounds into a sterile vehicle containing a basic dispersion medium and the required additives as described above. Sterile dry powders can be prepared by vacuum drying or freeze-drying a sterile solution containing the active compound and other required additives as described above.

[0065] Pharmaceutical forms suitable for injection use include sterile injectable solutions or dispersions and sterile powders for the preparation of sterile injectable solutions. Such forms must be stable under the conditions of manufacture and storage and can be protected against reduction, oxidation and microbial contamination. For injection, the composition can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0066] It is understood that the specific dosage level of the composition of the present invention for any particular subject will depend on a variety of factors, including, for example, the activity of the inhibitor used, the half-life of the inhibitor, the age, weight, general health and diet of the individual being treated, the timing of administration, the excretion rate, and combination with any other treatments or therapies. Single or multiple administrations can be performed with the dosage level and pattern selected by the treating physician. A wide range of dosages can be applicable. For example, about 0.1 mg to about 1 mg of the agent per kg of body weight per day can be administered, taking into account the patient. The dosage regimen can be adjusted to provide the optimal therapeutic response. For example, several divided doses can be administered daily, weekly, monthly or at other suitable time intervals, or the dosage can be proportionally reduced as indicated by the exigencies of the situation.

[0067] In exemplary embodiments of the present disclosure, it is contemplated that the inhibitor may be administered to a subject once a day or less than once a day, for example, once every two or three days, over the course of treatment required to achieve the desired result. Administration may be continuous, for example, every day or every other day, or intermittent, with dose intervals determined by the treating medical professional depending on the subject's response to treatment and the subject's progress over the course of treatment.

[0068] The present invention contemplates combination therapy in which the LIMK1 inhibitors described herein are co-administered with other suitable agents that can promote desired therapeutic or prophylactic results. "Co-administered" means co-administration in the same formulation, or in two different formulations via the same or different routes, or sequential administration by the same or different routes. "Simultaneously" means that the active agents are administered at substantially the same time. "Sequential" administration means a time difference of seconds to minutes, hours or days between the administration of the agents. Administration may be in any order.

[0069] Each embodiment described herein is intended to apply mutatis mutandis to every embodiment unless specifically stated otherwise.

[0070] Reference in this specification to any prior publication (or information derived therefrom) or to any known matter is not, and should not be taken as, an acknowledgement or admission, or any form of suggestion, that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavour to which this specification pertains.

[0071] The present disclosure will now be described with reference to the following specific examples, which should not be construed in any way as limiting the scope of the disclosure. EXAMPLES

[0072] The following examples are illustrative of the present disclosure and should not be construed in any way as limiting the general nature of the disclosure described throughout the specification.

[0073] General method Mouse. APP23 expresses the human Swedish mutant (K595N / M596L) amyloid-β precursor protein (APP) in neurons (Sturchler-Pierrat et al., 1997, Proc Natl Acad Sci USA 94, 13287-13292). Limk1 - / -Mice were previously described (Meng et al., 2002, Neuron 35, 121-133). All mice were on a pure C57Bl / 6 background. Mice were housed in standard individually ventilated cages with a 12-h light / dark cycle and standard chow and water available ad libitum. Both male and female mice were used unless otherwise specified. Investigators were blinded to genotype until data analysis was completed. All experiments were approved by the Macquarie University Animal Ethics Committee.

[0074] Memory testing. Memory of mice was assessed in the T-maze paradigm and Morris water maze following published protocols (Ittner et al., 2010, Cell 142, 387-397; Ke et al., 2015, Acta Neuropathol 130, 661-678). Chronic treatment of mice was performed by intraperitoneal injection of 10 mg / kg LIMKi or vehicle (0.5% hydroxypropylmethylcellulose 606 (HPMC 606), 0.5% polyvinylpyrrolidone k17 (PVP k17) and 0.1% tween 80) three or four times a week, starting at 3 months of age and continuing until 7 months of age. All mice were acclimated to the testing room before testing.

[0075] Electroencephalography. Hippocampal EEG recordings were performed using implanted telemetry electrodes (DSI) as previously described (Ittner et al., 2014, Acta neuropathologica communications 2, 149). All recordings were performed 1 week after transmitter implantation with mice individually housed in their home cages. Compound administration was performed intraperitoneally with mice temporarily restrained. The vehicle for the LIMK1 inhibitor LIMKi was hydroxypropyl methylcellulose 606 (HPMC 606) with polyvinylpyrrolidone k17 (PVP k17) and tween 80 at 0.5%, 0.5%, and 0.1%, respectively. Spike analysis was performed using the Neuroscore software module (DSI) as previously described (Ittner et al., 2016, Science 354, 904-908). Inter-frequency coupling of theta and gamma waves was performed as previously described (Ittner et al., 2016, Science 354, 904-908).

[0076] Histological staining. Staining of paraffin-embedded brain tissue was performed as previously described (Ittner et al., 2010, Cell 142, 387-397). Primary antibodies were against Aβ (6E10; Covance), p-ADF / cofilin (Sigma) and ADF / cofilin (Sigma). Nuclei were visualized with DAPI (Molecular Probes). ADF aggregates were counted in serial hippocampal sections.

[0077] LIMK1 activity assay. LIMK1 activity in the absence or presence of different concentrations of LIMKi (or DMSO) was determined using a commercially available assay according to the manufacturer's instructions (Promega).

[0078] Seizure model. Excitotoxic seizures were induced in mice by intraperitoneal injection of 50 mg / kg (body weight) pentylenetetrazole, followed by observation in a square area (40 cm × 40 cm). Seizure scoring was performed as previously described (Ittner et al., 2010, Cell 142, 387-397). For LIMKi-treated mice, scoring was adjusted by combining severe seizure scores of 5 to 7 into a single score of 5. For both scales, mild to moderate seizures are reflected by a score below 5.

[0079] Statistical Analysis. All statistical analyses were performed using Prism 7 software (GraphPad). All values ​​are presented as mean and standard error. A p-value of <0.05 was considered significant.

[0080] Example 1 - Limk1 depletion prevents memory deficits and neuronal network abnormalities Alzheimer's disease mouse models with transgenic expression of human mutant APP exhibit memory deficits, Aβ pathology, and early death, which are associated with excitotoxicity (Ittner and Gotz, 2011, Nat Rev Neurosci 12:67-72). - / - Mice show increased long-term potentiation but no fundamental alterations in memory formation (Meng et al., 2002, Neuron 35:121-133). To determine whether Limk1 functionally contributes to the deficits in Alzheimer's disease mice, we examined the function of Limk1 in Aβ-forming APP23 mice and in Limk1 - / - The strain was crossed with APP23 / Limk1 - / - Mice were obtained. Memory was assessed by standard Morris Water Maze (MWM) and T-maze tests. APP23 exhibited significant learning impairment in the MWM paradigm compared to non-transgenic littermate controls, and Limk1 - / - Interestingly, APP23 / Limk1 mice showed a tendency towards delayed learning. - / - The mouse is Limk1 - / -The APP23 / Limk1 mice showed a similar improvement in learning profile to the control mice (Figure 1A). These findings were confirmed by a T-maze test. Although acquisition performance was comparable across all genotypes, APP23 mice were unable to memorize the target arm in subsequent test sessions, whereas APP23 / Limk1 mice showed a similar improvement in learning profile to the control mice (Figure 1B). - / - , Limk1 - / - Transgenic and non-transgenic mice showed comparable memory in this simple task (Fig. 1B). Taken together, Limk1 depletion prevented the severe memory deficits in APP23 mice.

[0081] APP23 mice exhibit non-convulsive, subclinical seizure activity in electroencephalography (EEG) recordings, as well as disruption of cross-frequency coupling (CFC) of theta phase modulation of gamma power during spike-free episodes of EEG recordings (Ittner et al., 2014, Acta Neuropath Comm 2:149), a modality that is implicated in memory formation, including in humans. We used APP23 / Limk1 for recordings in freely moving mice. - / - Mouse and APP23 / Limk1 + / + , Limk1 - / - and Limk1 + / + Littermates were implanted with hippocampal electrode telemetry EEG transmitters. APP23 / Limk1 + / + Mice exhibited frequent hypersynchronous discharges in EEG recordings, whereas Limk1 - / - and Limk1 + / + In littermates, such events were hardly detected (Fig. 1C). - / - Mice showed a significant reduction in spike counts, but this was not significantly different from controls. + / + In mice, CFC during spike-free episodes was disrupted (data not shown). In contrast, APP23 / Limk1 - / - The mouse is Limk1 - / - and Limk1 + / +It showed the same CFC at 8 Hz as that detected in the littermate recordings. + / + A significant reduction in the modulation index (Figure 1D) and amplitude phase (Figure 1E) of APP23 / Limk1 mice was observed. - / - Limk1 in mice - / - and Limk1 + / + The results were normalized to the levels of littermates. In conclusion, the neuronal circuit abnormalities in APP23 mice were alleviated by Limk1 knockout.

[0082] Example 2 - Reduced pathology and Aβ toxicity in Limk1-deficient APP23 mice In brain sections from individuals with Alzheimer's disease, neurons near Aβ plaques stain prominently for phosphorylated ADF / cofilin (p-ADF / cofilin), a downstream target of LIMK1 (Figure 2A). Similarly, neurons near Aβ plaques in the brains of APP23 mice were labeled for p-ADF / cofilin. Similar intensive labeling of neurons was not seen in non-transgenic mice and human controls (data not shown). APP23 / Limk1 with ADF / cofilin antibody + / + Staining of the brain revealed numerous rod-shaped structures in the hippocampus (Fig. 2B). In contrast, APP23 / Limk1 - / - In the brain, such rod-like structures were hardly observed. - / - and Limk1 + / + The brain contained no such inclusions (data not shown).

[0083] Example 3 - Pharmacological LIMK1 inhibition protects neurons from Aβ toxicity and attenuates circuitry abnormalities in APP23 mice APP23 / Limk1 - / -Given the improvements seen in mice (Example 1), we determined whether treatment with a LIMK1 inhibitor could similarly prevent Aβ toxicity and ameliorate the deficits in APP23 mice. C57Bl / 6 primary mouse neurons were treated with Compound 1 (also referred to herein as LIMKi) shown below together with 0.05 μM oligomeric Aβ (oAβ) and dendritic spine loss and cell viability were determined. [ka]

[0084] Aβ-treated neurons exhibited spine loss and reduced viability. However, pretreatment with Compound 1 provided dose-dependent protection of cultured neurons from Aβ-induced dendritic spine loss and death. Next, we determined the potential short-term effects of Compound 1 in vivo. Administration of 10 mg / kg Compound 1 30 min before inducing excitotoxic seizures with PTZ significantly reduced the mean seizure severity and increased the latency to the occurrence of severe seizures in C57Bl / 6 mice (Figure 4A). Next, we tested the effects of acute Compound 1 administration on neuronal network activity in APP23 mice. Recordings before drug administration showed frequent hypersynchronous discharges in APP23 mice (Figure 4B). Similarly, continued recordings for 2 h after vehicle injection showed high spike activity. However, 30 min after administration of Compound 1, hypersynchrony was significantly reduced in Compound 1-treated APP23 mice compared to vehicle-treated APP23 mice and compared to pre-injection recordings (Figure 4B). Furthermore, CFC was re-established 1 h after administration of Compound 1 (data not shown). The significant reduction in modulation index (Figure 4C) and amplitude phase (Figure 4D) was normalized by Compound 1 administration in APP23 mice. Thus, inhibition of LIMK1 by Compound 1 provided protection from Aβ toxicity in neurons and excitotoxicity in vivo. Furthermore, treatment of APP23 with Compound 1 corrected the circuitry abnormalities, including CFC.

[0085] Example 4 - Pharmacological LIMK1 inhibition improves memory deficits in APP23 mice From the short-term effects of Compound 1 in neurons and APP23 mice (Example 3), we tested whether long-term treatment could improve memory deficits in APP23 mice. Three-month-old APP23 mice were treated for 4 months and then subjected to memory testing in the MWM. APP23 mice exhibit significant memory deficits at 3 months of age, which progress with age (Ittner et al., 2016, Science 354:904-908). An intermittent treatment schedule was chosen to avoid potential inhibition of astrocyte-mediated Aβ clearance, which could mask the effects of treatment. At 7 months of age, vehicle-treated APP23 mice exhibited significant memory deficits compared to vehicle-treated non-transgenic mice (Figure 4E). Limk1 - / - As in mice, chronic treatment with Compound 1 delayed learning in non-transgenic mice. Compound 1 administration improved memory in APP23 mice after 4 months of treatment.

Claims

1. In the manufacture of a medicament for treating, delaying the onset of, or ameliorating at least one symptom of dementia associated with β-amyloid (Aβ) accumulation, a compound of formula (I): 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof, The compound of formula (I) has the following structure: 【Chemistry 2】 To have, to use.

2. The use according to claim 1, wherein the dementia is Alzheimer's disease.

3. 2. The use according to claim 1, wherein the at least one symptom of dementia comprises memory deficits and / or abnormalities or disruptions in neural networks.

4. The use according to claim 3, wherein the abnormality or disruption of the neuronal network is associated with excitotoxicity or Aβ toxicity.

5. In the manufacture of a medicament for improving memory in a subject suffering from dementia associated with β-amyloid (Aβ) accumulation, 【Chemistry 3】 or a pharma- ceutically acceptable salt thereof, The compound of formula (I) has the following structure: 【Chemistry 4】 To have, to use.

6. In the manufacture of a medicament for reducing Aβ toxicity in neurons, 【Chemistry 5】 or a pharma- ceutically acceptable salt thereof, The compound of formula (I) has the following structure: 【Chemistry 6】 To have, to use.

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